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Identification of bacteria directly from positive blood culture samples by DNA pyrosequencing of the 16S rRNA gene


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Title: Identification of bacteria directly from positive blood culture samples by DNA pyrosequencing of the 16S rRNA gene
Authors: Motoshima, Maiko / Yanagihara, Katsunori / Morinaga, Yoshitomo / Matsuda, Junichi / Hasegawa, Hiroo / Kohno, Shigeru / Kamihira, Shimeru
Issue Date: Nov-2012
Publisher: Society for General Microbiology
Citation: Journal of Medical Microbiology, 61(11), pp.1556-1562; 2012
Abstract: Rapid identification of the causative bacteria of sepsis in patients can contribute to the selection of appropriate antibiotics and improvement of patients' prognosis. Genotypic identification is an emerging technology that may provide an alternative method to, or complement, established phenotypic identification procedures. We evaluated a rapid protocol for bacterial identification based on PCR and pyrosequencing of the V1 and V3 regions of the 16S rRNA gene using DNA extracted directly from positive blood culture samples. One hundred and two positive blood culture bottles from 68 patients were randomly selected and the bacteria were identified by phenotyping and pyrosequencing. The results of pyrosequencing identification displayed 84.3 and 64.7% concordance with the results of phenotypic identification at the genus and species levels, respectively. In the monomicrobial samples, the concordance between the results of pyrosequencing and phenotypic identification at the genus level was 87.0 %. Pyrosequencing identified one isolate in 60% of polymicrobial samples, which were confirmed by culture analysis. Of the samples identified by pyrosequencing, 55.7% showed consistent results in V1 and V3 targeted sequencing; other samples were identified based on the results of V1 (12.5 %) or V3 (31.8 %) sequencing alone. One isolate was erroneously identified by pyrosequencing due to high sequence similarity with another isolate. Pyrosequencing identified one isolate that was not detected by phenotypic identification. The process of pyrosequencing identification can be completed within ~4 h. The information provided by DNA-pyrosequencing for the identification of micro-organisms in positive blood culture bottles is accurate and could prove to be a rapid and useful tool in standard laboratory practice.
Keywords: RNA 16S / article / bacterium detection / bacterium identification / bacterium isolate / blood culture / DNA determination / human / major clinical study / nonhuman / phenotype / polymerase chain reaction / priority journal / pyrosequencing / species identification / Bacteria / Bacterial Infections / Bacteriological Techniques / Base Sequence / Candida albicans / Candidiasis / Diphosphates / DNA, Bacterial / DNA, Fungal / DNA, Ribosomal / Gene Expression Regulation, Bacterial / Humans / Polymerase Chain Reaction / RNA, Bacterial / RNA, Ribosomal, 16S / Sepsis / Sequence Analysis, DNA / Species Specificity
URI: http://hdl.handle.net/10069/31701
ISSN: 00222615
DOI: 10.1099/jmm.0.049163-0
Rights: © 2012 SGM.
Type: Journal Article
Text Version: author
Appears in Collections:Articles in academic journal

Citable URI : http://hdl.handle.net/10069/31701

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