Download Use of Antibiotic Resistance Marker Genes in

ENVIRONMENTAL RISK MANAGEMENT AUTHORITY
NGÄ KAIWHAKATÜPATO WHAKARARU TAIAO
Use of Antibiotic Resistance
Marker Genes in
Genetically Modified Organisms
Deborah Read
ERMA New Zealand
December 2000
IBSN 0-478-21515-0
$9.95 excl GST
1
Foreword
This is the first in a series of reference reports on key HSNO issues, which ERMA New Zealand
wishes to produce. The idea behind this so called ‘generic issues programme’ is that we need to take
time to properly research and consider matters which affect many HSNO applications. There either
isn’t the time or resourcing to do so in the context of individual applications, or the relevance is very
restricted for individual applications.
The current report provides a good example. Antibiotic resistance marker genes have been
commonly used in the development of GMOs, and the issue of what concerns this might raise
comes up repeatedly. The thorough analysis in this report should enable such concerns in the future
to be considered more quickly, more thoroughly but at lower cost than in the past. This makes sense
for everyone.
The pattern followed in the development of these reports will differ from case to case. However,
there is a commitment to making the process open, to enable a wide range of people to contribute or
comment if they have views or information to offer.
A second project in this series is underway, and it is to develop a better approach to dealing with
Māori cultural concerns over genetic modification. The subject matter is quite different and the
project will thus develop in its own way.
Regretfully no more projects in this series will be undertaken for the time being because we do not
have sufficient funding for this. However I am personally very keen to see this programme restarted
if funding issues can be restored.
Bas Walker
Chief Executive
ER-GI-01-1 12/00
2
Acknowledgements
This report was prepared by Dr Deborah Read, Senior Public Health Advisor,
ERMA New Zealand.
The report was externally peer reviewed by:
Dr Marion Healy,
Australia New Zealand Food Authority
Associate Professor Brian Jordan,
Massey University
Dr Maggie Brett,
Limited, and
Institute of Food, Nutrition and Human Health,
Kenepuru Science Centre, Institute of Environmental Science and Research
Professor Barry Scott,
Institute of Molecular BioSciences, Massey University.
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Table of Contents
Foreword ........................................................................................................................ 2
Acknowledgements ....................................................................................................... 3
Table of Contents........................................................................................................... 4
Terms of Reference ....................................................................................................... 9
Preface.......................................................................................................................... 10
Method .......................................................................................................................... 10
Summary ...................................................................................................................... 11
Evaluation of the implications of the use of antibiotic resistance marker genes in genetically
modified organisms ........................................................................................................................ 11
Antibiotics in New Zealand........................................................................................................ 11
Horizontal gene transfer ............................................................................................................ 12
Antibiotic resistance genes and human health....................................................................... 12
Antibiotic resistance marker genes and the environment ..................................................... 14
Comparative analysis of the risks of antibiotic resistance occurring by other means .......... 14
Pros and cons of alternative selectable markers, including removal of antibiotic resistance
marker genes from GMOs for release ........................................................................................... 16
Recommendations of agencies and their basis about the use of antibiotic resistance marker
genes ................................................................................................................................................ 16
Conclusions about the use of antibiotic resistance marker genes in GMOs, in the context of
applications under Part V of the Hazardous Substances and New Organisms (HSNO) Act
1996 ................................................................................................................................................... 17
1.
Introduction ......................................................................................................... 18
What is gene technology? .............................................................................................................. 18
What is a selectable marker? ......................................................................................................... 19
Antibiotic resistance marker genes............................................................................................... 19
What antibiotic resistance genes are used as markers? ............................................................ 20
Kanamycin resistance ............................................................................................................... 20
Hygromycin resistance .............................................................................................................. 22
4
Streptomycin resistance............................................................................................................ 22
Ampicillin resistance ................................................................................................................. 23
Others .......................................................................................................................................... 23
2.
Antibiotics and antibiotic resistance ................................................................. 25
What are antibiotics used for? ....................................................................................................... 25
Mechanisms of antibiotic resistance............................................................................................. 25
Development of antibiotic resistance............................................................................................ 25
The public health impact of antibiotic resistance ........................................................................ 27
Antibiotics in New Zealand............................................................................................................. 27
Aminoglycosides........................................................................................................................ 27
Other antibiotics ......................................................................................................................... 29
Antibiotic resistance in New Zealand ............................................................................................ 30
Is antibiotic resistance lost? .......................................................................................................... 31
3.
Antibiotic resistance marker genes in food ...................................................... 32
Introduction...................................................................................................................................... 32
The concept of substantial equivalence ....................................................................................... 32
The general basis for assessment of the potential health impact of marker genes and their
gene products .................................................................................................................................. 33
Antibiotic resistance genes and food safety ................................................................................ 33
Direct consequences of the antibiotic resistance gene ......................................................... 33
Direct consequences of the gene product encoded by the antibiotic resistance gene ..... 34
Indirect consequences of the effects of the antibiotic resistance gene or its gene product
...................................................................................................................................................... 36
Horizontal gene transfer in humans .............................................................................................. 37
Potential gene transfer to oral micro-organisms .................................................................... 37
Potential gene transfer to gut epithelial cells or micro-organisms....................................... 37
Inactivation of antibiotic by the gene product ........................................................................ 42
4.
Horizontal gene transfer mechanisms in bacteria............................................ 44
5
Transduction .................................................................................................................................... 44
Conjugation...................................................................................................................................... 45
Transformation ................................................................................................................................ 45
Evidence for gene transfer between bacteria............................................................................... 46
Prevalence of antibiotic resistance genes.................................................................................... 47
5.
Ecological issues ................................................................................................ 49
Introduction...................................................................................................................................... 49
Antibiotic resistance and weediness ............................................................................................ 49
Gene transfer .............................................................................................................................. 49
Antibiotics in soil........................................................................................................................ 50
Pleiotrophic effects ......................................................................................................................... 51
Use of antibiotic as a herbicide...................................................................................................... 51
Horizontal gene transfer in the environment................................................................................ 52
Prerequisites for transformation .............................................................................................. 53
Persistence of DNA .................................................................................................................... 53
Transformation of soil micro-organisms ................................................................................. 54
Approaches to evaluate possible horizontal gene transfer of plant DNA to soil microorganisms ................................................................................................................................... 58
6.
The New Zealand and international approach to antibiotic resistance marker
genes ............................................................................................................................ 62
The New Zealand approach to antibiotic resistance marker genes ........................................... 62
Environmental Risk Management Authority (ERMA).............................................................. 62
Antibiotic Resistance Expert Panel .......................................................................................... 62
Australia New Zealand Food Authority (ANZFA) .................................................................... 62
The approach of other countries and international organisations to antibiotic resistance
marker genes ................................................................................................................................... 63
Australia ...................................................................................................................................... 63
Norway......................................................................................................................................... 64
United Kingdom.......................................................................................................................... 64
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Codex Alimentarius Commission ............................................................................................. 66
European Commission .............................................................................................................. 66
Nordic Working Group on Food Toxicology and Risk Assessment ..................................... 66
United States............................................................................................................................... 67
World Health Organisation ........................................................................................................ 68
7.
Alternative strategies to antibiotic resistance marker genes.......................... 70
Characteristics of a selectable marker.......................................................................................... 70
Choice of a selectable marker........................................................................................................ 70
Disadvantages of antibiotic resistance marker genes ................................................................ 70
Herbicide resistance ....................................................................................................................... 71
Metabolic markers ........................................................................................................................... 72
Other selectable markers in plants................................................................................................ 74
Other selectable markers in mammalian cells ............................................................................. 74
Other markers in yeast and micro-organisms.............................................................................. 75
Alternatives to the selection of antibiotic resistance genes in bacteria.................................... 76
Elimination of the selectable marker ............................................................................................. 76
No selectable marker or reporter gene .................................................................................... 76
Reporter gene only..................................................................................................................... 77
Inactivation of the selectable marker gene.............................................................................. 77
Removal of the selectable marker gene................................................................................... 78
Modulation of gene expression ................................................................................................ 81
Intron-containing antibiotic resistance genes ........................................................................ 81
8.
Conclusion........................................................................................................... 83
9.
References ........................................................................................................... 86
10. Glossary ............................................................................................................... 96
11. Appendices .......................................................................................................... 97
Appendix I: Evaluation of the kanamycin resistance gene ......................................................... 97
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Impact of the gene product NPTII ............................................................................................. 97
Impact of the DNA in food ......................................................................................................... 98
Impact on the efficacy of antibiotics ........................................................................................ 99
Impact on the environment ..................................................................................................... 100
Appendix II: Genetically modified insect resistant maize ......................................................... 101
Appendix III: Summary of submissions ...................................................................................... 104
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Terms of Reference
Terms of Reference
1. The objective is to produce a reference report for the Environmental Risk Management
Authority that:
• Evaluates the implications of the use of antibiotic resistance marker genes in genetically
modified organisms (GMOs). This will include the technical mechanisms available for the
transfer of antibiotic resistance to other organisms, the likelihood of occurrence and under
what circumstances, and the practical effects of such transfers, ie the level and nature of risk
presented. The influence of the antibiotics involved will be considered.
• Provides some degree of comparative analysis of the risks of antibiotic resistance occurring
by other means, whether that represented by GMOs is additive/cumulative and is
significant in that context.
• Considers the pros and cons of alternative selectable markers, including removal of
antibiotic resistance marker genes from GMOs for release. This will include other risks that
may be increased by the alternatives.
2. The work will include a review of the relevant scientific literature, will include informed inputs
from sources within New Zealand and will consider the recommendations of other agencies, and
their basis, about the use of antibiotic resistance marker genes.
3. The final report will draw conclusions about the use of antibiotic resistance marker genes in
GMOs, in the context of applications under Part V of the Hazardous Substances and New
Organisms (HSNO) Act 1996. It will also include identification of the unresolved issues and the
level of certainty/uncertainty that can be attributed to the current state of knowledge.
Use of Antibiotic Resistance Marker Genes in GMOs
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Preface and Method
Preface
As most of the scientific literature focuses on antibiotic resistance marker genes in relation to
genetically modified (GM) plants the emphasis in this report is on plants, in particular food crops,
rather than other GMOs. The focus of the report is also on the implications of the use of antibiotic
resistance genes in GMOs for human health rather than the environment. An overview of horizontal
gene transfer in the environment is however included since gene transfer events in the environment
indirectly effect humans as a result of resistant micro-organisms contaminating food or water.
Method
A literature search of on-line bibliographic databases was undertaken using DIALOG for
publications in English concerning antibiotic resistance marker genes and alternative marker
strategies. The search period covered 10 years up until October 1999. Bibliographies of identified
papers were also examined. Some references published after the search period that have come to the
author’s attention have also been reviewed.
The final peer reviewed draft was available for public comment. Submissions are summarised in
Appendix III.
Use of Antibiotic Resistance Marker Genes in GMOs
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Summary
Summary
Risk assessment methods address the known and foreseeable risks derived from experience and from
extrapolation from experience. Risk assessment cannot offer an absolute guarantee of safety because
it is always carried out under some uncertainty and is limited by the state of available knowledge.
Antibiotic resistance marker genes are used to select transformed bacteria during the initial cloning
and manipulation of genes in the bacterium Escherichia coli (E. coli) and to select the few transformed
cells following introduction of the gene construct into the recipient host organism. Most antibiotic
resistance marker genes confer resistance by inactivating the antibiotic through modifying enzymes.
The most frequently used antibiotic resistance genes are the ampicillin resistance gene (bla) for
bacterial transformation and the kanamycin resistance gene (nptII) for plant transformation.
Antibiotic resistance marker genes serve no useful purpose in the GMO.
Key points from the report that address each of the Terms of Reference are summarised below.
Evaluation of the implications of the use of antibiotic resistance marker genes in
genetically modified organisms
Antibiotics in New Zealand
Annual antibiotic use in New Zealand is estimated to be 74.9 tonnes. Human use accounts for about
53 percent of this amount. In addition to antibiotic use in humans and animals about 1.2 tonnes of
streptomycin is used annually in horticulture (Antibiotic Resistance Expert Panel, 1999).
The main antibiotics currently used in New Zealand that are potentially affected by the use of
antibiotic resistance marker genes in GMOs are the aminoglycoside (eg kanamycin, neomycin) and
beta-lactam (eg ampicillin) antibiotics.
The estimated annual use of aminoglycosides in New Zealand is 2,242 kg (35 kg humans; 2,207 kg
animals) (Antibiotic Resistance Expert Panel, 1999). The amount used in molecular genetics research
is estimated as only hundreds of grams (B Scott, personal communication, May 2000).
Kanamycin is used only in serious systemic infections and when the infecting micro-organism is
resistant to other antibiotics. It is a reserve agent for tuberculosis. It is not absorbed by mouth and
has been given orally to reduce gut micro-organisms (eg preoperative bowel preparation).
Neomycin is used topically in the management of skin, eye and ear infections. It is poorly absorbed
by mouth and has been given orally to reduce gut micro-organisms (eg preoperative bowel
preparation). Although neomycin has limited clinical use it is an important antibiotic in veterinary
medicine.
Resistance mediated by the nptII gene is also demonstrable for some other aminoglycosides and
although these are not in widespread clinical or veterinary use they are still used for infections
resistant to other antibiotics.
Use of Antibiotic Resistance Marker Genes in GMOs
11
Summary
Ampicillin belongs to the clinically important group of antibiotics called the beta-lactams that include
the penicillins and the cephalosporins. The beta-lactams, in particular the penicillins, account for
about 67 percent of the estimated annual human antibiotic use (Antibiotic Resistance Expert Panel,
1999). It is given orally as well as intravenously or intramuscularly and is used in the treatment of a
variety of infections.
Ampicillin is also used in veterinary medicine. Beta-lactam antibiotics account for 26 percent of the
animal antibiotic use in New Zealand (Antibiotic Resistance Expert Panel, 1999).
Horizontal gene transfer
Horizontal gene transfer has been reported between distantly related bacteria, and from bacteria to
yeast, mammalian cells and plant cells.
The few examples of transfer from plants to bacteria indicated by DNA sequence comparisons and
the lack of experimental confirmation suggest that the frequency of evolutionary successful gene
transfer from plants to bacteria is extremely low. However this inference is based on a small number
of experimental studies and indications in the scientific literature.
Detection of horizontal gene transfer events is difficult due to the limitations of the techniques
available. Unequivocal proof requires isolation of the putative transformed bacteria for thorough
genetic characterisation.
The rate of gene transfer from plants to bacteria is insignificant compared to gene transfer between
micro-organisms. Almost any type of bacterium has the potential to transfer DNA to any other type
of bacterium if it contains a broad host range gene transfer element.
Antibiotic resistance genes and human health
The presence of the antibiotic resistance gene by itself is not associated with any adverse health
effects.
There is in vitro evidence that free DNA in human saliva is capable of transforming a naturally
competent human oral bacterium (Mercer et al, 1999). Since the regions preceding the stomach are
likely to have the highest concentrations of intact DNA entering with the diet further research is
needed to establish whether transformation of oral bacteria occurs at significant frequencies in vivo.
Although most ingested DNA is likely to be degraded and diluted in the human gastro-intestinal
tract, natural transformation of gut epithelial cells or micro-organisms cannot be completely ruled
out.
Research in mice indicates that DNA can survive digestion and uptake by gut epithelial cells occurs,
however at levels of DNA intake unlikely to be encountered in a normal diet (Schubbert et al, 1997).
The mechanism of DNA uptake by gut epithelial cells is unknown and its significance is unclear.
If DNA uptake does occur in humans critical factors are the presence of regulatory sequences that
allow gene expression and the presence of selective pressure. Without selective pressure it is highly
Use of Antibiotic Resistance Marker Genes in GMOs
12
Summary
unlikely that genes taken up by gut epithelial cells would be expressed even if they were integrated
into the genome.
Homology between the prokaryotic DNA sequences in an antibiotic resistance marker gene and the
recipient host’s DNA is more likely to be found in gut micro-organisms which are prokaryotic than
in gut epithelial cells which are eukaryotic. The probability of integration and expression of a marker
gene is therefore greater in gut micro-organisms than in gut epithelial cells.
Transformation is considered the only natural mechanism that can be involved in gene transfer from
plants to gut micro-organisms. Transformation requires access to free DNA that is present at the
time and place in which competent bacteria develop or reside.
Conditions in the mammalian intestine are probably more conducive to gene transfer than
conditions found elsewhere in nature. High concentrations of bacteria mean encounters between
different types of bacteria occur readily and residence time in the intestine is long enough to provide
opportunities for gene transfer. However there has been little direct evidence to support this
hypothesis.
The introduction of bacterial genes, bacterial regulatory sequences and bacterial origins of replication
into GM plants increases the degree of sequence homology between GM plant DNA and the
genomes of competent bacteria. It has been hypothesised that this could increase the probability of
DNA transfer from plants to bacteria by favouring homologous recombination in the recipient host
(Paget et al, 1998; Gebhard and Smalla, 1998).
If gene transfer and expression were to occur it is most likely from viable GM food micro-organisms
(or released GM micro-organisms that can be unintentionally ingested) followed by raw GM plant
material or the uncooked seed of GM plants. It is least likely from highly processed GM food microorganisms, plant or animal material.
Viable GM micro-organisms may remain intact through the gut and gene transfer could be achieved
through conjugation with gut micro-organisms.
It is generally considered that the probability of antibiotic resistance genes being transferred from
GM plant material or other eukaryotes to either gut epithelial cells or micro-organisms is extremely
low given the complexity of sequential steps required for gene transfer and gene expression.
However few experimental data are available to support this theoretical assessment.
Rare transfer events can be amplified very rapidly under selective pressure. The health impact would
be significant if a gene conferring resistance to a clinically important antibiotic was transferred and
expressed in a pathogenic micro-organism normally treated with that antibiotic.
Assuming that antibiotic resistant bacteria have not previously colonised the gastro-intestinal tract
there is no risk of compromised therapeutic efficacy of the antibiotic unless GM food containing the
particular antibiotic resistance gene is consumed at the time the antibiotic is administered orally.
From what is known about mechanisms of horizontal gene transfer between organisms and the
survival of intact DNA following processing and digestion it is concluded that the risk is highest for
Use of Antibiotic Resistance Marker Genes in GMOs
13
Summary
ingested viable GM micro-organisms, in particular if the antibiotic resistance gene confers resistance
to a clinically important antibiotic, and lowest for highly processed GM food.
Antibiotic resistance marker genes and the environment
Laboratory and field studies have been unable to confirm gene transfer occurs from plants or other
eukaryotes to naturally occurring soil or plant-associated bacteria. Two studies using artificially
introduced homology between plant and bacterium DNA have shown transfer of antibiotic
resistance marker genes from plants to bacteria under optimised laboratory conditions (Gebhard and
Smalla, 1998; de Vries and Wackernagel, 1998).
In the event that transfer from the plant genome to soil micro-organisms did occur in most cases
there would be no selective pressure. Exceptions are streptomycin use in horticulture or when
manure is used as fertiliser following in-feed antibiotic use for animal growth promotion and
prophylaxis.
Soil is a reservoir of micro-organisms with the capacity to produce antibiotics. There is a lack of
information about the extent of selective pressure of different antibiotics in soil and the soil
conditions that promote such selective pressure.
Extrapolation of transformation frequencies from microcosm studies to the environment could be
misleading because the concentrations of transforming DNA in situ are not known.
Lack of information on the prevalence of naturally competent bacteria in the environment, the
frequency of transformation and environmental factors triggering transformation impairs predictions
of the extent of horizontal gene transfer from plants to micro-organisms.
Comparative analysis of the risks of antibiotic resistance
occurring by other means
Antibiotics are used to eliminate bacteria and maintain the health of humans; animals, including fish;
and some plants. Global selective pressure has occurred as the same antibiotics are used in all three
areas.
Antibiotics have the undesirable side effect of selecting for the growth and spread of otherwise rare
resistant micro-organisms.
The extent of antibiotic resistance depends on the presence of the antibiotic and a resistance gene,
the transfer and expression of the resistance gene, and spread of resistant bacteria. Given the
presence of both the antibiotic and a resistance gene, antibiotic resistant bacteria will be selected and
propagated.
Resistance genes can be transferred between bacteria of the same or unrelated species.
Bacteria that develop resistance to one agent in a class typically show cross-resistance to other
antibiotics in the same class.
Use of Antibiotic Resistance Marker Genes in GMOs
14
Summary
The major source of the development and spread of antibiotic resistant micro-organisms in humans
is the human use (and often overuse) of antibiotics in both the community and hospitals. Within
hospitals person-to-person transmission is aided if infection control practices are less than ideal.
Some antibiotic resistant bacteria occur naturally in the environment but many are a result of
contamination with human and animal excreta in sewage, slurry and manure. Antibiotic resistance is
therefore also acquired through ingestion of resistant micro-organisms from animals or soil
contaminating food or water.
Enteric human pathogens (eg Salmonella) are commonly acquired from animals and in some instances
these micro-organisms acquire resistance to antibiotics used for growth promotion, prophylactic or
therapeutic use in animals.
Gaps in information about patterns of antibiotic resistance in New Zealand prevent the conclusion
that antibiotic use in animals is exacerbating antibiotic resistance in human pathogens.
Most overseas data support the concept that antibiotic resistance appeared only after the emergence
of strong selective pressure resulting from the massive use of industrially made antibiotics in human
and veterinary medicine and as food supplements for farm animals.
Antibiotic resistance is common in human commensal gut micro-organisms even in the absence of
concurrent or recent antibiotic consumption (Levy et al, 1988). Such resistant bacteria are a reservoir
of resistance genes that are potentially transferable, directly or indirectly, to human pathogens.
There are geographical variations within and between countries in the incidence of specific antibiotic
resistance. Global movement of people, breeding stock and food means resistance in one area can
spread to another.
Limited monitoring of some zoonotic micro-organisms from human, animal and food sources and
data from some hospitals indicate that there is a low level of acquired antibiotic resistance in New
Zealand compared to the United States, the United Kingdom and the European Union (Antibiotic
Resistance Expert Panel, 1999).
Untreatable infections due to bacteria that are resistant to all available antibiotics are still exceptional
but do occur. In New Zealand hospitals it is not uncommon to encounter bacterial infections that
are resistant to all but one or two antibiotics (Lang and Blackmore, 1999).
It is more likely that antibiotic resistance genes would be introduced into gut micro-organisms
through transfer between naturally occurring ingested contaminating micro-organisms and gut
micro-organisms than through transfer from DNA released during digestion of GM plant or animal
material. The probability that transfer occurs between ingested GM micro-organisms and gut microorganisms is likely to be the same as between non-GM micro-organisms and gut micro-organisms.
The potential impact of the use of antibiotic resistance genes in GMOs on the prevalence of
antibiotic resistance, though additive, is far less significant than the impact of the current use of
antibiotics in humans and animals in New Zealand. However antibiotic resistance is receiving
increasing scrutiny nationally and internationally, and there are an increasing number of strategies
being implemented with the aim of curbing all antibiotic use.
Use of Antibiotic Resistance Marker Genes in GMOs
15
Summary
Pros and cons of alternative selectable markers, including removal of antibiotic
resistance marker genes from GMOs for release
Alternative strategies to the use of antibiotic resistance genes in GMOs include no selectable marker
or reporter gene, a reporter gene only, inactivation of the selectable marker gene and removal of the
selectable marker gene. As yet these approaches are far from routine or from being generally
applicable.
Use of alternative markers, in particular genes concerned with various metabolic pathways, or
subsequent removal or inactivation of the antibiotic resistance gene is becoming more common.
Recently developed new marker strategies are based on the use of selectable genes that give the
transformed cells a metabolic advantage compared to the non-transformed cells which are starved
with a concomitant slow reduction in viability (eg the GUS gene and the manA gene with cytokinin
glucuronides and mannose respectively as selective agents).
Removal of selectable marker genes allows the same markers to be used repeatedly in subsequent
transformations into the same host and minimises the amount of foreign DNA to that involved in
conferring the desired traits. This will be important as successive rounds of genetic modification
become more prevalent.
Recommendations of agencies and their basis about the use of antibiotic
resistance marker genes
There appears to be an emerging international consensus to evaluate each GMO containing
antibiotic resistance marker genes on a case-by-case basis.
Marker genes that encode resistance to clinically important antibiotics should not be present in food
or feed in view of the potential for gene transfer and expression, albeit rare, to occur.
Viable GM food micro-organisms should not contain antibiotic resistance marker genes since gene
transfer to gut micro-organisms could occur as a result of conjugation.
The difference of opinion between countries about the presence of antibiotic resistance marker
genes in GMOs arises mainly over the question of whether extremely low but non-zero risks of
increased antibiotic resistance are acceptable.
Irrespective of the scientific conclusions removal of the antibiotic resistance gene from the final GM
plant or use of alternative strategies is now being recommended whenever feasible (Donaldson and
May, 1999; European Commission, 1999; WHO, 2000).
Elimination of antibiotic resistance marker genes could have positive effects on consumer
acceptance by alleviating perceived risks.
Use of Antibiotic Resistance Marker Genes in GMOs
16
Summary
Conclusions about the use of antibiotic resistance marker genes in GMOs, in the
context of applications under Part V of the Hazardous Substances and New
Organisms (HSNO) Act 1996
The scientific literature on the use of antibiotic resistance marker genes in GMOs is characterised by
many opinions and relatively few data.
Each GMO containing antibiotic resistance marker genes should be evaluated on a case-by-case
basis. Of particular relevance is the clinical importance of the antibiotic and the probability that the
antibiotic would be encountered.
The potential contribution to antibiotic resistance from antibiotic resistance marker genes is likely to
be very small in comparison to that arising from antibiotic use in medicine and agriculture, and
insignificant in terms of health impact unless the antibiotic is clinically important in New Zealand.
Limited data exist on the patterns of antibiotic resistance in New Zealand.
Studies that include selective pressure during exposure of competent bacteria with DNA are needed.
Although there is information about phenotypic resistance few studies have identified the gene
responsible. Monitoring should be at the genotypic rather than phenotypic level. Since all resistance
genes originate from micro-organisms monitoring should distinguish a gene that has been transferred
from a GM plant.
Relatively little consideration has been given to the potential cumulative consequences of a rare event
in a scenario of widespread cultivation of GM plants and ingestion of raw and unprocessed GM
plant material by millions of people and animals.
The overall importance of a value to be protected, such as human health, for the overall risk
assessment and the resulting regulatory constraints is frequently a matter of science policy rather than
a scientific issue (Doblhoff-Dier et al, 1999).
Although there is no scientific evidence that antibiotic resistance genes have transferred from GM
plants to pathogenic micro-organisms, and the probability of such an event is considered to be
extremely low, a precautionary approach could be adopted that recommends use of an alternative
marker such as the manA gene or removal of the antibiotic resistance gene from the final GMO.
Such an approach would take into account public perception of the role of antibiotic resistance
marker genes in antibiotic resistance.
Use of Antibiotic Resistance Marker Genes in GMOs
17
Introduction
1.
Introduction
What is gene technology?
1
Gene technology involves the isolation and subsequent introduction of discrete DNA
segments containing the gene(s) of interest into a recipient organism. It is also used to
control, increase or turn off specific functions within an organism.
2
Gene technology is characterised by the capacity to transfer genes between unrelated species
and to specify the genes that will be transferred. Its application to animals is still in its infancy
compared with its use in plants and micro-organisms.
3
Using special enzymes DNA from the donor organism that contains the desired gene is cut
into shorter fragments that are then separated and purified. The gene is then removed using
enzymes that cut the DNA in defined places.
4
In order to ensure that the desired gene is incorporated into the host cell nucleus as
efficiently as possible a carrier system or vector is normally used. A typical vector is made up
of a circular piece of DNA from a bacterium or virus that is cut using enzymes so that the
gene can be inserted. The vector carrying the gene is multiplied, usually in bacterial cells, to
produce a sufficient quantity of the gene construct. Cells are transformed by insertion of the
gene construct.
5
The vector must contain a promoter that enables the gene to function in the host. More
recently careful selection of the promoter is starting to allow gene expression to be targeted
(eg to the leaves and roots of plants).
6
Bacteria are transformed by direct DNA uptake. Plant cells are transformed by insertion of
the gene construct into the cell nucleus by one of several methods including:
• direct DNA uptake mediated by polyethylene glycol treatment or electroporation;
• micro-injection of DNA;
• particle bombardment (firing tiny particles coated with the DNA); and
• use of the soil bacterium Agrobacterium tumefaciens as a vehicle to carry the DNA.
7
Micro-injection into embryos is the standard method of transformation for mammals. The
embryo is transferred to a recipient mother and in a small proportion of injected embryos the
introduced DNA is integrated into the animal’s genome. Particle bombardment is also used
to transform animal cells.
8
Transformation is an inefficient process. The frequency with which transformed cells are
obtained varies from species to species and with various transformation methods but in
plants is often as low as 1 in 10,000 or 1 in 100,000 of the cells treated (WHO, 1993).
9
Recovery of the transformed cells depends on selection for the rare transformation event and
against the non-transformed cells. This is achieved by using a selectable marker.
Use of Antibiotic Resistance Marker Genes in GMOs
18
Introduction
What is a selectable marker?
10
A marker is used to indicate a successful genetic alteration in a cell in the early stages of
research. Markers are vital to the transformation process but usually secondary to the
objective of developing a GMO. The exception is herbicide resistance genes where the gene
is the marker and the herbicide resistant plant also has agronomic value. Expression of
selectable marker genes gives an almost absolute advantage to GMOs in the laboratory.
Generally no selective conditions are expected outside the laboratory with the exception of
herbicide resistance genes.
