Abstract

Sir, Streptococcus pneumoniae is the most common bacterial pathogen of the respiratory tract, primarily causing pneumonia, meningitis and acute otitis media. Infections caused by this pathogen are being further aggravated by the spread of resistance to several classes of antimicrobial agents including b-lactams, macrolides and tetracycline. Recently, respiratory fluoroquinolones, including levofloxacin, gemifloxacin and moxifloxacin, have been used for the treatment of community-acquired pneumonia. Respiratory fluoroquinolones, especially moxifloxacin, are still highly effective against S. pneumonia. However, with increasing use of respiratory fluoroquinolones worldwide, the number of clinical isolates of S. pneumoniae with fluoroquinolone resistance continues to increase. Fluoroquinolones inhibit DNA gyrase and topoisomerase IV, which are involved in bacterial DNA replication. Both enzymes are composed of two subunits: GyrA and GyrB in DNA gyrase, and ParC and ParE in topoisomerase IV. The most extensively described mechanism of resistance to fluoroquinolones is the development of point mutations in a particular region of each enzyme subunit, known as the quinolone resistance-determining region (QRDR). In clinical isolates of S. pneumoniae, resistance mutations in gyrA and parC are more common than in gyrB and parE. The most prevalent resistance mutations are S81F/Y or E85K in gyrA and S79Y/F in parC, all of which confer high-level resistance to ciprofloxacin and levofloxacin (MIC ≥16 mg/L) and low-level resistance to moxifloxacin (MIC 4–8 mg/L). There are high-level moxifloxacin-resistant clinical strains, but their resistance mechanisms are unknown. To study the progression of moxifloxacin resistance in S. pneumoniae, mutants of S. pneumoniae R6 were generated in a stepwise fashion by selecting colonies that grew at various antibiotic concentrations on gradient plates at each stage; the highest concentrations for each stage were 0.25, 8 and 16 mg/L. Three selection cycles were required to obtain the high-level moxifloxacin-resistant mutants, designated R6M-1, -2 and -3 respectively (Table 1). These moxifloxacin-resistant mutants also showed resistance to ciprofloxacin and levofloxacin. Interestingly, the increases in resistance were not equal for each antibiotic. The levels of resistance increased with the acquisition of additional mutations in gyrA, parC and parE (Table 1). The highly resistant R6M-3 strain contained common mutations in gyrA and parC, but it also had a mutation in parE (P454S in ParE). The mutation in ParE (P454S) was previously described in a clinical isolate in combination with mutations in ParC (S79Y) and GyrA (S81F); however, there was no evidence that this mutation was involved in fluoroquinolone resistance. PCR fragment transformation was used to test whether the ParE (P454S) mutation was involved in moxifloxacin resistance. The primers parE398 (5′-AAGGCGCGTGATGAGAGC-3′) and parE483 (5′-TCTGCTCCAACACCCGCA-3′) were used to amplify the QRDRs of parE from the highly moxifloxacin-resistant strain R6M-3 and from R6WT (where WT stands for wild-type). The resulting fragments (290 bp) were transformed into the strain R6M-2 by following the procedure described by Joloba et al. Transformants were selected in the presence of 16 mg/L moxifloxacin, and transformation was confirmed by PCR and sequence analysis. Thirty colonies were obtained following transformation with the PCR fragment carrying the ParE P454S mutation, and no colony was obtained following transformation with the PCR fragment without mutation. Sequencing analysis showed that all of the transformants carried a gene encoding the P454S mutation in ParE. Transformation efficiency was calculated to be 3.0×10 according to a previously described

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