FOODBORNE PATHOGENS AND DISEASE Volume 6, Number 5, 2009 ª Mary Ann Liebert, Inc. DOI: 10.1089=fpd.2008.0204
Molecular Characterization of Tetracycline-Resistant Genes and Integrons from Avirulent Strains of Escherichia coli Isolated from Catfish Mohamed Nawaz,1 Ashraf A. Khan,1 Saeed Khan,1 Kidon Sung,1 Khalil Kerdahi,2 and Roger Steele1
Abstract
A study was undertaken to investigate the occurrence of tetracycline-resistant genes and to characterize the integrons present in Escherichia coli isolated from catfish. Sixty-three tetracycline-resistant E. coli strains were isolated from the intestinal contents of 407 farm-raised catfish. All strains were resistant to multiple antibiotics. A polymerase chain reaction (PCR) assay detected tetA in the DNA of 15 of 63 (25.0%) isolates by amplifying a PCR amplicon measuring 957 bp. Oligonucleotide primers targeting a 436-bp region of tetB successfully amplified a PCR amplicon from 47 of 63 (77.0%) isolates, indicating that tetB was predominant. Oligonucleotide primers specific for tetC amplified a 589-bp PCR amplicon from 3 of 63 (5%) isolates. Eleven (17.0%) of the isolates contained both tetA and tetB genes. Class I integrons amplified from the genomic DNA of 14 of 63 (22.0%) isolates measured 1.6 and 1.8 kb. Sequence analysis of the 1.6 kb integrons indicated the presence of three different gene cassettes: a dfrA12, conferring resistance to trimethoprim; an open reading frame, orfF, a hypothetical protein of unknown function; and aadA2, conferring resistance to aminoglycosides. Sequence analysis of the 1.8-kb integron indicated the presence of dfrA17 and aadA5. PCR assays for the detection of the six predominant virulence genes failed to amplify any genes from the genomic DNA. Pulsed-field gel electrophoresis using XbaI identified 16 distinct macro restriction patterns among the 63 isolates. The dendrogram analysis indicated that the DNA from 4 of 16 isolates had a similarity index of 90.0%. Our results indicate that the use of oxytetracycline and Romet 30 (sulfadimethoxine and ormetoprim) in farm-raised catfish may select for multiple antibiotic–resistant E. coli that could serve as a reservoir of tetracycline, trimethoprim, and aminoglycoside resistance genes.
Introduction
S
eafood is an integral part of good human nutrition and the demand for these products has increased in the United States (Lipp and Rose, 1997). Ingestion of contaminated seafood has caused many foodborne disease outbreaks in the United States (Lipp and Rose, 1997; Feldhusen 2000). Although pathogenic Salmonella, Listeria, and Vibrio strains have been characterized from seafood (Lipp and Rose, 1997; Jorgensen and Huss, 1998; Heinitz et al., 2000), little is known about the role of Escherichia coli from seafood in the cause of foodborne disease outbreaks. These organisms are not present in large numbers in the intestinal tract of fish that require cool temperatures for growth (Matches and Abeyta, 1983; Ayulo et al., 1994). Catfish (Ictalurus punctatus) are cultured in the southeast part of the United States, where warm temperatures
(24–308C) favor culturing of the fish throughout the year (USDA, 2001) and may influence the colonization of E. coli in the gut of catfish. Since little is known about the prevalence of E. coli in catfish and the presence of virulence genes in these isolates, we isolated E. coli from the gastrointestinal tracts of catfish. We also, for the first time, cloned and sequenced class I integrons that harbor genes conferring resistance to sulfonamides and trimethoprim, which are key ingredients of Romet 30 (sulfadimethoxine and ormetoprim; Alpharma, Fort Lee, NJ), an antibiotic that is intensively used to curtail infectious diseases in catfish (Austin and Austin, 1993). Widespread use of antibiotics such as oxytetracycline and Romet 30 may result in the selection of tetracycline-, sulfonamide-, and trimethoprimresistant bacteria in the aquaculture environment. Little information is available on tetracycline- and sulfadimethoxine=
1
Division of Microbiology, National Center for Toxicological Research, Jefferson, Arkansas. Arkansas Regional Laboratory, Jefferson, Arkansas.