11
The selectable marker allows the researcher to readily identify and select the few transformed
cells and saves having to assay for incorporation of the desired gene by more complex and
expensive methods.
12
There are three classes of selectable marker genes. They are:
• genes that confer resistance to antibiotics
• genes that confer resistance to herbicides, and
• genes concerned with various metabolic pathways.
Antibiotic resistance marker genes
13
During the 1980s antibiotic resistance genes were introduced as markers for selection. The
ampicillin resistance gene is the most common antibiotic resistance marker for bacterial (E.
coli) transformation whereas for plant transformation markers largely comprise genes resistant
to the aminoglycoside antibiotics.
14
As a result of the presence of the antibiotic resistance gene the few cells that contain the
introduced DNA are able to grow and multiply in the presence of the antibiotic unlike the
surrounding non-transformed cells that either die or have severely inhibited growth. If plants,
the transformed cells are subsequently regenerated into GMOs.
15
Antibiotic resistance genes are used at two stages during the genetic modification of
eukaryotes. The first stage is in bacteria during the development of the gene construct
containing the genes to be introduced into the eukaryote and the second stage is in the
selection of transformed eukaryotic cells following introduction of the gene construct.
16
The bacterium E. coli is commonly used as the host organism during the development of the
gene construct. To allow identification of E. coli cells containing the construct it is necessary
to use a marker gene that is functional in the bacterium. It carries bacterial promoters that
will not be expressed or needed in an eukaryotic cell. The marker gene does not end up in the
GM plant or animal genome unless transformation methods using the entire construct are
used.
17
An antibiotic resistance marker gene may be included with the desired gene in the construct
that is introduced into an eukaryotic cell. Both genes will have eukaryotic promoters to allow
expression in eukaryotic cells. Although the marker gene is required only immediately after
Use of Antibiotic Resistance Marker Genes in GMOs
19
Introduction
the genetic modification procedure to facilitate identification of transformed cells it may
remain in most, if not all, cells of the resulting GM plant or animal, and be expressed.
18
Not all of the antibiotic resistance genes are equally effective for selecting transformed cells
due to the level of sensitivity of the plant species or variety, or the ability of the gene to
protect the transformed cells from the effects of the selective agent (WHO, 1993).
19
As a component of the genetic modification the antibiotic resistance gene(s) must be taken
into account when considering the release of the GMO and/or its product. Depending on
the nature of the organism, release may cause the gene and its product to be present in the
environment on a large scale. This has to be considered in the context of whether, and to
what extent, the gene is already present in the environment from other sources.
20
Antibiotic resistance genes are the most prevalent selectable marker genes. They were used in
31 of the 52 US Food and Drug Administration (FDA) consultations regarding GM crops to
the end of 1997. The kanamycin resistance gene (nptII) accounted for 27 of these and the
hygromycin resistance gene (hpt) the rest. Of the 30 consultations that were completed by the
end of 1997, in two cases nptII had been segregated out and the final GM variety did not
contain the resistance gene.
21
In 19 of the 52 FDA consultations the GM crop had one or more antibiotic resistance genes
under a bacterial promoter incorporated into the plant genome. In most cases the ampicillin
resistance gene (bla) was involved. In some cases only partial fragments of the genes were
incorporated and therefore the genes were not functional (US FDA, 1998).
22
The presence of antibiotic resistance genes in GMOs in New Zealand is currently confined
to GMOs developed in or imported into containment, and some imported processed GM
food.
What antibiotic resistance genes are used as markers?
23
Choice of antibiotic resistance marker genes that have been used in genetic modification has
been influenced by availability and familiarity.
Kanamycin resistance
24
The most commonly used selectable marker is the kanamycin resistance gene from a
transposon (Tn5) from E. coli K12.
25
The kanamycin resistance gene was one of the first markers to be developed and was
available from many laboratories. It is widely used as a selectable marker in the
transformation of organisms as diverse as bacteria, yeast, plants and animals. It was the first
marker used in plant genetic modification and the first GM food plant, the delayed softening
tomato Flavr Savr, commercialised in 1994, contains this gene. Many hectares of GM crops
containing this gene are being grown commercially in North America, Europe, Asia and
Australia (cotton only) (Conner, 1997).
Use of Antibiotic Resistance Marker Genes in GMOs
20
Introduction
26
At least 18 genes have been cloned which encode aminoglycoside modifying enzymes
conferring resistance to kanamycin (Nap et al, 1992). The enzymes catalyse three types of
reactions – acetylation, adenylation and phosphorylation. Four are acetyltransferases, three
are adenyltransferases and nine phosphotransferases. Of these the most commonly used is
aph(3')-IIa (or aphA-2, also called nptII) which encodes aminoglycoside 3'-phosphotransferase
II (APH(3')II, also called neomycin phosphotransferase II (NPTII)). Detailed biosafety
evaluations have been published on the nptII gene and its product (see Appendix I).
27
There is no structural similarity among the different classes of genes and enzymes. There are
even differences among the members of each type of enzymes (Karenlampi, 1996). The
relative occurrence of aminoglycoside modifying enzymes shows local and temporal
variations in populations. They are found in a variety of bacterial species and also in the
antibiotic-producing actinomycetes (Nap et al, 1992).
28
NPTII is not novel to humans because NPTII producing kanamycin resistant bacteria are
often present in the normal gut microflora. Cell death and lysis of these bacteria would result
in exposure of the immunocompetent cells of the gastro-intestinal tract to the same protein
as is present in the GMO (Karenlampi, 1996).
29
Many aminoglycosides are phosphorylated by NPTII but it does not confer resistance to all
aminoglycosides because of widely different phosphorylation rates for the different
substrates (Redenbaugh et al, 1993).
30
NPTII confers resistance to kanamycin, neomycin, paromomycin, ribostamycin, butirosin,
gentamicin A and B and geneticin (G418). Of these, kanamycin, neomycin and paromomycin
are used in human medicine. It does not confer resistance to gentamicin that is used
therapeutically, as gentamicin A and B are minor components of the commercial drug (US
FDA, 1994). Geneticin is used only for in vitro experimentation. Resistance is not conferred
to amikacin although enzymatic activity for this substrate is detectable in vitro. The resistance
profile of another enzyme (APH(3')-VI) includes amikacin (Karenlampi, 1996). The gene
used in the Flavr Savr tomato encodes an enzyme that confers resistance only to neomycin,
kanamycin and geneticin (Redenbaugh et al, 1994).
31
Kanamycin is normally used as the selective agent.
32
It has been suggested that neomycin phosphotransferase might modify intracellular
phosphorylation when introduced into mammalian cells. However several researchers have
expressed the kanamycin resistance gene in human cells and have introduced them into
people via gene therapy without reported adverse effects (Karenlampi, 1996).
33
There are no safety data on the other identified kanamycin resistance genes. It is possible that
several might be useful as selectable markers because of their ability to detoxify kanamycin in
the plant. The effects of these genes in a food plant can be expected to be similar to nptII
unless there are different substrates for the various enzymes (Karenlampi, 1996).
34
Practically the kanamycin resistance gene provides excellent selection because of the
properties of the antibiotic kanamycin and the resistance gene. With kanamycin, cell death is
slow enough to permit expression of the added marker gene and inactivation of the antibiotic
Use of Antibiotic Resistance Marker Genes in GMOs
21
Introduction
to occur before cell death. With some selective agents, exposure to the selective treatment
must be delayed to ensure that cells with the gene will survive. As a result cells are left longer
in tissue culture which is less desirable as somaclonal variation may occur.
35
Hygromycin and some of the herbicide resistance markers have similar advantages (WHO,
1993).
Hygromycin resistance
36
Hygromycin resistance genes aph(4)-Ia (or hph) and aph(4)-Ib (or hyg), also referred to as hpt
encode an aminoglycoside 4-phosphotransferase APH(4)-I, also called hygromycin
phosphotransferase. The aph(4)-Ia gene has been isolated from a strain of E. coli and aph(4)-Ib
has been isolated from a hygromycin B producing strain of Streptomyces.
37
The enzyme confers resistance to the aminoglycoside hygromycin B, an inhibitor of protein
synthesis in prokaryotic and eukaryotic cells.
38
There is no detailed safety evaluation available for these genes. Hygromycin is not in clinical
use therefore the potential for inactivation of an oral dose of antibiotic by consuming plant
material containing the marker gene simultaneously with the antibiotic is not an issue. It is
used in veterinary medicine (Karenlampi, 1996).
Streptomycin resistance
39
The streptomycin resistance gene provides a phenotypic marker facilitating non-lethal
screening of transformed cells based on differential colour. Sensitive plants are pale (but not
killed) as streptomycin inhibits protein synthesis and growth of the non-transformed plant
cells. The marker is used where preservation of both transformed and non-transformed cells
is an advantage. A problem in using this marker is that plants may also acquire streptomycin
resistance by mutation.
40
Several aminoglycoside modifying enzymes are known to confer resistance to streptomycin.
Of these two are adenyltransferases (eg ANT(3")-I (gene ant(3")-Ia or aadA )) and two are
phosphotransferases(eg APH(6)-I (genes aph(6)-Ia, aph(6)-Ib, aph(6)-Ic, aph(6)-Id)). The source
of the genes is the micro-organism that produces the antibiotic and resistance factors present
in bacteria.
41
ANT(3")-I confers resistance to streptomycin and spectinomycin.
42
There is no safety evaluation available for the streptomycin resistance genes. Although the
genes are not novel for humans as resistance factors carrying them are common and found
at high frequency in bacteria from clinical isolates, evaluation of allergenicity in humans and
pleiotrophic effects from the enzyme in different plants is required (Karenlampi, 1996).
Use of Antibiotic Resistance Marker Genes in GMOs
22
Introduction
Ampicillin resistance
43
The ampicillin resistance gene (bla, also referred to as amp) encodes a beta-lactamase enzyme.
Resistance to the clinically important group of antibiotics known as the beta-lactams, which
includes the penicillins and cephalosporins, is most frequently due to beta-lactamases.
44
The bla gene is present in many plasmids derived from the E. coli plasmid vector pBR322
including those of the pUC family which were used in the development of GM insect
resistant maize (Malik and Saroha, 1999). Appendix II discusses the controversy around the
use of this gene in GM insect resistant maize.
45
The bla gene on the pUC18 vector encodes an early form of beta-lactamase that has
subsequently undergone extensive evolution to form novel beta-lactamases with potent
activity against newer broad-spectrum beta-lactams. If such mutations occurred in the
resistance gene used as a selectable marker, and in the rare event that this gene was then
transferred and expressed in micro-organisms (eg Neisseria meningitidis carried in the human
nasopharynx), this could have implications for the treatment of serious bacterial infections
(eg meningococcal meningitis) (OECD, 2000a).
Others
46
Other antibiotic resistances that have been used include tetracycline, rifampicin, amikacin,
chloramphenicol, bleomycin and puromycin.
Tetracycline resistance
47
The tetracycline resistance gene (tet) is more abundant and widespread than the kanamycin
resistance gene. This is partly attributed to many years of use of tetracycline as a growth
promotant in animals and extensive use in human and veterinary medicine. The gene encodes
a protein that causes the active efflux of tetracycline from a bacterial cell (Pittard, 1997).
Chloramphenicol resistance
48
The chloramphenicol resistance gene (cat) encodes chloramphenicol acetyltransferase.
Resistance has been widely reported although its prevalence has tended to decline where use
of the antibiotic has become less frequent (Reynolds, 1996).
Amikacin resistance
49
The amikacin resistance gene confers resistance to a clinically important aminoglycoside
antibiotic. It has been used in a potato developed by Avebe to generate only the starch
mylopectin.
50
The European Commission Scientific Committee on Plants did not recommend the GM
potato for Commission approval for sale and cultivation throughout the European Union on
the grounds that the resistance gene could pose a risk to human and animal health (Scott,
1998).
Use of Antibiotic Resistance Marker Genes in GMOs
23
Introduction
Rifampicin resistance
51
The rifampicin resistance gene encodes an altered sub-unit of RNA polymerase and
transcription is no longer inhibited by rifampicin. Resistance occurs as a result of random
mutations at frequencies of about 10-9 in normal bacterial populations (Pittard, 1997).
52
Resistance to this antibiotic is not widespread but easily selected by the use of rifampicin. It is
used in the treatment of tuberculosis and leprosy in combination with other drugs to delay or
prevent the development of resistance.
Bleomycin (phleomycin) resistance
53
Sources of the bleomycin resistance gene are Streptoalloteichus hindustanus and E. coli. It is rarely
used in plant transformation. Bleomycin causes DNA breakdown resulting in the death of
rapidly growing cells and hence has a limited use in cancer treatment. It is not used as an
antibiotic (WHO, 1993).
Puromycin resistance
54
The puromycin resistance gene (pac) from Streptomyces alboniger encodes puromycin
acetyltransferase. This enzyme inactivates puromycin produced by Streptomyces alboniger. The
gene has been used as a marker for the selection of transformed mammalian cells (Vara et al,
1986).
55
Puromycin is a synonym for a tetracycline, achromycin, used topically in human medicine
(US Registry of Toxic Effects of Chemical Substances, 2000).
56
Streptomycin and chloramphenicol resistance genes are used as scoreable markers (or
reporter genes). These marker genes are normally used to detect the presence or assay the
level of expression of a transferred gene in a modified organism.
57
The application of reporter genes is generally confined to the laboratory or small-scale field
trials and is not primarily directed towards full-scale commercial use. However it has been
predicted that in the long term crops will be considered for commercial release while still
carrying reporter genes (Metz and Nap, 1997). The most frequently used reporter gene, the
beta-glucuronidase gene (GUS), is not an antibiotic resistance gene.
Use of Antibiotic Resistance Marker Genes in GMOs
24
Antibiotics and antibiotic resistance
2.
Antibiotics and antibiotic resistance
What are antibiotics used for?
58
Antibiotics are used both internally and externally in humans and animals to control, prevent
and treat bacterial infections, and in animals to enhance growth and feed efficiency in
situations where animals are intensively reared (eg chickens, pigs, some fish). They are also
used to maintain the health of some plants. Many of the same antibiotics are used in all three
areas.
59
Antibiotics have the undesirable side effect of selecting for the growth and spread of
otherwise rare resistant micro-organisms. Laboratory studies have demonstrated that
antibiotics increase the frequency of gene transfer between micro-organisms. Antibiotics can
also increase the mutation rate of micro-organisms thereby increasing the probability that a
new resistance determinant will arise (Heinemann, 1999).
Mechanisms of antibiotic resistance
60
Bacteria become resistant by one of the following mechanisms:
• production of enzymes that inactivate the antibiotic
• alteration of the cellular target, or
• active removal of the antibiotic from the bacterial cell (Antibiotic Resistance Expert
Panel, 1999).
Development of antibiotic resistance
61
Resistance to antibiotics resulting in therapeutic failure for bacterial pathogens was first
encountered soon after the introduction of antibiotics in the 1940s. Since then resistance has
emerged in response to the introduction of new or modified antibiotics although the time
taken has varied. Resistance has emerged for all known antibiotics in use (Levy, 1998). For
most antibiotics and classes of antibiotics antibiotic resistance genes have also entered the
bacterial population in the settings where antibiotics are used (eg hospitals, farms).
62
The major factors that contribute to resistance are the antibiotic itself and the resistance
genes being selected for. When an antibiotic is used to treat an infection the bacteria most
sensitive to the drug die or their growth is inhibited. Bacteria that have or acquire the ability
to resist the antibiotic persist and replace the sensitive bacteria. These bacteria may directly
cause human infections resistant to treatment.
63
Bacteria can also become resistant indirectly when resistance genes are passed on from other
bacteria by mechanisms that allow the transfer of genetic material. Antibiotic resistance genes
on plasmids and transposons flow to and from Gram-positive and Gram-negative bacteria,
and among bacteria that inhabit very different ecological niches. As a result resistance can be
Use of Antibiotic Resistance Marker Genes in GMOs
25
Antibiotics and antibiotic resistance
transferred between non-pathogenic and pathogenic bacteria, and from bacteria that usually
inhabit the gastro-intestinal tract of animals to those that infect humans.
64
Whenever an antibiotic is used bacteria will develop resistance, either by mutation, gene
transfer, or a combination of the two. Mutation occurs at a frequency of between 1 in 106
and 1 in 1012 bacteria (JETACAR, 1999). Gene transfer is more common than mutation.
Some resistance genes are located on plasmids and are transferable whereas others (eg
streptomycin, rifampicin, fluoroquinolones) are located on the chromosome and are not
transferable.
65
There is conflicting evidence with respect to the presence of antibiotic resistance in bacterial
pathogens before the advent of antibiotics. However it is likely that most of the antibiotic
resistance genes were already present. The biochemical diversity found among the types of
resistance mechanisms implies a diversity of origins. The most likely source of the resistance
genes is antibiotic-producing micro-organisms (eg Streptomyces) (Benveniste and Davies, 1973;
Trieu-Cuot et al, 1987). This has yet to be established conclusively (Davies, 1997).
66
The evolution and spread of antibiotic resistance depends on the selective pressure exerted in
the bacterial environment. Selective pressure is a general concept that refers to many factors
that create an environment and allow micro-organisms with novel mutations or newly
acquired characteristics to survive and proliferate. Micro-organisms resistant to antibiotics
were resistant before antibiotics were used but were not able to differentially proliferate; thus
both survival and proliferation are essential. Antibiotic exposure selectively amplifies resistant
bacteria and the resistance genes they carry. This increases the prevalence of resistant bacteria
in the total bacterial population and results in large reservoirs of resistant bacteria and
resistance genes where formerly they were rare. Resistance to specific antibiotics varies
geographically with considerable variation both between and within countries.
67
In small spatial compartments low level resistant bacteria may be selected by very small
quantities of antibiotics such as those created by antibiotic-producing micro-organisms, those
present in food as contaminants, or those present in the human body. Antibiotic
concentrations are often low in some compartments where the density of micro-organisms is
high (eg colon, oropharynx). Selection of low level resistant variants may increase the
possibility of further evolution towards higher resistance levels. Since antibiotic selection is
only part of the environment that results in the selection of antibiotic resistant bacteria it is
not always possible to correlate the use of antibiotics and resistance to antibiotics (Baquero et
al, 1998).
68
Most data support the concept that antibiotic resistance appeared only after the emergence of
strong selective pressure resulting from the massive use of industrially made antibiotics in
human and veterinary medicine and as food supplements for farm animals.
Use of Antibiotic Resistance Marker Genes in GMOs
26
Antibiotics and antibiotic resistance
The public health impact of antibiotic resistance
69
The extent of the clinical antibiotic resistance problem depends on the presence of the
antibiotic and a resistance gene, the spread of resistant bacteria and the cell-to-cell spread of
the resistance gene. Given the presence of both the antibiotic and a resistance gene, drug
resistant bacteria will be selected and propagated (Levy, 1997).
70
The prevalence of antibiotic resistant bacteria and the number of antibiotics to which they
are resistant are increasing because of the use of antibiotics. As a consequence the morbidity
and mortality of previously treatable bacterial diseases is increasing. Essential lifesaving
antibiotics are becoming less effective and there are fewer alternatives available for treatment
(JETACAR, 1999).
71
During the last decade the development of new antibiotics has become more difficult,
expensive and uncommon. This contrasts with previous decades following the discovery of
penicillin in 1940.
72
Emergence of resistance and control of antibiotic resistant pathogens are now major
challenges in both hospitals and the community. Multi-drug resistant pathogens such as
penicillin-resistant pneumococci, vancomycin-resistant enterococci, and methicillin-resistant
staphylococci have emerged as well as a variety of multi-resistant Gram-negative organisms
(eg Pseudomonas aeruginosa, Acinetobacter baumanii) (Levy, 1998).
73
Evaluation of the human health impact of antibiotic resistance depends on the importance of
the antibiotic or antibiotic class in medicine and potential human exposure (direct and
indirect) to resistant bacteria, in particular those that are human pathogens. However bacteria
that are not usually pathogenic may cause infections in some people (eg those who are
hospitalised or immunocompromised).
Antibiotics in New Zealand
74
Annual antibiotic use, excluding ionophores,1 in New Zealand is estimated to be 74.9 tonnes.
Human use accounts for about 53 percent of this amount. In addition to antibiotic use in
humans and animals about 1.2 tonnes of streptomycin is used annually in horticulture in New
Zealand (Antibiotic Resistance Expert Panel, 1999).
75
The main antibiotics currently used in New Zealand that are potentially affected by the use of
antibiotic resistance marker genes in GMOs are discussed below.
Aminoglycosides
76
Aminoglycoside antibiotics exert their effect on bacteria by binding to bacterial ribosomes
and inhibiting protein synthesis. Phosphorylation of the antibiotics by the aminoglycoside
modifying enzyme neomycin phosphotransferase interferes with binding and thus prevents
1
Ionophores have a different mode of action to other groups of antibiotics and are not used in human or veterinary medicine. They are not known to
select cross-resistance to antibiotics used in human or veterinary medicine.
Use of Antibiotic Resistance Marker Genes in GMOs
27
Antibiotics and antibiotic resistance
the antibiotic from inhibiting protein synthesis. In this way cells that contain the kanamycin
resistance gene and express neomycin phosphotransferase are resistant to the action of some
aminoglycoside antibiotics.
77
The estimated annual use of aminoglycosides in New Zealand is 2,242 kg (35 kg humans;
2,207 kg animals) (Antibiotic Resistance Expert Panel, 1999). The amount used in molecular
genetics research is estimated as only hundreds of grams (B Scott, personal communication,
May 2000).
78
In contrast in the Netherlands approximately one kilogram of kanamycin is used annually in
molecular genetics, 100 kg in humans and an estimated 20,000 kg of neomycin and 10,000 kg
of kanamycin in animals (Nap et al, 1992).
Kanamycin
79
Kanamycin is produced by the actinomycete Streptomyces kanamyceticus. It is active against
strains of Gram-negative bacteria, excluding Pseudomonas species, as well as some strains of
Staphylococcus and Mycobacterium, although resistant strains are widely distributed. A decline in
use has meant that resistance has become less prevalent.
80
It is used only in serious systemic infections and when the infecting micro-organism is
resistant to other antibiotics. It is a reserve agent for tuberculosis, in particular multi-drug
resistant tuberculosis the prevalence of which has increased dramatically in the last decade.
81
Kanamycin is given intramuscularly or intravenously in the treatment of severe infections
often in combination with another agent. It is not absorbed by mouth and has been given
orally to reduce gut micro-organisms (eg preoperative bowel preparation, hepatic
encephalopathy). The main side effects are ototoxicity and nephrotoxicity.
Neomycin
82
Neomycin is produced by the actinomycete Streptomyces fradiae. It is active against many strains
of Gram-negative bacteria, excluding Pseudomonas species, and against many strains of
Staphylococcus aureus. Its chemical properties are similar to kanamycin.
83
It is used topically in the management of skin, eye and ear infections. It is poorly absorbed by
mouth and has been given orally to reduce gut micro-organisms (eg preoperative bowel
preparation, hepatic encephalopathy, selective decontamination of the gastro-intestinal tract).
It is not used parenterally or systemically because of its ototoxicity and nephrotoxicity.
84
Although neomycin has limited clinical use it is an important antibiotic in veterinary
medicine.
Streptomycin
85
Streptomycin is produced by Streptomyces griseus. It is active against Mycobacterium tuberculosis as
well as against many Gram-negative bacteria, excluding Pseudomonas aeruginosa. It is used in the
treatment of tuberculosis in combination with other agents, and for other severe infections.
It is not absorbed by mouth. The main side effect is ototoxicity.
86
Streptomycin is also used in horticulture and veterinary medicine.
Use of Antibiotic Resistance Marker Genes in GMOs
28
Antibiotics and antibiotic resistance
87
The streptomycin resistance gene also confers resistance to spectinomycin. Spectinomycin is
used as an alternative agent in the treatment of uncomplicated gonorrhoea but is not
available in New Zealand.
Other aminoglycosides
88
The nptII gene also confers resistance to paromomycin. Paromomycin has a similar
antibacterial spectrum to neomycin and is given orally in the treatment of protozoal
infections such as amoebiasis. It is not available in New Zealand.
89
Gentamicin or tobramycin are the antibiotics of choice in the treatment of life-threatening
Gram-negative infections and are often used in association with other antibiotics eg betalactams. With the continuing emergence of aminoglycoside resistance amikacin and
netilmicin should be reserved for severe infections resistant to other aminoglycosides.
Amikacin, and to a lesser extent netilmicin, are not affected by most of the aminoglycoside
modifying enzymes eg NPTII that inactivate the aminoglycosides.
Other antibiotics
Ampicillin
90
Ampicillin is effective against Gram-positive and some Gram-negative bacteria. It has a
broader spectrum of activity than benzylpenicillin and is used in the treatment of a variety of
infections.
91
Ampicillin is a beta-lactam antibiotic. Beta-lactam antibiotics are the largest group of
antibiotics and include the penicillins and the cephalosporins. All have related structures and
the same mechanism of action. The beta-lactams, in particular the penicillins, account for
about 67 percent (26,289 kg) of the estimated annual human antibiotic use (Antibiotic
Resistance Expert Panel, 1999).
92
It is given orally as well as intravenously or intramuscularly and is often given with an
aminoglycoside for broad spectrum empirical therapy (Reynolds, 1996).
93
Ampicillin is also used in veterinary medicine. Beta-lactam antibiotics account for 26 percent
of the animal antibiotic use in New Zealand (Antibiotic Resistance Expert Panel, 1999).
Tetracyclines
94
The group of tetracycline antibiotics has a broad spectrum of activity against bacteria and
some protozoa. Tetracyclines have been used in the treatment of a large number of
infections. Although use has become more restricted with the emergence of resistance a
tetracycline is the usual antibiotic of choice in some infections (eg chlamydial and
mycoplasmal infections).
95
They are usually given orally.
96
Tetracyclines are also used in veterinary medicine.
Use of Antibiotic Resistance Marker Genes in GMOs
29
Antibiotics and antibiotic resistance
Rifampicin
97
Rifampicin is bactericidal against a wide range of micro-organisms and interferes with nucleic
acid synthesis by inhibiting RNA polymerase (Reynolds, 1996).
98
It is important in the treatment of tuberculosis and contacts of Haemophilus influenzae
meningitis and meningococcal disease to eradicate nasopharyneal carriage of H. influenzae and
Neisseria meningitidis respectively. This latter use is particularly important in New Zealand
which has been experiencing an epidemic of meningococcal disease for the last decade.
99
It is given orally as well as intravenously.
Chloramphenicol
100
Chloramphenicol is a broad-spectrum antibiotic effective against both Gram-negative and
Gram-positive bacteria as well as some other organisms (eg mycoplasmas). Use is limited by
its toxicity but it is used particularly in typhoid and other salmonellal infections and in the
treatment of bacterial meningitis. It is used orally or intravenously and widely used topically
in eye infections (Reynolds, 1996).
Antibiotic resistance in New Zealand
101
The potential contribution of antibiotic resistance acquired from consumption of antibiotic
resistance marker genes in GMOs needs to be considered in the wider context of antibiotic
resistance in New Zealand.
102
Untreatable infections due to bacteria that are resistant to all available antibiotics are still
exceptional but do occur. In New Zealand hospitals it is not uncommon to encounter
bacterial infections that are resistant to all but one or two antibiotics (Lang and Blackmore,
1999).
103
The major source of the development and spread of antibiotic resistant micro-organisms in
humans is the human use (and often overuse) of antibiotics in both the community and
hospitals. Within hospitals person-to-person transmission is aided if infection control
practices are less than ideal.
104
Antibiotic resistance is also acquired through ingestion of resistant micro-organisms from
animals or soil contaminating food or water. Enteric human pathogens (eg Salmonella) are
commonly acquired from animals and in some instances these micro-organisms acquire
resistance to antibiotics used for growth promotion, prophylactic or therapeutic use in
animals. Global movement of people, breeding stock and food also means resistance in one
area can spread to another.
105
Gaps in information about patterns of antibiotic resistance (eg in enteric micro-organisms
isolated from food animals) in New Zealand prevent the conclusion that antibiotic use in
animals is exacerbating antibiotic resistance in human pathogens. Limited monitoring of
some zoonotic micro-organisms from human, animal and food sources by the Institute of
Environmental Science and Research and data from some hospitals indicate that there is a
Use of Antibiotic Resistance Marker Genes in GMOs
30
Antibiotics and antibiotic resistance
low level of acquired antibiotic resistance in New Zealand compared to the United States, the
United Kingdom and the European Union (Antibiotic Resistance Expert Panel, 1999). The
Antibiotic Resistance Expert Panel considered that vigilance is required to ensure that in the
event that resistant strains of enteric zoonotic micro-organisms such as Campylobacter,
Enterococcus and pathogenic E. coli are introduced or emerge, action can be taken to limit their
spread.
106
There are no data on the antibiotic resistance patterns of bacteria found on fruit that may be
a result of streptomycin use in horticulture.
107
Whilst the potential contribution to antibiotic resistance from antibiotic resistance marker
genes is likely to be very small in comparison to that arising from antibiotic use in medicine
and agriculture, and insignificant in terms of health impact unless the antibiotic is clinically
important in New Zealand, it adds to the wider public health problem of increasing antibiotic
resistance. A number of strategies proposed by recent advisory bodies (eg European
Commission, 1998, 1999; JETACAR, 1999; Antibiotic Resistance Expert Panel, 1999) have
been or are being implemented to curb antibiotic use and halt further escalation of antibiotic
resistance.
Is antibiotic resistance lost?
108
Once resistance appears it is likely to decline slowly, if at all, once no antibiotic is present.
