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Antimicrobial Agents and Chemotherapy, March 2001, p. 723-726, Vol. 45, No. 3
Department of Medical Microbiology and
Parasitology,1 Avian
Medicine,2 and
Microbiology,3 The University of
Georgia, Athens, Georgia 30602, and Center for Veterinary
Medicine, Food and Drug Administration, Laurel, Maryland
207084
Received 23 May 2000/Returned for modification 11 September
2000/Accepted 7 December 2000
Many pathogenic and commensal organisms are multidrug resistant due
to exposure to various antibiotics. Often, this antimicrobial resistance is encoded by integrons that occur on plasmids or that are
integrated into the bacterial chromosome. Integrons are commonly associated with bacterial genera in the family
Enterobacteriaceae. We determined that class 1 integrases
were present in approximately 46% of the isolates from the family
Enterobacteriaceae; class 2 integrases were present only
among Escherichia coli and Salmonella isolates.
Seven percent of veterinary isolates were positive for class 3 integrase by DNA-DNA hybridization but could not be confirmed to be
positive by PCR. None of the veterinary isolates possessed the class 4 integrase gene. The distribution of these integrase genes was variable
within the members of the family Enterobacteriaceae when
some or all integrase classes were absent from a particular genus.
There was also considerable variability in the distribution of these
integrases within a species, depending on the animal host. Unlike the
class 1 integrases, the other integrase class, intI2,
appears to be more restricted in its distribution among the members of
the family Enterobacteriaceae. There is also considerable variability in the distribution of the class 1 integrases within E. coli strains isolated from different food animals. The
class 1 integrases are the most widely disseminated of the four classes among the members of the family Enterobacteriaceae from
both the clinical and normal flora of animals. This is the first report to closely examine the distribution of class 2 integrases in members of
the family Enterobacteriaceae isolated in the United States.
Transfer of antibiotic resistance
genes between different species of bacteria can be facilitated by
mobile DNA elements, such as transposons and plasmids. Integrons have
been identified on these mobile elements, and they often contain one or
more genes that encode antibiotic resistance (29). Four
classes of integrons have been described. The structure of the class 1 integron includes a 5' and 3' conserved segment (with respect to the
antibiotic resistance cassette insertion point) and a variable region
(15, 17, 21, 29). The 5' conserved segment consists of the
intI gene (integrase) and a promoter region expressing the
inserted gene(s) (29). The 3' conserved segment contains
the defective quaternary ammonium resistance gene qacE The family Enterobacteriaceae contains genera that inhabit
the intestinal tracts of humans and animals. These include both nonpathogenic species (the commensals) and pathogenic species (11). Because many species of this family coexist in the
intestinal tract and are frequently exposed to various antibiotics,
there is potential for the dissemination of antibiotic resistance
genes. Therefore, the presence of integrons in bacterial species that constitute the family Enterobacteriaceae is of interest with
respect to the growing dilemma of antibiotic resistance. We examined
the distribution of class 1 to 4 integrases among veterinary bacterial strains isolated from livestock, companion animals, and exotics.
Bacterial strains.
The bacterial isolates used in the
present study are members of various species of the following genera:
Citrobacter, Edwardsiella, Enterobacter, Escherichia (the species
Escherichia coli only), Hafnia,
Klebsiella, Proteus, Salmonella, and
Yersinia. Several isolates were obtained from archived
diagnostic specimens cultured in the 1980s and 1990s.
Edwardsiella tarda, Edwardsiella ictaluri, and
Yersinia ruckerii were isolated from diseased fish. Avian E. coli isolates were isolated from the tracheas, lungs, air
sacs, livers, and spleens of diseased chickens, quail, ostriches, and turkeys. Pathogenic E. coli strains were isolated from swine
and cattle with clinical cases of infection; the strains had been submitted to the North Dakota State University diagnostic laboratory. Avian Salmonella strains were isolated from either carcass
rinses or drag swabs of poultry houses in Georgia. Reptile
Salmonella strains were acquired from captive reptiles at
zoos in the southeastern United States.
Colony blots.
Probes for the genes of interest were created
by PCR with digoxigenin-labeled nucleotides by the procedure of Bass et
al. (2, 16, 33). The Molecular Genetics Instrumentation
Facility at the University of Georgia synthesized the oligonucleotides (Table 1). The following bacterial
strains served as positive controls and as templates for the generation
of PCR probes for DNA-DNA hybridization: E. coli
SK1593(pDU202), (intI1+) (32),
E. coli J53.3::Tn7
(intI2+), S. marcescens AK9373
(intI3+) (1), and V. cholerae (intI4+) (18). The
identity of the PCR product was confirmed by sequencing. All PCR
amplification products had 99% identity to the target gene sequences
that the primers were originally designed to amplify. DNA-DNA
hybridizations were performed as described by Sambrook et al.