2
553
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NAWAZ ET AL.
ormetroprim-resistant E. coli from catfish with a history of exposure to these antibiotics. Materials and Methods Isolation, characterization, and identification Bacteria were isolated from the intestines of 407 catfish collected from 16 commercial ponds (that use antibiotics in catfish cultivation) in Arkansas, Louisiana, and Texas. Bacteria were also isolated from 80 catfish that were cultured in ponds that did not receive antibiotics. These ponds were under the supervision of U.S. Department of Agriculture (USDA) Stuttgart National Aquaculture Research Center, Stuttgart, Arkansas. Typically, 1 g of the intestinal content of catfish was enriched for 6 hours in Luria broth (LB). Enriched samples were streaked on MacConkey agar plates supplemented with 30 mg=mL of tetracycline and incubated at 378C overnight. Presumptive colonies of E. coli were biochemically characterized and identified by the Vitek GNI þ card with VTKR07-01 software (bioMe´rieux Vitek, Hazelwood, MO) and by fatty acid methyl ester analysis (MIDI, Newark, DE). All isolates were stored in LB containing 20% glycerol at 708C. These isolates were grown at 378C on trypticase soy agar (TSA) plates supplemented with 5% sheep blood.
Detection of tetracycline resistance (tet) genes by polymerase chain reaction The three tet genes (tetA-C) were individually amplified by polymerase chain reaction (PCR) (Ozgumus et al., 2007) with oligonucleotide primers (Table 1). The universal PCR amplification included 30 thermal cycles of 30 seconds at 948C, 59 seconds at 558C, and 60 seconds at 728C, with an additional extension in the last cycle for 300 seconds at 728C. The amplified PCR products were maintained at 48C. A reagent blank contained all components of the reaction mixture except template DNA, for which sterile distilled water was substituted. The PCR products were subjected to electrophoresis on 1.2% agarose gels in 1 Tris-borate-EDTA (TBE) buffer. Confirmation of the PCR amplicon by restriction digestion Amplified PCR products were purified by the QIAquick PCR purification kit (Qiagen). The tetA, tetB, and tetC amplicons were digested for 4 hours at 378C with SalI, Sau3AI, and BamH1 (Promega Corporation, Madison, WI), respectively. The digested fragments were separated on a 1.5% agarose gel. A 100-bp DNA ladder (Invitrogen, San Jose, CA) was used as the size standard.
Antibiotic susceptibility testing by disk diffusion
PCR amplification of integrons
Antibiotic susceptibility of each E. coli isolate was determined by disk diffusion (Bauer et al., 1966) by the criteria specified by the National Committee for Clinical Laboratory Standards (NCCLS, 2002). Disks of bacitracin (10 mg), ampicillin (30 mg), penicillin (10 U), rifampicin (5 mg), sulfamethaxazole=trimethoprim (23:75; 1.25 mg), tetracycline (30 mg), chloramphenicol (30 mg), streptomycin (10 mg), nalidixic acid (30 mg), and ciprofloxacin (5 mg) were used. The sensitivity and resistance of each isolate were determined per the manufacturer’s (Becton Dickinson, Sparks, MD) instructions and by the criteria of the National Committee for Clinical Laboratory Standards.
The primers used for the amplification of the integrons (Ozgumus et al., 2007) are listed in Table 1. Thermal cycling conditions consisted of an initial denaturation cycle (948C for 4 minutes) followed by 30 cycles of denaturation (958C for 45 seconds), annealing (568C for 1 minute), extension (728C for 90 seconds), and a final cycle of amplification (728C for 7 minutes). The amplified DNA fragments were separated by electrophoresis using 1.2% agarose gels, stained with ethidium bromide (5 mg=mL), visualized with UV, and photographed using the Eagle Eye II gel documentation system (Stratagene, La Jolla, CA). Cloning and DNA sequencing of integron
Genomic DNA extraction Genomic DNA was extracted from cells grown overnight at 378C with the QIAamp DNA mini kit (Qiagen, Valencia, CA).
