Evolution of Mycobacterium abscessus in the human lung: Cumulative mutations and genomic rearrangement of porin genes in patient isolates.

BACKGROUND: Mycobacterium abscessus subspecies massiliense (M. massiliense) is increasingly recognized as an emerging bacterial pathogen, particularly in cystic fibrosis (CF) patients and CF centres' respiratory outbreaks. We characterized genomic and phenotypic changes in 15 serial isolates from two CF patients (1S and 2B) with chronic pulmonary M. massiliense infection leading to death, as well as four isolates from a CF centre outbreak in which patient 2B was the index case. RESULTS: Comparative genomic analysis revealed the mutations affecting growth rate, metabolism, transport, lipids (loss of glycopeptidolipids), antibiotic susceptibility (macrolides and aminoglycosides resistance), and virulence factors. Mutations in 23S rRNA, mmpL4, porin locus and tetR genes occurred in isolates from both CF patients. Interestingly, we identified two different spontaneous mutation events at the mycobacterial porin locus: a fusion of two tandem porin paralogs in patient 1S and a partial deletion of the first porin paralog in patient 2B. These genomic changes correlated with reduced porin protein expression, diminished 14C-glucose uptake, slower bacterial growth rates, and enhanced TNF-α induction in mycobacteria-infected THP-1 human cells. Porin gene complementation of porin mutants partly restored 14C-glucose uptake, growth rate and TNF-α levels to those of intact porin strains. CONCLUSIONS: We hypothesize that specific mutations accumulated and maintained over time in M. massiliense, including mutations shared among transmissible strains, collectively lead to more virulent, host adapted lineages in CF patients and other susceptible hosts.

Tissue specific diversification, virulence and immune response to Mycobacterium bovis BCG in a patient with an IFN-γ R1 deficiency.

Summary: We characterized Mycobacterium bovis BCG isolates found in lung and brain samples from a previously vaccinated patient with IFNγR1 deficiency. The isolates collected displayed distinct genomic and phenotypic features consistent with host adaptation and associated changes in antibiotic susceptibility and virulence traits. Background: We report a case of a patient with partial recessive IFNγR1 deficiency who developed disseminated BCG infection after neonatal vaccination (BCG-vaccine). Distinct M. bovis BCG-vaccine derived clinical strains were recovered from the patient's lungs and brain. Methods: BCG strains were phenotypically (growth, antibiotic susceptibility, lipid) and genetically (whole genome sequencing) characterized. Mycobacteria cell infection models were used to assess apoptosis, necrosis, cytokine release, autophagy, and JAK-STAT signaling. Results: Clinical isolates BCG-brain and BCG-lung showed distinct Rv0667 rpoB mutations conferring high- and low-level rifampin resistance; the latter displayed clofazimine resistance through Rv0678 gene (MarR-like transcriptional regulator) mutations. BCG-brain and BCG-lung showed mutations in fadA2, fadE5, and mymA operon genes, respectively. Lipid profiles revealed reduced levels of PDIM in BCG-brain and BCG-lung and increased TAGs and Mycolic acid components in BCG-lung, compared to parent BCG-vaccine. In vitro infected cells showed that the BCG-lung induced a higher cytokine release, necrosis, and cell-associated bacterial load effect when compared to BCG-brain; conversely, both strains inhibited apoptosis and altered JAK-STAT signaling. Conclusions: During a chronic-disseminated BCG infection, BCG strains can evolve independently at different sites likely due to particular microenvironment features leading to differential antibiotic resistance, virulence traits resulting in dissimilar responses in different host tissues.

Patients with Idiopathic Pulmonary Nontuberculous Mycobacterial Disease Have Normal Th1/Th2 Cytokine Responses but Diminished Th17 Cytokine and Enhanced Granulocyte-Macrophage Colony-Stimulating Factor Production.

