A Case of Mycobacterium Fortuitum Infection in the Left Thigh.

BACKGROUND: In March 2024, our hospital confirmed a case of Mycobacterium fortuitum infection in the left thigh. In January 2024, the patient underwent buttock augmentation surgery at a private plastic surgery hospital. One month after the surgery, the patient sought medical attention at the plastic surgery hospital, due to pain in both legs while sitting. Upon examination, two subcutaneous masses were found in the left thigh, the tumors were painful to pressure, with obvious redness and swelling and elevated skin temperature; therefore, the patient was treated with intravenous infusion (cephalosporin drugs), but after one month of treatment, no significant improvement was observed. In order to seek additional diagnosis and treatment, the patient came to our hospital for treatment. METHODS: Clinical treatment of the left lower limb included wound debridement, abscess incision and drainage, and photodynamic therapy with 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT). During surgery, subcutaneous tough tissue was taken for pathogen examination, including acid fast staining, bacterial culture, and identification. Additional auxiliary examinations: urine routine, blood routine, coagulation function, liver function, kidney function, blood lipids, and blood sugar. RESULTS: Bacterial acid-fast staining: positive. Bacterial Culture and Identification (MALDI-TOF MS): Mycobacterium fortuitum. Clinical treatment plan: clarithromycin 500 mg po bid, moxifloxacin 400 mg po qd, abscess incision and drainage, ALA-PDT. After 24 days of treatment, the patient's condition was good, the surgical incision healed well, there was no bleeding, exudation, or bruising, no redness, swelling, or tenderness, and the skin temperature was normal. The patient improved and was discharged. CONCLUSIONS: This article reports a case of Mycobacterium fortuitum infection in the left thigh. The Mycobacterium fortuitum was quickly and accurately identified by MALDI-TOF MS, and reasonable treatment measures were adopted clinically. The patient improved and was discharged. I hope that in the future, this study can provide assistance for the clinical diagnosis and treatment of Mycobacterium fortuitum infections.

Impact of bacterial co-isolation on treatment initiation and long-term prognosis of patients with nontuberculous mycobacterial pulmonary disease: analysis of a prospective cohort study.

BACKGROUND: Nontuberculous mycobacterial pulmonary disease (NTM-PD), a chronic respiratory condition, presents a growing challenge globally. Uncertainties exist regarding the impact of concurrent bacterial co-isolation on treatment initiation and long-term prognosis. METHODS: This study analysed data from participants enrolled in an ongoing prospective observational cohort study on NTM-PD (NCT01616745) between 1 July 2011, and 31 December 2022, who provided sputum samples for bacterial culture at enrolment. Identification of potential pathogenic microorganisms (PPMs) was defined as a positive bacterial culture. Clinical characteristics were compared between NTM-PD patients with Pseudomonas, non-pseudomonal PPMs, and those without PPM co-isolation. Cox proportional hazard regression models were employed to assess the association of bacterial co-isolation with rates of NTM-PD treatment initiation and all-cause mortality. RESULTS: Overall, 453 patients (median age, 62 years; 30% male) were included in the analysis. PPMs were co-isolated in 77 patients (17%), including 13 with Pseudomonas species. Co-isolation of Pseudomonas was associated with a significantly higher erythrocyte sedimentation rate (P = 0.02) and St. George's Respiratory Questionnaire score (P = 0.01). Non-pseudomonal PPM co-isolation was significantly associated with a higher likelihood of NTM-PD treatment initiation (adjusted hazards ratio [aHR], 1.56, 95% confidence interval [CI], 1.03-2.36, P = 0.036), whereas co-isolation of Pseudomonas was independently correlated with increased all-cause mortality (aHR, 3.25, 95% CI, 1.08-9.84, P = 0.037). CONCLUSIONS: Our findings emphasize the importance of microbial surveillance, as bacterial co-isolation affects treatment initiation and prognosis in patients with NTM-PD.

Facial cutaneous tuberculosis infected by non-tuberculous mycobacteria.

