Characterization of fluoroquinolone resistance in a clinical isolate of Pseudomonas stutzeri.

Sir, Pseudomonas stutzeri is a non-fluorescent denitrifying bacterium widely distributed in the environment and rarely isolated as an opportunistic pathogen from humans. This species accounts for 1%–2% of hospital-acquired Pseudomonas spp. infections. Pseudomonas stutzeri is susceptible to many more antibiotics than the human pathogen Pseudomonas aeruginosa, its most closely related species. This higher susceptibility may be explained by its reduced occurrence in the clinical environment and, consequently, its lower exposure to antibiotics. However, drug-resistant P. stutzeri clinical isolates have been recovered for almost all antibiotic families, with the noticeable exception of fluoroquinolones. We report herein a multidrug-resistant P. stutzeri (strain 13) isolated in 2007 at the VU Medical Center, Amsterdam, The Netherlands. P. stutzeri strain 13 was identified with the API32GN system (bioMérieux, Marcy l’Étoile, France) and PCR sequencing of 16S rDNA. Disc diffusion and broth microdilution methods were used to determine its antibiotic susceptibility, and results were interpreted according to the CLSI guidelines. P. stutzeri ATCC 17588 was used as a reference strain. P. stutzeri 13 was resistant to ticarcillin, piperacillin, piperacillin/tazobactam and imipenem, had reduced susceptibility to ceftazidime, cefepime and cefpirome, and was fully susceptible to aztreonam. A preliminary analysis showed that P. stutzeri isolate 13 expressed a metallo-b-lactamase named DIM-1 that was responsible for a high level of resistance to all b-lactams including carbapenems. In addition, P. stutzeri 13 was also resistant to gentamicin, tobramycin, rifampicin, chloramphenicol, tetracycline and, surprisingly, fluoroquinolones, remaining susceptible only to amikacin, netilmicin and colistin. Since resistance to fluoroquinolones had never been described in P. stutzeri so far, it was investigated in isolate P. stutzeri 13 showing high-level fluoroquinolone resistance (MIC of ciprofloxacin .32 mg/L) (Table 1). Since mutations in the DNA gyrase and topoisomerase IV genes are the most common mechanisms of fluoroquinolone resistance in Gramnegative organisms, whole-cell DNA of isolate 13 was extracted and used as a template for PCR amplification using primers able to amplify the corresponding quinolone resistance determining regions (‘QRDRs’) of the gyrA and parC genes of P. stutzeri ATCC 17588 (as control) and of P. stutzeri 13, and subsequently sequenced. The primers used were: GyrA-PstF (50-CAT GGG CGA ACT GGC CAA AG-30) and GyrA-PstR (50-CTG CCG GAA TCT GCT CGG TG-30) for gyrA (expected amplicon of 473 bp); and ParC-PstF (50-ATG AGC GAA TCC CTC GAC CTG-30) and ParC-PstR (50-GGT TCG TCC AGG GTG CCG TCG A-30) for parC (expected amplicon of 482 bp). The sequence of the quinolone-resistant isolate showed a single substitution in GyrA (S102I, corresponding to position S83 in P. aeruginosa) and a single substitution in ParC (S87I), compared with the P. stutzeri ATCC 17588 reference strain. These positions are the most frequent sites when mutations have been reported as a source of fluoroquinolone resistance in P. aeruginosa. Additionally, the QRDR regions of the gyrB and parE genes were also analysed. No change was found in the QRDR regions of those genes. In all cases, the sequence of the P. stutzeri ATCC 17588 reference strain was identical to those obtained from the GenBank database (P. stutzeri A1501, accession number NC_009434). In order to check for additional fluoroquinolone resistance mechanisms, plasmid-mediated quinolone resistance qnrA, qnrB, qnrS, qnrVC, qepA and aac(60)-Ib-cr genes were searched for, but no positive result was obtained. Considering that the overexpression of multidrug efflux systems plays an important role in fluoroquinolone resistance in P. aeruginosa, antimicrobial susceptibility testing was performed on Mueller–Hinton agar with or without the antibiotic efflux pump inhibitor phenylalanyl arginyl b-naphthylamide (PAbN) at 20 mg/L. The addition of PAbN significantly reduced the MICs of nalidixic acid, ofloxacin, norfloxacin, ciprofloxacin, levofloxacin, moxifloxacin, enrofloxacin and pefloxacin (Table 1), but also those of tetracycline, rifampicin, trimethoprim, chloramphenicol and several b-lactams (including meropenem, data not shown), suggesting that overexpression of efflux pumps may also be involved. No effect was observed for the aminoglycosides tobramycin and gentamicin. This could be related to the P. stutzeri TbtABM efflux pump, which has been Table 1. MICs of quinolones/fluoroquinolones for P. stutzeri 13 and P. stutzeri ATCC 17588