Contribution of ParE Mutation and Efflux to Ciprofloxacin Resistance in Pseudomonas aeruginosa Clinical Isolates

Abstract

Mechanisms of resistance to fluoroquinolone antibiotics in Pseudomonas aeruginosa are mainly comprised of: 1) alteration in DNA gyrase and topoisomerase IV owing to mutations clustered in the quinolone resistance-determining regions (QRDRs) 1-5 and 2) activation or enhancement of efflux pumps 6-8. We have previously reported gyrA, gyrB and parC mutations 3 in thirty clinical isolates of P. aeruginosa, collected at the Pitie-Salpetriere Hospital 3,9 and resistant to ciprofloxacin (MICs, 2 to 128 μg/ml). We observed that all isolates with a double gyrA-parC mutation displayed high ciprofloxacin MICs. However, some of the strains with a single mutation in gyrA or gyrB also had these high MICs. In the present work, other hypothetical mechanisms, such as mutation in parE and enhanced efflux, were investigated. Total DNA was extracted by the freeze-thaw procedure as previously described 3. parE QRDR was amplified and sequenced for all strains. Analysis of the entire parE sequence was carried out for strains with high ciprofloxacin MICs and no mutations in gyrB, parC or parE QRDR. Primers, nucleotide positions and annealing temperatures (Ta) are listed in Table 1. PCR amplifications were performed as previously described 3. For efflux assay, the MIC of ciprofloxacin was determined by the agar dilution method without and with 20 μg/ml of Phe-Arg-β-naphthylamide (PAβN) (Sigma-Aldrich). Eight of the thirty clinical isolates investigated for the ciprofloxacin resistance mechanism, harbored a mutation in the QRDR of ParE resulting in Asp-419to-Asn substitution (Table 2). These isolates were highly resistant to ciprofloxacin (MIC of 16-32 μg/ml) and harbored the GyrA alteration Thr-83-to-Ile but lacked GyrB and ParC alterations. Two strains were found to carry a ParE change outside the QRDR: one at position 473 (Ala-473-to-Val), and a novel one at position 200 (Val-200-to-Met). Finally, No ParE changes were found in the remaining strains, either those with a low level of resistance (ciprofloxacin MIC 1 μg/ml and 4 μg/ml. Multiple efflux pump systems that extrude quinolones have been described in P. aeruginosa 7,8. Investigation of efflux pump enhancement was undertaken by a phenotypic approach. PAβN has been described as an effective inhibitor of efflux pump in P. aeruginosa 14. For 27 clinical isolates, ciprofloxacin MICs decreased in the presence of PAβN by 2to 64fold. However, for only 7 strains, the decrease in ciprofloxacin MICs was more than 4-fold, indicating that most strains exhibited a natural or moderately enhanced efflux (Table 2). The effect of PAβN on ciprofloxacin MIC was higher in strains with patterns P1 and P3 (4to 64-fold decrease in MICs with 5 strains showing more than a 4-fold decrease) than in strains with patterns P4 to P8 (2to 8-fold reduction in MICs with 2 strains showing a 8-fold decrease). This result is concordant with a previous study on the PAβN effect on a large collection of levofloxacin-resistant clinical strains of P. aeruginosa 15. In conclusion, for most of the P. aeruginosa clinical strains, the high level of resistance to ciprofloxacin REPRINT