JAC Advance Access published online on July 7, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn286
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Original research |
Characterization of Salmonella enterica serovar Typhimurium conjugative plasmids transferring resistance to antibiotics and their interaction with the virulence plasmid
Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic
* Corresponding author. Tel: +420-533331201; Fax: +420-541211229; E-mail: rychlik{at}vri.cz
Received 18 April 2008; returned 12 May 2008; revised 18 June 2008; accepted 18 June 2008
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Abstract |
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Objectives: In this study, we analysed field isolates of Salmonella enterica serovar Typhimurium for the presence of conjugative plasmids transferring resistances to antibiotics.
Methods: Altogether 23 strains were analysed for the presence of conjugative R-plasmids. In the case of successful conjugation, the R-plasmids were characterized by PCR for antibiotic resistance genes, integrons and replicon typing. Variable regions of integrons were sequenced.
Results: Conjugation and transfer of antibiotic resistance was observed in 12 strains. Conjugative plasmids in these strains belonged to the IncI1 and IncHI1 replicons and four of them transferred antibiotic resistance associated with class I integrons. In two cases, resistance to tetracycline and/or ampicillin was not transferred by conjugation to 
10% of the transconjugants. Detailed characterization showed that the loss of both resistances was associated with the loss of Tn3 (blaTEM) and Tn1721 [tet(A)] from the conjugative plasmids p9046 and p9134. However, when only the tetracycline resistance was lost, the Tn1721 was replaced with a partial sequence of rck, and with complete coding sequences of srgA, srgB, ORF7 and pefI originating from the Salmonella Typhimurium virulence plasmid.
Conclusions: Two plasmids from our collection were capable of recombination with the virulence plasmid of Salmonella Typhimurium and subsequently spread both antibiotic resistance and virulence genes to the recipient.
Key Words: integrons , Tn3 , Tn1721 , HCM1.225 , rck
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Introduction |
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In Gram-negative bacteria, new resistance genes are most frequently acquired by the conjugational transfer of plasmids coding for antibiotic resistance (R-plasmids). The archetype of many current R-plasmids is the NR1 plasmid that contains three transposons, Tn9 with the catA1 gene, Tn21 with the aadA1 and sul1 genes, and Tn10 with the tet(B) gene. Plasmid NR1 with Tn21 and a class 1 integron is also an example of a composite genetic element, i.e. plasmid carrying transposons containing integrons.1 The conjugative transfer of a plasmid with a transposon harbouring an integron allows the simple movement of antimicrobial resistance from one bacterial cell to another, the transposition of the resistance genes from a plasmid to the chromosome and the instant ability to acquire new resistance gene cassettes due to the presence of integrons. Since plasmids are not essential for bacterial life, recombination events in these molecules are more frequent than in chromosomes. This is a reason for the appearance of new gene combinations including the recombination between different R-plasmids and serovar-specific virulence plasmids found in serovars Enteritidis, Typhimurium, Dublin, Choleraesuis or Gallinarum.2,3
In this study, we focused on a detailed analysis of R-plasmids found in Salmonella enterica serovar Typhimurium (Salmonella Typhimurium). Rather unexpectedly, in two conjugative plasmids, we repeatedly observed an incomplete transfer of the antibiotic resistance during conjugation and when we mapped the incomplete plasmids, we found that the apparent incomplete transfer of the antibiotic resistance was in fact a recombination between the R-plasmid and the virulence plasmid, leading to a generation of a new conjugative R-plasmid with virulence genes of Salmonella Typhimurium.
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Materials and methods |
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Bacterial strains
A total of 23 Salmonella Typhimurium strains previously characterized as possessing multidrug resistance4 were investigated in this study. The strains were selected based on the presence of antibiotic resistance genes that are not usually associated with the Salmonella Genomic Island 1 (SGI1). Salmonella Typhimurium F98 (serovar-specific virulence plasmid negative) and Salmonella Enteritidis 147 (serovar-specific virulence plasmid positive), both spontaneously resistant to nalidixic acid, were used as recipients in conjugation. To designate the conjugative plasmids, the prefix ‘p’ was used followed by the designation of the original host strain, e.g. conjugative plasmid pF8025 originated from Salmonella Typhimurium strain F8025.