There are no counter-selective measures against resistant bacteria. The slow loss of resistance
is linked to poorly reversible genetic and environmental factors (Levy, 1998). Resistance
genes may acquire new functions and also become maintained by factors other than the
antibiotic (Heinemann et al, 2000).
109
Expression of antibiotic resistance in bacteria may involve a fitness cost that is
disadvantageous compared with susceptible bacteria when no antibiotic is present and
resistance would therefore be gradually lost. This idea is supported by some laboratory
experiments and by a low prevalence of resistance in human and animal populations not
exposed to antibiotics. If this is the case then improving the management of antibiotics
should reduce the prevalence of resistance. However Schrag and Perrot (1996) demonstrated
that although resistance may initially impose a fitness cost, natural selection can result in
compensatory mutations that markedly reduce this cost by restoring physiological functions
impaired by resistance without altering the level of bacterial resistance. This suggests that
reduced antibiotic use may not lead to a decrease in the current prevalence of resistant
bacteria. Resistance can be maintained in bacterial populations in the apparent absence of
specific antibiotic selection for several years.
110
To restore efficacy to earlier antibiotics and maintain the success of new agents that are
introduced use needs to assure an ecological balance that favours the predominance of
susceptible bacteria (Levy, 1997).
Use of Antibiotic Resistance Marker Genes in GMOs
31
Antibiotic resistance marker genes in food
3.
Antibiotic resistance marker genes in food
Introduction
111
If food from GMOs becomes widely available there is the potential for antibiotic resistance
genes to be present in many everyday items in the diet. As a small number of different
marker genes are currently used on a regular basis it is feasible that the same marker proteins
may be present in several different GM components of the human diet thereby increasing the
total consumption of the marker gene protein (Kok et al, 1994). It is also foreseeable that
future GM food crops may contain several marker genes accumulated in consecutive
transformation events (Karenlampi, 1996).
112
However some antibiotic resistance marker genes, in particular the kanamycin resistance
gene, are not novel to the food supply. Such antibiotic resistance genes may be present in
contaminating bacteria on or in food.
113
A misconception occasionally associated with the introduction of GM food is that the
presence of antibiotic resistance marker genes means the GMO produces antibiotic. This is
incorrect. Consumers do not receive a dose of antibiotic when they eat such GM food.
114
Food safety regulation of GM food has been discussed by international expert committees
resulting in a number of advisory reports proposing different strategies for risk assessment.
The food safety of marker genes is discussed in most of these reports.
The concept of substantial equivalence
115
Assessment of the safety of food and food components derived by gene technology is based
on the concept of substantial equivalence that was elaborated by the Organisation for
Economic Cooperation and Development (OECD) in 1993 and subsequently endorsed by
the Food and Agriculture Organisation (FAO) and the World Health Organisation (WHO) in
1996. The concept of substantial equivalence compares the GMO to its traditional
counterpart as a means of identifying novel aspects that may affect its food safety. If a GM
food product is considered to be substantially equivalent to an analogous conventional food
product no additional safety concerns are expected.
116
Further evaluation is based on identified differences or, where no counterpart has been
previously consumed as food, on its composition and properties.
117
The safety assessment of GM food is by definition within the limits of current knowledge
(Jones, 1999). Marker genes are dealt with in the same way as other inserted genes.
118
Factors that influence whether the presence of a marker gene affects its substantial
equivalence to a conventional counterpart include:
• whether the marker encodes a protein product and, if so at what levels it would be
expected in the food, what its function is, and whether there are concerns about its
safety at the predicted levels;
Use of Antibiotic Resistance Marker Genes in GMOs
32
Antibiotic resistance marker genes in food
•
•
119
whether the marker encodes resistance to a clinically useful antibiotic and, if so does
ingestion at the same time as use of the antibiotic interfere with its clinical efficacy;
and
the probability of horizontal transfer of resistance genes to pathogens on or in the
food or in the consumer’s gastro-intestinal tract (OECD, 1993).
International consensus has not yet been obtained as regards interpretation and use of
substantial equivalence (Pascal, 1999).
The general basis for assessment of the potential health impact of marker genes
and their gene products
120
The structure and function of the gene and its expressed product are first compared to other
genes and proteins in order to evaluate the novelty of the marker.
121
If the gene is novel to humans the assessment also needs to consider the:
• structure of the gene and its product
• function of the gene product, and
• quantity of the gene and its product.
122
This includes the level of the gene and its product in food and the estimated daily intake, and
stability of the gene and its product in the gastro-intestinal tract (Karenlampi, 1996).
123
Other factors to consider are the availability of any required cofactor in the gastro-intestinal
tract and use of the antibiotic among those populations that eat the food.
Antibiotic resistance genes and food safety
124
Possible food safety problems from the use of antibiotic resistance genes include:
• direct consequences of the gene,
• direct consequences of the product encoded by the gene,
• indirect consequences of the effects of the gene or its product,
• possibility of horizontal gene transfer from ingested GMOs (and/or derived foods or
food components) into gut epithelial cells and/or gut micro-organisms, and
• inactivation of antibiotic by the gene product.
Direct consequences of the antibiotic resistance gene
125
DNA is present in the cells of all living organisms, including every plant and animal used for
food by humans or animals. The genes of incidental food contaminants are also consumed.
The large amount of DNA that passes the gastro-intestinal tract daily indicates that DNA
itself is not intrinsically toxic to humans.
Use of Antibiotic Resistance Marker Genes in GMOs
33
Antibiotic resistance marker genes in food
126
The DNA that makes up an antibiotic resistance gene has no unusual composition compared
to other genes (composed of four nucleotides common to all genes in all organisms in
varying amounts) and its presence poses no more health risk than the other DNA that is
ingested.
Direct consequences of the gene product encoded by the antibiotic resistance gene
127
The extent of normal exposure to protein variants is not well known but is easily in the tens
of thousands, and probably in the order of 100,000. An eukaryotic cell contains 5,000 to
10,000 different polypeptides that must be degraded to produce the amino acids required for
growth. When this number is multiplied by a factor to account for tissue-specific differences
and by the number of different species that are eaten the number of proteins in the diet
becomes very large. Genetic polymorphism also contributes to the total dietary protein array
(Kessler et al, 1992). These variants do not arise as totally new proteins but as incremental
changes of what was present previously.
128
Proteins derived from marker genes differ from proteins derived from other introduced
genes in a GM plant in two aspects. The same protein may be present in a number of GM
foods in the diet resulting in increased total marker gene protein consumption because of the
small number of marker genes currently used regularly. In addition as few tissue-specific or
developmental stage-specific promoters are yet available the gene will often be expressed in
more tissues and for a longer time period than necessary (Kok et al, 1994). It is however
likely that tissue-specific or developmental stage-specific promoters will become the norm.
129
Most proteins rapidly degrade upon consumption and exposure to the mammalian digestive
tract. The gastro-intestinal tract is specifically designed to digest ingested dietary proteins by
conversion to amino acids and small peptides that are absorbed by the intestinal tract.
130
If there is any risk from ingestion it should in most cases correlate with the potency of
functional protein available in the gastro-intestinal tract. This depends on the estimated daily
intake (and therefore the level of the protein in food) and stability of the protein in the
gastro-intestinal tract (Karenlampi, 1996). Food that potentially carries the greatest risk is
food consumed fresh (ie uncooked or unprocessed).
131
A protein is likely to be safe for consumption if based on experience proteins of the same
function have been safely consumed at similar levels. Proteins that are not functionally
similar to proteins known to be safely consumed need to be assessed relative to their
potential toxicity and allergenicity. The source, amino acid sequence and function of the gene
product can be used to identify proteins that would raise a safety concern.
132
Gene sequence information and evolutionary studies of proteins give useful guidance for
safety evaluations. Knowledge of the DNA sequence allows the use of computer algorithms
to identify other proteins with related sequences. An immunologically significant sequence
identity with known allergens requires a match of at least eight contiguous amino acids.
Criteria for comparing amino acid sequences may change or evolve over time with additional
research and insight into the molecular structure of allergens. Public domain sequence
databases include GenBank, EMBL, PIR and SwissProt.
Use of Antibiotic Resistance Marker Genes in GMOs
34
Antibiotic resistance marker genes in food
133
Physico-chemical and biological properties of the gene product can be compared to
properties of known allergenic proteins as a means of predicting allergenic potential. With
the exception of identifying known allergens transferred from allergenic sources there is
currently no single predictive property that can conclusively determine allergenic potential.
The key prerequisite for food protein allergenicity is resistance to heat denaturation and
proteolytic degradation. Typically allergens are 10 to 70 kDa in molecular weight and are
often glycosylated but there are exceptions to this generalisation (Metcalfe et al, 1996).
Relative abundance may also suggest a protein is allergenic. In general the proteins are not
likely to exceed 0.1 percent of the total soluble protein content of the GM plant material
(Metz and Nap, 1997).
134
Demonstration of the lack of amino acid sequence homology to known protein toxins or
allergens and their rapid proteolytic degradation under simulated mammalian digestive
conditions is considered appropriate to confirm safety. Demonstration of proteolytic
digestion under both gastric and intestinal conditions supports the expectation that the
protein is likely to be degraded during food consumption and digestion (FAO and WHO,
1996). To elicit an allergenic response the protein must survive the acid and proteolytic
environment of the gastro-intestinal tract to reach and be absorbed through the intestinal
mucosa and trigger a series of IgE-mediated responses.
135
In vitro testing is however not identical to the physiological conditions in the gastro-intestinal
tract. The conclusions drawn from such testing may not always give clear evidence on the
possible toxic or allergenic potential of peptides formed as breakdown products in the test
system. The absence of homology of a protein’s primary structure to a known allergen also
does not exclude the presence of allergenic epitopes formed by its secondary or tertiary
structure (OECD, 2000a).
136
Validated animal models are not yet available for evaluation or prediction of a novel protein’s
allergenicity or possible unintended effects. Traditional animal feeding studies are designed to
assess safety of substances that are an insignificant component of the diet such as food
additives. Such studies are inadequate for testing whole foods that are a substantial dietary
component due to the difficulty of feeding animals adequate doses of the test food. In many
cases meaningful information is unlikely to be produced (OECD, 2000a). Experiments on
the protein alone can provide more meaningful information than experiments on the whole
food.
137
New and better methods to evaluate GM foods are needed (Metz and Nap, 1997). Although
not considered necessary for the kanamycin resistance gene because of its evaluation to date
(see Appendix I) and history of human exposure, further evaluation is required for some
other resistance marker genes eg hygromycin, streptomycin (Karenlampi, 1996).
Consideration needs to be given to whether long term feeding studies are necessary to
provide greater information on potential allergenicity and toxicity (The Royal Society, 1998).
Re-examination of the methods for testing allergenicity and toxicity was also a
recommendation from the recent OECD conference on the scientific and health aspects of
GM foods (Krebs, 2000).
138
Many antibiotic resistance genes used in genetic modification are present on commercially
available plasmids whose characteristics are known and can therefore be taken into account
Use of Antibiotic Resistance Marker Genes in GMOs
35
Antibiotic resistance marker genes in food
in the assessment. For example, a gene with bacterial regulatory sequences that remains in a
construct with a high copy number bacterial replicon may dictate higher expression levels if it
is expressed in the GMO than those found from replicons currently present in nature
(ACNFP, 1996).
Indirect consequences of the effects of the antibiotic resistance gene or its gene product
139
There are no characteristics of marker genes or their products that suggest that their site of
insertion into the plant genome will result in specific secondary and/or pleiotrophic effects
that may in some way alter any of the organism’s toxicological or ecological characteristics.
Possible secondary effects in the plant due to insertion need to be determined on a case-bycase basis as they are expected to be highly dependent on the host plant and the site of
insertion of the marker gene. Secondary effects can be assessed by comparing key substances
to establish substantial equivalence (WHO, 1993).
140
Few data are available with respect to GM plants containing a selectable marker gene and no
methods yet exist to approach these issues in an undisputed way. Prediction can be
attempted to a limited extent. Pleiotrophic effects are therefore very difficult issues for safety
assessments. At present, for both the toxicology and ecology of GM plants it is unclear
whether unpredictable pleiotrophic effects, such as changes in a metabolite(s) or plant
growth and development, do occur to the extent that any effects can be measured in a
meaningful way. With respect to safety and biosafety regulations, general considerations seem
to allow the conclusion that putatively pleiotrophic effects will be of no or only minor
importance. However this view is controversial (Metz and Nap, 1997).
141
The food safety risks associated with development of GM plants need to be considered in
the context of the risks associated with plants developed using conventional breeding
methods. It has been argued that as gene technology is more precise and allows better
characterisation of the changes occurring developers are better placed to assess safety than
when using conventional methods (Smith, 2000).
Use of Antibiotic Resistance Marker Genes in GMOs
36
Antibiotic resistance marker genes in food
Horizontal gene transfer in humans
Potential gene transfer to oral micro-organisms
142
The regions preceding the stomach (ie mouth and oesophagus) are likely to have the highest
concentrations of intact DNA entering with the diet. Free DNA has been shown in vitro to
survive for 10 minutes (between 35 and 61 percent had been degraded) in human saliva and
to be capable of transforming a naturally competent human oral bacterium (Streptococcus
gordonii) to erythromycin resistance (Mercer et al, 1999). Further research is needed to
establish whether transformation of oral bacteria can occur at significant frequencies in vivo.
143
Pollen and other airborne sources such as dust from dry milling are also a potential source of
exposure to DNA from GM plants. The United Kingdom’s Advisory Committee on Novel
Foods and Processes (ACNFP) considers the likely levels of exposure and potential gene
transfer to oral and respiratory tract bacteria from GM plants in its assessment (OECD,
2000a). No references were found in the peer reviewed scientific literature that discussed this
risk.
Potential gene transfer to gut epithelial cells or micro-organisms
144
Although most of the ingested DNA will be degraded and diluted, natural transformation of
gut epithelial cells or micro-organisms cannot be excluded. It is conceivable that microenvironments exist where DNA is not degraded or that certain dietary components protect
against degradation (Flint and Chesson, 1999).
145
It is generally considered that the probability of antibiotic resistance genes being transferred
from GMO material to either gut epithelial cells or micro-organisms is extremely low.
However few experimental data are available to support this theoretical assessment (Kok et
al, 1994).
Degradation of DNA
146
Any gene is unlikely to be intact or functional after processing and/or cooking. In
unprocessed food the gene is likely to be intact when consumed but DNA is rapidly broken
down under normal gastro-intestinal conditions into fragments usually too small to be
functional.
147
Research in mice has however indicated that DNA can survive digestion. About two to four
percent of orally ingested foreign DNA was detected in the gastro-intestinal tract of mice and
fragments were detected in the faeces between one and seven hours after feeding. For
humans of average weights between 50 and 80 kg, an intake of about 50 to 80 mg of DNA in
the daily food would be needed to parallel the situation simulated in the mouse experiments
(Schubbert et al, 1994). This is unlikely to be encountered in a normal diet. For example,
Calgene Inc estimated the daily amount of ingested kanamycin resistance gene from fresh
Flavr Savr tomatoes was 0.33-1 pg (Karenlampi, 1996).
148
Although proteins are broken down to smaller peptides and amino acids by digestive
enzymes the possibility that the protein encoded by the gene remains intact needs to be
considered. In vitro models (eg Minekus et al, 1995) have been developed to simulate the
Use of Antibiotic Resistance Marker Genes in GMOs
37
Antibiotic resistance marker genes in food
human gastro-intestinal tract to study the fate of ingested compounds. They can be modified
to simulate atypical conditions (eg high gastric pH) that may be encountered in some people
that might affect protein degradation.
149
Seeds from some GM plants and some viable GM micro-organisms are notable exceptions to
DNA degradation. In seeds protected by a resilient seed coat (eg tomato) the DNA remains
intact through the gut but is not available for gene transfer. Even if transfer were to occur
expression is unlikely unless the regulatory sequences on the transferred sequence are
functional in the gut epithelial cell or gut micro-organism.
Potential DNA uptake
150
One of the public concerns associated with the introduction of GM food is the possibility
that genes from GMOs may be taken up by those eating such food and become part of their
genetic makeup. A cell would have to take up the DNA in question and integrate it into its
own genetic material. The view that DNA is unlikely to enter human cells is supported by the
limited success of gene therapy, even when conditions for transfer are optimised, carried out
during the last decade (Donaldson and May, 1999). The acquired gene must also have the
ability to be expressed in the cell (eg genes from bacteria are normally not expressed if
transferred to an eukaryotic cell).
151
Cells that may come into direct contact with the gene are the epithelial cells of the mucous
membrane that covers the surfaces of the gastro-intestinal tract. These cells are terminally
differentiated (ie do not divide) and have a relatively short life span of seven days.
152
There is some recent evidence in mice to show possible DNA uptake. Although after a single
feeding episode of phage M13 DNA in mice more than 95 percent of the foreign DNA was
lost after passing through the stomach, the findings suggest transport of very small quantities
of foreign DNA through the intestinal wall and Peyer’s patches to peripheral blood
leucocytes (white blood cells) and into several organs (spleen, liver). Fragments were found in
about 0.1 percent of the peripheral blood leucocytes up to eight hours after feeding and in
the spleen and liver up to 18 hours after feeding. The mechanism of foreign DNA uptake by
the gut epithelial cells is unknown. Foreign DNA was also detected up to 18 hours after
feeding in the caecum. However no foreign DNA was found in intestinal bacteria (Schubbert
et al, 1997).
153
Similar results to the study above were obtained by Schubbert et al using a plasmidcontaining gene for the green fluorescent protein (GFP) that was orally administered to mice.
It was detected in the intestinal contents, liver, spleen and kidney up to eight hours after
feeding and in the intestinal wall three to eight hours after feeding.
154
When foreign DNA was fed to pregnant mice DNA fragments were detected in various
organs of fetuses and newborn mice. Foreign DNA was not found in all the cells of the fetus
suggesting that the DNA entered the fetus transplacentally rather than by germ line
transmission (Schubbert et al, 1998).
155
There is some in vitro evidence that DNA can be transferred from invasive strains of bacteria
(Shigella flexneri and E. coli) to mammalian cells provided that they are able to penetrate the
host cell (Courvalin et al, 1995; Grillot-Courvalin et al, 1998). This direct gene transfer
Use of Antibiotic Resistance Marker Genes in GMOs
38
Antibiotic resistance marker genes in food
showed a broad host cell range and the vectors were stably inherited and expressed by the
cell progeny.
156
To date DNA uptake has not been reported for other DNA sources such as food and the
significance of the findings of Schubbert et al is unclear. DNA is consumed daily by most
people in most of the food (eg fruit, vegetables, cereals, meat) that they eat.
157
There are no published reports on the transfer, integration or expression of genes in human
gut epithelial cells (US FDA, 1998). Even if transformed, gut epithelial cells have a rapid
turnover and since the germ line is unaffected transformed cells could not be maintained or
spread in the human population. Many gut micro-organisms carry antibiotic resistance genes
but no problem associated with transfer to gut epithelial cells has ever been identified
(ACNFP, 1994).
158
Some experts participating in consultation carried out by the FDA cautioned that it should be
assumed that DNA can get into the gut epithelial cells but the critical factor is the lack of
selective pressure. Without selective pressure it is highly unlikely that genes taken up by these
cells would be expressed even if they were integrated into the genome (US FDA, 1998).
Potential transfer to gut micro-organisms
159
Homology between sequences in an antibiotic resistance marker gene that is prokaryotic in
origin and the recipient’s DNA is more likely to be found in gut micro-organisms which are
prokaryotic than in gut epithelial cells which are eukaryotic. The probability of integration
and expression of a marker gene is therefore greater in gut micro-organisms than in gut
epithelial cells.
160
Potential gene transfer scenarios to gut micro-organisms include from:
• micro-organisms in or on food
• GM food micro-organisms
• unintentional ingestion of GM micro-organisms released into the environment, and
• GM food (plant or animal).
161
For gene transfer to occur the following events are required:
• release of DNA from cells or tissue
• survival of DNA in the gastro-intestinal tract, including exposure to gastric acid and
nucleases,
• recipient micro-organisms are competent for transformation
• recipient micro-organisms bind the DNA to be transferred
• DNA penetrates the cell wall and translocates across the cell membrane
• DNA survives the restriction or modification system developed by the microorganism to degrade foreign DNA, and
• DNA is integrated into the host genome or plasmid (ACNFP, 1996).
162
If transfer is successful the gene must be expressed for it to have any impact.
Use of Antibiotic Resistance Marker Genes in GMOs
39
Antibiotic resistance marker genes in food
163
Viable GM micro-organisms may remain intact through the gut and gene transfer could be
achieved through conjugation with gut micro-organisms. Conjugation between lactic acid
bacteria and related species present in the gut has been recorded in vitro (ACNFP, 1994).
164
No data are available concerning the natural transformation of bacteria in the human gastrointestinal tract. The transit time of food in the mouth, pharynx, oesophagus, stomach and
duodenum is rapid and conditions in the stomach will prevent any natural transformation.
The most relevant location for possible gene transfer therefore is the lower part of the small
intestine (ileum) and the colon in which consumed food stays a relatively long time (Nap et
al, 1992).
165
There is concern about the potential consequence of transfer of an introduced gene from
GM food to gut micro-organisms in such a way that the gene can be successfully integrated
and expressed and impact on human health. The probability of transfer in the gastrointestinal tract has to be assessed in the light of the nature of the GMO and the
characteristics of the gene construct.
166
Conditions in the mammalian intestine are probably more conducive to gene transfer than
conditions found elsewhere in nature as the environment is warm, wet and contains
abundant nutrients. Concentrations of bacteria are so high that encounters between different
types of bacteria occur readily and residence time in the intestine is long enough to provide
many opportunities for gene transfer. However there has been little direct evidence to
support this hypothesis (Salyers, 1993).
167
When an antibiotic resistance gene is integrated into the plant genome the codon usage may
have been altered for more efficient expression in the plant and the gene may have picked up
the plant’s methylation patterns. If this DNA is now taken up by a bacterium it would be
recognised as foreign and degraded by the micro-organism’s restriction enzymes thus making
integration and expression even more unlikely (US FDA, 1998).
168
Since uptake is usually not sequence-specific an antibiotic resistance gene would also be
competing for transfer into a bacterium with the rest of the DNA in the plant genome and
DNA from other sources in the diet (US FDA, 1998).
169
The probability of integration into the genome of a gut micro-organism depends on the
degree of homology of the foreign DNA with that of the genomic DNA. At least 20 base
pairs in a complete homologous DNA sequence are required for significant recombination at
both ends of the foreign DNA. Antibiotic resistance genes are of bacterial origin and there
may be sufficient homology to allow integration of such genes into gut (or rumen) bacteria
under favourable circumstances. They are also propagated in E. coli and as a result there is
likely to be sufficient homology for plasmid or chromosomal integration into E. coli,
Enterobacter, Salmonella, Shigella or Pseudomonas to be feasible. It is possible that other less well
understood processes such as illegitimate recombination could also lead to plasmid or
chromosomal integration into gut (or rumen) bacteria (ACNFP, 1996).
170
The probability that foreign DNA would persist in a micro-organism is significantly
enhanced under conditions that exert selective pressure viz oral therapeutic use of the
relevant antibiotic. The antibiotic would not provide selective pressure if the gene is under
Use of Antibiotic Resistance Marker Genes in GMOs
40
Antibiotic resistance marker genes in food
the control of an eukaryotic promoter and therefore unable to be expressed in microorganisms (FAO and WHO, 1996). Rearrangements could however bring a prokaryotic
promoter in front of the gene leading to its expression (US FDA, 1998).
171
In January 1999 the New Scientist reported research in the Netherlands indicating that some
DNA can reach the large intestine intact and survive long enough for transfer to occur.
Using a computer-controlled artificial gut Havenaar and coworkers found that DNA from
GM bacteria containing an antibiotic resistance gene remains intact in the simulated large
intestine for a half-life of six minutes. If the modified bacteria were normally in the gut (eg
Enterococcus) each bacterium had a one in 10 million chance of transferring DNA containing
an antibiotic resistance gene to an indigenous gut bacterium. This suggests many bacteria
would be transformed given there are approximately 1014 bacteria present in the gastrointestinal tract. Transfer of antibiotic resistance was not detected from micro-organisms not
normally present in the gut (eg Lactobacillus) or the Flavr Savr tomato. The transfer rate
increased ten-fold when some normal gut micro-organisms were killed such as would occur
during antibiotic therapy (MacKenzie, 1999). To date this research has not been published in
the peer reviewed scientific literature.
172
Much of the literature relating to gene transfer relates to transfer in environments other than
the human gut. There are differences between the gastro-intestinal tract and other
environments such as soil that need to be taken into account in assessing possible food safety
concerns. These include:
• free DNA for uptake is continuously degraded in the gastro-intestinal tract;
• there are no authenticated reports of bacterial transformation in the human gastrointestinal tract;
• DNA degradation begins well before the arrival of material at the critical sites for
transformation (ie lower small intestine, caecum and colon);
• DNA degradation rapidly fractionates the DNA to sequences smaller than needed for
proper expression;
• for effective uptake of DNA by gut bacteria the plant or animal DNA should
undergo recombination and be expressed in the recipient bacteria.
173
In the case of bacteria a gene is most likely to be transferred if it is on a broad host range
gene transfer element such as a plasmid or transposon. Resistance to chloramphenicol,
streptomycin, ampicillin and neomycin is located on plasmids and transposons. The least
transmissible genes are those that are integrated into the chromosome and not linked with
gene transfer elements. For such genes to be transferred they must be acquired by a
transmissible element which requires strong selective pressure (Salyers, 1999).
174
A GM micro-organism intended for environmental release, where antibiotics are unlikely to
be encountered in concentrations sufficient to exert selective pressure, would be unlikely to
make a non-transmissible antibiotic resistance gene capable of transfer to other microorganisms. In the unlikely event that transfer did occur selection would be needed for it to be
incorporated and expressed in the new host. In contrast a GM micro-organism administered
to animals that were eating antibiotic-supplemented feed or to humans exposed to antibiotics
might experience the selective pressure needed to foster gene transfer. It is of note that
naturally occurring resistant strains (eg probiotic strains of Lactobacillus) could present as great
Use of Antibiotic Resistance Marker Genes in GMOs
41
Antibiotic resistance marker genes in food
or greater hazard as GM micro-organisms because their resistance genes are more likely to be
on transmissible elements (Salyers, 1999).
175
There is a relatively greater probability of transfer to gut micro-organisms from ingested GM
micro-organisms. If transfer of the intact gene occurred it is possible that the DNA could
become functional as the same or similar promoters used for expression in the GM microorganism may also be present in the gut micro-organism. It is also possible that intact
plasmid vectors from GM micro-organisms could be transferred to and maintained in gut
micro-organisms, and although less likely transposition could be achieved by systems used in
the recipient species (ACNFP, 1994).
176
Functional antibiotic resistance genes in GM probiotic bacteria would be expected to persist
in the gut since probiotic bacteria are intended to survive in and colonise the gut. The extent
to which they would compromise the use of the corresponding antibiotic would depend inter
alia on the level of gene expression and the extent of any cross-resistance to other antibiotics
(ACNFP, 1994).
177
In nature antibiotic resistance is widespread among contaminating micro-organisms found in
or on food. It is more likely that antibiotic resistance genes would be introduced into gut
micro-organisms through transfer between naturally occurring ingested contaminating microorganisms and gut micro-organisms than through transfer from DNA released during
digestion of GM plant or animal material. The probability that transfer occurs between
ingested GM micro-organisms and gut micro-organisms is likely to be the same as between
non-GM micro-organisms and gut micro-organisms (ACNFP, 1994).
Inactivation of antibiotic by the gene product
178
Under certain circumstances such as ingestion of viable GM micro-organisms there is a low
but finite probability that antibiotic resistance genes could be transferred. If transfer occurs
and the transformed micro-organisms survive, colonise the gut and express the protein
(whether or not it is expressed in the plant) an increase in gut micro-organisms resistant to
the specific antibiotic could result. Given sufficient selective pressure and because of the
short generation times of bacteria clonal expansion of the transformed bacteria could occur.
179
The gene involved may possibly interfere with antibiotic therapy either by the coincident
consumption of food containing the gene product with oral doses of the antibiotic or by the
transfer of the resistance gene to pathogenic gut micro-organisms treated with the particular
antibiotic.
180
The probability that all of the prerequisite events will occur resulting in a public health
problem is very unlikely but cannot be excluded. The clinical use and importance of the
antibiotic to which the gene confers resistance is therefore a central part of the safety
assessment.
181
Clinically important antibiotics include vancomycin and other glycopeptides, macrolides,
fluoroquinolones, gentamicin, amikacin, and amoxycillin and later derivatives of beta-lactam
antibiotics.
Use of Antibiotic Resistance Marker Genes in GMOs
42
Antibiotic resistance marker genes in food
182
A WHO workshop (1993) concluded that it is necessary to identify all oral uses of the
antibiotic and possible gastro-intestinal conditions (eg high gastric pH) that may interfere
with the normal degradation of DNA, and to determine the necessary enzyme cofactors and
their presence in the gut. Calculation of the extent of potential inactivation of an oral dose of
the antibiotic should be based on the maximum intake of the GM food that could be
consumed during oral antibiotic therapy.
183
Estimation of the extent of the antibiotic’s use indicates the therapeutic consequences should
the resistance become prevalent in bacterial pathogens. The potential for cross-resistance also
has to be taken into account as many genes are effective against antibiotics with similar
structures and mechanisms. Although the antibiotic used for selection may be rarely used it is
possible others in the group may be of therapeutic importance (eg amikacin). The prevalence
of the resistance gene in natural populations is also important (ACNFP, 1996).
