The increasing clinical significance of Mycobacterium abscessus is owed to its innate high-level, broad-spectrum resistance to antibiotics and therefore rapidly evolves as an important human pathogen. This warrants the identification of novel targets for aiding the discovery of new drugs or drug combinations to treat M. abscessus infections. This study is inspired by the drug-hypersensitive profile of a mutant M. abscessus (U14) with transposon insertion in MAB_1915. We validated the role of MAB_1915 in intrinsic drug resistance in M. abscessus by constructing a selectable marker-free in-frame deletion in MAB_1915 and complementing the mutant with the same or extended version of the gene and then followed by drug susceptibility testing. Judging by the putative function of MAB_1915, cell envelope permeability was studied by ethidium bromide accumulation assay and susceptibility testing against dyes and detergents. In this study, we established genetic evidence of the role of MAB_1915 in intrinsic resistance to rifampicin, rifabutin, linezolid, clarithromycin, vancomycin, and bedaquiline. Disruption of MAB_1915 has also been observed to cause a significant increase in cell envelope permeability in M. abscessus. Restoration of resistance is observed to depend on at least 27 base pairs upstream of the coding DNA sequence of MAB_1915. MAB_1915 could therefore be associated with cell envelope permeability, and hence its role in intrinsic resistance to multiple drugs in M. abscessus, which presents it as a novel target for future development of effective antimicrobials to overcome intrinsic drug resistance in M. abscessus.
OBJECTIVE: This study reports the role of a putative fadD (MAB_1915) in innate resistance to multiple drugs by M. abscessus, hence identifying MAB_1915 as a valuable target and providing a baseline for further mechanistic studies and development of effective antimicrobials to check the high level of intrinsic resistance in this pathogen.

UNASSIGNED: To investigate the prevalence of Mycobacterium abscessus complex (MABC), drug resistance characteristics, and the relationship between clarithromycin (CLA) susceptibility and MABC genotype in Chongqing, China.
UNASSIGNED: A total of 434 NTM patient isolates were collected between October 2018 and October 2019. Isolates confirmed to be non-tuberculous mycobacteria (NTM) were tested for minimal inhibitory concentrations of antimicrobial agents. In addition, rrl and erm(41) gene sequences were used to analyze the acquired macrolide resistance and inducible macrolide resistance.
UNASSIGNED: Overall, 17 different NTM species were detected, of which M. abscessus (22.6 %, 91/403) was most prevalent. Amikacin, CLA, azithromycin and cefoxitin exhibited potent activities against MABC organisms, but no significant differences were observed in drug resistance rates between M. abscessus and M. massiliense (P > 0.05). On day 3 of culture, the acquired resistance rate against CLA was 7.4 % (9/121). Of 41 MABC isolates with inducible CLA resistant, 95.1 % (39/41) isolates belonged to the erm(41) T28 sequevar, while the remaining 4.9 % (2/41) possessed the M. massiliense genotype. All erm(41) C28 sequevar isolates were sensitive to CLA on day3 and day 14 of culture. Meanwhile, of the 5 erm(41) T28 isolates with acquired resistance, all possessed rrl 2058/2059 mutations, including 3 isolates with A2058C mutation and 2 isolates with A2059G mutation. While 2 of the 4 M. massiliense isolates with acquired resistance possessed the A2059G mutation, and one isolate possessed the A2058G mutation.
UNASSIGNED: Erm(41) and rrl gene could serve as useful markers for predicting macrolide susceptibility of MABC complex isolates.

