Mycobacterium Abscessus Subspecies Research Articles

Notes from the Field: Potential Outbreak of Extrapulmonary Mycobacterium abscessus subspecies massiliense Infections from Stem Cell Treatment Clinics in Mexico - Arizona and Colorado, 2022.

Mycobacterium abscessus is an intrinsically drug-resistant, rapidly growing, nontuberculous mycobacterium; extrapulmonary infections have been reported in association with medical tourism (1). During November-December 2022, two Colorado hospitals (hospitals A and B) treated patient A, a Colorado woman aged 30-39 years, for M. abscessus meningitis. In October 2022, she had received intrathecal donor embryonic stem cell injections in Baja California, Mexico to treat multiple sclerosis and subsequently experienced headaches and fevers, consistent with meningitis. Her cerebrospinal fluid revealed neutrophilic pleocytosis and grew M. abscessus in culture at hospital A. Hospital A's physicians consulted hospital B's infectious diseases (ID) physicians to co-manage this patient (2).

Clinical characteristics and antimicrobial susceptibility profiles of Mycobacterium abscessus and Mycobacterium massiliense pulmonary infection

IntroductionMycobacterium abscessus complex (MABC) is the most common rapidly growing Mycobacterium species in structural pulmonary diseases and can be life-threatening. This study aimed to assess the clinical characteristics and drug susceptibility statuses of different Mycobacterium abscessus (MAB) subspecies. MethodsDNA sequencing was used to differentiate clinical MABC subspecies isolates. The Clinical and Laboratory Standards Institute guidelines were used to determine vitro susceptibility of imipenem-relebactam, omadacycline, and other conventional antibiotics. Patient's clinical characteristics were collected and analysed. ResultsThere were 139 M. abscessus, 39 M. massiliense, and 1 M. bolletii, accounting for 77.7%, 21.8%, and 0.5% of the MABC isolates, respectively. Patients with M. abscessus pulmonary disease (M.ab-PD) had higher proportions of older adults, tuberculosis history, chronic pulmonary disease, and malignancy than those with M. massiliense pulmonary disease (M.ma-PD). Patients with M.ab-PD had higher rates of bilateral middle- and lower-lobe involvement than patients with M.ma-PD. Both subspecies showed high resistance rates to doxycycline and moxifloxacin, and clarithromycin-induced resistance was more common in M.ab than in M.ma. Relebactam with imipenem resulted in a 2-fold reduction in the minimum inhibitory concentration (MIC) value compared to imipenem alone among MAB; furthermore, the MIC was lower in M.ab than in M.ma. Omadacycline and tigecycline had comparable in vitro susceptibility, and the MIC showed no statistically significant difference between M.ab and M.ma. ConclusionsM.ab is the most prevalent MABC subspecies in the Zhejiang Province. Patients with M.ab-PD have complex underlying diseases and broader lobar lesions. Imipenem-relebactam and omadacycline are promising antibiotics for MABC infection treatment.

Epidemiological Characteristics of Nontuberculous Mycobacterial Pulmonary Disease in South Korea: A Meta-Analysis of Individual Participant Data.

Despite the global increase in nontuberculous mycobacterial pulmonary disease (NTM-PD), there are geographical variations in clinical characteristics. We investigated the clinical characteristics of patients with NTM-PD in South Korea. We systematically reviewed articles about patients with NTM-PD in South Korea until February 2022. Individual participant data, regardless of treatment, were collected using a standard case report form. The data of 6,489 patients from 11 hospitals between 2002 and 2019 were analyzed. The mean age was 61.5 ± 11.7 years, and 57.7% were women. Mycobacterium avium (41.4%) and Mycobacterium intracellulare (38.4%) comprised most of the causative species, followed by Mycobacterium abscessus subspecies abscessus (8.6%) and Mycobacterium abscessus subspecies massiliense (7.8%). Bronchiectasis (59.4%) was the most common pulmonary comorbidity. Reported cases of NTM-PD increased over the years, but the proportions of causative species and radiologic forms remained similar. Distinct clinical characteristics were observed according to age and sex. Men were older at the time of diagnosis (median 63.8 vs. 59.9 years, P<0.001) and had more cavitary lesions than women (38.8% vs. 21.0%, P<0.001). The older group (≥65 years) had a higher proportion of patients with body mass index <18.5 kg/m2 (27.4% vs. 18.6%, P<0.001) and cavitary lesions (29.9% vs. 27.6%, P=0.009) than the younger group. We performed a meta-analysis of the clinical characteristics of patients with NTM-PD in South Korea. We found age- and sex-related differences in disease-specific severity. Further investigation would enhance our comprehension of the disease's nature and inherited and acquired host factors.

