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Molecular basis of rifampin resistance in Streptococcus pneumoniae.

ABSTRACT: Rifampin resistance among South African clinical isolates of Streptococcus pneumoniae was shown to be due to missense mutations within the rpoB gene. Sequence analysis of 24 rifampin-resistant isolates revealed the presence of mutations within cluster I as well as novel mutations in an area designated pneumococcus cluster III. Of the 24 isolates characterized, only 1 resistant isolate did not contain any mutations in the regions sequenced. Either the cluster I or the cluster III mutations separately conferred MICs of 32 to 128 microg/ml. Clinical isolate 55, for which the MIC was 256 microg/ml, was noted to contain 9 of the 10 mutations identified, which included the cluster I and cluster III mutations. As in Escherichia coli, it is possible that cluster I (amino acids 406 to 434) and cluster III (amino acids 523 to 600) of S. pneumoniae interact to form part of the antibiotic binding site, thus accounting for the very high MIC observed for isolate 55. PCR products containing cluster I or cluster III mutations were able to transform rifampin-susceptible S. pneumoniae to resistance. Although many of the isolates studied displayed identical sequences, pulsed-field gel electrophoresis analysis revealed that the isolates were not of clonal origin.

SUBMITTER: Padayachee T

PROVIDER: S-EPMC89483 | biostudies-literature | 1999 Oct

REPOSITORIES: biostudies-literature

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Molecular basis of rifampin resistance in Streptococcus pneumoniae.

Padayachee T T Klugman K P KP

Antimicrobial agents and chemotherapy 19991001 10

Rifampin resistance among South African clinical isolates of Streptococcus pneumoniae was shown to be due to missense mutations within the rpoB gene. Sequence analysis of 24 rifampin-resistant isolates revealed the presence of mutations within cluster I as well as novel mutations in an area designated pneumococcus cluster III. Of the 24 isolates characterized, only 1 resistant isolate did not contain any mutations in the regions sequenced. Either the cluster I or the cluster III mutations separa ...[more]

PMID: 10508007

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Project description:Ninety-two Streptococcus pneumoniae serotypes have been described so far, but the pneumococcal conjugate vaccine introduced in the Brazilian basic vaccination schedule in 2010 covers only the ten most prevalent in the country. Pneumococcal serotype-shifting after massive immunization is a major concern and monitoring this phenomenon requires efficient and accessible serotyping methods. Pneumococcal serotyping based on antisera produced in animals is laborious and restricted to a few reference laboratories. Alternatively, molecular serotyping methods assess polymorphisms in the cps gene cluster, which encodes key enzymes for capsular polysaccharides synthesis in pneumococci. In one such approach, cps-RFLP, the PCR amplified cps loci are digested with an endonuclease, generating serotype-specific fingerprints on agarose gel electrophoresis.In this work, in silico and in vitro approaches were combined to demonstrate that XhoII is the most discriminating endonuclease for cps-RFLP, and to build a database of serotype-specific fingerprints that accommodates the genetic diversity within the cps locus of 92 known pneumococci serotypes.The expected specificity of cps-RFLP using XhoII was 76% for serotyping and 100% for serogrouping. The database of cps-RFLP fingerprints was integrated to Molecular Serotyping Tool (MST), a previously published web-based software for molecular serotyping. In addition, 43 isolates representing 29 serotypes prevalent in the state of Minas Gerais, Brazil, from 2007 to 2013, were examined in vitro; 11 serotypes (nine serogroups) matched the respective in silico patterns calculated for reference strains. The remaining experimental patterns, despite their resemblance to their expected in silico patterns, did not reach the threshold of similarity score to be considered a match and were then added to the database.The cps-RFLP method with XhoII outperformed the antisera-based and other molecular serotyping methods in regard of the expected specificity. In order to accommodate the genetic variability of the pneumococci cps loci, the database of cps-RFLP patterns will be progressively expanded to include new variant in vitro patterns. The cps-RFLP method with endonuclease XhoII coupled with MST for computer-assisted interpretation of results may represent a relevant contribution to the real time detection of changes in regional pneumococci population diversity in response to mass immunization programs.

Dataset Information

Molecular basis of rifampin resistance in Streptococcus pneumoniae.

Publications

Molecular basis of rifampin resistance in Streptococcus pneumoniae.

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