Identification of mycobacteria by high-performance liquid chromatography

Abstract

Mycolic acids extracted from saponified mycobacterial cells were examined as p-bromophenacyl esters by high-performance liquid chromatography (HPLC). Standard HPLC patterns were developed for species of Mycobacterium by examination of strains from culture collections and other well-characterized isolates. Relative retention times of peaks and peak height comparisons were used to develop a differentiation scheme that was 98% accurate for the species examined. A rapid, cost-effective HPLC method which offers an alternative approach to the identification of mycobacteria is described.

Influence of the growth substrate on the mycolic acid profiles of mycobacteria

“…Furthermore, as mycobacteria appear to exhibit substrate-or substrate-class-dependent mycolic acid compositions, HPLC-based mycolic acid profiles can be used as diagnostic tools for the characterization of multiple substrate utilization in laboratory test systems. Our study also points out that the nature of the growth substrate is of critical importance for mycolic acid profiling as proposed method for the identification of Mycobacterium species as well as other actinomycetes (Butler and Kilburn, 1990;Butler et al ., 1991;Ritter et al ., 1996). (1) Cells grown on alkanes (depicted with open symbols; C w sat = 0.9-52 mg l -1 ) exhibited higher average retention times than cells grown on PAHs (C w sat = 62-980 mg l -1 ), glucose (C w sat = 9.1*10 8 mg l -1 ) or LB-broth [C w sat ª 1*10 8 mg l -1 ; C w sat was approximated at room temperature by gradual addition of LB broth (Fluka, Switzerland) to 20 ml of water and subsequent spectrophotometric analysis of the turbidity of the solution].…”

Lipoprotein processing is required for virulence of Mycobacterium tuberculosis†

“…For mycolic acid analyses by HPLC, M. tuberculosis was lyophilized, and the lyophilized cells (20 mg) were saponified by KOH. The free mycolic acids were extracted, transferred to their bromophenacyl esters and analysed by HPLC as described previously (Butler et al, 1991;Miller, 1997;Tortoli et al, 2001).…”

The Genomic Heterogeneity among Mycobacterium terrae Complex Displayed by Sequencing of 16S rRNA and hsp65 Genes

“…Other methods such as thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) are more powerful, but are cumbersome, expensive and limited by the need for standardized growth conditions. Currently, these methods are used in very few clinical laboratories (1,5,8).In addition to these phenotypic methods, genotypic and phylogenetic methods were introduced for polyphasic taxonomy and complete identification of microor-Abstract: The species identification within Mycobacterium terrae complex has been known to be very difficult. In this study, the genomic diversity of M. terrae complex with eighteen clinical isolates, which were initially identified as M. terrae complex by phenotypic method, was investigated, including that of three type strains (M. terrae, M. nonchromogenicum, and M. triviale).…”

“…Other methods such as thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) are more powerful, but are cumbersome, expensive and limited by the need for standardized growth conditions. Currently, these methods are used in very few clinical laboratories (1,5,8).…”