The amplified products (1.6 and 1.8 kb) were purified from agarose gels using the QIAquick gel extraction kit (Qiagen). Gel slice–extracted DNA was then reamplified, using the
Table 1. Oligonucleotide Primers Used in the Amplification of tet Genes and Integrons from Multiple Drug–Resistant E. coli Isolated from Catfish PCR conditionsa Primers
Nucleotide sequence
Denaturing
Annealing
Extension
Target
Size (bp)
TetAF TetAR TetBF TetBR TetCF TetCR IntF IntR
50 -GTAATTCTGAGCACTGTCGC-30 50 -CTGCCTGGACAACATTGCTT-30 50 -CTCAGTATTCCAAGCCTTTG-30 50 -ACTCCCCTGAGCTTGAGGGG-30 50 -GGTTGAAGGCTCTCAAGGGC-30 50 -CCTCTTGCGGGAATCGTCC-30 50 -GGCATCCAAGCAGCAAG-30 50 -AAGCAGACTTGACCTGA-30
948C, 30 s
558C, 60 s
728C, 60 s
tetA
957
948C, 30 s
568C, 60 s
728C, 60 s
tetB
436
948C, 45 s
568C, 60 s
728C, 60 s
tetC
589
948C, 30 s
558C, 60 s
728C, 60 s
Int
Variable
a tet genes were amplified after an initial denaturation at 948C for 3 minutes. The integrons were amplified after an initial denaturation of 948C for 5 minutes. PCR was performed with 30 cycles and after the completion of the 30 cycles, a final extension step of 10 minutes at 728C was included in all protocols. PCR, polymerase chain reaction.
E. COLI TETRACYCLINE RESISTANCE IN CATFISH
555
same reaction conditions to assess the quality of the gel excision step. The PCR product was then directly ligated into plasmid vector pCR2.1 (Invitrogen) and transformed according to the manufacturer’s recommendations. The clones were screened for inserts by isolating the recombinant plasmid, which was confirmed by digestion with EcoRI. The digested products were analyzed by agarose gel electrophoresis. Clones containing the different size inserts were purified for sequencing using the Qiagen plasmid purification kit. Sequencing of both strands was performed by using M13 primers with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems, Foster City, CA). DNA sequences were analyzed with Lasergene (DNASTAR, Inc., Madison, WI) software. PCR amplification of virulence markers from the tetracycline-resistant E. coli strains PCR assays for Shiga-like toxins (Stx1, Stx2, or their variants), the enterohemorrhagic hemolysin gene (hlyA), the bifunctional catalase peroxidase (KatP), the secreted serine protease (EspP), and eae, which is responsible for attaching and effacing lesions, were performed as described earlier (Khan et al., 2002). The primer sequence and PCR conditions are given in Table 2. The oligonucleotide primers were synthesized by Eurofins MWG Operon, Huntsville, AL. Pulsed-field gel electrophoresis Genomic DNA samples of the isolates were subjected to pulsed-field gel electrophoresis (PFGE) (On, 1998; Nawaz et al., 2006). DNA plugs were digested with 20 U of XbaI (New England Biolabs, Beverly, MA) at 378C for 5 hours. The digested DNA was separated on 1.0% SeaKem Gold (FMC Corp., Philadelphia, PA) agarose gel with Chef-Mapper III PFGE (BioRad Laboratories, Hercules, CA) system for 24 hours as described earlier (Nawaz et al., 2006). The gels were stained for 30 minutes with ethidium bromide, destained with distilled water, and photographed using the Eagle Eye II gel documentation system. The genetic relationship among the tetracycline-resistant E. coli isolates was analyzed using the Bionumerics software (Applied Maths, Kortrijk, Belgium).
