Streptococcus pneumoniae (S. pneumoniae) is a major pathogen of bacterial pneumonia, meningitis, sinusitis and otitis media. Antimicrobial resistance in S. pneumoniae, including macrolide resistance, has been a growing problem in recent years due to the increasing numbers of cases with treatment failures of infections caused by macrolide-resistant pneumococci (1).Macrolide resistance in S. pneumoniae is mediated by: the erm (B) gene encodes methylation of the ribosomal macrolide target sites and mef (A) encodes the drug efflux. Deterioration of riboproteins L4 and L22 and mutations in the 23S rRNA genes are involved in the other less common mechanisms (2).In this study, we analyzed the distribution of the phenotypes, genotypes, and clonal relatedness of macrolide-resistant S. pneumoniae strains isolated in our hospital.
MATERIALS AND METHODSEighty S. pneumoniae strains were collected between January 2008 and September 2009 in the Department of Microbiology of the Cerrahpaşa Faculty of Medicine. Ethics committee approval was received for this study from the Ethics Committee of Cerrahpaşa Faculty of Medicine. Strains were isolated mostly from sputum. The remaining isolates were from tracheal aspirates, bronchoalveolar lavage fluid, nasal swab and blood culture.S. pneumoniae isolates were identified based on conventional microbiological methods. Antibiotic susceptibility testing was performed with the disc diffusion method (3).Macrolide resistance phenotypes were determined by Montanari et al. (4) : 11.11.2013 Accepted: 23.11.2014 • DOI: 10.5152/balkanmedj.2015 Available at www.balkanmedicaljournal.org Cite this article as: Sirekbasan L, Gönüllü N, Sirekbasan S, Kuşkucu M, Midilli K. Phenotypes and genotypes of macrolide-resistant streptococcus pneumoniae. Balkan Med J 2015;32:84-8.
Copyright 2015 © Trakya University Faculty of MedicineBalkan Med J 2015;32:84-8 Original Article | 84MICs of macrolides (erythromycin, clarithromycin and azithromycin), lincosamides (clindamycin), streptogramins (quinupristin-dalfopristin) and penicillin G were measured by E-test (AB Biodisk; Solna, Sweden). The strain S. pneumoniae ATCC 49619 was used as a control strain.
Detection of erythromycin-resistance genesA PCR method was used for the detection of erythromycinresistance genes (5). Bacterial DNA was isolated using a DNA isolation kit (Roche Diagnostic; Mannheim, Germany). All PCR mixtures contain a volume of 25 µL with 5µL of target DNA, 0.5 µL of each primer (25 mM), 1.5 µL of 25 mM MgCl 2 , 0.5 µL of dNTP mixture (25 mM each), 0.125 µL of Taq DNA polymerase (5 U/µL) and 2.5 µL of 10X buffer (MBI; Fermentas, Lithuania). PCR for both genes was performed on PTC-200 (Peltier Thermal Cycler; MJ Research, USA). Cycling conditions for amplification were: 5 min at 94°C, followed by 40 cycles of 1 min at 94°C, 1 min at 48°C and 1.5 min at 72°C, with a final 10 min incubation at 72°C. PCR products were separated on ethidium bromide stained 1.5% agarose gels. The expected sizes of amplification products for erm (B) and mef (A) were ...