Analysis of penicillin-binding protein 1b and 2a genes from Streptococcus pneumoniae

Fifty clinical isolates (penicillin MICs, 0.03-8 mu g/mL) of Streptococcus pneumoniae were randomly selected from hospitals throughout South Africa, t ogether with seven strains isolated in Hungary (penicillin MICs, 16-32 mu g /mL). Penicillin-binding protein (pbp) Ib and 2a genes were amplified by PC R, and the purified DNA was digested with HinfI, StyI, and MseI + DdeI rest riction enzymes. The fragments were radioactively end-labeled and separated on polyacrylamide gels, and the DNA fingerprints were visualized following autoradiography. A collection of isolates was further selected for sequenc e analysis of pbp1b and 2a, DNA fingerprint analysis revealed a uniform pro file amongst all isolates for both genes. All isolates revealed a maximum o f only seven nucleotide substitutions in their pbp1b genes, resulting in a maximum of three amino acid substitutions in PBP 1B, ln the case of the pbp 2a gene, up to 13 nucleotide substitutions were observed randomly distribut ed amongst penicillin-susceptible and resistant isolates, revealing a maxim um of five amino acid substitutions in PBP 2A, No amino acid substitutions were found to be common amongst all penicillin-resistant isolates, Transfor mation experiments with pbp1b and 2a genes isolated from two resistant stra ins (MICs, 4 and 16 mu g/mL) failed to transform pneumococcal strains to in creased levels of penicillin resistance. These results show that the pbp1b and 2a genes examined here do not display the typical mosaic gene patterns observed in the pbp2x, 2b, and la genes of penicillin-resistant pneumococci . In addition, the transformation studies suggest that PBPs 1B and 2A may n ot play a role in the development of penicillin resistance in some pneumoco cci.