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ABSTRACT
LEMING, CHRISTOPHER LLOYD. Deducing the Biological Relevance and Transfer of
Antimicrobial Resistance Factors in Salmonella enterica serovar Typhimurium. (Under the
direction of Craig Altier)
Antimicrobial resistance in Salmonella can be encoded on plasmids or as
stable chromosomal elements. To study the nature of resistance transfer by Salmonella
enterica serovar Typhimurium strains of animal origin, we examined isolates obtained from
pigs in commercial swine operations. We found that one common phage type, DT193, was
most often resistant to five antimicrobials (ampicillin, kanamycin, streptomycin,
sulfonamides, and tetracycline). Isolates of this phage type typically encoded all of their
resistance genes on conjugative plasmids that could be efficiently transferred to E. coli. One
of the DT193 isolates (strain UT20), however, carried its resistance factors integrated into its
chromosome. Despite the chromosomal location of these resistance factors, they could still
be readily transferred by conjugation and resided in transconjugants on an autonomously
replicating plasmid. We also found a spontaneous mutant of UT20 that remained resistant,
but could not transfer resistance factors by conjugation, and found mutants having different
resistance patterns, each having lost regions of the chromosomally encoded resistance
element. Furthermore, the method of plasmid transfer was complex: approximately one-half
of E. coli transconjugants encoded all five resistances on a ~140 kb plasmid. Approximately
half encoded resistance to kanamycin, tetracycline, streptomycin, and sulfonamides on a
plasmid of ~126 kb, while less than 2% had a ~109 kb plasmid encoding resistance to
ampicillin alone. Restriction mapping of these three plasmids showed them to be closely
related, and all encoded their resistances with identical alleles. We propose that
recombinational events in the Salmonella host strain produce these three distinct derivative
plasmids.
Although the use of antimicrobials in food-producing animals has induced resistance
in pathogenic bacteria, it remains unclear whether the cessation of antimicrobial use would
eliminate these resistant strains. An important aspect to this question is the growth rate of
resistant strains in comparison to similar susceptible strains. To test the fitness for growth of
resistant Salmonella, we compared the growth rate in mixed cultures of multi-resistant
Salmonella isolates of the DT193 phage type to isogenic fully susceptible derivatives.
Competing strains were grown by serial transfer in antimicrobial-free media for 22 days, with
each strain in the population quantified every three days. Wild type strains varied greatly in
their relative fitness. UT20 displays a great fitness cost when it is in competition against its
isogenic, fully susceptible derivative. UT8, whose resistance factors are identical to those of
UT20 but are carried on a plasmid, displays an initial fitness cost that is much less severe and
appears to be surmountable. This difference in fitness is not due to differences in the genetic
backgrounds of the two strains since cured UT20 carrying the plasmid of UT8 also showed
only a mild fitness defect. Conjugation within competitions was measured and found not to
be an influencing factor on the persistence of resistance factors in a population.
It is our conclusion that the degree to which the fitness burden manifests itself is
dependent on the location of the resistant element. Naturally occurring multiple drug
resistant (MDR) strains of Salmonella enterica are at a large metabolic disadvantage
compared to susceptible isogenic strains if the resistance element is integrated into the
chromosome. It appears that if the resistance element is in plasmid form, then the fitness
burden is significantly less, as well as being surmountable through means as yet
undetermined.
BIOGRAPHICAL SKETCH
Christopher Lloyd Leming
Date of Birth:
January 12, 1979
Place of Birth:
Monterey, California, USA
Parents:
Michael Lloyd Leming, PhD, and Martha Reel Leming
Education:
2003
2001
Master of Science
Microbiology
College of Agriculture and Life Sciences
North Carolina State University
Bachelor of Science, cum laude
Microbiology
College of Agriculture and Life Sciences
North Carolina State University
Post Bachelor’s Course Work:
North Carolina State University
GN 701
Eukaryotic Molecular Genetics
MB 790x
Bacterial Pathogenesis
MB 751
Immunology
MB 758
Prokaryotic Molecular Genetics
BIT 815J
Introduction to Microarrays
BCH 703
Macromolecular Synthesis and Regulation
Employment:
2001 – present
Graduate Research Assistant
Department of Microbiology
North Carolina State University
2000 – 2001
Undergraduate Research Assistant
Department of Genetics
North Carolina State University
1999 – 2001
Head Swimming Coach
NCSU Faculty Club Swim Team
NCSU Faculty Club, Raleigh, NC
1999 – 2001
Assistant Swimming Coach
ii
New Wave Swim Team, Raleigh, NC
Teaching:
2003
MB 352
General Microbiology Laboratory
Peer-Reviewed Publications:
Suyemoto, MM, CL Leming, C Altier. 2003. Transfer of antimicrobial resistance by
Salmonella enterica serovar Typhimurium var. Copenhagen phage type DT193 of animal
origin. Antimicrob Agents Chemother submitted for publication
Leming, CL, MM Suyemoto, C Altier. 2003. Deducing the Biological Relevance of Multiple
Drug Resistance in Salmonella enterica serovar Typhimurium var. Copenhagen phage type
DT193 of animal origin. To be submitted for publication.
iii
TABLE OF CONTENTS
Page
LIST OF TABLES ................................................................................................................ vi
LIST OF FIGURES .............................................................................................................. vii
1. LITERATURE REVIEW ................................................................................................... 1
1.1 General Review ..................................................................................................... 1
1.2 Antimicrobial Use and Multiple Drug Resistant Salmonellae .............................. 1
1.3 Resistance Plasmids and Conjugal Transfer ......................................................... 4
1.4 Fitness Costs of Resistance Factors in Salmonella and E. coli .............................. 6
1.5 References ............................................................................................................. 9
2. TRANSFER OF ANTIMICROBIAL RESISTANCE BY SALMONELLA
ENTERICA SEROVAR TYPHIMURIUM VAR. COPENHAGEN
PHAGE TYPE DT193 OF ANIMAL ORIGIN ........................................................ 14
2.1 Abstract ............................................................................................................... 14
2.2 Introduction ......................................................................................................... 15
2.3 Materials and Methods ........................................................................................ 17
2.3.1 Strains and Growth Conditions ............................................................ 17
2.3.2 Conjugation of Resistance Plasmids .................................................... 17
2.3.3 Curing of Resistance Plasmids ............................................................. 18
2.3.4 DNA Preparation ................................................................................. 18
2.3.5 Electrophoresis and DNA Hybridization ............................................. 19
2.4 Results ................................................................................................................. 20
2.5 Discussion ........................................................................................................... 25
2.6 Acknowledgements ............................................................................................. 28
2.7 References ........................................................................................................... 28
3. DEDUCING THE BIOLOGICAL RELEVANCE OF MULTIPLE DRUG
RESISTANCE IN SALMONELLA ENTERICA SEROVAR TYPHIMURIUM
VAR. COPENHAGEN PHAGE TYPE DT193 OF ANIMAL ORIGIN ................. 35
3.1 Abstract ............................................................................................................... 35
3.2 Introduction ......................................................................................................... 36
3.3 Materials and Methods ........................................................................................ 39
3.3.1 Manipulation of Strains ........................................................................ 39
3.3.2 Fitness Assays ...................................................................................... 40
3.3.3 Statistical Analysis ............................................................................... 41
3.4 Results ................................................................................................................. 41
iv
TABLE OF CONTENTS cont.
3.4.1 Fitness of Naturally Occurring Strains ................................................ 41
3.4.2 Fitness of Exconjugates ....................................................................... 43
3.4.3 Assessing the Dynamic Influence of Conjugation ............................... 44
3.5 Discussion ........................................................................................................... 46
3.6 References ........................................................................................................... 50
v
LIST OF TABLES
Page
TABLE 3.1 Strains and Plasmids Used ................................................................................ 53
vi
LIST OF FIGURES
Page
FIGURE 2.1 Restriction maps of plasmids derived from UT20 .......................................... 31
FIGURE 2.2 Location of blaTEM in UT20 ............................................................................. 32
FIGURE 2.3 Chromosomal location of aph-Iab in UT20 .................................................... 33
FIGURE 2.4 Plasmid location and composition of UT20 and related strains ...................... 34
FIGURE 3.1 The proportion of nalidixic acid resistant bacteria in the competition
between wild type UT20 (AKSSuT) and CA905 (cured UT20, NalR) ..................... 54
FIGURE 3.2 The proportion of nalidixic acid resistant bacteria in the competition
between wild type UT8 (AKSSuT) and CA902 (cured UT8, NalR) ......................... 55
FIGURE 3.3 The proportion of nalidixic acid resistant bacteria in the competition
between CA905 (cured UT20, NalR) and CA906 (cured UT20 + pUT8,
AKSSuT) .................................................................................................................. 56
FIGURE 3.4 The proportion of nalidixic acid resistant bacteria in the competition
between CA905 (cured UT20, NalR) and CA909 (cured UT20 + pUT20,
AKSSuT) .................................................................................................................. 57
FIGURE 3.5 The proportion of exconjugates in all competitions ........................................ 58
vii
Chapter 1. Literature Review
1.1 General Review
Salmonellae were discovered in 1886 by veterinarians at the Bureau of Animal
Industry (Salmon and Smith 1886). The genus Salmonella has species distributed
worldwide. Some species are known to infect a wide variety of animals, including humans
(Murray 1991, Schwartz 1991, Cooper 1994). Serovars of S. enterica subspecies enterica are
the most common foodborne pathogens among the Salmonellae with 1435 of more than 2400
serovars belonging to this subspecies. Serovar Typhimurium is ubiquitous and does not
show host preference. (Farmer et al. 1999)
The United States Center for Disease Control and Prevention estimates that
approximately 40,000 cases of salmonellosis are reported in the United States every year.
Because many milder cases are not diagnosed or reported, the actual number of infections
may be thirty or more times greater
(http://www.cdc.gov/ncidod/dbmd/diseaseinfo/salmonellosis_g.htm). Out of the reported
cases, 95% are reported to be foodborne and each year more than 500 deaths are attributed to
foodborne salmonellosis (Mead et al. 1999).
1.2 Antimicrobial Use and Multiple Drug Resistant Salmonellae
The year 1936 ushered in the era of effective antimicrobial therapy (Colebrook and
Kenny 1936). In 1954, two million pounds of antimicrobials were produced in the United
States. Over the succeeding forty years, that number increased twenty-five fold (Levy 1998).
The addition of antimicrobials in animal feed began in the late 1950’s (ACSH report 1983).