OBJECTIVE: Although disseminated nontuberculous mycobacterial infection is attributed to defects in the interleukin (IL)-12/interferon-γ circuit, the immunophenotype of idiopathic pulmonary nontuberculous mycobacterial (PNTM) disease is not well defined. METHOD: We phenotyped Th1, Th2, Th17, and Treg cytokines and colony-stimulating factor production from patients with idiopathic PNTM disease. Data were compared with healthy donors, cystic fibrosis (CF), and primary ciliary dyskinesia (PCD) patients with PNTM disease. Both supernatant cytokine production and intracellular cytokines expressed by various leukocyte subpopulations following mitogen and antigen stimulation were assayed by electrochemiluminescence-based multiplex immunoassay and flow cytometry, respectively. RESULTS: Regardless of antigen or mitogen stimulation, neither intracellular nor extracellular Th1, Th2, and Treg cytokine levels differed between patients and controls. Th17 cells and IL-17A levels were lower in idiopathic PNTM patients, whereas monocyte granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in response to NTM stimulation was higher compared with healthy donors. Besides, distinct cytokine responses following stimulation by Mycobacterium abscessus and Mycobacterium avium were observed consistently within each group. CONCLUSIONS: The IL-12/IFN-γ circuit appeared intact in patients with idiopathic PNTM disease. However, idiopathic PNTM patients had reduced Th17 response and higher mycobacteria-induced monocyte GM-CSF expression.

STAT1 Gain-of-Function Mutations Cause High Total STAT1 Levels With Normal Dephosphorylation.

Signal transducer and activator of transcription (STAT1)1 gain of function (GOF) pathogenic variants have been associated with increased levels of phosphorylated STAT1 and STAT1-dependent cellular responses. Delayed dephosphorylation was proposed as the underlying mechanism leading to the characteristically raised pSTAT1 levels. We examined the levels of STAT1 protein and message as well as rates of STAT1 phosphorylation, dephosphorylation, and degradation associated with STAT1 GOF pathogenic variants. Fresh peripheral blood mononuclear cells (PBMC) from 14 STAT1 GOF patients carrying 10 different pathogenic variants in the coiled-coil, DNA binding, and SH2 domains and healthy donors were used to study STAT1 levels and phosphorylation (pSTAT1) following IFNγ and IFNα stimulation. STAT1 protein levels were measured by flow cytometry and immunoblot. STAT1 mRNA levels were measured using quantitative reverse transcription PCR. STAT1 protein degradation was studied using cycloheximide. Patient IFNγ and IFNα induced peak pSTAT1 was higher than in healthy controls. The velocity of pSTAT1 dephosphorylation after treatment of IFNγ stimulated CD14+ monocytes with the Janus Kinase (JAK)-inhibitor ruxolitinib was significantly faster in patient cells. STAT1 protein levels in patient CD14+ monocytes and CD3+ T cells were higher than in healthy donors. There was a strong and positive correlation between CD14+ STAT1 protein levels and peak pSTAT1 levels. Patient fresh PBMC STAT1 mRNA levels were increased at rest and after 16 h of incubation. STAT1 protein degradation was similar in patient and healthy volunteer cells. Patient IFNγ receptors 1 and 2 and JAK2 levels were normal. One patient in our cohort was treated with the oral JAK inhibitor ruxolitinib. Treatment was associated with normalization of both STAT1 protein and peak pSTAT1 levels. After JAK inhibitor treatment was stopped the patient's CD14+ monocyte STAT1 protein and peak phosphorylation levels increased proportionally. These findings suggest that patients with STAT1 GOF mutations have higher levels of total STAT1 protein, leading to high levels of pSTAT1 after stimulation, despite rapid STAT1 dephosphorylation and normal degradation.

Novel signal transducer and activator of transcription 1 mutation disrupts small ubiquitin-related modifier conjugation causing gain of function.

BACKGROUND: Sumoylation is a posttranslational reversible modification of cellular proteins through the conjugation of small ubiquitin-related modifier (SUMO) and comprises an important regulator of protein function. OBJECTIVE: We sought to characterize the molecular mechanism of a novel mutation at the SUMO motif on signal transducer and activator of transcription 1 (STAT1). METHODS: STAT1 sequencing and functional characterization were performed in transfection experiments by using immunoblotting and immunoprecipitation in STAT1-deficient cell lines. Transcriptional response and target gene activation were also investigated in PBMCs. RESULTS: We identified a novel STAT1 mutation (c.2114A>T, p.E705V) within the SUMO motif (702IKTE705) in a patient with disseminated Rhodococcus species infection, Norwegian scabies, chronic mucocutaneous candidiasis, hypothyroidism, and esophageal squamous cell carcinoma. The mutation is located in the tail segment and is predicted to disrupt STAT1 sumoylation. Immunoprecipitation experiments performed in transfected cells confirmed absent STAT1 sumoylation for E705V, whereas it was present in wild-type (WT) STAT1 cells, as well as the loss-of-function mutants L706S and Y701C. Furthermore, stimulation with IFN-γ led to enhanced STAT1 phosphorylation, enhanced transcriptional activity, and target gene expression in the E705V-transfected compared with WT-transfected cells. Computer modeling of WT and mutant STAT1 molecules showed variations in the accessibility of the phosphorylation site Y701, which corresponded to the loss-of-function and gain-of-function variants. CONCLUSION: This is the first report of a mutation in the STAT1 sumoylation motif associated with clinical disease. These data reinforce sumoylation as a key posttranslational regulatory modification of STAT1 and identify a novel mechanism for gain-of-function STAT1 disease in human subjects.

