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

• Published in: Journal of medical microbiology Vol. 61; no. 11; pp. 1556 - 1562
• Main Authors: MOTOSHIMA, Maiko, YANAGIHARA, Katsunori, MORINAGA, Yoshitomo, MATSUDA, Junichi, HASEGAWA, Hiroo, KOHNO, Shigeru, KAMIHIRA, Shimeru
• Format: Journal Article
• Language: English
• Published: Reading Society for General Microbiology 01.11.2012
• Subjects: Bacteria - classification; Bacteria - genetics; Bacteria - isolation & purification; Bacterial Infections - diagnosis; Bacterial Infections - microbiology; Bacteriological Techniques; Bacteriology; Base Sequence; Biological and medical sciences; Candida albicans - genetics; Candida albicans - isolation & purification; Candidiasis - diagnosis; Candidiasis - microbiology; Diphosphates - isolation & purification; Diphosphates - metabolism; DNA, Bacterial - chemistry; DNA, Fungal - chemistry; DNA, Ribosomal - chemistry; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial - physiology; Humans; Infectious diseases; Medical sciences; Microbiology; Miscellaneous; Polymerase Chain Reaction - methods; RNA, Bacterial - genetics; RNA, Ribosomal, 16S - genetics; Sepsis - microbiology; Sequence Analysis, DNA - methods; Species Specificity; 16S-RNA; Bacteria; Identification; Blood culture; Ribosomal DNA
• DOI: 10.1099/jmm.0.049163-0
• ISSN: 0022-2615

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.