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Several reports showed that every year approximately twenty million pounds of
antimicrobials are used in the production of swine, poultry, and beef (Levy et al. 1998,
Benbrook et al. 2001, Carneval 2001). Currently, more than forty percent of antimicrobials
produced are used for agricultural purposes, predominantly in animal husbandry (Institute of
Medicine 1998).
The animal production industry has become dependent on the use of antimicrobials.
A report published in 1981 estimated an economic loss of $3.5 billion per year in the United
States if antimicrobials were banned from use in animal production (CAST 1981). A report
published in 1997 estimated the annual medical cost of treatment for infections caused by
antimicrobial resistant organisms to be $4.0 billion (Cassell 1997). Direct comparisons
cannot be made due to the fact that the studies were conducted in different time periods.
Agricultural antimicrobial use (AAU) can have strong impacts on natural populations
by introducing new antimicrobial resistant (AR) strains earlier than they would appear
otherwise. The large impacts occur because the prevalence of AR increases at a time when
AR should be virtually absent, therefore, AAU substantially increases the rate at which new
AR strains appear in humans. Public health benefits may accumulate from restricting AAU
before AR bacteria emerge. Restricting AAU in new resistance classes would likely
maximize the time when AR in humans is rare, suggesting that the best time to regulate AAU
is before AR appears. (Smith et al. 2002)
One of the earliest reports of multiple drug resistant Salmonella Typhimurium
infection that was traced to animal origin was reported in the 1960’s. The presence of a drug
need not eliminate all the cells of a sensitive organism before transfer of resistance to that
drug occurs. Conversion of sensitive organisms to drug resistance with resistance factors
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may take place in the presence of therapeutic levels of the drug concerned. (Anderson et al.
1965a)
The Netherlands banned the use of antimicrobials at subtherapeutic levels in 1974. A
nationwide decrease was found in the level of tetracycline resistant Salmonella in human
beings and swine. The level of shedding tetracycline resistant organisms did not change in
calves. (van Leeuwen et al. 1979) In Denmark, avilamycin has been used primarily for
broilers. Since 1996, the level of use has decreased, almost immediately followed by a
decrease in resistance from 77.4% in 1996 to 4.8% in 2000. (Aarestrup et al. 2001)
Other studies, however, have shown contradictory results. Resistance genes are still
commonly found in areas where restrictions on antimicrobial use have been imposed
(Franklin, 1999). These findings have been supported by studies conducted at the genome
level. Compensatory mutations will ameliorate costs of resistance, enabling resistant
organisms to retain their resistances even in the absence of antimicrobial drugs. Such
compensatory mutations are often achieved by second site mutations. (Björkman et al. 1998,
Schrag and Perrot 1996)
Consistently high frequencies of antimicrobial resistance, mainly for tetracycline and
β-lactams, have been reported for Salmonella serovars isolated from swine (Benson et al.
1985, Murray et al. 1986, Takahashi et al. 1990, Lee et al. 1993, Seyfarth et al. 1997,
Farrington et al. 1999).
Multiple drug resistant (MDR) strains of Salmonella have become a major source of
foodborne salmonellosis in industrialized nations. The first MDR Salmonella outbreak was
recorded in the United Kingdom in 1965. The strain infected calves and humans (Anderson
1968). The first incidence of MDR Salmonella infection in North America occurred in
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Canada in 1965; the second in Connecticut, 1976. In both cases, strains with similar
resistance patterns were found in the animals and the humans who handled them (Fish et al.
1967, Lyons et al. 1980).
Hospitals and farms with high rates of antibiotic use are evolutionary incubators
where high-level AR bacteria and multidrug-resistant bacteria thrive. In such environments,
strong selection also favors the evolution of genetic mechanisms that increase the mobility of
genes. Some medical impacts may occur as a result of heavy veterinary therapeutic use, not
just for animal growth promotion; antimicrobial use selects for AR bacteria regardless of
why it is used. Many front-line antimicrobials are heavily used in animals therapeutically.
This may hasten the appearance of AR and decrease the efficacy of the drugs in humans.
One solution may be to regulate AAU before AR becomes a problem in medicine, and then
allow prudent use of AAU once clinically significant use of resistance has already developed.
(Smith et al. 2002)
1.3 Resistance Plasmids and Conjugal Transfer
Antimicrobial resistance factors are known to commonly cross species and genus
barriers by conjugation (Levy 1989). Similar tetracycline resistance genes have been found
between Gram-negative and Gram-positive organisms (Roberts 1997, Courvalin 1994). The
presence of plasmids carrying genes conferring multiple antimicrobial resistance has been
recorded since the 1960’s and 1970’s (Anderson 1968, Watanabe 1963, Hocmanova and
Krcmery 1974).
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If high-level AR genes exist, and if they can be transmitted on mobile genetic
elements, then the medical risks associated with AAU are much greater. AAU may provide
strong linkage to genes conferring resistance to other antimicrobial drugs. (Smith et al. 2002)
Sex-factors and phage elements are considered to be episomes, a term coined by
Jacob and Wollman (Jacob and Wollman 1958). Episomes are often infectious, either by
promoting their own transfer or by the production of infectious particles. Their most
important feature is that they can replicate in either one of two alternative states, that is,
independently in the cytoplasm or, after insertion, as an integral part of the bacterial
chromosome. R-factors, like sex-factors, are transferred by cell to cell contact. However,
they do not appear to have a stable chromosomal state, so a new term was coined, this time
by Joshua Lederberg (Lederberg 1952). The term plasmid describes all extrachromosomal
genetic structures that can reproduce autonomously, without regard to whether they could or
could not integrate into the host chromosome. All plasmids in bacteria are units of
replication or replicons. They are generally less than one-twentieth the size of the bacterial
chromosome, they contain the information for self-replication, and they often do so much
faster than the host chromosome. Only functions that ensure replication and replica division
are absolutely essential for plasmid survival. Such functions may occupy only a small
proportion of the entire plasmid gene complement. The remaining nonessential plasmid
functions are those which may be assumed to confer transient evolutional advantage on the
plasmid. (reviewed by Falkow 1975)
The frequency of transfer of R-factors from natural isolates to suitable recipient
bacteria is generally quite low, approximately 10-4 per R+ cell in one hour of mixed
incubation. After a cell receives the R-factor, it can replicate the incoming DNA, synthesize
5
the R-pilus, and be able to transfer the R-factor within 15 minutes after receipt. The
achievement of the transfer of an R-factor requires that the incoming autonomous replicon be
immune to the nucleases of the recipient cell and that its functions be properly transcribed
and translated by the host bacterium. (Falkow rev 1975)
R-factors occasionally bring about the transfer of the chromosomal genes of their
bacterial hosts at a frequency of about 10-8 per donor cell. Pearce and Meynell determined
two distinct differences in the behavior of an R-mediated chromosomal transfer and the
chromosomal transfer by a bona fide Hfr strain (Pearce and Meynell 1968). First, the
number of recombinant cells with an R-factor was significantly less than that observed with
an Hfr strain. Second, the recombinant cells almost invariably also received the R-factor,
whereas in Hfr-mediated transfer, the recombinant cells ordinarily do not receive F because it
is integrated at the chromosome terminus. (Falkow 1975)
1.4 Fitness Costs of Resistance Factors in Salmonella and E. coli
The evolution and spread of resistances can be attributed to the use and overuse of
antimicrobials (Guillemot 1999). Chromosomal mutations responsible for acquired
resistance in pathogenic bacteria are likely to be the same as those mutations that have little
or no cost observed experimentally (Levy 1992). Cases where resistance mutations and
accessory elements engender a cost, subsequent evolution in the absence of antibiotics
commonly results in the amelioration of those costs rather than reversion to drug sensitivity
(Andersson and Levin 1999). There are two reasons why amelioration by compensatory
mutation is more common than true reversion. Firstly, true reversion requires the nucleotide
that had mutated in order to confer resistance to mutate back to the original nucleotide that
6
was present. Compensatory mutations do not require a specific single nucleotide substitution
to occur and are, therefore, much more common. Additionally, compensatory evolution
establishes an adaptive valley that is difficult to traverse and thus return to the ancestral
genotype (Levin et al. 1999)
Secondly, population bottlenecks are associated with serial passage. Within a
population there are three subpopulations; the originally resistant bacteria, those that had
undergone a compensatory mutation, and those that had undergone true reversion. Naturally,
the originally resistant would be the largest percentage of the population. The compensatory
mutants will be the next largest percent, due to the fact that compensatory mutations occur
more frequently than true revertants, which would be the smallest percentage of the
population. A fraction of the stationary phase population is transferred and the population
growth cycle is repeated. Upon each repetition, the resistant bacteria that possess the
compensatory mutations will become a larger percentage of the population because it grows
faster than the original resistant bacteria. And because they are more common, the bacteria
with compensatory mutations will dominate the population over the susceptible bacteria that
had undergone reversion, so that before long, all that remains in the population is resistant
bacteria which have compensatory mutations that restore fitness. (Levin et al. 1999)
The failure of high fitness revertants to evolve is a consequence of two processes, the
rate of compensatory mutation exceeding that of reversion and the bottlenecks associated
with serial passage. Because of their higher mutation rate, compensatory mutations occur
earlier in the growth cycle within a transfer than revertants and increase in relative frequency.
While the revertants also occur and increase in frequency, the population reaches stationary
phase and stops growing before the revertant population becomes very large. At each
7
transfer, the frequency of compensatory mutants increases and eventually these bacteria
dominate the community. (Levin et al. 1999)
Reversion requires specific base substitutions. If all else were equal, one would
expect the rate of compensatory mutation to exceed that of reversion if there were more ways
to generate compensatory mutants then revertants (Levin et al. 1999). Evidence is present
that compensatory mutations have been fixed in long-term streptomycin resistant laboratory
strains of E. coli and may account for the persistence of rpsL mediated streptomycin
resistance in populations maintained for more than 10000 generations in the absence of the
antimicrobial (Schrag et al. 1997).
The environment in which the bacteria grow will influence what type of
compensatory mutation occurs. Compensatory mutations for rpsL mutants in S.
Typhimurium are only intragenic to the mutated gene when in mice. When in laboratory
medium, compensation occurs by extragenic suppressor mutations. (Björkman et al. 1998,
Björkman et al. 1999)
However, for fusR mutants, compensation is observed in the opposite pattern.
