Variable-Number Tandem-Repeat Analysis of Respiratory and Household Water Biofilm Isolates of "Mycobacterium avium subsp. hominissuis" with Establishment of a PCR Database.

"Mycobacterium aviumsubsp.hominissuis" is an important cause of pulmonary disease. It is acquired from environmental sources, but there is no methodology for large population studies. We evaluated the potential of variable-number tandem-repeat (VNTR) analysis. Clinical and household biofilmM. aviumisolates underwent molecular identification. Testing for IS901was done to separateM. aviumsubsp.aviumfromM. aviumsubsp.hominissuis VNTR types were defined using VNTR loci, and subtyping was performed using 3'hsp65and internal transcribed spacer (ITS) sequencing. Forty-nine VNTR types and eight subtypes ofM. aviumsubsp.hominissuis(IS901negative) were identified among 416 isolates ofM. aviumfrom 121 patients and 80 biofilm sites. Of those types, 67% were found only among patient isolates, 11% only among household water isolates, and 23% among both. Of 13 VNTR types that included ≥4 patients, the majority (61.5%) represented geographic clustering (same city). Most VNTR types with multiple patients belonged to the same 3'hsp65sequence code (sequevar). A total of 44 isolates belonging to fourM. aviumsubsp.hominissuisVNTR types (8%), including three with the rare Mav-F ITS sequence and 0/8 subspecies, produced amplicons with IS901PCR primers. By sequencing, all 44 amplicons were not IS901but ISMav6, which was recently observed in Japan but had not been previously described among U.S. isolates. VNTR analysis ofM. aviumsubsp.hominissuisisolates is easier and faster than pulsed-field gel electrophoresis. Seven VNTR loci separated 417 isolates into 49 types. No isolates ofM. aviumsubsp.aviumwere identified. The distributions of the VNTR copy numbers, the allelic diversity, and the low prevalence of ISMav6 differed from the findings for respiratory isolates reported from Japan.

Insertion site and distribution of a genomic island conferring DNA phosphorothioation in the Mycobacterium abscessus complex.

Nearly half of US clinical isolates of the emerging pathogen Mycobacterium abscessus were reported to exhibit smeared DNA during PFGE. This DNA degradation (Dnd) phenotype results from DNA phosphorothioation, a sulfur modification found in other bacteria and conferred by dnd genes located on mobile elements. Putative dnd genes are located on a 19.6 kbp genomic island (GI) in the M. abscessus type strain ATCC 19977. We confirmed that ATCC 19977(T) is Dnd-positive by PFGE and we developed a PCR assay to predict Dnd phenotype. Dnd-positive strains generated an amplicon from dndC whereas Dnd-negative strains generated a bridge amplicon that spanned the GI insertion site, indicating they lacked the entire 'Dnd-GI'. Comparative analyses of sequences from the bridge amplicon with ATCC 19977(T) revealed the Dnd-GI is flanked by 22 bp repeats in M. abscessus sensu stricto and inserted downstream of a tRNA-Ala gene and between inverted repeats. Regions flanking the Dnd-GI were highly conserved within the M. abscessus complex. Bioinformatics studies suggest the Dnd-GI inserted independently into a strain of Mycobacterium massiliense and that other species of mycobacteria also have dnd genes, supporting reports that the Dnd phenotype is common among actinomycetes. Within the M. abscessus complex, Dnd-positive clinical isolates were primarily M. abscessus sensu stricto, and tandem repeat typing indicated these isolates were highly related, confirming previous PFGE studies and revealing a widespread family of strains with significance in human disease.