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Characterization of Clinical Blood Isolates of Streptococcus agalactiae with Reduced Susceptibility to Levofloxacin

termined by broth microdilution method on MuellerHinton broth supplemented with 5% lysed horse blood according to the Clinical Laboratory standards institute (CLsi) guidelines 7 for the following antibiotics: levofloxacin, ciprofloxacin and norfloxacin (all from sigma-Aldrich, st. Louis, MO, UsA). Plates were incubated for 20-24 h in 5% CO2 at 35oC + 2 degrees. S. pneumoniae ATCC 49619 and S. agalactiae ATCC12386 were used as control strains. Dilutions of levofloxacin, ciprofloxacin and norfloxacin ranged from 0.06 to 32 mg/L. MiCs of levofloxacin were interpreted according to CLsi M100-s18 guidelines 8. Mutation alterations in the quinolone resistance determining regions (QRDRs) of the genes gyrA and parC were investigated by PCR with chromosomal DnA as template and subsequent DnA sequencing as described by Kawamura et al. 3. The Gen Bank accession numbers for the wild-type sequences used for comparison purposes were S. agalactiae A909 and S. agalactiae nEM316. Amplifications were performed in a DnA thermal cycler (Perkin-ElmerCetus, norwalk, CT, UsA). PCR reagents were purchased from Promega (Madison, Wi, UsA). Among the 48 GBs isolates collected from blood cultures no high-level fluoroquinolone resistant GBs (MiCs of levofloxacin >8 mg/L) were found. Levofloxacin, ciprofloxacin and norfloxacin showed a MiC50 and MiC90 of 0.5/1, 0.5/1 and 4/8 mg/L, a MiC range of 0.25-2, 0.25-2 and 4-16 mg/L and a MiC mode of 0.5, 0.5 and 4 mg/L, respectively. We selected 3 isolates tested with the higher MiCs to FQs (levofloxacin range 1-2 mg/l, ciprofloxacin range 1-2 mg/l and norfloxacin 16 mg/l) to perform further molecular studies in QRDRs of gyrA and parC. no mutations in the QRDRs of gyrA were detected in any isolate. Two GBs selected had MiCs of levofloxacin, ciprofloxacin and norfloxacin of 1, 1 and 16 mg/l respectively. One was a serotype iii GBs resistant to macrolides with cMLsB phenotype and harboring the erm(B) gene, tetracycline susceptible and three parC silent mutations were found at positions: Ala142(GCC≥GCA), Phe145 (TCC≥TTT) and Ala175 (GCA≥GCG). in the other GBs serotype ii selected with the same MiCs no mutations in the QRDR of parC and gyrA were found. in these two strains susceptible to quinolones the molecular analysis did not show relevant mutations, as expected. The third GBs selected was a serotype ii isolate, susceptible to macrolides and tetracyclines, with reduced susceptibility to FQs (MiCs of levofloxacin, ciprofloxacin and norfloxacin 2, 2 and 16 mg/l, respectively), and the sequence analysis of the parC gene showed a base pair chain TCT≥CCT at position 238 that resulted in an amino acid substitution ser-80-toPro (ser80Pro). This isolate was collected from a blood culture of a 59-year-old penicillin-allergic female with type ii diabetes as underlying condition with bacletteR