Compensatory mutations occur preferentially by intragenic suppression for bacteria grown in
LB and mainly by true reversion for bacteria evolved in mice. Even though revertants are
rarer than second-site suppressors, they were predominantly selected in mice because they
had fully restored fitness. (Björkman et al. 2000)
In the absence of antimicrobial selection, compensatory evolution, rather than
reversion to sensitivity, is expected to occur as long as the rate of compensatory mutations
exceeds that of reversion to sensitivity, and only a fraction of the population is transferred to
colonize a new host (Levin et al. 1997, Levin et al. 1999, Schrag et al. 1997). Nevertheless,
8
population models have shown that as long as populations of pathogenic microbes are
polymorphic and include susceptible as well as resistant lineages, and as long as these
susceptible strains have some competitive advantage over resistant strains, reductions in drug
use will increase the frequency of the sensitive strains and the likelihood of these lineages
replacing resistant lineages (Levin et al. 1997).
However, if the use of antimicrobials in animal husbandry is continued, the outcome
may be drastically different. Once the transfer of a resistance plasmid is affected, the
presence of the drug offers a survival advantage to the newly resistant organisms. The
further transmission of the drug resistance to human pathogens may then follow. The time
has clearly come for a re-examination of the whole question of the use of antimicrobials in
the rearing of livestock. (Anderson et al. 1965a)
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13
Chapter 2. Transfer of antimicrobial resistance by Salmonella enterica serovar
Typhimurium var. Copenhagen phage type DT193 of animal origin
2.1 ABSTRACT
Antimicrobial resistance in Salmonella can be encoded on plasmids or as stable
chromosomal elements. To study the nature of resistance transfer by Salmonella enterica
serovar Typhimurium strains of animal origin, we examined isolates obtained from pigs in
commercial swine operations. We found that one common phage type, DT193, was most
often resistant to five antimicrobials (ampicillin, kanamycin, streptomycin, sulfonamides, and
tetracycline). Isolates of this phage type typically encoded all of their resistance genes on
conjugative plasmids that could be efficiently transferred to E. coli. One of the DT193
isolates (strain UT20), however, carried its resistance factors integrated into its chromosome.
Despite the chromosomal location of these resistance factors, they could still be readily
transferred by conjugation and resided in transconjugants on an autonomously replicating
plasmid. We also found a spontaneous mutant of UT20 that remained resistant, but could not
transfer resistance factors by conjugation, and found mutants having different resistance
patterns, each having lost regions of the chromosomally encoded resistance element.
Further, the method of plasmid transfer was complex: approximately one-half of E. coli
transconjugants encoded all five resistances on a ~140 kb plasmid. Approximately half
encoded resistance to kanamycin, tetracycline, streptomycin, and sulfonamides on a plasmid
of ~126 kb, while less than 2% had a ~109 kb plasmid encoding resistance to ampicillin
14
alone. Restriction mapping of these three plasmids showed them to be closely related, and
all encoded their resistances with identical alleles. We propose that recombinational events
in the Salmonella host strain produce these three distinct derivative plasmids.
2.2 INTRODUCTION
The use of antimicrobials both for the treatment of disease and for growth promotion
has long raised concerns of inducing resistance in bacteria derived from food-producing
animals. With increasing frequency, Salmonella isolates obtained from food-producing
animals are resistant to one or more antimicrobials. Recent studies have shown that at least
half, and in some cases over 90%, of Salmonella isolates obtained from commercially raised
swine in the U.S. are multi-resistant (7, 9). A common resistance is to tetracyclines, an
antibiotic class used routinely in swine feed to improve feed efficiency and still an important
antibiotic for the treatment of a number of human diseases. In one study, more that 85% of
Salmonella isolates from swine were resistant to tetracycline (9), in another, greater than
95% were resistant to chlortetracycline (7). Resistance to several other antimicrobials has
been commonly found. A national survey of swine slaughter samples in 2000 showed
resistance to sulfamethoxazole, streptomycin, ampicillin, chloramphenicol, and kanamycin to
be common (12).
It has long been known that some Salmonella strains can transfer their resistance
determinants on conjugative plasmids. In fact, much of the early work that established the
existence of resistance factors on plasmids, and the very nature of plasmids themselves, was
performed in Salmonella (reviewed in 6). It has been shown that a single Salmonella strain
can harbor several plasmids carrying different resistance determinants. Further, some
15
resistance plasmids, even if they are themselves not self-transmissible, can move by
conjugation with the help of conjugative plasmids that co-exist in the same strain (2, 3).
Thus, the variety of resistance transfer mechanisms is great.
The pattern of resistance observed in Salmonella is often related to the phage type of
the isolate. A phage type commonly found among swine isolates and presenting a concern to
public health is DT193. Unlike other Salmonella serovar Typhimurium phage types,
however, DT193 includes a genetically diverse group of strains (4, 10). This phage type has
been reported to have various resistance patterns including resistance to tetracycline alone;
ampicillin, tetracycline, streptomycin, and sulfonamides; or most recently ampicillin,
kanamycin, streptomycin, sulfonamide, and tetracycline (AKSSuT) (9, 10). Isolates from
swine with the AKSSuT pentaresistance pattern, and more generally those with resistance to
kanamycin are often of the DT193 phage type (9). DT193 can encode resistance factors on
conjugative plasmids that can be readily transferred to other bacterial species (8), thus
presenting a risk for spread of resistance to human enteric organisms.
In this report, we examine the means by which multi-resistant Salmonella strains
derived from pigs encode and transfer resistance factors. We show that S. enterica serovar
Typhimurium can encode multiple resistance factors as part of a chromosomal element, but
that these factors can still be efficiently transferred to other organisms by conjugation,
existing as an episome in the recipient strains. Further, the process of resistance gene
transfer appears to be very dynamic, since resistance factors can be lost from the host strain
or transferred to the recipient in various combinations.
16
2.3 MATERIALS AND METHODS
2.3.1 Strains and growth conditions
All Salmonella strains used here with the UT designation are S. enterica serovar
Typhimurium var. Copenhagen of the DT193 phage type cultured from swine farms in North
Carolina as previously described (8). The E. coli strains used in conjugations were
spontaneous rifampicin- or nalidixic acid-resistant mutants of the K12 strain MG1655. For
comparisons with the Salmonella virulence plasmid, strain ATCC14028s was used. All
strains were grown in LB broth or on LB agar plates. Antibiotics were used at the following
concentrations: ampicillin, 100 µg/ml; kanamycin, 100 µg/ml; rifampicin, 100 µg/ml;
streptomycin, 100 µg/ml; tetracycline, 25 µg/ml; nalidixic acid, 50 µg/ml.
2.3.2 Conjugation of resistance plasmids
Multi-drug resistant donor strains were mated with a spontaneous rifampicin- or
nalidixic acid-resistant derivative of E. coli K12 strain MG1655 in a 1:10 ratio of donor to
recipient. Bacteria from mid-log cultures were centrifuged together and incubated for 3
hours at 37º C. The mixture was then transferred to a selective plate containing rifampicin
(or nalidixic acid) and one of the antimicrobials to be tested and incubated at 37º C overnight.
Transconjugants were confirmed as E. coli, rather than spontaneous rifampicin- or nalidixic
acid-resistant Salmonella, by growth on MacConkey agar. The presence of other resistance
markers was tested by patching individual colonies from the initial selection plate onto agar
plates containing the other antimicrobials to be tested and by the Kirby-Bauer disk
susceptibility method, performed in accordance with NCCLS guidelines (13).
17
2.3.3 Curing of resistance determinants
Resistance factors were cured from strain UT20 using a described protocol for
plasmid curing (5). Briefly, 5 ml of LB broth with SDS added to a final concentration of 1%
was inoculated with 100 µl of an overnight culture and bacteria were grown overnight with
aeration. Dilutions were plated onto LB agar, and isolated colonies were then patched onto
selective agar, each with a single antimicrobial to be tested, in order to screen for loss of
resistance.
2.3.4 DNA preparation
Plasmid DNA was prepared throughout using a Qiagen Plasmid Midi kit (Qiagen).
Genomic DNA was prepared by washing 25 ml of an overnight culture of bacteria in 0.1 M
Tris pH 8, 0.1 M EDTA, 0.15 M NaCl, and then re-suspending in 0.8 ml of the same buffer.
Samples were incubated with lysozyme at a final concentration of 1 mg/ml for 10 minutes at
37º C, 0.1 mg/ml DNAse-free RNAse was added for an additional 10 minutes, and then
samples were heated to 70º C for 3 minutes. N-lauryl sarcosine was added to a final
concentration of 2.2% with incubation at 70º C for 20 minutes and then 37º C for one hour.
Two mg of proteinase K was added and samples were incubated for 4 hours at 37º C. An
additional 2 mg of proteinase K was added, and samples were dialyzed overnight against 10
mM Tris pH 8, 10 mM EDTA, 0.15 M NaCl. DNA was extracted twice with water-saturated
phenol and twice with ether and was then dialyzed against TE pH 8. DNA concentration was
determined by absorbance at 260 nm.
18
2.3.5 Electrophoresis and DNA hybridization
Pulse-field gel electrophoresis was performed using 200 µl of bacteria grown
overnight adjusted to McFarland 3.0, mixed with an equal volume of agarose (InCert, FMC),
and dispensed into a mold to form agarose plugs. Bacteria embedded in the agarose plug
were then lysed with proteinase K and N-lauryl sarcosine and washed vigorously with Tris
EDTA. DNA was digested with XbaI and was separated using a CHEF-DRIII pulsed field
gel electrophoresis apparatus (Biorad) using the following conditions: voltage (6 V/cm),
initial switch time of 2.2 seconds and final switch time of 63.8 seconds for 18 hours. Probes
for aphA1-Iab and blaTEM were synthesized by PCR amplifying fragments of each gene
labeled with digoxigenin using a PCR DIG probe synthesis kit (Roche). The primers used
for amplification were: aphA1-Ia, 5’-aaacgtcttgctcgaggc and 5’-caaaccgttattcattcgtga; blaTEM,
5’-gcacgagtgggttacatcga and 5’-ggtcctccgatcgttgtcag. The entire pUT20A plasmid was
labeled as probe using random-primed labeling with digoxigenin of plasmid DNA derived
from an E. coli transconjugant. DNA hybridizations were performed as previously described
(14). Detection was performed according to manufacturer’s directions using
chemiluminescence and a Lumi-Imager (Roche). Plasmids from UT20 were restriction
mapped using BamHI, HindIII, and XbaI. DNA was digested both singly and with
combinations of enzymes and resulting fragments were separated by electrophoresis in 0.5 %
agarose. Large fragments were mapped by digesting plasmids with a single enzyme,
excising fragments from the gel and purifying them, and then digesting with a second
enzyme. Transformation of plasmids was performed using electroporation according to the
manufacturer’s recommendations (Bio-Rad).
