In vitro development of resistance to five quinolones and amoxicillin-clavulanate in Streptococcus pneumoniae

The ability of 50 sequential subcultures in subinhibitory concentrations of ciprofloxacin, levofloxacin, grepafloxacin, sparfloxacin, trovafloxacin, a nd amoxicillin-clavulanate to select for resistance was studied for six pen icillin-susceptible and four penicillin-intermediate pneumococci. Subcultur ing in ciprofloxacin, grepafloxacin, levofloxacin, and sparfloxacin led to selection of mutants requiring increased MICs for all 10 strains, with MICs rising from (i) 0.5 to 4.0 to (ii) 4.0 to 32.0 mu g/ml after 7 to 12 passa ges for ciprofloxacin, from (i) 0.06 to 0.25 to (ii) 0.5 to 8.0 mu g/ml aft er 5 to 23 passages for grepafloxacin, from (i) 0.5 to 1.0 to (ii) 1.0 to 6 4 mu g/ml after 14 to 49 passages for levofloxacin, and from (i) 0.125 to 0 .25 to (ii) 1.0 to 16.0 mu g/ml after 8 to 26 passages for sparfloxacin. Su bculturing in trovafloxacin led to increased MICs for eight strains, with M ICs rising from (i) 0.06 to 0.125 to (ii) 0.5 to 8.0 mu g/ml after 6 to 28 passages. Subculturing in amoxicillin-clavulanate led to raised MICs for on ly one strain, with the MIC rising from 0.015 to 0.125 mu g/ml after 24 pas sages. Double mutations in both ParC and GyrA led to high-level quinolone r esistance when ParC mutations were at S79. Trovafloxacin MICs were 1 to 2 m u g/ml in double mutants with ParC mutations at positions other than S79 (e .g., D83). Mutations in ParE (at D435, R447, and E474) and GyrB (at S405, D 406, and D435) were found in four and six mutants, respectively. In the pre sence of reserpine, 29 mutants had lower ciprofloxacin MICs (2 to 16 times lower), 8 mutants had lower levofloxacin MICs (2 times), and one mutant had a lower trovafloxacin MIC (2 times), suggesting the involvement of an effl ux mechanism. In contrast to the case for quinolones, subculturing in the p resence of amoxicillin-clavulanate did not select for resistance to this dr ug.