1 Molecular Diagnostics Unit, Cork Institute of Technology, Bishopstown, Cork; 3 Centre for Food Safety, University College Dublin, Faculties of Agri-Food & the Environment, Medicine and Veterinary Medicine, Belfield, Dublin 4, Ireland; 2 Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, viale Regina Elena 299, 00161 Rome, Italy
Received 4 October 2004; returned 12 November 2004; revised 6 December 2004; accepted 9 December 2004
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Abstract |
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Methods: A complete DNA sequence of the regions containing the resistance genes was obtained from the chromosome of the S. Typhimurium DT193 isolate and from the IncI plasmid of the S. Enteritidis isolate. The plasmid was also characterized by conjugation and incompatibility grouping.
Results: Two 10 kb multidrug resistance non-Salmonella Genomic Island 1 type clusters were independently identified in the S. Enteritidis plasmid and in the chromosome of the S. Typhimurium isolate. Detailed characterization identified an IP-type 2 integron containing a dfrA1-aadA1 gene cassette and other common resistance determinants derived from the RSF1010 plasmid.
Conclusions: These multidrug resistance regions originate following chromosomal integration of key resistance markers encountered on plasmids circulating in other Salmonella serotypes. This mechanism of marker acquisition may have future implications for the evolution of similar structures in previously susceptible serotypes, leading to an increased public health risk.
Keywords: antimicrobial resistance , resistance genes , Salmonella spp , integrons , plasmids
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Introduction |
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Previous genetic mapping experiments aimed at providing comparisons of the multidrug resistance determinants between S. Typhimurium DT104 and non-DT104 isolates in Ireland revealed the presence of chromosomally integrated integrons in other relevant S. Typhimurium phage types, such as DT193.2 In particular, these DT193 isolates were associated with one type of class 1 integron, designated IP-type 2, containing the dfrA1 and aadA1 gene cassettes, conferring resistance to trimethoprim and streptomycin/spectinomycin, respectively, which was also identified in the chromosome of S. Typhimurium DT104b.3
In this study, a new 10 kb resistance region, including the IP-type 2 integron, was identified integrated within the DT193 chromosome and compared with a similar resistance region found on a plasmid isolated in S. Enteritidis in Italy in 1997. Our findings demonstrate that the DT193 resistance region originates from the integration into the chromosome of particular resistance determinants that have been encountered on the S. Enteritidis plasmid.
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Materials and methods |
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In 1997, a multidrug-resistant S. Enteritidis isolate was isolated at the Istituto Superiore di Sanità in Italy from a human case of acute gastroenteritis, during routine surveillance activity. In 1998, a multidrug-resistant S. Typhimurium isolate was isolated from a food sample in Cork County Council Veterinary Laboratory, Ireland, and phage typed as DT193. Antibiotic susceptibility of both isolates was established by the disc diffusion method4 using 11 different antimicrobial drugs: ampicillin (AMP, 10 µg), cefalothin (30 µg) chloramphenicol (30 µg), ciprofloxacin (5 µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid (NAL, 30 µg), streptomycin (STR, 10 µg), sulphonamides (300 µg), tetracyclines (TET, 30 µg) and sulfamethoxazole/trimethoprim (SXT, 1.25/23.75 µg) were tested. The S. Enteritidis isolate showed NAL/STR/TET/SXT resistance. The S. Typhimurium isolate showed AMP/STR/TET/SXT resistance.
S. Enteritidis plasmid analysis
The S. Enteritidis plasmid was transferred by conjugation into the Escherichia coli CSH26RifR recipient strain, selecting transconjugants on LuriaBertani agar plates containing 100 mg/L of rifampicin and streptomycin (30 mg/L) or tetracyclines (30 mg/L). The STR/TET/SXT resistance pattern was transferred. Plasmid DNA from E. coli transconjugants was purified by the Concert Purification Midi Kit (Life Technologies, Milan, Italy) and used as the template for standard PCR amplification experiments (Promega Corporation, Madison, WI, USA), using the primer pairs listed in Table 1.
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S. Typhimurium DT193 resistance cluster characterization
A 10 579 bp resistance region was fully sequenced following detailed PCR-based gene mapping. Long PCR was achieved using the rTth XL kit from Perkin-Elmer (Warrington, UK). Initial primer sequences were based on individual resistance genes and additional primers were designed as sequence data became available. Any amplified PCR product of interest was cloned using the TOPO TA Cloning Kit (Invitrogen BV, The Netherlands) and the cloned inserts were sequenced (MWG Biotech, Ebersberg, Germany). Sequence data was initially analysed using the Gene Codes Corp. sequencing software package, DNA Sequencher (version 4.1) (Gene Codes Corp., Ann Arbor, MI, USA).
Nucleotide accession numbers
Comparative analysis of nucleotide sequences was performed by the advanced BLAST search program 2.0 (www.ncbi.nlm.nih.gov/blast/). CLUSTALW amino acid sequence alignments were produced for comparison (http://www.ebi.ac.uk/clustalw).
S. Enteritidis and S. Typhimurium DNA sequences are released under the accession numbers AJ628353 and AY524415, respectively.