19
2.4 RESULTS
We have investigated the nature and extent of antimicrobial resistance among
Salmonella that were derived from commercially raised swine, and thus present a possible
threat to human health through foodborne contamination. Common among Salmonella
isolates cultured from these animals was S. enterica serovar Typhimurium var. Copenhagen
(hereafter S. Copenhagen) of the DT193 phage type. This phage type was most often
resistant to ampicillin, kanamycin, tetracycline, streptomycin, and sulfonamides (AKSSuT).
In our studies, several isolates of this phage type were often cultured from the same group of
animals during a single sampling (8). In order to compare the means by which closely
related Salmonella isolates might differ in their expression and transfer of antimicrobial
resistance, we examined two such strains that were phenotypically identical and
genotypically very similar. These strains, UT08 and UT20, were two of ten S. Copenhagen
isolates cultured from the same sampling, were both of the DT193 phage type, and both had
the AKSSuT resistance pattern. Further, these two strains produced similar profiles on
amplified fragment length polymorphism analysis (AFLP), with 96% identity in their
banding patterns (8), indicating great genetic similarity.
We found that UT08 carried all five of its resistance factors on a single conjugative
plasmid that could be transferred to E. coli with an efficiency of 4x10-4
transconjugants/donor bacterium. The plasmid could be further transferred by conjugation
from an original E. coli recipient to other E. coli strains. Restriction mapping of the plasmid
showed it to have an apparent size of approximately 120 kb. Thus, UT08 carried a
conjugative resistance plasmid that was not unlike those reported in Salmonella strains for
many years. Strain UT20 was also capable of transferring all of its resistance factors to E.
20
coli by conjugation, although with an 80-fold lower efficiency than that of UT08, 5x10-6
transconjugants/donor bacterium. E. coli transconjugants produced by matings with UT20
carried a 140 kb plasmid encoding the AKSSuT resistance pattern (plasmid pUT20A; Figure
2.1). We could not, however, detect this plasmid in UT20, either by isolation of plasmid
DNA or by Southern hybridization using resistance alleles known to be present in UT20. To
determine the location of the UT20 resistance genes, we performed DNA hybridizations
using UT20 genomic DNA separated by pulse-field gel electrophoresis (PFGE). We found
that blaTEM, encoding ampicillin resistance in UT20, was located on a ~290 kb XbaI
fragment, much larger than the predicted size of the conjugative plasmid (Figure 2.2), and
suggesting that in UT20 ampicillin resistance is not encoded on this plasmid. In contrast,
three strains, UT08, UT12, and UT30, that are genotypically, phenotypically, and
epidemiologically closed related to UT20 (8) carried blaTEM on a ~48 kb XbaI fragment. We
further examined the location of aphA1-Iab, encoding kanamycin resistance in UT20. An
aphA1-Iab probe hybridized to 4.7 kb HindIII fragment derived from a digest of total
genomic DNA prepared from UT20. It failed, however, to hybridize to plasmid DNA from
that same strain provided at a six-fold higher molar concentration (Figure 2.3). Plasmid
DNA from UT08 prepared and hybridized in the same manner produced an obvious signal.
These results indicate that UT20 encodes at least these two resistance genes not on an
autonomous plasmid, but suggest that they reside instead within the bacterial chromosome.
We next determined whether the conjugative resistance plasmid found in E. coli
transconjugants after matings with UT20 existed in UT20 as an integrated chromosomal
element. We used the pUT20A plasmid as probe in DNA hybridizations. This plasmid was
prepared from an E. coli transconjugant, labeled, and hybridized to both plasmid and total
21
genomic DNA from UT20 cut with HindIII. As shown in Figure 2.4, pUT20 hybridized
strongly to UT20 genomic DNA. The hybridizing bands of UT20 genomic DNA
corresponded with those of the pUT20A plasmid itself, except for the loss of a ~13 kb
fragment and the addition of 12 kb and 11 kb fragments in UT20. This finding was
consistent with integration of the plasmid into the Salmonella genome, with the site of
integration within the 13 kb fragment. In contrast, UT20 plasmid DNA hybridized very
weakly with the pUT20A probe, despite the fact that plasmid DNA was provided in 32-fold
molar excess to the genomic DNA. Thus, these results show that, unlike UT08 and other
multi-resistant DT193 isolates we have studied, UT20 carries its conjugative resistance
element not as a plasmid, but instead integrated into its chromosome. It remains able,
however, to transfer its resistance factors by conjugation.
We also examined whether the integrated resistance plasmid of UT20 was similar to
the conjugative virulence plasmid found in many serovar Typhimurium strains (1). Figure
2.4 shows a single large band produced when the pUT20A probe was hybridized to genomic
DNA from strain ATCC14028s, known to carry the virulence plasmid. However, UT20
DNA did not hybridize to an spvA probe, representing a gene found on the virulence plasmid
that is important for systemic infection (data not shown). It is likely therefore that the UT20
integrated resistance plasmid shares homology with the virulence plasmid, but lacks
functions involved in virulence.
Although chromosomal resistance elements are generally stable, we determined next
whether the resistance element of UT20 could be readily lost. We found that treatment with
detergent (1% SDS) yielded derivatives that had lost all five resistance factors. Southern blot
analysis revealed that such a derivative had lost approximately 100 kb of the integrated
22
plasmid, retaining ~40 kb (Figure 2.4). In addition to this fully susceptible derivative, we
also found that detergent treatment yielded derivatives having two other distinct resistance
patterns: resistance to ampicillin alone (A), and resistance to the remaining four
antimicrobials (KSSuT). As shown in Figure 2.4, each strain lost approximately 100 kb of
the original integrated plasmid, and ~22 kb of the remaining DNA was in common between
the two strains. Additionally, we found that the KSSuT strain maintained a 4.7 kb HindIII
fragment that was lost in the A strain (Figure 2.4). Since that fragment had been shown
previously to include aphA1-Iab, encoding kanamycin resistance (Figure 2.3), it is likely that
the differing resistance patterns of these resulting strains are due to the loss of antimicrobial
resistance genes from the bacterial chromosome. In our studies, we also fortuitously
discovered a spontaneous derivative of UT20 that failed to conjugate its plasmid, but
remained resistant to all five antimicrobials. Southern analysis of this strain revealed that it
too had lost a large portion of the integrated plasmid, but produced a restriction pattern
different from any of the susceptible or partially susceptible strains (Figure 2.4). This strain
presumably arose from the loss of essential conjugation functions, but not antimicrobial
resistance genes. We made repeated attempts to cure this non-conjugative strain of its
resistance elements, but were unsuccessful. Thus, these results show that portions of the
integrated UT20 plasmid can readily be lost from the chromosome to produce strains with
different resistance profiles, but suggest that loss of the portion that includes conjugative
functions also results in a strain with stably encoded multi-resistance.
Although strain UT20 encoded its resistance factors as an integrated plasmid, it could
still transfer that plasmid by conjugation to E. coli, where in the recipient strain it resided as
an episome. Since UT20 could readily lose portions of its integrated plasmid as well, we
23
reasoned that it might also transfer derivative plasmids created as a result of DNA loss. To
test this, we mated UT20 to E. coli, selected for one resistance, and then screened for the
remaining resistances. We found that in all cases, resistance to kanamycin, tetracycline,
streptomycin, and sulfonamides was transferred together. However, only about one half of
transconjugants resistant to these four antimicrobials were also resistant to ampicillin. In
addition, the great majority of transconjugants selected originally on ampicillin were also
resistant to the other four antimicrobials, but approximately 2% were susceptible to all of
these antimicrobials. Thus, three distinct resistance patterns could be detected: AKSSuT and
KSSuT in approximately equal numbers, and A only, at about 2% of the total. One possible
explanation for this finding was that UT20 harbored two independent resistance elements,
one KSSuT and the other A, that could be transferred either separately or together. To test
this, we created restriction maps for plasmids having each of the three resistance patterns. As
shown in Figure 2.1, pUT20A, with the AKSSuT pattern, is approximately 140 kb in size.
pUT20B, which has the KSSuT pattern, shares a similar restriction map, but is missing a 14
kb portion that is present in pUT20A. pUT20C, resistant to ampicillin alone, is also similar
to pUT20A, but lacks a 31 kb region different from that missing in pUT20B. The similarities
among these three plasmids thus indicated that they were all derived from a common
structure. Therefore, the variable resistance pattern observed in transconjugants was not due
to the transfer of independent plasmids, but instead was the result of plasmid recombination
that excised one or more resistance factors.
The recombination events required to produce plasmids with different resistance
patterns could occur while the resistance element was integrated in the UT20 chromosome,
during conjugation, or within the recipient strain. To test whether the chromosomal location
24
of the UT20 resistance element was required for the loss of resistance factors from the
resulting plasmids, we studied the derivative of UT20 that had lost all five of its resistance
factors (Figure 2.4). We transformed this strain with pUT20A, restoring it to AKSSuT
resistance, and then conjugated it with E. coli. We found that resistance to all five
antimicrobials was fully linked in the transconjugants. We also found that pUT20A, when
transferred by conjugation from the original E. coli recipient into other strains, always
produced transconjugants with the AKSSuT pattern. These results therefore suggest that
chromosomal integration of the UT20 resistance plasmid is important for the production of
recombinant conjugative plasmids, and that the loss of resistance genes by autonomously
replicating plasmids is rare.
2.5 DISCUSSION
Antimicrobial resistance among Salmonella derived from food-producing animals has
presented a problem for nearly as long as antimicrobials have been used in animal
agriculture. A wide variety of resistance factors have been identified, often transferable on
conjugative plasmids. In this work, we have investigated the means by which Salmonella
cultured from commercial swineherds encode and transfer their resistance factors. We have
found that even strains that are genetically and epidemiologically closely related can encode
resistance factors differently (as integrated plasmids or episomally), can transfer those
resistance factors with varying efficiency, and can produce derivative plasmids having
different resistance patterns.
One of the strains studied here, UT20, carries its resistance factors on a plasmid
integrated into the Salmonella chromosome. Integrated resistance factors in Salmonella
25
strains such as phage type DT104 are thought to present a threat to the control of
antimicrobial-resistant organisms. Since these resistance factors reside as chromosomal
elements, they are likely to be stably maintained, even if the selective pressure of
antimicrobial use is absent. Although both chromosomally encoded resistance genes and
integrated plasmids are widely known, UT20 is unusual. The UT20 integrated plasmid
appears to be highly plastic, since it remains transferable by conjugation and is subject to the
independent loss of several of its functions. UT20 is thus capable of producing derivative
strains with different resistance patterns or without the ability to conjugate resistance factors
to other strains. Further, the loss of conjugative functions also appears to have made the
integrated element more stable, since we were unable to cure resistance factors from a
derivative strain that had lost the ability to conjugate. It is likely that conjugative functions
are linked to those required for plasmid excision, making the chromosomal resistance
element remaining in the non-conjugative strain a stable part of the Salmonella genome.