In vitro susceptibility of nontuberculous mycobacteria in China

ObjectivesThis study aimed to measure the prevalence of resistance to antimicrobial agents, and explore the risk factors associated with drug resistance by using nontuberculous Mycobacteria (NTM) isolates from China.MethodsA total of 335 NTM isolates were included in our analysis. Broth dilution method was used to determine in vitro drug susceptibility of NTM isolates.ResultsClarithromycin (CLA) was the most potent drug for Mycobacterium intracellulare (MI). The resistance rate of 244 MI isolates to CLA was 21%, yielding a minimum inhibitory concentrations (MIC)50 and MIC90 of 8 and 64 mg/L, respectively. 51% of 244 MI isolates exhibited resistance to amikacin (AMK). For 91 Mycobacterium abscessus complex (MABC) isolates, 6 (7%) and 49 (54%) isolates were categorized as resistant to CLA at day 3 and 14, respectively. The resistance rate to CLA for Mycobacterium abscessus subspecies abscessus (MAA) was dramatically higher than that for Mycobacterium abscessus subspecies massiliense (MAM). Additionally, the percentage of patients presenting fever in the CLA-susceptible group was significantly higher than that in the CLA-resistant group.ConclusionsOur data demonstrate that approximate one fifth of MI isolates are resistant to CLA. We have identified a higher proportion of CLA-resistant MAA isolates than MAM. The patients caused by CLA-resistant MI are at low risk for presenting with fever relative to CLA-susceptible group.

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

ABSTRACT 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.

Evaluation of cleaning and sterilization of liposuction cannulas after intentional contamination with human fat, Mycobacterium abscessus subspecies bolletii, and Geobacillusstearothermophilus

The complex design of cannulas is a major challenge for reprocessing due to the retention and accumulation of fat residues. To evaluate the cleaning of liposuction cannulas and the protective effects of the remaining fat residues on Mycobacterium abscessus subspecies bolletii (MASB) and Geobacillus stearothermophilus inactivation during steam sterilization. In phase 1, six standard operating procedures were evaluated for cleaning of liposuction cannulas. In phase 2, the sectioned lumens of the liposuction cannulas were contaminated with the largest and smallest volume of human fat found in phase 1, with the addition of MASB. In phase 3, the same volumes of human fat from phase 2 were used to contaminate paper strips containing G.stearothermophilus. In phase 1 the residual amount of fat ranged from 6 to 52mg. In phases 2 and 3, the minimum and maximum amounts of fat (6 and 50mg) protected micro-organisms during steam sterilization at 134°C for 1.5 and 3min. Effective cleaning and sterilization of liposuction cannulas intentionally contaminated with human fat, MASB and G.stearothermophilus could not be performed.

The synergetic effect of sitafloxacin–arbekacin combination in the Mycobacterium abscessus species

Mycobacterium abscessus species (MABS) is the most commonly isolated rapidly growing mycobacteria (RGM) and is one of the most antibiotic-resistant RGM with rapid progression, therefore, treatment of MABS is still challenging. We here presented a new combination treatment with sitafloxacin that targeted rough morphotypes of MABS, causing aggressive infections. Thirty-four clinical strains of MABS were isolated from various clinical samples at the Juntendo university hospital from 2011 to 2020. The susceptibility to a combination of sitafloxacin and antimicrobial agents was compared to that of the antimicrobial agents alone. Out of 34 MABS, 8 strains treated with sitafloxacin–amikacin combination, 9 of sitafloxacin–imipenem combination, 19 of sitafloxacin–arbekacin combination, and 9 of sitafloxacin–clarithromycin combination showed synergistic effects, respectively. Sitafloxacin–arbekacin combination also exhibited the synergistic effects against 10 of 22 Mycobacterium abscessus subspecies massiliense (Mma) strains and 8 of 11 Mycobacterium abscessus subspecies abscessus (Mab) strains, a highly resistant subspecies of MABS. The sitafloxacin–arbekacin combination revealed more synergistic effects in rough morphotypes of MABS (p = 0.008). We demonstrated the synergistic effect of the sitafloxacin–arbekacin combination against MABS. Further, this combination regimen might be more effective against Mab or rough morphotypes of MABS.

