Quantitative Contributions of Target Alteration and Decreased Drug Accumulation to Pseudomonas aeruginosa Fluoroquinolone Resistance

Abstract

Quinolone antibiotics constitute a clinically successful and widely used class of broad-spectrum antibiotics; however, the emergence and spread of resistance increasingly limits the use of fluoroquinolones in the treatment and management of microbial disease. In this study, we evaluated the quantitative contributions of quinolone target alteration and efflux pump expression to fluoroquinolone resistance in Pseudomonas aeruginosa. We generated isogenic mutations in hot spots of the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, and parC and inactivated the efflux regulator genes so as to overexpress the corresponding multidrug resistance (MDR) efflux pumps. We then introduced the respective mutations into the reference strain PA14 singly and in various combinations. Whereas the combined inactivation of two efflux regulator-encoding genes did not lead to resistance levels higher than those obtained by inactivation of only one efflux regulator-encoding gene, the combination of mutations leading to increased efflux and target alteration clearly exhibited an additive effect. This combination of target alteration and overexpression of efflux pumps was commonly observed in clinical P. aeruginosa isolates; however, these two mechanisms were frequently found not to be sufficient to explain the level of fluoroquinolone resistance. Our results suggest that there are additional mechanisms, independent of the expression of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and/or MexXY-OprM efflux pump, that increase ciprofloxacin resistance in isolates with mutations in the QRDRs.

Predicting antimicrobial resistance in Pseudomonas aeruginosa with machine learning‐enabled molecular diagnostics

“…Particularly, two SNPs in the quinolone resistance-determining region (QRDR) of gyrA and parC had the strongest impact on the classification (Dataset EV3). This is an expected finding as quinolone antibiotics act by binding to their targets, gyrase, and topoisomerase IV (Bruchmann et al, 2013); and target-mediated resistance caused by specific mutations in the encoding genes is the most common and clinically significant form of resistance (del Barrio-Tofiño et al, 2017 Each panel depicts the results for a different anti-pseudomonal drug (CAZ: ceftazidime; CIP: ciprofloxacin; MEM: meropenem; TOB: tobramycin) for the best data type combination (GPA+EXPR/SNPs). Misclassified and correctly classified samples for the training dataset (80%) were inferred in a 10-fold cross-validation.…”

The genetic basis of the fitness costs of antimicrobial resistance: a meta‐analysis approach

“…The classic paradigm is that resistance evolution occurs by a few mutations of large effect. However, there is growing evidence that high levels of antibiotic resistance evolve by the substitution of multiple mutations that confer resistance to the same antibiotic (Weinreich et al 2006;Bruchmann et al 2013;Farhat et al 2013;Zhang et al 2013). When resistance evolves by mutations in multiple genes, epistatic interactions between resistance mutations have the potential to influence the overall cost associated with resis-tance.…”

Transcriptome Profiling of Antimicrobial Resistance in Pseudomonas aeruginosa

“…This opportunistic pathogen plays a dominant role as an infectious agent affecting the lungs of cystic fibrosis patients (3)(4)(5) and has emerged as one of the most important human pathogens involved in nosocomial infections (6). P. aeruginosa is known not only for its high intrinsic resistance to a broad spectrum of antimicrobial compounds (7,8), but also for its remarkable ability to acquire new resistances via horizontal gene transfer and via the adoption of drug resistance-associated mutations during the course of infection (9)(10)(11)(12)(13). In particular, the accelerating development of multidrug-resistant P. aeruginosa strains represents a great diagnostic and therapeutic challenge in modern medicine (14,15) and, with the lack of new antibiotic options, emphasizes the need for the optimization of current diagnostics, therapies, and prevention of the spread of these organisms.…”