Download Monitoring and Identifying Antibiotic Resistance Mechanisms in

Monitoring and Identifying Antibiotic Resistance Mechanisms in Bacteria
M. T. Roe and S. D. Pillai
Food Safety & Environmental Microbiology Program, Poultry Science Department,
Texas A&M University, College Station, Texas 77843-2472
ABSTRACT Sub-therapeutic administration of antibiotics to animals is under intense scrutiny because they
contribute to the dissemination of antibiotic-resistant bacteria into the food chain. Studies suggest that there is a
link between the agricultural use of antibiotics and antibiotic-resistant human infections. Antibiotic-resistant organisms from animal and human wastes reenter the human and animal populations through a number of pathways including natural waters, irrigation water, drinking
water, and vegetables and foods. Antibiotic usage in the
United States for animal production (disease prevention
and growth promotion) is estimated to be 18 million
pounds annually. As much as 25 to 75% of the antibiotics
administered to feedlot animals are excreted unaltered
in feces. Because about 180 million dry tons of livestock
and poultry waste is generated annually in the United
States, it is not surprising that animal-derived antibioticresistant organisms are found contaminating groundwater, surface water, and food crops. It is extremely important to clearly understand the molecular mechanisms
that could potentially cause lateral or horizontal gene
transfer of antibiotic resistance genes among bacteria.
Once the mechanisms and magnitude of resistance gene
transfer are clearly understood and quantified, strategies
can be instituted to reduce the potential for dissemination
of these genes.
(Key words: integrons, antimicrobial resistance, bacteria, environment, characterization)
2003 Poultry Science 82:622–626
This paper provides an overview of integrons, which
are considered to be one of the primary mechanisms
by which antimicrobial resistance genes are transferred
between bacteria. The paper focuses on integrons in
poultry production and processing and in natural ecosystems.
INTRODUCTION
The use of antimicrobials as feed supplements is a
common practice in livestock production. Studies suggest that there may be links between this practice and
the development of antimicrobial resistance among the
microflora of these animals, especially the enteric bacterial pathogens (Aarestrup, 1995; Witte, 1998; Smith et
al., 1999; Wegener et al., 1999; Endtz et al., 1990; McEwen
and Fedorka-Cray, 2002). Antimicrobial resistance in
poultry-related pathogens is of concern because many
of these compounds are no longer useful for treating
bacterial infections in poultry (Bass et al., 1999; Schwarz
and Chaslus-Dancla, 2001). Even though animal growers
argue that the use of antimicrobials in animal feed has
beneficial effects such as promoting growth and reducing cost, regulatory agencies such as the United States
Food and Drug Administration, the Centers for Disease
Control and Prevention, and the World Health Organization all argue that sub-therapuetic use of antimicrobials in animal feed should be restricted (Feinman, 1998;
Harrison and Lederberg, 1998).
ANTIMICROBIAL RESISTANCE
MECHANISMS
Microbial populations develop resistance to antimicrobials through several mechanisms. The rate at which
an individual gene mutates to express an antimicrobial
resistance phenotype is a complex phenomenon in
which environment, cell physiology, bacterial genetics,
and population dynamics all play roles (Martinez and
Baquero, 2000). In addition, for full resistance to occur,
mutations must develop within multiple genes because
of genetic redundancy in the antimicrobial targets. A
primary example of this is the fluoroquinolone target
gene gyrA, gyrB, parA, and parC that are all targets for
the fluoroquinolone antimicrobial, and all must have
mutations for full resistance phenotypes to develop
(Martinez and Baquero, 2000). Bacteria can also acquire
antimicrobial resistance genes through horizontal or lateral gene transfer.
Bacterial cells can acquire genetic sequences from
other organisms through several processes. First, there
is the take up of naked DNA from their immediate sur-
2003 Poultry Science Association, Inc.
Received for publication October 21, 2002.
Accepted for publication December 18, 2002.
1
To whom correspondence should be addressed: spillai@poultry.
tamu.edu.
622
SYMPOSIUM: USE OF ANTIMICROBIALS IN PRODUCTION
roundings by a process termed transformation. The frequency with which bacteria acquire DNA from the environment depends on several factors including cell wall
structure and bacterial species with transfer frequencies
being as low as 10−7. Bacterial cells have to be “competent” to acquire extraneous DNA by transformation.
