Interplay between intrinsic and acquired resistance to quinolones in Stenotrophomonas maltophilia

Abstract

To analyse whether the mutation-driven resistance-acquisition potential of a given bacterium might be a function of its intrinsic resistome, quinolones were used as selective agents and Stenotrophomonas maltophilia was chosen as a bacterial model. S. maltophilia has two elements - SmQnr and SmeDEF - that are important in intrinsic resistance to quinolones. Using a battery of mutants in which either or both of these elements had been removed, the apparent mutation frequency for quinolone resistance and the phenotype of the selected mutants were found to be related to the intrinsic resistome and also depended on the concentration of the selector. Most mutants had phenotypes compatible with the overexpression of multidrug efflux pump(s); SmeDEF overexpression was the most common cause of quinolone resistance. Whole genome sequencing showed that mutations of the SmeRv regulator, which result in the overexpression of the efflux pump SmeVWX, are the cause of quinolone resistance in mutants not overexpressing SmeDEF. These results indicate that the development of mutation-driven antibiotic resistance is highly dependent on the intrinsic resistome, which, at least for synthetic antibiotics such as quinolones, did not develop as a response to the presence of antibiotics in the natural ecosystems in which S. maltophilia evolved.

Vitamin K 3 Induces the Expression of the Stenotrophomonas maltophilia SmeVWX Multidrug Efflux Pump

“…The MBS287 strain, a mutant derived from the wild-type S. maltophilia D457 strain, constitutively expresses high levels of smeVWX due to a mutation (Gly266Asp) in the gene encoding its local regulator SmeRv. This strain can be used as a control for measuring the expression levels of smeVWX when the efflux pump is overexpressed (22). To test the smeVWX sensor, the fluorescence levels given by the PBT02 and PBT06 strains and the growth of both strains were measured for 18 h. As mentioned above, the expression levels of SmeVWX are very low; consequently, it is expected that the fluorescence given by the expression of YFP under the control of the smeVWX promoter in the PBT02 strain is lower than the one obtained in the overexpressing derivative strain PBT06.…”

“…Expression of these determinants is usually repressed by specific regulators encoded by genes located upstream of the operons that contain the structural genes for these efflux pumps (19). In the opportunistic pathogen S. maltophilia, some of these RND multidrug efflux pumps, such as SmeDEF, SmeIJK, SmeOP, and SmeYZ, contribute to bacterial intrinsic resistance (4,6,9,14), while others, such as SmeVWX, have very low levels of expression and its overexpression alone leads to acquired resistance (11,22). In addition to genetic alterations, resistance can also be achieved by the presence of effectors or conditions that trigger the expression of multidrug efflux pumps, leading to the acquisition of transient phenotypic antibiotic resistance (25)(26)(27).…”

“…Fifty nanograms of cDNA was used in each reaction. Primers SmeV-RT.fw and SmeV-RT.rv, which amplify the smeV gene (22), were used at 400 nM. Primers FtsZ1 and FtsZ2 were used to amplify the housekeeping gene ftsZ (22).…”

“…Primers SmeV-RT.fw and SmeV-RT.rv, which amplify the smeV gene (22), were used at 400 nM. Primers FtsZ1 and FtsZ2 were used to amplify the housekeeping gene ftsZ (22). Differences in the relative amounts of mRNA were determined according to the 2 ϪΔΔCT method (59,60).…”

“…The operon is regulated by the LysR-type transcriptional regulator SmeRv (11). SmeVWX does not participate in S. maltophilia intrinsic resistance, likely because its expression levels are too low (11); nevertheless, it is known that this efflux pump contributes to the acquisition of resistance mediated by mutations in the transcriptional regulator SmeRv, leading to the overexpression of the efflux system (22). Further, quinolone-resistant clinical isolates overexpressing this efflux pump have been found, indicating that SmeVWX overexpression may have clinical relevance (12,22).…”