To Study The Relationship Between The Pbp1a And The Penicillin Resistant Streptococcus Pneumoniae

Objective:This thesis is to explore the mutation of pbp1a/pbp2b and then to find out the relationship of drug resistance against penicillin for streptococcus pneumoniae and thus to illustrate the variant mechanism of drug resistance of streptococcus pneumoniae. It is also to explore the variant routes of solving penicillin-resistance of streptococcus pneumoniae by gene so as to investigate the mutated penicillin resistant streptococcus pneumoniae and provide an experimental evidence for its infection.Methods:1.Pneumococci were isolated from the secretions of the children infected in the respiratory tract. The minimal inhibitory concentration(MIC)of penicillin was determined by the method of M-H serial dilution. Penicillin resistant S.pneumoniae were screened.2.pbp2b and pbp1a of PRSP were amplified by nPCR. DNA sequences of their PCR products were detected and compared with the DNA sequences of SPR6 strain sensitive to penicillin. The quality of mutation of pbp2b and pbp1a was comprehended deeply and the structural variety of PBP2b and PBP1a was analyzed.3. The pbp1a DNA in STMK of S.pneumoniae was biosynthesized, and cloned into pCU19 expressive plasmid after it was identified. Finally recombinant plasmid pUC19-pbp1a containing Ampr andβ- galactosidase was constructed.4. Competence of S.pneumoniae.was induced by CSP and pUC19-pbp1a was transformed into S.pneumoniae. The sensitivity of S.pneumoniae for penicillin was detected.Results:1. In 169 isolates of S.pneumoniae, 6 strains were died, 75 strains were susceptible, 17 strains were intermediate-susceptible and 71 strains were resistant to penicillin.2.In the 71 strains of PRSP, 58 strains presenting pbp2b mutation were detected. Among the 58 strains, 56 strains presented point mutation and 2 presented CCT insertion mutation. All of the 58 strains revealed the lower degree of drug fast. Among the 58 strains with bpb2b mutation, 21 strains had pbp1a mutation and revealed the high degree of drug fast. Most of the proteins with varied PBP2B were Thr252→Ala, and most of the proteins with varied PBP1A were ThR371→Ala.3. Recombinant plasmid pUC19-pbp1a containing Ampr andβ- galactosidase was successfully constructed. After pUC19-pbp1a was sequenced, it was found out that the recombinant plasmid was in right sequence and gene insertion was in right direction.4. Competence of S.pneumoniae was successfully induced and pUC19-pbp1a was transformed into S.pneumoniae. pbp1a was amplified and sequenced by PCR and was in right sequence, having no mutation. It was found out that the drug fast of the transforming strain was altered.Conclusion:1. The results show the intimate correlation between the gene mutation of pbp1a and pbp2b and penicillin resistance. The different degrees of penicillin resistance attribute to the different gene mutations of pbp1a and pbp2b.2. Expressing plasmidpUC19-pbp1a was successfully constructed.3. Transforming strains have improved their sensitivity to penicillin but just a little bit. It was proved that the resistance of pbp2b mutation was aggravated by pbp1a mutation.