Bacteria such as Campylobacter are thought to be naturally competent. The process of transformation is complex, and there are differences in the process among
gram-positive and gram-negative bacteria. The process
involves the specific recognition sequences in order for
the new DNA to be taken up by the bacteria (Elkins et
al., 1991).
Bacteria can also exchange and acquire genetic material through conjugation of self-replicating extra chromosomal DNA or plasmids. This requires physical contact between cells, which allows the plasmids to be exchanged between donor and recipient cells. An example
of this is the floR gene encoding florofenicol resistance
in Escherichia coli found in cattle (Cloeckaert et al., 2000).
The floR antimicrobial resistance gene encodes a 1,122
amino acid protein that confers resistance to florofenicol
through an unknown mechanism (Arcangioli et al.,
1999).
A third mechanism of horizontal gene transfer is the
introduction of genetic material into a bacterium by a
bacteriophage, or transduction. In this method, the virus
attaches and injects its own nucleic acids to the bacterial
cell, which in some cases facilitates the introduction of
new genes into the bacterial genome. This concept has
been used in molecular cloning by introducing vector
DNA, including antimicrobial resistance, through bacteriophage λ for some time (Sambrook et al., 1989).
Transposons, which are genetic elements conferring
a selectable phenotype flanked by two insertion sequences, are involved in horizontal gene transfer events
between bacteria. Transposons are unique in that they
have the ability of excising themselves from one genetic
locus and moving to another, whether it is within the
same bacteria or bacteria in another taxa (Ochman et
al., 2000). Transposons can be transferred through all
of the methods mentioned above namely, conjugation,
transformation, and transduction. Transposons play a
significant role in antimicrobial resistance development
because they often contain gene sequences mediating
antimicrobial resistance called integron gene sequences.
Integrons are thought to play a significant role in the
rapid dissemination of antimicrobial resistance among
bacteria (Ochman et al., 2000).
INTEGRONS AND ANTIMICROBIAL
RESISTANCE
First identified by Stokes and Hall (1989), the integron
gene sequence has become identified as a primary
method by which bacteria acquire antimicrobial resistance. The integron gene sequence is unique in that it
acts as a site-specific recombination system capable of
capturing or excising novel genetic elements called gene
623
cassettes. These gene cassettes are promoterless genes
with a recombination site known as a 59-base element
located downstream of the gene. The gene cassette codes
for a wide range of antimicrobial resistant determinants
(Recchia and Hall, 1995). Integrons are also thought to
play a role in the transfer of pathogenic elements (Mazel
et al., 1998). These circular gene cassettes are exchanged
between bacteria and are linearized by the integrase
enzyme before being inserted at the integration site.
Multiple gene cassettes can be inserted into the integron
gene sequence to confer a multiple antimicrobial resistant phenotype to the bacteria (Hall and Collis, 1995;
Rowe-Magnus and Mazel, 1999; Ochman et al., 2000).
The integron has several unique genetic components
that allow them to insert gene cassettes. One is the 5′
intI gene, which codes for the site-specific recombinase
(integrase) enzyme. The second is the Pant promoter site
that acts as the promoter governing inserted gene cassettes. The third unique element is the attI, which is an
inverted repeated sequence, that is the recombination
site for the lineraized gene cassettes (Stokes and Hall,
1989; Hall and Collis, 1995; Rowe-Magnus and Mazel,
1999). Multiple gene cassettes can be integrated at one
locus on the chromosome or on a plasmid to form the
integron gene sequence array (Recchia and Hall, 1995,
1997).
Four different classes of integrons, namely class 1,
class 2, class 3, and class 4, have been described to date,
each of which has several distinctive traits (Hall and
Collis, 1995; Mazel et al., 1998). The four classes of integrons differ primarily by the sequence of their integrase
gene. The integrase genes of class 2 and class 3 integrons
(intI2 and intI3) share only 45% and 60% amino acid
similarity respectively with the class 1 integrase, intI1
(Hall and Collis, 1995). The class 1 integron designated
In2 is most commonly associated with the Tn21 transposon (Liebert et al., 1999) and contains a 3′ conserved
sequence composed of the qacE∆1 and sul1 resistance
genes with an unknown open reading frame, orf5, located further downstream. In the class 1 integron In16
located on transposon Tn402, however, the 3′ conserved
segment genes qacE∆1, sul1, and orf5 are replaced by
the tni genes (Partridge et al., 2001).
