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The Contribution of Efflux Systems to Levofloxacin Resistance in Stenotrophomonas maltophilia Clinical Strains Isolated in Warsaw, Poland

ABSTRACT:

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Fluoroquinolones, mainly levofloxacin, are considered an alternative treatment option of Stenotrophomonas maltophilia infections to trimethoprim/sulfamethoxazole. However, an increase in the number of levofloxacin-resistant strains is observed worldwide. The fluoroquinolone resistance in S. maltophilia is usually caused by an overproduction of various multidrug efflux pumps, which are able to extrude antibiotics and chemotherapeutics from the bacterial cells. The purpose of the study was to analyze the contribution of efflux systems to levofloxacin resistance in S. maltophilia clinical strains, isolated in Warsaw, by phenotypic and molecular methods. Previously, the occurrence of genes encoding various ten efflux pumps was shown in 94 studied isolates. Additionally, 44 of 94 isolates demonstrated reduction in susceptibility to levofloxacin. In this study, in the presence of efflux pump inhibitors, an increase in levofloxacin susceptibility was observed in 13 isolates. The overexpression of genes encoding two efflux pump system, such as SmeDEF and Sme VWX (in five and one isolate, respectively), was demonstrated. Sequencing analysis revealed an amino acid change in the local regulators of these efflux pump operons. Our data indicate that the overproduction of the SmeVWX efflux system, unlike SmeDEF, plays a significant role in the levofloxacin resistance of the clinical isolates.

Abstract

Levofloxacin is considered an alternative treatment option of Stenotrophomonas maltophilia infections to trimethoprim/sulfamethoxazole. The fluoroquinolone resistance in S. maltophilia is usually caused by an overproduction of efflux pumps. In this study, the contribution of efflux systems to levofloxacin resistance in S. maltophilia clinical isolates was demonstrated using phenotypic (minimal inhibitory concentrations, MICs, of antibiotics determination ± efflux pump inhibitors, EPIs) and molecular (real-time polymerase-chain-reaction and sequencing) methods. Previously, the occurrence of genes encoding ten efflux pumps was shown in 94 studied isolates. Additionally, 44/94 isolates demonstrated reduction in susceptibility to levofloxacin. Only 5 of 13 isolates (with ≥4-fold reduction in levofloxacin MIC) in the presence of EPIs showed an increased susceptibility to levofloxacin and other antibiotics. The overexpression of smeD and smeV genes (in five and one isolate, respectively) of 5 tested efflux pump operons was demonstrated. Sequencing analysis revealed 20–35 nucleotide mutations in local regulatory genes such as smeT and smeRv. However, mutations leading to an amino acid change were shown only in smeT (Arg123Lys, Asp182Glu, Asp204Glu) for one isolate and in smeRv (Gly266Ser) for the other isolate. Our data indicate that the overproduction of the SmeVWX efflux system, unlike SmeDEF, plays a significant role in the levofloxacin resistance.

SUBMITTER: Zajac O

PROVIDER: S-EPMC9311822 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

Project description:Clinical strains of Stenotrophomonas maltophilia are often highly resistant to multiple antibiotics, although the mechanisms of resistance are generally poorly understood. Multidrug resistant (MDR) strains were readily selected by plating a sensitive reference strain of the organism individually onto a variety of antibiotics, including tetracycline, chloramphenicol, ciprofloxacin, and norfloxacin. Tetracycline-selected MDR strains typically showed cross-resistance to erythromycin and fluoroquinolones and, in some instances, aminoglycosides. MDR mutants selected with the other agents generally displayed resistance to chloramphenicol and fluoroquinolones only, although two MDR strains (e.g., K1385) were also resistant to erythromycin and hypersusceptible to aminoglycosides. Many of the MDR strains expressed either moderate or high levels of a novel outer membrane protein (OMP) of ca. 50 kDa molecular mass, a phenotype typical of MDR strains of Pseudomonas aeruginosa hyperexpressing drug efflux systems. Indeed, the 50-kDa OMP of these S. maltophilia MDR strains reacted with antibody to OprM, the outer membrane component of the MexAB-OprM MDR efflux system of P. aeruginosa. Similarly, a ca. 110-kDa cytoplasmic membrane protein of these MDR strains also reacted with antibody to the MexB component of the P. aeruginosa pump. The outer and cytoplasmic membranes of several clinical S. maltophilia strains also reacted with the anti-OprM and anti-MexB antibodies. N-terminal amino acid sequencing of a cyanogen bromide-generated peptide of the 50-kDa OMP of MDR strain K1385, dubbed SmeM (Stenotrophomonas multidrug efflux), revealed it to be very similar to a number of outer membrane multidrug efflux components of P. aeruginosa and Pseudomonas putida. Deletion of the L1 and L2 beta-lactamase genes confirmed that these enzymes were responsible for the bulk of the beta-lactam resistance of K1385 and its parent. Still, overexpression of the MDR efflux mechanism in an L1- and L2-deficient derivative of K1385 did yield a modest increase in resistance to a few beta-lactams. These data are consistent with the MDR efflux mechanism(s) playing a role in the multidrug resistance of S. maltophilia.

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The Contribution of Efflux Systems to Levofloxacin Resistance in Stenotrophomonas maltophilia Clinical Strains Isolated in Warsaw, Poland

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