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: Denmark14 ST230, Portugal19F ST177 and Spain9V 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.

Genetic diversity of Streptococcus pneumoniae in Tunisia.

OBJECTIVES: This study aimed to explore the genetic diversity of Streptococcus pneumoniae isolates in a Tunisian pneumology hospital. METHODS: A total of 141 S. pneumoniae strains isolated between 2009-2016 in the microbiology laboratory at A. Mami Hospital of Pneumology were investigated. Antimicrobial susceptibility testing was performed the disk diffusion method. MICs of penicillin G, amoxicillin and cefotaxime were determined by Etest. Serotyping was inferred from the results of multiplex PCR targeting 40 serotypes. Sequence types (STs) were determined by multilocus sequence typing (MLST). RESULTS: Among the 141 S. pneumoniae isolates, 98 (69.5%) were resistant to erythromycin. Evaluation of ß-lactam susceptibility showed that 90 strains (63.8%) were non-susceptible to penicillin, whereas 48 (34.0%) had decreased susceptibility to amoxicillin and 21 (14.9%) to cefotaxime. Twenty-five serotypes were detected, and 10 isolates were classified as non-typeable. Vaccine coverage was 56.7%, 60.3% and 75.2% for pneumococcal conjugate vaccine 7 (PCV7), PCV10 and PCV13, respectively. Overall, 73 STs were identified, including 23 described for the first time. The most frequent STs were ST179 (n = 17), ST3772 (n = 14), ST2918 (n = 10) and ST4003 (n = 5), related to serotypes 19F, 19A, 14 and 23F, respectively. Moreover, 110 strains were classified within 45 STs. Three international antimicrobial-resistant clones were found, including Denmark14-ST230 (n = 22), Spain9V-ST156 (n = 22) and Portugal19F-ST177 (n = 20). CONCLUSION: This study emphasises the clonal and international dissemination of antimicrobial-resistant S. pneumoniae clones. Significant differences in genetic variation were documented by MLST within the various serotypes identified.

Is sequencing better than phenotypic tests for the detection of pyrazinamide resistance?

SETTING: Phenotypic tests used to detect pyrazinamide (PZA) resistance are slow and have a high rate of false resistance. OBJECTIVE: To evaluate the accuracy of pncA sequencing for the detection of PZA resistance in Mycobacterium tuberculosis strains isolated in Tunisia. DESIGN: A total of 82 isolates, 41 resistant and 41 susceptible to PZA on BACTEC™ MGIT™ 960, were sequenced for pncA. Whole genome sequencing was performed for strains that were phenotypically resistant and had wild-type pncA in addition to MGIT retesting with a modified protocol. RESULTS: Twenty-three strains resistant to PZA with negative pyrazinamidase (PZase) activity harboured a mutation in the promoter or coding region of pncA. However, 18 strains resistant to PZA did not present any mutation. Repeat MGIT 960 showed that 16 of 18 M. tuberculosis isolates were falsely resistant to PZA. Compared with MGIT, PZase activity assay and pncA sequencing both presented a sensitivity of 92.0% (95%CI 73.9-99.0) and a specificity of respectively 96.5% (positive predictive value [PPV] 92.0%, negative predictive value [NPV] 96.5%) and 100.0% (PPV 100.0%, NPV 96.6%). CONCLUSION: The standard MGIT assay showed a high rate of false resistance to PZA, and the PZase activity assay is slow. pncA sequencing could therefore represent a rapid, accurate, alternative test to detect PZA resistance.