A simple chemical test to distinguish mycobacteria from other mycolic-acid-containing actinomycetes

Abstract

Two hundred and fifty-two representatives of the genera Cuvynebacteuium, Gorduna, Mycobactevium, Nocardia, Rkoducuccus and Tsukamurella were degraded by alkaline hydrolysis and their mycolic acids extracted as methyl esters following phase-transfer-catalysed esterification. When the mycolic acid methyl esters were treated with a mixture of acetonitrile and toluene all mycobacterial mycolates formed copious white precipitates whereas all but 5 out of the 106 non-mycobacterial mycolates remained in solution. The precipitated methyl mycolates and the dried soluble mycolates were compared by pyrolysis gas chromatography and silica gel thin-layer chromatography. O n pyrolysis, the precipitated methyl mycolates from mycobacteria yielded fatty acid methyl esters with 20 to 26 carbon atoms whereas those from the remaining taxa produced shorter-chain esters. Mycobacteria and Tsukamurella paurometabolu gave multispot mycolic acid patterns on thin-layer chromatography of their methyl esters whereas those from the remaining strains gave single spots. Our results indicate that Rhodococcus chlouophanolicus strains contain mycolic acids typical of mycabacteria, It can be concluded that the mycolic acid precipitation test provides a simple and reliable way of distinguishing mycobacteria from all other prokaryotes, notably from other mycolic-acid-containing taxa.

The medically important aerobic actinomycetes: epidemiology and microbiology

“…In addition, such hydrolysates may contain diagnostic sugars, which are also of diagnostic value in identification. For routine work, the simplified techniques of Becker et al (46) (239). Mycolic acid methyl esters can be identified on thin-layer chromatograms because they are not removed when plates are subsequently washed with methanol-water (5:2 [vol/vol]) (393,398).…”

“…Tsukamurella wratislaviensis strains produced single spots with Rf values comparable to those of the single spots produced by gordonae and nocardiae. The corynebacteria and rhodococci also produced single spots, with slightly lower RJ values (239). Although the nonpathogenic nocardiae-N. amarae, N. brevicatena, N. camea, and Nocardia vaccinii-have chemotaxonomic properties in common with the pathogenic species assigned to the genus Nocardia, the nonpathogenic species can be distinguished from the pathogenic species by use of a combination of biochemical and morphologic properties (330).…”

“…by quantitative mycolic acid analysis done by high-performance liquid chromatography (11,88,239). By combining information from this chemotaxonomic marker with other key characteristics, including arylsulfatase production and metabolism of glucose, differentiation can be made among Rhodococcus, Mycobacterium, Corynebacterium, Gordona, and Tsukamurella organisms (Table 4).…”

“…(560). The species assigned to the genus Gordona include the former Rhodococcus bronchialis, Rhodococcus rubropertinctus, Rhodococcus sputi, and Rhodococcus terrae, which contain mycolic acids with between 48 and 66 carbon atoms and large amounts of a dihydrogenated menaquinone with nine isoprene units (239). The remaining Rhodococcus spp.…”

“…The remaining Rhodococcus spp. contain shorter-chain mycolic acids (C34 to C52) and a dihydrogenated menaquinone with eight isoprene units as the major isoprenoic quinone (119,239). Of interest, it was recently shown that Rhodococcus chubuensis, listed as a species incertae sedis in Bergey's Manual ofSystematic Bacteriology (198), contains a mycolic acid pattern similar to that of G. bronchialis (134).…”