184
Theoretical calculations of the probability of a transfer event based on a number of
assumptions have shown that the chances of this occurring are extremely small and probably
insignificant when compared with naturally occurring resistance. Experiments to test whether
these assumptions and calculations are correct although difficult to perform would be
extremely valuable. Studies with human volunteers could be considered (Kok et al, 1994).
Preliminary results from a study by Heritage and coworkers of gene transfer from GM maize
to chicken gut bacteria have found no evidence of transfer of the ampicillin resistance gene
to normal flora (Coghlan, 2000). This study will also investigate whether gene transfer occurs
from GM maize to sheep gut bacteria.
185
The types of experiments carried out should be commensurate with the importance of the
antibiotic that may be compromised. While an in vitro model is sufficient for an antibiotic that
is relatively unimportant clinically, studies in animals may be warranted for important
antibiotics. Some scientists have suggested that it could be concluded that transfer does not
occur if a large number of animals were fed GM plants containing an antibiotic resistance
gene under strong selective pressure and new resistant micro-organisms with this genotype
were not detected (US FDA, 1998).
Use of Antibiotic Resistance Marker Genes in GMOs
43
Horizontal gene transfer mechanisms in bacteria
4.
Horizontal gene transfer mechanisms in bacteria
186
The significance of horizontal gene transfer for bacterial evolution was not recognised until
the 1950s when multi-drug resistance emerged on a global scale (Ochman et al, 2000).
187
Bacteria transfer resistance genes through three main mechanisms. DNA can enter the cell
on plasmids through cell-to-cell contact between two cells (conjugation), by bacteriophage
introduction (transduction), or by cellular uptake of free (extracellular) DNA
(transformation). Gene transfer crosses species and genus barriers and groups of resistance
genes tend to travel together. This has contributed largely to the efficiency by which
antibiotic resistance has spread.
188
The relative importance of the gene transfer mechanisms in nature is not known. The
specific requirements of each mechanism suggest different probabilities for their occurrence
in different natural habitats. Conjugation has the greatest requirements. Each transfer
mechanism is also characterised by its specific host range.
189
Barriers to gene transfer between bacterial species include different microhabitats, host
ranges of genetic exchange vectors and barriers that block the establishment of the acquired
genetic information.
190
To become replicable and stably inherited, transferred DNA must become integrated into the
recipient chromosome. Efficiency of integration by homologous recombination depends on
the genomic sequence divergence between species. Once recombination is initiated genetic
integrity is controlled by the mismatch-repair system that inhibits recombination.
191
The frequency of recombination in natural populations depends upon the recombination rate
and natural selection. If the transformants are selectively disadvantageous they will be lost
from the population and never observed. If they are selectively neutral most may be lost but
some may be retained though at a lower frequency than those that are selectively
advantageous. The rate of interspecies recombination is very low but the rare transformants
can survive in situations of strong selective pressure (Matic et al, 1996).
192
Genes that do not provide a meaningful function also tend to be lost from bacterial genomes
(Ochman et al, 2000).
Transduction
193
Transduction can occur in a wide range of bacteria. In transduction bacteriophages pick up
genetic material from one bacterial cell and place it in another. As part of their life cycle
bacteriophages attach to bacteria and inject their DNA. This DNA then serves as the
blueprint for making more copies of the bacteriophage which are released from the infected
bacterium and go on to infect other cells. Sometimes some of the new particles carry
bacterial instead of viral DNA and deliver it to a second bacterium that incorporates it into
its chromosome. Bacteriophages are capable of transferring whole plasmids and pieces of
chromosomes between hosts. Transduction depends on the survival of the transducing
Use of Antibiotic Resistance Marker Genes in GMOs
44
Horizontal gene transfer mechanisms in bacteria
particles but not of the donor bacterium. Most bacteriophages infect only one species of
bacteria and most bacteriophages in the wild infect only bacteria that are native to the
bacteriophage’s habitat (Miller, 1998).
Conjugation
194
Conjugation requires cell-to-cell contact and is a process by which plasmids or transposons
transfer DNA from donor to recipient cells. It occurs in both Gram-positive and Gramnegative bacteria and can involve distantly related species. Some plasmids can transfer DNA
between very unrelated species: between Gram-negative and Gram-positive bacteria, from
bacteria (E. coli) to yeast, and from bacteria (A. tumefaciens) to plants (Droge et al, 1998).
Conjugation requires live metabolically active donor and recipient cells.
195
The main limitation is the host range in which the element can express its genes and
replicate. Since many of the conjugative elements exhibit a broad host range of transfer and
autonomous replication, conjugation is regarded as an important factor for gene transfer
among bacteria.
196
Conjugal transfer of resistance genes between the normal microflora of animals and humans
can be demonstrated under laboratory conditions (Shoemaker et al, 1992). Bacterial gene
transfer studies in model ecosystems or in the environment have identified environmental
hot-spots, particularly the phytosphere, where conditions increased the probability of
conjugative transfer among bacteria (Pukall et al, 1996; Droge et al, 1998).
Transformation
197
Transformation is the process by which a competent bacterial cell takes up, integrates and
expresses foreign DNA. Transformation in both Gram-negative and Gram-positive bacteria
requires that the free DNA remains stable and that potential recipient cells become
competent to take it up (ie the recipients must display specialised surface proteins that bind
to the DNA and internalise it).
198
Transformation does not require a living donor cell because release of DNA during death
and cell lysis suffices to provide free DNA. Therefore it can potentially occur with DNA
from any source. Transformation in bacteria is assumed to be limited to those situations
where the incoming DNA can reconstitute to form a self-replicating entity (eg plasmid), or
where there is sufficient sequence homology with the recipient chromosome to allow
insertion by recombination.
199
Currently only about 40 species are known to be capable of developing the competence for
transformation (Gebhard and Smalla, 1998). The natural competence to act as recipients has
been identified in several genera including Acinetobacter, Haemophilus, Pneumococcus, Streptococcus,
Bacillus, Pseudomonas and Neisseria. Since some of these bacterial species are promiscuous in
their uptake of free DNA, gene transfer by transformation between even distantly related
bacteria is possible (Droge et al, 1998).
Use of Antibiotic Resistance Marker Genes in GMOs
45
Horizontal gene transfer mechanisms in bacteria
200
Experiments under controlled conditions suggest there may be species-specific and even
strain-specific responses to environmental factors with regard to competence development
and transformation efficiency. How long the competent state of cells can be maintained once
it is achieved is important for the overall probability that a cell will become transformed.
201
There is no specific model micro-organism that is representative of other naturally
transformable bacteria in a given habitat.
Evidence for gene transfer between bacteria
202
Little is known about natural transformation among gut bacteria for most gut species. An in
vivo experiment by Griffith in 1928 in mice in which the non-pathogenic form of S.
pneumoniae was transformed by dead cells from the pathogenic form was the first
demonstration of gene transfer by transformation. There is no other direct experimental
evidence for transformation in vivo. However results of co-cultivation experiments suggest
that substantial intra- and interspecies transfer of virulence determinants via transformation
occurs in Neisseria species. An increasing body of nucleotide sequence data of chromosomal
genes in transformable species further suggests the occurrence of frequent genetic exchange
within and between species (Lorenz and Wackernagel, 1994).
203
There is also evidence in several naturally transformable bacteria (eg Neisseria meningitidis) of
antibiotic resistance arising from transfer of DNA, including from different species, and
formation of mosaic genes from the original susceptible alleles and introduced resistant
alleles. These mosaic genes express enzymes with decreased affinity for the antibiotic
(Maiden, 1998).
204
Transfer of a plasmid containing an antibiotic resistance gene between Enterococcus faecalis and
Lactobacillus species has been demonstrated in the murine intestinal tract (McConnell et al,
1991). Although this gene transfer event involved members of minor populations of
intestinal bacteria it shows that such transfers do occur in the intestine.
205
Discovering that antibiotic resistance genes in two different genera of bacteria are 99 percent
identical at the DNA sequence level suggests that a horizontal transfer event occurred
sometime during the past million years but not that the transfer event was recent. In contrast
the finding of 100 percent sequence identical tetQ genes in two different human colonic
Bacteroides species suggests transfer has occurred recently. However more systematic studies
are needed to establish that such transfers occur frequently and to determine whether their
frequency is related to the widespread use of antibiotics (Salyers, 1993).
206
Failure to demonstrate transfer of a particular marker gene from a bacterial donor to a
bacterial recipient under laboratory conditions does not mean that it is non-transmissible.
Transfer could be regulated and if so the proper inducer may not have been used.
Alternatively the marker gene could be on an element that is mobilised by some other gene
transfer element that is not present in the particular donor (Salyers, 1993).
207
From laboratory studies transformation and conjugation are recognised as the gene transfer
mechanisms that have the broadest host range. It has been argued that in the environment
Use of Antibiotic Resistance Marker Genes in GMOs
46
Horizontal gene transfer mechanisms in bacteria
transformation may be a rare event except under defined conditions whereas conjugation
may be favoured because the genetic material to be transferred is protected from attack by
free nucleases. In addition conjugal DNA transfer has been shown to partially overcome host
restriction systems due to the transfer of single stranded DNA that may be refractory to
nuclease activity in the recipient.
208
Understanding of the mechanisms and factors involved in gene transfer in natural bacterial
populations is very limited (Davies, 1997). Probably not only the physical characteristics of
the population but also other factors play important roles in natural gene transfers. For
example, some antibiotics have been shown to promote conjugation of resistance genes.
209
Low doses of tetracycline increased conjugative transfer of a transposon conferring resistance
to kanamycin, erythromycin and tetracycline from E. faecalis to Listeria monocytogenes in the
gastro-intestinal tract of mice (Doucet-Populaire et al, 1991).
Prevalence of antibiotic resistance genes
210
Resistance genes reside in commensal (normally harmless) bacteria as well as pathogens.
Some antibiotic resistant bacteria occur naturally in the environment but many arise by
contamination with human and animal excreta in sewage, slurry and manure. This in turn
contaminates water and agricultural land to become a source of resistant bacteria for animals
and humans. They may be ingested as contaminants of water and food or in the case of
animals by licking their environment. Even people not receiving antibiotics or on a
vegetarian diet may be colonised by large numbers of resistant bacteria (Linton, 1986).
211
Enterococci and streptococci that contain resistance plasmids or transposons are common in
the gastro-intestinal tract of humans and animals. These bacteria might serve as a reservoir of
resistance genes for other bacteria (Doucet-Populaire et al, 1991).
212
In addition a number of antibiotics are contaminated with DNA encoding antibiotic
resistance from the producing organism (Webb and Davies, 1993).
213
A US study found high level aminoglycoside resistance in aquatic environmental isolates of
enterococci (Rice et al, 1995). The occurrence of high level resistance is an example of
acquired rather than intrinsic resistance.
214
A study by Smalla et al (1993) showed that resistance to kanamycin is widely distributed
among culturable bacteria isolated from different habitats. Since culturable bacteria represent
only a minor proportion of the bacterial populations of the tested habitats the total DNA
extracted directly from environmental samples was used to get information about the natural
occurrence of the nptII gene and the transposon Tn52 in non-culturable bacteria. Bacteria
may be naturally non-culturable or become so as a result of environmental stress. This
approach gives information on the presence of a certain sequence in a sample but not on the
localisation of the detected sequence (ie in culturable, non-culturable or dead bacteria, or in
free DNA).
2
The nptII gene is commonly associated with transposon Tn5.
Use of Antibiotic Resistance Marker Genes in GMOs
47
Horizontal gene transfer mechanisms in bacteria
215
Bacteria containing Tn5 or nptII were not found in soil and were primarily obtained from
sewage samples. Evidence for the occurrence of nptII was obtained for sewage, pig manure
slurry, river water and some soil samples via polymerase chain reaction (PCR) analysis of
environmental DNA extracts. Tn5 was not detectable via PCR in any of these extracts but it
was found in soil samples taken from a field two and four weeks after release of a Tn5containing GMO. Although the kanamycin resistance phenotype is widespread among
environmental bacteria Smalla et al (1993) concluded that Tn5 or nptII are scarce.
216
Using a marker-rescue system de Vries and Wackernagel (1998) found nptII without the need
for amplification by PCR in all four soil samples tested.
217
Henschke and Schmidt (1990) found a high proportion of antibiotic resistant strains among
soil bacteria.
218
A US study of the natural frequency of antibiotic resistance genes (to seven different
antibiotics) in the commensal gut microflora of people in the community and in hospital
found resistance was common in the absence of concurrent or recent antibiotic use. In more
than a third of the faecal samples resistant strains constituted a major proportion of the total
microflora (Levy et al, 1988).
219
In people with no recent history of antibiotic use at least 10 percent of the total microflora
was resistant to at least one antibiotic in 63 percent of samples. Thirty-eight percent of the
samples showed resistance(s) in half or more of the microflora. This level of resistance may
be the result of slow loss of resistance genes acquired from previous antibiotic use, ingestion
of resistant bacteria in food, or other unknown factors that favour persistence of resistance
genes.
220
Individuals taking antibiotics produced more samples with a higher proportion (≥ 50 percent)
of resistant bacteria and these samples also had a significantly greater number of different
resistance genes.
221
Data from a multiple sample group revealed changes in resistance patterns were very
common and occurred frequently within a two week period. Ninety percent of individuals
showed a gain and/or loss of one or more detectable resistances (Levy et al, 1988).
222
About 106 to 108 E. coli are generally found per gram of human faeces. Given about 100g of
faeces is excreted per day, an individual with a 10 percent frequency of a resistance gene
produces 107 to 109 resistant bacteria per day. Even at a one percent resistance level relatively
large quantities of resistance genes are being excreted. This represents a large reservoir of
resistant bacteria and resistance genes potentially transferable, directly or indirectly to human
pathogens (Levy et al, 1988). Notably this study preceded the release of GMOs containing
antibiotic resistance genes into the US food chain. This refutes the claims of Ho who links
the commercialisation of gene technology to the spread of antibiotic resistance. No direct
evidence is provided for the hypothesis but the claim is deduced from the evidence that
horizontal gene transfer spreads antibiotic resistance (Ho, 1998).
Use of Antibiotic Resistance Marker Genes in GMOs
48
Ecological issues
5.
Ecological issues
Introduction
223
Specific ecological concerns with respect to antibiotic resistance marker genes relate to the
possibility that if the gene is expressed in the plant antibiotic resistance might result in a plant
or one of its wild relatives becoming a weed, or might disturb the ecological relationships of
the plant in another unknown way. The antibiotic resistance gene could also potentially be
transferred from the GMO to soil micro-organisms. Any increase in antibiotic resistant soil
micro-organisms could lead to a potential increase in human exposure to antibiotic resistant
micro-organisms from ingesting them as contaminants of food and water.
Antibiotic resistance and weediness
Gene transfer
224
The antibiotic resistance gene might spread from the GM plant to sexually compatible
species or to other organisms that as a result become a weed. Gene transfer by sexual
hybridisation to plants of the same or related species depends on the degree of sexual
compatibility between the donor and recipient species as well as the physical distance
between the two, the duration of pollen viability, and factors such as temperature. The donor
and recipient plants should produce receptive flowers at the same time in the presence of any
necessary pollinating agent. If fertilisation successfully occurs it may not always result in a
plant that is able to grow successfully, or if a plant is produced then it may not compete well
with other species in the environment. If the resulting plant produces pollen that goes on to
fertilise other plants then the inserted gene will reach an equilibrium in the overall population
if there is no selective advantage for the plants that contain it.
225
The spread of an antibiotic resistance gene to relatives via pollen depends on a variety of
ecological and genetic factors and stochastic events. Factors concerning the plant itself and
the ecological relationships with other species of that plant at the particular location need to
be taken into account to be able to estimate the probability of cross-pollination. Interspecies
gene transfer is much less frequent than intervarietal exchanges (Thuriaux, 1999). It is
prudent to assume that an antibiotic resistance gene may spread by cross-pollination in some
conditions and at some locations with a certain probability that can be estimated (Nap et al,
1992).
226
There is little information relating to the transfer distances of pollen from GM plants
containing marker genes and the dynamics of its spread in a natural population (Harding,
1999).
227
Antibiotic resistance would only be able to contribute to the weed characteristics of a plant if
the environment exerts selective pressure. Even then it may have no effect on weediness.
Similarly, relatives that receive the gene would only become less controllable than the parent
plant in the context of selective antibiotic conditions.
Use of Antibiotic Resistance Marker Genes in GMOs
49
Ecological issues
228
Monocotyledonous plants are already highly resistant to kanamycin probably because it
cannot enter the cell in sufficient amounts. Kanamycin resistance may therefore develop as a
result of spontaneous mutations and does not seem to require the transgene. As a result
phenotypic kanamycin resistance should not be considered a novel characteristic for an
ecosystem and will not contribute to enhanced weediness of a plant or its relative (Nap et al,
1992).
229
The only situation in which antibiotic resistance feasibly contributes to a selective advantage
of the GM plant is when selective antibiotic concentrations are found in the environment as
a result of natural production by soil micro-organisms or addition of antibiotic to soil.
Antibiotics in soil
230
Soil is a reservoir of micro-organisms with the capacity to produce antibiotics. Kanamycin
and neomycin are produced by soil micro-organisms but it is unclear whether they are only
produced under laboratory and industrial conditions or also in the natural environment.
Actual concentrations of antibiotics in natural soils are also unclear and difficult to estimate,
since with few exceptions antibiotics are rarely isolated from soil. This is probably a
methodological problem since, depending on the specific antibiotic, antibiotics are presumed
to bind to varying degrees to soil and hence become inactivated (Nielsen et al, 1998).
231
There is a lack of information about the extent of selective pressure of different antibiotics in
soil and the soil conditions that promote such selective pressure. The selective advantage of
expressing antibiotic resistance genes in soil is unclear and estimation of the selection of
putative transformants receiving antibiotic resistance genes from GM plants is currently not
possible (Nielsen et al, 1998).
232
A study by de Oliveira et al (1995) showed evidence for selective pressure exerted by
streptomycin in soil influencing the survival of an introduced Pseudomonas fluorescens strain
carrying the streptomycin resistance encoding transposon Tn5 and for lack of selection by
kanamycin. The effect of streptomycin in soil on the Tn5 carrying bacteria depended on
conditions such as soil type.
233
Recorbet et al (1992) assessed the occurrence of a selective advantage associated with
antibiotic resistance encoded by nptII by adding kanamycin and neomycin. No selection was
found suggesting kanamycin is inactivated via adsorption to soil.
234
Veterinary use is a potential source of antibiotic addition to soil through the application of
manure. Kanamycin and neomycin are poorly absorbed in the gastro-intestinal tract and an
estimated 97 percent leaves the body unchanged in faeces.
235
Most substances (eg nucleic acids, proteins, clay) can form complexes with aminoglycoside
antibiotics and therefore reduce their biological activity. Most, if not all, soils are therefore
able to inactivate substantial amounts by adsorption or desorption. Although kanamycin and
neomycin are relatively stable compounds it is also likely that the concentration is limited by
biodegradation. Consequently it is unlikely that soil will be able to accumulate kanamycin or
neomycin in concentrations that are selective for antibiotic resistant plants (Nap et al, 1992).
Use of Antibiotic Resistance Marker Genes in GMOs
50
Ecological issues
236
It has been argued that particularly in the absence of selection, the burden of an extra but
useless gene would reduce the fitness of the plant suggesting that the gene may decrease
weediness. Data from laboratory studies and field trials do not support this view (Nap et al,
1992).
Pleiotrophic effects
237
As well as inactivating the antibiotic, the antibiotic resistance gene product could interfere
with existing metabolic pathways. Substrate specificity of neomycin phosphotransferase is
high and available data show that the enzyme does not interfere with the basic functions of
growth and development of a plant. The probability of the kanamycin resistance gene
undergoing mutation resulting in a novel and functional enzyme with other substrate
specificity in such a way that plant metabolism is disturbed and the plant has a selective
advantage has been judged as negligible (Nap et al, 1992).
238
However the effect of the enzymatic activity (eg phosphorylation) of the marker gene in the
plant’s biochemical environment is unknown. Plant cells produce a diverse range of
metabolites. Many are chemically ill-defined with no known function. Some may have a
chemically similar structure to aminoglycoside antibiotics making them possible targets for
phosphorylation by neomycin phosphotransferase. No activity of phosphorylation products
has yet been identified but the potential of this non-specific phosphorylation to chemically
modify plant metabolites not normally phosphorylated and their metabolite breakdown
products is likely to vary among plants. As the range of GM plant species increases the
potential for non-specific phosphorylation of metabolites is also likely to increase. This may
have consequences for insects that come in contact with plant exudates such as sap or pollen
(Harding and Harris, 1997).
239
The presence of an antibiotic resistance gene or its product may have pleiotrophic effects
that in some way alter any of the ecological relationships of the plant itself, any wild relative
derived from outcrossing, or any micro-organism derived from horizontal gene transfer
(Metz and Nap, 1997).
Use of antibiotic as a herbicide
240
The presence of kanamycin resistance in crops means it is possible to use kanamycin as a
herbicide. Apart from the relatively high costs of production, kanamycin and neomycin are
not likely to be used as a herbicide because of their limited efficacy, the intrinsic resistance of
various plants, and the likely public unacceptability of such a practice. Even if large amounts
were used it is considered that their physico-chemical characteristics would prevent the
establishment of selective conditions (Nap et al, 1992).
Use of Antibiotic Resistance Marker Genes in GMOs
51
Ecological issues
Horizontal gene transfer in the environment
241
Horizontal gene transfer in the environment includes from plant to plant via pollen, from
micro-organism to micro-organism, from micro-organism to plant, and from plant (or
animal) to micro-organism.
242
Horizontal gene transfer into plants from the soil bacterium Agrobacterium tumefaciens that is a
plant pathogen is well documented and occurs in nature. This is the best studied example of
gene transfer from a micro-organism to a plant. In contrast, research has not detected
transfer of plant DNA to Agrobacterium (OECD, 2000b).
243
As plants do not have any identified mechanism to facilitate broad host range gene transfer
(except for pollen hybridisation with related species) the possibilities and barriers of
horizontal gene transfer from plants to micro-organisms have been approached within the
framework of known mechanisms of horizontal gene transfer within bacteria.
244
Horizontal transfer of genetic information between bacteria has been extensively
demonstrated both in vitro and in natural systems (Droge et al, 1999). It is unclear to what
extent all possible mechanisms of gene transfer in bacteria have been identified since less
than one percent of the bacteria present in the natural environment have been described at
species level (Nielsen et al, 1998).
245
Transduction is unlikely as viruses that function in both plants and bacteria, and thereby
possibly facilitate gene transfer from plants to bacteria have not yet been identified.
246
Mechanisms that support conjugative gene transfer from higher plants to bacteria are
unknown, and transposons that function in both plants and prokaryotes have not been
identified (Nielsen et al, 1998).
247
Transformation is a mechanism by which plant DNA could be transferred to microorganisms. Since some competent bacterial species take up free DNA independently of its
sequence, transformation theoretically facilitates gene transfer from plants to bacteria. It is
considered the only natural mechanism that can be involved in promoting the uptake of plant
DNA by bacteria (Bertolla and Simonet, 1999).
248
Several barriers that restrict gene transfer between distantly related organisms in the
environment have been proposed. The main ones are probably transfer and establishment
barriers (Nielsen et al, 1998).
249
During plant growth or decay or animal feeding on plants the antibiotic resistance gene could
become available and transfer to micro-organisms. Plant cells contain DNases that are
released when cells are damaged resulting in the breakdown of the plant’s DNA. Soil microorganisms would increase the degradation process and further reduce the probability of
transfer from decaying material. If DNA was available then the level of transformation of
soil micro-organisms would depend upon the proportion of micro-organisms that were
naturally transformable. DNA transfer between micro-organisms is supposed to occur
frequently in soil, but the available data are related to conjugative transfer of plasmids. Plant
Use of Antibiotic Resistance Marker Genes in GMOs
52
Ecological issues
DNA will not be available as plasmids so conjugative DNA transfer can be excluded (Nap et
al, 1992).
250
Assessment of potential impact must also consider bacterial ecology. Bacterial populations
found in different sites differ markedly in composition, metabolic activities and their
environmental conditions (Salyers, 1999). Studies have shown that natural transformation can
occur in soils but the major limiting factor is development of competence by bacteria.
However there are niches in which bacteria may find more favourable conditions for
developing the specific metabolism required for transformation (eg the gut of warm-blooded
animals) (Bertolla and Simonet, 1999).
Prerequisites for transformation
251
There are a number of steps for transformation to occur under natural conditions. These are:
• release of DNA into the environment;
• adsorption onto soil for protection against enzymatic activity;
• presence of genetically adapted bacterial genotypes for natural transformation;
• appropriate conditions for the development of competence;
• efficient adsorption of the DNA to the bacterial cell surface;
• efficient DNA uptake;
• chromosomal integration via recombination or autonomous replication of the
transforming DNA; and
• expression of the gene by the recipient bacterium (Bertolla and Simonet, 1999).
252
Transformation requires access to free DNA which must be present at the time and place in
which competent bacteria develop or reside.
253
The probability that bacterial transformation will occur is greater the slower the rate of
turnover of the DNA in soil. Free DNA in soil is derived from lysis of cells either following
the death of plants, animals and micro-organisms or as a result of excretion of plasmid or
chromosomal DNA by some micro-organisms. Most free DNA is rapidly degraded but a
proportion escapes as a result of adsorption onto soil particles. DNA degradation rates differ
significantly among various habitats.
254
Both plant and bacterial DNA have been shown to persist in soil for weeks or months.
Persistence of DNA in soil over time does not necessarily imply that the DNA is in a
physical or chemical condition that makes it available for transformation. However bacterial
DNA adsorbed to soil particles has been shown in microcosm experiments to be able to
transform competent bacteria (Sikorski et al, 1998).
Persistence of DNA
255
Different abiotic and biotic factors seem to affect the persistence of free DNA in soil making
general predictions on the persistence of plant DNA in soils difficult. Plant DNA can persist
for months in soil in particular those rich in organic matter and clay on which nucleic acids
can adsorb without inhibiting their availability to competent bacteria. Adsorption provides
partial protection from degradation by nucleases. This is considered the most important
Use of Antibiotic Resistance Marker Genes in GMOs
53
Ecological issues
factor determining DNA persistence in the environment. In addition, high bacterial activity
accompanied by an enhanced presence of bacterial DNase affects the persistence of free
DNA in soil (Gebhard and Smalla, 1999).
256
Analysis of DNA extracted directly from soil samples by PCR showed long term persistence
of GM plant DNA in soil samples collected at the release site of GM sugar beet. The soil
samples were taken about six months after GM sugar beet was incorporated into the soil
after shredding. None of the more than 2,000 kanamycin resistant bacteria screened for the
presence of nptII by dot blot hybridisations contained the GM plant DNA (Smalla et al,
1994).
257
Paget et al (1998) carried out a field experiment with GM tobacco containing a bacterial
gentamicin resistance marker gene (aacC1). The fate of plant DNA in soil was monitored for
up to three years. Most previous studies have relied on laboratory systems. Results indicated
that plant DNA is released into the soil and can persist there for months but no longer than
three years under natural conditions. (Results were negative three years after harvesting the
GM tobacco). The gene was detected by PCR in all soil samples suggesting that soil contains
non-culturable bacteria containing sequences very similar to the aacC1 gene.
258
Under most experimental conditions DNA is initially degraded at a high rate. In controlled
laboratory systems using mixtures of ground plant tissue and soil a small proportion of the
added DNA resisted degradation and nptII was detectable for several months. This DNA
may have been adsorbed to soil particles and therefore protected from complete degradation.
Low temperature and low moisture were found to have stabilising effects on DNA in nonsterile soil (Widmer et al, 1996).
259
An investigation of the persistence of nptII in the field showed differences in DNA stability
that may be related to the different plant types and different experimental conditions (GM
tobacco leaves were buried and residual GM potato plant litter was obtained from the soil
surface). Results indicated that residual plant tissue might be a location where relatively large
amounts of detectable DNA persist for several months at a field site (Widmer et al, 1997).
260
Gebhard and Smalla (1999) have demonstrated long term persistence of GM sugar beet
DNA under field conditions (up to two years) and in soil microcosms with introduced free
GM sugar beet DNA (up to six months).
261
Research findings of rapid initial degradation of plant DNA in different soil and field systems
indicate that a possible transfer frequency of the kanamycin resistance gene and most likely
other genes from plants to micro-organisms may be very low and restricted to microhabitats
that contain residual plant tissues and DNA adsorbed on soil particles.
Transformation of soil micro-organisms
262
Predictions of the extent of horizontal gene transfer from plants to bacteria are currently
hindered by lack of information on the abundance of naturally competent bacteria in the
environment, frequency of transformation and environmental factors triggering competence
and transformation (Gebhard and Smalla, 1999).
Use of Antibiotic Resistance Marker Genes in GMOs
54
Ecological issues
263
Induction of competence is usually related to the physiological stage of the cells and/or to
accumulation of an environmental factor. Knowledge of environmental factors that affect the
regulation of bacterial competence is scarce (Lorenz and Wackernagel, 1994).
264
Adsorption of DNA to the bacterial cell surface is considered the first step of the
transformation process.
265
Some soil micro-organisms may be naturally transformable and may take up and incorporate
DNA causing genomic rearrangements that might help them occupy particular ecological
niches.
266
Recombination events in bacteria with environmentally regulated transient deficiencies in
their DNA repair and recombination system or mutations, illegitimate recombination events,
homologous recombination, and origin of replication based plasmid rescue are potential
means to stabilise DNA from GMOs if transferred to micro-organisms (Nielsen et al, 1998).