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Results |
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An S. Enteritidis isolate showed the STR/TET/SXT resistance pattern by the presence of a conjugative 80 kb plasmid. This plasmid was assigned to the I1 incompatibility group, by Southern-blot hybridization5 performed with the inc/rep plasmid bank, as previously described.6 The IncI plasmid was further analysed by PCR for the integrase gene (intI1), integron-borne gene cassettes7 and specific antibiotic resistance genes (Table 1). The IncI plasmid was positive for intI1, tet(A) and strA-strB genes, but the 5'CS/3'CS primer pair failed to generate an amplicon. By Southern-blot hybridization5 the intI1, tet(A) and strA-strB genes were all identified within a unique 20 kb BglII fragment (data not shown), which was cloned and sequenced (Figure 1).
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Comparison of the 12 kb IncI S. Enteritidis and the 10 kb chromosomal S. Typhimurium DT193 resistance gene regions
The chromosomal resistance region of the multidrug-resistant S. Typhimurium DT193 showing the presence of the IP-type 2 integron, was completely characterized and sequenced starting with primers for both the integron and the blaTEM-1 sequences, previously identified in this isolate.2 This analysis revealed the presence of an strB-strA-sul2-repC-repA region derived from the RSF1010 plasmid, flanked by the IP-type 2 integron and by an inverted IS26 element, preceded by a putative transposase gene (tnpB) located upstream of the blaTEM-1 gene. The tnpB region was also noted in the RSF1010 plasmid sequence, adjacent to the strB gene (Figure 1).
Based on the DNA sequence comparison, a high level of structural similarity between the 10 kb chromosomally integrated resistance gene region of S. Typhimurium DT193 and the IncI plasmid-region was noted. Both structures contained common elements of class 1 integrons, including intI1 and most of the variable gene cassette region. However, the former gene is preceded by the tnpM gene, known to be part of the transposase machinery of the Tn21-like transposons (Database accession no. AF071413) in the IncI plasmid, whereas the tnpM gene is missing in the DT193 resistance island. A further difference between both sequences consists of opposite orientations with respect to the integrons and also the integron located on the S. Enteritidis plasmid is devoid of its 3'CS.
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Discussion |
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Based on the DNA sequence comparison, our findings suggest that the development of multidrug resistance in these isolates occurred through the acquisition of conserved and highly diffused genetic traits, by independent rounds of insertion and recombination. The resistance genes included in these regions are very common and have been previously described in many unrelated bacteria isolated from humans and animals.8 These genes are often located on the small broad-host-range non-conjugative plasmid, RSF1010, or on a similar plasmid, for example pBP1, whereas in isolates of plant origin they are carried on large conjugative plasmids characterized by the presence of the transposon Tn5393.9,10
The flanking sequences would appear to dictate the genetic routes by which these markers are transferred between different bacteria. In our case, the mechanism of mobilization would appear to be mediated by IS26 elements. Although a DNA segment flanked by direct IS26 copies, but not by inverted IS26 copies, can transpose to a recipient replicon,11 the presence of these insertion sequences may have contributed to the intrinsic instability of the resistance regions, resulting in deletions and inversions.
In conclusion, our findings support the hypothesis that the versatility of plasmids and the exchange of resistance genes by vertical and horizontal gene transfer may have largely contributed to the spread of these antimicrobial resistance traits.
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Footnotes |
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Acknowledgements |
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References |
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2
.
Daly, M. & Fanning, S. (2000). Characterisation and chromosomal mapping of antimicrobial resistance genes in Salmonella enterica serotype Typhimurium. Applied and Environmental Microbiology 66, 48428.
3
.
Daly, M., Buckley, J., Power, E. et al. (2004). Evidence for a chromosomally-located third integron in Salmonella Typhimurium DT104b. Antimicrobial Agents and Chemotherapy 48, 13502.
4 . National Committee for Clinical Laboratory Standards (2003). Performance Standards for Antimicrobial Disk Susceptibility TestsEighth Edition: Approved Standard M2-A8. NCCLS, Wayne, PA, USA.
5 . Sambrook, J. E., Fritsch, F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
6 . Couturier, M., Bex, F., Bergquist, P. L. et al. (1988). Identification and classification of bacterial plasmids. Microbiological Reviews 52, 37595.[ISI]
7 . Stokes, H. W. & Hall, R. M. (1989). A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons. Molecular Microbiology 3, 166983.[ISI][Medline]
8 . Sundin, G. W. & Bender, C. L. (1996). Dissemination of the strA-strB streptomycin-resistance genes among commensal and pathogenic bacteria from humans, animals and plants. Molecular Ecology 5, 13343.[ISI][Medline]
9 . Schlüter, A., Heuer, H., Szczepanowski, R. et al. (2003). The 64 508 bp IncP-1ß antibiotic multiresistance plasmid pB10 isolated from a waste-water treatment plant provides evidence for recombination between members of different branches of the IncP-1ß group. Microbiology 149, 313953.[CrossRef][ISI][Medline]
10 . Sundin, G. W., Monks, D. E. & Bender, C. L. (1995). Distribution of the streptomycin-resistance transposon Tn5393 among phylloplane and soil bacteria from managed agricultural habitats. Canadian Journal of Microbiology 41, 7929.[ISI][Medline]
11 . Doroshenko, V. G. & Livshits, V. A. (2004). Structure and mode of transposition of Tn2555 carrying sucrose utilization genes. FEMS Microbiology Letters 233, 3539.[CrossRef][ISI][Medline]
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