ABSTRACT
A qnrB19 gene variant, carried by an IncL/M-like plasmid, was detected in a multidrug Salmonella enterica serovar Typhimurium human strain with reduced susceptibility to ciprofloxacin. The genetic environment around the gene was fully sequenced (20 kb). A large gene cluster, containing the aph, qnrB19, and blaSHV-12-like resistance genes, is inserted inside a Tn3 transposon.
Fluoroquinolones represent a choice treatment for invasive and systemic salmonellosis. Fluoroquinolone resistance usually arises spontaneously due to mutations within the DNA gyrase and topoisomerase IV genes (15), but the following plasmid-mediated quinolone resistance mechanisms reducing ciprofloxacin activity have been recognized: qnr (2, 16, 18), aac(6′)-Ib-cr (19), qepA (26), and oqxAB (14). The most common qnr genes (qnrA, qnrB, and qnrS) (17, 21) were identified in plasmids ranging from 54 to >180 kb in Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, and Salmonella spp., often associated with extended-spectrum β-lactamase genes (1, 5, 8, 10, 11, 18, 22).
In Italy, data on the occurrence of plasmid-mediated quinolone resistance in Salmonella enterica are not available, and this study evaluates its frequency in 1,811 epidemiologically unrelated human isolates of Salmonella collected in the framework of the Enternet surveillance system during the period from 2003 to 2006 (http://www.iss.it/ente ) (12).
Three strains showed resistance (MIC = 4 μg/ml; two S. enterica serovar Kentucky and one S. enterica serovar Typhimurium) and 89 showed reduced susceptibility (MIC = 0.125 μg/ml and <1 μg/ml) to ciprofloxacin (13) when tested using a standardized agar dilution method (6). Resistance to nalidixic acid was observed in 85 out of the 92 strains examined.
In the three resistant strains, mutations in the gyrA (Ser83Phe and Asp87Asn) and parC (Thr57Ser and Ser80Ile) genes were identified (7, 25). All the strains showing reduced susceptibility for ciprofloxacin were screened by PCR for qnrA, qnrB, qnrS, aac(6′)-Ib-cr, and qepA genes (3, 19, 26).
A strain of S. Typhimurium, STYM61/9, was positive for qnrB, identified as a qnrB19 gene variant by DNA sequencing (23). The STYM61/9 strain, isolated from a domestic case of gastroenteritis in 2004, was resistant to ampicillin, cefotaxime, ceftazidime, kanamycin, and trimethoprim-sulfamethoxazole and susceptible to nalidixic acid (the MICs for the quinolones, obtained using the Etest method, are in Table 1). No mutations in the gyrA, gyrB, parC, and parE genes were found. PCR amplification, using primers specific for blaSHV and blaCTX-M (20), and sequencing identified the blaSHV-12-like gene in this strain (K94→E with respect to the blaSHV-12 gene).
To localize the qnrB19 and blaSHV-12 genes, the plasmid of STYM61/9 was transferred by conjugation to E. coli CSH26RifR (24). The transconjugants (conjugation frequency, 5 × 10−7) showed the same resistance phenotype of the donor, except for trimethoprim-sulfamethoxazole resistance, with a ciprofloxacin MIC of 0.19 μg/ml (Table 1).
The plasmid obtained (the p61/9 plasmid) was analyzed by KpnI digestion and Southern blot hybridization with the blaSHV-12 and qnrB19 amplicons as probes that colocalized on a KpnI fragment of approximately 20 kb. A KpnI library was constructed, and a 20-kb DNA sequence insert was determined and extended at its 5′ end by ca. 1 kb, using ABI 3730 DNA sequencing instruments (23). The consensus map is shown in Fig. 1 (GenBank accession no. FJ790886).
The p61/9 cloned fragment contained the replication region (repA, repB, and repC genes) showing 97% and 96% sequence identity to those of the pCTX-M3 (GenBank accession no. NC_004464) and pMU407.1 (GenBank accession no. U27345) plasmids, respectively, both classified as IncL/M plasmids.
