bThe erm(41) gene confers inducible macrolide resistance in Mycobacterium abscessus subsp. abscessus, calling into question the usefulness of macrolides for treating M. abscessus subsp. abscessus infections. With an extended incubation (14 days), isolates with MICs of >8 g/ml are considered macrolide resistant by current CLSI guidelines. Our goals were to determine the incidence of macrolide susceptibility in U.S. isolates, the validity of currently accepted MIC breakpoints, and the erm(41) sequences associated with susceptibility. Of 349 isolates (excluding those with 23S rRNA gene mutations), 85 (24%) had clarithromycin MICs of <8 g/ml. Sequencing of the erm(41) genes from these isolates, as well as from isolates with MICs of >16 g/ml, including ATCC 19977 T , revealed 10 sequevars. The sequence in ATCC 19977 T was designated sequevar (type) 1; most macrolide-resistant isolates were of this type. Seven sequevars contained isolates with MICs of >16 g/ml. The T28C substitution in erm(41), previously associated with macrolide susceptibility, was identified in 62 isolates (18%) comprising three sequevars, with MICs of <2 (80%), 4 (10%), and 8 (10%) g/ml. No other nucleotide substitution was associated with macrolide susceptibility. We recommend that clarithromycin susceptibility breakpoints for M. abscessus subsp. abscessus be changed from <2 to <4 g/ml and that isolates with an MIC of 8 g/ml have repeat MIC testing or erm sequencing performed. Our studies suggest that macrolides are useful for treating approximately 20% of U.S. isolates of M. abscessus subsp. abscessus. Sequencing of the erm gene of M. abscessus subsp. abscessus will predict inducible macrolide susceptibility.T hree closely related taxa of rapidly growing mycobacteria (RGM) with a controversial species/subspecies status, i.e., Mycobacterium abscessus subsp. abscessus, "M. abscessus subsp. massiliense," and M. abscessus subsp. bolletii, comprise the heterogeneous M. abscessus group (1, 2). A 2013 phylogenetic analysis based on genomic sequencing corroborated previous recommendations that the three taxa should be separated into three subspecies (3). Of these three subspecies, M. abscessus subsp. abscessus and M. abscessus subsp. bolletii, a relatively rare subspecies in the United States, demonstrate the presence of inducible macrolide resistance conferred by a novel erm gene, erm(41) (4).In contrast, the erm(41) gene is generally present in isolates of M. abscessus subsp. massiliense but contains a large, 397-bp deletion (that includes position 28T) that results in a nonfunctional erm(41) gene, and thus isolates do not show inducible macrolide resistance (2).In 2009, Nash and colleagues described two strains (MAB30 and MC1028) of M. abscessus subsp. abscessus which contained a nonfunctional erm gene (4); the strains were not inducibly resistant to clarithromycin, and even after extended incubation, they demonstrated susceptible clarithromycin MICs. The loss of function of the erm genes was associated with a T-to-C substitution at position 28 of th...

e Amikacin is a major drug used for the treatment of Mycobacterium avium complex (MAC) disease, but standard laboratory guidelines for susceptibility testing are not available. This study presents in vitro amikacin MICs for 462 consecutive clinical isolates of the MAC using a broth microdilution assay. Approximately 50% of isolates had amikacin MICs of 8 g/ml, and 86% had MICs of <16 g/ml. Of the eight isolates (1.7%) with MICs of 64 g/ml, five had an MIC of 32 g/ml on repeat testing. Ten isolates (2.1%) had an initial amikacin MIC of >64 g/ml, of which seven (1.5%) had MICs of >64 g/ml on repeat testing. These seven isolates had a 16S rRNA gene A1408G mutation and included M. avium, Mycobacterium intracellulare, and Mycobacterium chimaera. Clinical data were available for five of these seven isolates, all of which had received prolonged (>6 months) prior therapy, with four that were known to be treated with amikacin. The 16S mutation was not detected in isolates with MICs of <64 g/ml. We recommend primary testing of amikacin against isolates of the MAC and propose MIC guidelines for breakpoints that are identical to the CLSI guidelines for Mycobacterium abscessus: <16 g/ml for susceptible, 32 g/ml for intermediate, and >64 g/ml for resistant. If considered and approved by the CLSI, this will be only the second drug recommended for primary susceptibility testing against the MAC and should facilitate its use for both intravenous and inhaled drug therapies.

