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 μg/ml) (11). Against A. urinae isolates, moxifloxacin (MIC50, 0.12 μg/ml) was 4- and 16-fold more active than ciprofloxacin/levofloxacin (MIC50, 0.5 μg/ml) and ofloxacin (MIC50, 2 μg/ml), respectively (Table 1). Against A. sanguinicola isolates, moxifloxacin (MIC50, 0.25 μg/ml) was also 4- and 16-fold more active than ciprofloxacin/levofloxacin (MIC50, 1 μg/ml) and ofloxacin (MIC50, 4 μg/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).
TABLE 1.
Susceptibility to fluoroquinolones and mutations in QRDRs of gyrA, gyrB, parC, and parE genes of A. urinae and A. sanguinicola
| Organism (no. of strains) | MIC (μg/ml) ofa: |
QRDR mutation(s) |
|||||||
|---|---|---|---|---|---|---|---|---|---|
| OFX | CIP | CIP+R | LVX | MXF | gyrA | gyrB | parC | parE | |
| A. urinae strains (19) | |||||||||
| HM 335 | ≥32 | ≥32 | ≥32 | ≥32 | ≥32 | S84L | S79R | ||
| HM 365 | 2 | 0.5 | 0.5 | 0.25 | 0.12 | ||||
| HM 384 | 0.5 | 0.12 | 0.12 | 0.12 | 0.12 | ||||
| HM 525 | 0.5 | 0.25 | 0.25 | 0.12 | 0.06 | ||||
| HM 580 | 1 | 0.25 | 0.25 | 0.25 | 0.03 | ||||
| HM 693 | 2 | 0.5 | 0.5 | 0.5 | 0.25 | ||||
| HM 704 | 4 | 1 | 1 | 1 | 0.25 | ||||
| HM 713 | 2 | 0.5 | 0.25 | 0.5 | 0.12 | ||||
| HM 743 | 2 | 0.5 | 0.25 | 0.5 | 0.12 | ||||
| HM 827 | 1 | 0.25 | 0.25 | 0.25 | 0.06 | ||||
| HM 834 | ≥32 | ≥32 | ≥32 | ≥32 | 2 | E83K | |||
| HM 867 | 2 | 0.5 | 0.5 | 0.5 | 0.12 | ||||
| HM 915 | 0.5 | 0.12 | 0.12 | 0.12 | 0.03 | ||||
| HM 963 | 2 | 0.5 | 0.25 | 0.5 | 0.12 | ||||
| HM 980 | 1 | 0.25 | 0.25 | 0.25 | 0.12 | ||||
| HM 1010 | 0.5 | 0.25 | 0.25 | 0.25 | 0.06 | ||||
| HM 1062 | 2 | 1 | 1 | 0.5 | 0.12 | ||||
| HM 1107 | 2 | 0.5 | 0.25 | 0.5 | 0.06 | ||||
| HM 1230 | 2 | 1 | 1 | 0.5 | 0.25 | ||||
| A. sanguinicola strains (8) | |||||||||
| HM 826 | ≥32 | 8 | 8 | 8 | 1 | D78N, S79T | |||
| HM 833 | 4 | 1 | 0.5 | 1 | 0.5 | ||||
| HM 946 | 1 | 0.5 | 0.5 | 0.5 | 0.25 | ||||
| HM958 | ≥32 | 16 | 16 | 8 | 1 | D83G | |||
| HM 962 | 4 | 1 | 1 | 1 | 0.25 | ||||
| HM 1014 | 2 | 1 | 1 | 1 | 0.25 | ||||
| HM 1036 | 4 | 2 | 1 | 1 | 0.5 | ||||
| HM 1273 | 1 | 1 | 0.5 | 1 | 0.25 | ||||
OFX, ofloxacin; CIP, ciprofloxacin; CIP+R, ciprofloxacin plus reserpin (10 μg/ml), LVX, levofloxacin; MXF, moxifloxacin.
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 A. sanguinicola was the presence of an aspartate residue at position 83 in ParC, as described in Streptococcus pneumoniae (9). Whereas all susceptible strains possessed no mutation, at least one mutation was found in gyrA and/or parC in all four resistant strains (Table 1). Except for S79T, similar amino acid changes in hot spot positions of ParC have been identified in other Gram-positive bacteria; these are S79R in E. faecalis and Enterococcus faecium, E83K in E. faecium and Staphylococcus aureus, and D78N and D83G in S. pneumoniae (1, 5, 7, 9). Concerning GyrA, an identical mutation (S84L) has also been identified in S. aureus, E. faecium, and Streptococcus agalactiae (6, 9). These findings suggest that topoisomerase IV seems to be the primary target of fluoroquinolones in Aerococcus spp., as previously described in other Gram-positive bacteria (4, 9).
TABLE 2.
Deoxynucleotide primers used in this study
| Species | Primera | Sequence (5′-3′) | Gene | Annealing temp (°C) | Product size (bp) |
|---|---|---|---|---|---|
| A. urinae | gyrA-uri-F | TCT CAA ACC CGT CCA CCG | gyrA | 55 | 232 |
| gyrA-uri-R | GGC TTG GTC CCC GTC GA | ||||
| gyrB-uri-F | TTG AAG GGC AAA CCA AGA TG | gyrB | 52 | 486 | |
| gyrB-uri-R | TCA CCA ATT TAT GGT AAC GG | ||||
| parC-uri-F | TAT ATT ATT CAA GAA CGC GC | parC | 50 | 333 | |
| parC-uri-R | GTC CTT AAG TAG TTC ATC GG | ||||
| parE-uri-Fb | ATT TGA AGG TCA AAC CAA GG | parE | 50 | 504 | |
| parE-uri-Rb | GCA TCG GTC ATG ATG ATC AC | ||||
| A. sanguinicola | |||||
| gyrA-san-F | GGG ATG AAA CCT GTC CAC CG | gyrA | 55 | 244 | |
| gyrA-san-R | GCT AAG CGG CAG CCT GGT C | ||||
| gyrB-san-F | CAG ACC CAC AGT TTG AAG G | gyrB | 52 | 521 | |
| gyrB-san-R | ATC GAC ATC GGC ATC AGT C | ||||
| parC-san-F | TAC ATT ATT CAA GAA CGG GC | parC | 52 | 389 | |
| parC-san-R | GGT TCT TCC TCA GTA TCA TC |
F, sense primer; R, antisense primer.
Amplification of the parE gene from A. sanguinicola was also performed using primers parE-uri-F and parE-uri-R.
Nucleotide sequence accession numbers.
The QRDR sequences of the gyrA, gyrB, parC, and parE genes of A. urinae HM 384 (susceptible strain) have been submitted to GenBank under accession no. HM744700, HM744701, HM744702, and HM744703, respectively, as have those of A. sanguinicola HM 1273 (susceptible strain), under accession no. HM744704, HM744705, HM744706, and HM744707, respectively.
Footnotes
Published ahead of print on 15 November 2010.
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