Results Isolation and identification of E. coli from catfish samples Two hundred eighty-three bacterial isolates that fermented lactose on MacConkey agar were picked for further characterization and identification. The colonies of these isolates, which typically were purplish-pink, were restreaked on TSA plates with 5% blood to obtain pure cultures. These isolates were rod-shaped and Gram-negative, and they produced lysine decarboxylase and ornithine decarboxylase but not arginine dihydrolase. The isolates were able to utilize lactose, glucose, maltose, sucrose, rhamnose, sorbitol, and arabinose, but failed to utilize raffinose or metabolize urea or citrate. Based on these characteristics, 63 of the 283 (22.0%) bacterial isolates were identified as E. coli. Antibiotic resistance profiles Based on the method used to detect isolates, all isolates were resistant to tetracycline. Twenty-two of 63 (35%) isolates were also resistant to penicillin, streptomycin, bacitracin, and rifampicin, and 20 of 63 (32%) were resistant to ampicillin, penicillin, streptomycin, bacitracin, and rifampicin. Fourteen of 63 (22%) were resistant to ampicillin, penicillin, chloramphenicol, sulfamethaxazole=trimethoprim, streptomycin, bacitracin, and rifampicin; and 7 of 63 (11%) were resistant to ampicillin, penicillin, chloramphenicol, streptomycin, bacitracin, and rifampicin. None of the isolates were resistant to either nalidixic acid or ciprofloxacin. Overall, nine E. coli were isolated from catfish cultured in ponds that did not receive any antibiotics. All nine of these isolates were sensitive to tetracycline, sulfamethaxazole=trimethoprim, streptomycin, nalidixic acid, ciprofloxacin, chloramphenicol, and rifampicin. These isolates were not further characterized. PCR amplification of tet genes PCR amplification of tet genes from genomic DNA of all 63 tetracycline-resistant E. coli isolates from catfish was performed. The oligonucleotide primers for tetA amplified the 957-bp PCR amplicon (Fig. 1A, lane 2) from 15 of 63 (23.8%)
Table 2. Oligonucleotide Primers and PCR Protocols Used to Amplify Virulence Genes from E. coli Isolates from Catfish PCR conditionsa Primers
Nucleotide sequence
Denaturing
Annealing
Extension
Target
Size (bp)
EVT1 EVT2 EVS1 EVC2 hlyA1 hlyA4 wkat-B wkat-F D1 D13R EAE1 EAE2
50 -CAACACTGGATGATCTCAG-30 50 -CCCCCTCAACTGCTAATA-30 50 -ATCAGTCGTCACTCACTGGT-30 50 -CTGCTGTCACAGTGACAAA-30 50 -GGTGCAGCAGAAAAAGTTGTAG-30 50 -TCTCGCCTGATAGTGTTTGGTA-30 50 -CTTCCTGTTCTGATTCTTCTGG-30 50 -AACTTATTTCTCGCATCATCC-30 50 -CGTCAGGAGGATGTTCAG-30 50 -CGACTGCACCTGTTCCTGATTA-30 50 -AAACAGGTGAAACTGTTGCC-30 50 -CTCTGCAGATTAACCTCTGC-30
948C, 60 s
558C, 60 s
728C, 60 s
stx1
349
948C, 60 s
558C, 60 s
728C, 60 s
stx2
110
948C, 30 s
578C, 60 s
728C, 90 s
hlyA
1551
948C, 30 s
568C, 60 s
728C, 150 s
katP
2125
948C, 30 s
528C, 60 s
728C, 90 s
etpD
1062
948C, 60 s
558C, 90 s
728C, 90 s
eae
350
a PCR was started after an initial denaturation at 948C for 3 minutes. The protocol was performed with 30 cycles and after the completion of the 30 cycles, a final extension step of 10 minutes at 728C was carried out. PCR, polymerase chain reaction.
