Comparison of multidrug resistance gene regions between two geographically unrelated Salmonella serotypes

M. Daly1,{dagger}, L. Villa2, C. Pezzella2, S. Fanning1,3 and A. Carattoli2,*

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


* Corresponding author. Tel: +39-06-4990-3128; Fax: +39-06-4938-7112; Email: alecara{at}iss.it

Received 4 October 2004; returned 12 November 2004; revised 6 December 2004; accepted 9 December 2004


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The aim of this study was to identify chromosomally integrated genes conferring multidrug resistance to a Salmonella enterica (S.) serotype Typhimurium isolate, phage type DT193, isolated in Ireland and to compare them with resistance genes conferring plasmid-mediated multidrug resistance to a S. Enteritidis isolate from Italy.

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


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Current global trends show increasing numbers of resistant Salmonella spp. in humans and animals. Resistance genes in Salmonella have been often located within integrons, sometimes associated with transposons and insertion sequences. These genetic elements essentially favour the exchange of resistance genes between plasmids and the bacterial chromosome by (independent) transposition events. A well described example of chromosomally integron-borne resistance genes is represented by the pandemic Salmonella enterica (S.) serotype Typhimurium definitive type 104 (DT104). This clone is resistant to five different classes of antimicrobials including ampicillin, chloramphenicol/florfenicol, streptomycin/spectinomycin, sulphonamides and tetracyclines. All five resistance determinants were mapped to the chromosome and located on the distal end of the 43 kb Salmonella Genomic Island 1.1

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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

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 Luria–Bertani 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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Table 1. Primers used in PCR amplification

 
A BglII library was constructed by ligation of BglII-digested plasmid DNA of S. Enteritidis at the BamHI site of the pUC18 cloning vector, transforming competent cells of E. coli DH5{alpha}.5 Tetracycline-resistant transformants were selected on LB agar plates containing 10 mg/L tetracyclines, obtaining the pTet2 recombinant plasmid. Four subclones were obtained from the pTet2 construct by cloning SacI and SalI restriction fragments into the respective site of the pUC18 vector. The DNA sequence of the 20 kb BglII was completely determined by primer walking on the pTet2 clone and subclones.

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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The 12 kb resistance gene region in the IncI S. Enteritidis plasmid

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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Figure 1. Consensus map of the S. Enteritidis plasmid and S. Typhimurium DT193 resistance regions. Open reading frames in the consensus map are indicated by solid arrows orientated with respect to the direction of transcription; genes derived from the RSF1010 plasmid, whose map is shown below, are indicated. White arrows represent integron-associated genes. The short lines below the map indicate the 5'- and 3'-conserved segments of the integrons (5'CS and 3'CS). The IRi and IRt integron flanking sequences are indicated. Each IS26 element is indicated with a grey-shaded box, showing the transposase tnpA gene flanked by the right (IRR) and left (IRL) inverted repeats. A white box shows the tnpB locus on the S. Typhimurium resistance region and RSF1010 plasmid.

 
The analysis of the DNA sequence revealed the presence of a multiresistance gene region carrying the strA, strB, sul2, repC and repA genes derived from the RSF1010 plasmid (EMBL accession no. M28829), flanked by two inverted IS26 elements. The distal IS26 element probably caused the deletion of the 5' end of the repA gene of RSF1010. To the right of this element a putative recombinase gene and the tetR-tet(A) genes, conferring tetracycline resistance, were identified (Figure 1). To the 5'-end of the proximal IS26 element was located an IP-type 2 integron, carrying the dfrA1-aadA1 gene cassette. The latter element probably caused a deletion at the 3'-end of the aadA1 gene ({Delta}aadA1 in the Figure 1), removing the 3'-conserved segment (3'CS) of the integron, resulting in failure to amplify this region as a composite cassette. The IRi and IRt, initial and terminal inverted repeats, characteristic of class 1 integrons7 were identified within the sequence, the latter located upstream of the tetR gene (Figure 1).

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.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, the characterization of two multidrug resistance gene regions, showing a high level of structural similarity, found on an S. Enteriditis IncI plasmid and in the chromosome of S. Typhimurium DT193, are reported.

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.


    Footnotes
 
{dagger} Present address. Dairy Products Research Centre, Teagasc-Moorepark, Fermoy, Co. Cork, Ireland. Back


    Acknowledgements
 
We wish to thank Dr Jim Buckley and Dr Giuliano Imperiali for supporting this work. In addition, the financial support of ISS funds 2003–2004, C3MD-cap 524 (A.C.) and Irish Government TSR-3-2000 (S.F.) is gratefully acknowledged.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Boyd, D., Peters, G. A., Cloeckaert, A. et al. (2001). Complete nucleotide sequence of a 43-kilobase genomic island associated with the multidrug resistance region of Salmonella enterica serovar Typhimurium DT104 and its identification in phage type DT120 and serovar Agona. Journal of Bacteriology 183, 5725–32.[Abstract/Free Full Text]

2 . Daly, M. & Fanning, S. (2000). Characterisation and chromosomal mapping of antimicrobial resistance genes in Salmonella enterica serotype Typhimurium. Applied and Environmental Microbiology 66, 4842–8.[Abstract/Free Full Text]

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, 1350–2.[Abstract/Free Full Text]

4 . National Committee for Clinical Laboratory Standards (2003). Performance Standards for Antimicrobial Disk Susceptibility Tests—Eighth 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, 375–95.[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, 1669–83.[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, 133–43.[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, 3139–53.[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, 792–9.[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, 353–9.[CrossRef][ISI][Medline]





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