First molecular characterization of fluoroquinolone resistance in Aerococcus spp.

Aerococcus urinae and Aerococcus sanguinicola are emerging Gram-positive pathogens responsible for urinary tract infections, especially in elderly patients (2). Although they seem to be intrinsically susceptible to fluoroquinolones (3, 8, 10), acquired fluoroquinolone resistance has not been yet reported. Resistance to fluoroquinolones in Gram-positive bacteria is mainly due to point mutations in the quinolone-resistance determining regions (QRDRs) of the GyrA and GyrB subunits of the DNA gyrase and QRDRs of ParC and ParE subunits of the topoisomerase IV (4). Decreased accumulation of fluoroquinolones is a second resistance mechanism that is mediated by the overexpression of efflux pump systems (4). Since QRDR sequences of A. urinae and A. sanguinicola are not available, the aim of this study was to elucidate the mechanisms associated with the fluoroquinolone resistance. Nineteen A. urinae and 8 A. sanguinicola urinary isolates, previously identified by 16S rRNA sequencing, were studied (2). The MICs of ofloxacin, ciprofloxacin, levofloxacin, and moxifloxacin were established using the Etest method (AB Biodisk, Solna, Sweden) on Mueller-Hinton agar supplemented by 5% horse blood. The MICs of ciprofloxacin were also determined in the presence of reserpin, an efflux pump inhibitor, incorporated in the medium (10 g/ml) (11). Against A. urinae isolates, moxifloxacin (MIC50, 0.12 g/ml) was 4and 16-fold more active than ciprofloxacin/levofloxacin (MIC50, 0.5 g/ml) and ofloxacin (MIC50, 2 g/ml), respectively (Table 1). Against A. sanguinicola isolates, moxifloxacin (MIC50, 0.25 g/ ml) was also 4and 16-fold more active than ciprofloxacin/ levofloxacin (MIC50, 1 g/ml) and ofloxacin (MIC50, 4 g/ml), respectively (Table 1). The potent activity of moxifloxacin against Aerococcus spp. is concordant with data previously reported (8). Finally, active efflux did not seem to play a major role in fluoroquinolone resistance in aerococci, since the MICs of ciprofloxacin were similar in the absence or presence of reserpin (Table 1). Following the use of degenerate primers, the DNA fragments corresponding to QRDRs of GyrA, GyrB, ParC, and ParE were amplified using standard PCR conditions with novel specific primers (Table 2). The sequences of GyrA, GyrB, ParC, and ParE of A. urinae were 100%, 98%, 88%, and 93% identical to those of A. sanguinicola, respectively. In A. urinae, a serine residue and a glutamate residue were found at positions 84 and 88 (corresponding to 83 and 87 in Escherichia coli numbering) in GyrA and also at positions 79 and 83 (corresponding to 80 and 84 in E. coli numbering) in ParC, as described in Enterococcus faecalis (9). The unique difference with