f Recent studies have shown that respiratory isolates from pulmonary disease patients and household water/biofilm isolates of Mycobacterium avium could be matched by DNA fingerprinting. To determine if this is true for Mycobacterium intracellulare, household water sources for 36 patients with Mycobacterium avium complex (MAC) lung disease were evaluated. MAC household water isolates from three published studies that included 37 additional MAC respiratory disease patients were also evaluated. Species identification was done initially using nonsequencing methods with confirmation by internal transcribed spacer (ITS) and/or partial 16S rRNA gene sequencing. M. intracellulare was identified by nonsequencing methods in 54 respiratory cultures and 41 household water/biofilm samples. By ITS sequencing, 49 (90.7%) respiratory isolates were M. intracellulare and 4 (7.4%) were Mycobacterium chimaera. In contrast, 30 (73%) household water samples were M. chimaera, 8 (20%) were other MAC X species (i.e., isolates positive with a MAC probe but negative with species-specific M. avium and M. intracellulare probes), and 3 (7%) were M. avium; none were M. intracellulare. In comparison, M. avium was recovered from 141 water/biofilm samples. These results indicate that M. intracellulare lung disease in the United States is acquired from environmental sources other than household water. Nonsequencing methods for identification of nontuberculous mycobacteria (including those of the MAC) might fail to distinguish closely related species (such as M. intracellulare and M. chimaera). This is the first report of M. chimaera recovery from household water. The study underscores the importance of taxonomy and distinguishing the many species and subspecies of the MAC. P revious studies have suggested household water (especially from bathroom showers) as a source of the Mycobacterium avium complex (MAC), which causes chronic lung disease (1, 2, 3, 4, 5). Both M. avium and M. intracellulare have been recovered from sputum, sinus, and household or potable water samples in multiple countries, including the United States and Japan (1, 4-8). In the majority of these studies, either hybridization probe methods (AccuProbe; Hologic Gen-Probe, Inc., San Diego, CA) or a multiplex 16S rRNA gene PCR was used for identification (1, 5, 9, 10). Isolates positive with the MAC probe but negative with the species-specific M. avium and M. intracellulare probes are collectively referred to as MAC X species.Currently We recently completed a study of variable-number tandemrepeat (VNTR) typing of clinical isolates of M. intracellulare using ITS sequencing to confirm the species designation (7). We then began household water biofilm studies for some of our patients and performed species identification and VNTR genotyping for MAC isolates as needed.Preliminary studies suggested that respiratory isolates of M. intracellulare indeed belonged to that species based on ITS sequencing (7) but that biofilm isolates thought to be M. intracellulare were actually M. chimaera ...

Lung disease caused by nontuberculous mycobacteria (NTM) is an emerging infectious disease of global significance. Epidemiologic studies have shown the Hawaiian Islands have the highest prevalence of NTM lung infections in the United States. However, potential environmental reservoirs and species diversity have not been characterized. In this cross-sectional study, we describe molecular and phylogenetic comparisons of NTM isolated from 172 household plumbing biofilms and soil samples from 62 non-patient households and 15 respiratory specimens. Although non-uniform geographic sampling and availability of patient information were limitations, Mycobacterium chimaera was found to be the dominant species in both environmental and respiratory specimens. In contrast to previous studies from the continental U.S., no Mycobacterium avium was identified. Mycobacterium intracellulare was found only in respiratory specimens and a soil sample. We conclude that Hawai’i’s household water sources contain a unique composition of Mycobacterium avium complex (MAC), increasing our appreciation of NTM organisms of pulmonary importance in tropical environments.

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