Identification of Mycobacterium abscessus Subspecies by MALDI-TOF Mass Spectrometry and Machine Learning.

Mycobacterium abscessus is one of the most common and pathogenic nontuberculous mycobacteria (NTM) isolated in clinical laboratories. It consists of three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. Due to their different antibiotic susceptibility pattern, a rapid and accurate identification method is necessary for their differentiation. Although matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has proven useful for NTM identification, the differentiation of M. abscessus subspecies is challenging. In this study, a collection of 325 clinical isolates of M. abscessus was used for MALDI-TOF MS analysis and for the development of machine learning predictive models based on MALDI-TOF MS protein spectra. Overall, using a random forest model with several confidence criteria (samples by triplicate and similarity values >60%), a total of 96.5% of isolates were correctly identified at the subspecies level. Moreover, an improved model with Spanish isolates was able to identify 88.9% of strains collected in other countries. In addition, differences in culture media, colony morphology, and geographic origin of the strains were evaluated, showing that the latter had an impact on the protein spectra. Finally, after studying all protein peaks previously reported for this species, two novel peaks with potential for subspecies differentiation were found. Therefore, machine learning methodology has proven to be a promising approach for rapid and accurate identification of subspecies of M. abscessus using MALDI-TOF MS.

Rapid and Accurate Discrimination of Mycobacterium abscessus Subspecies Based on Matrix-Assisted Laser Desorption Ionization-Time of Flight Spectrum and Machine Learning Algorithms.

Mycobacterium abscessus complex (MABC) has been reported to cause complicated infections. Subspecies identification of MABC is crucial for adequate treatment due to different antimicrobial resistance properties amid subspecies. However, long incubation days are needed for the traditional antibiotic susceptibility testing (AST). Delayed effective antibiotics administration often causes unfavorable outcomes. Thus, we proposed a novel approach to identify subspecies and potential antibiotic resistance, guiding early and accurate treatment. Subspecies of MABC isolates were determined by secA1, rpoB, and hsp65. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) spectra were analyzed, and informative peaks were detected by random forest (RF) importance. Machine learning (ML) algorithms were used to build models for classifying MABC subspecies based on spectrum. The models were validated by repeated five-fold cross-validation to avoid over-fitting. In total, 102 MABC isolates (52 subspecies abscessus and 50 subspecies massiliense) were analyzed. Top informative peaks including m/z 6715, 4739, etc. were identified. RF model attained AUROC of 0.9166 (95% CI: 0.9072-0.9196) and outperformed other algorithms in discriminating abscessus from massiliense. We developed a MALDI-TOF based ML model for rapid and accurate MABC subspecies identification. Due to the significant correlation between subspecies and corresponding antibiotics resistance, this diagnostic tool guides a more precise and timelier MABC subspecies-specific treatment.

Isolation and Antimicrobial Susceptibility of Nontuberculous Mycobacteria in a Tertiary Hospital in Korea, 2016 to 2020.

There is a global increase in isolation of nontuberculous mycobacteria (NTM). The aim of the study was to analyze longitudinal trends of NTM identification and pattern of antimicrobial susceptibility testing. NTM recovery rates, distribution of NTM species identification, and antimicrobial susceptibility pattern of NTM at Pusan National University Yangsan Hospital between January 2016 and December 2020 were retrospectively analyzed. A total of 52,456 specimens from 21,264 patients were submitted for mycobacterial culture, of which 2,521 from 1,410 patients were NTM positive over five years (January 2016 to December 2020). NTM isolation showed an increasing trend from 2016 to 2020 (p&lt;0.001, test for trend) mainly caused by Mycobacterium avium complex. The vast majority of M. avium complex were susceptible to key agents clarithromycin and amikacin. For Mycobacterium kansasii, resistance to rifampin and clarithromycin is rare. Amikacin was the most effective drug against Mycobacterium abscessus subspecies abscessus and Mycobacterium subspecies massiliense. Most of M. subspecies massiliense were susceptible to clarithromycin, while the majority of M. abscessus subspecies abscessus were resistant to clarithromycin (p&lt;0.001). There was an increasing trend of NTM isolation in our hospital. Resistance to key drugs was uncommon for most NTM species except for M. abscessus subspecies abscessus against clarithromycin.