Mycobacterium abscessus (M. abscessus) is a multidrug-resistant nontuberculous mycobacterium (NTM) that is responsible for a wide spectrum of infections in humans. The lack of effective bactericidal drugs and the formation of biofilm make its clinical treatment very difficult. The FDA-approved drug library containing 3048 marketed and pharmacopeial drugs or compounds was screened at 20 μM against M. abscessus type strain 19977 in 7H9 medium, and 62 hits with potential antimicrobial activity against M. abscessus were identified. Among them, bithionol, a clinically approved antiparasitic agent, showed excellent antibacterial activity and inhibited the growth of three different subtypes of M. abscessus from 0.625 μM to 2.5 μM. We confirmed the bactericidal activity of bithionol by the MBC/MIC ratio being ≤4 and the time-kill curve study and also electron microscopy study. Interestingly, it was found that at 128 μg/mL, bithionol could completely eliminate biofilms after 48h, demonstrating an outstanding antibiofilm capability compared to commonly used antibiotics. Additionally, bithionol could eliminate 99.9% of biofilm bacteria at 64 μg/mL, 99% at 32 μg/mL, and 90% at 16 μg/mL. Therefore, bithionol may be a potential candidate for the treatment of M. abscessus infections due to its significant antimicrobial and antibiofilm activities.

OBJECTIVE: Non-tuberculous mycobacterial pulmonary disease (NTM-PD) is caused by an imbalance between pathogens and impaired host immune responses. Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) are the two major pathogens that cause NTM-PD. In this study, we sought to dissect the transcriptomes of peripheral blood immune cells at the single-cell resolution in NTM-PD patients and explore potential clinical markers for NTM-PD diagnosis and treatment.
METHODS: Peripheral blood samples were collected from six NTM-PD patients, including three MAB-PD patients, three MAC-PD patients, and two healthy controls. We employed single-cell RNA sequencing (scRNA-seq) to define the transcriptomic landscape at a single-cell resolution. A comprehensive scRNA-seq analysis was performed, and flow cytometry was conducted to validate the results of scRNA-seq.
RESULTS: A total of 27,898 cells were analyzed. Nine T-cells, six mononuclear phagocytes (MPs), and four neutrophil subclusters were defined. During NTM infection, naïve T-cells were reduced, and effector T-cells increased. High cytotoxic activities were shown in T-cells of NTM-PD patients. The proportion of inflammatory and activated MPs subclusters was enriched in NTM-PD patients. Among neutrophil subclusters, an IFIT1+ neutrophil subcluster was expanded in NTM-PD compared to healthy controls. This suggests that IFIT1+ neutrophil subcluster might play an important role in host defense against NTM. Functional enrichment analysis of this subcluster suggested that it is related to interferon response. Cell-cell interaction analysis revealed enhanced CXCL8-CXCR1/2 interactions between the IFIT1+ neutrophil subcluster and NK cells, NKT cells, classical mononuclear phagocytes subcluster 1 (classical Mo1), classical mononuclear phagocytes subcluster 2 (classical Mo2) in NTM-PD patients compared to healthy controls.
CONCLUSIONS: Our data revealed disease-specific immune cell subclusters and provided potential new targets of NTM-PD. Specific expansion of IFIT1+ neutrophil subclusters and the CXCL8-CXCR1/2 axis may be involved in the pathogenesis of NTM-PD. These insights may have implications for the diagnosis and treatment of NTM-PD.

OBJECTIVE: Mycobacterium abscessus is a non-tuberculous mycobacterial pathogen that causes pulmonary and skin infections globally. Clarithromycin plays a pivotal role in treating M. abscessus infections, with resistance often leading to treatment failure. While canonical mutations in the 23S rRNA residue 2270/2271 are recognized as the primary mechanism for acquired clarithromycin resistance, resistant isolates lacking these mutations have been widely reported. This study aims to identify new mechanisms of clarithromycin resistance in M. abscessus.
METHODS: We selected spontaneous resistant mutants derived from two parental strains characterized by erm(41) T28 and C28 sequevars, respectively. Whole-genome sequencing was performed on mutants lacking the 23S rRNA 2270/2271 mutations. Site-directed mutagenesis was used to confirm the resistance phenotypes of newly identified mutations. Bioinformatic analysis of publicly available genomes was conducted to evaluate the presence of these mutations in clinical isolates. The spatial localization of these mutations in the ribosome was analyzed to investigate potential mechanisms of resistance.
RESULTS: A total of 135 resistant mutants were selected from the parental strains. Sequencing of the 78 mutants lacking the 23S rRNA 2270/2271 mutations identified mutations within the peptidyl-transferase center and hairpin loops 35, 49, and 74 of the 23S rRNA. These noncanonical mutations were identified in 57 of 1875 genomes of clinical isolates. Thirteen representative mutations were introduced into the bacterial genome, and their contributions to macrolide resistance were confirmed. The newly identified mutations all localized at the entrance of the nascent peptide exit tunnel, potentially contributing to resistance by disrupting the macrolide binding pocket.
CONCLUSIONS: Several noncanonical 23S rRNA mutations conferring clarithromycin resistance were identified. These mutations enhance our understanding of macrolide resistance in M. abscessus and could serve as important markers for diagnosing clarithromycin resistance.