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FIG. 1. Detection of the tetracycline resistance genes tetA–C by polymerase chain reaction (PCR) in E. coli isolates from catfish. (A) Lane 1, 100-bp molecular weight marker; lanes 2–3, 957-bp tetA amplified from E.coli genomic DNA and the SalI restriction digested fragments (728 and 229 bp). (B) Lane 1, 100-bp molecular weight marker; lanes 2–3, 436-bp tetBPCR product amplified from E. coli genomic DNA and Sau3A1 restriction digested fragments (271 and 165 bp. (C) Lane 1, 100-bp molecular weight marker, lanes 2–3, 589-bp tetC PCR product amplified from the genomic DNA of E. coli and the BamH1 restriction fragments (305 and 284 bp). isolates. The identity of the 957-bp PCR amplicon was confirmed by restriction digestion by SalI, which produced the predicted 728- and 229-bp DNA fragments (Fig. 1A, lane 3). PCR amplification of the genomic DNA was performed with tetB- and tetC-specific oligonucleotide primers. The oligonucleotide primers were able to amplify the 436-bp tetB PCR product (Fig. 1B, lane 2) from 47 of 63 (74.6%) isolates. The identity of the 436-bp PCR amplicon was confirmed by restriction digestion of the PCR amplicon with Sau3A1, which produced the predicted 271- and 165-bp DNA fragments (Fig. 1B, lane 3). Primers specific for the tetC amplified a 589-bp product in 3 of 63 (4.8%) isolates (Fig. 1C, lane 2). The identity of the 589-bp tetC PCR product was established by restriction digestion of the amplicon with BamH1, which produced the predicted 305and 284-bp DNA fragments (Fig. 1C, lane 3). Eleven of the 63 (17.5%) strains harbored both tetA and tetB. None of the strains harbored all three tet determinants.
NAWAZ ET AL.
FIG. 2. Amplification of class I integrons from genomic DNA of tetracycline-resistant E.coli isolates from catfish. Lanes 1 and 5, 1.0 kb DNA ladder; 2, integrons measuring 1.6 kb amplified from 5 of 14 isolates; 4, integrons measuring 1.8 kb amplified from the genomic DNA from 9 of 14 isolates. called dhfrXII), which confers resistance to trimethoprim antibiotic, an open reading frame, orfF, a hypothetical protein of unknown function; and an aadA1 gene encoding an aminoglycoside adenyltransferase, which confers resistance to streptomycin and spectinomycin. The gene cassettes dhfrA12 and ofrF contained a 59-base element recombination site (Fig. 3A). The sequence analysis of the 1.6 kb integron is deposited with GenBank (accession number EU650399). The aligned nucleotide sequencing analysis of the 1.8 kb integrons indicated that these DNA segments contained two prominent antibiotic resistance insert gene cassettes: dhfrA17, encoding dihydrofolate reductase type12, conferring resistance to trimethoprim, and aadA5, encoding an aminoglycoside adenyltransferase (Fig. 3B). The two gene cassettes contained a 59-base element recombination site. The sequence analysis of the 1.8-kb integron is deposited with GenBank (accession number EU 650403). Amplification of the virulence genes by PCR
Amplification of the Class 1 integron One pair of synthetic class I integron-specific oligonucleotide primers was used to screen for integrons from the genomic DNA of the isolates (Table 1). The primers were able to amplify the integrons from 14 of 63 (22.0%) isolates. These integrons measured 1.6 kb (Fig. 2, lane 2) and 1.8 kb (Fig. 2, lane 3). The integrons measuring 1.8 kb were dominant, occurring in 9 of 14 (64.0%) integron-positive isolates. The integrons measuring 1.6 kb occurred in 5 of 14 integron-positive isolates (36.0%). The aligned analysis of nucleotide sequencing indicated that the 1.6 kb integrons contained three different gene inserts (Fig. 3A): a dihydrofolate reductase type A12, dfrA12 (previously
Genomic DNA from the 63 tetracycline-resistant E. coli strains were screened for the virulence genes stx1 and stx2, hlyA, KatP, etpD, and eae by PCR. Oligonucleotide primers specific for the amplification of the 349-bp and 110-bp regions of stx1 and stx2, a 1551-bp region of hlyA, a 2125-bp region of KatP, a 1067-bp region of etpD, and a 350-bp region of the eae gene failed to produce specific PCR amplicons from the template DNA of any of the isolates. Pulsed-field gel electrophoresis All 63 tetracycline-resistant E. coli isolated from catfish were typeable by the PFGE methodology used. XbaI-PFGE
E. COLI TETRACYCLINE RESISTANCE IN CATFISH
A
attI 1
557
59-be dfrA12
59-be orfF
aadA2
B 59-be dfrA17
59-be aadA5
