GENETIC STUDIES OF PENICILLIN-BINDING PROTEIN GENES FROM STREPTOCOCCUS ORALIS

• Main Authors: HUANG, MIN-SE, 黃敏瑟
• Other Authors: LEE-JENE TENG
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/37409829581557291712

碩士 === 國立臺灣大學 === 醫事技術學系 === 86 === Viridans group streptococci are members of the normal mouth flora and are associated with subacute bacterial endocarditis (SBE). Among them, Streptococcus oralis has been found to be one of the major pathogens. Penicillin treatment is considered as the first-choice antimicrobial drug. However, the appearance and spread of penicillin- resistant isolates become a worldwide problem. Resistance to -lactam in both viridans group streptococci and pneumococcus has been shown to be due to the development of altered penicillin-binding proteins (PBPs) with lowered affinity for penicillin. Horizontal transfer of these variant PBP genes enables bacteria to evolve rapidly by the acquisition of novel determinants of resistance. So far, correlation between PBP genes and resistance to -lactam in S. oralis is still unresolved. We studied the thirty-four clinical isolates of S. oralis (MICs, 0.032 to 6 μg/ml) obtained from National Taiwan University Hospital (NTUH). Species identification was rechecked with both API rapid ID32 STREP system and 16S rRNA gene. The polymerase chain reaction was used to amplify the penicillin-binding domain of PBPs 2b and 2x from the chromosomal DNAs of the bacteria. PCR products were then digested with restriction enzymes. On the electrophoretic profiling of PBPs 2b and 2x genes, the DNA fingerprintings of S. oralis were found to be highly heterogeneous. They were quite different from those obtained from S. pneumoniae which has been studied. Cloning and sequencing were also performed to analyze the nucleotides and amino acid composition around the homology boxes on PBP genes. In the case of PBP 2b, a substitution of Gln by His at position 457 (Gln457His), closed to the homology box Ser442SerAsn was frequently observed in the resistant isolates. The particular Thr445Ala mutation of S. pneumoniae seems to be not necessarily present in S. oralis. In PBP 2x, Asn417Lys and Asp 506Glu were found between the homology box Ser395SerAsn and Lys547SerGly. It remains to be clarified whether these substitutions effect a change in PBP affinity. Our results show that pbp fingerprinting in clinical isolates was almost different with each strain. Some isolates showing indistinguishable pbp patterns were further studied by genomic typing method for whole chromosome. RAPD (random amplified polymorphic DNA) with three different primers was performed with clinical isolates. RAPD from unrelated isolates was found very heterogenous. However, both pbp fingerprinting and RAPD patterns of isolates recovered from same individual were the same. The emergence of phenotypic variation in clinical isolates is another problem which often confused with species identification in clinical laboratory. Two types of colonies derived from one pure culture were observed. They had distinguishable colonial appearance. However, same genetic backgrounds were observed.