Thymol tolerance in Escherichia coli induces morphological, metabolic and genetic changes

Background Thymol is a phenolic compound used for its wide spectrum antimicrobial activity. There is a limited understanding of the antimicrobial mechanisms underlying thymol activity. To investigate this, E. coli strain JM109 was exposed to thymol at sub-lethal concentrations and after 16 rounds of exposure, isolates with a 2-fold increased minimal inhibitory concentration (MIC) were recovered (JM109-Thyr). The phenotype was stable after multiple sub-cultures without thymol. Results Cell morphology studies by scanning electron microscopy (SEM) suggest that thymol renders bacterial cell membranes permeable and disrupts cellular integrity. 1H Nuclear magnetic resonance (NMR) data showed an increase in lactate and the lactic acid family amino acids in the wild type and JM109-Thyr in the presence of thymol, indicating a shift from aerobic respiration to fermentation. Sequencing of JM109-Thyr defined multiple mutations including a stop mutation in the acrR gene resulting in a truncation of the repressor of the AcrAB efflux pump. AcrAB is a multiprotein complex traversing the cytoplasmic and outer membrane, and is involved in antibiotic clearance. Conclusions Our data suggests that thymol tolerance in E. coli induces morphological, metabolic and genetic changes to adapt to thymol antimicrobial activity.

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

ABSTRACTWe have analyzed the contribution of different efflux components to glutathione-mediated abrogation of ciprofloxacin's activity inEscherichia coliand the underlying potential mechanism(s) behind this phenomenon. The results indicated that glutathione increased the total active efflux, thereby partially contributing to glutathione-mediated neutralization of ciprofloxacin's antibacterial action inE. coli. However, the role of glutathione-mediated increased efflux becomes evident in the absence of a functional TolC-AcrAB efflux pump.

Characterization of a Novel Pyranopyridine Inhibitor of the AcrAB Efflux Pump of Escherichia coli

ABSTRACTMembers of the resistance-nodulation-division (RND) family of efflux pumps, such as AcrAB-TolC ofEscherichia coli, play major roles in multidrug resistance (MDR) in Gram-negative bacteria. A strategy for combating MDR is to develop efflux pump inhibitors (EPIs) for use in combination with an antibacterial agent. Here, we describe MBX2319, a novel pyranopyridine EPI with potent activity against RND efflux pumps of theEnterobacteriaceae. MBX2319 decreased the MICs of ciprofloxacin (CIP), levofloxacin, and piperacillin versusE. coliAB1157 by 2-, 4-, and 8-fold, respectively, but did not exhibit antibacterial activity alone and was not active against AcrAB-TolC-deficient strains. MBX2319 (3.13 μM) in combination with 0.016 μg/ml CIP (minimally bactericidal) decreased the viability (CFU/ml) ofE. coliAB1157 by 10,000-fold after 4 h of exposure, in comparison with 0.016 μg/ml CIP alone. In contrast, phenyl-arginine-β-naphthylamide (PAβN), a known EPI, did not increase the bactericidal activity of 0.016 μg/ml CIP at concentrations as high as 100 μM. MBX2319 increased intracellular accumulation of the fluorescent dye Hoechst 33342 in wild-type but not AcrAB-TolC-deficient strains and did not perturb the transmembrane proton gradient. MBX2319 was broadly active againstEnterobacteriaceaespecies andPseudomonas aeruginosa. MBX2319 is a potent EPI with possible utility as an adjunctive therapeutic agent for the treatment of infections caused by Gram-negative pathogens.

Molecular mechanisms of antimicrobial resistance in fecal Escherichia coli of healthy dogs after enrofloxacin or amoxicillin administration

Escherichia coli respond to selective pressure of antimicrobial therapy by developing resistance through a variety of mechanisms. The purpose of this study was to characterize the genetic mechanisms of antimicrobial resistance in fecal E. coli after the routine use of 2 popular antimicrobials. Fourteen resistant E. coli isolates, representing predominant clones that emerged in healthy dogs’ feces after treatment with either amoxicillin (11 E. coli isolates) or enrofloxacin (3 E. coli isolates), were tested for mutations in DNA gyrase (gyrA and gyrB) and in topoisomerase IV (parC) and for the presence of β-lactamases (blaTEM, blaSHV, blaPSE-1 and blaCTX-M) and plasmid-mediated quinolone resistance (qnrA, qnrB, qnrS, aac(6′)-Ib, and qepA), by polymerase chain reaction. Escherichia coli isolates cultured following amoxicillin therapy only expressed single-drug resistance to β-lactams, while the isolates cultured from dogs receiving enrofloxacin therapy expressed multidrug resistance (MDR). The use of RND efflux pump inhibitors increased the susceptibility of the 3 MDR E. coli isolates to doxycycline, chloramphenicol, enrofloxacin, and ciprofloxacin, which indicates a role of the efflux pump in the acquisition of the MDR phenotype. Amplification and sequencing of AcrAB efflux pump regulators (soxR, soxS, marR, and acrR) revealed only the presence of a single mutation in soxS in the 3 MDR isolates.

Characterization of RarA, a Novel AraC Family Multidrug Resistance Regulator in Klebsiella pneumoniae

ABSTRACTTranscriptional regulators, such as SoxS, RamA, MarA, and Rob, which upregulate the AcrAB efflux pump, have been shown to be associated with multidrug resistance in clinically relevant Gram-negative bacteria. In addition to the multidrug resistance phenotype, these regulators have also been shown to play a role in the cellular metabolism and possibly the virulence potential of microbial cells. As such, the increased expression of these proteins is likely to cause pleiotropic phenotypes.Klebsiella pneumoniaeis a major nosocomial pathogen which can express the SoxS, MarA, Rob, and RamA proteins, and the accompanying paper shows that the increased transcription oframAis associated with tigecycline resistance (M. Veleba and T. Schneiders, Antimicrob. Agents Chemother. 56:4466–4467, 2012). Bioinformatic analyses of the availableKlebsiellagenome sequences show that an additional AraC-type regulator is encoded chromosomally. In this work, we characterize this novel AraC-type regulator, hereby called RarA (Regulator of antibiotic resistance A), which is encoded inK. pneumoniae,Enterobactersp. 638,Serratia proteamaculans568, andEnterobacter cloacae. We show that the overexpression ofrarAresults in a multidrug resistance phenotype which requires a functional AcrAB efflux pump but is independent of the other AraC regulators. Quantitative real-time PCR experiments show thatrarA(MGH 78578 KPN_02968) and its neighboring efflux pump operonoqxAB(KPN_02969_02970) are consistently upregulated in clinical isolates collected from various geographical locations (Chile, Turkey, and Germany). Our results suggest thatrarAoverexpression upregulates theoqxABefflux pump. Additionally, it appears thatoqxR, encoding a GntR-type regulator adjacent to theoqxABoperon, is able to downregulate the expression of theoqxABefflux pump, where OqxR complementation resulted in reductions to olaquindox MICs.

Interplay between Drug Efflux and Antioxidants in Escherichia coli Resistance to Antibiotics

ABSTRACT We investigated possible cross talk between endogenous antioxidants glutathione, spermidine, and glutathionylspermidine and drug efflux in Escherichia coli. We found that cells lacking either spermidine or glutathione are less susceptible than the wild type to novobiocin and certain aminoglycosides. In contrast, exogenous glutathione protects against both bactericidal and bacteriostatic antibiotics. The glutathione protection does not require the AcrAB efflux pump but fails in cells lacking TolC because exogenous glutathione is toxic to these cells.