FIG. 3. (A) Schematic representation of the dfrA12-orfF-aadA2 gene cassettes cloned and sequenced from integrons measuring 1.6 kb. The dfrA12, orfF, and aadA2 reading frames are represented by arrows. The 59-base elements and the recombination site attl 1 are marked. (B) Schematic representation of the dfrA17-aadA5 gene cassettes cloned and sequenced from integrons measuring 1.8 kb. The dfrA12 and aadA5 reading frames are represented by arrows. identified 16 distinct macro restriction patterns among the 63 isolates (Fig. 4). Dendrogram analysis indicated that the XbaIdigested profiles of strain ECTa3 and ECT721 had a similarity index of ca. 90.0%. Similarly, strain ECT711 had a similarity index of ca. 90.0% with the XbaI digest of strain ECT660. Lower similarity (85.0%) was observed between strains ECT90 and ECT720. The XbaI-PFGE of strain ECT900 had a similarity index of ca. 80.0% with the profile of strain ECT154. The XbaI-PFGE profile of all other strains were at or below 80.0% similarity (Fig. 4). Discussion Although several investigators have reported on the isolation and characterization of tetracycline-resistant E. coli from food-producing animals (Lanz et al., 2003; Sengelov et al., 2003), little information is available on the characterization of
tetracycline=sulfmethaxazole=trimethoprim-resistant E. coli from aquaculture samples with a history of exposure to these antibiotics. Furushita et al. (2003) studied the prevalence of tet determinants in Gram-negative bacteria isolated from seafood. They reported that 43% (29 of 66) of the genomic DNA from Gram-negative bacteria isolated from yellowtail, amberjack, and bluefin tuna carried the tetB determinant followed by tetC and tetD. Recently, an Australian study (Akinbowale et al., 2007) indicated that tetM (50%) was the dominant tetracycline resistance determinant, followed by tetE (45%), in Gram-negative bacteria isolated from aquaculture sediments. Results from our study on the prevalence of tetracycline resistance genes indicated that tetB was the dominant determinant, present in 75% of the isolates, followed by tetA (24%) and tetC (5%). The occurrence and prevalence of more than 14 different tet determinants in various food producing animals and their environments may be
FIG. 4. XbaI pulsed-field gel electrophoresis of the genomic DNA of selected E. coli isolates from catfish and a dendrogram analysis of the macro restriction patterns by the Bionumeric software.
558 either species specific or ecosystem specific. The tet determinants in E. coli may help the bacteria survive from the inhibitory effects of tetracycline produced by other aquatic microflora or protect the bacteria from the adverse effects of tetracycline drugs added to the ponds. Multiple antibiotic resistance integrons are important contributors to the development of antibiotic resistance in the Enterobacteriaceae (Stokes and Hall, 1989; Levesque et al., 1995; Carattoli 2001; Warturangi et al., 2003; Gestal et al., 2005; Sunde 2005). More than 100 different antibiotic resistance gene cassettes have been found within integrons and a majority encode antibiotic resistance phenotypes (Stokes and Hall, 1989; Sunde 2005). Sequence analysis of the 1.6-kb integron observed in this study was identical to cassette array reported in strain of E. coli isolated from sputum (GenBank accession number EF450247), except for the base substitution, G?A at nucleotide position 525 resulting in amino acid replacement valine?isoleucine of dihydrofolate reductase type A12. In addition, a 59-base element recombination site was not present on the open reading frame, orfF in the sequence. The sequence analysis of the two-gene cassette array observed in the 1.8-kb integron in this study is identical to sequence reports to other bacteria such as Staphylococcus epidermidis (GenBank accession number AB291061), E. coli (accession number AM886293), Pseudomonas aeruginosa (DQ838665), and Salmonella (AY263739). Our inability to detect the occurrence of sul genes responsible for sulfonamide resistance may be due to the shortcomings of the design or use of PCR primers which may have missed the DNA fragments containing sul genes. Characterization of pathogenic bacteria responsible for the outbreak of infectious diseases by phenotypic and genotypic methods can provide useful epidemiological data (Tenover et al., 1995; On, 1998; Nawaz et al., 2006). These methods may provide valuable information on antibiotic resistance profiles, rates of transmission, reservoirs