Mutations in gyrB play an important role in ciprofloxacin-resistant Pseudomonas aeruginosa

Abstract

Purpose: To investigate the main molecular resistance mechanisms to fluoroquinolones (FQs) in Pseudomonas aeruginosa and also to investigate the effect of time and concentration on mutations in resistance genes.

Materials and methods: The clinical isolates of P. aeruginosa which are sensitive to ciprofloxacin (CIP) or levofloxacin (LEV) were collected. The isolates were incubated with different concentrations of CIP or LEV for 5 days and the minimal inhibitory concentrations (MICs) of CIP, LEV and ofloxacin (OFX) were measured. The MIC of FQs to P. aeruginosa was measured by the agar dilution method. FQ resistance determining regions of gyrA, gyrB, parC and parE were amplified by PCR, and mutations in four genes were explored using sequence analysis with the Snapgene software. The relative expression levels of two efflux pumps genes (mexA and mexE) were measured by quantitative reverse transcription PCR.

Results: A total of eleven isolates were collected from the Second Hospital of Shanxi Medical University. Amino acid alterations in gyrA and gyrB were mainly detected in resistant mutants, and the percentage of strains with amino acid alterations in gyrB was significantly higher than that in gyrA (P<0.001). MICs of strains with mutations both in gyrA and gyrB were not significantly higher than those of strains with mutations only in gyrB (P>0.05). No amino acid alterations were detected in genes of parC and parE. In both gyrA and gyrB, the number of amino acid alterations increased with incubation time prolonged and increased with increasing incubation concentration.

Conclusion: CIP was more competent than LEV in making P. aeruginosa resistant to in vitro selection. Mutations occurring in gyrB played an important role in FQ resistance of P. aeruginosa in vitro selection.

Keywords: Pseudomonas aeruginosa; fluoroquinolones; in vitro; molecular resistance mechanisms.

Figure 1

The flowchart describing the experimental design. Abbreviations: MIC, minimum inhibitory concentration; RT-PCR, reverse transcription PCR.

Figure 2

Simple effect analysis of the relationship between groups and MICs to CIP, LEV and OFX. Notes: (A) CIP. (B) LEV. (C) OFX. 3/0, 5/0: MICs on day 3 or 5 divided by the original MICs, which means the MIC incremental quantity on day 3 or 5. 3/1, 5/1: MICs on day 3 or 5 divided by the MICs on day 1, which means the MIC incremental speed on day 3 or 5. 1/0: MICs on day 1 divided by the original MICs, which means the MIC incremental quantity and speed on day 1. 5/3: MICs on day 5 divided by MICs on day 3, which means the MIC incremental speed on day 5. There is a statistical difference between the CIP group and the LEV group for the same MIC increment and the same drug (*P<0.05). There is a statistical difference between the CIP group and the LEV group for the same MIC increment and the same drug (**P<0.001). Abbreviations: CIP, ciprofloxacin; LEV, levofloxacin; MIC, minimum inhibitory concentration; OFX, ofloxacin.

Figure 3

The comparison of resistance rates of strains to CIP, LEV and OFX between the two groups. Notes: (A) CIP. (B) LEV. (C) OFX. There was a statistical difference between the two groups (*P<0.05). Abbreviations: CIP, ciprofloxacin; LEV, levofloxacin; MIC, minimum inhibitory concentration; OFX, ofloxacin.

Figure 4

The relative expression of two efflux pumps genes – MexA and MexE. Notes: (A) MexA. (B) MexE. The efflux pump was highly expressed if the relative expression of MexA was higher than 3.00. The efflux pump was highly expressed if the relative expression of MexE was higher than 10.00. Abbreviation: MIC, minimum inhibitory concentration.