The Study Of Molecular Typing And Antibiotic Resistance Of Streptococcus Pneumoniae

ObjectiveTo establish a method based on PCR for serotyping of Streptococcus pneumoniae isolates. PCR serotyping method was applied for investigating the major epidemic serotype/groups and understanding the serotype/groups distribution of S. pneumoniae. To establish and optimize the method of PFGE for analyzing S. pneumoniae. PFGE method was applied for understanding molecular features of S. pneumoniae in China. Sensitivity test was applied for understanding the susceptibility and changes in antibiotic resistance of clinical isolates of S. pneumoniae. To establish the databank covering the information of serology, molecular characteristics, antibiotic resistance of S. pneumoniae.MethodsIn this work,144 isolates of S. pneumoniae were collected and serogrouped firstly.14 pairs of primers were synthesized for detecting specific genus gene of S. pneumoniae and specific serotype or serogroup genes. Reaction concentrations of each pair of primers and reaction system were optimized. We built one rapid, simple, and cost-effective PCR-based method used for typing of pneumococci. Based on the published literature of PFGE for S. pneumoniae typing, we optimized the conditions of PFGE and used it for molecular typing of S. pneumoniae. The sensitivities of all isolates to 6 antibiotics were determined by K-B (Kirby-Bauer) method according to the standard of CLIS. To provide the basis for the clinical treatment, we analyzed the resistant spectrum of isolates.ResultsThe result showed high degree specificity of all primers among species and serogroups with sensitivity reaching 1-10pg/μl All 144 isolates could be divided into 10 serotype/groups by PCR method. The results of PCR typing were similar to serotyping. PFGE was used for analyzing the genotyping characteristics of all isolates.92 PFGE types indicated that no prevalent clone of S. pneumoniae was found in this study. Same serogroup isolates showed similar PFGE patterns. We detected the susceptibility of pneumococcus to 6 antibiotics such as Minocycline HC1, Ofloxacin, Chloramphenicol, Azithromycin, Rifampin, Sulfamethoxazole/Trimethoprim. We found the most of isolates were resistant to more than one antibiotic.Conclusions We have developed one simple, reliable and economical method for S. pneumoniae serotyping, which can be used for testing 13 serotypes of S. pneumoniae prevailing among people. PFGE has great discriminatory ability for subtyping of S. pneumoniae, and showed better epidemiological survey capacity. The antibiotic resistance of isolates is very complex. In addition to OFX, the isolates resistant to other 5 antibiotics were found. Moreover most of isolates are resistant to more than 2 antibiotics.