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Identification of Abiotrophia adiacens and Abiotrophia defectiva by 16S rRNA gene PCR and restriction fragment length polymorphism analysis.

ABSTRACT: Abiotrophia adiacens and Abiotrophia defectiva, previously referred to as nutritionally variant streptococci, Streptococcus adjacens and Streptococcus defectivus, respectively, are causes of infective endocarditis. We describe a method of identifying these two species and also of distinguishing them from 15 other major etiological pathogens of infective endocarditis by means of 16S rRNA gene PCR amplification followed by restriction fragment length polymorphism analysis (PCR-RFLP). The 16S rRNA genes were successfully amplified with a set of universal primers from all 17 species of bacteria examined, including viridans group streptococci. The RFLP patterns of A. adiacens and A. defectiva obtained by HaeIII or MspI digestion were readily distinguished from each other and from those of other bacteria. When PCR analysis was performed with the supernatant of a suspension of a boiled colony, the 16S rRNA genes of 80 of 82 isolates (97%) of A. adiacens and all isolates (11 of 11) of A. defectiva were amplified. The HaeIII RFLP patterns of the isolates were the same as those of the corresponding type strains, although 28% of A. adiacens isolates revealed intraspecies polymorphism. The detection limit of this method was 0.1 pg of genomic DNA, as assessed by using the digoxigenin-labeling DNA detection system. Thus, the PCR-RFLP analysis that we developed is applicable for the routine detection of Abiotrophia from clinical specimens.

SUBMITTER: Ohara-Nemoto Y

PROVIDER: S-EPMC229992 | biostudies-other | 1997 Oct

REPOSITORIES: biostudies-other

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Identification of Abiotrophia adiacens and Abiotrophia defectiva by 16S rRNA gene PCR and restriction fragment length polymorphism analysis.

Ohara-Nemoto Y Y Tajika S S Sasaki M M Kaneko M M

Journal of clinical microbiology 19971001 10

Abiotrophia adiacens and Abiotrophia defectiva, previously referred to as nutritionally variant streptococci, Streptococcus adjacens and Streptococcus defectivus, respectively, are causes of infective endocarditis. We describe a method of identifying these two species and also of distinguishing them from 15 other major etiological pathogens of infective endocarditis by means of 16S rRNA gene PCR amplification followed by restriction fragment length polymorphism analysis (PCR-RFLP). The 16S rRNA ...[more]

PMID: 9316889

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Project description:ObjectiveBacteremia due to lactobacilli is uncommon, yet it is increasing in frequency, especially among immunosuppressed patients. In the clinical laboratory, lactobacilli must be subcultured from positive blood cultures before identification by traditional biochemical methods. Delays in diagnosis are significant because the organisms are inherently resistant to vancomycin, a drug frequently prescribed for empiric therapy for gram-positive bacteremia. Recently, we developed a rapid terminal-restriction fragment length polymorphism (T-RFLP) diagnostic assay based on species-specific variations in the bacterial 16S rRNA gene. We sought to apply this technique to the identification of Lactobacillus spp. from three cases of bacteremia.DesignThe results of the T-RFLP analysis are compared with two standard biochemical identification methods.MethodsLactobacillus strains were isolated from positive clinical blood cultures. Initial suspect cultures were subcultured and characterized using an automated substrate hydrolysis system and Lactobacillus carbohydrate fermentation profiles. Further biochemical and molecular analyses were performed from isolates propagated in Lactobacillus MRS broth. DNA was extracted and the 16S rRNA gene sequenced. Two sets of fluorescent labeled primers targeting the 16S rRNA gene were used for polymerase chain reaction (PCR) with chromosomal preparations from reference strains and blood isolates. The PCR products were digested with restriction enzymes and terminal-restriction fragment profile analysis performed.ResultsT-RFLP analysis correctly identified the Lactobacillus species in each case. T-RFLP analysis could be completed within 8 hours of obtaining a positive blood culture as compared to more than the 24 to 48 hours required for traditional culturing and biochemical characterizations.ConclusionT-RFLP analysis allows for rapid identification of Lactobacillus directly from positive blood cultures and circumvents the requirement for subculture. Reduced diagnostic time has implications for duration of infection, the cost of patient care, length of hospitalization, development of broad-spectrum antibiotic resistance, and mortality due to bacteremia. T-RFLP profiling represents a highly reproducible and predictive source for identification of many organisms associated with bacteremia.

Dataset Information

Identification of Abiotrophia adiacens and Abiotrophia defectiva by 16S rRNA gene PCR and restriction fragment length polymorphism analysis.

Publications

Identification of Abiotrophia adiacens and Abiotrophia defectiva by 16S rRNA gene PCR and restriction fragment length polymorphism analysis.

Similar Datasets

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