The replication region was followed by a Tn3 transposon derivative containing truncated transposase (ΔtnpA), resolvase (tnpR), and blaTEM-1 genes (100% identity to Tn3; GenBank accession no. AY214164). Within the truncated tnpA gene, the aphI, qnrB19, and blaSHV-12-like resistance genes were flanked by IS26 or ISEcp1 elements (Fig. 1). In particular, one of these IS26 elements, followed by the aphI gene, caused the deletion of 47 bp inside the tnpA gene of Tn3. Within this region, the Tn2012 transposon, comprising the ISEcp1C element and the qnrB19 gene, was identified. The latter transposon showed 100% and 99% identity to those identified in plasmids from K. pneumoniae and E. coli, respectively (4, 8). Since the Tn2012 transposon of the p61/9 plasmid was bracketed by two inverted repeat (IR) sequences (IRL and IRR2) but lacked the 5-bp duplication at the target site (direct repeat [DR]), we can assume that it was acquired by illegitimate recombination rather than by ISEcp1-mediated transposition. Downstream, the Tn2012 transposon, a region bracketed by two IS26 sequences, was detected which encodes two open reading frames (ORF33 and ORF40) and the SHV-12-like protein. This segment showed 99% identity with two different regions of the p1658/97 plasmid (GenBank accession no. AF550679) identified in one E. coli strain carrying the blaSHV-5 gene. This IS26-flanked segment in p61/9 likely derived by the assembly of two noncolinear regions of p1658/97 and by the acquisition of mutations in the blaSHV-5 gene leading to the blaSHV-12-like gene. Downstream, the Tn3 transposon, a truncated integron containing the aadB gene cassette and lacking the N-terminal amino acid sequence of the integrase gene and the qacΔE1 and sul1 genes, was detected. The DNA sequence up to the KpnI cloning site showed 98% identity to the trbC gene of the pCTX-M3 plasmid. A duplication of the Tn3 integration target site was identified close to the repC and trbC genes, but the acquisition of the truncated integron shifted the DR far from the IR of the Tn3 transposon (Fig. 1A).
The resistance region of p61/9 clearly arose from multiple insertion events. It is likely that the Tn3 transposon, carrying the integron at the right arm, was first acquired by an IncL/M-like plasmid between the replication and the trbC genes and that subsequently, the integration of the region flanked by IS26 elements disrupted the tnpA gene of Tn3 (Fig. 1A). Consecutive recombination events lead to the construction of the IS26-bracketed region (Fig. 1A to C); in particular, the integration of the Tn2012 transposon into Tn4352B occurred, leading to a novel resistance region carrying relevant genes such as qnrB19 and blaSHV-12.
Recently, an IncN plasmid carrying the qnrB19 gene has been identified in a human strain of S. Typhimurium isolated in The Netherlands (9); however, to our knowledge, this is the first description of the physical association in a cluster of the qnrB19 and blaSHV-12 genes and the first report of an S. Typhimurium clinical isolate harboring a qnrB19 gene in Italy.
In conclusion, this study confirms the low frequency of Qnr and extended-spectrum β-lactamase determinants among Salmonella human isolates in Italy. Nevertheless, the presence of these genes on a mobile element flanked by IS26 elements could lead to easier dissemination among plasmids and the bacterial chromosome, contributing to the horizontal transfer and spread among unrelated species.
Model for the hypothetical evolution of the resistance gene region of plasmid p61/9, via Tn3 transposition and the IS26 and ISEcp1C elements on the IncL/M-like plasmid, through the steps depicted in panels A and B. (C) Graphic presentation of the 21-kb multiresistant region containing the qnrB19 gene. The white and the black bars indicate the 5-bp DRs and the IRs at the boundaries of the Tn3 transposon, respectively. The schematic map is not to scale.
MICs of fluoroquinolones for S. Typhimurium STYM61/9, transconjugant p61/9T, and the recipient strain E. coli CSH26RifR
ACKNOWLEDGMENTS
We are grateful to A. Carattoli at Istituto Superiore di Sanità for providing us with the qnrA and aac(6′)-Ib-cr positive control strains and for encouraging discussion and helpful suggestion. We thank D. J. Mevius of the Central Institute for Animal Disease Control, Levystad, The Netherlands, for providing us with the qnrB and qnrS positive control strains.
This work was partially funded by the European Community Network of Excellence Med-Vet-Net Workpackages 21 (contract no. FOOD-CT-2004-506122).
A.M.D. and C.L. contributed equally to this study.
FOOTNOTES
- Received 4 March 2009.
- Returned for modification 8 May 2009.
- Accepted 9 June 2009.
- Copyright © 2009 American Society for Microbiology
