Integrons and Transposons on the Salmonella enterica Serovar Typhimurium Virulence Plasmid

Abstract

A virulence plasmid was identified in a multidrug-resistant Salmonella enterica serotype Typhimurium strain carrying the spvC, rck, and pefA virulence genes and two class 1 integrons linked to the Tn21 and Tn1696 transposons. A novel trimethoprim resistance gene, designated dfrA23, was also identified within the integron region. The association of multidrug resistance and virulence determinants represents an interesting example of virulence plasmid evolution.

Antibiotic resistance is formally an adaptive mechanism contributing to bacterial virulence. Resistance and virulence genes have features common, both being located in the bacterial chromosome, as well as on plasmids, associated in gene clusters to form resistance or pathogenicity islands, transferred by mobile elements or phages. The presence of virulence and resistance genes on the same plasmid may generate coselection of both types of determinants (1, 15).

In Salmonella enterica serotypes Enteritidis, Typhimurium, Dublin, and Choleraesuis, several virulence genes have been located on plasmids; these are the spvC (Salmonella plasmid virulence), rck (resistance to complement killing), and pefA (plasmid encoded fimbria) genes (5, 7, 8). Salmonella virulence plasmids range in size from 50 to 285 kb and belong to incompatibility group IncFII (5, 9).

In this report, we describe the molecular characterization of a Salmonella virulence plasmid that has acquired multiple resistance determinants. This virulence plasmid has been found in the previously described strain S. enterica serotype Typhimurium ST4 (3). This strain was isolated in Italy in 1997 from a human with salmonellosis and showed resistance to ampicillin, streptomycin, spectinomycin, sulfonamides, gentamicin, kanamycin, and trimethoprim. The ST4 strain contained two class 1 integrons, hereafter referred to as In-t7 and In-t8 (3, 10, 18). By PCR amplification and DNA sequencing, In-t7 was found to carry the aadB gene cassette, conferring gentamicin and kanamycin resistance, while In-t8 carried the bla_oxa-30 and aadA1 gene cassettes, conferring ampicillin and streptomycin-spectinomycin resistance, respectively (3, 12).

In order to determine the localization of the integrons in strain ST4, conjugation experiments were performed with the nalidixic acid-resistant strain Escherichia coli CSH26-N as the recipient strain (14). No transconjugants were obtained by analyzing 10⁸ recipient cells on Luria-Bertani agar medium containing ampicillin (50 μg/ml) or kanamycin (30 μg/ml) and nalidixic acid (40 μg/ml). However, the entire resistance profile of strain ST4 was successfully transferred by transformation of E. coli DH5α competent cells (Invitrogen-Life Technologies, Groningen, The Netherlands) with purified plasmid DNA (High Purity Plasmid Purification Systems, Marligen Bioscience Inc.). A unique plasmid (t-ST4) of approximately 110 kb, carrying the In-t7 and In-t8 integrons, was identified in the E. coli transformants and then analyzed by replicon typing with the complete repertoire of inc and rep probes specific for the major incompatibility groups (6). The t-ST4 plasmid strongly hybridized to the rep^FII probe (data not shown), namely, to the same replicon characterizing the Salmonella virulence plasmids (5, 9). This result suggested that t-ST4 might be a virulence plasmid.

To confirm this hypothesis, we analyzed the t-ST4 plasmid for the presence of virulence determinants with four sets of previously described primers (7, 9). By PCR amplification, this plasmid was positive for the spvC (7), rck, and pefA virulence genes (9), as well as for the traT gene (9), one of the genes of the tra locus mediating conjugative transfer of the S. enterica serotype Typhimurium virulence plasmids (5). These results confirmed that t-ST4 is a Salmonella virulence plasmid.