BACKGROUND: Cutaneous infections caused by non-tuberculous mycobacteria (NTM) are extremely rare, particularly when they are localized to the facial area. This condition presents significant diagnostic challenges due to its unusual presentation and the need for precise microbiological identification. CASE PRESENTATION: A two-year-old male patient presented with a progressively enlarging reddish-brown mass on the left side of his face. Despite the absence of systemic symptoms, the lesion's growth warranted investigation due to its growth. Ultrasonography showed a hypoechoic mass in the dermis, indicating an underlying abscess. The subsequent aspiration resulted in pale yellow pus, which upon testing and culture, confirmed the presence of Mycobacterium avium complex infection, a species of NTM. This case exemplifies the synergy between imaging modalities and microbiological analysis, highlighting the crucial role of both in achieving favorable clinical outcomes in patients with suspected cutaneous NTM infections. Ultrasound can expedite diagnosis, improve treatment planning, and enhance patient care by enabling targeted interventions and monitoring response to therapy in these scenarios. However, it is the combination of pathogen-specific diagnostics that ensures accurate etiological attribution and appropriate antimicrobial stewardship. CONCLUSION: Although rare, facial cutaneous infections caused by NTM still deserve thorough investigation to determine the exact cause. Ultrasound is used to identify cutaneous lesions, measure their extent, and guide surgical procedures. The ultimate diagnosis is based on microbiological confirmation.

Successful treatment of Keratitis caused by Mycobacterium chelonae and an overview of previous cases in Europe.

INTRODUCTION AND PURPOSE: Mycobacterium (M.) chelonae is responsible for a half of relatively rare nontuberculous mycobacteria (NTM) keratitis. We report a case of M. chelonae keratitis in a woman following sclerocorneal suture extraction after cataract surgery. RESULTS: A 70-year-old woman presented with a red eye and corneal infiltration of her left eye six weeks following sclerocorneal suture extraction after an elective cataract surgery in another institute. She complained of a sharp, cutting pain and photophobia. Since initial corneal scrapes and conjunctival swabs proved no pathogen using culture and PCR methods, non-specific antibiotics and antifungal agents were administered. As keratitis was complicated by an inflammation in the anterior chamber and vitreous, samples of the vitreous fluid were sent for microbiologic examination. DNA of Epstein-Barr virus (EBV) was repeatedly detected. Since the intrastromal abscess had formed, corneal re-scrapings were performed and M. chelonae was detected using culture, MALDI-TOF MS and PCR methods. Therapy was changed to a combination of oral and topical clarithromycin, intravitreal, topical and intracameral amikacin, and oral and topical moxifloxacin. The successful therapy led to stabilization. The optical penetrating keratoplasty was performed and no signs of the infection recurrence were found. CONCLUSIONS: The diagnosis of nontuberculous mycobacterial keratitis is difficult and often delayed. An aggressive and prolonged antimicrobial therapy should include systemic and topical antibiotics. Surgical intervention in the form of corneal transplantation may be required in the active and nonresponsive infection. In the presented case this was necessary for visual rehabilitation due to scarring.

Effect of comorbidities on mortality in patients with nontuberculous mycobacterial infection in Korea: National Health Insurance Service-National Sample Cohort data.

Patients with nontuberculous mycobacteria (NTM) infection have multiple comorbidities, but the impact of comorbidities on mortality are not well known. We aimed to compare the mortality between people with and without NTM infection and associated comorbidities and their prognostic value on mortality using National Health Insurance Service-National Sample Cohort data from 2006 to 2019. In this matched cohort study, people with and without NTM infection aged 20-89 years were matched 1:4 by sex, age, region, and income. The hazard ratios (HRs) with 95% confidence intervals (CIs) of mortality in patients with NTM infection were estimated using a Cox proportional hazard regression model. In total, 2421 patients with NTM infection (mean age, 54.8 years) and 9684 controls were included. NTM-infected patients had a significantly increased risk of mortality than matched controls in the multivariable model adjusted for age, sex, region, income, and Charlson comorbidity index (aHR = 1.88, 95% CI 1.65-2.14). Among patients with NTM infection, respiratory comorbidities including chronic obstructive pulmonary disease, asthma, interstitial lung disease, and moderate to severe liver disease and malignancy were positively associated with mortality. NTM infection was independently associated with an increased risk of mortality, and mortality risk in patients with NTM infection may be increased by coexisting comorbidities.

