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Genetic characterization of three qnrS1-harbouring multidrug-resistance plasmids and qnrS1-containing transposons circulating in Ho Chi Minh City, Vietnam - PubMed

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Genetic characterization of three qnrS1-harbouring multidrug-resistance plasmids and qnrS1-containing transposons circulating in Ho Chi Minh City, Vietnam

Vien Le et al. J Med Microbiol. 2015 Aug.

Abstract

Plasmid-mediated quinolone resistance (PMQR) refers to a family of closely related genes that confer decreased susceptibility to fluoroquinolones. PMQR genes are generally associated with integrons and/or plasmids that carry additional antimicrobial resistance genes active against a range of antimicrobials. In Ho Chi Minh City (HCMC), Vietnam, we have previously shown a high frequency of PMQR genes within commensal Enterobacteriaceae. However, there are limited available sequence data detailing the genetic context in which the PMQR genes reside, and a lack of understanding of how these genes spread across the Enterobacteriaceae. Here, we aimed to determine the genetic background facilitating the spread and maintenance of qnrS1, the dominant PMQR gene circulating in HCMC. We sequenced three qnrS1-carrying plasmids in their entirety to understand the genetic context of these qnrS1-embedded plasmids and also the association of qnrS1-mediated quinolone resistance with other antimicrobial resistance phenotypes. Annotation of the three qnrS1-containing plasmids revealed a qnrS1-containing transposon with a closely related structure. We screened 112 qnrS1-positive commensal Enterobacteriaceae isolated in the community and in a hospital in HCMC to detect the common transposon structure. We found the same transposon structure to be present in 71.4 % (45/63) of qnrS1-positive hospital isolates and in 36.7 % (18/49) of qnrS1-positive isolates from the community. The resulting sequence analysis of the qnrS1 environment suggested that qnrS1 genes are widely distributed and are mobilized on elements with a common genetic background. Our data add additional insight into mechanisms that facilitate resistance to multiple antimicrobials in Gram-negative bacteria in Vietnam.

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Figures

Fig. 1
Fig. 1. Linear DNA sequence alignments of pE66An, pK18An and pK1HV against plasmid pK245 centred at the qnrS1-encoding region. Regions of DNA identity of 99 % or greater are linked by red blocks. The open box is the region containing the qnrS1-harbouring transposon, which is identical in all four plasmids except for a 980 bp insert in pK18An. The sizes of the plasmids are shown, with each shaded/open block representing 10 kbp.
Fig. 2
Fig. 2. Annotated circular plasmid maps of pE66An, pK18An and pK1HV orientated from the origin of replication. (a) pE66An; (b) pK18An; (c) pK1HV. Identified and annotated ORFs are colour coded on each of the three plasmids: red, plasmid replication; dark blue, inorganic/metal/UV resistance; sky blue, conjugal transfer; dark pink, antimicrobial resistance; light green, unknown; light blue, regulators; orange, conserved hypothetical; brown, pseudogenes or partial genes; light pink, IS elements. The arrow annotation shows the strand on which the ORF is located. The inner circle shows the GC skew ([GC]/[G+C]) and the next outer circle shows G+C (mol%) plot. Fragments with substantial DNA homology to other sequenced plasmids and the qnrS-encoding region are highlighted.
Fig. 3
Fig. 3. A schematic representation of sequenced qnrS1-containing transposons. Graphical representation of the synteny between the qnrS-containing transposons between the three plasmids sequenced here (pK18An, pE66An and pK1HV) and other sequenced fragments containing the qnrS-encoding region. The plasmids and the host organism in which they were first identified are given. The region with the greatest DNA homology is identified and includes the highlighted ORFs for qnrS (red), a putative IS2 element (grey), a gene encoding a putative resolvase protein (ydaA) and three other ORFs encoding hypothetical proteins of unknown function. Additional genes are colour coded: blue, bla LAP-2; grey, IS elements; white, ORFs without a name encoding hypothetical uncharacterized proteins. The locations of the binding sites for PCR amplification of the transposon are highlighted.
Fig. 4
Fig. 4. EcoRI digestion of qnrS-encoding plasmids hybridized with the qnrS1 and bla LAP-2 probes. Upper panel: agarose gel electrophoresis of qnrS-encoding plasmids after digestion with EcoRI. The resulting patterns are duplicate digestions from five plasmids after conjugation. Lanes: 2 and 8, isolate LTMV18; 3 and 9, isolate LTMV33; 4 and 10, isolate LTMV6; 5 and 11, isolate LTMV30; 6 and 12, isolate LTMV1. The ladder (lane 1) is 1 kb Plus (Invitrogen) with sizes shown in kb. Lanes 7 and 13 contain the PCR amplicons of qnrS and bla LAP-2 as positive controls. Bottom panel (left): hybridization against the qnrS1 probe after Southern blotting. All five plasmids contained fragments corresponding to probe qnrS1. Bottom panel (right): hybridization against the bla LAP-2 probe after Southern blotting.

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