Class 2 integrons are commonly associated with the
Tn7 transposon and its derivatives all of which contain
a 3′ conserved segment composed of the tns gene module
which contain five genes that encode products involved
in the movement of the transposon. The class 3 integron
has only been identified in Serratia marcescens and is not
commonly associated with a transposon carrier (Arakawa et al., 1995). The class 4 integron gene sequence
has been classified only in Vibrio cholerae genome and
is associated with the Vibrio cholerae repeated sequences
(VCR) but not a transposon sequence (Mazel et al., 1998).
INTEGRONS IN POULTRY PROCESSING
AND RETAIL CHICKEN PRODUCTS
Hofacre et al. (2001) have linked multiple antimicrobial resistance phenotype in bacteria isolated from ani-
624
ROE AND PILLAI
mal feed to integron sequences. They isolated gramnegative bacterial isolates from 165 rendered animal
products (meat, bone, fish, and feather meal), which
were used as protein sources for animal feed and determined their resistance patterns. The colonies were
probed with DNA probes specific to the intI1 gene coding for the class 1 integrase. Resistance profiles showed
that multiple antimicrobial resistance (resistance to five
or more antimicrobials tested) was observed in three of
the 36 isolates tested (8%) among the gram-negative
isolates. Resistance was more prevalent in meat and
bone meal than in feather meal. The class 1 integrase
gene was found in nine of the 24 isolates (37%).
Integrons have also been shown to be associated with
multiple antimicrobial resistance in isolates taken from
on-farm poultry (Bass et al., 1999). This study utilized
100 bacterial isolates of pathogenic E. coli of poultry
origin, which were screened for antimicrobial resistance
to the four major families of antimicrobials, namely βLactams, chloramphenicols, tetracyclines, and aminoglycosides. They reported that class 1 integrons were
found in 63% of multiple antimicrobial-resistant E. coli
of poultry origin. The isolates were also screened for
the presence of the genes intI1 and qacE∆1 using DNAspecific probes. Furthermore, they identified the presence of the aadA1 (aminoglycoside resistance) and merA
(mercury resistance) gene cassettes in 50% of the isolates.
They acknowledged that the antimicrobial resistance
phenotypes of the isolates could not be explained by the
class 1 integron gene sequences solely. Because merA is
harbored on the transposon Tn21, the authors suggested
that the class 1 integrons are actually components on a
Tn21 like transposon placing the class 1 integrons in the
In2 family.
Goldstein et al. (2001) examined the prevalence of
class 1 and class 2 integrons in 588 bacterial isolates
from clinical and poultry isolates using specific DNA
probes. Multiple drug resistance phenotype (resistant
to five or more antimicrobials tested) was evident in
56% (64/115) of the isolates. Of the poultry isolates
tested, 73% possessed the class 1 integrase gene intI1.
The class 2 integrase gene intI2 was found in 8% of
the isolates, and the intI1 and intI2 genes were found
together in 7% of the isolates. The class 3 integrase gene
was only found in a total of 12% of all isolates that
contained an integrase gene. To confirm the class 3 integrase presence, the authors utilized DNA PCR to amplify
the intI3 gene; however, the isolates did not amplify.
Class 4 integron genes were not found in any of the
isolates tested.
Hudson et al. (2000) examined the antimicrobial resistance profiles and the prevalence of the class 1 integron
genes intI1, merA, and aadA1 within 22 isolates of Salmonella enterica serovar Typhimurium DT104 in non-domesticated birds (birds of the passerine species such as
cowbirds, and English sparrows, which are not grown
as companion animals or for food). By using probes
specific for the class 1 integron associated genes, namely
intI1, aadA1, and merA, they found that these genes were
present in various combinations in 41% (9/22) of isolates examined.
White et al. (2001) examined the prevalence of antimicrobial-resistant Salmonella spp. from retail ground
meats. Out of 200 meat samples that were tested, 22%
contained Salmonella spp., and 53% of these isolates were
resistant to three or more antimicrobials. When the sulfamethoxazole-resistant isolates were screened for the
class 1 integron gene sequence (because class 1 integrons
typically contain the sul1 gene mediating sulfamethoxazole resistance), most them had integrons with genes
mediating resistance to aminoglycosides, sulfonamides,
trimethoprim, and β-lactams.