Nontuberculous mycobacterial pulmonary disease (NTM PD) incidence trends in the United States, 2010-2019.

BACKGROUND: Nontuberculous mycobacteria (NTM) are ubiquitous environmental bacteria that cause chronic lung disease. Rates of NTM pulmonary disease (NTM PD) have increased over the last several decades, yet national estimates in the United States (US) have not been assessed since 2015. METHODS: We used a nationally representative population of Medicare beneficiaries aged ≥ 65 years to assess rates of NTM PD in a high-risk population from 2010 to 2019. Poisson generalized linear models were used to assess the annual percent change in incidence in the overall population and among key demographic groups such as sex, geography, and race/ethnicity. We evaluated the relative prevalence of various comorbid conditions previously found to be associated with NTM PD. RESULTS: We identified 59,724 cases of incident NTM PD from 2010 to 2019 from an annual mean population of 29,687,097 beneficiaries, with an average annual incidence of 20.1 per 100,000 population. NTM PD incidence was overall highest in the South and among women, Asian individuals, and persons aged ≥ 80 years relative to other studied demographic groups. The annual percent change in NTM PD incidence was highest in the Northeast, at 6.5%, and Midwest, at 5.9%, and among women, at 6.5%. Several comorbid conditions were highly associated with concurrent NTM diagnosis, including allergic bronchopulmonary aspergillosis, bronchiectasis, and cystic fibrosis. CONCLUSIONS: Here we provide current estimates of NTM PD incidence and prevalence and describe increasing trends in the US from 2010 to 2019. Our study suggests a need for improved healthcare planning to handle an increased future caseload, as well as improved diagnostics and therapeutics to better detect and treat NTM PD in populations aged ≥ 65 years.

Nontuberculous Mycobacterial Pulmonary Disease: Patients, Principles, and Prospects.

Nontuberculous mycobacterial pulmonary disease (NTM-PD) is increasing in incidence globally and challenging to manage. The 2020 multisociety treatment guideline and the 2022 consensus recommendations provide comprehensive evidence-based guides to manage pulmonary diseases caused by the most common NTM. However, with >190 different NTM species that may require different multidrug regimens for treatment, the breadth and complexity of NTM-PD remain daunting for both patients and clinicians. In this narrative review, we aim to distill this broad, complex field into principles applicable to most NTM species and highlight important nuances, specifically elaborating on the presentation, diagnosis, principles of patient-centered care, principles of pathogen-directed therapy, and prospects of NTM-PD.

Comparative analysis of non-tuberculous mycobacterial lung disease and lung colonization: a case-control study.

BACKGROUND: Non-tuberculous mycobacteria (NTM) are common opportunistic pathogens, and the most common infection site is lung. NTM are found commonly in the environment. Many patients have NTM lung colonization (NTM-Col). NTM lung disease (NTM-LD) have no specific sympotms, though it is hard to differentiate NTM-LD and NTM-Col under this circumstance. The aim of this study is to explore the differences between NTM-LD and NTM-Col for future clinical diagnosis and treatment. METHODS: We retrospectively enrolled patients who had a history of NTM isolated from respiratory specimens in Peking Union Medical College Hospital (PUMCH) from January 1st, 2013 to December 31st, 2022. Patients were classified into NTM-LD group and NTM-Col group. Demographic characteristics, clinical manifestations, laboratory tests and imaging findings of the two groups were compared. Comparative analysis was also performed in peripheral blood lymphocyte subsets among three groups. RESULTS: A total of 127 NTM-LD patients and 37 NTM-Col patients were enrolled. Proportion of patients with bronchiectasis was higher in NTM-LD group than in NTM-Col group (P = 0.026). Predominant NTM isolates were Mycobacterium avium complex (MAC). NTM-LD group had a higher proportion of Mycobacterium intracellulare (P = 0.004). CD4+ T cells counts was lower in NTM-LD group (P = 0.041) than in NTM-Col group. Imaging finding of bronchiectasis (P = 0.006) was higher in NTM-LD group than in NTM-Col group. Imaging findings of bronchiectasis (OR = 6.282, P = 0.016), and CD4+ T cell count (OR = 0.997, P = 0.012) were independent associated factors for differential diagnosis between NTM-LD and NTM-Col. CONCLUSION: NTM isolates from both NTM-LD and NTM-Col patients were predominantly MAC, with a higher Mycobacterium intracellulare isolation rate in NTM-LD group. Imaging findings of bronchiectasis and lower peripheral blood CD4+ T cell count may be helpful to separate the diagnosis of NTM-LD from NTM-Col.