Transcriptional Response of Respiratory Epithelium to Nontuberculous Mycobacteria.

The incidence of pulmonary nontuberculous mycobacteria (NTM) disease is increasing, but host responses in respiratory epithelium infected with NTM are not fully understood. In this work, we aimed to identify infection-relevant gene expression signatures of NTM infection of the respiratory epithelium. We infected air-liquid interface (ALI) primary respiratory epithelial cell cultures with Mycobacterium avium subsp. avium (MAC) or Mycobacterium abscessus subsp. abscessus (MAB). We used cells from four different donors to obtain generalizable data. Differentiated respiratory epithelial cells at the ALI were infected with MAC or MAB at a multiplicity of infection of 100:1 or 1,000:1, and RNA sequencing was performed at Days 1 and 3 after infection. In response to infection, we found down-regulation of ciliary genes but upregulation of genes associated with cytokines/chemokines, such as IL-32, and cholesterol biosynthesis. Inflammatory response genes tended to be more upregulated by MAB than by MAC infection. Primary respiratory epithelial cell infection with NTM at the ALI identified ciliary function, cholesterol biosynthesis, and cytokine/chemokine production as major host responses to infection. Some of these pathways may be amenable to therapeutic manipulation.

Risks of Ruxolitinib in STAT1 Gain-of-Function-Associated Severe Fungal Disease.

Heterozygous STAT1 gain-of-function (GOF) mutations are associated with chronic mucocutaneous candidiasis and a broad spectrum of infectious, inflammatory, and vascular manifestations. We describe therapeutic failures with the Janus Kinase (JAK) inhibitor ruxolitinib in 2 STAT1 GOF patients with severe invasive or cutaneous fungal infections.

Progressive Multifocal Leukoencephalopathy in Primary Immune Deficiencies: Stat1 Gain of Function and Review of the Literature.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) is a rare, severe, otherwise fatal viral infection of the white matter of the brain caused by the polyomavirus JC virus, which typically occurs only in immunocompromised patients. One patient with dominant gain-of-function (GOF) mutation in signal transducer and activator of transcription 1 (STAT1) with chronic mucocutaneous candidiasis and PML was reported previously. We aim to identify the molecular defect in 3 patients with PML and to review the literature on PML in primary immune defects (PIDs). METHODS: STAT1 was sequenced in 3 patients with PML. U3C cell lines were transfected with STAT1 and assays to search for STAT1 phosphorylation, transcriptional response, and target gene expression were performed. RESULTS: We identified 3 new unrelated cases of PML in patients with GOF STAT1 mutations, including the novel STAT1 mutation, L400Q. These STAT1 mutations caused delayed STAT1 dephosphorylation and enhanced interferon-gamma-driven responses. In our review of the literature regarding PML in primary immune deficiencies we found 26 cases, only 54% of which were molecularly characterized, the remainder being syndromically diagnosed only. CONCLUSIONS: The occurrence of PML in 4 cases of STAT1 GOF suggests that STAT1 plays a critical role in the control of JC virus in the central nervous system.

New Real-Time PCR Assays for Detection of Inducible and Acquired Clarithromycin Resistance in the Mycobacterium abscessus Group.