Primers for the detection of the left junction of SGI1 and the genes typical for SGI1 [blaPSE1, floR, aadA2, sul1 and tet(G)], for the detection of integrons (intI1 and 5'CS–3'CS) and for the detection of non-SGI1 genes [blaTEM, cat, strA, sul2, tet(A), tet(B) or tet(C)] have been described previously.4,5 Primers targeted at HI1, HI2, I1, X, N, A/C and FII plasmid incompatibility groups (those previously recorded in Salmonella spp.) were designed according to Carattoli et al.6 The kanamycin-resistant strains were analysed for the presence of the aphAI-IAB gene using primers 5'-AAACGTCTTGCTCGAGGC-3' and 5'-CAAACCGTTATTCATTCGTGA-3'. The PCR was carried out using PCR Master Mix (Qiagen, Germany) as described previously.4,5 PCR products generated with the 5'CS–3'CS primer pair that differed in size from 1 and 1.2 kb fragments typical for the SGI1 were sequenced using the BigDye Terminator v1.1 Sequencing Standard Kit (Applied Biosystems), and obtained sequences were compared at the GenBank web site using BLAST.
Ten microlitres of 16 h culture of both the donor and recipient strains were inoculated into 4 mL of LB medium, and conjugation was performed at either 24 or 37°C with gentle shaking. The resulting transconjugants were selected on agar plates containing nalidixic acid and one of the antibiotics to which the donor was resistant. The following day, 10 colonies growing on each of the plates, e.g. ampicillin and nalidixic acid, were subcultured on the remaining antibiotic plates (e.g. tetracycline and chloramphenicol) to find out which of the antibiotic resistances were transferred simultaneously during a single conjugation event.
Incomplete conjugational transfer of antibiotic resistance
For visualization of the plasmids, PFGE was applied. Total genomic DNA was isolated and immediately separated by PFGE as described previously.7
To characterize the deletions in conjugative plasmids of the Salmonella Typhimurium 9046 and 9134 strains in detail, PCR amplification together with sequencing of the amplification products and microarray analysis were done. Using the PCR, the whole region between the tet(A) and blaTEM genes was amplified and sequenced. As a next step, using the sequence of blaTEM and tet(A), primers for inverse PCR were designed from the left and right end of this sequence, and sequences immediately upstream or downstream of these loci were identified.
In parallel, microarray analysis was used to determine the genes transferred during conjugation. Microarray chips were prepared by spotting The Salmonella Genus AROS V1.0 (Operon, Germany) as described previously.8 Since this set also contains oligonucleotides complementary to the pHCM1 and pHCM2 plasmids from Salmonella Typhi coding for the resistances to antibiotics, we expected that these genes, in particular, would enable us to at least partially characterize the conjugative plasmids. For the microarray hybridization, total DNA was purified using the DNeasy Tissue Kit from Qiagen, Germany. Labelling of purified DNA with the Cy3 and Cy5 fluorescent dyes, hybridization and microarray scanning were performed exactly as described previously.8 In the microarray hybridization, the labelled DNA from the transconjugants was always hybridized together with the DNA purified from the recipient.
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Results and discussion |
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Of the 23 strains examined, conjugation was observed in 12 (Table 1). Conjugation occurred with both Salmonella Typhimurium F98 and Salmonella Enteritidis 147 used as recipients. We never observed the transfer of any of the genes localized on SGI1. The remaining resistance genes were mostly transferred together, although in strain S17/1 we did not observe the transfer of blaTEM. In strains 115/6 and B71, the tet(C) gene was never transferred by conjugation and the sul2 gene could not be conjugated from three donor strains (115/0, B71 and F8538). Six strains conjugated preferentially at 24°C and another six strains at 37°C. In strains 9046 and 9134, the analysis of transconjugants demonstrated that although the full set of non-SGI1 resistance genes was usually transferred, in
10% of the transconjugants, only cat, strA and sul2 but not tet(A), or tet(A) together with blaTEM, were transferred (see below).