Case report: Mycobacterium chimaera-induced lymph node infection in a patient with chronic myeloproliferative neoplasm misdiagnosed as tuberculous lymphadenitis.

Herein, we report a case of lymphadenitis caused by Mycobacterium chimaera. A 54-year-old woman with chronic myeloproliferative neoplasm was admitted to the hospital with cervical lymphadenopathy. After preliminary exclusion of various diseases such as lymphoma, Epstein-Barr virus infection, and autoimmune disease, a lymph node biopsy specimen showed epithelioid granulomatous lymphadenitis with caseous necrosis, epithelial-like cells, and multinucleated giant cells as seen in tuberculosis (TB). Although Mycobacterium tuberculosis was never isolated, diagnostic anti-TB treatment was commenced. Following over 9 months of treatment, there was no significant reduction in the size of her cervical lymph nodes, and she continued to experience recurrent low-grade fevers. One sample from the fourth lymph node biopsy tested negative for metagenomic next-generation sequencing (mNGS), and another sample tested positive in the BACTEC MGIT960 liquid culture system, identifying the strains as Mycobacterium chimaera. Anti-non-tuberculous mycobacteria (NTM) therapy was initiated, and the patient achieved symptom improvement. In conclusion, NTM lymphoid infection is easily misdiagnosed as long-term etiologic negativity.

Non-Tuberculous Mycobacterial Pulmonary Disease with Detection of Mycobacterium Tuberculosis in Pleural Fluid.

BACKGROUND: Tuberculous pleurisy (TP) is one of the most common types of extrapulmonary tuberculosis, often secondary to tuberculosis (TB). Clinical and imaging manifestations of non-tuberculous mycobacterial pulmonary diseases (NTM-PD) are usually similar to those of tuberculosis. Because of their similarity and the high incidence of tuberculosis, non-tuberculous mycobacterial infections are often overlooked for a long time. Especially in people without immunodeficiency. METHODS: Mycobacterium tuberculosis (MTB) in pleural effusion was found by metagenomic next-generation sequencing (mNGS). During anti-tuberculosis treatment, mNGS of lung tissue by ultrasound-guided percutaneous lung puncture revealed that this patient had combined NTM-PD. RESULTS: Mycobacterium chelonae (M. chelonae) was detected by mNGS, and after anti-NTM treatment, the patient's chest CT showed that the inflammation was absorbed more than before, and the patient's symptoms improved. CONCLUSIONS: When TB is poorly treated with standardized anti-tuberculosis therapy, comorbid non-tuberculous mycobacterial infections may be considered, and mNGS may complement traditional pathogenetic testing.

The Intriguing Pattern of Nontuberculous Mycobacteria in Bulgaria and Description of Mycobacterium bulgaricum sp. nov.

