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. 2014 Jul;58(7):4162-71.
doi: 10.1128/AAC.02649-14. Epub 2014 May 12.

Putrescine Reduces Antibiotic-Induced Oxidative Stress as a Mechanism of Modulation of Antibiotic Resistance in Burkholderia Cenocepacia

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Putrescine Reduces Antibiotic-Induced Oxidative Stress as a Mechanism of Modulation of Antibiotic Resistance in Burkholderia Cenocepacia

Omar M El-Halfawy et al. Antimicrob Agents Chemother. .
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Abstract

Communication of antibiotic resistance among bacteria via small molecules is implicated in transient reduction of bacterial susceptibility to antibiotics, which could lead to therapeutic failures aggravating the problem of antibiotic resistance. Released putrescine from the extremely antibiotic-resistant bacterium Burkholderia cenocepacia protects less-resistant cells from different species against the antimicrobial peptide polymyxin B (PmB). Exposure of B. cenocepacia to sublethal concentrations of PmB and other bactericidal antibiotics induces reactive oxygen species (ROS) production and expression of the oxidative stress response regulator OxyR. We evaluated whether putrescine alleviates antibiotic-induced oxidative stress. The accumulation of intracellular ROS, such as superoxide ion and hydrogen peroxide, was assessed fluorometrically with dichlorofluorescein diacetate, while the expression of OxyR and putrescine synthesis enzymes was determined in luciferase assays using chromosomal promoter-lux reporter system fusions. We evaluated wild-type and isogenic deletion mutant strains with defects in putrescine biosynthesis after exposure to sublethal concentrations of PmB and other bactericidal antibiotics. Exogenous putrescine protected against oxidative stress induced by PmB and other antibiotics, whereas reduced putrescine synthesis resulted in increased ROS generation and a parallel increased sensitivity to PmB. Of the 3 B. cenocepacia putrescine-synthesizing enzymes, PmB induced only BCAL2641, an ornithine decarboxylase. This study reveals BCAL2641 as a critical component of the putrescine-mediated communication of antibiotic resistance and as a plausible target for designing inhibitors that would block the communication of such resistance among different bacteria, ultimately reducing the window of therapeutic failure in treating bacterial infections.

Figures

FIG 1
FIG 1
Putrescine reduces ROS production induced by PmB in B. cenocepacia K56-2. ROS were detected by DCF. n = 6 from 2 independent experiments. **, P < 0.01; ***, P < 0.001.
FIG 2
FIG 2
BCAL2641 is the only putrescine synthesis enzyme in B. cenocepacia involved in reduced susceptibility to PmB. (A) Putrescine synthesis pathway in B. cenocepacia K56-2 together with the enzymes involved. ADC, arginine decarboxylase; ODC, ornithine decarboxylase. (B) TLC plate showing the lack of production of putrescine in the ΔBCAM1111-1112 Prha-BCAL2641 conditional mutant under nonpermissive conditions. Put, putrescine; Cad, cadaverine; Spd, spermidine; Spn, spermine. (C to E) Sensitivity of the wild-type and ΔBCAL2641 (OME11) and ΔBCAM1111-1112 (OME12) putrescine synthesis mutants to 2,048 μg/ml PmB as determined turbidimetrically. n = 3 from a representative experiment. (C) Low initial inoculum in LB medium; (D) high initial inoculum in LB medium; (E) M9 minimal medium.
FIG 3
FIG 3
BCAL2641 is the main ornithine decarboxylase responsible for reduction of ROS accumulation. ROS production in response to 1 mg/ml PmB in wild-type strain K56-2 compared to the ΔBCAL2641 (OME11) and ΔBCAM1111-1112 (OME12) putrescine synthesis mutants detected by DCF. n = 6 from 2 independent experiments. ns, not significant. ***, P < 0.001.
FIG 4
FIG 4
(A) Induction of OxyR expression as an indicator of ROS accumulation in the wild type (OME56) compared to the ΔBCAL2641 (OME57) and ΔBCAM1111-1112 (OME58) putrescine synthesis mutants in response to 500 μg/ml PmB with or without 10 mM putrescine determined by the luciferase expression assay at 3 h. Results are shown as percentage of relative light units (RLU)/OD600 relative to the OME56 control (K56-2 background). The mean RLU/OD600 of the control is 0.09567. The percentages of OD600 are shown in Fig. S2 in the supplemental material. n = 9 from 3 different clones. ns, not significant. *, P < 0.05; and ***, P < 0.001. (B) In vitro antioxidant activity of putrescine. n = 6 from 2 independent experiments.
FIG 5
FIG 5
Luciferase expression assay of the different putrescine-synthesizing enzymes in response to 500 μg/ml PmB at 3 h. Results are shown as percentage of relative light units (RLU)/OD600 relative to the control (untreated K56-2 background). The percentages of OD600 are shown in Fig. S3 in the supplemental material. (A) Expression of BCAL2641 in the wild-type (OME50) and ΔBCAM1111-1112 (OME51) backgrounds. n = 6 from 2 different clones. The mean RLU/OD600 of the control is 1.4829. (B) Expression of BCAM1111 in the wild-type (OME52) and ΔBCAL2641 (OME53) backgrounds. n = 6 from 2 different clones. The mean RLU/OD600 of the control is 1.5585. (C) Expression of BCAM1112 in the wild-type (OME54) and ΔBCAL2641 (OME55) backgrounds. n = 7 from 2 different clones. The mean RLU/OD600 of the control is 0.2423. ns, not significant. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.
FIG 6
FIG 6
Role of putrescine in the bactericidal antibiotic-mediated ROS accumulation in B. cenocepacia K56-2. n = 9 from 3 independent experiments. The 4 tested antibiotics alone significantly (P < 0.001) induced the accumulation of ROS compared to the effect in control cells. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.
FIG 7
FIG 7
Effects of different antibiotics on the expression of BCAL2641 (in OME50), oxyR (in OME56), and BCAM1111 (in OME52), determined using a luciferase expression assay at 3 h. Results are shown as percentage of relative light units (RLU)/OD600 relative to the control (untreated K56-2 background). The percentages of OD600 are shown in Fig. S5 in the supplemental material. n = a minimum of 6 from at least 2 different clones. The mean RLU/OD600 values of the control are 1.0759 for BCAL2641, 0.1087 for oxyR, and 1.4723 for BCAM1111. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.
FIG 8
FIG 8
Model summarizing the role of putrescine in protecting B. cenocepacia from antibiotic-induced stress. Nor, norfloxacin; Rif, rifampin.

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