Abstract
Fluoroquinolone resistance in Pseudomonas aeruginosa typically arises through site-specific mutations and overexpression of efflux pumps. In this study, we investigated the dynamics of different resistance mechanisms in P. aeruginosa populations that have evolved under fluoroquinolone pressure, as well as the interactions between these mechanisms in evolutionary trajectories. Bacteria of strain ATCC27853 were selected under different concentrations of ciprofloxacin and levofloxacin for six parallel lineages, followed by amplification of four target genes in the quinolone-resistance determining region (QRDR) and Sanger sequencing to identify the mutations. The expression of four efflux pump proteins was evaluated by real-time polymerase chain reaction using the relative quantitation method, with the ATCC27853 strain used as a control. We found that ciprofloxacin killed P. aeruginosa sooner than did levofloxacin. Further, we identified five different mutations in three subunits of QRDRs, with gyrA as the main mutated gene associated with conferring fluoroquinolone resistance. Additionally, we found a larger number of mutations appearing at 2 mg/L and 4 mg/L of ciprofloxacin and levofloxacin, respectively. Moreover, we identified the main efflux pump being expressed as MexCD-OprJ, with initial overexpression observed at 0.25 mg/L and 0.5 mg/L of ciprofloxacin and levofloxacin, respectively. These results demonstrated gyrA83 mutation and MexCD-OprJ overexpression as the primary mechanism conferring ciprofloxacin and levofloxacin resistance in P. aeruginosa. In addition, we also show that ciprofloxacin exhibited a stronger ability to kill the bacteria while potentially rendering it more susceptible to resistance.
Highlights
Pseudomonas aeruginosa is a Gram-negative, opportunistic human pathogen and is considered to be one of the main pathogens associated with nosocomial infections
Epidemiological analyses indicate that high-level resistance to FQ requires quinolone-resistance determining region (QRDR) mutations in at least two genes [4], and that overexpression of the MexEF-OprN efflux pump represents a major mechanism by which P. aeruginosa can acquire higher FQ-resistance levels [5]
minimum inhibitory concentration (MIC) determination and selection of FQ-resistant P. aeruginosa populations The MICs of both ciprofloxacin and levofloxacin in P. aeruginosa ATCC27853 were 0.5 mg/L, which is within the ranges defined for ciprofloxacin (0–1 mg/L) and levofloxacin (0–2 mg/L), according to the 2015 Clinical & Laboratory Standards Institute (CLSI)
Summary
Pseudomonas aeruginosa is a Gram-negative, opportunistic human pathogen and is considered to be one of the main pathogens associated with nosocomial infections. Epidemiological analyses indicate that high-level resistance to FQ requires QRDR mutations in at least two genes [4], and that overexpression of the MexEF-OprN efflux pump represents a major mechanism by which P. aeruginosa can acquire higher FQ-resistance levels [5]. Microbial resistance is an evolutionary response to antibiotic pressure, and evolutionary steps result in alteration of drug susceptibility to clinical resistance. Recent studies have established causal links between antibiotic deployment therapies and the course/timing of mutations, the cost of resistance, and the likelihood of developing compensating mutations [6]. The main limitation of using experimental animal models to investigate resistance mechanisms is the risk of spreading the plasmid to the surroundings. A combination of drugs is usually used in the therapy, which may interfere with the experiments investigating the mechanisms associated with FQ resistance in P. aeruginosa. The purpose of this study was to develop an understanding of chromosome-mediated resistance using an in vitro-selection model to block communication between P. aeruginosa and the environment
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