We investigated the rise of nontuberculous mycobacteria (NTM) infections in Bulgaria, focusing on species identification and distribution from 2018 to 2022. Utilizing advanced diagnostic tools, including the Hain Mycobacterium CM/AS method, Myco-biochip assay, and whole-genome sequencing, the study identifies and characterizes a diverse range of Mycobacterium species from clinical samples. While M. avium, M. gordonae, M. fortuitum, and M. chelonae were dominating, a number of rare species were also found. They include such species as M. marseillense and M. celatum. Moreover, the noticeable prevalence of M. terrae complex species missed by conventional testing was observed. We identified a rare species, highly homologous to previously described strains from Japan; based on genome-genome distance data, we propose its reannotation as a new species. Further, a novel species was identified, which is significantly distinct from its closest neighbor, M. iranicum, with ANI = 87.18%. Based on the SeqCode procedure, we propose to name this new species Mycobacterium bulgaricum sp. nov. Dynamic changes in NTM species prevalence in Bulgaria observed from 2011 to 2022 highlight the emergence of new species and variations tied to environmental and demographic factors. This underscores the importance of accurate species identification and genotyping for understanding NTM epidemiology, informing public health strategies, and enhancing diagnostic accuracy and treatment protocols.

Dual spatial host-bacterial gene expression in Mycobacterium abscessus respiratory infections.

Co-localization of spatial transcriptome information of host and pathogen can revolutionize our understanding of microbial pathogenesis. Here, we aimed to demonstrate that customized bacterial probes can be successfully used to identify host-pathogen interactions in formalin-fixed-paraffin-embedded (FFPE) tissues by probe-based spatial transcriptomics technology. We analyzed the spatial gene expression of bacterial transcripts with the host transcriptomic profile in murine lung tissue chronically infected with Mycobacterium abscessus embedded in agar beads. Customized mycobacterial probes were designed for the constitutively expressed rpoB gene (an RNA polymerase ß subunit) and the virulence factor precursor lsr2, modulated by oxidative stress. We found a correlation between the rpoB expression, bacterial abundance in the airways, and an increased expression of lsr2 virulence factor in lung tissue with high oxidative stress. Overall, we demonstrate the potential of dual bacterial and host gene expression assay in FFPE tissues, paving the way for the simultaneous detection of host and bacterial transcriptomes in pathological tissues.

Nationwide evaluation of treatment outcomes and survival of patients with non-tuberculous mycobacterial pulmonary disease.

Treatment outcomes and long-term survival of non-tuberculous mycobacterial pulmonary disease (NTM-PD) in a real-world setting are difficult to assess, especially for species other than Mycobacterium avium complex (MAC).This was a retrospective cohort study on all Croatian residents with respiratory NTM isolates from 2006 to 2015, with follow-up to 2020.Therapy was started in 98/137 (71.5%) of patients, significantly more often in patients with fibrocavitary disease and/or sputum smear positivity. Unsuccessful treatment outcomes were recorded in 39/98 (39.8%) patients (14 deaths and 25 treatment failures). One-year and 5-year all-cause mortality were respectively 18.2% and 37.6%. Guideline-based treatment (GBT) was started in 50/98 (51%) of treated patients and followed for the recommended duration in 35.7% (35/98). This resulted in a higher chance of cure (OR 3.79, 95% CI 1.29 to 11.1; P = 0.012) than inadequately treated/untreated patients. For Mycobacterium xenopi disease, high cure rates (>80%) were achieved both with GBT and non-GBT treatment regimens.Guideline-based therapy resulted in a four-time higher chance of being cured. The impact of GBT on treatment outcomes was clear for MAC disease, but no apparent effect was observed for patients with M. xenopi disease..

Complex trauma sequelae: Mycobacterium goodii and Priestia endophytica Hardware infection in a patient with Ehlers-Danlos syndrome.

A 26-year-old man with Ehlers-Danlos syndrome, recurrent otitis externa, and chronic otitis media sustained a left lower extremity amputation and open femur fracture with internal hardware fixation after a motor vehicle collision in Arizona. He presented to the emergency department at our institution with severe left leg pain and purulent discharge despite receiving two unidentified antibiotics upon discharge. Evaluations revealed an abscess and malunion of the femur. Initial cultures yielded scant Priestia endophytica, leading to daptomycin treatment. His condition worsened until Gram-positive bacilli identified as Mycobacterium goodii, a rare nosocomial mycobacterial species, were found. Significant improvement occurred with appropriate antibiotics. This case highlights the challenges in diagnosing and managing M. goodii infections in immunocompromised patients with orthopedic complications and notes P. endophytica as a previously unreported, possibly opportunistic human pathogen.