Treatment Outcome in Patients with Mycobacterium abscessus Complex Lung Disease: The Impact of Tigecycline and Amikacin.

Background: The contemporary guidelines have recommended multiple antimicrobial therapies along with oral macrolides for the treatment of Mycobacterium abscessus complex lung disease (MABC-LD). However, there is little evidence supporting the parenteral tigecycline-containing regimens against MABC-LD. Therefore, we conducted this study to evaluate the effect of intravenous tigecycline-containing regimens on the treatment of MABC-LD. Methods: A retrospective study was conducted in 6 medical centers. Patients with MABC-LD that were followed up at ≥12 months were enrolled. Mycobacterium abscessus subspecies were identified by hsp65, rpoB, secA1 gene PCR, and sequencing. Antimicrobial susceptibility was determined for 34 patients using broth microdilution methods following the Clinical and Laboratory Standards Institute (CLSI) guideline. The microbiology and treatment outcomes were defined as either success or failure. The impacts of tigecycline and amikacin were adjusted for age, comorbidities, surgical resection, and radiologic scores. Results: During the study period, seventy-one patients were enrolled for final analysis. The microbiology failure rate was 61% (43/71) and the treatment failure rate was 62% (44/71). For M. abscessus complex, 97% (33/34) of tigecycline MIC were ≤1 mg/L. Amikacin also demonstrated great susceptibility (94.1%; 32/34). Treatment with regimens containing tigecycline plus amikacin provided better microbiology success (adjusted OR 17.724; 95% CI 1.227–267.206) and treatment success (adjusted OR 14.085; 95% CI 1.103–166.667). Conclusion: The outcome of MABC-LD is always unsatisfactory. Treatment regimens with oral macrolide in combination with tigecycline and amikacin were correlated with increased microbiology success and less treatment failure.

Treatment outcome of continuation of intravenous amikacin for Mycobacterium abscessus pulmonary disease with a persistent culture positivity after the treatment initiation

IntroductionWhether prolonged intravenous amikacin treatment would lead to better treatment results in patients with Mycobacterium abscessus subspecies abscessus (M. abscessus) pulmonary disease (PD) is unknown. We investigated the efficacy of continued amikacin treatment for the microbiological outcome of M. abscessus PD patients with persistent culture positivity after treatment initiation. MethodsWe retrospectively evaluated 62 patients with M. abscessus PD who were treated with intravenous amikacin and beta-lactams along with a macrolide-based regimen at 3 tertiary referral centers in South Korea. The intravenous antibiotic treatment duration was determined by the attending physician. ResultsThe median treatment durations with amikacin and beta-lactam in the 62 patients were 25.1 and 8.2 weeks, respectively. The overall microbiological cure rate was 29.0%. Among the 62 patients, 44 showed persistent culture positivity at 8 weeks after treatment with an amikacin-containing multidrug regimen. The median parenteral amikacin treatment duration after 8 weeks in these patients was 18.0 weeks. The conditional probability of microbiological cure with continuation of the amikacin-containing regimen in these patients was 18.2% (95% confidence interval 8.2–32.7). Additionally, the conditional probability of microbiological cure in the 34 patients with persistent culture positivity at 12 weeks was 8.8% (95% confidence interval 1.9–23.7). After 16 weeks, the conditional probability of microbiological cure decreased further, reaching 0% at 28 weeks after treatment initiation. ConclusionThe continuation of intravenous amikacin therapy was usually not followed by culture conversion in M. abscessus PD patients with persistent sputum culture positivity after treatment initiation.

Investigating Nontuberculous Mycobacteria Transmission at the Colorado Adult Cystic Fibrosis Program.