267
Transformation rates can be markedly affected by widespread bacterial restriction and
modification systems protecting the host DNA against contamination by foreign sequences.
The fact that DNA uptake involves a single strand step in most naturally competent bacteria
indicates that transforming DNA will escape the restriction systems, allowing successful
transformation of the bacterium. Saturating amounts of DNA or inefficient restriction
systems could also lead to successful transformation.
268
Stable maintenance of GM plant DNA in bacteria requires linkage to an origin of replication
such as by integration of this DNA into the bacterial chromosome, or its autonomous
replication based on the presence of replication functions and an origin of replication in the
DNA.
269
Homologous recombination can only occur when the donor DNA and the host genome
share DNA sequence similarities. The effectiveness of homologous recombination may also
vary as a function of the insertion site of the transforming DNA.
270
Recombination is also under the control of the mismatch repair system involved in
correction of replication errors and base modifications. This mechanism prevents
heterologous recombination and allows the cell to maintain some level of genetic stability
(Matic et al, 1996). However one percent of natural isolates of pathogenic and commensal
enterobacteria (E. coli and Salmonella species) display mutations that could favour
recombination with heterologous transforming DNA (Bertolla and Simonet, 1999).
Subpopulations of bacterial communities might show an enhanced frequency for such
recombination.
271
Illegitimate recombination events occur in different organisms. Construction of all GMOs is
currently based on illegitimate recombination events with random sites of insertion of the
genes in the host genome (Nielsen et al, 1998).
272
With the introduction of bacterial genes, bacterial promoter and terminator sequences, and
bacterial origins of replication into GM plants, the degree of sequence homology between the
genomes of competent bacteria and GM plant DNA increases. The probability of gene
Use of Antibiotic Resistance Marker Genes in GMOs
55
Ecological issues
transfer is therefore increased (Gebhard and Smalla, 1998). Homologous recombination
between a recombinant sequence in the plant chromosome and the natural sequence in
competent bacteria can result in the stable insertion of the DNA.
273
Short regions of homology can mediate recombination that includes incorporation of
adjacent non-homologous sequences. Short repetitive sequences are commonly found
dispersed in bacterial genomes and although speculative, these may, if integrated into the
genomes of GMOs also mediate the transfer of adjacent non-homologous gene sequences to
bacteria. Vector development that facilitates the transfer of large DNA fragments may
introduce longer bacterial sequences in GMOs (Nielsen et al, 1998).
274
Recently a new class of mobile genetic elements in bacteria, the gene cassettes, have been
described. Gene cassettes are usually found integrated adjacent to integrons which can
mediate the expression of the cassettes and their movement. Many antibiotic resistance genes
have been identified as functional gene cassettes including some selectable markers in GMOs
(Hall, 1997). Although not generally used as cassettes in GM work use of a gene cassette in a
GMO may circumvent the requirement for homologous recombination based stabilisation of
DNA in bacteria since integration of the cassette can be encoded by the integron.
275
Stabilisation of GM DNA in bacteria is also feasible if the plant DNA contains replication
functions and a bacterial origin of replication facilitating its autonomous replication.
Construction of GM monocotyledonous plants such as cereals, rice and maize is usually
facilitated by electroporation or the use of particle guns that result in the integration of whole
plasmids with intact replication functions. If fragments of such DNA become recircularised
following their uptake in bacterial recipients they might become stabilised by a plasmid
rescue-like mechanism.
276
Due to DNA cloning methods, eukaryotic genes inserted into GMOs do not normally have
introns which probably enhances their expression if transferred downstream of promoters in
bacteria. Although the promoters inserted into GMOs usually display low activity in
prokaryotic hosts some are also active in bacteria (eg the frequently used cauliflower mosaic
virus 35S promoter expresses in E. coli). Insertion of whole plasmids into the GMO may lead
to the presence of bacterially expressed vector sequences like the ampicillin resistance gene
located on the vector pUC18.
277
Random insertion of protein encoding sequences from GM DNA into existing regulatory
sequences in the genome of the bacterium may also mediate gene expression after gene
transfer from plants.
278
Uptake and recombination with GM DNA fragments rather than whole genes might also
influence gene expression and variability in bacteria. For example, if a deletion is restored in
an antibiotic resistance gene, or its expression is upregulated after recombination, this would
lead to a stronger antibiotic resistance in the bacterium. Similarly recombination may also
alter the specificity of the enzyme conferring the antibiotic resistance (Nielsen et al, 1998).
279
A concentration of DNA per hectare from GM tomatoes was calculated assuming inter alia
that 10 percent of the DNA was released and 10 percent of the released DNA was intact.
With this concentration and assuming 10 percent of soil micro-organisms are transformable,
Use of Antibiotic Resistance Marker Genes in GMOs
56
Ecological issues
five percent of which are competent and a natural transformation frequency of 0.01 percent,
a transformation frequency of 8.7 x 10-12 was estimated. If gene transfer results in 8.7 x 10-5
transformants per gram of soil containing 107 viable micro-organisms of which 1600 were
already kanamycin resistant, the contribution to natural kanamycin resistance would be 5.4 x
10-6 percent (Nap et al, 1992).
280
Transfer of the kanamycin resistance gene will not immediately result in kanamycin resistance
because the gene carries regulatory sequences that will generally not work in microorganisms. Upon transfer, recombination therefore needs to occur to make the gene
functional, the micro-organism should be present in a natural environment in which a
selective advantage for kanamycin resistance occurs, and should be able to outcompete the
kanamycin resistant organisms already present. The probability for the subsequent
occurrence of all these events is negligible and as a result it was concluded that horizontal
transfer of the kanamycin resistance gene will not alter or disturb a soil ecosystem (Nap et al,
1992).
281
Calgene Inc calculated the probability of transfer of the kanamycin resistance marker gene
from the Flavr Savr tomato to indigenous soil micro-organisms. The theoretical gene transfer
model was developed based on the putative transfer from the plant to B. subtilis which is
naturally transformable but shows no homology to the GM DNA. Alternatively transfer of
DNA from the plant to A. tumefaciens was considered. It is not naturally transformable but
contains the plasmid carrying the same T-DNA border regions inserted into the plant
genome.
282
The models estimated a worst case scenario (eg 100 percent of soil bacteria are
transformable, every fragment of the plant DNA in soil contains nptII, the transformed gene
is expressed after integration into the recipient genome and the gene product is active and
stable in the bacterial cells) or the more likely scenario (eg only 10 percent of soil bacteria are
transformable, the nptII gene constitutes 10-5 of the tomato genome, the gene is not always
integrated and expressed). The estimates for the Bacillus type transformation system were 9 x
105 transformants per acre in the worst case scenario and two transformants per acre in the
more likely case. Estimates were three orders of magnitude less for the Agrobacterium type
transformation system. From these calculations it was concluded that in the worst case
kanamycin resistant Bacillus transformants will constitute about 10-7 of the kanamycin
resistant soil bacteria and Agrobacterium transformants will constitute 10-10 of the kanamycin
resistant soil bacteria (Droge et al, 1998).
283
Transformation frequency of competent E. coli from GM insect resistant maize containing
the ampicillin resistance gene was estimated to be 1 in 6.8 x 1019. Some experts consulted by
the FDA said if transformation were to occur it would be more likely in experiments using
competent bacteria in the laboratory than in nature because competent bacteria have the
highest transformation frequency. If transformation was not observed in the laboratory in
either Gram-negative or Gram-positive bacteria the results suggest that gene transfer may not
occur in the natural environment to the extent that health or safety concerns would arise.
Other experts stated that they did not have much confidence in an in vitro experiment
because it does not reflect the complex natural ecosystem. In addition a monoculture of E.
coli is an artificial system that is not a strong basis on which to assess risk (US FDA, 1998).
Use of Antibiotic Resistance Marker Genes in GMOs
57
Ecological issues
Approaches to evaluate possible horizontal gene transfer
of plant DNA to soil micro-organisms
284
Although experimental approaches in both field and laboratory studies have not been able to
confirm the occurrence of gene transfer to naturally occurring bacteria a few studies have
shown transfer of marker genes from plants to bacteria based on homologous
recombination. These results and the few examples of gene transfer indicated by DNA
sequence comparisons suggest that the frequency of stable gene transfer from plants to
bacteria is extremely low. However this inference is based on a small number of experimental
studies and indications found in the literature (Nielsen et al, 1998).
Comparison of DNA sequences
285
A few cases suggestive of horizontal gene transfer from plants to bacteria have been
identified after comparison of DNA sequences between plants and bacteria. The low number
of examples suggests that the frequency of stable gene transfer from plants to bacteria is
extremely low (Droge et al, 1998).
Screening of bacteria from environmental samples (fields or microcosms with introduced GM plants)
286
Only about 10 percent of soil bacteria are assessable via cultivation techniques. The expected
low frequency of transfer under natural conditions impedes screening bacteria from
environmental samples as the number of putative transformants has been suggested to be
below the limit of detection. Some of the biases involved in the isolation of bacteria from soil
can be circumvented by analysing total DNA extracted from soil samples (eg Smalla et al,
1993).
287
The few studies of gene transfer from GM plants to bacteria in soil in natural (eg Paget et al,
1998) or soil microcosm conditions have not been able to show such transfer, indicating that
transfer did not occur, or transfer frequencies and expression were too low to be detected, or
the techniques used were not appropriate for its detection.
Experimental studies under optimised laboratory conditions
288
Gene transfer from GM plants to bacteria has only been investigated experimentally with the
hypothesis that such gene transfer takes place by transformation. Reported studies have all
been done in the laboratory with readily culturable, Gram-negative, soil or plant-associated
bacteria.
• Studies that have been conducted to assess the potential for gene transfer from GM
plants to soil or plant-associated micro-organisms:
(1)
Horizontal gene transfer from GM potato to the plant pathogen Erwinia
chrysanthemi was not detected under conditions mimicking a natural infection.
Gradual stepwise alteration of laboratory conditions to natural conditions revealed
a gradual decrease of the potential transformation frequency from 6.3 x 10-2 (one
transformant per 630 bacteria) under optimal laboratory conditions to a calculated
2.0 x 10-17 under natural conditions. The latter estimate is far below the detection
limit. However the natural competence of Erwinia was low and the presence and
stability of released plant DNA with transforming activity from the lysed potato
was not demonstrated (Schluter et al, 1995).
Use of Antibiotic Resistance Marker Genes in GMOs
58
Ecological issues
(2)
Broer et al (1996) used the plant pathogen A. tumefaciens as a recipient for GM
tobacco. Gene transfer was not detected. Transformation frequency was found to
be below the detection limit (6 x 10-12). Development of competence for natural
transformation has not been shown for A. tumefaciens and its ability to take up
linear DNA was not shown in this study.
(3)
Acinetobacter are naturally competent bacteria and characterised in vitro by high
transformation rates. Nielsen et al (1997) did not detect any transformant using
Acinetobacter and GM potato and GM sugar beet. Frequencies decreased to 10-11 in
vitro and less than 10-16 in soil far below the detection limit. There was no sequence
similarity between the two genomes which could prevent homologous
recombination occurring. The donor DNA also did not possess any functional
replication origin to permit the potentially circularised DNA to replicate
autonomously.
(4)
Gebhard and Smalla (1998) and de Vries and Wackernagel (1998) developed a
model based on the construction of recipient strains with a deleted sequence of
the marker gene. Under optimised in vitro conditions they demonstrated that plant
DNA could successfully transform Acinetobacter and restore an intact gene in the
recipient strain that could express kanamycin. Frequencies were low - 5.4 x 10-9
(Gebhard and Smalla, 1998) and 3 x 10-8 (de Vries and Wackernagel, 1998) and
decreased to 1.5 x 10-10 when the pure plant DNA solution was replaced by a
crushed leave suspension (Gebhard and Smalla, 1998). Transformation might
occur in soil if homologous sequences are present in competent bacteria though
the frequency is likely to be lower than under laboratory conditions.
Transformation might not have been previously demonstrated experimentally
because of an absence of homologous sequences in the bacteria or use of less
efficiently transformable bacteria (Gebhard and Smalla, 1998).
(5)
The plant pathogen Ralstonia solanacearum is naturally transformable. To overcome
natural genetic barriers (only DNA of R. solanacearum transforms this bacterium)
GM donor plants and recipient R. solanacearum were constructed to deal with
homologous recombination requirements. Transformation did not occur (Bertolla
and Simonet, 1999).
289
The only published experimental evidence that demonstrates that gene transfer of
heterologous genes occurs from GM plants to naturally occurring soil or plant-associated
bacteria are the two studies mentioned above that used artificially introduced homology
between the DNA of the plant donor and recipient bacterium.
290
Horizontal gene transfer has been reported from plants to plant-associated fungi. In a study
using the pathogenic fungus Aspergillus niger and GM Brassica and GM Datura innoxia plants
some fungi acquired antibiotic resistance during co-cultivation but lost this resistance during
further cultivation even under selective pressure. One isolate exhibited stable resistance
suggesting gene transfer but the mechanism of such a transfer in fungi is unknown (Hoffman
et al, 1994).
Use of Antibiotic Resistance Marker Genes in GMOs
59
Ecological issues
291
A review by Bertolla and Simonet (1999) concluded that research is a long way from
demonstrating that plant-bacterium transfer does occur under natural conditions. Even under
optimised conditions such an event only occurs at very low frequencies. Dilution of the
marker gene in the genome of GM plants makes the transformation rate lower than that
found under optimised laboratory conditions. In addition the GM DNA is potentially
transferable only to a very limited number of bacteria (those that develop competence and
show tolerance to foreign DNA). When a transfer event does occur it is unlikely to confer
any selective advantage to the recipient micro-organism and could even be considered a
genetic burden.
292
Studies of gene transfer have mainly been done without selective pressure during the
exposure time with DNA. There is a need for studies that incorporate selective pressure
during the exposure of competent bacteria with selectable DNA to be designed (Nielsen et
al, 1998).
293
Extrapolation of transformation frequencies from microcosms to the environment could be
misleading because concentrations of transforming DNA in situ are not known.
Transformation frequencies differ considerably among species (Lorenz and Wackernagel,
1994). Conditions influencing occurrence of gene transfer in the natural environment also
might remain unidentified in laboratory studies thus generating under- or over-estimates of
transformation frequencies.
294
Some experts consulted by the FDA felt it was imprudent to increase the availability of
resistance genes in the environment because this may reduce the typical four to five year time
lag between first use of a new antibiotic and emergence of resistance in hospitals. Others felt
the risk of transfer from plant genome to soil micro-organisms is not significant as there is
no selective pressure in most cases. Exceptions include the use of streptomycin as a pesticide
in horticulture or use of manure as fertiliser following use of antibiotics as growth
promotants in animals (US FDA, 1998).
295
Detection of gene transfer events is difficult due to the limitations of the techniques
available. Transfer of antibiotic resistance genes is also difficult to document due to high
levels of resistance that already exist. Unequivocal proof of gene transfer requires isolation of
the putative transformants for thorough genetic characterisation. However the strategy to
monitor the transfer of complete genes of larger DNA fragments might fail because
transformation often involves the stable integration of short DNA fragments resulting in
gene mosaics.
296
The high prevalence of naturally occurring antibiotic resistant bacteria in the environment
has often been used as an argument for the low impact of potential transfer of antibiotic
resistance genes, particularly nptII, from GMOs. However the description of a phenotypically
observed resistance pattern does not address the natural presence of the antibiotic resistance
genes in these bacteria. Many mechanisms can be involved in bacterial antibiotic resistance. A
clearer distinction needs to be made between observed phenotype and the corresponding
genotype. Although there is information about phenotypic resistance few studies have
identified the gene responsible (US FDA, 1998; Salyers, 1999).
Use of Antibiotic Resistance Marker Genes in GMOs
60
Ecological issues
297
Monitoring should be at the genotypic rather than the phenotypic level and given that all
resistance genes originate from micro-organisms it should distinguish a gene that has been
transferred from a GM plant (Salyers, 1999).
298
Examining areas with a high concentration of GM plants would increase the chance of
finding the rare transfer event. Monitoring markers where the antibiotic is used in animal
feed as a growth promotant would also increase the chance of finding a transformant because
there would be selective pressure (US FDA, 1998).
299
The present knowledge of bacterial ecology in soil environments is unable to predict and
quantify factors in soil that affect the selection of bacterial transformants receiving novel
genes. Widespread introduction of GM plants into the environment will generate a
continuous exposure of bacteria to high numbers of transgenes and may as a result enhance
the probability of the amplification of these genes after integration in bacterial hosts.
Use of Antibiotic Resistance Marker Genes in GMOs
61
The New Zealand and international approach to
antibiotic resistance marker genes
6.
The New Zealand and international approach to antibiotic
resistance marker genes
300
The emphasis in this section is on the approach of national and international regulatory
agencies, advisory bodies and other organisations to the potential health rather than
environmental impact of the use of antibiotic resistance genes in GMOs.
The New Zealand approach to antibiotic resistance marker genes
Environmental Risk Management Authority (ERMA)
301
To date the Environmental Risk Management Authority has not considered any applications
for release of a GMO in New Zealand. Field trials of GM sugar beet, potato, sheep, and
cattle containing an antibiotic resistance gene have been approved. The gene was either the
kanamycin (neomycin) or puromycin resistance gene.
Antibiotic Resistance Expert Panel
302
The Antibiotic Resistance Expert Panel that reported to the Ministry of Agriculture and
Forestry Antibiotic Resistance Steering Group on antibiotic resistance and in-feed use of
antibiotics in New Zealand agreed with the conclusion of the Australian Joint Expert
Technical Advisory Committee on Antibiotic Resistance (JETACAR) report that the
probability of antibiotic resistance marker gene transfer to gut micro-organisms is extremely
remote. If transfer did occur there would then have to be concurrent or subsequent selection
pressure by the presence of the antibiotic in the gut before it could become a health issue.
However as a precautionary approach the Panel recommended avoidance of antibiotic
resistance marker genes in the development of GMOs intended for wide release (Antibiotic
Resistance Expert Panel, 1999).
Australia New Zealand Food Authority (ANZFA)
303
The Australia New Zealand Food Authority considers the possibility of gene transfer and its
consequence for human health in its safety assessments of GM foods.
304
It recommends that vectors should be modified to minimise the probability of gene transfer
and marker genes that confer resistance to clinically useful antibiotics (eg vancomycin) should
not be used (ANZFA, 1998).
305
ANZFA considers the overall risk of gene transfer affecting the clinical use of antibiotics in
humans to be effectively zero. However the issue is considered on a case-by-case basis
(OECD, 2000a).
306
To date 20 applications seeking approval for GM foods have been received by ANZFA. One
application has been withdrawn and a complete safety assessment has been completed for
two – insect resistant cotton and glyphosate tolerant soybean. The cotton contains the
kanamycin resistance gene (nptII) and streptomycin resistance gene (aad). Only cottonseed oil
Use of Antibiotic Resistance Marker Genes in GMOs
62
The New Zealand and international approach to
antibiotic resistance marker genes
and cellulose from processed linters, both highly refined products, are used as food. The final
modification of soybeans does not include an antibiotic resistance marker gene.
307
In 2000 ANZFA released draft safety assessments for public comment on a further eight
applications. Three applications (glyphosate tolerant maize, insect resistant maize and
glyphosate tolerant canola) have no antibiotic resistance genes in the final GM plant. Of the
other five applications, glyphosate tolerant cotton contains the kanamycin and streptomycin
resistance genes; insect resistant potato (with the exception of one line), insect and potato
leafroll virus resistant potato and insect and potato virus-Y resistant potato contain the
kanamycin and/or streptomycin resistance genes; and high oleic acid soybean contains the
ampicillin resistance gene (bla).
308
The aad and bla genes are under the control of bacterial promoters and therefore are not
expressed in GM plant cells.
The approach of other countries and international organisations to antibiotic
resistance marker genes
309
The stance adopted internationally toward the use of antibiotic resistance marker genes varies
according to the level of risk that is regarded as acceptable. Public consultation has not been
a feature of these determinations.
Australia
Genetic Manipulation Advisory Committee (GMAC)
310
The Genetic Manipulation Advisory Committee is a non-statutory body that oversees
development and use of novel genetic manipulation techniques in Australia.3 It provides
expert technical advice on specific biosafety matters to organisations using these techniques
and to regulatory agencies.
311
Decisions are made on a case-by-case basis.
312
GMAC has concluded that there is no significant biosafety risk associated with the use of the
kanamycin (neomycin) resistance gene. The probability that the antibiotic resistance gene
could be transferred intact from a GM plant to a pathogenic micro-organism, and expressed
in that micro-organism is extremely remote. In addition resistance to the antibiotics is already
widespread and the contribution that GM plants would make to existing levels of resistance
would be negligible.
Joint Expert Technical Advisory Committee on Antibiotic Resistance (JETACAR)
313
The Joint Expert Technical Advisory Committee on Antibiotic Resistance predominantly
examined the relationship between the use of antibiotics in food-producing animals and
antibiotic resistant bacteria in animals and humans. However it also considered the use of
antibiotic resistance genes as markers in genetic modification. It acknowledged that most
GMOs to be released into the environment in the next 10 years will be plants and many of
3
GMAC will be replaced by a statutory authority, the Office of the Gene Technology Regulator in 2001.
Use of Antibiotic Resistance Marker Genes in GMOs
63
The New Zealand and international approach to
antibiotic resistance marker genes
these will contain the kanamycin resistance gene. The Committee concluded that even if
improbably kanamycin resistance was transferred to gut micro-organisms in humans or
animals it would be of minimal consequence to human health due to the existence already of
intestinal kanamycin resistant bacteria (JETACAR, 1999).
Norway
314
Legislation on GM foods is divided in Norway between the Ministry of Environment (Gene
Technology Act 1993) and the Ministry of Health and Social Affairs (Food Control Act).
315
In Norway since 1997 three GM food plants have been banned from being marketed
because of the presence of antibiotic resistance marker genes.
316
Regulations are being developed that will ban the production, import and sale of GM foods
and feed that contain antibiotic resistance genes introduced during genetic modification
(OECD, 2000a).
United Kingdom
Advisory Committee on Novel Foods and Processes (ACNFP)
317
The Advisory Committee on Novel Foods and Processes is an independent expert
committee that advises British Health and Agriculture Ministers on the safety of novel foods
and processes, including GM food organisms and derived products.
318
In 1991 the ACNFP advised that the developer would need to submit detailed information
on the method of selection, including details of any antibiotic resistance marker genes used,
and if the genes encoded for resistance to clinically useful antibiotics evidence that they have
been removed or inactivated would normally be necessary.
319
The ACNFP concluded that of the potential food safety problems that could arise from the
use of antibiotic resistance markers only the possibility of transfer and subsequent expression
of the marker genes in gut micro-organisms is of significance. The possibility of such transfer
and expression occurring following ingestion is extremely low and if it were to occur is most
likely from live GM bacteria used as starter cultures or probiotics. It is less likely to occur
from ingested raw GM plant material or from the uncooked seed of GM plants, and least
likely from highly processed GM food micro-organisms and plant material.
320
The Committee recommended that GM food micro-organisms intended to be ingested live
(eg lactic acid bacteria) should not contain antibiotic resistance marker genes and food or
feed from GM plants and non-viable GM micro-organisms should be evaluated on a case-bycase basis (ACNFP, 1994). These recommendations were reaffirmed in a subsequent report
(ACNFP, 1996).
321
As decisions are made on a case-by-case basis ACNFP does not publish prescriptive lists of
acceptable and unacceptable antibiotic resistance marker genes.
322
Any further increase, however small, in resistant micro-organisms through transfer of
antibiotic resistance genes from GM food would be undesirable. Researchers developing
Use of Antibiotic Resistance Marker Genes in GMOs
64
The New Zealand and international approach to
antibiotic resistance marker genes
GMOs for food should be encouraged to develop and use alternative markers and/or
methods to excise the antibiotic resistance genes used (ACNFP, 1994).
323
However the Committee recognises that many GM crops that are currently being developed
as a food source were developed before alternative marker genes were available and that
commercial considerations have required the continued use of proven selection techniques
including the use of antibiotic resistance marker genes.
324
The ACNFP considers that the very low probability of transfer and expression of an
antibiotic resistance gene in gut or rumen micro-organisms is of little concern for markers
with plant promoters but not those with bacterial promoters. Where transfer is considered to
be possible and subsequent expression likely and the potentially affected antibiotic is
clinically important (eg ampicillin) use of the marker would be unlikely to be approved.
325
The ACNFP has recommended rejection of three applications submitted to it on the
grounds that there was a very small, though finite, risk of transfer of the resistance gene to
intestinal micro-organisms of animals fed unprocessed plant material which could
compromise human use of the antibiotic. These GM plants were maize containing the
ampicillin resistance marker gene and two cottons containing a gene resistant to streptomycin
and spectinomycin (OECD, 2000a).
326
The Committee is unlikely to have concerns about markers with bacterial regulatory
sequences for which it can be proved that the gene has been disrupted or truncated (ACNFP,
1996).
The Royal Society
327
The Royal Society has also expressed concern about the use of antibiotic resistance marker
genes in food and endorsed the ACNFP’s conclusions.
328
Any further increase in the use of such markers in the human or animal food chain would be
undesirable and any further increase, however small, in antibiotic resistant micro-organisms
through transfer of markers from GM food should be avoided.
329
It is no longer acceptable to have antibiotic resistance genes present in a new GM crop under
development for potential food use and researchers should not produce GM plants
containing genes that confer resistance to antibiotics that are used to treat infections in
animals or humans. Such genes if used in future should be removed at an early stage in
development of the GM plant and where possible alternative marker systems should be used
(The Royal Society, 1998).
British Medical Association (BMA)
330
A report from the BMA concluded that the use of antibiotic resistance marker genes in GM
food is an unacceptable risk, however small, to human health. Since the risk to human health
from antibiotic resistance developing in micro-organisms is one of the major public health
threats that will be faced in the 21st century, and the risk of antibiotic resistance being passed
on to bacteria affecting humans through marker genes in the food chain cannot at present be
ruled out, the BMA recommended a ban on their use in GM food. It also recommended
Use of Antibiotic Resistance Marker Genes in GMOs
65
The New Zealand and international approach to
antibiotic resistance marker genes
further research on the health risks arising from antibiotic resistance (British Medical
Association, 1999).
Other
331
A report to the UK Ministerial Group on Biotechnology from the British Government’s
Chief Medical Adviser and Chief Scientific Adviser recommended that developers of GM
food should be encouraged to phase out the use of antibiotic resistance marker genes as soon
as is feasible. The report also stated that it is unacceptable for a viable GM micro-organism
containing an antibiotic resistance gene to be eaten (Donaldson and May, 1999).
Codex Alimentarius Commission
332
The Codex Alimentarius, an instrument of the FAO and the WHO, develops international
food safety standards. Its primary objective is to protect the health of consumers and to
ensure fair practices in international food trade. An ad hoc Intergovernmental Task Force on
Foods derived from biotechnology has been set up by Codex and met for the first time in
March 2000. It has agreed to develop specific guidance on risk assessment of GM foods.
European Commission
Scientific Steering Committee on Antimicrobial Resistance
333
The European Commission’s Scientific Steering Committee on Antimicrobial Resistance
stated that although the risk of gene transfer is extremely small each plant containing
antibiotic resistance marker genes should be evaluated on a case-by-case basis. Emphasis
should mainly be on evaluation of the selection pressure acting on the bacterial recipients
after possible gene transfer.
334
Although there is no evidence that antibiotic resistance marker genes have transferred from
GM plants to pathogenic bacteria and the possibility of such an event has been argued to be
remote, the Committee considered it appropriate to recommend that markers should be
removed from plant cells before commercialisation whenever feasible. Failure to remove
markers should be justified by the developer and use of genes that might have the capacity to
express and confer resistance against clinically important antibiotics should be avoided.
335
It also recommended that assessment of the potential for transfer of marker genes from a
plant into micro-organisms should be examined more closely (European Commission, 1999).
Nordic Working Group on Food Toxicology and Risk Assessment
336
The concept of a positive list of selectable marker genes refers to a list of acceptable marker
genes with respect to biosafety. Development of a positive list was initially discussed at a
WHO workshop in 1993. It was considered that it was not possible at that time to develop a
list that did not cause food safety concerns. Further development work was carried out by
the Nordic Working Group on Food Toxicology and Risk Assessment under the auspices of
the Nordic Council of Ministers (Karenlampi, 1996).
337
The Nordic Working Group proposed the kanamycin resistance gene and glyphosate
resistance gene for the list.
Use of Antibiotic Resistance Marker Genes in GMOs
66
The New Zealand and international approach to
antibiotic resistance marker genes
338
Inclusion of a marker gene in a positive list indicates that the marker gene itself, regardless of
any promoter and terminal sequences, and its gene product are considered safe for human
consumption irrespective of the host plant in which the gene is inserted. Secondary effects
are not evaluated as part of the procedure for inclusion because these may differ according to
the site of insertion.
339
A marker gene can be included in the list without restrictions or with specific conditions (eg
amount of the gene or its product present in the GM plant) for a specific plant family. Before
acceptance on a positive list marker genes should be shown to be safe in at least two to three
different plant families (Karenlampi, 1996).
United States
International Food Biotechnology Council
340
The International Food Biotechnology Council concluded in 1990 that the use of antibiotic
resistance marker genes does not pose any risks unless selection pressure occurs at the time
of the probably very rarely occurring gene transfer event from the plant genome to gut
micro-organisms.
