Genomic analyses of DNA transformation and penicillin resistance in Streptococcus pneumoniae clinical isolates

Alterations in penicillin-binding proteins, the target enzymes for ß-lactam antibiotics, are recognized as primary penicillin resistance mechanisms in Streptococcus pneumoniae. Nonetheless, evidence is available for the involvement of additional resistance mechanisms. We reconstructed penicillin resistance by serial genome transformation of the DNAs derived from three penicillin resistant clinical isolates into a sensitive S. pneumoniae background. The genome sequence of the three transformants T2-18209, T5-1983 and T3-55938 revealed that 16.2 kb, 82.7kb and 137.2 kb of their genomes had been replaced with 3, 10 and 23 multiple-gene recombinant sequence segment (RSSs) of the respective parental clinical isolates, documenting the extent of DNA transformation between strains. Sequencing of the transformants with similar minimal inhibitory concentrations for penicillin as the parent clinical strains also confirmed the importance of mosaic PBP2x, 2b and 1a as a driving force in penicillin resistance. A role in resistance for mosaic PBP2a was also observed for two of the clinical resistant isolates. We also report a new role for a cytoplasmic alpha amylase in conferring moderate resistance to penicillin in the presence of altered penicillin-binding proteins.