Open Access Highly Accessed Methodology article

Accurate identification of fastidious Gram-negative rods: integration of both conventional phenotypic methods and 16S rRNA gene analysis

Maria G de Melo Oliveira12, Susanne Abels13, Reinhard Zbinden1, Guido V Bloemberg1 and Andrea Zbinden1*

Author Affiliations

1 Institute of Medical Microbiology, University of Zurich, 8006 Zurich, Switzerland

2 Present address, Policlínica Nascente, Prefeitura Municipal de Fortaleza, Fortalez-Ceará, Brasil

3 Present address, Institute for Hygiene and Public Health, University Hospital, 53105 Bonn, Germany

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BMC Microbiology 2013, 13:162  doi:10.1186/1471-2180-13-162

Published: 16 July 2013

Abstract

Background

Accurate identification of fastidious Gram-negative rods (GNR) by conventional phenotypic characteristics is a challenge for diagnostic microbiology. The aim of this study was to evaluate the use of molecular methods, e.g., 16S rRNA gene sequence analysis for identification of fastidious GNR in the clinical microbiology laboratory.

Results

A total of 158 clinical isolates covering 20 genera and 50 species isolated from 1993 to 2010 were analyzed by comparing biochemical and 16S rRNA gene sequence analysis based identification. 16S rRNA gene homology analysis identified 148/158 (94%) of the isolates to species level, 9/158 (5%) to genus and 1/158 (1%) to family level. Compared to 16S rRNA gene sequencing as reference method, phenotypic identification correctly identified 64/158 (40%) isolates to species level, mainly Aggregatibacter aphrophilus, Cardiobacterium hominis, Eikenella corrodens, Pasteurella multocida, and 21/158 (13%) isolates correctly to genus level, notably Capnocytophaga sp.; 73/158 (47%) of the isolates were not identified or misidentified.

Conclusions

We herein propose an efficient strategy for accurate identification of fastidious GNR in the clinical microbiology laboratory by integrating both conventional phenotypic methods and 16S rRNA gene sequence analysis. We conclude that 16S rRNA gene sequencing is an effective means for identification of fastidious GNR, which are not readily identified by conventional phenotypic methods.

Keywords:
Fastidious Gram-negative rods; 16S rRNA gene; Conventional phenotypic methods