Food and Drug Administration (FDA)
341
In 1992 the FDA issued a policy statement on foods derived from GM plant varieties. Both
the antibiotic resistance gene and its product, unless removed, are expected to be present in
foods derived from such plants. Selectable marker genes that produce enzymes that inactivate
clinically useful antibiotics theoretically may reduce the therapeutic efficacy of the antibiotic
when taken orally if the enzyme in food inactivates the antibiotic. The FDA stated that it will
be important to evaluate such concerns with respect to commercial use of antibiotic
resistance genes in food in particular those that will be widely used (US FDA, 1992).
342
In 1994 the FDA amended its food additive regulations to permit the use of the kanamycin
resistance gene and its expression product in the development of new varieties of tomato,
oilseed rape and cotton. It concluded that if gene transfer did occur from plants to microorganisms there would be no significant increase in antibiotic resistant human pathogens.
Some members of the FDA Food Advisory Committee, although convinced that gene
transfer from tomato plants to soil micro-organisms was improbable, were concerned about
the use of the kanamycin resistance gene in other crops that may be grown on a wide scale.
The FDA concluded that gene transfer from crops to micro-organisms, as well as other
antibiotic resistance genes, should be evaluated on a case-by-case basis (US FDA, 1994).
343
The FDA carried out consultation with external experts between November 1996 and
February 1997 to determine whether circumstances exist under which the FDA should
recommend that a given antibiotic resistance gene not be used in food crops and if so, to
identify the nature of those circumstances.
344
The kanamycin resistance gene was regarded as acceptable for use by these scientific experts.
Some suggested hygromycin and others included the beta-lactamase gene of pUC18 and the
tetracycline resistance gene. Any potential transfer was felt unlikely to add to the existing high
levels of resistance in any meaningful way. It was suggested that use of genes other than
Use of Antibiotic Resistance Marker Genes in GMOs
67
The New Zealand and international approach to
antibiotic resistance marker genes
kanamycin and hygromycin might be acceptable on the basis of studies to address potential
transfer and post-market surveillance for transfer of the gene in question. Surveillance would
give regulatory agencies an opportunity for early intervention to prevent an adverse impact
on public health (US FDA, 1998).
345
The potential effects due to ingestion of enzymes encoded by antibiotic resistance genes as
food components raised little concern in comparison to potential health effects from gene
transfer to micro-organisms.
346
The FDA concluded that the probability of gene transfer from plants to gut micro-organisms
(human or animal) and to micro-organisms in the environment is remote. Several barriers
operate against such transfer. The rate of such transfer, if any, would be insignificant when
compared to transfer between micro-organisms and in most cases would not add to existing
levels of resistance in bacterial populations in any meaningful way. Caution should be the rule
for marker genes that encode resistance to antibiotics that are the only drug available to treat
certain infections (eg vancomycin); they should not be used in GM plants (US FDA, 1998).
347
Developers should evaluate the use of antibiotic resistance genes in crops on a case-by-case
basis taking into account information on the therapeutic importance of the antibiotic,
frequency of use, route of administration, uniqueness, whether there would be selective
pressure for transformation to take place, and the level of resistance to the antibiotic present
in bacterial populations. If the gene or its gene product in food or feed could compromise
the use of the relevant antibiotic it should not be present in the finished food or feed (US
FDA, 1998).
Environmental Protection Agency (EPA)
348
The kanamycin resistance gene nptII and its enzyme neomycin phosphotransferase are
considered inert ingredients by the EPA when they are introduced into a plant in order to
ensure or confirm the presence of a plant pesticide and are exempted from the requirement
of a tolerance in or on all raw agricultural commodities (US EPA, 1994).
World Health Organisation
349
In 1991 a FAO/WHO assessment of biotechnology in food production and processing
concluded that the risk of transfer of antibiotic resistance genes from food in the gut to gut
micro-organisms can be considered insignificant in comparison with the risk of the microorganisms becoming resistant to antibiotics by other mechanisms.
350
However vectors should be modified so as to minimise the probability of transfer of
antibiotic resistance genes to other micro-organisms and genes that encode resistance to
clinically useful antibiotics should not be used in micro-organisms intended to be present as
living organisms in food (eg yoghurt). Food components obtained from micro-organisms
containing antibiotic resistance marker genes should be demonstrated to be free of viable
cells and genetic material that could encode antibiotic resistance (WHO, 1991).
351
These earlier recommendations relating to safety assessment of food and food components
derived from GM micro-organisms were endorsed in 1996 (FAO and WHO, 1996).
Use of Antibiotic Resistance Marker Genes in GMOs
68
The New Zealand and international approach to
antibiotic resistance marker genes
352
A WHO workshop on the health aspects of marker genes considered the probability of gene
transfer from plants to micro-organisms to be extremely small. It concluded that there is no
substantial evidence for gene transfer from ingested plant material to gut micro-organisms. If
transfer were to occur, the nature of the gene and its product and the conditions in the
gastro-intestinal tract will determine whether or not it is a food safety problem. Evaluation
should be on a case-by-case basis (WHO, 1993).
353
Joint consultation by FAO and WHO concluded that as the probability of gene transfer is
very low data on such gene transfer will only be needed when the nature of the marker gene
is such that if transfer were to occur it would give rise to a health concern. In assessing
potential health impact the human or animal use of the antibiotic and presence and
prevalence of resistance to the same antibiotic in gut micro-organisms should be considered
(FAO and WHO, 1996). These conclusions were reiterated recently but the use of alternative
strategies to antibiotic resistance marker genes, if shown to be safe, was also encouraged
(WHO, 2000).
Use of Antibiotic Resistance Marker Genes in GMOs
69
Alternative strategies to antibiotic resistance marker genes
7.
Alternative strategies to antibiotic resistance marker genes
354
Questions raised with respect to the use of antibiotic resistance marker genes have led to the
development of alternative strategies. Many other strategies for selecting transformed cells
now exist and where antibiotic resistance genes are used, strategies exist to remove them
from the final GMO.
355
Alternative strategies to antibiotic resistance marker genes must also be acceptable to the
public.
Characteristics of a selectable marker
356
Characteristics of a useful selectable marker system include:
• a minimum of non-transformed cells or tissue escape the selection;
• selection results in a large number of independent transformation events and does
not significantly interfere with regeneration;
• it works well in many species; and
• an assay is available to confirm that the marker is present (ACNFP, 1994).
Choice of a selectable marker
357
Most selectable marker genes confer resistance to an antibiotic, herbicide or other toxic
agent.
358
Choice is usually based on:
• The effectiveness of the agent in limiting the growth of non-transformed cells.
This is determined by the mechanism of toxicity and uptake of the agent by the target
tissue.
• The efficacy of the selectable marker gene in providing resistance.
Efficacy is determined by the mode and site of action of the gene product, the activity
of the promoter directing gene expression, efficiency of translation and stability of the
gene product.
• The availability of vectors (Langridge, 1997).
359
However use of alternative selectable marker genes or subsequent deletion of the antibiotic
resistance gene is becoming more common (Donaldson and May, 1999).
Disadvantages of antibiotic resistance marker genes
360
Disadvantages of using antibiotic resistance genes as selectable markers include:
• Transformed cells convert the selective agent (ie antibiotic) to a detoxified compound
that may still have negative effects on cell proliferation and differentiation.
Use of Antibiotic Resistance Marker Genes in GMOs
70
Alternative strategies to antibiotic resistance marker genes
• It is difficult to carry out recurrent transformations using the same selectable marker
to stack the genes of interest.
• Only a limited number of antibiotic resistance genes are available for practical use.
• Some plant species are insensitive or tolerant of the selective agents.
• Public perception of the risk of antibiotic resistance.
Herbicide resistance
361
Herbicide resistance due to detoxification or degradation of a specific broad spectrum
herbicide may be relatively independent of the plant species. The resistance gene is therefore
useful in genetic modification of a variety of plants. Herbicide resistance can also be used as a
marker in the yeast Saccharomyces cerevisiae. Herbicide resistance markers include tolerance to
glufosinate, glyphosate, chlorsulfuron, or bromoxynil.
362
Glufosinate inhibits amino acid biosynthesis. Glufosinate tolerance genes include bar (cloned
from the soil bacterium Streptomyces hygroscopicus), pat (isolated from Streptomyces
viridochromogenes) and synthetic pat. The genes encode the enzyme phosphinothricin
acetyltransferase (PAT). Phosphinothricin acetyltransferase detoxifies phosphinothricin
which inhibits glutamine synthetase causing rapid accumulation of ammonia and plant cell
death. PAT is not an endogenous enzyme in humans. It has no homology to known toxins
or allergens and is rapidly degraded in simulated digestion studies (Karenlampi, 1996).
363
Glyphosate prevents synthesis of the aromatic amino acids that are essential for protein
synthesis. Glyphosate tolerance genes include epsps from E. coli, Salmonella typhimurium, and
Agrobacterium and mutated epsps in plants. The epsps gene encodes the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). All plant, microbial and fungal food
sources contain EPSPS protein. EPSPS has no significant homology to known toxins or
allergens, is rapidly degraded in simulated digestion studies and displays no enzymatic activity
in the stomach. No adverse effects were found in acute mouse gavage studies. Low levels of
the protein are expressed in GM plants (Karenlampi, 1996).
364
Chlorsulfuron is a sulfonylurea and interferes with amino acid biosynthesis. Chlorsulfuron
tolerance genes are mutated als genes isolated from various plants (eg tobacco, maize, sugar
beet). The als gene is present in all plants and encodes the enzyme acetolactate synthase
(ALS). The enzyme is not novel in the human diet. Interest in its use in genetic modification
decreased with the appearance of spontaneous resistance to chlorsulfuron.
365
Glyphosate and chlorsulfuron markers differ from the glufosinate marker in that the genes
conferring tolerance encode proteins normally present in all plants, except that they are
mutated (single base pair substitution). The mutated enzymes have retained their normal
physiological function in amino acid biosynthesis but have obtained an altered affinity to the
herbicide. Consequently they are not considered novel components of food plants. They are
considered good candidates for a positive list of marker genes if the enzyme concentrations
in the transformed plants are not markedly different from the concentrations in herbicide
sensitive plants (Karenlampi, 1996).
Use of Antibiotic Resistance Marker Genes in GMOs
71
Alternative strategies to antibiotic resistance marker genes
366
Health concerns relate to potential new metabolites or herbicide residues that could occur as
a result of the introduced resistance and interaction between the gene product and the
herbicide. If resistance is introduced only for herbicide use as a selective agent and there is no
field application of the herbicide, direct plant exposure to the herbicide does not occur and
metabolites and/or residues of the herbicide do not raise food safety issues. However the use
of herbicide resistance genes in plants should be avoided for use only as a selectable marker if
the gene will be treated for regulatory purposes as if field application of herbicide will occur
(Langridge, 1997).
Metabolic markers
367
A large number of genes other than antibiotic and herbicide resistance genes have been
isolated for use as selectable markers. Some have been important markers in insect (eg
organophosphate resistance) and fungal (eg benomyl resistance) transformation but are of
minimal use for plants. Of those used in plants, many are based on resistance to toxic agents
(eg methotrexate) that do not occur in the natural environment.
368
The use of a toxic agent as a selectable marker as an alternative to an antibiotic resistance
gene in food plants and animals may not be acceptable among consumers and may arouse
similar concerns.
369
Resistance to heavy metal ions has been used in GM micro-organisms. Genes conferring
resistance to arsenite and mercuric salts or organomercurials have been used as selectable
markers in Tn5- and Tn10-derived transposon vectors (de Lorenzo, 1992).
370
Resistance to copper has been used as a marker in yeasts. Metal resistance, through insertion
of a mammalian metallothionein gene can be used as a selectable marker in plants. It has
been suggested that crop plants containing a metal resistance marker that encodes for a
protein that binds heavy metals might become toxic through the accumulation of metals but
this has not been tested experimentally (ACNFP, 1994).
371
Tryptophan decarboxylase (TDC) catalyses the conversion of L-tryptophan into non-toxic
tryptamine. The tdc gene has been used as a selectable marker in the transformation of
species like Nicotiana tabacum that have no detectable endogenous TDC activity. A possible
disadvantage is accumulation of tryptamine in the transformed tissue. Its applicability in
other species will depend on endogenous TDC activity and their tolerance to elevated tdc
gene-directed tryptamine levels (Goddijn et al, 1993).
372
A recent development is based on the use of selectable marker genes that give the
transformed cells a metabolic advantage compared to the non-transformed cells which are
starved with a concomitant slow reduction in viability. One method depends on conversion
by the E. coli beta-glucuronidase gene (GUS) of the inactive form of the plant hormone
cytokinin to a biologically active form that stimulates the transformed cells to regenerate. In
this selection system the GUS gene functions as a selectable gene as well as a reporter gene
(Okkels et al, 1997). The method uses cytokinin glucuronides as selective agents and is based
on the fact that in vitro grown plant tissue cultures are auxotrophic in several aspects. In
particular exogenously applied cytokinin significantly enhances regeneration and growth.
Use of Antibiotic Resistance Marker Genes in GMOs
72
Alternative strategies to antibiotic resistance marker genes
373
Higher transformation frequencies (1.7-2.9 fold higher) are obtained than with kanamycin
selection. This could be related to the fact that most cells in the explants die during
traditional antibiotic resistance selection. The effect of necrotic tissue during selection is
presumably reduced mitotic activity of the transformed cells resulting in less GM shoots
emerging from the explants (Joersbo and Okkels, 1996).
374
Another method involves the use of mannose which cannot be metabolised by many plant
species as a selective agent. After uptake it is phosphorylated to the sugar mannose-6phosphate that accumulates resulting in severe growth inhibition. The manA gene encodes
the enzyme phosphomannose isomerase (PMI) that converts mannose-6-phosphate into
fructose-6-phosphate which is easily metabolised. The gene exists in humans, many animals
and many species of gut bacteria. It cannot work in all plants (eg soya) as they already have
the manA gene (Coghlan, 1999). It has been successfully used to date in wheat, maize and
sugar beet.
375
Joersbo et al (1998) obtained transformation frequencies about 10-fold higher compared to
kanamycin selection and at least 80-90 percent of the isolated shoots contained the transgene.
High selection efficiency means the use of a reporter gene is not required. Rooting of the
GM shoots was markedly improved using mannose instead of kanamycin selection. Nontransformed explants and shoots grew very slowly, lost vigour and acquired a light brown
colour. The higher transformation frequency may occur because transformed cells are
actively encouraged to grow rather than just allowed to survive. As a result the adverse
effects of dying cells are to a large extent avoided. The assay for PMI activity is also more
convenient than for traditional selectable genes in particular nptII.
376
It is considered that this selection system should replace antibiotic resistance and other
markers currently in use (Malik and Saroha, 1999).
377
The xylose isomerase gene (xylA) has been used as an alternative selectable marker. It also
favours regeneration and growth of the transformed cells while non-transformed cells are
starved but not killed. The gene enables transformed cells to use xylose as a carbohydrate
source. Even without optimisation, transformation efficiency is higher than kanamycin
selection and fully grown plants can be produced about two weeks earlier than kanamycin
selected plants because of vigorous shoot development. It is inexpensive and considered to
be applicable to a broad range of species since the plant species (potato, tomato, tobacco)
tested produced GM plants in the first transformation experiment (Haldrup et al, 1998).
378
Biosynthesis of the amino acids lysine, threonine and S-aminoethyl L-cysteine is controlled
by aspartate kinase and dihydrodipicolinate synthase both of which are subject to feedback
inhibition. Bacterial genes for these enzymes are less sensitive to inhibition than their
counterparts in plants and have been used for genetic modification of plants. Genetically
modified plants containing E. coli genes for their expression can be grown on media
containing lysine and threonine, and cysteine respectively (ACNFP, 1994).
379
The isopentenyltransferase gene (ipt) has been used as an alternative marker for Agrobacteriummediated transformation. The gene of interest is linked to the gene from Agrobacterium that
encodes isopentenyltransferase, an enzyme involved in cytokinin biosynthesis. Cytokinins
Use of Antibiotic Resistance Marker Genes in GMOs
73
Alternative strategies to antibiotic resistance marker genes
stimulate shoot formation from plant cells so only cells containing the introduced DNA form
shoots and differentiate into mature plants. An inducible system has been developed to
carefully control ipt expression so that it is only turned on when needed for shoot formation
and not long enough to cause abnormalities. Future refinements to the approach could avoid
introducing the bacterial ipt gene, instead using controlled expression of shoot-inducing
genes from plants as a marker (Kunkel et al, 1999).
380
Metabolic markers function by interfering with the metabolism of the host plant. They may
reduce the sensitivity of the plant to high concentrations of substances normally found in the
plant. There could be changes in some important components of the plant (eg vitamin or
natural toxin content) because of the direct interference with the plant’s metabolism. The
extent of interference to a metabolic pathway that might occur from the product of the
inserted metabolic marker gene can be expected to vary considerably between different plant
species. As a result it may not be possible to extrapolate from the results of a safety
evaluation of a metabolic marker gene in one plant species to another (WHO, 1993).
Other selectable markers in plants
381
Negative selection systems include root inhibition markers and induced toxicity markers. The
concept of negative selection is based on the expression of a marker gene that causes
immediate or conditional cell death. It can be used when a particular class of cells needs to be
eliminated.
382
The aux2 or tms2 gene derived from Agrobacterium species encode the enzyme amidohydrolase
that catalyses conversion of indole acetamide and naphthalene acetamide to indole acetic acid
(IAA) and naphthalene acetic acid (NAA) respectively. Modified plants containing these
genes overproduce IAA and NAA when grown on a medium containing indole or
naphthalene acetamide that inhibits root system formation (Harding, 1999).
383
The bacterial cytosine deaminase gene (codA) encodes the enzyme cytosine deaminase.
Cytosine deaminase activity is not usually detectable in higher organisms. Its absence in many
plant species indicates wide applicability as a negative selection marker. Cytosine deaminase
converts the selective agent 5-fluorocytosine to toxic 5-fluorouracil which kills the GM plants
(Kobayashi et al, 1995).
384
When plants are grown in the presence of ammonium instead of nitrate the endogenous
nitrate reductase gene is expressed at very low levels and is not sensitive to chlorate, an
inhibitor of nitrate reductase. In GM plants containing endogenous and introduced nitrate
reductase genes the modified gene is constitutively expressed in the presence of ammonium
so that GM plants are killed by chlorate and only non-GM plants survive (Harding and
Harris, 1997).
Other selectable markers in mammalian cells
385
Glutamine synthetase catalyses the formation of glutamine from glutamate and ammonia
which is essential to the cell for growth. It has been used as a marker for mammalian cells in
Use of Antibiotic Resistance Marker Genes in GMOs
74
Alternative strategies to antibiotic resistance marker genes
the presence of the glutamine synthetase inhibitor, methionine sulphoximine (Harding,
1999).
386
Micro-organisms catalyse synthesis of tryptophan from indole and serine. The bacterial trpB
gene produces tryptophan synthase and its transfer into mammalian cells allows the positive
selection of such cells on medium containing indole (Harding, 1999).
387
The bacterial hisD gene produces histidinol dehydrogenase which catalyses oxidation of
histidinol to histidine. When this gene is transferred into mammalian cells it allows the
selection of cells on growth medium containing histidinol only. This is a double selection
system since histidinol is toxic and histidinol dehydrogenase detoxifies it while providing
histidine for growth (Harding, 1999).
Other markers in yeast and micro-organisms
388
L-canavanine is an amino acid analogue that can be included in culture media to select for
transformed L-canavanine resistant cells. The marker has been used in S. cerevisiae (ACNFP,
1994).
389
Complementation systems involving auxotrophic markers have been used widely for the
yeast S. cerevisiae and have been described for E. coli (ACNFP, 1994). The success of
transformation in yeast is largely due to the ease of selection for transformants. As laboratory
strains of yeast are generally haploid, a mutation or deletion of a gene in an important
biosynthetic pathway can be complemented with the wild type gene in a transformation
vector. Since a large number of such auxotrophic mutants are available and many of the wild
type genes have been cloned this provides a ready series of selectable markers. A similar
approach is possible in diploid species but the target for the transformation must be made
homozygous for the mutation. Problems with the method are that homozygous auxotrophic
mutants and the cloned wild type gene must be available and it is restricted to these defined
genotypes (Langridge, 1997).
390
Sugar catabolism markers that use the presence of enzymes involved in breakdown of
specific sugars have been used to identify transformed bacteria.
391
Alternative markers for GM micro-organisms include resistance to the antimicrobial, nisin
and resistance to bacteriophage. Nisin is found naturally in some lactic acid bacteria and is
used in food preservation. Resistance does not involve enzyme production; its mechanism is
unknown but is thought to involve either tolerance or receptor resistance. If the nisin
resistance marker were acquired by a spoilage micro-organism this could result in spoilage of
unprocessed food as the micro-organism would continue to grow in the presence of nisin.
Resistance to bacteriophage is a potential selectable marker for lactic acid bacteria (ACNFP,
1994).
Use of Antibiotic Resistance Marker Genes in GMOs
75
Alternative strategies to antibiotic resistance marker genes
Alternatives to the selection of antibiotic resistance genes in bacteria
392
Use of antibiotic resistance genes is a convenient but not an absolute requirement for cloning
genes in E. coli. Selection vectors are now available based on the control of cell death. The
gene sequences for biosynthesis of amino acids, purines and pyrimidines are routinely used
for cloning genes in yeast and fungi and similar markers can be used for cloning genes in E.
coli. Such markers have appeal because the construction of the recipient host by homologous
recombination with deletion of selectable marker sequences is possible. The marker gene can
also be cleaved out of the recombinant plasmid before it is introduced into the plant tissue.
Cotransformation with a plasmid that can be used to select the transformed cells and later
eliminated is also a possibility (Malik and Saroha, 1999).
393
Specialised host-vector systems in which an essential gene that has been deleted from the
host chromosome is supplied by a recombinant plasmid have been developed. As a result
direct selection of recombinant clones is possible without addition of a selective agent. It has
been successfully tested in construction of live vaccine candidates. Broad host range plasmids
carrying the thyA+/thymidylate synthase autoselective marker have been constructed which
use a thyA-- mutant strain as the host (de Lorenzo, 1992).
394
Ideally if a gene is to have broad application as a marker it should be expressed in a variety of
organisms. The thymidylate synthase gene (thyA) is from a species of bacteria used routinely
for the manufacture of cheese. Mutants have been isolated from many micro-organisms.
They lack thymidylate synthase activity and rely on an alternate pathway of DNA synthesis
from exogenous thymidine or thymine.
395
In a thyA mutant background the thyA gene can become a positive marker for strain
construction. Plasmids marked with the thyA gene are likely to be stably maintained in thyA
mutant strains, in media or environments lacking or limiting thymine or thymidine. It is
unlikely that natural environments have sufficient thymidine or thymine to interfere with the
effectiveness of the thyA gene marker system although further experimentation is required
(Ross et al, 1990).
Elimination of the selectable marker
396
Except for herbicide resistance, selectable marker genes are not aimed at any change or
improvement of the agronomic or other characteristics of the organism involved.
Elimination of the gene is therefore an alternative strategy.
No selectable marker or reporter gene
397
Direct introduction of DNA into a cell with the potential to develop into a whole organism is
the best method of genetic modification for the release of organisms into the environment.
Micro-injection has been applied to a wide range of higher organisms in particular animals,
including fish, and some plant species, and avoids the use of markers (Harding, 1999). Direct
DNA transfer via micro-injection of DNA into cells of microspore-derived embryoids may
improve the efficiency of gene transfer to acceptable values. Cell finder systems in which a
Use of Antibiotic Resistance Marker Genes in GMOs
76
Alternative strategies to antibiotic resistance marker genes
computer-controlled microscope allows easy positioning and relocation of cultured cells and
protoplasts, in combination with improved gene delivery techniques, may also develop into a
system that allows the identification and isolation of GM plants only carrying the desired
gene (Metz and Nap, 1997).
398
Currently it is not practical to avoid the use of marker genes. Foreign DNA can be readily
identified by PCR based assay systems and methods are available that do not require direct
isolation of DNA so large numbers of cells can be screened rapidly. However transformation
efficiency is still very low. A single GM plant is also rarely enough; several GM plants are
usually sought for each construct due to variation in transgene expression levels and stability
(Langridge, 1997).
Reporter gene only
399
When marker genes are used for the rapid analysis of promoter activity they are called
reporter genes. Another way to eliminate selectable marker genes is to use a reporter gene to
monitor transformation. For plant cells the use of reporter genes to identify or tag GM cells
is feasible only when transformation efficiency is high. Although the currently most
frequently used reporter gene, the GUS gene requires assays that destroy the GM material,
reporter genes that encode firefly luciferase and the jellyfish Aequorea victoria green fluorescent
protein can be assayed non-destructively. These genes allow the identification of transformed
cells through a change in their colour. Non-destructive assay raises the possibility of
monitoring transformation during the development of the GM shoot and selection of the
shoot in a very early stage (Ghorbel et al, 1999).
400
Green fluorescent protein has been expressed in a wide variety of organisms (eg E. coli, yeast,
Drosophilia, mammals, worms and plants) and only needs ultraviolet or blue light to fluoresce.
Detection of other commonly used reporter genes requires enzymes, cofactors or exogenous
substrates. Expression of GFP is cell autonomous and independent of cell type and location
(Sheen at al, 1995).
401
It is possible that the frequency of recovery of transformants will be increased by the use of
colour markers relative to selection based on a toxic agent. In selecting for transformed cells
in the presence of a toxic agent it is unclear whether or not transformed but poorly
expressing GM cells are being killed or whether the regeneration rates are being decreased by
having the GM cells surrounded by cells that are dead or dying (Langridge, 1997).
402
Considerable effort is being expended on developing and testing transformation procedures
based on colour markers.
Inactivation of the selectable marker gene
403
Limiting the expression of the selectable marker gene to the stages at which selection for
transformation is applied will result in GM plants in which the transgene is present but not
the transgene encoded protein. It has been shown that the wound-inducible promoter
AoPR1 isolated from asparagus, when fused to the kanamycin resistance gene, allows
selection during transformation but results in very low levels of the transgene product in the
mature plant (Metz and Nap, 1997).
Use of Antibiotic Resistance Marker Genes in GMOs
77
Alternative strategies to antibiotic resistance marker genes
404
Gene silencing may possibly be developed into a method to obtain plants without selectable
marker gene activity (Metz and Nap, 1997). Schmulling and Rohrig (1995) have
demonstrated in GM tobacco hybrids that single genes could be selectively inactivated on
T-DNAs harbouring several genes. They concluded that it is likely that several factors
determine the probability of interaction between different loci and therefore influence the
differences in the kinetics of inactivation and restoration of gene expression (eg accessibility
of the loci for pairing and relative positions of the inserts in the genome).
Removal of the selectable marker gene
405
Removal of the selectable marker gene allows the same marker to be used repeatedly in
subsequent transformations into the same host. As successive rounds of transformation with
additional genes of interest become prevalent this will be an important attribute given the
limited availability of selectable markers. Removal also minimises the amount of foreign
DNA to that which is actually involved in conferring the desired trait(s) and could have
positive effects on consumer acceptance (Ow and Medberry, 1995).
406
When using Agrobacterium T-DNA transfer as the means of transformation the selectable
marker gene, being on the same T-DNA as the desired gene, is linked to the desired gene.
When unlinked the gene can be segregated from the selectable marker gene and plants
carrying only the desired gene can be identified with routine procedures.
407
Several strategies have been used to unlink the selectable marker gene from the desired gene
and generate marker-free GM plants. They include cotransformation of two T-DNA
molecules (Komari et al 1996), site-specific recombination (Dale and Ow, 1991) and
transposition-mediated repositioning of the marker gene (Goldsbrough et al, 1993). These
strategies require sexual crossing to eliminate the marker genes and hence their applicability
to plant species that have a long generation time is limited. However some recently
developed systems can generate marker-free GM plants without the need to sexually cross
plants eg the multi-autotransformation (MAT) vector system (Ebinuma et al, 1997).
Cotransformation
408
The cotransformation systems appear to be the simplest of the strategies to eliminate marker
genes and have been widely used in direct transformation methods. In these systems the
marker gene and desired gene are introduced into the plant genome on separate vectors.
Following several generations of crossing the marker gene will be eliminated in some
progeny through genetic segregration.
409
Another advantage of cotransformation systems is that the construction of the separate
molecules for the marker gene and desired gene is less tedious than creation of linked DNA
fragments.
410
Komari et al (1996) obtained GM tobacco and rice plants free from selectable markers by a
relatively simple procedure consisting of Agrobacterium-mediated cotransformation and
segregation of the progeny. Since the vector system functioned efficiently both in a
dicotyledon, tobacco, and a monocotyledon, rice, this system is potentially useful for a wide
range of plant species.
Use of Antibiotic Resistance Marker Genes in GMOs
78
Alternative strategies to antibiotic resistance marker genes
411
The feasibility of cotransformation is disputed but it has been suggested that the method
would be suitable for transformations using Agrobacterium and cloned gene transfer strategies.
Frequencies of cotransformation are low and not potentially useful for species that are
difficult to transform (ACNFP, 1994).
412
Biolistics with independent DNA molecules may also result in unlinked cotransformation
(Metz and Nap, 1997).
Site-specific recombination
413
Site-specific recombination is the enzyme-mediated cleavage and ligation of two defined
deoxynucleotide sequences. Once transformants with the desired gene are obtained the
selectable marker gene is no longer necessary. Removal of such unnecessary DNA has been
demonstrated using the Cre/lox recombination system (Dale and Ow, 1991).