Rationale: Healthcare-associated transmission of nontuberculous mycobacteria (NTM) among people with cystic fibrosis (pwCF) has been investigated at CF centers worldwide, with conflicting conclusions. We investigated transmission at the Colorado Adult CF Program. Objectives: To systematically investigate healthcare-associated transmission and/or acquisition of NTM to determine similarity among respiratory and environmental isolates, and to compare home residence watershed mapping among pwCF having genetically similar NTM isolates. Methods: Whole-genome sequencing of NTM isolates from 80 pwCF was conducted to identify genetically similar isolate clusters (⩽30 SNP differences). Epidemiology, comparison of respiratory and environmental isolates, and home residence watershed mapping were analyzed. Measurements and Main Results: Whole-genome sequencing analysis revealed 11 clusters of NTM [6 Mycobacterium abscessus subspecies (ssp.) abscessus, 1 M. abscessus ssp. massiliense, 2 Mycobacterium avium, and 2 Mycobacterium intracellulare] among pwCF. Epidemiologic investigation demonstrated opportunities for healthcare-associated transmission in two M. abscessus and two M. avium clusters. Respiratory and healthcare environmental isolate comparisons revealed no genetic similarity. Individuals comprising one M. abscessus cluster, with no plausible healthcare-associated transmission, resided in the same watershed. Conclusions: This study suggests healthcare-associated transmission of M. abscessus is rare and includes a report of potential healthcare-associated transmission of M. avium among pwCF. One M. abscessus cluster possibly had common acquisition arising from residing in the same watershed. The presence of genetically similar isolates is insufficient to demonstrate healthcare-associated NTM transmission. Standardizing epidemiologic investigation, combined with environmental sampling and watershed analysis, will improve understanding of the frequency and nature of healthcare-associated NTM transmission among pwCF.

A first case report of nasopharyngeal Mycobacterium abscessus subspecies massiliense infection

BackgroundMycobacterium abscessus subspecies massiliense is a non-tuberculous mycobacteriosis and was subdivided from Mycobacterium abscessus in 2006. This article is the first report on nasopharyngitis caused by Mycobacterium abscessus subspecies massiliense.Case presentationA 45-year-old woman had an 18-month history of recurrent nasopharyngitis and presented with pain in the throat. Mycobacterial tissue culture and polymerase chain reaction testing revealed the presence of Mycobacterium abscessus subspecies massiliense in the nasopharyngeal tissue. This patient underwent surgery, followed by multiple rounds of chemotherapy with oral and intravenous antibiotic agents for 16 weeks. She has had no recurrence during the 56 weeks since treatment.ConclusionIt is difficult to detect the presence of Mycobacterium abscessus subspecies massiliense in a culture from the swabbing sample. The tissue culture from a biopsy specimen is mandatory for the identification of the species. Currently, no definite treatment policy is available and only empirical treatment is applied. This case is an important for the diagnosis and treatment of this bacterial infection on nasopharynx.

Genomic characterization of sporadic isolates of the dominant clone of Mycobacterium abscessus subspecies massiliense

Recent studies have characterized a dominant clone (Clone 1) of Mycobacterium abscessus subspecies massiliense (M. massiliense) associated with high prevalence in cystic fibrosis (CF) patients, pulmonary outbreaks in the United States (US) and United Kingdom (UK), and a Brazilian epidemic of skin infections. The prevalence of Clone 1 in non-CF patients in the US and the relationship of sporadic US isolates to outbreak clones are not known. We surveyed a reference US Mycobacteria Laboratory and a US biorepository of CF-associated Mycobacteria isolates for Clone 1. We then compared genomic variation and antimicrobial resistance (AMR) mutations between sporadic non-CF, CF, and outbreak Clone 1 isolates. Among reference lab samples, 57/147 (39%) of patients with M. massiliense had Clone 1, including pulmonary and extrapulmonary infections, compared to 11/64 (17%) in the CF isolate biorepository. Core and pan genome analyses revealed that outbreak isolates had similar numbers of single nucleotide polymorphisms (SNPs) and accessory genes as sporadic US Clone 1 isolates. However, pulmonary outbreak isolates were more likely to have AMR mutations compared to sporadic isolates. Clone 1 isolates are present among non-CF and CF patients across the US, but additional studies will be needed to resolve potential routes of transmission and spread.