(26), with hybridizations and washes done at 68°C for all probes with the exception of the intI2 probe, for which
a temperature of (60°C) was used. Hybridization was detected with the
antidigoxigenin antibody-alkaline phosphatase conjugate provided with
the Genius System (Boehringer Mannheim).
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.3.723-726.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Incidence of Class 1 and 2 Integrases in Clinical
and Commensal Bacteria from Livestock, Companion Animals, and
Exotics
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
1
and the sul1 gene, which encodes resistance to sulfonamides
(21, 29). The variable region, located between the two
conserved segments, is the site for the insertion of antibiotic
resistance gene cassettes and includes aatC (also known as
the 59-base element), which participates in the recombination mechanism
(7, 10, 30). Class 1 integrons have also been identified
on transposable elements such as mercury resistance transposon
Tn21 (8). Class 2 integrons are present on
transposon Tn7 and contain a dihydrofolate reductase gene
cassette (6, 29). Class 3 integrons have recently been
characterized in Serratia marcescens, isolated in Japan, by
the identification of the blaIMP gene, which
encodes broad-spectrum 
-lactam antibiotic resistance. The
blaIMP gene is flanked by a 59-base element and an integrase-like gene and the aac(6')-Ib gene, which
encodes aminoglycoside resistance (1). Class 4 integrons
have been characterized only in Vibrio cholerae to date.
This novel class contains the intI4 gene, which encodes a
new integrase which makes tandem arrays of V. cholerae
repeated sequences similar to the arrays of antibiotic resistance gene
cassettes found in class 1 integrons (18).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
TABLE 1.
PCR primers
Antimicrobial susceptibility determination. The antimicrobial MICs for the bacterial isolates tested were determined with the Sensititre automated antimicrobial susceptibility system (Trek Diagnostic Systems, Westlake, Ohio), and the results were interpreted according to National Committee Clinical Laboratory Standards guidelines for broth microdilution methods (19, 20). Sensititre susceptibility testing was performed according to the manufacturer's instructions. The following antimicrobials were assayed: ampicillin, chloramphenicol, gentamicin, kanamycin, streptomycin, sulfamethoxazole, and tetracycline.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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We examined the susceptibilities of veterinary isolates to a
battery of antibiotics including ampicillin, chloramphenicol, gentamicin, kanamycin, streptomycin, sulfamethoxazole, and
tetracycline. Veterinary E. coli isolates were generally
resistant to five or more antibiotics, while other gram-negative
enteric organisms varied in their patterns of susceptibility to these
antibiotics (Table 2). Isolates belonging
to the genus Hafnia were susceptible to all seven
antibiotics tested. We had previously determined that multiple-drug
resistance exhibited by avian E. coli isolates correlated
with the incidence of class 1 integrons in this population (2). Could the differences in antibiotic susceptibilities
observed among our veterinary isolates be due to the prevalence of
integrons among the members of the family
Enterobacteriaceae?
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Five hundred eighty-eight organisms (representing nine bacterial
genera) were screened for the presence of class 1 through 4 integrases.
Two hundred ninety-four (52%) of the 555 Enterobacteriaceae isolates contained either class 1 or class 2 integrases (Table 3). Class 1 integrases were the dominant
class identified, with 271 (92%) of these 294 integrase-positive
isolates having intI1 (Table 3). Seventy-three and 61% of
the E. coli and Salmonella isolates,
respectively, contained class 1 integrases, while only 10 and 13% of
Hafnia and Enterobacter isolates (two closely
related commensal genera), respectively, were positive for
intI1. Integrase genes were not identified in any of the
aquatic pathogens (Table 3).
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Forty (14%) of the 294 integrase-positive isolates of the family Enterobacteriaceae had the class 2 integrase. The class 2 integrases were restricted in their distributions, being identified only in E. coli and Salmonella isolates. Among the Salmonella isolates, the incidence of intI1 ranged from 17% (reptile) to 79% (poultry), depending on the source of the isolate. Class 2 integrases were also variable in their distributions among the Salmonella isolates, with 2% of poultry isolates and 17% of reptile isolates being positive for intI2. As for the Salmonella isolates, the distribution of the class 2 integrases was variable in E. coli, depending on the animal source of the isolate. E. coli strains isolated from swine and companion animals were negative for intI2, but this gene was present in 14 and 23% of the E. coli strains from poultry and beef cattle, respectively. None of the veterinary isolates tested contained the class 4 integrase gene (intI4). The PCR amplification products originally used as DNA probes for Southern were confirmed through DNA sequencing of these PCR amplicons. The DNA sequences of the intI2 PCR amplicons from three veterinary isolates, an avian E. coli isolate, a bovine E. coli isolate, and an avian Salmonella isolate, (Genbank accession nos. AF318070, AF318071, and AF318072, respectively), also had 99% identities to the sequence of the intI2 integrase present in Tn7.