414
Using the Cre/lox recombination system a plant is transformed with the desired gene using a
selectable marker gene flanked by lox recombination sites. The cre gene accompanied by a
second marker gene is introduced through sexual hybridisation or a second transformation
round. The initial marker is excised from the genome through the action of cre recombinase
(bacterial recombination enzyme) which acts at the lox sites. After its removal plants are left
to flower and set seed and the progeny are screened for segregation of the cre gene which is
linked to the remaining marker gene. Plants without the cre and marker genes are selected and
propagated.
415
Gleave et al (1999) have developed a plant transformation vector incorporating the Cre/lox
site-specific recombination system to facilitate the elimination of marker genes from GM
plants and the cytosine deaminase gene (codA). Transient expression of cre recombinase is
used to mediate excision of lox flanked marker genes from GM plants and the codA gene is
used to select plants that have undergone Cre-mediated recombination. Using this approach
marker-free plants can be produced without sexual crossing. It should be applicable to many
established perennial horticultural cultivars requiring vegetative propagation to maintain their
elite genome.
416
Several DNA site-specific recombination systems have been shown to function in higher
eukaryotic cells. Strategic placement of the recognition sites into the plant genome has
permitted the deletion, inversion, integration and translocation of host and introduced DNA
fragments. Recombinase-based strategies afford precise and predictable engineering of the
plant genome (Ow and Medberry, 1995). The approach is also relevant to genetic
modification of animals and cell lines. In the case of cell lines, where loss of the cre locus
cannot be achieved by sexual segregation, transient expression of the cre gene or direct
introduction of purified cre protein could be used (Dale and Ow, 1991).
417
It can be applied to a wide range of plants but is not feasible in vegetatively propagated crops
with low fertility and seed selection might scramble elite genomes in clonally propagated
plants (Harding, 1999).
418
A lot of progress has been made since the first reports of site-specific recombination in
plants. However recombinase-based technology is still an academic exercise. It has yet to be
Use of Antibiotic Resistance Marker Genes in GMOs
79
Alternative strategies to antibiotic resistance marker genes
seen if and when it will have an impact on crop improvement (Ow and Medberry, 1995).
Two other recombination systems (FLP-frt and R-rs) are also being evaluated in plants (Metz
and Nap, 1997).
419
Zubko et al (2000) have developed a system to remove selectable marker genes from tobacco
based on intrachromosomal homologous recombination between two homologous
sequences using a vector containing nptII and attachment regions from bacteriophage λ. It
does not require removal of recombinase by genetic segregation and is therefore less time
consuming.
Transposition-mediated repositioning
420
Transposition-mediated repositioning requires the introduction of the selectable marker gene
and the desired gene on a vector containing a transposase function. The selectable marker
gene moves away from the gene or vice versa to become unlinked from the gene (eg
transformation system using the Ac/Ds maize transposable element) (Metz and Nap, 1997).
Large numbers of progeny plants are needed for selection and the construction of the vector
has to allow transposition to occur at a sufficient distance from the selectable marker gene to
ensure that the selectable marker gene and desired gene are separated during recombination
(ACNFP, 1994).
421
Removal of the selectable marker gene has also been achieved by linking the marker to a
negative selectable marker eg codA gene; human herpes simplex virus thymidine kinase type 1
gene (HSVtk) on the same DNA molecule. Plant cells expressing cytosine deaminase will
convert 5-fluorocytosine to toxic 5-fluorouracil. The HSVtk encoded enzyme inhibits
growth in the presence of the antiviral drug, ganciclovir. All plants carrying the antibiotic
resistance selectable marker will also carry the negative selectable marker and will be killed by
treatment with 5-fluorocytosine or ganciclovir. Only plants without the selectable marker will
survive and about half will carry the desired gene. These can be identified by PCR assays
(Langridge, 1997).
422
Ebinuma et al (1997) have developed a plant vector system for repeated transformation
(called multi-autotransformation (MAT)) in which a chimaeric ipt gene inserted into a
transposable element Ac is used as a selectable marker. It is easy to detect visually GM plants
that carry a functional ipt gene due to shoot formation. Chimeric ipt genes are not commonly
used as markers because the resulting GM plants lose apical dominance and are unable to
root due to overproduction of cytokinins. Hence Ac was used to remove the ipt gene after
transformation although the frequency of marker-free GM plants obtained was relatively low.
423
The vector system may provide an alternative approach to regenerate some plant species that
have been difficult to transform and provides a way of bypassing the problem of long
generation times (eg tree species). The system allows removal of a marker gene from a
vegetatively propagated crop without crossing. Other transformation systems for eliminating
marker genes cannot be applied to those crops because they need sexual crosses to produce
marker-free plants and to be able to carry out successive transformation.
424
Strategies to obtain GM plants without a selectable marker gene have been successful in
some cases. However these approaches are far from routine or from being generally
applicable and are more time consuming. These techniques could be optimised further for
Use of Antibiotic Resistance Marker Genes in GMOs
80
Alternative strategies to antibiotic resistance marker genes
more routine GM plant production. Approaches using protoplasts are likely to suffer from
disadvantages compared to Agrobacterium-mediated plant transformation due to somaclonal
variation. Approaches that are based on segregating away the unlinked selectable marker gene
require plants that are relatively homozygous and that can be easily backcrossed. These
approaches will be essentially impossible for some plants (eg potato).
Removal of marker genes from micro-organisms
425
Removal methods from GM micro-organisms involve use of enzymic excision or of
homologous recombination.
426
During transformation, target sites are inserted to flank the selectable marker gene. Selected
GM micro-organisms are then further modified to include sequences which code for
enzymes active at the target sites and the action of these enzymes excises the selectable
marker gene.
427
The delivery vector which is unable to replicate in the recipient organism carries the desired
gene flanked by DNA homologous to that on the recipient chromosome and antibiotic
resistance and beta-galactosidase (lacZ) marker genes. Crossover of homologous DNA
integrates the vector into the recipient chromosome, indicated by antibiotic resistance. The
vector DNA, including the markers, then separates from the recipient chromosome and is
lost, indicated by the absence of lacZ activity. The desired gene remains in the recipient
chromosome.
428
A site-specific recombination vector has been developed that selectively removes the
antibiotic resistance marker gene from Bacillus thuringiensis (Bt) after introduction of the
plasmid into the host strain (Sanchis et al, 1997).
Modulation of gene expression
429
Antisense technology could be applied to marker genes. When an antisense gene is
introduced into a cell it encodes antisense RNA that is complementary to the messenger
RNA of the original marker gene and blocks its expression.
430
Catalytic RNA such as ribozymes cleaves either itself or other RNA molecules. It has created
new opportunities to repress the expression of selectable marker genes (Harding, 1999).
Expression of a ribozyme gene directed towards the target mRNA of the npt marker gene in
plants resulted in a reduction of the gene product expression (Steinecke et al, 1992).
431
Genetically modified plants containing selectable markers could be further modified with
genes that encode antibodies against the gene product (Hiatt et al, 1989).
Intron-containing antibiotic resistance genes
432
Most of the antibiotics used for selection of transformed plant tissues can also inhibit
Agrobacterium growth. This inhibition is usually not effective against Agrobacterium during the
plant transformation process because the antibiotic resistance gene used for plant selection is
expressed in Agrobacterium.
Use of Antibiotic Resistance Marker Genes in GMOs
81
Alternative strategies to antibiotic resistance marker genes
433
Introns have been inserted into the hygromycin resistance gene (hph) coding region to abolish
its expression in Agrobacterium and hence minimise the problem of Agrobacterium overgrowth
during plant transformation. Control of overgrowth reduces the amount of labour and
increases the chance of success of each experiment. Introns also enhanced the expression
level of the hph gene and as a result either maintained or enhanced the selection efficiency of
the hph gene during transformation. Better quality transgene mRNA and GM lines with low
copy numbers of the transgene were also produced.
434
An advantage of using an intron-disrupted antibiotic resistance gene as a selectable marker is
a reduced probability of antibiotic resistance being acquired by gut bacteria following
consumption of GM material by humans or animals (Wang et al, 1997).
Use of Antibiotic Resistance Marker Genes in GMOs
82
Conclusion
8.
Conclusion
435
People already consume naturally occurring micro-organisms on or in food or water that
contain antibiotic resistance genes and also acquire antibiotic resistant micro-organisms as a
result of antibiotic treatment. These are the major sources of antibiotic resistance in humans
and relate to decades of widespread antibiotic use in humans and animals.
436
The scientific literature on the use of antibiotic resistance genes in GMOs is characterised by
many opinions and relatively few data. Many different scientific disciplinary approaches have
assessed the uncertainty and the available evidence. Opinion is polarised between scientists
who are concerned about what might happen based on extrapolations from laboratory
experiments and those who maintain the weight of evidence for successful gene transfer is
not sufficiently convincing for there to be any concern. However often the overall
importance of a value to be protected such as human health is more a matter of science
policy, to be determined in the New Zealand context by the ERMA, rather than a scientific
issue.
437
Scientific concern about the use of antibiotic resistance genes in GMOs relates more to
potential health effects from gene transfer to micro-organisms than to potential health effects
due to ingestion of the antibiotic resistance gene or its gene product or transfer to gut
epithelial cells.
438
As there is no evidence that antibiotic resistance marker genes have transferred from GM
plants to pathogenic micro-organisms, and experimental evidence suggests the probability of
such an event is remote, scientists generally consider that the risk of antibiotic resistance
arising from the presence of antibiotic resistance genes in GMOs is extremely low. Even if
the probability of horizontal gene transfer is extremely low this should not however be
confounded with the potential outcome. Rare transfer events can be amplified rapidly under
selective pressure. The health impact would be significant if a gene conferring resistance to a
clinically important antibiotic was transferred and expressed in a pathogenic micro-organism
normally treated with that antibiotic. From what is known about mechanisms of horizontal
gene transfer between organisms and the survival of intact DNA following processing and
digestion it is concluded that the risk is highest for ingested viable GM micro-organisms, in
particular if the antibiotic resistance gene confers resistance to a clinically important
antibiotic, and lowest for highly processed GM food.
439
However there are gaps in scientific knowledge in this area. Assessment of the potential for
transfer of antibiotic resistance marker genes from GM plants or other eukaryotes into
micro-organisms needs to be examined more closely. Studies are needed that incorporate
selective pressure during the exposure of competent bacteria with DNA. Data are also
limited about the antibiotic resistance genes with the exception of the kanamycin resistance
gene.
440
Relatively little consideration has been given to date to the potential cumulative
consequences of a rare event in a scenario of widespread cultivation of GM plants and
ingestion of raw and unprocessed GM plant material by millions of people and animals.
Use of Antibiotic Resistance Marker Genes in GMOs
83
Conclusion
441
Widespread disagreement exists on what constitutes a tolerable risk when it comes to
antibiotic resistance. There is a dichotomy of opinion between the view that any further
increase, irrespective of how small, in the prevalence of antibiotic resistant micro-organisms
through the transfer of antibiotic resistance genes from GMOs would be undesirable and the
view that the consequence would not be meaningful due to existing levels of resistance in the
human gut and environment.
442
The concept of the precautionary approach as a matter to be taken into account under the
HSNO Act 1996, though not defined, takes into account scientific uncertainty and conflict. It
also allows for consideration of the social construct of tolerable or acceptable risk.
443
Risk assessment reports have argued that there are no scientific, health or safety reasons to
restrict the use of the nptII gene and even if methods for removal are available and prove
generally applicable, removal of the nptII gene should not be required (Appendix I). These
conclusions have also been broadened by some to there being no scientific reason to prohibit
or limit the use of antibiotic resistance marker genes, nor to encourage or require their
removal from GM plants. The arguments put forward to justify the inclusion of antibiotic
resistance marker genes do not necessarily apply to all pathogenic micro-organisms and to all
geographical locations. In this respect it is important to consider the antibiotic involved: how
it is administered, its uses, the potential for cross-resistance to other antibiotics, the
prevalence of resistance, and the existence of alternative therapeutic agents.
444
The commonly used kanamycin resistance marker gene confers resistance against kanamycin
and neomycin. These antibiotics have limited clinical use because of their side effects and are
not often used orally. Although there are no New Zealand data on the prevalence of
kanamycin resistance it is unlikely to differ markedly from that found in other developed
countries. Kanamycin is however used as a reserve drug in the treatment of tuberculosis.
445
The ampicillin resistance gene used in bacterial transformation is often not present intact in
the final GMO. If it is present the probability of transfer from the GMO to pathogenic
micro-organisms and gene expression needs to be considered in a New Zealand context of
high use of beta-lactams, the clinically important group of antibiotics to which ampicillin
belongs.
446
In the environment if transfer from the plant genome to soil micro-organisms did occur in
most cases there would be no selective pressure. Exceptions are streptomycin use in
horticulture or when manure is used as fertiliser following in-feed antibiotic use for animal
growth promotion and prophylaxis.
447
There appears to be an emerging international consensus among regulatory agencies to
evaluate each GMO containing antibiotic resistance marker genes on a case-by-case basis and
a move to advocating restriction of introduced genes in the final GMO to those genes
needed to confer the desired trait(s).
448
Irrespective of the scientific conclusions removal of the antibiotic resistance gene from the
final GM plant or use of alternative strategies is now being recommended whenever feasible.
It is consumer acceptance of a product that governs its market performance. Elimination of
antibiotic resistance marker genes could have positive effects on consumer acceptance by
Use of Antibiotic Resistance Marker Genes in GMOs
84
Conclusion
alleviating perceived risks. In addition antibiotic resistance marker genes serve no useful
purpose in the final genetically modified organism.
449
Post-market surveillance for transfer of the antibiotic resistance gene has been suggested as a
mechanism to safeguard against occurrence of antibiotic resistance from the use of marker
genes other than hygromycin (which is not used in human medicine) and kanamycin. It
would provide regulatory agencies an opportunity for early intervention to prevent an
adverse impact on public health.
450
The potential impact of the use of antibiotic resistance genes in GMOs on the prevalence of
antibiotic resistance is far less significant than the impact of the current use of antibiotics in
humans and animals in New Zealand. However antibiotic resistance is receiving increasing
scrutiny nationally and internationally, and there are an increasing number of strategies being
implemented with the aim of curbing all antibiotic use. In addition alternative strategies to
antibiotic resistance marker genes such as gene removal or other selectable markers are
increasing, some of which are looking feasible in terms of safety and practicability.
Use of Antibiotic Resistance Marker Genes in GMOs
85
References
9.
References
Abbott A. Germany holds up cultivation of GM maize. Nature 2000; 403: 821.
Advisory Committee on Novel Foods and Processes. Report on the use of antibiotic resistance
markers in genetically modified food organisms. London: Ministry of Agriculture, Fisheries and
Food, July 1994.
Advisory Committee on Novel Foods and Processes. The use of antibiotic resistance markers in
genetically modified plants for human food: clarification of principles for decision-making. London:
Ministry of Agriculture, Fisheries and Food, July 1996.
Antibiotic Resistance Expert Panel. Antibiotic resistance and in-feed use of antibiotics in New
Zealand. Report to the Ministry of Agriculture and Forestry Antibiotic Resistance Steering Group.
July, 1999.
Australia New Zealand Food Authority. Guidelines for the safety assessment of foods to be included
in Standard A18 – food produced using gene technology. Canberra: Australia New Zealand Food
Authority, August 1998.
Baquero F, Negri MC, Morosini MI, Blazquez J. Antibiotic-selective environments. Clin Infect Dis
1998; 27(Suppl 1): S5-11.
Benveniste R, Davies J. Aminoglycoside antibiotic-inactivating enzymes in Actinomycetes similar to
those present in clinical isolates of antibiotic-resistant bacteria. Proc Natl Acad Sci USA 1973; 70:
2276-80.
Bertolla F, Simonet P. Horizontal gene transfers in the environment: natural transformation as a
putative process for gene transfers between transgenic plants and microorganisms. Res Microbiol
1999; 150(6): 375-84.
British Medical Association. The impact of genetic modification on agriculture, food and health. An
interim statement. London: British Medical Association, 1999.
Broer I, Droge-Laser W, Gerke M. Examination of the putative horizontal gene transfer from
transgenic plants to Agrobacteria. In Schmidt ER, Hankeln T (eds). Transgenic organisms and
biosafety, horizontal gene transfer, stability of DNA and expression of transgenes. Heidelberg:
Springer-Verlag, 1996: 67-70.
Coghlan A. On your markers. New Scientist 20 November 1999: 10.
Coghlan A. So far so good. New Scientist 25 March 2000: 4.
Conner AJ. Genetically engineered crops. Environmental and food safety issues. Miscellaneous series
39. Wellington: The Royal Society of New Zealand, 1997.
Use of Antibiotic Resistance Marker Genes in GMOs
86
References
Courvalin P, Goussard S, Grillot-Courvalin C. Gene transfer from bacteria to mammalian cells. CR
Acad Sci Paris 1995; 318: 1207-12.
Dale EC, Ow DW. Gene transfer with subsequent removal of the selection gene from the host
genome. Proc Natl Acad Sci USA 1991; 88: 10558-62.
Davies JE. Origins, acquisition and dissemination of antibiotic resistance determinants. In Chadwick
DJ, Goode J (eds). Antibiotic resistance: origins, evolution, selection and spread. Ciba Foundation
Symposium 207. Chichester: John Wiley and Sons, 1997: 15-35.
de Lorenzo V. Genetic engineering strategies for environmental applications. Curr Opin Biotechnol
1992; 3(3): 227-31.
de Oliveira RD, Wolters AC, van Elsas JD. Effects of antibiotics in soil on the population dynamics
of transposon Tn5 carrying Pseudomonas fluorescens. Plant Soil 1995; 175: 323-33.
de Vries J, Wackernagel W. Detection of nptII (kanamycin resistance) genes in genomes of transgenic
plants by marker-rescue transformation. Mol Gen Genet 1998; 257: 606-13.
Doblhoff-Dier O, Bachmayer H, Bennett A, Brunius G, et al. Safe biotechnology 9: values in risk
assessment for the environmental application of microorganisms. Trends Biotechnol 1999; 17(8):
307-11.
Donaldson L, May R. Health implications of genetically modified food. Report to the UK Ministerial
Group on Biotechnology. London: Department of Health, 1999.
Doucet-Populaire F, Trieu-Cuot P, Dosbaa I, Andremont A, et al. Inducible transfer of conjugative
transposon Tn1545 from Enterococcus faecalis to Listeria monocytogenes in the digestive tracts of
gnotobiotic mice. Antimicrob Agents Chemother 1991; 35: 185-7.
Droge M, Puhler A, Selbitschka W. Horizontal gene transfer as a biosafety issue: a natural
phenomenon of public concern. J Biotechnol 1998; 64(1): 75-90.
Droge M, Puhler A, Selbitschka W. Horizontal gene transfer among bacteria in terrestrial and aquatic
habitats as assessed by microcosm and field studies. Biol Fertil Soils 1999; 29(3): 221-45.
Ebinuma H, Sugita K, Matsunaga E, Yamakado M. Selection of marker-free transgenic plants using
the isopentenyl transferase gene. Proc Natl Acad Sci USA 1997; 94: 2117-121.
European Commission. Opinion of the Economic and Social Committee on the ‘Resistance to
antibiotics as a threat to public health’. Official Journal of the European Communities. 1998/C
407/2.
European Commission. Opinion of the Scientific Steering Committee on Antimicrobial Resistance.
European Commission Directorate-General XXIV, 1999.
Flavell RB, Dart E, Fuchs RL, Fraley RT. Selectable marker genes: safe for plants? Bio/technology
1992; 10(2): 141-4.
Use of Antibiotic Resistance Marker Genes in GMOs
87
References
Flint HJ, Thomson AM. Deoxyribonuclease activity in rumen bacteria. Lett Appl Microbiol 1990; 11:
18-21.
Flint HJ, Chesson A. Safety implications of genetically modified organisms. Feed Mix 1999; 7(3): 325.
Food and Agriculture Organisation, World Health Organisation. Biotechnology and food safety:
Report of a joint FAO/WHO consultation. Geneva: World Health Organisation, 1996.
Fuchs RL, Ream JE, Hammond BG, Naylor MW, et al. Safety assessment of the neomycin
phosphotransferase II (NPTII) protein. Bio/technology 1993; 11: 1543-47.
Gebhard F, Smalla K. Transformation of Acinetobacter sp. strain BD413 by transgenic sugarbeet
DNA. Appl Environ Microbiol 1998; 64(4): 1550-4.
Gebhard F, Smalla K. Monitoring field releases of genetically modified sugar beets for persistence of
transgenic plant DNA and horizontal gene transfer. FEMS Microbiol Ecol 1999; 28(3): 261-72.
Ghorbel R, Juarez J, Navarro L, Pena L. Green fluorescent protein as a screenable marker to increase
the efficiency of generating transgenic woody fruit plants. Theor Appl Genet 1999; 99(1-2): 350-8.
Gleave AP, Mitra DS, Mudge SR, Morris BA. Selectable marker-free transgenic plants without sexual
crossing: transient expression of cre recombinase and use of a conditional lethal dominant gene. Plant
Mol Biol 1999; 40(2): 223-35.
Goddijn OJ, van der Duyn Schouten PM, Schilperoort RA, Hoge JH. A chimaeric tryptophan
decarboxylase gene as a novel selectable marker in plant cells. Plant Mol Biol 1993; 22: 907-12.
Goldsbrough AP, Lastrella CN, Yoder JI. Transposition mediated repositioning and subsequent
elimination of marker genes from transgenic tomato. Bio/technology 1993; 11: 1286-92.
Grillot-Courvalin C, Goussard S, Huetz F, Ojcius DM, et al. Functional gene transfer from
intracellular bacteria to mammalian cells. Nat Biotechnol 1998; 16: 862-6.
Haldrup A, Petersen SG, Okkels FT. The xylose isomerase gene from Thermoanaerobacterium
thermosulfurogenes allows effective selection of transgenic plant cells using D-xylose as the selection
agent. Plant Mol Biol 1998; 37(2): 287-96.
Hall RM. Mobile gene cassettes and integrons: moving antibiotic resistance genes in Gram-negative
bacteria. In Chadwick DJ, Goode J (eds). Antibiotic resistance: origins, evolution, selection and
spread. Ciba Foundation Symposium 207. Chichester: John Wiley and Sons, 1997: 192-205.
Harding K, Harris PS. Risk assessment of the release of genetically modified plants: a review. AgroFood-Industry Hi-Tech 1997; 8(6): 8-13.
Harding K. Biosafety of selectable marker genes. Dundee: Crop Genetics, Scottish Crop Research
Institute, 1999.
Use of Antibiotic Resistance Marker Genes in GMOs
88
References
Heinemann JA. How antibiotics cause antibiotic resistance. DDT 1999; 4: 72-9.
Heinemann JA, Ankenbauer RG, Amabile-Cuevas CF. Do antibiotics maintain antibiotic resistance?
DDT 2000; 5: 195-204.
Henschke RB, Schmidt FRJ. Screening of soil bacteria for plasmids carrying antibiotic resistance.
Biol Fertil Soils 1990; 9: 257-60.
Hiatt A, Cafferkey R, Bowdish K. Production of antibodies in transgenic plants. Nature 1989; 342:
76-8.
Ho M-W. Genetic engineering - dream or nightmare? The brave new world of bad science and big
business. Bath: Gateway Books, 1998.
Hoffmann T, Golz C, Schieder O. Foreign DNA sequences are received by a wild-type strain of
Aspergillus niger after co-culture with transgenic higher plants. Curr Genet 1994; 27: 70-6.
International Food Biotechnology Council. Biotechnologies and food: assuring the safety of foods
produced by genetic modification. Washington: International Food Biotechnology Council, 1990.
Joersbo M, Okkels FT. A novel principle for selection of transgenic plant cells: positive selection.
Plant Cell Rep 1996; 16(3-4): 219-21.
Joersbo M, Donaldson I, Kreiberg J, Petersen S, et al. Analysis of mannose selection used for
transformation of sugar beet. Mol Breed 1998; 4: 111-7.
Joint Expert Technical Advisory Committee on Antibiotic Resistance. The use of antibiotics in foodproducing animals: antibiotic-resistant bacteria in animals and humans. Canberra: Commonwealth
Department of Health and Aged Care and Commonwealth Department of Agriculture, Fisheries and
Forestry - Australia, 1999.
Jones L. Genetically modified foods. BMJ 1999; 318: 581-4.
Karenlampi S. Health effects of marker genes in genetically engineered food plants. Copenhagen:
Nordic Council of Ministers, 1996.
Kessler DA, Taylor MR, Maryanski JH, Flamm EL, et al. The safety of foods developed by
biotechnology. Science 1992; 256: 1747-9.
Kobayashi T, Hisajima S, Stougaard J, Ichikawa H. A conditional negative selection for Arabidopsis
expressing a bacterial cytosine deaminase gene. Jpn J Genet 1995; 70: 409-22.
Kok EJ, Noteborn HPJM, Kuiper HA. Food safety assessment of marker genes in transgenic crops.
Trends Food Sci Technol 1994; 5: 294-8.
Use of Antibiotic Resistance Marker Genes in GMOs
89
References
Komari T, Hiei Y, Saito Y, Murai N, et al. Vectors carrying two separate T-DNAs for
cotransformation of higher plants mediated by Agrobacterium tumefaciens and segregation of
transformants free from selection markers. Plant J 1996; 10(1): 165-74.
Krebs J. GM food safety: facts, uncertainties, and assessment. The OECD Edinburgh conference on
the scientific and health aspects of genetically modified foods. 28 February-1 March 2000.
Chairman's report. Paris: Organisation for Economic Cooperation and Development, April 2000.
Kunkel T, Niu Q-W, Chan Y-S, Chua N-H. Inducible isopentenyl transferase as a high-efficiency
marker for plant transformation. Nat Biotechnol 1999; 17(9): 916-9.
Lang S, Blackmore T. Agricultural use of antibiotics. Is human health threatened? New Ethicals
1999; 2(9): 9-13.
Langridge P. Markers for the selection of transgenic plants. In McLean GD, Waterhouse PM, Evans
G, Gibbs MJ (eds). Commercialisation of transgenic crops: risk, benefit and trade considerations.
Proceedings of a workshop, Canberra, 11-13 March 1997. Canberra: Cooperative Research Centre
for Plant Science and Bureau of Resource Sciences, 1997: 179-91.
Levy SB, Marshall B, Schluederberg S, Rouse D, et al. High frequency of antimicrobial resistance in
human fecal flora. Antimicrob Agents Chemother 1988; 32: 1801-6.
Levy SB. Antibiotic resistance: an ecological imbalance. In Chadwick DJ, Goode J (eds). Antibiotic
resistance: origins, evolution, selection and spread. Ciba Foundation Symposium 207. Chichester:
John Wiley and Sons, 1997: 1-14.
Levy SB. Multi-drug resistance - a sign of the times. N Engl J Med 1998; 338(19): 1376-8.
Linton A. Flow of resistance genes in the environment and from animals to man. J Antimicrob
Chemother 1986; 18(Suppl C): 189-97.
Lorenz MG, Wackernagel W. Bacterial gene transfer by natural genetic transformation in the
environment. Microbiol Rev 1994; 58(3): 563-602.
MacKenzie D. Gut reaction. New Scientist 1999 30 January: 4.
Maiden MCJ. Horizontal genetic exchange, evolution, and spread of antibiotic resistance in bacteria.
Clin Inf Dis 1998; 27(Suppl 1): S12-20.
Malik VS, Saroha MK. Marker gene controversy in transgenic plants. J Plant Biochem Biotechnol
1999; 8: 1-13.
Matic I, Taddei F, Radman M. Genetic barriers among bacteria. Trends Microbiol 1996; 4: 69-73.
McAllan AB. The degradation of nucleic acids in, and the removal of breakdown products from the
small intestine of steers. Br J Nutr 1980; 44: 99-112.
McAllan AB. The fate of nucleic acids in ruminants. Proc Nutr Soc 1982; 41(3): 309-17.
Use of Antibiotic Resistance Marker Genes in GMOs
90
References
McConnell MA, Mercer AA, Tannock GW. Transfer of plasmid pAMβ1 between members of the
normal microflora inhabiting the murine digestive tract and modification of the plasmid in a
Lactobacillus reuteri host. Microbial Ecol Health Dis 1991; 4: 343-55.
Mercer DK, Scott KP, Bruce-Johnson WA, Glover LA, et al. Fate of free DNA and transformation
of the oral bacterium Streptococcus gordonii DL1 by plasmid DNA in human saliva. Appl Environ
Microbiol 1999; 65(1): 6-10.
Metcalfe DD, Astwood JD, Townsend R, Sampson HA, et al. Assessment of the allergenic potential
of foods derived from genetically engineered crop plants. Crit Rev Food Sci Nutr 1996; 36(S): S16586.
Metz PL, Nap JP. A transgene-centred approach to the biosafety of transgenic plants: overview of
selection and reporter genes. Acta Bot Neerl 1997; 46(1): 25-50.
Miller RV. Bacterial gene swapping in nature. Sci Am 1998; 278: 66-71.
Minekus M, Marteau P, Havenaar R, Huis in’t Veld JHJ. A multicompartmental dynamic computercontrolled model simulating the stomach and small intestine. Alta-Altern Lab Anim 1995; 23(2): 197209.
Nap JP, Bijvoet J, Stiekema WJ. Biosafety of kanamycin-resistant transgenic plants. Transgenic Res
1992; 1: 239-49.
Nielsen KN, Gebhard F, Smalla K, Bones AN, et al. Evaluation of possible horizontal gene transfer
from transgenic plants to the soil bacterium Acinetobacter calcoaceticus BD413. Theor Appl Genet 1997;
95: 815-21.
Nielsen KM, Bones AM, Smalla K, van Elsas JD. Horizontal gene transfer from transgenic plants to
terrestrial bacteria – a rare event? FEMS Microbiol Rev 1998; 22: 79-103.
