Phenotypic and molecular characterization of macrolide resistance mechanisms among Streptococcus pneumoniae isolated in Tunisia

Introduction . Streptococcus pneumoniae is responsible for many community infections, with the main ones being pneumonia and meningitis. Pneumococcus has developed increased resistance to multiple classes of antibiotics. The evolution of antibiotic resistance in pneumococcus was influenced by changes in serotype distribution under vaccine selection pressure. Aim . The aim of this study was to determine the genes involved in macrolide resistance, the antimicrobial susceptibility, the serotype distribution and the spread of international antibiotic-resistant clones among clinical isolates of S. pneumoniae . Methodology . We investigated 86 erythromycin-resistant S. pneumoniae strains isolated from respiratory ( n =74) or non-respiratory ( n =12) samples in Tunisia. Antimicrobial susceptibility was tested using the disk diffusion method. Macrolide-resistant strains were analysed by polymerase chain reaction (PCR) for ermA , ermB , mefA and msrD . We also investigated the macrolide resistance mechanisms in eight isolates (9.3%) by sequencing the L4 and L22 riboprotein-coding genes, plus relevant segments of the three 23S rRNA genes. Capsular serotypes were detected by multiplex PCR. Sequence types (STs) were explored using multilocus sequence typing (MLST). Results . Among the 86 studied strains, 70 (81.4 %) were resistant to penicillin G. The prevalent serotypes were 19F, 14, 19A and 23F. We observed that the cMLSB phenotype (66/86, 76.7%) was the most common in these pneumococci. In addition, ermB was the most frequent resistance gene. No mutation in ribosomal protein L22 or L4 or 23S rRNA was detected. Overall, 44 STs were identified in this study, including 16 that were described for the first time. Resistance to lincomycin, tetracycline and trimethoprim/sulfamethoxazole was observed in 55 (64 %), 34 (39.5 %) and 31 (36 %) isolates, respectively. Furthermore, an increase in fluoroquinolone use in particular may lead to the emergence of levofloxacin-resistant strains. Multidrug resistance was observed in 83 isolates (96.5%). Three global antibiotic-resistant clones were identified: Denmark^{14} ST230, Portugal^{19F} ST177 and Spain^{9V} ST156. Conclusion . This study shows that macrolide resistance among S. pneumoniae isolated in Tunisia is mainly related to target site modification. Our observations demonstrate a high degree of genetic diversity and capsular types among strains resistant to macrolides.