To further characterize this plasmid, t-ST4 was analyzed by EcoRI and SphI digestion (Fig. 1A and D) and by Southern blot hybridization (19) with the traT and spvC amplicons as probes. A susceptible S. enterica serotype Enteritidis strain carrying the virulence plasmid (SE81/10) was used as a control in the hybridization experiments. Both plasmids positively hybridized to the spvC probe on the same EcoRI fragment, demonstrating that this locus was conserved in t-ST4 (Fig. 1B). The traT probe recognized only the t-ST4 plasmid (Fig. 1C), as expected since the S. enterica serotype Enteritidis virulence plasmid is known to lack the tra locus (5). These results indicated that t-ST4 is related to the S. enterica serotype Typhimurium virulence plasmid. The t-ST4 plasmid was also hybridized with a tnpM-specific probe obtained by PCR amplification of the tnpM gene from Tn21 as previously described (22). This hybridization revealed the presence of the transposase machinery of the Tn21 transposon on t-ST4 (Fig. 1E, lane 1). Additional bands were detected in the spvC and tnpM hybridizations, likely as a result of low contamination of partially digested DNAs or cross-hybridization of the probes.

FIG. 1.

Restriction and Southern blot hybridization analysis of t-ST4. Restricted plasmids were separated by 0.8% agarose gel electrophoresis. (A) plasmid restriction profiles generated by EcoRI digestion of virulence plasmids t-ST4 (lane 1) and SE81/10 (lane 2). (B and C) Southern blot hybridization (19) of the EcoRI-digested DNAs with the spvC and traT probes, respectively. The arrows indicate the expected EcoRI fragments recognized by the spvC and traT probes, respectively, as deduced from the DNA sequence of the Salmonella LT2 virulence plasmid (EMBL accession no. AE006471). (D) Plasmid restriction profiles generated by SphI digestion of t-ST4 (lane 1) and pACYC184::Tn21 (lane 2), used as a positive control for tnpM hybridization. (E) Southern blot hybridization of the SphI-digested DNA with the tnpM probe. The arrow indicates the expected SphI fragment recognized by the tnpM probe (EMBL accession no. AF071413). Lanes M, 1Kb plus DNA ladder (Invitrogen-Life Technologies).

The aadB, bla_oxa-30, aadA1, and sul-1 genes, previously identified in the In-t7 and In-t8 integrons, accounted for the entire resistance pattern conferred by the t-ST4 plasmid, except for trimethoprim resistance, which was not identified as an integron-borne gene cassette (3). To clone the trimethoprim resistance gene, an SphI DNA library of t-ST4 was constructed in the pUC18 vector (Amersham Biosciences). The library was introduced by transformation in E. coli DH5α competent cells, selecting transformants on Muller-Hinton agar plates containing 30 μg of trimethoprim per ml. We achieved several positive transformants, all carrying an insert of 11.8 kb. Among them, the p-ST4Tp clone was chosen as the prototypic clone for further characterization. The complete DNA sequence of the p-ST4Tp clone was determined, and the consensus map obtained is shown in Fig. 2. The sequence revealed that the In-t7 integron shows a common region of 2,154 bp at the 3′ conserved segment (17) containing an open reading frame named orf513 that was previously described for several other class 1 integrons, including In7 (GenBank accession no. L06418), In6 (GenBank accession no. L06822), In34 (GenBank accession no. AY123253), and In-t1 (GenBank accession no. AJ310778). orf513 was followed by an open reading frame, encoding a novel putative predicted protein of 186 amino acids, that we designated dfrA23. The translated polypeptide showed 70 and 35% homology with amino acid sequences of the DfrA19 (EMBL accession no. CAC81324) and DfrA10 (EMBL accession no. AAA92749) dihydrofolate reductases, respectively (Fig. 2), supporting the identification of the unknown protein as a novel trimethoprim resistance gene. The dfrA23 start codon is at positions 7,303 to 7,301, and the TAA stop codon is at positions 6,745 to 6,743 (EMBL accession no. AJ746361), showing the opposite direction of transcription with respect to the integron-borne gene cassettes and to orf513. The dfrA23 gene is located near the transposase (tnpA) and resolvase (tnpR) genes of the Tn1696 transposon. At the 5′ end of the tnpR_Tn1696 gene there is the 5′ conserved segment of the In-t8 integron, cloned in the p-ST4Tp construct up to the SphI site in the integrase gene (Fig. 2). The complete DNA sequence of the In-t7 and In-t8 integrons was obtained by sequencing three PCR amplicons (PCR1, PCR2, and PCR3 in Fig. 2) obtained with the previously described primer pairs intA-ant3A, ant3S-orf5RV, and tnpA-3′CS (3, 22). The positions of In-t7 and In-t8 with respect to the tnpR genes of Tn21 and Tn1696, respectively, are identical to those of other integrons previously located in these transposons (16, 17, 22).