Lucey et al. (2000) studied the contribution of class 1
integron in Campylobacter spp. by using isolates from
poultry and porcine carcass samples collected at slaughter. Most (62.3%) of the isolates were resistant to sulfonamide. Using PCR analysis, they found that 40% (40/
55) of the isolates tested contained the class 1 integron.
Individual amplicons ranging in size from 230 bp to 1.47
kb were detected with a 350-bp fragment common to
all but one of the isolates tested. Several amplified ‘gene
cassette patterns’ or variable regions were identified
from each isolate, and the sul1 and qacE∆1 were also
identified using PCR amplification to confirm their presence in these isolates.
INTEGRONS IN NATURAL ECOSYSTEMS
The occurrence of integrons in natural ecosystems is
important because the environment apparently serves
as a source and sink for agriculture. However, studies
on the prevalence of the integron gene sequence within
natural environments have been limited to date. Rosser
and Young (1999) examined the prevalence and characterization of the class 1 integron gene sequence in bacteria from an estuary of the Tay and Earn River west of
Dundee, United Kingdom. In this study, 3,000 gramnegative isolates were collected over 2 mo. The colonies
were analyzed using colony blot analysis with DNA
probes targeted against the class 1 integrase gene intI1
and the quaternary ammonium compound resistance
gene qacE∆1, both of which are linked to the class 1
integron. Of the 3,000 isolates screened for the class 1
integron gene sequence, 3.6% of the isolates hybridized
to the intI1 probe using the colony blot hybridization
technique.
Eighty-five isolates containing the class 1 integron
were examined for other gene cassettes using PCR amplification utilizing primers for gene cassettes commonly found in class 1 integron variable regions. Amplified cassettes were cloned and sequenced to map the
variable region. The authors found broad diversity of
gene cassettes present in the variable regions studied
including the aadA1 and aadA2 gene cassettes, which
encode resistance to streptomycin and spectinomycin
antimicrobials. Of the 85 integron-positive isolates that
were analyzed, 26 were Pseudomonas spp., 28 were Vibrio
spp., and 31 were coliforms. This study illustrated that
SYMPOSIUM: USE OF ANTIMICROBIALS IN PRODUCTION
class 1 integrons are present in natural surface waters
and are distributed among a variety of bacterial genera.
Integron sequences have also been isolated from soils
(Nield et al., 2001). Using culture-independent methods,
they detected several novel integrase genes. However,
complete gene cassettes were not identified.
In studies in our laboratory, out of 322 E. coli isolates
originally isolated from irrigation water and associated
sediments, 32 isolates (10%) were resistant to two or
more antimicrobial compounds. Of these 32 multidrugresistant isolates, four isolates (13%) contained class 1specific integron sequences (class 1 integron variable
region and the class 1 integrase genes). Only one isolate
contained class 2 integron-specific sequences (class II
integron variable and class II integrase genes). Nucleotide sequence analysis showed that the class I integron
bearing strains contained a conserved 780-bp aadA gene
cassette, whereas the class II integron bearing strain contained two distinct gene cassettes, sat-1 and aadA. Even
though all integron-bearing strains harbored the aadA
sequence, none of the strains were resistant to streptomycin. The results suggest that irrigation water and sediments contaminated with fecal waste can be sources of
class 1 and class 2 integron-bearing Escherichia coli and
that environmental factors can cause a lack of phenotypic expression in these strains (Roe, unpublished
data). Pillai and Pepper (1990) in their work with transposable elements had also reported on this similar lack
of phenotypic expression in bacterial cells in cells, even
when the genotypic potential exists.
In conclusion, studies suggest that poultry products at
the farm, processing, and retail levels harbor antibioticresistant organisms and the genetic determinants that
could potentially disseminate these genes. Relying
solely on the phenotypic characterization of antibiotic
resistance may result in underestimation of the true genetic potential of bacteria to resist antimicrobial compounds. Culture-independent assays such as analysis of
total microbial community nucleic acids for resistancecoding genes will provide a better picture of the genetic
potential of a microbial community to acquire or transmit resistance genes. Nucleotide sequence analysis of
the antimicrobial resistance gene determinants will help
identify whether certain agronomic or processing methods are selecting for specific genotypes.