Impact of Susceptibility to Injectable Antibiotics on the Treatment Outcomes of Mycobacterium abscessus Pulmonary Disease

BackgroundCurrent guidelines recommend a susceptibility-based regimen for Mycobacterium abscessus subspecies abscessus pulmonary disease (MAB-PD), but the evidence is weak. We aimed to investigate the association between treatment outcomes and in vitro drug susceptibility to injectable antibiotics in MAB-PD patients.MethodsWe enrolled MAB-PD patients treated with intravenous amikacin and beta-lactams for ≥4 weeks at 4 referral hospitals in Seoul, South Korea. Culture conversion and microbiological cure at 1 year were evaluated based on susceptibility to injectable antibiotics among patients treated with those antibiotics for ≥2 weeks.ResultsA total of 82 patients were analyzed. The mean age was 58.7 years, and 65.9% were women. Sputum culture conversion and microbiological cure were achieved in 52.4% and 41.5% of patients, respectively. Amikacin was the most common agent to which the M. abscessus subspecies abscessus isolates were susceptible (81.7%); 9.8% and 24.0% of the isolates were resistant to cefoxitin and imipenem, respectively. The clarithromycin-inducible resistance (IR) group (n = 65) had a lower microbiological cure rate than the clarithromycin-susceptible group (35.4% vs 64.7%). The treatment outcomes appeared to be similar regardless of in vitro susceptibility results with regard to intravenous amikacin, cefoxitin, imipenem, and moxifloxacin. In the subgroup analysis of the clarithromycin-IR group, the treatment outcomes did not differ according to antibiotic susceptibility.ConclusionsWe did not find evidence supporting the use of susceptibility-based treatment with intravenous amikacin and beta-lactams in patients with MAB-PD. Further research is required.

A novel DNA chromatography method to discriminate Mycobacterium abscessus subspecies and macrolide susceptibility.

BackgroundThe clinical impact of infection with Mycobacterium (M.) abscessus complex (MABC), a group of emerging non-tuberculosis mycobacteria (NTM), is increasing. M. abscessus subsp. abscessus/bolletii frequently shows natural resistance to macrolide antibiotics, whereas M. abscessus subsp. massiliense is generally susceptible. Therefore, rapid and accurate discrimination of macrolide-susceptible MABC subgroups is required for effective clinical decisions about macrolide treatments for MABC infection. We aimed to develop a simple and rapid diagnostic that can identify MABC isolates showing macrolide susceptibility.MethodsWhole genome sequencing (WGS) was performed for 148 clinical or environmental MABC isolates from Japan to identify genetic markers that can discriminate three MABC subspecies and the macrolide-susceptible erm(41) T28C sequevar. Using the identified genetic markers, we established PCR based- or DNA chromatography-based assays. Validation testing was performed using MABC isolates from Taiwan.FindingWe identified unique sequence regions that could be used to differentiate the three subspecies. Our WGS-based phylogenetic analysis indicated that M. abscessus carrying the macrolide-susceptible erm(41) T28C sequevar were tightly clustered, and identified 11 genes that were significantly associated with the lineage for use as genetic markers. To detect these genetic markers and the erm(41) locus, we developed a DNA chromatography method that identified three subspecies, the erm(41) T28C sequevar and intact erm(41) for MABC in a single assay within one hour. The agreement rate between the DNA chromatography-based and WGS-based identification was 99·7%.InterpretationWe developed a novel, rapid and simple DNA chromatography method for identification of MABC macrolide susceptibility with high accuracy.FundingAMED, JSPS KAKENHI

Repurposing drugs for treatment of Mycobacterium abscessus: a view to a kill.

