ABSTRACT
Streptococcus pyogenes are one of the most important pathogens that cause human diseases. They colonize the throat or skin and cause a wide variety of infections in children and adults. They are the major bacterial cause of pharyngitis and tonsillitis. Penicillin is the treatment of choice, but in cases of penicillin allergy or therapy failure, macrolides are alternative options. However, there has been a global rise in the emergence of macrolide resistance among these organisms. The aim of this study was to determine the prevalence and mechanisms of antibiotic resistance in isolates of S. pyogenes from patients with throat infections in Benin City, Nigeria.
Throat swabs were collected aseptically from 197 subjects and immediately taken to the laboratory. The samples were inoculated on blood agar containing 5% defibrinated sheep blood and incubated at 37oC for 20 hr. Colonies that showed β-haemolysis were then sub-cultured on fresh blood agar plates, to obtain a pure culture of isolates, and then incubated at 37°C for 20 hr. Streptococcus pyogenes isolates were identified by standard microbiological methods and confirmed by bacitracin sensitivity test and latex agglutination test. Streptococcus pyogenes isolates were then tested for susceptibility to eight antibiotics belonging to 7 classes, including β-lactams (Penicillin G 10unit and Ceftriaxone 30 µg), macrolides (Erythromycin 15 µg), lincosamides (Clindamycin 2 µg), fluoroquinolones (Levofloxacin 5 µg), glycopeptides (Vancomycin 30 µg), phenicols (Chloramphenicol 30 µg), and oxazolidinone (Linezolid 30 µg) using the Kirby–Bauer disc diffusion method and the results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The presence of macrolide, lincosamide and streptogramin B (MLSB) resistant phenotypes was then tested on the resistant isolates using the double disk diffusion method (D-zone test). Phenotypic detection of efflux activity was carried out on the macrolide resistant isolates using the Ethidium Bromide-Agar (EtBr-Agar) cartwheel method. The results provided by the EtBr-agar cartwheel method was further analysed by determining the effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) and menadione on the activity of Erythromycin.
Results revealed that out of the 197 specimens collected, nineteen (19) were identified and confirmed as S. pyogenes, of which 11 (57.89%) were from males and 8 (42.11%) were from females. The prevalence of S. pyogenes in the study population was 9.65%. The results from susceptibility test showed that all the isolates were susceptible to penicillin, ceftriaxone, vancomycin, levofloxacin, linezolid and chloramphenicol. Four (4) isolates were resistant to erythromycin, with the inducible MLSB (i.e., target site modification) the predominant phenotype (75%) followed by the M phenotype (i.e., active efflux) which accounted for 25% of the resistance. Inducible clindamycin resistance was observed in 3 (15.79%) of the isolates. For the phenotypic detection of efflux activity, a range of fluorescent bacteria were detected after incubation, depending on their ability to efflux ethidium bromide. Whereas isolate 190 (Reference strain), 22, 78, and 114 (iMLSB phenotypes) fluoresced at a concentration of 1.0 mg/l of EtBr, isolate 101 (M phenotype) did not fluoresce even with a concentration of EtBr as high as 2.0 mg/l. The minimum inhibitory concentration (MIC) of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the isolates was found to be 8µg/ml while plates containing menadione showed growth even at a concentration of up to 128 µg/ml. In the presence of CCCP at 4 µg/ml, the MIC of erythromycin was decreased by 2 to 4-fold. On the other hand, menadione was found to reduce the MIC of erythromycin by up to 2-fold. Thus, menadione can potentiate the activity of erythromycin against Streptococcus pyogenes in vitro.
