Novel mycolic acid-containing bacteria in the family Segniliparaceae fam. nov., including the genus Segniliparus gen. nov., with descriptions of Segniliparus rotundus sp. nov. and Segniliparus rugosus sp. nov.

Four strains of novel, rapidly growing, acid-alcohol-fast-staining bacteria were characterized with a polyphasic approach. Isolates were received by the Centers for Disease Control and Prevention from domestic health department laboratories for reference testing as unidentifiable, clinical mycobacteria. Bacteria were rod-shaped and produced non-pigmented (white to beige), non-photochromogenic, smooth or wrinkled-rough colonies on Middlebrook 7H10 and 7H11 media at 33 degrees C. The smooth and wrinkled colony forms were representative of two species with 68.0 and 72.0 mol% DNA G+C content. The cell wall contained meso-diaminopimelic acid and mycolic acids. Species were characterized by cellular fatty acids of C10:0, C14:0, C16:1omega9t, C16:0, C18:1omega9c and 10-methyl C18:0 (tuberculostearic acid). HPLC analysis of mycolic acids produced a novel late-emerging, genus-specific mycolate pattern. TLC analysis demonstrated a novel alpha(+)-mycolate. Species were 98.9% similar by comparison of 16S rRNA gene sequences; however, the DNA-DNA association was <28 %. Phylogenetic analysis of 16S rRNA gene sequences demonstrated an association with Rhodococcus equi, although a DNA-DNA relatedness value of 2% did not support a close relationship. PCR analysis of a proposed, selected actinomycete-specific 439 bp fragment of the 65 kDa heat-shock protein was negative for three of the four isolates. The creation of Segniliparaceae fam. nov. is proposed to encompass the genus Segniliparus gen. nov., including two novel species, the type species Segniliparus rotundus sp. nov. and Segniliparus rugosus sp. nov., with the respective type strains CDC 1076(T) (=ATCC BAA-972(T)=CIP 108378(T)) and CDC 945(T) (=ATCC BAA-974(T)=CIP 108380(T)).

Taxonomic variation in the Mycobacterium fortuitum third biovariant complex: description of Mycobacterium boenickei sp. nov., Mycobacterium houstonense sp. nov., Mycobacterium neworleansense sp. nov. and Mycobacterium brisbanense sp. nov. and recognition of Mycobacterium porcinum from human clinical isolates.

The Mycobacterium fortuitum third biovariant complex (sorbitol-negative and sorbitol-positive) contains unnamed taxa first characterized in 1991. These organisms can cause respiratory infections, a spectrum of soft tissue and skeletal infections, bacteraemia and disseminated disease. To evaluate this group of organisms, clinical reference isolates and the type strains of M. fortuitum third biovariant complex sorbitol-negative (n = 21), M. fortuitum third biovariant complex sorbitol-positive (n = 3), M. fortuitum (n = 3), Mycobacterium peregrinum (pipemidic acid-susceptible) (n = 1), Mycobacterium porcinum (n = 1), Mycobacterium senegalense (n = 2) and Mycobacterium septicum (n = 1) were characterized by using conventional phenotypic (morphological, physiological and antimicrobial susceptibilities), chemotaxonomic (HPLC and cellular fatty acids) and genotypic [RFLP of the rRNA gene (ribotyping), PCR-RFLP of a 439 bp segment of the 65 kDa hsp gene (PCR restriction analysis) and 16S rRNA gene sequence] analysis, DNA G + C content and DNA-DNA relatedness analyses. The results of these studies indicated that the strains comprised M. porcinum (n = 13), M. septicum (n = 1) and four novel closely related genetic groups within the M. fortuitum third biovariant complex: Mycobacterium boenickei sp. nov. (n = 6), Mycobacterium houstonense sp. nov. (n = 2), Mycobacterium neworleansense sp. nov. (n = 1) and Mycobacterium brisbanense sp. nov. (n = 1), with type strains ATCC 49935T (= W5998T = DSM 44677T), ATCC 49403T (= W5198T = DSM 44676T) ATCC 49404T (= W6705T = DSM 44679T) and ATCC 49938T (= W6743T = DSM 44680T), respectively.

Characterization of a novel rapidly growing Mycobacterium species associated with sepsis.

A rapidly growing mycobacterium was isolated five times from blood cultures from a 6-year-old female patient with relapsed pre-B-cell acute lymphocytic leukemia. All five isolates had identical nucleotide sequences for the first 500 bp of the 16S rRNA gene, indicative of a single species. High-performance liquid chromatography analysis of mycolic acids indicated that the species was similar to Mycobacterium smegmatis. Sequence analysis of the 16S rRNA gene (1,455 bp) for one isolate demonstrated that the species was closely related to Mycobacterium diernhoferi. Based on the phenotypic features and phylogenetic analysis, it was concluded that the isolates represented a novel rapidly growing Mycobacterium species. The name "Mycobacterium hackensackense" is proposed for this unique strain, 147-0552(T), which was deposited in the American Type Culture Collection as ATCC BAA-823(T).

Mycobacterium shottsii sp. nov., a slowly growing species isolated from Chesapeake Bay striped bass (Morone saxatilis).

Slowly growing, non-pigmented mycobacteria were isolated from striped bass (Morone saxatilis) during an epizootic of mycobacteriosis in the Chesapeake Bay. Growth characteristics, acid-fastness and results of 16S rRNA gene sequencing were consistent with those of the genus Mycobacterium. A unique profile of biochemical reactions was observed among the 21 isolates. A single cluster of eight peaks identified by analysis of mycolic acids (HPLC) resembled those of reference patterns but differed in peak elution times from profiles of reference species of the Mycobacterium tuberculosis complex. One isolate (M1 75T) was placed within the slowly growing mycobacteria by analysis of aligned 168S rRNA gene sequences and was proximate in phylogeny to Mycobacterium ulcerans and Mycobacterium marinum. However, distinct nucleotide differences were detected in the 16S rRNA gene sequence among M175T, M. ulcerans and M. marinum (99.2% similarity). Isolate M175T could be differentiated from other slowly growing, non-pigmented mycobacteria by its inability to grow at 37 degrees C, production of niacin and urease, absence of nitrate reductase and resistance to isoniazid (1 microg ml(-1)), thiacetazone and thiophene-2-carboxylic hydrazide. Based upon these genetic and phenotypic differences, isolate M175T (=ATCC 700981T =NCTC 13215T) is proposed as the type strain of a novel species, Mycobacterium shottsii sp. nov.