FIG. 2.

Consensus map of the t-ST4 resistance island and alignment of the deduced DfrA23 protein sequence. The open reading frames identified within the t-ST4 resistance region are shown by arrows indicating their directions of transcription. EcoRI (E) and SphI (S) restriction sites are indicated. The bars below the map represent the positions of the p-ST4Tp construct and the sequenced amplicons (PCR1 to -3, flanked by arrows). The position of the tnpM probe used in Southern blot hybridization experiments is indicated. The amino acid sequence alignment below the map compares the deduced sequence of the dfrA23-encoded protein with the DfrA19 and DfrA10 protein sequences. Amino acid similarities are indicated by shaded boxes; darker shading indicates amino acid identities among the three sequences; gaps are marked by dashes.

In order to confirm that the novel dfrA23 gene was a functional gene, the 789-bp amplicon, containing the entire dfrA23 coding sequence flanked by 126 bp upstream of the ATG codon and 113 bp at the 3′ end (positions 7430 to 6641 of the sequence with GenBank accession no. AJ746361), was cloned into the pCR2.1 vector (Invitrogen-Life Technologies) in the opposite orientation with respect to the P_lac promoter's direction of transcription. InvF′α E. coli (Invitrogen-Life Technologies) transformants carrying this construct, but not those carrying the pCR2.1 vector alone, were capable of growing on Mueller-Hinton agar plates containing up to 100 μg of trimethoprim per ml, thus confirming the functional activity of the novel dfrA23 gene.

The assembled map obtained by DNA sequencing and Southern blot hybridization results shows that t-ST4 carries two integrons and two transposons in a head-to-tail configuration to form a complex array of resistance genes. This resistance island has many traits in common with other previously described resistance determinants. The In-t7 integron and the adjacent common region strongly resembled In34, an integron carrying the aadB gene cassette and previously associated with the Tn1696 transposon (17), and is also very similar to In7, which is found in the E. coli pDGO100 plasmid (16, 21). In-t8 is similar to other integrons carrying the bla_oxa-30 or bla_oxa-1-aadA1 gene cassette (bla_oxa-30 differs from bla_oxa-1 by a single point mutation), previously described in association with the Tn21-derivative transposons (9, 11, 20, 22). One of the latter integrons has been recently identified on S. enterica serotype Typhimurium virulence plasmid pUO-StVR2 (9). To our knowledge, there are very few reports describing resistance genes and integrons localized on virulence plasmids (4, 7, 8, 13) and this is the first example of the coexistence of two transposons (Tn21 and Tn1696) and two integrons (In-t7 and In-t8) on a Salmonella virulence plasmid.

Our findings suggest that virulence plasmids may evolve through the acquisition of multiple resistance genes. It is also interesting that such resistance determinants are often colocalized in the same chromosomal or plasmid site to form a resistance island. The t-ST4 resistance island strongly resembled those previously described in S. enterica serotype Typhimurium DT104 salmonella genomic island 1 and on the S. enterica serotype Typhimurium IncFI plasmid (2, 22). This association is probably the result of many recombination- and transposition-mediated processes that, in the case of t-ST4, occurred between the virulence plasmid and other resistance donor plasmids.

Nucleotide sequence accession number.

The nucleotide sequence of the 16,410 bp from the t-ST4 resistance region was submitted to the EMBL database and assigned accession no. AJ746361.