| Summary: | 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 (Gln457His), closed to the homology box Ser442SerAsn was
frequently observed in the resistant isolates. The particular Thr445Ala
mutation of S. pneumoniae seems to be not necessarily present in S. oralis.
In PBP 2x, Asn417Lys and Asp 506Glu 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.
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