The effect of smoking on nontuberculous mycobacterial pulmonary disease and tuberculosis: a nationwide retrospective cohort study.

Although smoking is an established risk factor for Mycobacterial infection, the association between smoking and nontuberculous mycobacterial pulmonary disease (NTM-PD) remains unclear. We evaluated the association between smoking and NTM-PD and tuberculosis (TB) using a population-based South Korean nationwide cohort. Using the Korean National Health Insurance Database, we screened individuals over 20 years of age who underwent the national health screening program in 2009. Out of 3,774,308 eligible populations, we identified 2,964 and 26,112 cases of newly developed NTM-PD and TB, respectively. We used multivariate Cox proportional hazards models to estimate the adjusted hazard ratios (aHRs) of risk factors for NTM-PD and TB. The incidence rates for developing NTM-PD and TB were 0.08 and 0.68 per 1,000 person-years, respectively. Current smokers (aHR 0.63, 95% confidence interval [CI] 0.56-0.71) and current heavy smokers (≥ 20 pack-years, aHR 0.74, 95% CI 0.63-0.86) were at lower risk for NTM-PD development than never smokers. On the contrary, current smokers (aHR 1.19, 95% CI 1.15-1.23) and current heavy smokers (aHR 1.27, 95% CI 1.22-1.33) had a higher risk for TB development than never smokers. These trends were augmented if individuals started smoking before age 20 years. In subgroup analyses stratified by age, these trends were prominent in the 40-64 years age range. Current smoking was associated with a decreased risk of NTM-PD and increased risk of TB. These risks were augmented by early smoking initiation and in the middle age population.

A novel hematological score (HS) and its related nomogram model to predict nontuberculous mycobacterial pulmonary disease in patients with suspected multidrug-resistant pulmonary tuberculosis.

BACKGROUND: Nontuberculous mycobacteria pulmonary disease (NTM-PD) exhibits clinical and radiological characteristics similar to those of pulmonary tuberculosis (PTB). This study aimed to develop a novel hematological score (HS) and its related nomogram model to identify NTM-PD in patients with suspected multidrug-resistant pulmonary tuberculosis (SMDR-PTB) due to lack of response to first-line anti-TB treatment (ATT). METHODS: We retrospectively recruited patients with SMDR-PTB from Wuhan Jinyintan Hospital between January 2014 and January 2022. These patients were divided into NTM-PD and MDR-PTB groups based on pathogen test results. Participants were randomly allocated to training and validation set in a 7:3 ratio. The ROC and LASSO regression were employed to select variables. Multivariate logistic analysis was conducted on the training set to develop the HS and its related nomogram models, followed by internal validation on the validation set. RESULTS: The HS was constructed and developed on CKMB, ADA, GGT, LDL, and UHR, demonstrating good predictive value with AUCs of 0.900 and 0.867 in the training and validation sets, respectively. The HS-based nomogram model consists of Age, Gender, DM, HIV infection, ILD and HS, and exhibited strong discriminative ability, accuracy, and clinical utility in two sets. The AUCs were 0.930 and 0.948 in the training set and validation set, respectively. CONCLUSION: HS may be a useful biomarker for identifying NTM-PD in patients with SMDR-PTB. The HS-based nomogram model serves as a convenient and efficient tool for guiding the treatment of SMDR-PTB patients.

Mycobacterium abscessus Cutaneous Infection in the Immunosuppressed: A Case Report on an Atypical Pathogen.

