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Mechanisms of Action: Physiological Effects

The SmeYZ Efflux Pump of Stenotrophomonas maltophilia Contributes to Drug Resistance, Virulence-Related Characteristics, and Virulence in Mice

Yi-Tsung Lin, Yi-Wei Huang, Shiang-Jiuun Chen, Chia-Wei Chang, Tsuey-Ching Yang
Yi-Tsung Lin
aDivision of Infectious Diseases, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
bSchool of Medicine, National Yang-Ming University, Taipei, Taiwan
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Yi-Wei Huang
cDepartment of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan
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Shiang-Jiuun Chen
dDepartment of Life Science, Institute of Ecology and Evolutionary Biology and TechComm-5, College of Life Science, National Taiwan University, Taipei, Taiwan
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Chia-Wei Chang
cDepartment of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan
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Tsuey-Ching Yang
cDepartment of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan
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DOI: 10.1128/AAC.00372-15
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    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.

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    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.

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    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.

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    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.

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    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.

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    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.

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  • TABLE 1

    Susceptibility tests of S. maltophilia KJ and KJΔYZ

    AntimicrobialMIC (μg/ml)
    KJKJΔYZ
    β-Lactams
        Ampicillin>2,048>2,048
        Piperacillin1,0241,024
        Cefuroxime2,048>2,048
        Cefoxitin1,0241,024
        Aztreonam>2,048>2,048
    Chloramphenicol88
    Nalidixic acid88
    Tetracycline168
    Aminoglycosides
        Amikacin1,0248
        Gentamicin1,0248
        Kanamycin2568
    Macrolides
        Erythromycin64128
        Leucomycin256128
    Trimethoprim-sulfamethoxazole32/1602/10
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The SmeYZ Efflux Pump of Stenotrophomonas maltophilia Contributes to Drug Resistance, Virulence-Related Characteristics, and Virulence in Mice
Yi-Tsung Lin, Yi-Wei Huang, Shiang-Jiuun Chen, Chia-Wei Chang, Tsuey-Ching Yang
Antimicrobial Agents and Chemotherapy Jun 2015, 59 (7) 4067-4073; DOI: 10.1128/AAC.00372-15

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The SmeYZ Efflux Pump of Stenotrophomonas maltophilia Contributes to Drug Resistance, Virulence-Related Characteristics, and Virulence in Mice
Yi-Tsung Lin, Yi-Wei Huang, Shiang-Jiuun Chen, Chia-Wei Chang, Tsuey-Ching Yang
Antimicrobial Agents and Chemotherapy Jun 2015, 59 (7) 4067-4073; DOI: 10.1128/AAC.00372-15
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