Small-Colony Mutants of Staphylococcus aureus Allow Selection of Gyrase-Mediated Resistance to Dual-Target Fluoroquinolones

Abstract

Fluoroquinolones acting equally through DNA gyrase and topoisomerase IV in vivo are considered desirable in requiring two target mutations for emergence of resistant bacteria. To investigate this idea, we have studied the response of Staphylococcus aureus RN4220 to stepwise challenge with sparfloxacin, a known dual-target agent, and with NSFQ-105, a more potent sulfanilyl fluoroquinolone that behaves similarly. First-step mutants were obtained with both drugs but only at the MIC. These mutants exhibited distinctive small-colony phenotypes and two-to fourfold increases in MICs of NSFQ-105, sparfloxacin, and ciprofloxacin. No changes were detected in the quinolone resistance-determining regions of the gyrA, gyrB, grlA, or grlB gene. Quinoloneinduced small-colony mutants shared the delayed coagulase response but not the requirement for menadione, hemin, or thymidine characteristic of small-colony variants, a subpopulation of S. aureus that is often defective in electron transport. Second-step mutants selected with NSFQ-105 had gyrA(S84L) alterations; those obtained with sparfloxacin carried a gyrA(D83A) mutation or a novel gyrB deletion (⌬RKSAL, residues 405 to 409) affecting a trypsin-sensitive region linking functional domains of S. aureus GyrB. Each mutation was associated with four-to eightfold increases in MICs of NSFQ-105 and sparfloxacin, but not of ciprofloxacin, which we confirm targets topoisomerase IV. The presence of wild-type grlB-grlA gene sequences in second-step mutants excluded involvement of topoisomerase IV in the small-colony phenotype. Growth revertants retaining mutant gyrA or gyrB alleles were quinolone susceptible, indicating that resistance to NSFQ-105 and sparfloxacin was contingent on the small-colony mutation. We propose that small-colony mutations unbalance target sensitivities, perhaps through altered ATP or topoisomerase levels, such that gyrase becomes the primary drug target. Breaking of target parity by genetic or physiological means eliminates the need for two target mutations and provides a novel mechanism for stepwise selection of quinolone resistance.Staphylococcus aureus is a key gram-positive pathogen that causes life-threatening systemic infections, including pneumonia, septicemia, endocarditis, and osteomyelitis. Although several effective antistaphylococcal agents have been developed, their use has been compromised by the emergence of resistant strains. Thus, the introduction of penicillins was followed by the rapid selection of ␤-lactamase-expressing strains (5). Similarly, strains resistant to methicillin through expression of the mecA gene are widespread in many hospitals, and such isolates frequently express a multiple-resistance phenotype (5). In addition, recent work suggests that small-colony variants (SCVs) of S. aureus associated with persistent and refractory infections may play a role in clinical resistance (22,37). SCVs are naturally occurring variants with a normal microscopic morphology but a small colony size and a complex phenotype, including reduced expres...

Evolution of Ciprofloxacin-Resistant Staphylococcus aureus in In Vitro Pharmacokinetic Environments

“…Others have also reported resistant S. aureus variants lacking the expected mutations in the QRDRs of target genes (12,43). In one of our experiments we found a mutation (corresponding to A176G) outside the traditional QRDR that has recently been reported to be associated with fluoroquinolone resistance (25).…”

Comparative Mutant Prevention Concentrations of Pradofloxacin and Other Veterinary Fluoroquinolones Indicate Differing Potentials in Preventing Selection of Resistance

“…It remains to be analyzed, however, how the vast differences in MPCs (especially for staphylococci) translate into treatment success and prevention of FQ resistance. It has been stated that the ideal FQ should have similar affinities for both targets, DNA gyrase and topoisomerase IV (6,17,33). However, as both enzymes differ in mode of expression as well as the molecular mechanisms (23), this may be feasible for specific FQs only, e.g., clinafloxacin (33).…”

Molecular Evaluation of Antibiotic Susceptibility: Tropheryma whipplei Paradigm

“…In contrast, a serine residue is found in M. fortuitum, Mycobacterium peregrinum, and Mycobacterium aurum, which are naturally more susceptible to these antibiotics (8,9,26). Other critical positions in the GyrA QRDR have been described as positions 84 and 87 in E. coli (31) and corresponding positions in other species (23,31,32). Highlevel resistance to fluoroquinolones has been described in M. tuberculosis due to amino acid substitution in the wild-type GyrA sequence at the critical positions 83, 84, and 87, with MICs being more than 100-fold those for wild-type strains when these mutations accumulate (16).…”