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Integrons different from those associated with SGI1 were found in five strains (F8025, F8475, B71, 115/0 and 116/1) and, only in strain 116/1, the integron containing sat and aadA1 gene cassettes could not be transferred by conjugation. Plasmids pB71 and p115/0 possessed identical integrons that contained aadA1 and qacE
1 gene cassettes, pF8025 coded for the integron containing dfrA1 and aadA1 gene cassettes and plasmid pF8475 transferred the integron containing dfrA12 and aadA2 gene cassettes. Since the recipient strain Salmonella Typhimurium F98 is free of any plasmids including the virulence plasmid, replicon typing of the conjugative R-plasmids could be easily performed in all 12 transconjugants. Among the R-plasmids, only two different incompatibility groups were identified. Six plasmids belonged to the IncI1 replicon and four plasmids belonged to IncHI1. In two transconjugants, none of the PCRs yielded positive results and therefore the plasmid incompatibility group could not be determined (Table 1).
In general, the conjugative plasmids were similar to those described in previous studies. They belonged to two incompatibility groups, IncI1 and IncHI1, which have been already associated with antibiotic resistance9,10 and four of them transferred the class 1 integrons described previously.2 Three plasmids, pB71, p109/9 and pF8475, were quite unstable in Salmonella Typhimurium F98 after conjugational transfer in the absence of selective pressure. Salmonella Enteritidis 147 pF8025 (but not Salmonella Enteritidis 147), Salmonella Typhimurium F98 pF8025 (but not Salmonella Typhimurium F98) and also the original donor Salmonella Typhimurium F8025 were capable of biofilm formation on the air–LB broth interface if grown statically for 3 days in LB broth.
In two strains and their conjugative plasmids (p9046 and p9134), in 10% of the transconjugants, an incomplete transfer of antibiotic resistance was observed. Both the donor strains as well as the transconjugants were found to contain a single R-plasmid of 130 kb (see Figure 1a, for p9134). In transconjugants, in which either tet(A) was absent or tet(A) together with blaTEM were absent, a decrease in the molecular weight of the plasmids could be observed (Figure 1a). The incomplete conjugational transfer was highly reproducible and we proved it in more than five independent conjugational events for each donor strain. In Salmonella Typhimurium 9046, we only observed reduced frequency of loss of both the ampicillin and tetracycline resistance.
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concatemers; lane 1, Salmonella Typhimurium 9134 donor strain with the virulence plasmid of 94 kb and the conjugative p9134 plasmid; lane 2, p9134 plasmid in Salmonella Typhimurium F98 transconjugant; lane 3, p9134Mapping of the plasmids p9046 and p9134 showed that the locus coding for ampicillin and tetracycline resistance was exactly the same and consisted of two transposons, Tn3 (coding for blaTEM) and Tn1721 [coding for tet(A)], Tn3 being inserted in ORF294 of Tn1721 (Figure 1b). Similar, although not identical, association of Tn3 with Tn1721 has been described by Pasquali et al.11 As determined by microarray analysis, plasmid p9134 also harboured the gene PSLT059 found in the virulence plasmid of Salmonella Typhimurium, HCM1.225 (putative transposase) localized on the pHCM1 plasmid of Salmonella Typhi and STY0114 coding for a transposase found in the chromosome of the same bacterium. These genes were also found in plasmids transferring the incomplete set of antibiotic resistance, and by PCR in p9046.