of infection, and mechanisms of infection. The 63 E. coli described in this investigation can be divided into four different groups based on their antibiograms and three different groups based on the presence of tet determinants. Based on the results of XbaI-PFGE, the tetracycline-resistant E. coli isolates from catfish could be put into 16 different groups. Our study detected nonvirulent E. coli which carried resistance to multiple antibiotics in farm raised catfish exposed to antibiotics. It is possible that the intense use of oxytetracycline and Romet 30 in catfish ponds may play a role in selecting E. coli resistant to these antibiotics. This is supported by our findings that in catfish taken from ponds where antibiotics were not used, all E. coli isolates were sensitive to tetracycline. It is thus important that these two antibiotics be appropriately used in catfish ponds to preserve the efficacy of these drugs. In addition, the presence of class I integrons in some of these strains of E. coli may be a public health risk because commensal bacteria in aquaculture ecosystem may become an important reservoirs of antibiotic-resistant genes and may play a role in the transfer the resistance determinants to other bacteria associated with humans. Acknowledgment We thank Drs. Carl E. Cerniglia, J.B. Sutherland, and Carina M. Jung for critical review of the manuscript. Views pre-
NAWAZ ET AL. sented here do not necessarily reflect those of the U.S. Food and Drug Administration. This work was supported by the National Center for Toxicological Research, U.S. Food and Drug Administration. Disclosure Statement No competing financial interests exist. References Akinbowale OL, Peng H, and Barton MD. Diversity of tetracycline resistance genes in bacteria from aquaculture sources in Australia. J Appl Microbiol 2007;103:2016–2025. Austin B and Austin DA. Bacterial Fish Pathogens: Disease in Farmed and Wild Fish, 2nd edition. Chichester, United Kingdom: Ellis Horwood, 1993. Ayulo AMR, Machado RA, and Scussel VM. Enterotoxigenic Escherichia coli and Staphylococcus aureus in fish and seafood from southern region of Brazil. Int J Food Microbiol 1994; 24:171–178. Bauer AW, Kirby WMM, Sherris JC, et al. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966;45:493–496. Carattoli A. Importance of integrons in the diffusion of resistance. Vet Res 2001;32:243–259. Feldhusen F. The role of seafood in bacterial foodborne diseases. Microbes Infect 2000;2:1651–1660. Furushita M, Shiba T, Maeda T, et al. Similarity of tetracycline resistance genes isolated from fish farm bacteria to those from clinical isolates. Appl Environ Microbiol 2003;69:5336–5342. Gestal AM, Stokes HW, Partridge SR, et al. Recombination between dfra12-orfF-aadA2 cassette array and an aadA1 gene cassette creates a hybrid cassette, aadA8b. Antimicrob Agents Chemother 2005;49:4771–4774. Heinitz ML, Ruble RD, Wagner DE, et al. Incidence of Salmonella in fish and seafood. J Food Prot 2000;63:579–592. Jorgensen LV and Huss HH. Prevalence and growth of Listeria monocytogenes in naturally contaminated seafood. Int J Food Microbiol 1998;42:127–131. Khan A, Das SC, Ramamurthy T, et al. Antibiotic resistance, virulence gene, and molecular profiles of Shiga toxinproducing Escherichia coli isolates from diverse sources in Calcutta, India. J Clin Microbiol 2002;40:2009–2015. Lanz R, Kuhnert P, and Boerlin P. Antimicrobial resistance and resistance gene determinants in clinical Escherichia coli from different animal species in Switzerland. Vet Microbiol 2003;91: 73–84. Levesque C, Piche L, Larose C, et al. PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob Agents Chemother 1995;39:185–191. Lipp EK and Rose JB. The role of seafood in food-borne disease in the US of America. Revue Scientifique et Technique, Office International des Epizooties 1997;16:620–640. Matches JR and Abeyta C. Indicator organisms in fish and shellfish. Food Technol 1983;37:114–117. Nawaz MS, Sung K, Khan SA, et al. Biochemical and molecular characterization of tetracycline-resistant Aeromonas veronii isolates from catfish. Appl Environ Microbiol 2006;72:6461– 6466. [NCCLS] National Committee for Clinical Laboratory Standards. Development of in vitro susceptibility testing criteria and quality control parameters for veterinary antimicrobials agents. Approved guideline M37-A2. Wayne, PA: National Committee for Clinical Standards, 2002.
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