It is critical for the poultry, beef, and porcine industries to understand the underlying mechanistic processes of the dissemination of antimicrobial resistance
during preharvest and processing of their products. Possible research areas include the role of quorum sensing
on exposure to sub-therapeutic antibiotic concentrations
and acquisition of antibiotic resistance and the impacts(s) that specific poultry processing steps on retarding the transfer or resistance determinants (Lu et
al., 2003). Current agronomic practices may need to be
optimized or modified to limit the dissemination of antimicrobial gene cassettes and yet maintain an economically viable poultry industry.
625
ACKNOWLEDGMENTS
This work was supported by the USDA/CSREES project 2001-34461-10405 and the Texas Agricultural Experiment Station Hatch project H-8708.
REFERENCES
Aarestrup, F. M. 1995. Occurrence of glycopeptide resistance
among Enterococcus faecium isolates from conventional and
ecological poultry farms. Microb. Drug Resist. 1:255–257.
Arakawa, Y., M. Murakami, K. Suzuki, H. Ito, R. Wacharotayankun, S. Ohsuka, N. Kato, and M. Ohta. 1995. A novel
integron-like element carrying the metallo-beta-lactamase
gene blaIMP. Antimicrob. Agents Chemother. 39:1612–1615.
Arcangioli, M. A., S. Leroy-Setrin, J. L. Martel, and E. ChaslusDancla. 1999. A new chloramphenicol and florfenicol resistance gene flanked by two integron structures in Salmonella
typhimurium DT104. FEMS Microbiol. Lett. 174:327–332.
Bass, L., C. A. Liebert, M. D. Lee, A. O. Summers, D. G. White,
S. G. Thayer, and J. J. Maurer. 1999. Incidence and characterization of integrons, genetic elements mediating multipledrug resistance, in avian Escherichia coli. Antimicrob. Agents
Chemother. 43:2925–2929.
Cloeckaert, A., S. Baucheron, G. Flaujac, S. Schwarz, C. Kehrenberg, J. L. Martel, and E. Chaslus-Dancla. 2000. Plasmidmediated florfenicol resistance encoded by the floR gene
in Escherichia coli isolated from cattle. Antimicrob. Agents
Chemother. 44:2858–2860.
Elkins, C., C. E. Thomas, H. S. Seifert, and P. F. Sparling. 1991.
Species-specific uptake of DNA by gonococci is mediated
by a 10-base-pair sequence. J. Bacteriol. 173:3911–3913.
Endtz, H. P., R. P. Mouton, T. van der Reyden, G. J. Ruijs,
M. Biever, and B. van Klingeren. 1990. Fluoroquinolone
resistance in Campylobacter spp. isolated from human stools
and poultry products. Lancet 335:787.
Feinman, S. E. 1998. Antibiotics in animal feed-drug resistance
revisited. ASM News 64:24–30.
Goldstein, C., M. D. Lee, S. Sanchez, C. Hudson, B. Phillips,
B. Register, M. Grady, C. Liebert, A. O. Summers, D. G.
White, and J. J. Maurer. 2001. Incidence of class 1 and 2
integrases in clinical and commensal bacteria from livestock, companion animals, and exotics. Antimicrob. Agents
Chemother. 45:723–726.
Lu, L., M. E. Hume, and S. D. Pillai. 2003. Quorum sensing
in Escherichia coli in response to subtherapeutic antibiotic
exposure. 103rd General Meetings of the American Society
for Microbiology. American Society for Microbiology,
Washington, DC.
Hall, R. M., and C. M. Collis. 1995. Mobile gene cassettes
and integrons: Capture and spread of genes by site-specific
recombination. Mol. Microbiol. 15:593–600.
Hofacre, C. L., D. G. White, J. J. Maurer, C. Morales, C. Lobsinger, and C. Hudson. 2001. Characterization of antibioticresistant bacteria in rendered animal products. Avian Dis.
45:953–961.
Harrison, P. F., and J. Lederberg. 1998. Antimicrobial resistance: issues and options. Page 128 in Forum on Emerging
Infections. Institute of Medicine, National Academy Press,
Washington, DC.
Hudson, C. R., C. Quist, M. D. Lee, K. Keyes, S. V. Dodson,
C. Morales, S. Sanchez, D. G. White, and J. J. Maurer. 2000.
Genetic relatedness of Salmonella isolates from nondomestic
birds in southeastern United States. J. Clin. Microbiol.
