Application of the real-time pcr method for genotypic identification of β-lactam resistance in isolates from invasive pneumococcal diseases

We sought to identify genotypic resistance classes by real-time PCR in 300 Streptococcus pneumoniae isolates from invasive pneumococcal diseases. Primers and molecular beacon probes were designed for the lytA gene, 3 pbp genes, and the mefA/ermB genes. Targeted sequences of pbp1a, pbp2x, and pbp2b genes in susceptible strain R6 corresponded to those of penicillin G-nonsusceptible strains, including sites within or adjacent to conserved amino acid motifs. If amplification did not occur, the corresponding penicillin-binding protein (PBP) was considered to possess amino acid substitution(s) affecting minimal inhibitory concentrations (MICs) of β-lactam antibiotics. Real-time PCR required 90min or less. Strains were assigned to six genotypic classes: Genotypic penicillin-susceptible S. pneumoniae (gPSSP) with 3 normal genes (22.3%); genotypic penicillin-intermediate S. pneumoniae (gPISP) (pbp2x) with an abnormal pbp2x gene (25.3%); gPISP (pbp2b) with an abnormal pbp2b gene (7.3%); gPISP (pbp1a+2x) with abnormal pbp1a+2x genes (11.3%); gPISP (pbp2x+2b) with abnormal pbp2x+2b genes (4.7%); or genotypic penicillin-resistant S. pneumoniae (gPRSP) with 3 abnormal PBP genes (29.0%). Sensitivity and specificity of real-time PCR compared with those of conventional PCR were high, 73.7-100% and 97.7-100%, respectively. As for relationships between genotype and β-lactam MICs, 90% of MICs for every resistance class were distributed within three serial dilutions for almost all antibiotics. MICs of each β-lactam antibiotic were estimated with high probability from genotypic patterns. In conclusion, determination of genotypic classes of S. pneumoniae using rapid real-time PCR is useful in selecting effective therapeutic agents for patients with pneumococcal infection.