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 90 min 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.
Laboratory of Molecular Epidemiology for Infectious Agents, Kitasato Institute for Life Sciences, Kitasato University , 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
Naoko
N
Chiba
Miyuki
M
Morozumi
Kimiko
K
Ubukata
D001426:Bacterial Proteins / Q000235:genetics; D005838:Genotype; D006801:Humans; D008826:Microbial Sensitivity Tests; D046915:Penicillin-Binding Proteins / Q000235:genetics*; D010406:Penicillins / Q000494:pharmacology; D011008:Pneumococcal Infections / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013296:Streptococcus pneumoniae / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D018440:beta-Lactam Resistance / Q000235:genetics*; D047090:beta-Lactams / Q000494:pharmacology
D001426:Bacterial Proteins; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D047090:beta-Lactams