JAC Advance Access originally published online on October 26, 2006
Journal of Antimicrobial Chemotherapy 2006 58(6):1308-1310; doi:10.1093/jac/dkl416
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Correspondence |
Characterization of class 1 integron resistance gene cassettes in Salmonella enterica serovars Oslo and Bareily from imported seafood
1 Division of Microbiology, US Food and Drug Administration, National Center for Toxicological Research Jefferson, AR 72079, USA 2 US Food and Drug Administration/PRL-SW Irvine, CA, USA
*Corresponding author. Tel: +1-870-543-7601; Fax: +1-870-543-7307; E-mail: Ashraf.khan{at}fda.hhs.gov
Keywords: S. enterica , antibiotic resistance , mobile genetic elements
Sir,
Recently, numerous outbreaks have been caused by the multidrug-resistant Salmonella enterica serovar Typhimurium definitive phage type DT104 with resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulphonamides and tetracycline (ACFSSuT), but increasingly other multidrug-resistant Salmonella serovars have been responsible for infections in humans.1 In the United States, there are an estimated 800 000 to 4 million Salmonella infections annually, and approximately 500 of the cases are fatal.2 A variety of food including poultry, beef, pork, eggs, milk, cheese, fish, shellfish, fruits, juice and vegetables have been implicated as vehicles transmitting salmonellosis to humans.2
Mobile genetic elements, such as plasmids, transposons and the more recently explored integrons, which are able to disseminate antibiotic resistance genes by horizontal or vertical transfer, have been shown to play an important role in the evolution and dissemination of multidrug resistance in Gram-negative bacteria.3
The aim of this study was to determine integron-mediated antibiotic resistance in a diverse sample set of S. enterica serovars isolated from imported seafood. A total of 105 S. enterica strains were isolated from imported seafoods from 20 countries in the US from 2000 to 2005. The strains were identified, serotyped and tested for levels of resistance to antibiotics commonly used in either clinical or veterinary medicine (ampicillin, amoxicillin, amikacin, ceftiofur, ceftriaxone, cefoxitin, chloramphenicol, ciprofloxacin, florfenicol, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfisoxazole, tetracycline and trimethoprim/sulfamethoxazole) by using methods described previously.4 Two S. enterica strains (serovars Bareily and Oslo) that originated from two different countries (Vietnam and India) were resistant to trimethoprim/sulfamethoxazole, sulfisoxazole, ampicillin, tetracycline and chloramphenicol and were characterized for class 1 integron. Escherichia coli ATCC 25922, which is susceptible to all the drugs, was used as the quality control strain.
To amplify class 1 integron gene cassettes primers CSL1 (5'-GGCATCCAAGCAGCAAGC-3') and CSR1 (5'-AAGCAGACTTGACCTGAT-3') were used. To confirm other antibiotic resistance genes we used the primers and PCR methods described previously.4,5 The annealing temperatures ranged from 50 to 60°C. For a positive control Salmonella Typhimurium DT104 strain DT74 was used. As a negative control, E. coli cells or DNA was used. A reagent blank was included in each PCR reaction, which contained distilled water instead of template DNA. To identify SGI1 in these strains, PCR was performed by using primers U7-L12 (5'-ACACCTTGAGCAGGGCAAAG-3')/LJ-R1 (5'-AGTTCTAAAGGTTCGTAGTCG-3') and 104-RJ (5'-TGACGAGCTGAAGCGAATTG-3')/104D (5'-ACCAGGGCAAAACTACACAG-3') corresponding to the left and right junctions of SGI1 in the Salmonella chromosome as described previously.5 PCR was also performed to detect the presence or absence of the retron sequence, which is located downstream of SGI1 only in serovar Typhimurium DT104.5 The aadA2 gene was amplified by using the same PCR conditions as described previously5 except the annealing temperature was 57°C and the primers were aadA2F1 (5'-ATGAGGGTAGCGGTGACCATC-3') and aadA2R1 (5'-TCATTTACCAACTGACTTGATG-3'). The amplified integrons were purified from agarose gels and cloned into pCR2.1 plasmid. Clones containing a 1.24 kb insert were screened for insert and purified for sequencing. Sequencing of both strands was performed. The GenBank nucleotide sequence accession numbers of the S. enterica serovar Bareily (61) and Oslo (64) dfrA1-orfC cassettes are DQ641476 [GenBank] and DQ641477 [GenBank] .
The Salmonella strains used in this study belonged to 36 different serovars and 20 different serogroups. The most predominant serovars were Weltevreden, Newport, Saintpaul, Senftenberg, Lexington, Enteritidis and Bareily. Twenty isolates were found to be resistant to at least one of the sixteen antibiotics tested. Five strains of Salmonella sp. [61 (serovar Bareily, serogroup C1), 64 (serovar Oslo, serogroup C1), 77 (serovar Hadar, serogroup C2), 611 (serovar Weltevreden, serogroup E1) and 725 (serovar Rissen, serogroup C1)] resistant to two or more antibiotics were selected for integron analysis (Table 1). All five strains were resistant to tetracycline and sulfisoxazole. Three isolates (61, 64 and 611) were resistant to ampicillin. Three isolates (61, 64 and 77) were resistant to chloramphenicol. Only two isolates (61 and 64) were resistant to five antibiotics: ampicillin, tetracycline, sulfisoxazole, chloramphenicol and trimethoprim/sulfamethoxazole. All five strains were susceptible to florfenicol. Antimicrobial resistance was not solely associated with particular Salmonella serogroups, but three strains, 61, 64 and 725, are of serogroup C1. Of the five multidrug-resistant Salmonella isolates tested, two isolates [61 (serovar Bareily, serogroup C1) and 64 (serovar Oslo, serogroup C1)] amplified a 1.24 kb PCR product by using the primers CSL1 and CSR1, suggesting the presence of class 1 integrase (intI1). PCR results were negative for the left and right junctions of SGI1 as well as the retron sequence. We did not find any plasmids in either strain, which suggests these integrons are located on the chromosome. Since both strains were resistant to trimethoprim/sulfamethoxazole, PCR was performed to screen for dfrA1, dfrA5, dfrA7 and dfrB1 genes as described previously by Frech et al.6 Both strains (61 and 64) were positive for only one trimethoprim/sulfamethoxazole resistance gene (dfrA1), amplifying a 414 bp amplicon. Both strains (61 and 64) amplified the entire coding region (792 bp) of the streptomycin resistance gene aadA2 gene by PCR but strain 61 did not show phenotypic resistance to streptomycin.