38:1860–1865.
Liebert, C. A., R. M. Hall, and A. O. Summers. 1999. Transposon Tn21, flagship of the floating genome. Microbiol. Mol.
Biol. Rev. 63:507–522.
Lucey, B., D. Crowley, P. Moloney, B. Cryan, M. Daly, F. O’Halloran, E. J. Threlfall, and S. Fanning. 2000. Integronlike
626
ROE AND PILLAI
structures in Campylobacter spp. of human and animal origin. Emerg. Infect. Dis. 6:50–55.
Martinez, J. L., and F. Baquero. 2000. Mutation frequencies
and antibiotic resistance. Antimicrob. Agents Chemother.
44:1771–1777.
Mazel, D., B. Dychinco, V. A. Webb, and J. Davies. 1998. A
distinctive class of integron in the Vibrio cholerae genome.
Science 280:605–608.
McEwen, S. A., and P. J. Fedorka-Cray. 2002. Antimicrobial
use and resistance in animals. Clin. Infect. Dis. 34(Suppl.
3):S93–S106.
Nield, B. S., A. J. Holmes, M. R. Gillings, G. D. Recchia, B.
C. Mabbutt, K. M. Nevalainen, and H. W. Stokes. 2001.
Recovery of new integron classes from environmental
DNA. FEMS Microbiol. Lett. 195:59–65.
Ochman, H., J. G. Lawrence, and E. A. Groisman. 2000. Lateral
gene transfer and the nature of bacterial innovation. Nature
405:299–304.
Partridge, S. R., H. J. Brown, H. W. Stokes, and R. M. Hall.
2001. Transposons Tn1696 and Tn21 and their integrons
In4 and In2 have independent origins. Antimicrob. Agents
Chemother. 45:1263–1270.
Pillai, S. D., and I. L. Pepper. 1990. Transposon Tn5 as an
identifiable marker in rhizobia: Survival and genetic stability of Tn5 mutant bean rhizobia under temperature stressed
conditions in desert soils. Microb. Ecol. 21:21–33.
Recchia, G. D., and R. M. Hall. 1995. Gene cassettes: A new
class of mobile element. Microbiology 141:3015–3027.
Recchia, G. D., and R. M. Hall. 1997. Origins of the mobile gene
cassettes found in integrons. Trends Microbiol. 5:389–394.
Rosser, S. J., and H. K. Young. 1999. Identification and characterization of class 1 integrons in bacteria from an aquatic
environment. J. Antimicrob. Chemother. 44:11–18.
Rowe-Magnus, D. A., and D. Mazel. 1999. Resistance gene
capture. Curr. Opinion Microbiol. 2:483–488.
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular
Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor
Laboratory Press, New York.
Schwarz, S., and E. Chaslus-Dancla. 2001. Use of antimicrobials
in veterinary medicine and mechanisms of resistance. Vet.
Res. 32:201–225.
Smith, K. E., J. M. Besser, C. W. Hedberg, F. T. Leano, J. B.
Bender, J. H. Wicklund, B. P. Johnson, K. A. Moore, and
M. T. Osterholm. 1999. Quinolone-resistant Campylobacter
jejuni infections in Minnesota, 1992–1998. Investigation
Team. N. Eng. J. Med. 340:1525–1532.
Stokes, H. W., and R. M. Hall. 1989. A novel family of potentially mobile DNA elements encoding site-specific geneintegration functions: integrons. Mol. Microbiol. 3:1669–
1683.
Wegener, H. C., F. M. Aarestrup, L. B. Jensen, A. M. Hammerum, and F. Bager. 1999. Use of antimicrobial growth
promoters in food animals and Enterococcus faecium resistance to therapeutic antimicrobial drugs in Europe. Emerg.
Infect. Dis. 5:329–335.
White, D. G., S. Zhao, R. Sudler, S. Ayers, S. Friedman, S.
Chen, P. F. McDermott, S. McDermott, D. D. Wagner, and
J. Meng. 2001. The isolation of antibiotic-resistant Salmonella
from retail ground meats. N. Eng. J. Med. 345:1147–1154.
Witte, W. 1998. Medical consequences of antibiotic use in agriculture. Science 279:996–997.