Dataset Information

Overexpression of the Efflux Pumps SmeVWX and SmeDEF Is a Major Cause of Resistance to Co-trimoxazole in Stenotrophomonas maltophilia.

ABSTRACT: Co-trimoxazole is one of the antimicrobials of choice for treating Stenotrophomonas maltophilia infections. Most works on the molecular epidemiology of the resistance to this drug combination are based on the analysis of sul genes. Nevertheless, the existence of clinical co-trimoxazole-resistant S. maltophilia isolates that do not harbor sul genes has been reported. To investigate potential mutations that can reduce the susceptibility of S. maltophilia to co-trimoxazole, spontaneous S. maltophilia co-trimoxazole-resistant mutants isolated under different co-trimoxazole concentrations were studied. All mutants presented phenotypes compatible with the overexpression of either SmeVWX (94.6%) or SmeDEF (5.4%). Indeed, the analysis of a selected set of strains showed that the overexpression of either of these efflux pumps, which was due to mutations in their regulators smeRv and smeT, respectively, was the cause of co-trimoxazole resistance. No other efflux pump was overexpressed in any of the studied mutants, indicating that they do not participate in the observed resistance phenotype. The analysis of mutants overexpressing or lacking SmeDEF or SmeVWX shows that SmeDEF contributes to the intrinsic and acquired resistance to co-trimoxazole in S. maltophilia, whereas SmeVWX only contributes to acquired resistance. It is important to highlight that all mutants were less susceptible to other antibiotics, including chloramphenicol and quinolones. Since both SmeVWX and SmeDEF are major determinants of quinolone resistance, the potential cross-selection of resistance to co-trimoxazole and quinolones, when either of the antimicrobials is used, is of particular concern for the treatment of S. maltophilia infections.

SUBMITTER: Sanchez MB

PROVIDER: S-EPMC5971588 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Overexpression of the Efflux Pumps SmeVWX and SmeDEF Is a Major Cause of Resistance to Co-trimoxazole in Stenotrophomonas maltophilia.

Sánchez María Blanca MB Martínez José Luis JL

Antimicrobial agents and chemotherapy 20180525 6

Co-trimoxazole is one of the antimicrobials of choice for treating Stenotrophomonas maltophilia infections. Most works on the molecular epidemiology of the resistance to this drug combination are based on the analysis of sul genes. Nevertheless, the existence of clinical co-trimoxazole-resistant S. maltophilia isolates that do not harbor sul genes has been reported. To investigate potential mutations that can reduce the susceptibility of S. maltophilia to co-tr ...[more]

PMID: 29610195

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Project description:SmeYZ efflux pump is a critical pump responsible for aminoglycosides resistance, virulence-related characteristics (oxidative stress susceptibility, motility, and secreted protease activity), and virulence in Stenotrophomonas maltophilia. However, the regulatory circuit involved in SmeYZ expression is little known. A two-component regulatory system (TCS), smeRySy, transcribed divergently from the smeYZ operon is the first candidate to be considered. To assess the role of SmeRySy in smeYZ expression, the smeRySy isogenic deletion mutant, KJ?RSy, was constructed by gene replacement strategy. Inactivation of smeSyRy correlated with a higher susceptibility to aminoglycosides concomitant with an increased resistance to chloramphenicol, ciprofloxacin, tetracycline, and macrolides. To elucidate the underlying mechanism responsible for the antimicrobials susceptibility profiles, the SmeRySy regulon was firstly revealed by transcriptome analysis and further confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and promoter transcription fusion constructs assay. The results demonstrate that inactivation of smeRySy decreased the expression of SmeYZ pump and increased the expression of SmeDEF pump, which underlies the ?smeSyRy-mediated antimicrobials susceptibility profile. To elucidate the cognate relationship between SmeSy and SmeRy, a single mutant, KJ?Ry, was constructed and the complementation assay of KJ?RSy with smeRy were performed. The results support that SmeSy-SmeRy TCS is responsible for the regulation of smeYZ operon; whereas SmeSy may be cognate with another unidentified response regulator for the regulation of smeDEF operon. The impact of inverse expression of SmeYZ and SmeDEF pumps on physiological functions was evaluated by mutants construction, H2O2 susceptibility test, swimming, and secreted protease activity assay. The increased expression of SmeDEF pump in KJ?RSy may compensate, to some extents, the SmeYZ downexpression-mediated compromise with respect to its role in secreted protease activity.

Project description:Stenotrophomonas maltophilia, a nonfermenting Gram-negative rod, is frequently isolated from the environment and is emerging as a multidrug-resistant global opportunistic pathogen. S. maltophilia harbors eight RND-type efflux pumps that contribute to multidrug resistance and physiological functions. Among the eight efflux pumps, SmeYZ pump is constitutively highly expressed. In our previous study, we demonstrated that loss-of-function of the SmeYZ pump results in pleiotropic phenotypes, including abolished swimming motility, decreased secreted protease activity, and compromised tolerance to oxidative stress and antibiotics. In this study, we attempted to elucidate the underlying mechanisms responsible for ΔsmeYZ-mediated pleiotropic phenotypes. RNA-seq transcriptome analysis and subsequent confirmation with qRT-PCR revealed that smeYZ mutant experienced an iron starvation response because the genes involved in the synthesis and uptake of stenobactin, the sole siderophore of S. maltophilia, were significantly upregulated. We further verified that smeYZ mutant had low intracellular iron levels via inductively coupled plasma mass spectrometry (ICP-MS). Also, KJΔYZ was more sensitive to 2,2'-dipyridyl (DIP), a ferrous iron chelator, in comparison with the wild type. The contribution of SmeYZ, SmeDEF, and SbiAB pumps to stenobactin secretion was suggested by qRT-PCR and further verified by Chrome Azurol S (CAS) activity, iron source utilization, and cell viability assays. We also demonstrated that loss-of-function of SmeYZ led to the compensatory upregulation of SbiAB and SmeDEF pumps for stenobactin secretion. The overexpression of the SbiAB pump resulted in a reduction in intracellular iron levels, which may be the key factor responsible for the ΔsmeYZ-mediated pleiotropic phenotypes, except for antibiotic extrusion. IMPORTANCE Efflux pumps display high efficiency of drug extrusion, which underlies their roles in multidrug resistance. In addition, efflux pumps have physiological functions, and their expression is tightly regulated by various environmental and physiological signals. Functional redundancy of efflux pumps is commonly observed, and mutual regulation occurs among these functionally redundant pumps in a bacterium. Stenotrophomonas maltophilia is an opportunistic pathogen that shows intrinsic multi-drug resistance. In this study, we demonstrated that SmeYZ, SbiAB, and SmeDEF efflux pumps of S. maltophilia display functional redundancy in siderophore secretion. Inactivation of smeYZ led to the upregulation of smeDEF and sbiAB. Unexpectedly, sbiAB overexpression resulted in the reduction of intracellular iron levels, which led to pleiotropic defects in smeYZ mutant. This study demonstrates a previously unidentified connection between efflux pumps, siderophore secretion, and intracellular iron levels in S. maltophilia.

Dataset Information

Overexpression of the Efflux Pumps SmeVWX and SmeDEF Is a Major Cause of Resistance to Co-trimoxazole in Stenotrophomonas maltophilia.

Publications

Overexpression of the Efflux Pumps SmeVWX and SmeDEF Is a Major Cause of Resistance to Co-trimoxazole in Stenotrophomonas maltophilia.

Similar Datasets

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