Members of the Mycobacterium abscessus group (MAG) cause lung, soft tissue, and disseminated infections. The oral macrolides clarithromycin and azithromycin are commonly used for treatment. MAG can display clarithromycin resistance through the inducible erm(41) gene or via acquired mutations in the rrl (23S rRNA) gene. Strains harboring a truncation or a T28C substitution in erm(41) lose the inducible resistance trait. Phenotypic detection of clarithromycin resistance requires extended incubation (14 days), highlighting the need for faster methods to detect resistance. Two real-time PCR-based assays were developed to assess inducible and acquired clarithromycin resistance and tested on a total of 90 clinical and reference strains. A SYBR green assay was designed to distinguish between a full-length and truncated erm(41) gene by temperature shift in melting curve analysis. Single nucleotide polymorphism (SNP) allele discrimination assays were developed to distinguish T or C at position 28 of erm(41) and 23S rRNA rrl gene mutations at position 2058 and/or 2059. Truncated and full-size erm(41) genes were detected in 21/90 and 69/90 strains, respectively, with 64/69 displaying T at nucleotide position 28 and 5/69 containing C at that position. Fifteen isolates showed rrl mutations conferring clarithromycin resistance, including A2058G (11 isolates), A2058C (3 isolates), and A2059G (1 isolate). Targeted sequencing and phenotypic assessment of resistance concurred with molecular assay results. Interestingly, we also noted cooccurring strains harboring an active erm(41), inactive erm(41), and/or acquired mutational resistance, as well as slowly growing MAG strains and also strains displaying an inducible resistance phenotype within 5 days, long before the recommended 14-day extended incubation.

Clonal Diversification and Changes in Lipid Traits and Colony Morphology in Mycobacterium abscessus Clinical Isolates.

The smooth-to-rough colony morphology shift in Mycobacterium abscessus has been implicated in loss of glycopeptidolipid (GPL), increased pathogenicity, and clinical decline in cystic fibrosis (CF) patients. However, the evolutionary phenotypic and genetic changes remain obscure. Serial isolates from nine non-CF patients with persistent M. abscessus infection were characterized by colony morphology, lipid profile via thin-layer chromatography and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), sequencing of eight genes in the GPL locus, and expression level of fadD23, a key gene involved in the biosynthesis of complex lipids. All 50 isolates were typed as M. abscessus subspecies abscessus and were clonally related within each patient. Rough isolates, all lacking GPL, predominated at later disease stages, some showing variation within rough morphology. While most (77%) rough isolates harbored detrimental mutations in mps1 and mps2, 13% displayed previously unreported mutations in mmpL4a and mmpS4, the latter yielding a putative GPL precursor. Two isolates showed no deleterious mutations in any of the eight genes sequenced. Mixed populations harboring different GPL locus mutations were detected in 5 patients, demonstrating clonal diversification, which was likely overlooked by conventional acid-fast bacillus (AFB) culture methods. Our work highlights applications of MALDI-TOF MS beyond identification, focusing on mycobacterial lipids relevant in virulence and adaptation. Later isolates displayed accumulation of triacylglycerol and reduced expression of fadD23, sometimes preceding rough colony onset. Our results indicate that clonal diversification and a shift in lipid metabolism, including the loss of GPL, occur during chronic lung infection with M. abscessus. GPL loss alone may not account for all traits associated with rough morphology.

Heme Oxygenase-1 Regulation of Matrix Metalloproteinase-1 Expression Underlies Distinct Disease Profiles in Tuberculosis.

Pulmonary tuberculosis (TB) is characterized by oxidative stress and lung tissue destruction by matrix metalloproteinases (MMPs). The interplay between these distinct pathological processes and the implications for TB diagnosis and disease staging are poorly understood. Heme oxygenase-1 (HO-1) levels were previously shown to distinguish active from latent TB, as well as successfully treated Mycobacterium tuberculosis infection. MMP-1 expression is also associated with active TB. In this study, we measured plasma levels of these two important biomarkers in distinct TB cohorts from India and Brazil. Patients with active TB expressed either very high levels of HO-1 and low levels of MMP-1 or the converse. Moreover, TB patients with either high HO-1 or MMP-1 levels displayed distinct clinical presentations, as well as plasma inflammatory marker profiles. In contrast, in an exploratory North American study, inversely correlated expression of HO-1 and MMP-1 was not observed in patients with other nontuberculous lung diseases. To assess possible regulatory interactions in the biosynthesis of these two enzymes at the cellular level, we studied the expression of HO-1 and MMP-1 in M. tuberculosis-infected human and murine macrophages. We found that infection of macrophages with live virulent M. tuberculosis is required for robust induction of high levels of HO-1 but not MMP-1. In addition, we observed that CO, a product of M. tuberculosis-induced HO-1 activity, inhibits MMP-1 expression by suppressing c-Jun/AP-1 activation. These findings reveal a mechanistic link between oxidative stress and tissue remodeling that may find applicability in the clinical staging of TB patients.

