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Mechanisms of Resistance

Involvement of Antibiotic Efflux Machinery in Glutathione-Mediated Decreased Ciprofloxacin Activity in Escherichia coli

Manish Goswami, Mahesh Subramanian, Ranjeet Kumar, Jana Jass, Narendra Jawali
Manish Goswami
aMolecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, India
cThe Life Science Center, School of Science and Technology, Örebro University, Örebro, Sweden
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Mahesh Subramanian
bBio-Organic Division, Bhabha Atomic Research Centre, Trombay, Mumbai, India
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Ranjeet Kumar
cThe Life Science Center, School of Science and Technology, Örebro University, Örebro, Sweden
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Jana Jass
cThe Life Science Center, School of Science and Technology, Örebro University, Örebro, Sweden
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Narendra Jawali
aMolecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, India
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DOI: 10.1128/AAC.00414-16
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    FIG 1

    Growth kinetics of JW0452, JW0451, and JW5503 compared to those of the wild-type parent strain BW25113. The growth curves were generated by incubating 1% overnight inocula of strains BW25113, JW0452, JW0451, and JW5503 in LB medium (control) (A), LB medium supplemented with 10 mM GSH (B), LB medium with 4 ng/ml ciprofloxacin (subinhibitory ciprofloxacin concentration for BW25113) (C), LB medium with 10 mM GSH and 16 ng/ml ciprofloxacin, and LB medium with 16 ng/ml ciprofloxacin (inhibitory concentration for BW25113). The growth kinetics experiment was performed for two biological replicates. Generation times of the strains were calculated from the data of a single representative set.

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    FIG 2

    Total efflux activities of wild-type E. coli BW25113 and efflux mutants JW0452, BW0451, and JW5503 (carrying acrA, acrB, and tolC deletions, respectively) determined in presence and absence of GSH. (A) Representative flow cytometric histograms depicting the extent of intracellular fluorescence due to Hoechst 33342 retention in different bacterial strains in the presence and absence of GSH (n = 3 per group). In each of the histograms, the x axis represents the signal acquired from the FL4 channel, and the y axis represents the count. (B) Bar chart showing the amount of Hoechst 33342 retained inside the bacterial cell (n = 3 per group). Data points show means ± SEM. One-way Analysis of variance (ANOVA) followed by Tukey's posttest was used to determine the statistical significance. *, compared with control (P < 0.01); #, compared with the absence of GSH for a given bacterial strain (P < 0.05).

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    FIG 3

    Ciprofloxacin accumulation inside wild-type E. coli BW25113 (A) and efflux mutants JW0451 (B), BW452 (C), and JW5503 (D) in the presence and absence of 10 mM GSH. Separate bars depict the extents of fluorescence due to ciprofloxacin accumulation in the presence and absence of GSH (n = 3 per group). Error bars represent ± SEM. *, significantly different compared to the corresponding absence of GSH treatment (P < 0.05).

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  • TABLE 1

    Ciprofloxacin susceptibilities of wild-type E. coli BW25113 and efflux mutants in the presence and absence of GSHa

    Strain nameMIC of ciprofloxacin (ng/ml)GSH-mediated increase in MIC (ratio of without GSH/with GSH) (fold change)
    Without GSHWith GSH
    BW251131625616
    JW55034328
    JW04524328
    JW04514328
    • ↵a Only those strains where a change in MIC was observed in the presence or absence of GSH compared with that for the wild-type parent strain BW25113 are shown. The ciprofloxacin MICs for all remaining efflux pump mutants (mentioned in Table S1 in the supplemental material) were unaltered compared with that for BW25113, both in the presence and absence of 10 mM GSH.

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      Supplemental Tables S1 and S2 and Figure S1

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Involvement of Antibiotic Efflux Machinery in Glutathione-Mediated Decreased Ciprofloxacin Activity in Escherichia coli
Manish Goswami, Mahesh Subramanian, Ranjeet Kumar, Jana Jass, Narendra Jawali
Antimicrobial Agents and Chemotherapy Jun 2016, 60 (7) 4369-4374; DOI: 10.1128/AAC.00414-16

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Involvement of Antibiotic Efflux Machinery in Glutathione-Mediated Decreased Ciprofloxacin Activity in Escherichia coli
Manish Goswami, Mahesh Subramanian, Ranjeet Kumar, Jana Jass, Narendra Jawali
Antimicrobial Agents and Chemotherapy Jun 2016, 60 (7) 4369-4374; DOI: 10.1128/AAC.00414-16
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