In incomplete p9134 transconjugants lacking tet(A) and blaTEM, both Tn3 and Tn1712 were excised, together with an additional 7 kb of genetic information including the aac(3)-IV gene. The original plasmid sequence recovered at the HMC1.225 gene (Figure 1b). However, the incomplete transconjugants lacking only tet(A) were of quite an unusual structure. The sequence of the original plasmid p9046 or p9134 was terminated downstream from blaTEM and the original plasmid sequence was recovered at HMC1.225. However, instead of a simple deletion, the lost sequence was replaced with a partial sequence of rck, and with complete coding sequences of srgA, srgB, ORF7 and pefI originating from the Salmonella Typhimurium virulence plasmid (Figure 1b). Although we did not investigate it further, microarray analysis together with confirmatory PCR showed that an additional three genes PSLT024, PSLT025 and PSLT026 originally from the virulence plasmid were transferred by the p9134Tet, but not by full-sized p9134 or p9134TetAmp. Plasmid recombinants between the virulence and R-plasmids have already been described in Salmonella; however, none appears to be similar to ours.2,3 All of this shows that the exposure of Salmonella Typhimurium to R-plasmid may lead to their subsequent recombination with the virulence plasmid and the spread of both antibiotic resistance and virulence genes to a new recipient.
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Funding |
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This work was funded by projects 1B44019 and MZE0002716201 of the Czech Ministry of Agriculture.
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Transparency declarations |
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None to declare.
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Acknowledgements |
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We wish to acknowledge the excellent technical assistance of Michaela Dekanova and Neysan Donelli for English language corrections.
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References |
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1 . Liebert CA, Hall RM, Summers AO. Transposon Tn21, flagship of the floating genome. Microbiol Mol Biol Rev (1999) 63:507–22.
2
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Guerra B, Soto S, Helmuth R, et al. Characterization of a self-transferable plasmid from Salmonella enterica serotype Typhimurium clinical isolates carrying two integron-borne gene cassettes together with virulence and drug resistance genes. Antimicrob Agents Chemother (2002) 46:2977–81.
3
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Chu C, Chiu CH, Wu WY, et al. Large drug resistance virulence plasmids of clinical isolates of Salmonella enterica serovar Choleraesuis. Antimicrob Agents Chemother (2001) 45:2299–303.
4
.
Faldynova M, Pravcova M, Sisak F, et al. Evolution of antibiotic resistance in Salmonella enterica serovar Typhimurium strains isolated in the Czech Republic between 1984 and 2002. Antimicrob Agents Chemother (2003) 47:2002–5.
5 . Matiasovicova J, Adams P, Barrow PA, et al. Identification of putative ancestors of the multidrug-resistant Salmonella enterica serovar Typhimurium DT104 clone harboring the Salmonella genomic island 1. Arch Microbiol (2007) 187:415–24.[CrossRef][Web of Science][Medline]
6 . Carattoli A, Bertini A, Villa L, et al. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods (2005) 63:219–28.[CrossRef][Web of Science][Medline]
7 . Hradecka H, Kolackova I, Karpiskova R, et al. An outbreak of human salmonellosis caused by ampicillin-resistant Salmonella enterica serovar Enteritidis PT13 in the Czech Republic. Epidemiol Infect (2006) 134:737–40.[CrossRef][Medline]
8
.
Sebkova A, Karasova D, Crhanova M, et al. aro mutations in Salmonella enterica cause defects in cell wall and outer membrane integrity. J Bacteriol (2008) 190:3155–60.
9
.
Hopkins KL, Liebana E, Villa L, et al. Replicon typing of plasmids carrying CTX-M or CMY β-lactamases circulating among Salmonella and Escherichia coli isolates. Antimicrob Agents Chemother (2006) 50:3203–6.
10 . Maher D, Taylor DE. Host range and transfer efficiency of incompatibility group HI plasmids. Can J Microbiol (1993) 39:581–7.[Web of Science][Medline]
11
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Pasquali F, Kehrenberg C, Manfreda G, et al. Physical linkage of Tn3 and part of Tn1721 in a tetracycline and ampicillin resistance plasmid from Salmonella Typhimurium. J Antimicrob Chemother (2005) 55:562–5.
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