Molecular epidemiology and variants of the multidrug-resistant Streptococcus pneumoniae Spain14-5 international clone among Spanish clinical isolates

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Abstract
Objective: To analyse the molecular structure of several antimicrobial resistance determinants in isolates of the Spain14-5 clone to better understand its emergence and spread. Methods: The distinct genes and mechanism of resistance to penicillin, erythromycin, clindamycin, tetracycline, chloramphenicol and trimethoprim were studied in an apparently homogeneous group of 117 isolates of the multidrug-resistant Spain14-5 major clone isolated in Spain between 1981 and 2004. Results: Several genotyping techniques such as PFGE, BOX-PCR and multilocus sequence typing (MLST) revealed a high degree of homogeneity among these isolates over time. Nevertheless, distinct variants of the clone could be established according to the restriction fragment length polymorphism (RFLP) patterns of the penicillin-binding protein (pbp) genes and the sequences of the dihydrofolate reductase (dhfr) gene. In addition, an association between the pbp2b RFLP patterns, the ddl alleles identified by MLST and the dhfr alleles was found. The emergence of variants of the Spain14-5 clone, which had lost macrolide and tetracycline resistance, while harbouring the ins and xis genes of the Tn916–Tn1545 family of conjugative transposons, was documented. Two different tet(M) alleles were detected in isolates of the clone, one of them with a mosaic structure. Conclusions: The finding of different patterns or alleles of the genes responsible for antibiotic resistance among isolates of the Spain14-5 clone from different Spanish cities indicates different evolutionary events within isolates of a unique Streptococcus pneumoniae clone.