The current treatment regimens recommended for Mycobacterium abscessus subspecies abscessus (Mab) pulmonary disease are not effective. We identified 16 drugs with potential to build new regimens, translating to 560 possible three-drug combination regimens. To determine MICs and efficacy of drugs from different antibiotic classes for treatment against Mab, in order to winnow down the potential drugs for combination therapy to tractable numbers, for future use in hollow-fibre studies. The MICs of levofloxacin, minocycline, meropenem, imipenem, tedizolid, bedaquiline, azithromycin, clarithromycin, amikacin, vancomycin, delafloxacin, tebipenem/avibactam and omadacycline were determined for 20 Mab isolates. In addition, concentration-response studies with tedizolid, bedaquiline, clarithromycin, amikacin, tebipenem/avibactam, cefdinir, faropenem, omadacycline and daunorubicin were performed and data were fitted to the inhibitory sigmoid Emax model. Efficacy was defined as maximal kill, expressed as cfu/mL kill below day 0 burden. The lowest MICs among the 13 antibiotics were of bedaquiline, tebipenem/avibactam and omadacycline. The antibiotics that killed Mab below the day 0 burden were the anticancer agent daunorubicin (3.36 log10 cfu/mL), cefdinir (1.85 log10 cfu/mL), faropenem (2.48 log10 cfu/mL) and tebipenem/avibactam (1.71 log10 cfu/mL kill). The EC50 values of these drugs were 11.67, 9.52, 48.2 and 0.33 mg/L, respectively, below peak concentrations of these drugs. The low MICs and efficacy at clinically achievable concentrations mean that tebipenem/avibactam, daunorubicin, omadacycline and bedaquiline give a view of components of a three-drug regimen likely to effectively kill Mab. We propose pharmacokinetic/pharmacodynamic studies to identify such a regimen and the doses to be combined.

Kinetic Characterization of a Novel Fosfomycin Resistance Enzyme from Mycobacterium Bolletii

Fosfomycin is a small antibiotic that is effective against both Gram-positive and Gram-negative bacteria via inhibition of cell wall formation. Fosfomycin resistance enzymes have been observed in both Gram-positive and Gram-negative bacteria: FosA, a M2+-dependent glutathione (GSH)-S-transferase found in Gram-negative species, FosB, a M2+- and K+-dependent bacillithiol (BSH)-S-transferase found in Gram-positive species, and the evolutionary precursor FosX, a M2+-dependent hydrolase also found in Gram-negative species. Each of these enzymes is activated by Mn2+ and inhibited by Zn2+. We have discovered, expressed, and purified a novel enzyme from Mycobacterium abscessus subspecies Bolletii that shares 55% sequence identity with FosX. Preliminary time trace kinetic data using 31P NMR suggest that this enzyme, which we call FosM, is also a M2+-dependent thiol transferase. Kinetic activity has been observed with both GSH and L-cysteine as thiol substrates. However, Mycobacteria do not produce GSH or BSH, and we believe FosM uses the natural thiol found in mycobacteria, mycothiol (MSH), as its substrate. Preliminary data also suggest that FosM is activated by Mn2+ and inhibited by Zn2+ similar to both FosA and FosB. Hydrolytic activity has also been observed, reinforcing that FosM may be an evolutionary descendant of FosX. We are currently trying to obtain MSH to demonstrate that FosM is a new class of fosfomycin resistance enzyme from Mycobacteria.

Aerosolization of Mycobacterium avium and Mycobacterium abscessus from a household ultrasonic humidifier.

To measure the aerosolization of Mycobacterium avium subspecies hominissuis and Mycobacterium abscessus subspecies abscessus from ultrasonic humidifiers. An ultrasonic humidifier was filled with sterile tap water and inoculated with water-acclimated cells of either the M. avium or M. abscessus strains to achieve a range of densities similar to those of mycobacteria found in drinking waters. During operation of the humidifier, aerosols were collected using an Andersen 6-Stage Cascade Sampler. Cells of the M. avium and M. abscessus strains were readily aerosolized and recovered in particles (1-5 µm diameter); small enough to enter the furthest reaches of the human lung. Aerosolization of M. abscessus was significantly reduced in the presence of a normal drinking water bacterial flora. Significantly greater numbers of M. avium cells were aerosolized from high-density suspensions (1200 c.f.u. ml-1), than from low-density (120 c.f.u. ml-1) and very low-density (12 c.f.u. ml-1) suspensions. This report documents the potential for M. avium subspecies hominissuis and M. abscessus subspecies abscessus cells in drinking water to be aerosolized from one type of portable humidifier; an ultrasonic humidifier. Care should be taken in using an ultrasonic humidifier where an individual at risk for mycobacterial pulmonary disease could be exposed.