Thus, UT20 might represent a strain in transition from one that harbors a transferable
plasmid to one with its resistance factors as a permanent part of its chromosome.
One important feature of the strain studied here is its ability to transfer by conjugation
plasmids with three different resistance patterns. Early work on resistance showed that
Salmonella strains can harbor several resistance plasmids that can be transferred either singly
or as co-integrate plasmids, producing transconjugants with differing resistance phenotypes
(2, 3, 11). In the case of UT20, however, the acquisition of different resistance factors was
not due to the transfer of multiple plasmids, but instead to the production of three similar
plasmids derived from the integrated resistance element of UT20. Restriction mapping
showed the three to be closely related, but to differ by the loss of discreet regions of the
26
plasmid likely to encode resistance genes. It is therefore clear that this strain can generate a
variety of resistance plasmids that can be further transferred to other strains by conjugation,
increasing the diversity of resistance phenotypes observed in the bacterial population. Our
previous work has also shown a closely related plasmid (that of UT30) to have acquired a
class I integron and gained new resistance factors (8). Thus, these S. Copenhagen DT193
strains of animal origin are capable of altering their resistance profiles and of transferring a
variety of resistance factors to other strains.
The mechanism by which UT20 can produce and transfer plasmids with different
resistance patterns remains unclear. It is likely however that the chromosomal location of the
plasmid is important for this process. We found that further transfer of the AKSSuT
pUT20A plasmid from E. coli transconjugants did not result in plasmids with fewer
resistance factors. Also, a strain closely related to UT20 but carrying its resistance factors on
a plasmid, strain UT08, very rarely produced plasmids without the full complement of
resistance factors (not shown). The simplest model for the behavior of UT20 is that multiple
sites for recombination exist within its integrated plasmid. These sites could consist of
repeated sequence elements sufficient to allow homologous recombination, or might be the
targets of a site-specific recombinase. Excision from the chromosome could therefore occur
using one of a number of site combinations, giving rise to at least three derivative plasmids
capable of conjugal transfer. It remains possible as well that additional undetected excised
plasmids exist, including those without antimicrobial resistance markers. The spontaneous
excision of portions of the chromosomal element could give rise to plasmids capable of
transfer by conjugation. If such excision events do occur, they must be of low frequency,
since we were unable to detect DNA homologous to that of the pUT20A plasmid in
27
preparations of plasmid DNA from UT20. The requirement for excision from the
chromosome prior to conjugation might however explain the reduced efficiency of
conjugation observed in UT20 as compared to a similar strain bearing its resistance factors
on a plasmid. Finally, the question remains of the frequency of chromosomal integration by
the resistance plasmid of DT193 strains. This phage type is one of the most commonly
isolated from swineherds in our studies (8, 9), and has been shown in the past to encode its
resistance factors on plasmids (10). It remains to be studied whether a large number of
isolates have chromosomally integrated plasmids and whether any are likely to be stably
encoded resistance elements.
2.6 ACKNOWLEDGMENTS
This work was supported by funding from the FDA (FD-U-001880-01) to C.A. We thank
Paul Orndorff for the critical review of this manuscript.
2.7 REFERENCES
1. Ahmer B.M., M. Tran, and F. Heffron. 1999. The virulence plasmid of Salmonella
typhimurium is self-transmissible. J. Bacteriol. 181:1364-1368.
2. Anderson, E.S., and M.J. Lewis. 1965a. Drug resistance and its transfer in
Salmonella typhimurium. Nature 206:579-583.
3. Anderson, E.S., and M.J. Lewis. 1965b. Characterization of a transfer factor
associated with drug resistance in Salmonella typhimurium. Nature 208:843-849.
28
4. Baquar, N., E.J. Threlfall, B. Rowe, and J. Stanley. 1994. Phage type 193 of
Salmonella typhimurium contains different chromosomal genotypes and multiple
IS200 profiles. FEMS Microbiol. Lett. 115:291-295.
5. El-Mansi, M., K.J. Anderson, C.A. Inche, L.K. Knowles, and D.J. Platt. 2000.
Isolation and curing of the Klebsiella pneumoniae large indigenous plasmid using
sodium dodecyl sulphate. Res. Microbiol. 151:201-208.
6. Falkow, S. 1975. Infectious Multiple Drug Resistance. Pion Limited, London.
7. Farrington, L.A., R.B. Harvey, S.A. Buckley, R.E. Droleskey, D.J. Nisbet, and
P.D. Inskip. 2001. Prevalence of antimicrobial resistance in Salmonellae isolated
from market-age swine. J. Food Prot. 64:1496-1502.
8. Gebreyes, W.A., and C. Altier. 2002. Molecular characterization of multi-drug
resistance determinants among Salmonella enterica serovar Typhimurium isolates
collected from swine. J. Clin. Micro. 40:2813-2822.
9. Gebreyes, W.A., P. R. Davies, W.E. Morrow, J. A. Funk , and C. Altier. 2000.
Antimicrobial Resistance of Salmonella Isolated from Swine. J. Clin. Microbiol.
38:4633-4636.
10. Hampton, M.D., E.J. Threlfall, J.A. Frost, L.R. Ward, and B. Rowe. 1995.
Salmonella typhimurium DT 193: differentiation of an epidemic phage type by
antibiogram, plasmid profile, plasmid fingerprint and salmonella plasmid virulence
(spv) gene probe. J. Appl. Bacteriol. 78:402-408.
11. Helmuth, R., R. Stephan, E. Bulling, W.J. van Leeuwen, J.D. van Embden, P.A.
Guinee, D. Portnoy, and S. Falkow. 1981. R-factor cointegrate formation in
Salmonella typhimurium bacteriophage type 201 strains. J. Bacteriol. 146:444-452.
29
12. National Antimicrobial Resistance Monitoring System. 2000. Annual Report.
http://www.cdc.gov/narms/annual/2000/annual_pdf.htm.
13. National Committee for Clinical Laboratory Standards. 1999. Performance
standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated
from animals; approved standard M31-A. National Committee for Clinical
Laboratory Standards, Wayne, Pa.
14. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: a
Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
30
KTSSu
B
H
B
H
X
B
H
B
BX
H HHH
H
A
B
H
X
B
H H
10 kb
Figure 2.1 Restriction maps of plasmids derived from strain UT20. The restriction map
shown is that of plasmid pUT20A, encoding AKSSuT resistance. The black bar above shows
the size and location of the region missing in pUT20C, with resistance to ampicillin (A)
alone, while the white bar above shows the region missing in pUT20B, with KTSSu
resistance. B, BamHI; H, HindIII; X, XbaI.
31
UT UT U U
08 12 T20 T30
UT UT U U
08 12 T20 T30
-291kb-
-48.5kb-
Figure 2.2 Location of blaTEM in strain UT20. Genomic DNA from four related strains
was cut with XbaI and separated by pulse-field electrophoresis, and then blaTEM was detected
by DNA hybridization.
32
UT
pU
20
UT
20
P l a Ge
n
T2
0A s m i d omi
c
UT
08
Pla
sm
id
4.7 kb
Figure 2.3 Chromosomal location of aph-Iab in strain UT20. Total genomic and
plasmid DNA were isolated from UT20, cut with HindIII, and probed for aph-Iab. pUT20A
is the AKSSuT plasmid derived from UT20 and prepared from an E. coli transconjugant.
Plasmid DNA from UT20 produced no signal, although it was provided in six-fold molar
excess over UT20 genomic DNA. Plasmid DNA prepared by the same method from a
related strain, UT08, hybridized to the aph-Iab probe.
33
Fu
T C Non
lly
K
C
co
pU
T
su
sc
T2 UT U UT 140 nju SSu
ep
T
2
2
g
0A 0 2 08 8s a
o
tib
Pl Gen 0 Pl Pl Pla tive nly
le
U
as o as as
U
UT
s
m m m mi mi T2 T2
20
id i c id d
d
0
0
A
A
ly
on
0
T2
U
23
12
10
8
6
4
2
Figure 2.4 Plasmid location and composition of UT20 and related strains. DNA was cut
with HindIII and hybridized to labeled pUT20A plasmid as probe. Lanes marked as plasmid
are plasmid DNA preparations, while unmarked lanes and those marked as genomic are total
genomic DNA preparations. Molecular weight is shown as kb on the left.
34
Chapter 3. Deducing the Biological Relevance of Multiple Drug Resistance in
Salmonella enterica serovar Typhimurium var. Copenhagen phage type DT193 of
animal origin
3.1 ABSTRACT
Salmonella enterica serovar Typhimurium is a food-borne pathogen that can cause
disease in humans. It exists in food producing animals, such as swine and poultry, without
clinical signs. In the US, antimicrobials are used extensively in animal feed as growth
promoters, therefore, promoting the occurrence and persistence of multi-drug resistance in
enteric organisms, including Salmonella enterica.
Although the use of antimicrobials in food-producing animals has induced resistance
in pathogenic bacteria, it remains unclear whether the cessation of antimicrobial use would
eliminate these resistant strains. An important aspect to this question is the growth rate of
resistant strains in comparison to similar susceptible strains. To test the fitness for growth of
resistant Salmonella, we compared the growth rate in mixed cultures of multi-resistant
Salmonella isolates of the DT193 phage type (resistant to ampicillin, kanamycin,
streptomycin, and sulfonamides, and tetracycline) to isogenic fully susceptible derivatives.
Competing strains were grown by serial transfer in antimicrobial-free media for 22 days, with
each strain in the population quantified every three days. Wild type strains varied greatly in
their relative fitness. Strain UT20, whose resistance factors have been inserted into the
chromosome, displays a great fitness cost when it is in competition against its isogenic, fully
susceptible derivative. UT8, whose resistance factors are identical to those of UT20 but are
carried on a plasmid, displays an initial fitness cost that is much less severe and appears to be
35
surmountable. This difference in fitness is not due to differences in the genetic backgrounds
of the two strains since cured UT20 carrying the plasmid of UT8 also showed only a mild
fitness defect. Conjugation within competitions was measured and found not to be an
influencing factor on the persistence of resistance factors in a population.
