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Simple Summary: 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. 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. [ABSTRACT FROM AUTHOR]

Simple Summary: Nosocomial infections caused by Stenotrophomonas maltophilia have been increasing worldwide. These bacteria are intrinsically resistant to most antibiotics. The underestimated resistance mechanism of Gram-negative rods is an overexpression of multidrug-resistant (MDR) efflux pumps. The aim of this study was to analyze the genetic diversity of isolates derived from various clinical materials, including blood, and the prevalence of MDR efflux pump genes and susceptibility profiles to the anti-S. maltophilia drugs. The research was conducted on 94 S. maltophilia isolates derived from hospitalized patients and outpatients in Warsaw, Poland. All isolates were susceptible to trimethoprim-sulfamethoxazole and minocycline, while 44/94 isolates demonstrated reduction in susceptibility to levofloxacin. A large genetic variation was observed among these isolates. However, a clonal relationship was revealed among two groups of bloodstream isolates from one hospital ward: (1) nine isolates, (2) six isolates. Moreover, the presence of genes encoding ten different efflux pumps from the resistance-nodulation-division family and the ATP-binding cassette family was shown in the majority of the 94 isolates. The obtained knowledge about the prevalence of efflux pump genes in clinical S. maltophilia strains makes it possible to predict the scale of the risk of resistance emergence in strains as a result of gene overexpression. An increase of nosocomial infections caused by Stenotrophomonas maltophilia strains has recently been observed all over the world. The isolation of these bacteria from the blood is of particular concern. In this study we performed the phenotypic and genotypic characterization of 94 S. maltophilia isolates, including isolates from patients hospitalized in a tertiary Warsaw hospital (n = 79) and from outpatients (n = 15). All isolates were found to be susceptible to trimethoprim-sulfamethoxazole and minocycline, while 44/94 isolates demonstrated a reduction in susceptibility to levofloxacin. A large genetic variation was observed among the isolates tested by pulsed-field gel electrophoresis. A clonal relationship with 100% similarity was observed between isolates within two sub-pulsotypes: the first included nine bloodstream isolates and the second involved six. Multilocus sequence typing showed two new sequence types (ST498 and ST499) deposited in public databases for molecular typing. Moreover, the presence of genes encoding ten different efflux pumps from the resistance-nodulation-division family and the ATP-binding cassette family was shown in the majority of the 94 isolates. The obtained knowledge about the prevalence of efflux pump genes in clinical S. maltophilia strains makes it possible to predict the scale of the risk of resistance emergence in strains as a result of gene overexpression. [ABSTRACT FROM AUTHOR]