The SmeYZ efflux pump of Stenotrophomonas maltophilia contributes to drug resistance, virulence-related characteristics, and virulence in mice

Abstract

The resistance-nodulation-division (RND)-type efflux pump is one of the causes of the multidrug resistance of Stenotrophomonas maltophilia. The roles of the RND-type efflux pump in physiological functions and virulence, in addition to antibiotic extrusion, have attracted much attention. In this study, the contributions of the constitutively expressed SmeYZ efflux pump to drug resistance, virulence-related characteristics, and virulence were evaluated. S. maltophilia KJ is a clinical isolate of multidrug resistance. The smeYZ isogenic deletion mutant, KJΔYZ, was constructed by a gene replacement strategy. The antimicrobial susceptibility, virulence-related physiological characteristics, susceptibility to human serum and neutrophils, and in vivo virulence between KJ and KJΔYZ were comparatively assessed. The SmeYZ efflux pump contributed resistance to aminoglycosides and trimethoprim-sulfamethoxazole. Inactivation of smeYZ resulted in attenuation of oxidative stress susceptibility, swimming, flagella formation, biofilm formation, and secreted protease activity. Furthermore, loss of SmeYZ increased susceptibility to human serum and neutrophils and decreased in vivo virulence in a murine model. These findings suggest the possibility of attenuation of the resistance and virulence of S. maltophilia with inhibitors of the SmeYZ efflux pump.

FIG 1

Bacterial growth curves, viability, and colony sizes. (A) Growth curves. The overnight-cultured bacteria were inoculated into fresh LB broth at the initial OD₄₅₀ of 0.15. The bacterial growth was monitored by recording the OD₄₅₀ every 3 h. Data are the means from three independent experiments. Error bars indicate the standard deviations for three triplicate samples. (B) Bacterial viability was monitored by counting the numbers of CFU. Data are the means from three independent experiments. Error bars indicate the standard deviations for three triplicate samples. (C) Single colony isolation. The single colonies of KJ and KJΔYZ cells were isolated by the three-phase streaking method on LB agar. (D) Bacterial surface hydrophobicity values. The bacterial surface hydrophobicity values were determined by partitioning of cells in two-phase systems (water-hexadecane). Data are the means from three independent experiments. Error bars indicate the standard deviations for three triplicate samples. *, P < 0.05, significance calculated by Student's t test.

FIG 2

Oxidative stress susceptibility assay. Data are the means from three independent experiments. Error bars indicate the standard deviations for three triplicate samples. *, P < 0.05, significance calculated by Student's t test. (A) H₂O₂ susceptibility. The MH agar was uniformly spread with bacterial cell suspension. Sterile filter paper with 20 μl of 10% H₂O₂ was placed onto MH agar, and the diameter of a zone of growth inhibition was measured after 24 h of incubation at 37°C. (B) Menadione tolerance assay. Serial dilutions of the logarithmic-phase bacterial cells were plated onto LB containing 0, 10, 20, and 30 μg/ml menadione. The CFU were scored after 24 h of incubation at 37°C. The percentage of relative survival was defined as the CFU ratio of mutant to wild type.

FIG 3

Motility ability and flagella formation. (A) Motility ability. Five microliters of bacterial cell suspension was inoculated into the swimming agar (1% tryptone, 0.5% NaCl, and 0.15% agar). Results were expressed as diameters (millimeters) of swimming zones after 48 h of incubation at 37°C. Data are the means from three independent experiments. Error bars indicate the standard deviations for three triplicate samples. *, P < 0.05, significance calculated by Student's t test. (B) Flagella formation. The flagella were negatively stained with 1% phosphotungstic acid (pH 7.4) and observed by TEM.

FIG 4

Biofilm formation and secreted protease activity assay results. Data are the means from three independent experiments. Error bars indicate the standard deviations for three triplicate samples. *, P < 0.05, significance calculated by Student's t test. (A) Biofilm formation. Each microtiter well was inoculated with 200 μl of the OD₄₅₀ 0.1 bacterial culture and incubated at 37°C for 48 h. The total cell biomass was estimated by enumerating the CFU. The amount of biofilm was determined by crystal violet staining. The stained biofilms were quantified by measuring the A₅₇₀ of dissolved crystal violet. (B) Secreted protease activity assay. Forty microliters of bacterial cell suspension was dipped onto LB agar containing 1% skim milk. After incubation at 37°C for 72 h, the proteolytic activity of the bacteria was assessed by measuring the transparent zones around the bacteria.

FIG 5

Susceptibility to human serum and neutrophils. Data represent the means from 3 independent trials. Error bars represent the standard deviations. (A) Nonimmune healthy human serum sensitivity assays of the wild-type KJ and KJΔYZ strain. **, P < 0.0001; *, P < 0.001. (B) Bacterial susceptibilities to killing by human neutrophils of the wild-type KJ and KJΔYZ strains. Survival rates indicate the percentages of survival of wild-type or mutant strains calculated on the basis of viable counts relative to those for the no-neutrophil controls.

FIG 6

Virulence to mice. Mouse lethality following challenges with wild-type KJ and KJΔYZ strains is presented. Male C57BL/6 mice (n = 9 from the summary of two independent experiments) were inoculated by intraperitoneal injection with 1 × 10⁹ CFU of the wild-type KJ and KJΔYZ strains. Survival was assessed for 14 days following infection.