It is our conclusion that the degree to which the fitness burden manifests itself is
dependent on the location of the resistant element. Naturally occurring multiple drug
resistant (MDR) strains of Salmonella enterica are at a large metabolic disadvantage
compared to susceptible isogenic strains if the resistance element is integrated into the
chromosome. It appears that if the resistance element is in plasmid form, then the fitness
burden is significantly less, as well as being surmountable by means as yet undetermined.
3.2 INTRODUCTION
A wide range of antimicrobial classes are used as growth promoters for livestock.
However, in addition to promoting the growth of the animals and increasing feed efficiency,
the extensive use of antimicrobials also promotes the occurrence and persistence of
antimicrobial resistance in the commensal enteric bacteria. Antimicrobial resistance in
Salmonella enterica, as an enteric organism in swine, poses a huge problem to the potential
health of any human with which it comes in contact. Salmonella is a dangerous human
pathogen, and fighting infection could be exceedingly difficult if the offending strain is
resistant to multiple antimicrobial agents. The United States Center for Disease Control and
Prevention estimates that approximately 40,000 cases of salmonellosis are reported in the
United States every year. Because many milder cases are not diagnosed or reported, the
actual number of infections may be thirty or more times greater
36
(http://www.cdc.gov/ncidod/dbmd/diseaseinfo/salmonellosis_g.htm). Out of the reported
cases, 95% are reported to be foodborne, and each year more than 500 deaths are attributed to
foodborne salmonellosis (Mead et al. 1999). Foodborne outbreaks of non-typhoidal
Salmonella of various serovars have been recorded in large numbers in different parts of the
world (Aserkoff et al. 1970; Fontaine et al. 1978; Taylor et al. 1982; Holmberg et al. 1984;
Spika et al. 1987). Therefore, it was of great concern when multiple drug resistant (MDR)
Salmonella strains were discovered in large concentrations on local swine farms in North
Carolina. Of the two thousand, nine hundred seventy-six isolates, fully 84% were found to
be resistant to tetracycline. Ampicillin resistance consisted of 47% of the isolates, and 30%
were resistant to chloramphenicol. (Gebreyes et al. 2000)
Whether or not acquired resistance factors will dissipate from the environment once
selective pressures are removed remains to be seen. The resolution must depend on the
fitness cost imposed on the bacteria by the genetic elements conferring antimicrobial
resistance. The literature suggests that there is indeed a fitness burden associated with
antimicrobial resistance, however, amelioration of these costs through compensatory
mutations instead of true reversion seems to be the predominate resolution. (Andersson and
Levin 1999, Levin et al. 1999, Schrag and Perrot 1996) In E. coli and Salmonella,
spontaneous resistance to streptomycin can be conferred by a single base pair mutation in the
rpsL gene (Schrag and Perrot 1996, Björkman, et al. 1998). By finding the relative rates of
growth and survival between resistant and susceptible isogenic strains, the fitness burden of
antimicrobial resistance can theoretically be deduced. However, the cases discussed to date
in the literature are examples of spontaneous mutations that confer resistance in model
strains, and are not indicative of the genotypes found in natural field isolates.
37
Resistances in bacteria of animal origin are often the result of acquired resistance
factors, encoded either on plasmids or integrated into the bacterial chromosome, rather than
the result of spontaneous mutation. Additionally, these plasmid-encoded resistance factors
are known to be transmissible by conjugation. Most of the early work done to prove the
existence of resistance factors and to determine the very nature of plasmids was done in
Salmonella (reviewed in Falkow 1975). It has been shown that a single Salmonella strain can
harbor several plasmids carrying different resistance determinants. Some resistance
plasmids, even if they themselves are not self-transmissible, can move by conjugation with
the help of conjugative plasmids that co-exist in the same strain (Anderson and Lewis, 1965).
Therefore, under the proper selection, conjugative plasmids can spread rapidly through an
environment where antimicrobials are present and quickly dominate a population.
We have performed several competition assays using naturally occurring field isolates
of multiple drug resistant Salmonella enterica and their cured isogenic strains. UT20 and
UT8 are phenotypically and epidemiologically identical, and are genotypically very similar
strains of Salmonella enterica serovar Typhimurium phage type DT193 that are resistant to
ampicillin, kanamycin, streptomycin, sulfonamides, and tetracycline. UT8 has its resistances
in a plasmid about 140 kb in size. UT20 has its resistances as cis-elements in the
chromosome (Suyemoto et al. 2003). Both strains, UT8 and UT20, can easily conjugate with
other Salmonella and E. coli, and the predominant product of both conjugation events is a
plasmid of about 140 kb. (Suyemoto et al. 2003) It is in our finding that maintaining
resistance factors in the chromosome confers a greater fitness burden to the bacteria than
maintaining the same resistance factors in plasmid form. In addition, resistance factors
within the population can perpetuate themselves by conjugation, but this event does not
38
appear to have a major impact on the total resistance found in a population grown in the
absence of antimicrobial agents.
3.3 MATERIALS AND METHODS
3.3.1 Manipulation of Strains
Strains used in this study are shown in Table 3.1. All Salmonella strains with the UT
designation are S. enterica serovar Typhimurium var. Copenhagen of the DT193 phage type
cultured from swine farms in North Carolina as previously described (Gebreyes 2002). They
are resistant to ampicillin, kanamycin, streptomycin, sulfonamides, and tetracycline
(AKSSuT). UT8 has the five resistances encoded in a conjugative plasmid of about 140 kb.
UT20 possesses the identical resistances, but the plasmid has integrated into the
chromosome. UT20 can still conjugate with E. coli and other Salmonella, but the
exconjugates all possess the resistances on a plasmid (Gebreyes et al. 2000). Strain CA882
is a spontaneous mutant of UT20 that maintains all of its resistance factors, but has lost its
ability to conjugate.
Resistance factors were cured from strains UT20 and UT8 using a described protocol
for plasmid curing (El-Mansi et al). Five ml of LB broth with 1% SDS was inoculated with
100 µl of an overnight culture. Bacteria were grown overnight with aeration. Dilutions were
plated onto LB agar, and isolated colonies were then patched onto selective agar, in order to
screen for the loss of resistance. The strain CA879 is UT8 cured of all its resistances. The
strain CA885 is similarly cured UT20. CA906 is cured UT20 plus the plasmid of UT8,
therefore, it is resistant to AKSSuT. CA909 is cured UT20 plus the plasmid of UT20, also
resistant to AKSSuT.
39
In experiments where a measurement of conjugation was necessary, spontaneous
nalidixic acid resistant markers were created by selection on agar plates containing 50µg/ml
of nalidixic acid. CA902 is the NalR derivative of cured UT8. CA905 is the NalR derivative
of cured UT20.
3.3.2 Fitness Assays
Competitions between two strains were carried out in LB broth in the absence of
antimicrobials. Pure cultures of individual strains were grown overnight to stationary phase
under the proper antimicrobial selection. The cultures where plated on selective media the
next day (Day 0) to titer them. One ml of culture was spun down and washed with fresh,
antimicrobial-free media. This volume was then used for inoculation purposes.
Approximately equal proportions of competing strains were inoculated into liquid LB broth.
Cultures were passaged by 1:1000 serial transfer (i.e. 10 µl in 10ml). The cultures were
plated at Day 0, Day 1, and every three days succeeding thereafter for 22 days, on LB agar
containing the appropriate selecting agent. Tetracycline, which has a plating efficiency near
100%, was used to select for the bacteria with the AKSSuT resistance pattern. We compared
the fitness of a resistant strain with one that had been cured of its resistance factors, but had
been made nalidixic acid resistant as a positive selection. Nalidixic acid selection was used
to culture the cured derivatives. Nalidixic acid resistance is at or near neutral fitness (this
study). In combination with tetracycline, nalidixic acid was used to select for the bacteria
that were products of conjugation. Antimicrobials were used at the following concentrations:
tetracycline, 25 µg/ml; nalidixic acid, 50 µg/ml.
40
3.3.3 Statistical Analysis
Within each competition, we measured the proportion of nalidixic acid resistant
bacteria, the proportion of tetracycline resistant bacteria, and the proportion of bacteria
doubly resistant to nalidixic acid and tetracycline. In order to gain an accurate count of the
total bacteria within the population, we added the number of nalidixic acid resistant bacteria,
N, to the number of tetracycline resistant bacteria, T, then subtracted the number of nalidixic
acid and tetracycline resistant bacteria, NT. This formed the denominator to our proportions,
(N+T-NT). The proportion of nalidixic acid resistant, cured susceptible strains was found by
the equation: [(N-NT)/(N+T-NT)]. The equation to the proportion of the AKSSuT resistant
strains was: [(T-NT)/(N+T-NT)]. The proportion of exconjugates within the population was
represented by the equation: [(NT)/(N+T-NT)].
3.4 RESULTS
3.4.1 Fitness of Naturally Occurring Strains
Due to the dominant presence of multiple drug resistant (MDR) Salmonella enterica
found in naturally occurring bacterial populations, it was our intent to determine whether
possessing multiple antimicrobial resistance determinants incurs a metabolic burden, and
therefore, a fitness cost to the host bacterium. We used for these studies Salmonella enterica
serovar Typhimurium var. Copenhagen strains of the DT193 phage type that are known to be
epidemiologically and phenotypically identical, and genotypically closely related (Gebreyes
and Altier, 2002). UT8 and UT20 were both isolated from a swine farm in North Carolina.
Both are resistant to ampicillin, kanamycin, streptomycin, sulfonamides, and tetracycline
(AKSSuT). The resistance element was found to be encoded on a 140 kb plasmid in UT8.
41
However, in the case of UT20, these resistances, and indeed the entire plasmid, were found
integrated into the chromosome (Suyemoto, 2003). Nevertheless, both are capable of
conjugation with at least Salmonella and E. coli.
In order to determine if the resistances caused a fitness burden, we maintained either
UT8 or UT20 in co-culture with its resistance-cured, fully susceptible derivative in the
absence of antimicrobials by serial passage daily for a period of 22 days, and assessed every
three days the relative abundance of each strain. Wild type UT20 diminished from the
population precipitously, leaving behind its susceptible derivative (CA905) as the strain
dominating the population (Figure 3.1). This became apparent as early as Day 1, when the
mean proportion of CA905 in the total bacterial population was 0.82. The difference was
even greater on Day 4 when the mean proportion of CA905 was 0.92. By Day 22, the final
day of the experiment, the proportion of CA905 was 0.96. This gave us a strong indication
that the resistances produced a large metabolic cost for the host bacteria.
