To Study the Relationship between the pbp1a and the Penicillin Resistant Streptococcus Pneumoniae
|School||Chongqing Medical University|
|Keywords||Streptococcus pneumoniae Gene Mutation Penicillin Resistance Carrier|
Objective: To investigate the relationship between the pbp1a and pbp2b gene mutation Streptococcus pneumoniae resistant to penicillin, and further elucidate the mechanism of the Streptococcus pneumoniae resistance variation. Way to explore the genetic level to solve the variability of β-lactam antibiotics of the penicillin-resistant Streptococcus pneumoniae, Streptococcus pneumoniae resistance to penicillin antibiotics for the prevention and treatment of infection to provide experimental basis. Method: 1. Isolated sputum samples from children with respiratory infections, Streptococcus pneumoniae, liquid medium serial dilution assay penicillin the minimum inhibitory concentration (MIC) of Streptococcus pneumoniae, screening penicillin-resistant Streptococcus pneumoniae strains. The 2. Nested polymerase chain reaction (nPCR) the amplification penicillin-resistant strains pbp2b, and pbp1a gene. The amplification products of DNA sequencing, the measurement sequence with penicillin-sensitive strains (SPN R6) Comparison of gene sequences, the mutation in the nature of a deeper understanding and analysis of the expression of the protein structural changes. 3 biosynthesis containing District the Streptococcus pneumoniae pbp1a fragment STMK identified and cloned into the expression vector pUC19, to construct recombinant plasmid pUC19-pbp1a of the ampicillin resistance gene (Ampr) and β-galactosidase screening systems. 4 CSP induced Streptococcus pneumoniae competent transforming the pUC19-pbp1a carrier detection of Streptococcus pneumoniae to penicillin sensitivity. Results: 1. Isolated from sputum samples out of 169 Streptococcus pneumoniae, save in six died, the remaining 163, including 75 penicillin-susceptible strains, 17 moderately susceptible strains resistant 71. 2. In 71 resistant pbp2b amplification results in 58 to exist the different locations pbp2b mutation. 56 for the different positions of the point mutations, two for CCT insertion mutation, most of them appear in the low-level resistant. In the 58 pbp2b mutants, 21 a pbp1a mutations exist in the high levels of drug-resistant strains. The structural changes in protein expression pbp2b mutation Thr252 → Ala, the main expression of protein structural changes pbp1a mutated Thr371 → Ala. Successfully constructed the screening system containing the ampicillin resistance gene (Ampr) and β-galactosidase pbp1a fragment expression vector (pUC19-PBP1A), identified by double digestion sequencing, the sequence of the recombinant plasmid is correct gene insertion in the right direction. Successfully induced Streptococcus pneumoniae competent, transforming the pUC19-pbp1a plasmid, PCR amplification and sequencing correct sequence in pbp1a, no mutation. And measuring the resistance of the transformed bacteria changed. Conclusions: 1. Streptococcus pneumoniae clinical isolates pbp2b, and pbp1a gene mutation and its resistant to penicillin is closely related to different types of mutations related to the degree of penicillin-resistant. 2. Successfully constructed the pUC19-pbp1a expression plasmid. 3. Improved sensitivity of recombinant bacteria to penicillin, confirmed pbp1a mutation may aggravate pbp2b resistance mutations.