Thirty-four (12%) of the 294-integrase positive Enterobacteriaceae isolates were found to be positive for intI3 by DNA-DNA hybridization. When several isolates, identified by DNA-DNA hybridization as having intI3, were screened by PCR with intI3-specific primers, only the control produced the expected 600-bp PCR amplicon. It is possible that a single mismatch involving the terminal 3' nucleotide of either the forward or the reverse primer and its target might prohibit PCR detection of the intI3 genes in these isolates (5). This is especially relevant with the recent report of a hybrid class 2 integrase in Acinetobacter baumannii (24). We may also be dealing with a variant hybrid class 3 integrase. We are in the process of cloning and sequencing DNA fragments that hybridize with the intI3-specific probe to resolve the discrepancy in our results between the DNA-DNA hybridizations and PCR.
Several isolates possessed multiple integrase classes. Class 1 and 2 integrases were present together in E. coli (10%) and Salmonella (2%) isolates. Interestingly, class 1 and 2 integrases were most prevalent among veterinary E. coli isolates that displayed resistance to five or more antibiotics.
Class 1 integrons and integrases have been identified in a number of different bacterial genera and appear to be prevalent in nature (2, 3, 22, 28, 31, 34). We found that the same bacterial genera or species associated with the normal flora in animals contained the same class 1 integrase gene as their human counterpart bacteria (4, 9, 23, 25). Our isolates of Edwardsiella and Yersinia were negative for the class 1 integrase gene. This is in contrast to a study by Petersen et al. (22), who reported class 1 integrases among bacteria isolated from trout. The lack of integrases in these two fish pathogens may be due to differences in bacterial species, host species, geography, or fish management practices with regard to antibiotic usage. The dissemination of the class 1 integrons, in many instances, has been attributed to the spread of an integron-containing transposon, Tn21 (2, 34). We have recently found that class 1 integrons in avian E. coli strains contain a single antibiotic resistance gene, aadA1 (streptomycin-spectinomycin resistance), and that this integron is part of a Tn21-like element (2). However, there are clearly cases in which class 1 integrons contain other resistance genes (9, 14, 23, 27) and map to plasmids (25, 31) or transposons other than Tn21 (14). The nature of the class 1 integrons in many of these veterinary isolates is not known, but we do expect to find differences due to the dissimilarities in both the animal microflora and the therapeutic as well as prophylactic usage of antibiotics among these different animal species. It is interesting that several bacterial isolates from nondomestic, free-ranging animals contained integrases. We have observed similar results for Salmonella strains isolated from wild, nondomestic birds in the southeastern United States (12). Does the presence of these integrases and integrons represent the natural frequency of these mobile elements in nature, or does it signify human contamination of the environment?
The low incidence of intI2 in select veterinary isolates (6%) was similar to the distribution of the class 2 integron (Tn7) in human, urinary isolates of E. coli, Enterobacter spp., and Klebsiella spp. examined (3). The distribution of Tn7 in clinical isolates correlates with the frequency of trimethoprim resistance (3, 13). Trimethoprim resistance is due to the dihydrofolate reductase enzyme encoded by the dhfr gene in Tn7 (6). Differences in the frequency of intI2 among our veterinary isolates might be attributed to differences in the prevalence of Tn7 and usage of the antibiotic trimethoprim or the related veterinary analogue ormetoprim.
To the best of our knowledge this is the first study to look at the distributions of two new classes of integrases, class 3 and 4 integrases, in a large-scale study. None of the veterinary isolates assayed contained the class 4 integrase originally identified in V. cholerae (18). This integron therefore appears to be more limited in its distribution. The identification of three classes of integrases in multiple bacterial genera comprising both pathogenic and commensal organisms indicates that integrons have the capability to disseminate antimicrobial resistance genes within bacteria associated with diverse animal production environments. We are currently characterizing the integrons associated with both commensal and pathogenic bacteria such as Salmonella to determine whether the animal's microbiota can serve as a reservoir for antimicrobial resistance genes.
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ACKNOWLEDGMENTS |
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The National Science Foundation's Prokaryotic Diversity Research Training Grant at the University of Georgia made this work possible. The Wallace Research Foundation and the International Academy of Oral Medicine and Toxicology support work in the laboratory of A.O.S. USDA NRICGP grant 99-35212-8680 supports work in the laboratory of J.J.M.
We thank Komanci Payne, Kathleen Keyes, and Amy Stallings for participation in the preparation of the PCR probes used in this study. We also thank Marie Maier for technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Avian Medicine, The University of Georgia, Athens, GA 30602. Phone: (706) 542-5071. Fax: (706) 542-5630. E-mail: jmaurer{at}calc.vet.uga.edu.
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