In order to determine if the fitness burden imposed by a plasmid encoded element was
different from that of a chromosomally encoded element, we competed UT8 against its cured
counterpart, CA902. UT8 showed only a slight fitness burden throughout our experiment,
remaining relatively close in proportion with its fully susceptible derivative, CA902 (Figure
3.2). The mean proportion of susceptible bacteria, inoculated initially at one half the
population, rose to 0.79 by Day 4. The susceptible proportion remained around 0.8 for the
succeeding days, then returned to 0.51 by Day 22, the end of the experiment. These findings
suggest that there is little to no fitness burden imposed by the plasmid as an episome,
however, there is a substantial cost when a similar plasmid, that of UT20, integrates into the
chromosome.
42
3.4.2 Fitness of Exconjugates
One possible explanation for the lack of a serious fitness burden due to UT8
resistance elements is an extragenic compensatory mutation. A compensatory mutation in
UT8 could have rendered the strain invulnerable to the negative effects of possessing its large
plasmid. To determine if this were indeed the case, we created derivatives of UT20 that were
cured of all resistance elements in the chromosome, but possessed either the plasmid of UT8
(thus labeled CA906) or the plasmid derivative of the chromosomally encoded resistance
element of UT20 (thus labeled CA909). If the plasmids did cause a metabolic cost, CA906
and CA909 would be expected to display that cost, since both are based upon the UT20
background strain, rather than UT8, in which the putative compensatory mutation would
have occurred. Both strains retained the same resistance markers, AKSSuT, on 140 kb
plasmids, and both possessed the ability to be transferred by conjugation.
The outcome of competing cured UT20 with either CA906 or CA909 was very
similar to that of wild type UT8 versus cured UT8. By the end of Day 22, the proportion of
the resistant subpopulation to the susceptible subpopulation was nearly equal in both cases.
Figure 3.3 shows the competition of CA905 (UT20 cured, NalR) versus CA906 (UT20 cured,
plus the plasmid of UT8). The proportion of resistant bacteria on Day 1 was actually greater
than the proportion of susceptible bacteria (the resistant subpopulation was initially in the
majority because slightly more of these bacteria were initially inoculated into the test
medium). However, despite the initial numerical advantage of resistant bacteria, the number
of bacteria of the susceptible strain still rose and peaked at Day 10 with a mean proportion of
0.93. Then, similar to the previous experiment with UT8, the number of resistant bacteria in
the population began to rise again, so that by Day 22, the mean proportion of the susceptible
43
bacteria was 0.61 and the mean proportion of the resistant bacteria was 0.22. The reason that
these proportions do not add to 1.00 is due to the proportion of exconjugates, 0.16, within the
population.
Figure 3.4 shows the competition of CA905 (UT20 cured, NalR) versus CA909
(UT20 cured, plus the plasmid derivative of UT20). Again, the susceptible bacteria in the
population were at a numerical disadvantage at the start of the experiment, and, again, the
proportion of susceptible bacteria initially rose and peaked at Day 10 with a mean proportion
of 0.94. The proportion of the susceptible population then slowly dropped, so that by Day
22, the proportion of the susceptible bacteria was 0.69, while the proportion of resistant
bacteria was 0.26. The proportion of exconjugates in this population was 0.05.
These results were nearly identical to the competition assay involving UT8 described
above, and indicate that no pre-existing compensatory mutation was necessary to maintain
the fitness of strains carrying the resistance determining plasmid. Indeed, these results imply
that possession of the multiple antimicrobial resistance factors is only a major fitness burden
when the resistance plasmid has been inserted into the chromosome.
3.4.3 Assessing the Dynamic Influence of Conjugation
As described earlier in this work, both wild types UT8 and UT20 are capable of
conjugating their resistance elements to other bacteria. Our initial experiments left open the
possibility that exconjugates in the co-culture (initially susceptible bacteria receiving the
resistance plasmid by conjugation) were being counted with the initial resistant strain,
thereby artificially inflating the proportion of that resistant strain in the population. It
became clear that we needed to quantify the exconjugates within each competition assay.
We therefore marked our cured strains with nalidixic acid resistance by spontaneous
44
mutation. In order to ensure that the addition of nalidixic acid resistance itself caused no
fitness burden, we performed a competition assay between CA885 (UT20 cured) and CA905
(UT20 cured, NalR), and between CA879 (UT8 cured) and CA902 (UT8 cured, NalR) by the
same methods as described above. These competitions proved that strains with nalidixic acid
resistance were at or near neutral fitness (data not shown).
Figures 3.5a and 3.5b show either wild type UT20 or UT8 competing against its
cured, NalR derivative (i.e. UT20 vs. CA905 in 5a, and UT8 vs. CA902 in 5b). The
proportion of exconjugates, defined as bacteria resistant to both nalidixic acid and
tetracycline (NalRTetR), varied slightly in the population between competitions. The mean
proportion of exconjugates within the UT20 vs. CA905 (UT20 cured, NalR) competition was
0.005. The mean proportion of exconjugates within the UT8 vs. CA902 (UT8 cured, NalR)
competition was 0.003. We observed a steady increase in the proportion of tetracycline
resistant bacteria in the competition of UT8 vs. UT8 cured, NalR, but we did not see a
concomitant increase in the proportion of NalRTetR bacteria during the same experiment.
This shows that conjugation between wild type antibiotic resistant and susceptible strains has
little or no effect on the competition assay and on the population as a whole.
Similar results for the proportion of NalRTetR bacteria were found when CA905
(UT20 cured, NalR) was in competition against either CA906 (UT20 cured, plus the plasmid
of UT8) in Fig. 5c or CA909 (UT20 cured, plus the plasmid derivative of UT20) in Fig. 5d.
However, the proportion of exconjugates in either competition was higher than in
competitions with the wild types. The mean proportion of exconjugates throughout the
experiment with UT20 cured NalR vs. UT20 cured carrying the plasmid pUT8 was 0.049.
The mean proportion of exconjugates within UT20 cured NalR vs. UT20 cured with the
45
plasmid pUT20 was 0.021. The reason for the apparently higher efficiency of conjugation is
not known, but the results remain clear. Conjugation has little effect on the total count of
antimicrobial resistant cells within a wild type population not under constant selection for the
presence of antimicrobial resistant phenotypes.
To further corroborate our results, we performed a competition in which conjugation
was not a compounding factor. A spontaneous mutant of UT20 was found, labeled CA882,
that was incapable of conjugation, yet retained its resistance element. With the exception of
CA882, all of the previously mentioned MDR strains were capable of donating their
resistances by conjugation, and all of the cured strains were competent to receive the
resistances by conjugation.
We competed CA882 (UT20, AKSSuT, no conjugation) with CA905 (UT20 cured,
NalR). The mean proportion of CA905 by Day 7 was 0.98. By Day 22 it was 0.999. The
AKSSuT resistant strain, CA882, receded from the population rapidly. These results are
nearly identical to those represented by Figure 3.1. We continued to test for the presence of
exconjugates but found none (data not shown). Therefore, we were able to definitively
conclude that transfer of resistance elements did not affect the difference between resistant
and susceptible strains in competition.
3.5 DISCUSSION
In this work we explored the possibility that episomally encoded multiple
antimicrobial resistance factors incurred a metabolic burden and fitness cost to the host
bacteria. We found that the Salmonella enterica serovar Typhimurium strain UT20, whose
multiple antimicrobial resistance factors are chromosomally encoded, suffered a large fitness
46
cost associated with its possession of the resistance factors. While it did not vanish from the
population, the proportion of UT20 in the population diminished very rapidly such that it was
approaching zero. Were this experiment to be carried out for a longer time frame, the
proportion of UT20 might very well reach the limit of detection.
The possible reasons for these observations are numerous and require further
exploration. One hypothesis for the lack of fitness in UT20 is that its integration into the
bacterial chromosome disrupted a locus required for efficient growth. If a critical gene or
operon were made nonfunctional, then the strain would be at a severe disadvantage when not
in the selective environment that prevented other bacteria from surviving.
Another possibility is that interference with replication could cause UT20 to have a
large disadvantage. The resistance factor could be interrupting or preventing the proper
replication process during repeated insertion and excision from the chromosome. The
topoisomerases involved in replication could be preoccupied with the insertion/excision
process, slowing down the process of unbinding the concatamers of replicated DNA.
It has been ruled out that the UT20 background is fundamentally different from the
UT8 background. Cured UT20 plus either the plasmid of UT8 or the plasmid derivative of
UT20 behaved in a very similar manner to that of cured UT8 when it was in competition
against wild type UT8. The resistance factor, when in plasmid form, did not cause an
extensive fitness burden, as it did when it was intragenic to the chromosome. This
experiment also ruled out the possibility of any intrinsic evolution that UT8 may have
undergone on its own in order to ameliorate the possible costs of plasmid ownership. It is
entirely possible that both UT8 and UT20 could have undergone compensatory mutations
during the competition assays that reduced any fitness burden caused by the resistance factor.
47
This could explain the initial drop in proportions of resistance bacteria at the beginning of
some competitions and their successive rise in proportions later in the experiment.
Regardless of why UT20 is less fit than its cured, isogenic strain, the results clearly indicate
that resistance factor integration into the chromosome is not stable and not favored by
evolutionary processes unless there is a specific selective pressure, such as persistent
antimicrobial treatment, to promote its existence.
According to our results, it seems apparent that UT8, while initially displaying a
slight fitness cost as a result of possessing the resistance determinant, can overcome those
costs fairly rapidly, conceivably by compensatory mutations. There is no disruption in the
chromosome with which UT8 must contend. Neither is there a chromosomal replication
issue, as the plasmid in question replicates autonomously.
In vivo experimentation needs to be performed in order to prove how much of a
fitness burden plasmid mediated acquired resistance factors incur in an agriculturally relevant
environment, but it appears that compensatory mutations can occur in vitro that will
ameliorate the cost of the metabolic burden.
The resistance factor’s ability to be transferred by conjugation, regardless of its
location relative to the chromosome, was explored and proved to have a minimal effect on
the population as a whole. Never, in the competitions involving wild type strains, did the
exconjugates reach more than 0.5% of the population. This indicates that transfer of
resistance in the agricultural realm is not a major concern in the absence of antimicrobial
agents. In fact, it seems that the presence of antimicrobial agents puts selective pressure on
the population to promote the occurrence of the transfer event. However, caution must still
be exercised. In hospital settings, the extent of transfer of resistance within a patient has not
48
been measured. As of this writing, it is still unknown how often a MDR pathogen might
conjugate its resistances to the enteric organisms in the host, human or non-human, during
the infection. Consider this hypothetical situation. Under specific circumstances, MDR
bacteria could infect a hospitalized patient. Transfer of the resistance factor(s) may not take
place very often, but it wouldn’t have to. Were the patient to need antimicrobial treatment,
all but the initial bacteria from the infection and the exconjugates would be killed, thereby
placing the same selective pressure on the host’s enteric organism population that has already
been placed on the enteric organism populations of livestock. Therefore, by destroying all
the susceptible bacteria, the proportion of resistant bacteria and their exconjugates would rise
dramatically.