Mycobacterium abscessus complex (MAbc) is a rapidly growing nontuberculous mycobacterium that represents an increasingly prevalent cause of skin infections. This report describes an atypical presentation of MAbc to heighten physician awareness of the pathogen. A 69-year-old woman with immunocompromised status presented with a 4-month history of a solitary, nonhealing ulcer on her right lower extremity after an insect bite. After no improvement following oral amoxicillin/clavulanate and topical mupirocin for the initial diagnosis of cellulitis, biopsy and culture of the lesion revealed MAbc. Microscopic examination revealed reactive cutaneous inflammation without evidence of malignancy. Acid-fast bacteria (AFB) stain was negative, and no granulomas were noted histologically. Clarithromycin and doxycycline were initiated while awaiting susceptibility testing results. Final culture showed MAbc sensitive to amikacin, cefoxitin, and clarithromycin. Unfortunately, before antibiotic therapy could be modified, the patient died. The presentation of a solitary lower-extremity ulcer is rare compared with current literature. This case occurred after a suspected insect bite rather than instrumentation. In addition, this case demonstrated negative AFB stain and absence of granulomas on histologic analysis. The patient's death did not allow for evaluation of treatment efficacy. Existing literature characterizing MAbc is sparse. Most cases present as multiple papules, nodules, and abscesses with positive AFB staining and granulomas; it is possible for deviations to exist depending on host immune status. Considering the highly drug-resistant nature of M abscessus, prompt diagnosis and treatment are crucial. For this to occur, clinicians must maintain high clinical suspicion for M abscessus infection in any chronic, nonhealing wound failing to respond to initial treatment.

Contribution of machine learning for subspecies identification from Mycobacterium abscessus with MALDI-TOF MS in solid and liquid media.

Mycobacterium abscessus (MABS) displays differential subspecies susceptibility to macrolides. Thus, identifying MABS's subspecies (M. abscessus, M. bolletii and M. massiliense) is a clinical necessity for guiding treatment decisions. We aimed to assess the potential of Machine Learning (ML)-based classifiers coupled to Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) MS to identify MABS subspecies. Two spectral databases were created by using 40 confirmed MABS strains. Spectra were obtained by using MALDI-TOF MS from strains cultivated on solid (Columbia Blood Agar, CBA) or liquid (MGIT®) media for 1 to 13 days. Each database was divided into a dataset for ML-based pipeline development and a dataset to assess the performance. An in-house programme was developed to identify discriminant peaks specific to each subspecies. The peak-based approach successfully distinguished M. massiliense from the other subspecies for strains grown on CBA. The ML approach achieved 100% accuracy for subspecies identification on CBA, falling to 77.5% on MGIT®. This study validates the usefulness of ML, in particular the Random Forest algorithm, to discriminate MABS subspecies by MALDI-TOF MS. However, identification in MGIT®, a medium largely used in mycobacteriology laboratories, is not yet reliable and should be a development priority.

Atypical Mycobacterium abscessus BlaRI Ortholog Mediates Regulation of Energy Metabolism but Not ß-Lactam Resistance.

Mycobacterium abscessus (Mab) is highly drug resistant, and understanding regulation of antibiotic resistance is critical to future antibiotic development. Regulatory mechanisms controlling Mab's ß-lactamase (BlaMab) that mediates ß-lactam resistance remain unknown. S. aureus encodes a prototypical protease-mediated two-component system BlaRI regulating the ß-lactamase BlaZ. BlaR binds extracellular ß-lactams, activating an intracellular peptidase domain which cleaves BlaI to derepress blaZ. Mycobacterium tuberculosis (Mtb) encodes homologs of BlaRI (which we will denote as BlaIR to reflect the inverted gene order in mycobacteria) that regulate not only the Mtb ß-lactamase, blaC, but also additional genes related to respiration. We identified orthologs of blaIRMtb in Mab and hypothesized that they regulate blaMab. Surprisingly, neither deletion of blaIRMab nor overexpression of only blaIMab altered blaMab expression or ß-lactam susceptibility. However, BlaIMab did bind to conserved motifs upstream of several Mab genes involved in respiration, yielding a putative regulon that partially overlapped with BlaIMtb. Prompted by evidence that respiration inhibitors including clofazimine induce the BlaI regulon in Mtb, we found that clofazimine triggers induction of blaIRMab and its downstream regulon. Highlighting an important role for BlaIRMab in adapting to disruptions in energy metabolism, constitutive repression of the BlaIMab regulon rendered Mab highly susceptible to clofazimine. In addition to our unexpected findings that BlaIRMab does not regulate ß-lactam resistance, this study highlights the novel role of mycobacterial BlaRI-type regulators in regulating electron transport and respiration.