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Sequence analysis of 1.24 kb clones of 61 and 64 revealed the presence of dfrA1 conferring resistance to trimethoprim/sulfamethoxazole and an open reading frame orfC gene cassette of unknown function. The gene cassette showed 100% identity with the dfrA1 and orfC genes encoding resistance to trimethoprim/sulfamethoxazole and unknown function. This cassette array is identical to reports in E. coli (GenBank accession no. AB161449 [GenBank] ), Vibrio cholerae7 (AF455254 [GenBank] ) and Salmonella spp. (AB186122 [GenBank] , AY963803 [GenBank] ).
Non-typhoidal Salmonella spp. are common food-associated pathogens, and Salmonella infections account for a large proportion of deaths associated with food-related illness.8 Recently Brands et al.9 have observed the prevalence of the human pathogen S. enterica serovar Newport in oysters harvested in the United States. Use of antimicrobials (for both therapeutic and non-therapeutic purposes) in food animals is the dominant factor in increased reports of drug-resistant isolates. To our knowledge, this is the first report of this class of integron variant in these two serovars (Bareily and Oslo) of S. enterica. S. enterica serovar Emek, which had a similar integron cassette array but had SGI1 sequences, was isolated from humans.5 Class 1 integrons on transferable plasmids are considered to be the main mechanism for the rapid spread of multidrug-resistant phenotypes among Gram-negative bacteria.5 However we did not find any plasmid in these strains. These two serovars lack the SGI1 sequence and show higher similarity to V. cholerae integrons. V. cholerae are found to have acquired resistance for trimethoprim/sulfamethoxazole and streptomycin from an SXT constin, a conjugative, self-transmissible, integrating element harbouring resistance determinants to trimethoprim/sulfamethoxazole, streptomycin, sulfamethoxazole and chloramphenicol.5 These two serovars are isolated from seafood and may have acquired resistance in a similar fashion to V. cholerae.
The SGI1 variant antibiotic resistance gene clusters have been reported in several serovars of S. enterica that have dfrA1 and dfrA105 encoding trimethoprim resistance. These variant antibiotic resistance gene clusters were probably generated by recombinational events such as insertions and deletions. The serovars Bareily and Oslo of our study represent the example in which gene replacement took place in one of the integron structures. The dfrA1 and orfC gene cassettes array found instead of aadA2 may have been introduced by homologous recombination with a class 1 integron containing the same array of gene cassettes from another bacterium.5,7 Another possibility involves exchange between aadA2 and the two gene cassettes, which would imply excision of aadA2 and its replacement by the other gene cassettes. The array of gene cassettes found in the integron of serovar Oslo and Bareily strains was the same as that reported in integrons of V. cholerae isolated in Thailand and India.7 Considering the origin of the serovar Oslo and Bareily strains (exported seafood from India and Vietnam), a possible explanation could be the exchange of antibiotic resistance gene cassettes between epidemic multidrug-resistant V. cholerae strains and Salmonella strains. In Asia multidrug-resistant V. cholerae epidemics in humans might be responsible for the spread of antibiotic resistance genes. Human colonization by V. cholerae creates a hyper-infectious bacterial state, which is perpetuated even after purging into natural aquatic reservoirs and may contribute to epidemic spread of cholera. These aquatic reservoirs may be an ecological niche where antibiotic resistance gene exchange takes place between different pathogenic Enterobacteriaceae.
This study indicates that antimicrobial-resistant S. enterica serovars are prevalent in imported seafoods. The use of antimicrobials in aquaculture farming in Southeast Asia may be selecting for antimicrobial-resistant Salmonella species that can contaminate imported seafoods coming into the United States. It is thus important that antimicrobials be appropriately used in aquaculture on a global basis to preserve the efficacy of the existing drugs and to limit the risk of transfer of resistant food-borne pathogens to humans.
Transparency declarations
None to declare.
Acknowledgements
We thank Dr Carl E. Cerniglia, Dr Chris A. Elkins and Dr Huizhong Chen for critical review of the manuscript. This work was supported by the National Center for Toxicological Research, US Food and Drug Administration. Views presented here do not necessarily reflect those of the FDA.
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Levings RS, Lightfoot D, Partridge SR, et al. (2005) The genomic island SGI1, containing the multiple antibiotic resistance region of Salmonella enterica serovar Typhimurium DT104 or variants of it, is widely distributed in other S. enterica serovars. J Bacteriol 187:4401–9.
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