Pulmonary infection due to Mycobacterium abscessus complex (MABC) usually occurs in children with underlying risk factors including cystic fibrosis (CF), chronic lung disease, and immunocompromised status, but rarely in immunocompetent children without underlying lung disease, especially in infants. We present a case of MABC pulmonary disease (MABC-PD) in an otherwise healthy 53-day-old male infant with one week of cough and respiratory distress. Computed tomography showed multiple masses across both lungs. Isolated mycobacteria from his bronchoalveolar lavage fluid were identified as MABC. We describe our complete evaluation, including immunodeficiency evaluation incorporating whole exome sequencing and our therapeutic process given complicated susceptibility pattern of the M. abscessus isolate, and review literature for MABC-PD in immunocompetent children. The infant was successfully treated through prolonged treatment with parenteral Amikacin, Cefoxitin, Linezolid, and Clarithromycin, combined with inhaled Amikacin.

BACKGROUND: Psoralea corylifolia L. (Fabaceae) is a traditional medicinal herb used to treat various diseases, including kidney disease, asthma, psoriasis and vitiligo.
OBJECTIVE: To explore the antibacterial activity of Psoralea corylifolia L. and its bioactive components against Mycobacterium abscessus (M. abscessus).
METHODS: Ultra high performance liquid chromatography was utilized to analyze the bioactive fractions and compounds present in 30%, 60%, and 90% ethanol extracts of Psoralea corylifolia L.. The antibacterial effects of Psoralea corylifolia L. and potential active ingredients were determined by minimum inhibitory concentration (MIC). The bactericidal activity of the active ingredient isobavachalcone was evaluated and then scanning electron microscopy was used to explore the bactericidal mechanism of isobavachalcone.
RESULTS: The 90% ethanol extracts of Psoralea corylifolia L. showed significant antibacterial activity against M. abscessus, with an MIC of 156 μg/mL. Isobavachalcone was identified as the bioactive ingredient, and testing of 118 clinical isolates of M. abscessus indicated their MICs ranged from 2 to 16 μg/mL, with an average MIC of 8 μg/mL. Furthermore, the minimum bactericidal concentration/MIC ratio and the time-kill test indicated rapid bactericidal activity of isobavachalcone against M. abscessus. Finally, we found that the bactericidal mechanism of isobavachalcone involved damage to the bacterial cell membrane, causing wrinkled and sunken cell surface and a noticeable reduction in bacterial length.
CONCLUSIONS: Psoralea corylifolia L. ethanol extracts as well as its active component isobavachalcone show promising antimicrobial activity against M. abscessus.