The caveat to all this is the fact that these experiments have been performed in a
laboratory setting. The results may not directly correlate with the actual events that occur in
nature. The reason proposed earlier as to why the proportion of UT8 initially drops and then
rises again is that UT8 may have developed compensatory mutations. This assumes that UT8
is not evolved to survive best in laboratory media. The question arises that if UT8 could
develop compensatory mutations now, why then did it not develop them before it was
introduced into the laboratory media. If those mutations could have occurred previously,
then they must have at some point in time. The fact that we don’t see them immediately
means they must themselves have some kind of cost and that it is not beneficial to have those
mutations at all times. This means that our laboratory media itself is something of a selective
agent, promoting mutations that could allow wild type strains to grow better in laboratory
media than they would normally.
49
Regardless to the differences between in vivo and in vitro experimentation, UT20
would probably not survive in a natural antimicrobial-free environment in the long term. It
seems to be at too much of a metabolic disadvantage to endure without the selective agents in
the environment to eliminate competing organisms.
It is our conclusion that naturally occurring MDR strains of Salmonella enterica are
at a metabolic disadvantage to their susceptible isogenic strains. The degree to which the
fitness burden manifests itself is dependent on the location of the resistant element relative to
the host chromosome. A resistance element that has been inserted into the chromosome can
impose a great metabolic burden to the bacterium, such that when it is in an unselective
environment it will not endure. However, when the resistance element is extrachromosomal,
the metabolic burden is significantly less. Additionally, this disadvantage is surmountable.
Although this fitness burden remains slight, the only factor that absolutely ensures it
persistence in the natural environment is the continued use of antibiotics in animal
husbandry.
3.6 REFERENCES
Anderson, ES, and Lewis MJ. 1965a. Drug resistance and its transfer in Salmonella
typhimurium. Nature 206:579-583
Anderson, ES, and Lewis MJ. 1965b. Characterization of a transfer factor associated with
drug resistance in Salmonella typhimurium. Nature 208:843-849
Andersson, DI and Levin BR. (1999) The biological cost of antibiotic resistance. Cur Op
Microbiol 2:489-493.
Aserkoff B, Schroeder SA, Brachman PS. 1970. Salmonellosis in the United States50
-a five-year review. Am J Epidemiol. 92:13-24.
Björkman, J, Hughes D, Andersson DI. (1998) Virulence of antibiotic-resistant Salmonella
typhimurium. PNAS 95:3949-3953
El-Mansi, M, Anderson KJ, Inche CA, Knowles LK, and Pratt DJ. (2000) Isolation and
curing of the Klebsiella pneumoniae large indigenous plasmid using sodium dodecyl sulfate.
Res Microbiol 151:201-208
Fontaine RE, Arnon S, Martin WT, Vernon TM Jr, Gangarosa EJ, Farmer JJ
3rd, Moran AB, Silliker JH, Decker DL. 1978. Raw hamburger: an
interstate common source of human salmonellosis. Am J Epidemiol. 107: 36-45
Falkow, S. (1975) Infectious Multiple Drug Resistance. Pion Limited, London
Gebreyes, WA, Funk JA, Davies P, and Altier C. (2000) Antimicrobial resistance
of Salmonella isolates from swine. J Clin Microbiol 38:4633-4636.
Gebreyes, WA and Altier C. (2002) Molecular characterization of multi-drug resistance
determinates among Salmonella enterica serovar Typhimurium isolates collected from swine.
J Clin Microbiol 40:2813-2822
Holmberg SD, Wells JG, Cohen ML. 1984. Animal-to-man transmission of
antimicrobial-resistant Salmonella: investigations of U.S. outbreaks, 1971-1983.
Science. 225:833-5
Levin, BR, Perrot V, and Walker N. (1999) Compensatory mutations, antibiotic resistance
and the population genetics of adaptive evolution in bacteria. Genetics 154:985-997
Salmon DE, Smith T. 1886. The bacterium of swine plague. Am. Mon. Microbiol. J.
7:204
Schrag S and Perrot V. (1996) Reducing antibiotic resistance. Nature 381:120-121
51
Schrag SJ, Perrot V, and Levin BR. (1997) Adaptation to the fitness costs of antibiotic
resistance in Escherichia coli. Proc. R. Soc. Lond. B 264:1287-1291
Spika JS, Waterman SH, Hoo GW, St Louis ME, Pacer RE, James SM, Bissett
ML, Mayer LW, Chiu JY, Hall B, et al. 1987. Chloramphenicol-resistant
Salmonella newport traced through hamburger to dairy farms. A major
persisting source of human salmonellosis in California. N Engl J Med.
316:565-70
Taylor DN, Wachsmuth IK, Shangkuan YH, Schmidt EV, Barrett TJ, Schrader
JS, Scherach CS, McGee HB, Feldman RA, Brenner DJ. 1982.
Salmonellosis associated with marijuana: a multistate outbreak traced by
plasmid fingerprinting. N Engl J Med. 306: 1249-53
52
Table 3.1 Strains and Plasmids Used
Strains
Phenotype
Source or Reference
UT8
wt, AKSSuT (plasmid)
Gebreyes, et al.
UT20
wt, AKSSuT (chromosome)
Gebreyes, et al.
CA879
UT8 cured
this study
CA882
UT20 no conjugation, AKSSuT
this study
CA885
UT20 cured
this study
CA902
UT8 cured, NalR
this study
CA905
UT20 cured, NalR
this study
CA906
UT20 cured + pUT8, AKSSuT
this study
CA909
UT20 cured + pUT20, AKSSuT
this study
Plasmids
Genotype
Source or Reference
pUT8
AKSSuT, 140kb
this study
pUT20
AKSSuT, 140kb
this study
53
Fig 3.1 UT20 vs UT20cNal
1
0.8
0.6
0.4
0.2
0
Day 0 Day 1 Day 4 Day 7 Day
10
Day
13
Day
16
Day
19
Day
22
Figure 3.1 The proportion of nalidixic acid resistant bacteria in the competition
between wild type UT20 (AKSSuT) and CA905 (cured UT20, NalR). This graph shows
the rapid rise in the number of cured bacteria and their domination in the population over the
wild type, resistant bacteria. Graphed is the mean proportion of cured bacteria at each day
assessed. The error bars represent the standard deviation at each point. n = 3.
54
Proportion Nalidixic Acid
Resistant Bacteria
Fig 3.2 UT8 vs UT8cNal
1
0.8
0.6
0.4
0.2
0
Day 0 Day 1 Day 4 Day 7 Day
10
Day
13
Day
16
Day
19
Day
22
Figure 3.2 The proportion of nalidixic acid resistant bacteria in the competition
between wild type UT8 (AKSSuT) and CA902 (cured UT8, NalR). This graph shows an
initial rise in the number of cured bacteria, followed by a decrease in that proportion.
Towards the end of the experiment, the proportion of wild type, resistant bacteria began to
rise again. On the final day, the ratio of cured to resistant strains was 1:1. Graphed is the
mean proportion of cured bacteria at each day assessed. The error bars represent the standard
deviation at each point. n = 3.
55
Proportion Nalidixic Acid Resistant
Bacteria
Fig 3.3 UT20cNal vs UT20c+pUT8
1
0.8
0.6
0.4
0.2
0
Day 0 Day 1 Day 4 Day 7
Day
10
Day
13
Day
16
Day
19
Figure 3.3 The proportion of nalidixic acid resistant bacteria in the competition
between CA905 (cured UT20, NalR) and CA906 (cured UT20 + pUT8, AKSSuT). This
graph is similar to Figure 3.2, but the mean proportion of cured bacteria attains a higher level
before it drops to 0.62. Graphed is the mean proportion of cured bacteria at each day
assessed. The error bars represent the standard deviation at each point. n = 3.
56
Proportion Nalidixic Acid Resistant
Bacteria
Fig 3.4 UT20cNal vs UT20c+pUT20
1
0.8
0.6
0.4
0.2
0
Day 0 Day 1 Day 4 Day 7
Day
10
Day
13
Day
16
Day
19
Figure 3.4 The proportion of nalidixic acid resistant bacteria in the competition
between CA905 (cured UT20, NalR) and CA909 (cured UT20 + pUT20, AKSSuT). This
graph is nearly identical to Figure 3.3 and similar to Figure 3.2. The mean proportion of
cured bacteria rises to a higher level than in Figure 3.2, then it drops to a proportion of 0.69.
Graphed is the mean proportion of cured bacteria at each day assessed. The error bars
represent the standard deviation at each point. n = 3.
57
Fig 3.5b UT8 vs UT8cNal
1
0.8
0.6
0.4
0.2
0
Day 0 Day 1 Day 4 Day 7 Day
10
Day
13
Day
16
Day
19
Day
22
Proportion Nal&Tet Resistant
Bacteria
Proportion Nal&Tet Resistant
Bacteria
Fig 3.5a UT20 vs UT20cNal
1
0.8
0.6
0.4
0.2
0
Day 0 Day 1 Day 4 Day 7 Day
10
Day
13
Day
16
Day
19
0.8
0.6
0.4
0.2
0
Day 0 Day 1 Day 4 Day 7 Day
10
Day
13
Day
16
Day
19
Day
22
Fig 3.5d UT20cNal vs UT20c+pUT20
Day
22
Proportion Nal&Tet Resistant
Bacteria
Proportion Nal&Tet Resistant
Bacteria
Fig 3.5c UT20cNal vs UT20c+pUT8
1
1
0.8
0.6
0.4
0.2
0
Day 0 Day 1 Day 4 Day 7 Day
10
Day
13
Day
16
Day
19
Day
22
Figure 3.5 The proportion of exconjugates in all competitions. (a) wild type UT20
versus CA905 (cured UT20, NalR). (b) wild type UT8 versus CA902 (cured UT8, NalR). (c)
CA905 (cured UT20, NalR) versus CA906 (cured UT20 + pUT8, AKSSuT). (d) CA905
(cured UT20, NalR) versus CA909 (cured UT20 + pUT20). All graphs show the effect of
conjugation on a population in a nonselective environment. Graphed is the mean proportion
of cured bacteria at each day assessed. The error bars represent the standard deviation at
each point. n = 3.
58