Opposing roles of STAT1 and STAT3 in IL-21 function in CD4+ T cells.

IL-21 is a type I cytokine essential for immune cell differentiation and function. Although IL-21 can activate several STAT family transcription factors, previous studies focused mainly on the role of STAT3 in IL-21 signaling. Here, we investigated the role of STAT1 and show that STAT1 and STAT3 have at least partially opposing roles in IL-21 signaling in CD4(+) T cells. IL-21 induced STAT1 phosphorylation, and this was augmented in Stat3-deficient CD4(+) T cells. RNA-Seq analysis of CD4(+) T cells from Stat1- and Stat3-deficient mice revealed that both STAT1 and STAT3 are critical for IL-21-mediated gene regulation. Expression of some genes, including Tbx21 and Ifng, was differentially regulated by STAT1 and STAT3. Moreover, opposing actions of STAT1 and STAT3 on IFN-γ expression in CD4(+) T cells were demonstrated in vivo during chronic lymphocytic choriomeningitis infection. Finally, IL-21-mediated induction of STAT1 phosphorylation, as well as IFNG and TBX21 expression, were higher in CD4(+) T cells from patients with autosomal dominant hyper-IgE syndrome, which is caused by STAT3 deficiency, as well as in cells from STAT1 gain-of-function patients. These data indicate an interplay between STAT1 and STAT3 in fine-tuning IL-21 actions.

High-level relatedness among Mycobacterium abscessus subsp. massiliense strains from widely separated outbreaks.

Three recently sequenced strains isolated from patients during an outbreak of Mycobacterium abscessus subsp. massiliense infections at a cystic fibrosis center in the United States were compared with 6 strains from an outbreak at a cystic fibrosis center in the United Kingdom and worldwide strains. Strains from the 2 cystic fibrosis outbreaks showed high-level relatedness with each other and major-level relatedness with strains that caused soft tissue infections during an epidemic in Brazil. We identified unique single-nucleotide polymorphisms in cystic fibrosis and soft tissue outbreak strains, separate single-nucleotide polymorphisms only in cystic fibrosis outbreak strains, and unique genomic traits for each subset of isolates. Our findings highlight the necessity of identifying M. abscessus to the subspecies level and screening all cystic fibrosis isolates for relatedness to these outbreak strains. We propose 2 diagnostic strategies that use partial sequencing of rpoB and secA1 genes and a multilocus sequence typing protocol.

Inhaled amikacin for treatment of refractory pulmonary nontuberculous mycobacterial disease.

RATIONALE: Treatment of pulmonary nontuberculous mycobacteria, especially Mycobacterium abscessus, requires prolonged, multidrug regimens with high toxicity and suboptimal efficacy. Options for refractory disease are limited. OBJECTIVES: We reviewed the efficacy and toxicity of inhaled amikacin in patients with treatment-refractory nontuberculous mycobacterial lung disease. METHODS: Records were queried to identify patients who had inhaled amikacin added to failing regimens. Lower airway microbiology, symptoms, and computed tomography scan changes were assessed together with reported toxicity. MEASUREMENTS AND MAIN RESULTS: The majority (80%) of the 20 patients who met entry criteria were women; all had bronchiectasis, two had cystic fibrosis and one had primary ciliary dyskinesia. At initiation of inhaled amikacin, 15 were culture positive for M. abscessus and 5 for Mycobacterium avium complex and had received a median (range) of 60 (6, 190) months of mycobacterial treatment. Patients were followed for a median of 19 (1, 50) months. Eight (40%) patients had at least one negative culture and 5 (25%) had persistently negative cultures. A decrease in smear quantity was noted in 9 of 20 (45%) and in mycobacterial culture growth for 10 of 19 (53%). Symptom scores improved in nine (45%), were unchanged in seven (35%), and worsened in four (20%). Improvement on computed tomography scans was noted in 6 (30%), unchanged in 3 (15%), and worsened in 11 (55%). Seven (35%) stopped amikacin due to: ototoxicity in two (10%), hemoptysis in two (10%), and nephrotoxicity, persistent dysphonia, and vertigo in one each. CONCLUSIONS: In some patients with treatment-refractory pulmonary nontuberculous mycobacterial disease, the addition of inhaled amikacin was associated with microbiologic and/or symptomatic improvement; however, toxicity was common. Prospective evaluation of inhaled amikacin for mycobacterial disease is warranted.

