Molecular characterisation of multidrug-resistant pneumococcal clones colonising healthy children in Mérida, Venezuela.

Genetic mechanisms of resistance, clonal composition and the occurrence of pili were analysed in 48 multidrug-resistant (MDR) pneumococci isolated from healthy children in Mérida, Venezuela. Intermediate resistance to penicillin was related to variations in pbp2b and pbp2x. High-level resistance to penicillin as well as low susceptibility to cephalosporins and carbapenems were associated with alterations in pbp1a, pbp2b and pbp2x. Non-ß-lactam resistance was associated with Tn3872, Tn5253, Tn6002 and Tn2010 transposons. Macrolide-resistant strains carried ermB or mefE, but not mefA. Tetracycline- and chloramphenicol-resistant pneumococci carried tetM and cat, respectively. MDR pneumococci were related to six clonal complexes (CCs), largely CC156 or CC15. Limited diversity in pbp2a,2b,2x-RFLP profiles within each clone was observed. Conversely, detection of non-ß-lactam resistance and transposons revealed clear genetic diversity within clones. A group of non-typeable/cpsA-negative pneumococci related to the null capsule clade 1 (NCC1) carrying a Tn2009 element was found. Each NCC1-related strain showed a novel MLST allelic combination and a different pbp2a,2b,2x-RFLP profile. PI-1 (pilus type 1 rrgC gene) was present in most of the MDR pneumococci and its occurrence was commonly homogeneous within each clone. PI-1 was present in all CC242 and CC320 pneumococci, whereas it was absent in all CC37, CC81 and NCC1 isolates. CC156 and CC15 isolates showed variations in the occurrence of PI-1. Both PI-1 and the islet for pilus type 2 were present in CC320 isolates. We provide useful data to follow the evolution of clonal composition, mechanisms of antibiotic resistance and the occurrence of pili among pneumococci circulating in Mérida.

Evaluation of Two Supplemented Culture Media for Long-Term, Room-Temperature Preservation of Streptococcus pneumoniae Strains.

OBJECTIVE: To produce two supplemented agar types in order to store pneumococci for several months at room temperature. METHODS: Todd-Hewitt/Hemoglobin/Yeast/Charcoal/Agar (TH-HYC) and Todd-Hewitt/Skim-Milk/Yeast/Charcoal/Agar (TH-SYC) were used to prepare two supplemented agar types. Nineteen pneumococci isolated from patients or asymptomatic carriers displaying diverse serotypes and multilocus sequence types (MLST) were subcultured and stored onto supplemented agar types, in four different tests, at room temperature. FINDINGS: At the end of all tests (4-6 months) all noncontaminated subcultures were viable and maintained all phenotypic characteristics. Survival-time curves revealed a slow decrease of viable CFU over time on agar types, but at the end the number of viable CFU was satisfactory (≥2+ of growth). Decreasing of CFU was significantly higher for clinical versus nasopharyngeal isolates. Subcultures contamination rates were 6.25% and 14.58% after 2 and 6 months of storage, respectively. CONCLUSION: TH-HYC and TH-SYC agar types allowed the viability of pneumococci with several serotypes, MLST, and genetic profiles, after 6 months of storage at room temperature. We consider that these agar types are a valid alternative to preserve pneumococci over an extended period, especially when methods as cryopreservation or lyophilization are not available, and are useful for transporting strains between laboratories.