We describe here the characterization of five isolates of Mycobacterium simiae-like organisms representing a novel group based on whole-cell fatty acid analysis and genotypic evaluation. Two of the five isolates in this study, W55 and W58, were previously considered to belong to M. simiae serotype 2. Analysis of cellular fatty acids by gas-liquid chromatography indicated a close clustering of this group, which was well differentiated from the other M. simiae-like species. Molecular characterization was performed by nucleic acid sequencing of the small subunit rRNA gene and the gene encoding the 65-kDa heat shock protein and genomic DNA hybridization. Sequence analysis of the entire 16S rRNA gene showed a unique sequence most closely related to those of M. triplex and M. simiae. The hsp65 partial gene sequence was identical for the five isolates, with 97% identity to the M. simiae type strain. However, qualitative whole genomic DNA hybridization analysis confirmed that this group is genetically distinct from M. simiae and M. triplex. Antimicrobial susceptibilities for this group resemble those of M. simiae and M. lentiflavum. We conclude that this group represents a unique Mycobacterium species for which we propose the name Mycobacterium sherrisii sp. nov.The genus Mycobacterium consists of a diverse group of acidfast bacilli that exist in the environment and cause infections in humans and animals (22). Conventional methods for identification of these organisms are well established and inexpensive, but these methods are also time-consuming and labor-intensive and can provide inconsistent results, leading to species identifications based on a "best-fit" method. The introduction of several molecular tools and improved techniques in the last two decades has not only revolutionized the identification process by reducing the time to identification but also has provided clues to the presence of novel strains. Mycolate analysis has proven to be a powerful phenotypic method for determining the presence of new species based on novel patterns (9, 25). Fatty acid analysis techniques such as high-performance liquid chromatography (HPLC) and gas-liquid chromatography (GLC) were developed to analyze the mycolic acid and whole-cell fatty acid profiles, respectively, to identify characteristic patterns of established mycobacterial species (7,10,12,30).The advent of nucleotide probes designed to hybridize to specific rRNA gene sequences allowed rapid identification of the most common mycobacterial species (16). Lately, genotypic analyses aimed at identifying nucleotide sequences of several chromosomal genes in the Mycobacterium genus coding for 16S rRNA, internal transcribed spacer 1, and 65-kDa heat shock protein (hsp65) have proven to be useful in the molecular characterization of well-established species and in identifying new ones, including previously misidentified groups (8,15,17,26). Analysis of the hypervariable region in the 16S rRNA gene sequence has a high species specificity and also has been useful to assign new spec...

A distinct group of slowly growing mycobacteria was identified on the basis of growth characteristics, biochemical and lipid profiles, and nucleic acid analyses. The isolates showed growth at 22 to 37؇C, yellow pigmentation, and negative tests for Tween 80 hydrolysis, nicotinic acid, nitrate reductase, and urease; tests for arylsulfatase, pyrazinamidase, and heat-stable catalase were variable. Analysis of cellular fatty acids by gas-liquid chromatography and mycolic acids by thin-layer chromatography and high-performance liquid chromatography indicated a distinctive pattern which was unlike those of other species. Determination of the 16S rRNA gene sequence showed a unique sequence closely related to Mycobacterium simiae and M. genavense. On the basis of DNA homology studies, we suggest that these organisms are representatives of a novel species, for which the name M. lentiflavum sp. nov. is proposed.