Hypoxia increases antibiotic resistance in Pseudomonas aeruginosa through altering the composition of multidrug efflux pumps

Abstract

Antibiotic resistance is a significant and developing problem in general medical practice and a common clinical complication in cystic fibrosis patients infected with Pseudomonas aeruginosa. Such infections occur within hypoxic mucous deposits in the cystic fibrosis lung; however, little is known about how the hypoxic microenvironment influences pathogen behavior. Here we investigated the impact of hypoxia on antibiotic resistance in P. aeruginosa. The MICs of a selection of antibiotics were determined for P. aeruginosa grown under either normoxic or hypoxic conditions. The expression of mRNAs for resistance-nodulation-cell division (RND) multidrug efflux pump linker proteins was determined by real-time PCR, and multidrug efflux pump activity was inhibited using Phe-Arg β-naphthylamide dihydrochloride. The MIC values of a subset of clinically important P. aeruginosa antibiotics were higher for bacteria incubated under hypoxia than under normoxia. Furthermore, hypoxia altered the stoichiometry of multidrug efflux pump linker protein subtype expression, and pharmacologic inhibition of these pumps reversed hypoxia-induced antibiotic resistance. We hypothesize that hypoxia increases multidrug resistance in P. aeruginosa by shifting multidrug efflux pump linker protein expression toward a dominance of MexEF-OprN. Thus, microenvironmental hypoxia may contribute significantly to the development of antibiotic resistance in P. aeruginosa infecting cystic fibrosis patients.

Fig 1

Altered antibiotic resistance in P. aeruginosa under hypoxic conditions. (A) Mueller-Hinton agar plates were inoculated with ATCC 27853, which was grown for 20 h in a normoxic (N) or a hypoxic (H) (1% oxygen) environment. (B) Determination of MICs for ATCC 27853 at 21% (N) and 1% (H) oxygen by broth microdilution of 21 antibiotics using a Sensititre GNX2F susceptibility plate. AMI, amikacin; GEN, gentamicin; TOB, tobramycin; FOT, cefotaxime; TAZ, ceftazidime; FEP, cefepime; AZT, aztreonam; TIM, ticarcillin-clavulanic acid; P/T, piperacillin-tazobactam; MERO, meropenem; ETP, ertapenem; DOR, doripenem; IMI, imipenem; COL, colistin; POL, polymyxin B; LEVO, levofloxacin; CIP, ciprofloxacin; DOX, doxycycline; MIN, minocycline; TGC, tigecycline; SXT, trimethoprim-sulfamethoxazole. Data are mean MICs ± standard errors of the means for 3 independent experiments.

Fig 2

Altered stoichiometry of RND multidrug efflux pump composition under hypoxic conditions. The expression of the genes (mexA, mexC, and mexE) encoding the linker molecules for the RND multidrug efflux pumps MexAB-OprM, MexCD-OprJ, and MexEF-OprN in P. aeruginosa under normoxia (N) and hypoxia (H) was investigated by real-time PCR. (A and B) ATCC 27853 was grown under N or H for 6 h (A) or 24 h (B). Expression of the mexA, mexC, and mexE genes was detected by real-time PCR. Relative gene expression was normalized and expressed as mexE/mexA and mexE/mexC ratios. Values are means ± standard errors of the means for 3 independent experiments (*, P < 0.05). (C) Clinical P. aeruginosa strains were exposed to N and H for 6 h, and expression of the genes encoding the linker molecules was detected by real-time PCR. Relative gene expression was expressed as mexE/mexA and mexE/mexC ratios. Values are means ± standard errors of the means for 2 independent experiments (*, P < 0.05).

Fig 3

Increased antibiotic resistance in hypoxia is reversed by a multidrug efflux pump inhibitor. MICs for ATCC 27853 were determined by broth microdilution using a Sensititre GNX2F susceptibility plate under normoxia (N), hypoxia (H), or hypoxia in the presence of the efflux pump inhibitor (H+EPI) Phe-Arg β-naphthylamide dihydrochloride (20 μg/ml). A selection of CF-relevant antibiotics for which the EPI decreased the MIC under hypoxia is shown. Values are means ± standard errors of the means for 3 independent experiments (*, P < 0.05).

Fig 4

Hydroxylase inhibition fails to increase antibiotic resistance. MICs for ATCC 27853 were determined by broth microdilution using a Sensititre GNX2F susceptibility plate in the presence of the hydroxylase inhibitor DMOG (1 mM) or the vehicle control (DMSO). Data are mean MICs ± standard errors of the means for 3 independent experiments.