Draft Genome Sequences of Burkholderia cenocepacia ET12 Lineage Strains K56-2 and BC7.

The Burkholderia cepacia complex (BCC) is a group of closely related bacteria that are responsible for respiratory infections in immunocompromised humans, most notably those with cystic fibrosis (CF). We report the genome sequences for Burkholderia cenocepacia ET12 lineage CF isolates K56-2 and BC7.

Adaptability and persistence of the emerging pathogen Bordetella petrii.

The first described, environmentally isolated, Bordetella petrii was shown to undergo massive genomic rearrangements in vitro. More recently, B. petrii was isolated from clinical samples associated with jaw, ear bone, cystic fibrosis and chronic pulmonary disease. However, the in vivo consequences of B. petrii genome plasticity and its pathogenicity remain obscure. B. petrii was identified from four sequential respiratory samples and a post-mortem spleen sample of a woman presenting with bronchiectasis and cavitary lung disease associated with nontuberculous mycobacterial infection. Strains were compared genetically, phenotypically and by antibody recognition from the patient and from inoculated mice. The successive B. petrii strains exhibited differences in growth, antibiotic susceptibility and recognition by the patient's antibodies. Antibodies from mice inoculated with these strains recapitulated the specificity and strain dependent response that was seen with the patient's serum. Finally, we characterize one strain that was poorly recognized by the patient's antibodies, due to a defect in the lipopolysaccharide O-antigen, and identify a mutation associated with this phenotype. We propose that B. petrii is remarkably adaptable in vivo, providing a possible connection between immune response and bacterial evasion and supporting infection persistence.

Clinical and therapeutic features of pulmonary nontuberculous mycobacterial disease, Brazil, 1993-2011.

To identify clinical and therapeutic features of pulmonary nontuberculous mycobacterial (PNTM) disease, we conducted a retrospective analysis of patients referred to the Brazilian reference center, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, who received a diagnosis of PNTM during 1993­2011 with at least 1 respiratory culture positive for NTM. Associated conditions included bronchiectasis (21.8%), chronic obstructive pulmonary disease (20.7%), cardiovascular disease (15.5%), AIDS (9.8%), diabetes (9.8%), and hepatitis C (4.6%).Two patients had Hansen disease; 1 had Marfan syndrome. Four mycobacterial species comprised 85.6% of NTM infections: Mycobacterium kansasii, 59 cases (33.9%); M. avium complex, 53 (30.4%); M. abscessus, 23 (13.2%); and M. fortuitum, 14 (8.0%). A total of 42 (24.1%) cases were associated with rapidly growing mycobacteria. In countries with a high prevalence of tuberculosis, PNTM is likely misdiagnosed as tuberculosis, thus showing the need for improved capacity to diagnose mycobacterial disease as well as greater awareness of PNTM disease prevalence.

New rapid scheme for distinguishing the subspecies of the Mycobacterium abscessus group and identifying Mycobacterium massiliense isolates with inducible clarithromycin resistance.

Mycobacterium abscessus (M. abscessus sensu lato, or the M. abscessus group) comprises three closely related taxa whose taxonomic statuses are under revision, i.e., M. abscessus sensu stricto, Mycobacterium bolletii, and Mycobacterium massiliense. We describe here a simple, robust, and cost-effective PCR-based method for distinguishing among M. abscessus, M. massiliense, and M. bolletii. Based on the M. abscessus ATCC 19977(T) genome, regions that discriminated between M. abscessus and M. massiliense were identified through array-based comparative genomic hybridization. A typing scheme using PCR primers designed for four of these locations was applied to 46 well-characterized clinical isolates comprising 29 M. abscessus, 15 M. massiliense, and 2 M. bolletii isolates previously identified by multitarget sequencing. Interestingly, 2 isolates unequivocally identified as M. massiliense were shown to have a full-length erm(41) gene instead of the expected gene deletion and showed inducible clarithromycin resistance after 14 days. We propose using this PCR-based typing scheme combined with erm(41) PCR for straightforward identification of M. abscessus, M. massiliense, and M. bolletii and the assessment of inducible clarithromycin resistance. This method can be easily integrated into a routine workflow to provide subspecies-level identification within 24 h after isolation of the M. abscessus group.