Two-laboratory collaborative study on identification of mycobacteria: molecular versus phenotypic methods

Abstract

Previous studies have indicated that the conventional tests used for the identification of mycobacteria may (i) frequently result in erroneous identification and (ii) underestimate the diversity within the genus Mycobacterium. To address this issue in a more systematic fashion, a study comparing phenotypic and molecular methods for the identification of mycobacteria was initiated. Focus was given to isolates which were difficult to identify to species level and which yielded inconclusive results by conventional tests performed under day-today routine laboratory conditions. Traditional methods included growth rate, colonial morphology, pigmentation, biochemical profiles, and gas-liquid chromatography of short-chain fatty acids. Molecular identification was done by PCR-mediated partial sequence analysis of the gene encoding the 16S rRNA. A total of 34 isolates was included in this study; 13 of the isolates corresponded to established species, and 21 isolates corresponded to previously uncharacterized taxa. For five isolates, phenotypic and molecular analyses gave identical results. For five isolates, minor discrepancies were present; four isolates remained unidentified after biochemical testing. For 20 isolates, major discrepancies between traditional and molecular typing methods were observed. Retrospective analysis of the data revealed that the discrepant results were without exception due to erroneous biochemical test results or interpretations. In particular, phenotypic identification schemes were compromised with regard to the recognition of previously undescribed taxa. We conclude that molecular typing by 16S rRNA sequence determination is not only more rapid (12 to 36 h versus 4 to 8 weeks) but also more accurate than traditional typing.

Identification ofMycobacteriumspecies by comparative analysis of thednaAgene

“…Several methods based on molecular biological techniques have been reported. The sequences that have been reported include hsp65, 16S rRNA gene, and ITS (Plikaytis et al, 1992;De Smet et al, 1995;Springer et al, 1996;Messer & Weigel, 1997;Roth et al, 1998;Brunello et al, 2001). Each gene has several advantages and disadvantages.…”

A glimpse into the past and predictions for the future: the molecular evolution of the tuberculosis agent

“…The hundreds of mycobacterial species are mostly harmless saprophytes, which is especially true for the so-called fast growing mycobacteria that contain only a handful of opportunistic pathogens, such as Mycobacterium abscessus (Ripoll et al, 2009). The slow growing mycobacteria, in contrast, form a subcluster in the 16S rRNA tree (Springer et al, 1996) and harbour major human and animal pathogens, whose phenotypic characteristics are summarized in a recently updated edition of Bergey's Manual of Systematic Bacteriology (Magee and Ward, 2012). With an average generation time of 20-24 h M. tuberculosis (Cole et al, 1998) is part of this subcluster that also includes the leprosy bacillus Mycobacterium leprae (Cole et al, 2001), the agent of Buruli ulcer Mycobacterium ulcerans (Stinear et al, 2007), the fish pathogen Mycobacterium marinum (Stinear et al, 2008), the agent of Johne's disease Mycobacterium avium paratuberculosis (Li et al, 2005) or the opportunistic human pathogen Mycobacterium kansasii (Veyrier et al, 2011).…”

The influence of culture conditions on the identification ofMycobacteriumspecies by MALDI-TOF MS profiling

“…Conventional methods for mycobacterial identification and species differentiation rely on their biochemical profiles and phenotypic traits, such as morphological features and growth rates. However, identification based on these phenotypic features may, quite often, result in erroneous identification (Springer et al, 1996).…”