Gradient plate method to induce Streptococcus pyogenes resistance

In recent years, increasing numbers of Streptococcus pyogenes (GAS) strains displaying resistance to macrolides have been reported in Finland, Japan, Asia and Spain. Antibiotic use has been shown to be a risk factor for infection with and carriage of drug-resistant streptococci. The aim of this study was to compare in-vitro development of resistance of streptococci to beta-lactams (penicillin, amoxycillin, cefotiam and cefuroxime) and erythromycin by serial passages in subinhibitory concentrations of antibiotics (subMICs) by gradient plate method. Three clinical strains of GAS were tested. Two were susceptible to erythromycin (MIC = 0.015 mg/L and 0.013 mg/L) and one resistant. Serial passages were performed daily by gradient plate method until a four-fold increase of the MIC was achieved. GAS variants obtained after serial passages in beta-lactams had MICs increased at least four-fold. They remained susceptible to these antibiotics. With erythromycin, final MICs reached intermediate and resistant level. Results obtained in this study with erythromycin are in good correlation with clinical studies showing that prior exposure to macrolides may help to facilitate the emergence of drug-resistant strains of streptococci.