Mycobacterium abscessus (M. abscessus) inherently displays resistance to most antibiotics, with the underlying drug resistance mechanisms remaining largely unexplored. Efflux pump is believed to play an important role in mediating drug resistance. The current study examined the potential of efflux pump inhibitors to reverse levofloxacin (LFX) resistance in M. abscessus. The reference strain of M. abscessus (ATCC19977) and 60 clinical isolates, including 41 M. abscessus subsp. abscessus and 19 M. abscessus subsp. massilense, were investigated. The drug sensitivity of M. abscessus against LFX alone or in conjunction with efflux pump inhibitors, including verapamil (VP), reserpine (RSP), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or dicyclohexylcarbodiimide (DCC), were determined by AlarmarBlue microplate assay. Drug-resistant regions of the gyrA and gyrB genes from the drug-resistant strains were sequenced. The transcription level of the efflux pump genes was monitored using qRT-PCR. All the tested strains were resistant to LFX. The drug-resistant regions from the gyrA and gyrB genes showed no mutation associated with LFX resistance. CCCP, DCC, VP, and RSP increased the susceptibility of 93.3% (56/60), 91.7% (55/60), 85% (51/60), and 83.3% (50/60) isolates to LFX by 2 to 32-fold, respectively. Elevated transcription of seven efflux pump genes was observed in isolates with a high reduction in LFX MIC values in the presence of efflux pump inhibitors. Efflux pump inhibitors can improve the antibacterial activity of LFX against M. abscessus in vitro. The overexpression of efflux-related genes in LFX-resistant isolates suggests that efflux pumps are associated with the development of LFX resistance in M. abscessus.

OBJECTIVE: This study aims to estimate the overall in vitro activity of bedaquiline (BDQ) against clinical isolates of Mycobacterium abscessus complex (MABS) and M. avium complex (MAC), considering BDQ as a repurposed drug for non-tuberculous mycobacteria (NTM) infections.
METHODS: We conducted a systematic review of publications in PubMed/ MEDLINE, Web of Science, and Embase up to 15 April 2023. Studies were included if they followed the Clinical and Laboratory Standards Institute (CLSI) criteria for drug susceptibility testing (DST). Using a random effects model, we assessed the overall in vitro BDQ resistance rate in clinical isolates of MABS and MAC. Sources of heterogeneity were analysed using Cochran\'s Q and the I2 statistic. All analyses were performed using CMA V3.0.
RESULTS: A total of 24 publications (19 reports for MABS and 11 for MAC) were included. Using 1 µg/mL and 2 µg/mL as the breakpoint for BDQ resistance, the pooled rates of in vitro BDQ resistance in clinical isolates of MABS were found to be 1.8% (95% confidence interval [CI], 0.7-4.6%) and 1.7% (95% CI, 0.6-4.4%), respectively. In the case of MAC, the pooled rates were 1.7% (95% CI, 0.4-6.9%) and 1.6% (95% CI, 0.4-6.8%) for 1 µg/mL and 2 µg/mL, respectively.
CONCLUSIONS: This study reports the prevalence of BDQ resistance in clinical isolates of MABS and MAC. The findings suggest that BDQ holds potential as a repurposed drug for treating MABS and MAC infections.

UNASSIGNED: Mycobacterium abscessus is ubiquitous in the environment and seldom causes infections in immunocompetent individuals. However, skin and soft tissue infections caused by M. abscessus have been reported in recent years. Additionally, the cutaneous infections or outbreaks post cosmetic surgery caused by M. abscessus have been increasing due to the popularity of plastic surgery. The main modes of transmission are through contaminated saline, disinfectants, or surgery equipment, as well as close contact between patients. This article describes three patients who were admitted to our hospital between November 2019 and October 2020. They presented with long-term non-healing wounds caused by M. abscessus infection after undergoing plastic surgery. Symptoms presented by the three patients included swelling, ulceration, secretion, and pain. After identification of M. abscessus with Ziehl-Neelsen staining and MALDI-TOF MS system, the patients were treated with surgical debridement and clarithromycin.
UNASSIGNED: It is important to note that a long-term wound that does not heal, especially after plastic surgery, should raise suspicion for M. abscessus infection. The infection mechanism in these three patients may have been due to exposure to surgical equipment that was not properly sterilized or due to poor sterile technique by the plastic surgeon. To prevent such infections, it is important to ensure proper sterilization of surgical equipment and saline.