Burden of unidentifiable mycobacteria in a reference laboratory

Abstract

Modern identification techniques at the genomic level have greatly improved the taxonomic knowledge of mycobacteria. In adjunct to nucleic acid sequences, mycobacterial identification has been endorsed by investigation of the lipidic patterns of unique mycolic acids in such organisms. In the present investigation, the routine use of high-performance liquid chromatography (HPLC) of mycolic acids, followed by the sequencing of the 16S rRNA, allowed us to select 72 mycobacterial strains, out of 1,035 screened, that do not belong to any of the officially recognized mycobacterial species. Most strains (i.e., 47) were isolated from humans, 13 were from the environment, 3 were from animals, and 9 were from unknown sources. The majority of human isolates were grown from the respiratory tract and were therefore most likely not clinically significant. Some, however, were isolated from sterile sites (blood, pleural biopsy, central venous catheter, or pus). Many isolates, including several clusters of two or more strains, mostly slow growers and scotochromogenic, presented unique genetic and lipidic features. We hope the data reported here, including the results of major conventional identification tests, the HPLC profiles of strains isolated several times, and the whole sequences of the 16S rRNA hypervariable regions of all 72 mycobacteria, may encourage reporting of new cases. The taxonomy of the genus Mycobacterium is, in our opinion, still far from being fully elucidated, and the reporting of unusual strains provides the best background for the recognition of new species. Our report also shows the usefulness of the integration of novel technology to routine diagnosis, especially in cases involving slow-growing microorganisms such as mycobacteria.

FIG. 1

Mycolic acid profile in HPLC of several unidentified mycobacteria isolated more than once. LMMIS is the low-molecular-mass internal standard. HMMIS is the high-molecular-mass internal standard.

FIG. 2

Unusual mycolic acid profiles in HPLC of several unidentified mycobacteria. LMMIS is the low-molecular-mass internal standard. HMMIS is the high-molecular-mass internal standard.

FIG. 3

Sequences of hypervariable regions A and B within the 16S rRNA of 72 unidentified mycobacteria. Positions are indicated by E. coli alignment. Only nucleotides different from M. tuberculosis are shown. Dashes indicate deletions. Groups: i, M. terrae-like; ii, thermotolerant rapid growers; iii, “historical” slow growers; iv, M. simiae-like; v, rapid growers.

FIG. 3

Sequences of hypervariable regions A and B within the 16S rRNA of 72 unidentified mycobacteria. Positions are indicated by E. coli alignment. Only nucleotides different from M. tuberculosis are shown. Dashes indicate deletions. Groups: i, M. terrae-like; ii, thermotolerant rapid growers; iii, “historical” slow growers; iv, M. simiae-like; v, rapid growers.

FIG. 4

Phylogenetic tree of 16S rRNAs of the 72 unidentified mycobacteria and of the major known species.