Complete nucleotide sequence of a small qnrS1-carrying plasmid from Salmonella enterica subsp. enterica Typhimurium DT193

Sir, In a recent study on transferable quinolone resistance among Salmonella enterica strains isolated in the UK, qnrS1 genes were identified on plasmids in the four serotypes Typhimurium, Stanley, Virchow and Virginia.1 Since no complete sequence of a qnrS1-carrying plasmid has been available so far, we decided to sequence completely the smallest type of qnrS1-carrying plasmid to gain insight into the structure and putative origin of this plasmid. The plasmid chosen, TPqnrS-1a, was obtained from a multiresistant Salmonella Typhimurium DT193 strain and previously shown to mediate only decreased susceptibility to ciprofloxacin.1 The plasmid was subjected to extended restriction analyses using the enzymes BglII, ClaI, DraI, EcoRI, EcoRV, HindIII, HpaI, KpnI, PstI, PvuI, PvuII and SmaI in single and double digests. On the basis of these results, a restriction map was constructed. The four HindIII fragments of ∼1.0, 2.0, 2.6 and 4.4 kb were cloned into pBluescript II SK+ (Stratagene, Amsterdam, The Netherlands) and the recombinant plasmids transformed into Escherichia coli JM109. The different fragments were sequenced on both strands by primer walking. Initial sequence analyses were conducted with standard M13 universal and reverse primers and completed with primers derived from the sequences obtained with the standard primers (MWG, Ebersberg, Germany). Sequence analysis was performed with the BLAST programs blastn and blastp (http://www.ncbi.nlm.nih.gov/BLAST) and with the ORF Finder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The nucleotide sequence of the plasmid TPqnrS-1a has been deposited in the EMBL database under accession number AM746977. With a size of 10 066 bp, plasmid TPqnrS-1a proved to be slightly smaller than initially estimated1 and consisted of two different segments (Figure 1). The initial 4666 bp exhibited 89% nucleotide sequence identity to the 4669 bp plasmid pEC278 isolated from a pathogenic E. coli strain (accession no. AY589571). Within this region, three partially overlapping reading frames were detected, which code for the mobilization proteins MobA, MobB and MobC. The gene mobA codes for a 516-amino-acid protein, which showed 92% identity to the 499-amino-acid MobA protein of pEC278. The 161-amino-acid MobB and the 107-amino-acid MobC proteins exhibited 93% and 94% identity, respectively, to the same-sized MobB and MobC proteins of pEC278. Upstream of the mobC gene, an origin of replication similar to that of the E. coli plasmid p15A (accession no. V00309) was found. The adjacent 2830 bp showed 99% identity to plasmid pINF5 from Salmonella Infantis2 and included the qnrS1 resistance gene area, a truncated IS2 insertion sequence and an incomplete Tn5058-related resolvase gene. Further downstream, another 2044 bp region with 98% identity to pINF5 was detected. This region was orientated in the opposite direction in TPqnrS-1a when compared with the Salmonella Infantis plasmid. Between these two pINF5-homologous segments, a 526 bp region without significant homology to database entries was detected (Figure 1). Poirel et al.3 recently also observed the aforementioned inversion in the qnrS1 downstream region of plasmid pS5-1 from Enterobacter cloacae. Comparison of the sequenced parts of plasmids pAH0376 from S. flexneri6 and pINF5 from Salmonella Infantis2 with the complete plasmids TPqnrS-1a from Salmonella Typhimurium DT193 and pEC278 from E. coli. A distance scale in kb is presented below each map. The reading frames are shown as arrows with the arrowhead indicating the direction of transcription. The arrows marked as 213, 259, 213 and 196 in the map of pINF5 indicate reading frames for hypothetical proteins of 213, 259, 213 and 196 amino acids, respectively. The 5 bp direct repeats at the boundaries of the Tn3 elements in pAH0376 and pINF5 as well as the 4 bp direct repeats at the outside boundaries of the truncated IS26 are shown in boxes. The black, striped and stippled boxes indicate the terminal inverted repeats of Tn3, IS26 and IS2, respectively. The areas of homology between plasmids pAH0376, pINF5 and/or TPqnrS-1a as well as between TPqnrS-1a and pEC278 are shown by grey shading. The qnrS1 gene of TPqnrS-1a codes for a 218-amino-acid protein indistinguishable from the QnrS1 proteins previously detected in the S. enterica serovars Infantis (accession no. CAJ84117), Bovismorbificans (accession no. ABF47469), Stanley (accession nos ABI63549 and ABI63550), Virchow (accession nos ABI63552 and ABI63553) and Virginia (accession no. ABI63554) and also from those of Shigella flexneri (accession nos BAD88772 and BAD88776), Klebsiella pneumoniae (accession no. ABG56870) as well as Proteus mirabilis (accession no. ABP88832). Identity to the two so far known QnrS2 proteins from S. enterica serovar Anatum (accession no. ABF47470) and to that of the 8469 bp plasmid pGNB2 obtained from unknown activated sludge bacterium (accession no. ABE98197) was 92%. It should be noted that the qnrS2-carrying plasmid pGNB24 and the qnrS1-carrying plasmid TPqnrS-1a do not share any obvious structural similarities. A comparison between the TPqnrS-1a-associated QnrS1 protein and the 218-amino-acid proteins from Vibrio splendidus (accession no. EAP95542) and Vibrio spp. (accession no. EAQ55748)—the latter two considered as a natural reservoir of qnrS genes5—revealed 83% and 82% amino acid identity, respectively, and 91% amino acid similarity. Previous studies revealed that the qnrS1 gene is often located on large plasmids that carry additional resistance genes.1–3,6 Although plasmids pAH0376 from S. flexneri6 and pINF5 from Salmonella Infantis2 carried a complete Tn3 with a blaTEM-1 β-lactamase gene, recently discovered plasmids of E. cloacae identified the qnrS1 gene in close proximity to the novel β-lactamase gene blaLAP-1.3 In the present study, we characterized a comparatively small plasmid that carried the qnrS1 gene as the sole resistance gene. To the best of our knowledge, plasmid TPqnrS-1a is the first qnrS1-carrying plasmid for which the complete sequence is available. A detailed sequence analysis of this plasmid suggested that it has most likely derived from an interplasmid recombination event between a qnrS1-carrying plasmid such as pINF5 from Salmonella Infantis2 or pS5-1 from E. cloacae3 and a small mobilizable plasmid similar to pEC278 from E. coli. The identification of qnrS1 genes on structurally diverse plasmids points towards the dissemination of these genes and their adaptation in new hosts as relevant factors in the emergence of transferable quinolone resistance.7 This study was financially supported by internal funding provided by the Institut für Tierzucht (FAL). We thank Vera Nöding for excellent technical assistance. None to declare.