In Vitro Development of Resistance to Six Quinolones in Streptococcus pneumoniae, Streptococcus pyogenes , and Staphylococcus aureus

Abstract

Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus isolates were exposed to subinhibitory MICs of ciprofloxacin, sparfloxacin, gatifloxacin, moxifloxacin, clinafloxacin, and gemifloxacin during a 10-day period. Subculturing led to resistance development, regardless of the initial potencies of the quinolones. None of the quinolones was associated with a significantly slower rate of resistance development.Fluoroquinolone resistance in gram-positive cocci is related to mutations in the DNA gyrase and topoisomerase IV genes (8-12, 16, 23) and the active efflux of agents (1, 3, 13, 21-25, 31, 44). Because fluoroquinolones differ in both their target affinity (8,16,(33)(34)(35)(36)39) and their activation of efflux pumps (7,13,21,22,24,25,31,42), one can speculate that the phenotypic expression of quinolone resistance will also differ. Studies have shown that fluoroquinolone resistance can be selected for in pneumococci and staphylococci (5,6,37).In order to analyze the ability of newer fluoroquinolones to cause resistance development in Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus, we repeatedly exposed six clinical strains of each species to ciprofloxacin, sparfloxacin, gatifloxacin, moxifloxacin, clinafloxacin, and gemifloxacin.Approximately 5 ϫ 10 7 CFU of each of the 18 strains was added to tubes containing 9.9 ml of appropriate broth containing antibiotic concentrations ranging from 3 doubling dilutions above to 3 doubling dilutions below the MIC of each of the six agents. The tubes were then incubated for 24 h at 37°C. Aliquots from the test tubes containing the highest drug concentration that permitted visible growth were used following a 1:100 dilution to inoculate a second set of serial drug dilutions. After overnight incubation, the bacteria were transferred again. Finally, after 10 serial transfers, the bacteria for which the MICs were the highest were collected, stored, and also subcultured on quinolone-free agar for 10 days to assess the stability of resistance.MICs were determined by the microdilution methodology according to NCCLS guidelines (29, 30). Ciprofloxacin MIC determinations were conducted in the presence and absence of reserpine (20 g/ml; tests were repeated three times) for all of the original isolates (n ϭ 18) as well as for all of the selected mutants (n ϭ 108) (7).S. pneumoniae and S. aureus isolates were analyzed before and after transfers for mutations in the quinolone-resistance determining regions (QRDRs) of parC or grlA and gyrA, respectively (19,40,41).The MIC results from subculturing as well as the mutations in the QRDRs of S. pneumoniae and S. aureus are summarized in the Tables 1 to 3. Subculturing with newer quinolones led to resistance development in all three species. This is in line with previous reports with regard to cephalosporins, macrolides, and older quinolones in pneumococci (5, 6, 37).Resistance was stable in all cases; i.e., the MICs for the 108 selected mutants remained within 1 doubling dilution after 10 tr...

An intrinsic pattern of reduced susceptibility to fluoroquinolones in pediatric isolates of Streptococcus pyogenes

“…The point mutation patterns among the pediatric isolates are similar to those previously documented in adult isolates and somewhat similar to those key residue replacements in S. pneumoniae but with much less variety of amino acid substitutions (Boos et al, 2001;Janoir et al, 1996). By comparison with published sequence data from naturally occurring and laboratory generated fluoroquinolone-resistant isolates, a significant increase in MIC appears to require mutations in both parC and gyrA, especially in residue serine-79 and serine-81, respectively, which are compatible to the development of fluoroquinolone resistance in S. pneumoniae (Boos et al, 2001;Janoir et al, 1996;Billal et al, 2007).…”

Mutant Selection Window in Levofloxacin and Moxifloxacin Treatments of Experimental Pneumococcal Pneumonia in a Rabbit Model of Human Therapy

“…Of note, all of these new in vivo mutants harbored a mutation in the gyrase gene, a finding which is concordant with in vitro data (6,32,33,35,46,55). In vitro, the mutation frequency of pneumococcal strains, with or without the presence of a parC mutation, is ca.…”

“…However, further information concerning the in vivo efficacy of new fluoroquinolones is needed. Especially, the risk of in vivo mutations has to be investigated, taking into account the high in vitro rate of mutation of pneumococci when exposed to quinolones (1,6,41,42,45,46,55,66,68,74) compared to beta-lactam agents.…”

Emerging fluoroquinolone-non-susceptible group A streptococci in two different paediatric populations

“…A higher level of resistance is reached by the accumulation of additional mutations in the secondary target (DNA gyrase, gyrA gene) [4]. Efflux of the FQ via membrane-associated pumps also contributes to resistance [5,6].…”