Ochman H, Lawrence JG, Groisman EA. Lateral gene transfer and the nature of bacterial
innovation. Nature 2000; 405: 299-304.
Okkels FT, Ward JL, Joersbo M. Synthesis of cytokinin glucuronides for the selection of transgenic
plant cells. Phytochem 1997; 46: 801-4.
Organisation for Economic Cooperation and Development. Safety evaluation of foods derived by
modern biotechnology: concepts and principles. Paris: Organisation for Economic Cooperation and
Development, 1993.
Organisation for Economic Cooperation and Development. Report of the Task Force for the Safety
of Novel Foods and Feeds. Paris: Organisation for Economic Cooperation and Development, May
2000a.
Use of Antibiotic Resistance Marker Genes in GMOs
91
References
Organisation for Economic Cooperation and Development. Report of the Working Group on
Harmonisation of Regulatory Oversight in Biotechnology. Paris: Organisation for Economic
Cooperation and Development, May 2000b.
Ow DW, Medberry SL. Genome manipulation through site-specific recombination. Crit Rev Plant
Sci 1995; 14: 239-61.
Paget E, Lebrun M, Freyssinet G, Simonet P. The fate of recombinant plant DNA in soil. Eur J Soil
Biol 1998; 34(2): 81-8.
Pascal G. Transgenic plants and food safety. In Kahn A (ed). Transgenic plants in agriculture. Ten
years experience of the French Biomolecular Engineering Commission. Paris: John Libbey Eurotext,
1999: 43-50.
Pittard AJ. The use of antibiotic resistance markers in transgenic plants and microorganisms which
are to be released into the environment. In McLean GD, Waterhouse PM, Evans G, Gibbs MJ (eds).
Commercialisation of transgenic crops: risk, benefit and trade considerations. Proceedings of a
workshop, Canberra, 11-13 March 1997. Canberra: Cooperative Research Centre for Plant Science
and Bureau of Resource Sciences, 1997: 173-8.
Pukall R, Tschape H, Smalla K. Monitoring the spread of broad host and narrow host range
plasmids in soil microcosms. FEMS Microbiol Ecol 1996; 20: 53-66.
Recorbet G, Givaudan A, Steinberg C, Bally R, et al. Tn5 to assess soil fate of genetically marked
bacteria: screening for aminoglycoside-resistance advantage and labelling specificity. FEMS Microbiol
Ecol 1992; 86: 187-94.
Redenbaugh K, Berner T, Emlay D, Frankos B, et al. Regulatory issues for commercialization of
tomatoes with an antisense polygalacturonase gene. In Vitro Cell Dev Biol 1993; 29P: 17-26.
Redenbaugh K, Hiatt W, Martineau B, Lindeman J, et al. Aminoglycoside 3'-phosphotransferase II
(APH(3')II): review of its safety and use in the production of genetically engineered plants. Food
Biotechnol 1994; 8: 137-65.
Reynolds JE (ed). Martindale. The Extra Pharmacopoeia. 31st ed. London: The Royal Pharmaceutical
Society, 1996.
Rice EW, Messer JW, Johnson CH, Reasoner DJ. Occurrence of high-level aminoglycoside
resistance in environmental isolates of enterococci. Appl Environ Microbiol 1995; 61(1): 374-6.
Ross P, O’Gara F, Condon S. Thymidylate synthase gene from Lactococcus lactis as a genetic marker:
an alternative to antibiotic resistance genes. Appl Environ Microbiol 1990; 56: 2164-9.
Salyers A. Gene transfer in the mammalian intestinal tract. Curr Opin Biotechnol 1993; 4: 294-8.
Salyers A. Genetically engineered foods: safety issues associated with antibiotic resistance genes.
Urbana: Department of Microbiology, University of Illinois, 1999.
Use of Antibiotic Resistance Marker Genes in GMOs
92
References
Sanchis V, Agaisse H, Chaufaux J, Lereclus D. A recombinase-mediated system for elimination of
antibiotic resistance gene markers from genetically engineered Bacillus thuringiensis strains. Appl
Environ Microbiol 1997; 63(2): 779-84.
Schluter K, Futterer J, Potrykus I. “Horizontal” gene transfer from a transgenic potato line to a
bacterial pathogen (Erwinia chrysanthemi) occurs – if at all – at an extremely low frequency.
Bio/technology 1995; 13(10): 1094-8.
Schmulling T, Rohrig H. Gene silencing in transgenic tobacco hybrids: frequency of the event and
visualization of somatic inactivation pattern. Mol Gen Genet 1995; 249(4): 375-90.
Schrag SJ, Perrot V. Reducing antibiotic resistance. Nature 1996; 381: 120-1.
Schubbert R, Lettmann C, Doerfler W. Ingested foreign (phage M13) DNA survives transiently in
the gastrointestinal tract and enters the bloodstream of mice. Mol Gen Genet 1994; 242(5): 495-504.
Schubbert R, Renz B, Schmitz B, Doerfler W. Foreign (M13) DNA ingested by mice reaches
peripheral leukocytes, spleen, and liver via the intestinal wall mucosa and can be covalently linked to
mouse DNA. Proc Natl Acad Sci USA 1997; 94(3): 961-6.
Schubbert R, Hohlweg U, Renz D, Doerfler W. On the fate of orally ingested foreign DNA in mice:
chromosomal association and placental transmission to the fetus. Mol Gen Genet 1998; 259(6): 56976.
Scott A. EU Committee refuses first crop. Chem Week 1998; 160(43): 21.
Sheen J, Hwang S, Niwa Y, Kobayashi H, et al. Green-fluorescent protein as a new vital marker in
plant cells. Plant J 1995; 8: 777-84.
Shoemaker NB, Wang G-R, Salyers A. Evidence for natural transfer of a tetracycline resistance gene
between bacteria from the human colon and bacteria from the bovine rumen. Appl Environ
Microbiol 1992; 58(4): 1313-20.
Sikorski J, Graupner S, Lorenz MG, Wackernagel W. Natural genetic transformation of Pseudomonas
stutzeri in a non-sterile soil. Microbiol 1998; 144: 569-76.
Smalla K, van Overbeek LS, Pukall R, van Elsas JD. Prevalence of nptII and Tn5 in kanamycinresistant bacteria from different environments. FEMS Microbiol Ecol 1993; 13: 47-58.
Smalla K, Gebhard F, van Elsas JD, Matzk A, et al. Bacterial communities influenced by transgenic
plants. In Jones DD (ed). Proceedings of the 3rd International Symposium on the biosafety results of
field tests of genetically modified plants and microorganisms. Oakland: University of California,
1994: 157-67.
Smith N. Seeds of opportunity: an assessment of the benefits, safety, and oversight of plant
genomics and agricultural biotechnology. Report of the Subcommittee on Basic Research to the
Committee on Science, US House of Representatives for the 106th Congress 2nd session. Washington,
DC: 13 April 2000.
Use of Antibiotic Resistance Marker Genes in GMOs
93
References
Steinecke P, Herget T, Schreier PH. Expression of a chimeric ribozyme gene results in
endonucleolytic cleavage of target mRNA and a concomitant reduction of gene expression in vivo.
EMBO J 1992; 11(4): 1525-30.
The Royal Society. Genetically modified plants for food use. UK: The Royal Society, 1998.
Thuriaux P. Gene flow. In Kahn A (ed). Transgenic plants in agriculture. Ten years experience of the
French Biomolecular Engineering Commission. Paris: John Libbey Eurotext, 1999: 89-97.
Trieu-Cuot P, Arthur M, Courvalin P. Origin, evolution and dissemination of antibiotic resistance
genes. Microbiol Sci 1987; 4: 263-6.
US Environmental Protection Agency. Neomycin phosphotransferase II: tolerance exemption.
Federal Register 28 September 1994; 59: 49351.
US Food and Drug Administration. Statement of Policy: Foods derived from new plant varieties.
Federal Register 29 May 1992; 57: 22984-23005.
US Food and Drug Administration. Secondary direct food additives permitted in food for human
consumption; food additives permitted in feed and drinking water of animals; aminoglycoside 3'phosphotransferase II. Federal Register 23 May 1994; 59: 26700-11.
US Food and Drug Administration. Guidance for industry: use of antibiotic resistance marker genes
in transgenic plants. Rockville: US FDA, September 1998. Draft report.
US Registry of Toxic Effects of Chemical Substances. Puromycin. TOMES CPS (TM) System.
Micromedex, Inc. 2000; 45.
Vara JA, Portela A, Ortin J, Jimenez A. Expression in mammalian cells of a gene from Streptomyces
alboniger conferring puromycin resistance. Nucleic Acids Res 1986; 14(11): 4617-24.
Wang MB, Upadhyaya NM, Brettell RIS, Waterhouse PM. Intron-mediated improvement of a
selectable marker gene for plant transformation using Agrobacterium tumefaciens. J Genet Breed 1997;
51(4): 325-34.
Webb V, Davies J. Antibiotic preparations contain DNA: a source of drug resistance genes?
Antimicrob Agents Chemother 1993; 37: 2379-84.
Widmer F, Seidler RJ, Watrud LS. Sensitive detection of transgenic plant marker gene persistence in
soil microcosms. Mol Ecol 1996; 5: 603-13.
Widmer F, Seidler RJ, Donegan KK, Reed GL. Quantification of transgenic marker gene persistence
in the field. Mol Ecol 1997; 6: 1-7.
World Health Organisation. Strategies for assessing the safety of foods produced by biotechnology.
Report of a joint FAO/WHO consultation. Geneva: World Health Organisation, 1991.
Use of Antibiotic Resistance Marker Genes in GMOs
94
References
World Health Organisation. Health aspects of marker genes in genetically modified plants: report of
a WHO workshop. Geneva: World Health Organisation, 1993.
World Health Organisation. Safety aspects of genetically modified foods of plant origin. Report of a
joint FAO/WHO expert consultation on foods derived from biotechnology. Geneva: World Health
Organisation, 2000.
Zubko E, Scutt C, Meyer P. Intrachromosomal recombination between attP regions as a tool to
remove selectable marker genes from tobacco transgenes. Nat Biotechnol 2000; 18: 442-5.
Use of Antibiotic Resistance Marker Genes in GMOs
95
Glossary
10.
Glossary
Auxotrophic markers
Bacteriophage
Cytokinin
DNA
Eukaryote
Gene
Horizontal gene transfer
Intron
Mismatch-repair system
Phytosphere
Pleiotrophic effects
Polymerase chain reaction
Probiotics
Prokaryote
Promoter
Replicon
Transposon
Zoonosis
Allow cells to synthesise an essential component, usually an amino
acid, a vitamin or other minor nutrient in media that lack that
component.
A virus that infects bacteria.
Plant growth hormone.
Deoxyribonucleic acid, which is present in all living cells and contains
the information for cell structure, organisation and function.
An organism that contains a nucleus like plants and animals.
An ordered sequence of nucleotide bases comprising a segment of
DNA.
Non-sexual or parasexual transfer of genetic material between
genomes of organisms of the same or different species.
Regions within genes that do not code for protein sequences.
Comprises enzymes that recognise and process mispaired and nonpaired bases.
Plant environment ie leaves and roots.
Multiple effects resulting from a single genetic change.
Artificial amplification of a DNA sequence by repeated cycles of
replication and strand separation.
Products that are consumed intentionally to alter the number and type
of gut bacteria.
An organism without a nucleus ie bacteria.
DNA sequence that receives specialised proteins that bind and switch
on a gene.
Length of DNA that behaves as an autonomous unit during DNA
replication.
Mobile segment of DNA that has the capacity to move from one site
in the genome to another. Transposons vary in size and often contain
antibiotic resistance genes as well as genes encoding for functions
concerned with their mobility.
Disease of animals that may be transmitted to humans.
Use of Antibiotic Resistance Marker Genes in GMOs
96
Appendices
11.
Appendices
Appendix I: Evaluation of the kanamycin resistance gene
451
Detailed evaluations have been published on the kanamycin resistance gene (nptII) and its
product neomycin phosphotransferase (NPTII) that indicate that NPTII produced in GM
plants presents no discernible food or feed safety or environmental concerns (Nap et al,
1992; Flavell et al, 1992; Fuchs et al, 1993; Redenbaugh et al, 1994; US FDA, 1994).
452
A review on the biosafety of kanamycin resistant GM plants concluded that use of the
kanamycin resistance gene is an excellent choice because of the high substrate specificity of
the enzyme encoded. Physico-chemical properties of the antibiotic exclude the existence of
selective conditions in the environment and hence the expression of nptII in GMOs will not
give the organism any selective advantage outside the laboratory because of this gene
compared to the non-GM parent organism (Nap et al, 1992).
453
Nap et al (1992) also concluded that even if methods are available and become generally
applicable, removal of the nptII gene from the final GMO should not be required.
454
NPTII does not contain any properties that would distinguish it toxicologically from any
other phosphorylating enzymes that historically have been part of the food supply without
adverse consequences.
455
The kanamycin resistance gene has been used in clinical studies reported to the National
Institutes of Health involving human gene therapy. They represent a human in vivo safety
evaluation of the marker gene. No adverse effects of the gene or gene product were observed
(Redenbaugh et al, 1994). However this research provides little information concerning the
safety of the gene and its product in food.
Impact of the gene product NPTII
456
The fate of the protein during digestion was assessed using a simulated in vitro mammalian
digestion model. No protein was detected at 10 seconds in gastric fluid and 50 percent
degradation had occurred after two to five minutes in intestinal fluid. Enzymatic activity was
lost by two minutes in gastric fluid and 15 minutes in intestinal fluid. If analysis had been
carried out at earlier time points loss of functional activity would have been detected sooner
(Fuchs et al, 1993). These data confirmed findings submitted to the FDA by Calgene Inc
(developer of the Flavr Savr tomato) and suggested consumption should not pose any
significant allergenic concerns.
457
Solid food empties from the stomach by about 50 percent in two hours while liquid empties
by 50 percent in about 25 minutes. Intestinal transit times of NPTII were measured using
isotopically labelled chromate which is not absorbed. It was first detected in the faeces at
four to 10 hours and last at 68 to 165 hours. Hence there is minimal, if any, potential for
NPTII to reach the intestinal mucosa to trigger an IgE-mediated response. The protein also
has no homology to any reported allergen (Fuchs et al, 1993).
Use of Antibiotic Resistance Marker Genes in GMOs
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Appendices
458
Since proteins that are toxic produce toxic effects following acute exposure an acute
toxicological study was carried out. An acute mouse gavage study confirmed that the protein
caused no adverse effects when administered by gavage at a cumulative target dosage of up
to 5,000 mg/kg of body weight. This dosage correlates to at least a million-fold safety factor
relative to the average daily consumption of potato or tomato assuming that all the potatoes
or tomatoes consumed contained the protein. In other words a 5,000 mg/kg body weight
dose is equivalent to an average human consuming in one day more than one million
tomatoes or potatoes expressing the level of NPTII previously reported for these GM crops.
Whole food feeding studies also showed no adverse effects. Rats were fed potatoes or
tomatoes at a level equivalent to an average human consuming 40 raw potatoes or 100
tomatoes per day for 28 days with no adverse effects (Fuchs et al, 1993).
459
Human exposure to the protein can also be assumed to occur as a result of the background
of kanamycin resistant micro-organisms in the environment and the human gut.
460
Humans continually ingest kanamycin resistant micro-organisms. The diet, particularly raw
vegetables is the major source. It has been conservatively estimated that a human ingests 1.2
x 106 kanamycin resistant micro-organisms daily (Flavell et al, 1992).
461
With respect to the possibility of increased intestinal absorption of proteins in neonates and
people with certain conditions (eg ulcers) the FDA concluded there is no reason to expect
that absorption of intact or partially digested NPTII protein would present a safety concern
different from absorption of any other protein in the diet.
462
Phosphorylation of kanamycin or neomycin by NPTII requires the cofactor ATP. ATP is
unstable in the low pH of the digestive system and endogenous concentrations in the
stomach are below that required for catalytic activity. It is susceptible to inactivation by heat
and by enzymes (eg intestinal alkaline phosphatases). The primary source of ATP in the
gastro-intestinal tract is uncooked fruits and vegetables.
463
Dietary exposure of NPTII was estimated by Calgene Inc as very low - 480 µg per person per
day or 0.16 parts per million in the diet based on a 100 percent market share for tomatoes
containing NPTII. The exposure estimate was based on several conservative assumptions.
464
High temperature treatment denatures proteins and inactivates enzymes and therefore
processed products that contain tomatoes with the kanamycin resistance gene are unlikely to
contain any enzymatically active NPTII. Purified oils essentially do not contain protein.
Intact DNA including the kanamycin resistance gene are not expected to survive production
of oils and animal feeds from cottonseed and rapeseed because of release of degradative
enzymes normally present within the cell from mechanical grinding and enzyme inactivation
from high temperatures and solvent extraction (US FDA, 1994).
Impact of the DNA in food
465
Most of the DNA remaining after digestion would be smaller than the kanamycin resistance
gene which is about 1,000 base pairs long.
Use of Antibiotic Resistance Marker Genes in GMOs
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Appendices
466
Calgene Inc calculated that for consumption of fresh GM tomatoes containing nptII at the
90th percentile the transformation frequency of intestinal micro-organisms will be about 3 x
10-15 transformants per day.4 This is more than five orders of magnitude less than the
frequency of mutation to kanamycin resistance per bacterial replication (ie 10-9). For every
300,000 bacteria that mutate to kanamycin resistance per replication (generally a matter of
hours) there would be at most under worst case conditions one kanamycin resistant
bacterium per day added to that number due to gene transfer. The number of kanamycin
resistant micro-organisms that would result from potential gene transfer in the human gastrointestinal tract is therefore negligible with respect to the number of intrinsically resistant
micro-organisms already present (Nap et al, 1992).
Impact on the efficacy of antibiotics
467
To be of clinical significance the nptII gene would have to be transferred to and expressed in
a pathogenic micro-organism that is treated with kanamycin or neomycin.
468
Calgene Inc reported that studies that simulated abnormal gastric conditions (eg patients
treated with antacids) showed that NPTII is not degraded in neutralised (pH 7) simulated
gastric fluid and therefore may remain active. In this situation the concentration of ATP
which the enzyme requires to inactivate kanamycin and neomycin would be limiting.
469
Using a worst case scenario Calgene Inc concluded that compromised oral antibiotic efficacy
due to ingestion of GM food containing NPTII would be extremely unlikely. The worst case
scenario was based on a number of assumptions several of which are extremely unlikely to
occur. Assumptions made were:
• 95th percentile consumption at one sitting of fruits or vegetables with high ATP
content;
• stoichiometric reaction of 100 percent of the ATP in ingested food with orally
administered neomycin (highly unlikely);
• administration of neomycin simultaneously with consumption of GM food
containing NPTII and with other fruits or vegetables rich in ATP;
• presence of intact functional NPTII which requires a buffered stomach environment
(pH 7); and
• stability of ATP in the stomach.
470
Oral kanamycin or neomycin is administered only for hepatic encephalopathy and
preoperative bowel preparation. During preoperative preparation for bowel surgery it is
highly unlikely that patients would be consuming any solid foods. For patients with hepatic
encephalopathy who consume fresh fruit and vegetables Calgene Inc calculated that 1.5
percent of a 1 g dose of antibiotic would be inactivated by consuming NPTII as a
component of fresh tomatoes (Redenbaugh et al, 1994). This conclusion was supported by
data from an in vitro study that showed no significant inactivation of kanamycin when tomato
extract containing NPTII and kanamycin was incubated over a four hour period.
A model showed that 3 mg of tomato genomic DNA (from consumption of 250 g tomatoes) in 1500 ml (volume of fluid in the small intestine) would
generate 0.0024 transformants, an increase of 2.4 x 10-13 percent of the transformable intestinal microflora or 2.4 x 10-15 percent of the total intestinal
microflora (Nap et al, 1992).
4
Use of Antibiotic Resistance Marker Genes in GMOs
99
Appendices
471
None of the bacterial species known to be present in the gastro-intestinal tract has been
found capable of acquiring foreign DNA by natural transformation. Calgene Inc noted that
although they developed their transformation model for certain Streptococcus species they were
not aware of any information indicating that Streptococcus found in the gastro-intestinal tract
can be naturally transformed.
472
E. coli contains DNA segments homologous with part of the kanamycin resistance construct
because the construct contains part of an E. coli gene. Although E. coli constitutes one of the
predominant species of aerobic gastro-intestinal tract bacteria it is not transformation
competent under normal gastro-intestinal conditions.
473
The FDA concluded that the therapeutic efficacy of neomycin in animal feed would not be
affected since Calgene Inc found no significant inactivation of neomycin in feed
manufactured using GM cottonseed or rapeseed during an eight week storage period.
474
In the event that DNA was not completely degraded by processing during feed production
any remaining DNA would be degraded by digestion. Studies have shown that nucleic acids
entering the rumen are rapidly degraded (McAllan, 1980; 1982). Also many rumen bacterial
strains have nuclease activity which degrades DNA and provides another barrier to
transformation (Flint and Thomson, 1990).
475
The probability of generating a kanamycin resistant organism in cattle as a result of gene
transfer from GM feed was concluded as being equally negligible (Nap et al, 1992).
Impact on the environment
476
It is unlikely that as a result of the increase in resistance genes that the gene product could
reach environmental concentrations that may be toxic to plants, soil micro-organisms or
wildlife because of a lack of persistence and low toxicity of most proteins in the environment
and narrow substrate specificity of most enzymes including NPTII. Generally natural organic
materials are rapidly degraded in the soil (Redenbaugh et al, 1994).
477
Kanamycin resistant micro-organisms represent a small proportion of the total soil
microflora but are present at detectable background levels. The probability of a bacterium
obtaining the nptII gene from plant DNA compared to from another bacterium is extremely
low.
478
Calgene Inc calculated that at worst kanamycin resistant transformants resulting from plant
DNA left in the field would represent no more than one micro-organism in 10 million
existing kanamycin resistant soil micro-organisms. This is the estimated number of
kanamycin resistant soil micro-organisms present in one hectare (Redenbaugh et al, 1994).
479
It was concluded that even if horizontal gene transfer occurred release of NPTII into the
environment would not be of concern since calculations using the most favourable
assumptions and probabilities do not show significant changes in the potential numbers of
micro-organisms containing the resistance gene or potential impact on the level of NPTII in
the environment (Redenbaugh et al, 1994).
Use of Antibiotic Resistance Marker Genes in GMOs
100
Appendices
Appendix II: Genetically modified insect resistant maize
480
Considerable controversy has been associated with the inclusion of the ampicillin resistance
gene (bla) that encodes resistance to ampicillin in insect resistant maize developed by CibaGeigy (now Novartis) and Dekalb for animal feeds.
481
This controversy centres on the scenario that as a result of feeding unprocessed GM maize
containing the bla gene to animals, the gene may be transferred to bacteria in the animals’
guts and ampicillin resistant bacteria may subsequently be transmitted to humans via the food
chain.
482
Genetically modified maize varieties have been produced by transformation with directly
introduced linearised plasmid DNA containing the desired gene. An antibiotic resistance
gene is usually present as a result of the use of antibiotic resistance to select recombinant
bacteria in the laboratory.
483
The Novartis and Dekalb maize contains an intact bla gene with a promoter and an origin of
replication (ori) derived from the pUC18 cloning vector. The pUC ori generates over 600
copies per cell (compared to the four to 18 copies per cell of the ColE1 ori vector found in
nature).
484
Little information is available on the state of the pUC18 DNA that is integrated in the
Novartis or Dekalb maize genome. It is not certain that the bla gene and the region of the
plasmid necessary for plasmid replication are intact. There is no selection for maintenance of
an intact bla gene or pUC18 ori in a plant cell because pUC18 does not replicate in an
eukaryotic host and the bla gene does not have an eukaryotic promoter. Maize is naturally
resistant to penicillins so there is no selective advantage even if the gene were to be
expressed. As a result of lack of selection parts of pUC18 could have undergone deletions or
other modifications during or after integration (Salyers, 1999).
485
The smallest contiguous DNA fragment from pUC18 that could contain the bla gene is about
900 base pairs. The fragment size necessary to contain both the gene and the origin of
replication is about 1600 base pairs. It has been found experimentally that during DNA
isolation within one hour of tissue disruption all DNA is degraded to fragments of less than
500 base pairs, the majority to less than 150 base pairs. It is therefore unlikely that DNA
fragments of sufficient size would survive plant nuclease activity (Malik and Saroha, 1999).
486
Although DNA can be partially protected from digestion when associated with particulate
matter (eg soil, plant) it would then also be unavailable for binding to proteins on the
bacterial cell surface, a prerequisite for DNA uptake into the bacterial cell. Multiple copies of
the intact DNA fragment would need to aggregate at the same binding site on the bacterial
cell surface for uptake to occur. This is considered highly improbable. There is also a very
large quantity of other DNA fragments competing for the binding sites that further reduces
the opportunity for successful bacterial transformation (Malik and Saroha, 1999).
487
If pUC18 and the bla gene are intact and unaltered, and one or more tandem duplications of
the plasmid occurs less than one in 10,000 cells would contain the circular form of the
plasmid as a result of homologous recombination (Salyers, 1999).
Use of Antibiotic Resistance Marker Genes in GMOs
101
Appendices
488
It is highly unlikely but not impossible that the bla gene could be incorporated as a single
copy via illegitimate recombination into the bacterial chromosome of E. coli or its relatives.
The frequency of illegitimate recombination is less than that of homologous recombination
(Salyers, 1999).
489
The bla gene is expressed in E. coli or its close relatives such as Salmonella species but not in
ruminal or intestinal anaerobes. A strong selective pressure would be necessary to maintain
the gene in a gut bacterium in the competitive environment of the rumen or intestine
(Salyers, 1999).
490
In 1996 the ACNFP recommended approval be declined for unprocessed insect resistant
maize for animal feeds. Although transfer of the bla gene to ruminal or intestinal bacteria
would be a very rare event there was concern that long term high level consumption by
animals, in particular ruminants, might allow such an event to occur and as a result of the
presence of a promoter and the pUC ori on the gene such transfer would have serious
consequences for therapy with beta-lactam antibiotics (US FDA, 1998).
491
Initially the European Union (EU) did not approve the GM maize. Subsequently the
European Commission Scientific Committee for Food concluded that the risk of bacterial
transformation from GM maize is extremely low. If transfer did occur it would have no
detectable additional effect as the bla gene is already widespread in nature including the
human and animal gastro-intestinal tracts. The European Commission Scientific Committee
for Animal Nutrition also concluded that the probability of transfer of a functional bla gene
into bacteria is virtually zero, and if it did occur it would be clinically insignificant.
492
A group of scientists at a conference in September 1996 sponsored by Tufts University and
the Foundation for Nutritional Advancement also concluded that use of the bla gene
constitutes an insignificant to near zero risk of causing ampicillin resistance in animals or
humans. This is because the probability of DNA survival in fragments large enough to be
taken up by bacteria is very low, the probability of bacteria taking up or incorporating DNA
into the bacterial genome is virtually zero, if it was incorporated there is a low probability
that it would be expressed, and the clinical significance is virtually zero because ampicillin
resistance is widespread and can be overcome by antibiotics other than ampicillin (US FDA,
1998).
493
The majority scientific opinion was that the presence of the bla gene in the maize genome
posed no significant antibiotic resistance risk even if large amounts were ingested regularly by
animals over prolonged periods. As a result in December 1996 the EU authorised the sale
and cultivation of insect resistant maize.
494
This decision has been challenged by some countries on grounds including health (eg France,
Austria, Luxembourg, Italy). French regulatory authorities claimed that the proportion of
ampicillin resistant gut bacteria was much lower than that stated by proponents of GM maize
(Salyers, 1999). More recently Germany halted the approval process for commercial
cultivation citing the need for further research with respect to the transfer of antibiotic
resistance (Abbott, 2000).
Use of Antibiotic Resistance Marker Genes in GMOs
102
Appendices
495
The difference of opinion arises mainly over the question of whether extremely low but nonzero risks of increased antibiotic resistance are acceptable.
496
Preliminary results from a study by Heritage and coworkers of gene transfer from GM maize
to chicken gut bacteria have found no evidence of transfer of the bla gene to normal flora.
However this study has yet to be completed and published in the peer reviewed scientific
literature (Coghlan, 2000).
Use of Antibiotic Resistance Marker Genes in GMOs
103
Appendices
Appendix III: Summary of submissions
The following submissions were received as a result of the draft report’s availability for public
comment and were considered in the preparation of the final version.
Submission
Wendy Johnson, Friends of the Earth (NZ)
Jim Waters, Ministry of Health
Comments
Issues were raised that are outside the Terms of Reference
of this report:
• Extension of the report to include all possible
scenarios of ingestion of genetically engineered
DNA
• Intestinal permeability - its association with
certain diseases, causes, and social and public
health costs
• Approach of the ERMA to applications involving
GM foods
•
The unique narrow specificity of certain
aminoglycosides means that in spite of their side
effects they continue to be important antibiotics
in an era of complex medical care with greater
risk of complications from infection due to
resistant micro-organisms.
•
Development of resistance to antibiotics needed
for the control of meningococcal disease and to
the aminoglycosides needs to be prevented in
New Zealand.
•
The Ministry supports reducing unnecessary
antibiotic use in the interest of reducing selection
pressure around antibiotic resistance. A side
benefit of this is a reduction in selection of
antibiotic resistance marker genes but the main
focus is other sources of resistance.
•
Issues were raised that relate to the Authority’s
decision-making process and are outside the
Terms of Reference of this report.
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