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.

Nontuberculous mycobacteria in cystic fibrosis and non-cystic fibrosis bronchiectasis.

Increasing numbers of cystic fibrosis (CF) and non-CF bronchiectasis patients are affected by pulmonary nontuberculous mycobacteria (NTM) infection worldwide. Two species of NTM account for up to 95% of the pulmonary NTM infections: Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MABSC). Diagnosis of pulmonary NTM infection is based on criteria specified in the 2007 American Thoracic Society/Infectious Disease Society of America (ATS/IDSA) guidelines. While many initial positive cultures do not progress to active NTM disease, even a single positive NTM sputum culture obtained from higher risk groups such as classic CF or older women with bronchiectasis and very low body mass index should be closely monitored for progressive disease. Macrolides remain the most effective agents available against MAC and MABSC. Infection with MABSC may be associated with worse clinical outcomes, as more than half of MABSC isolates have inducible macrolide resistance conferred by an active erm(41) gene. Of growing concern in CF is that MABSC is becoming more common than MAC, seems to target younger patients with classic CF, and is more difficult to manage, often requiring prolonged courses of intravenous antibiotics. Recurrence rates of NTM after initial successful treatment remain high, likely due to nonmodifiable risk factors raising the question of whether secondary prophylaxis is feasible. More rapid and readily available methods for detecting inducible macrolide resistance and better in vitro susceptibility testing methods for other drugs that correlate with clinical responses are needed. This is crucial to identify more effective regimens of existing drugs and for development of novel drugs for NTM infection.

Attitudinal analysis of vaccination effects to lead endemic phases.

To achieve endemic phases, repeated vaccinations are necessary. However, individuals may grapple with whether to get vaccinated due to potential side effects. When an individual is already immune due to previous infections or vaccinations, the perceived risk from vaccination is often less than the risk of infection. Yet, repeated rounds of vaccination can lead to avoidance, impeding the establishment of endemic phases. We explore this phenomenon using an individual-based Monte Carlo simulation, validating our findings with game theory. The Nash equilibrium encapsulates individuals' non-cooperative behavior, while the system's optimal value represents the societal benefits of altruistic cooperation. We define the difference between these as the price of anarchy. Our simulations reveal that the price of anarchy must fall below a threshold of 12.47 for endemic phases to be achieved in a steady state. This suggests that for a basic reproduction number of 10, a consistent vaccination rate greater than 89% is required. These findings offer new insights into vaccination-related decision-making and can inform effective strategies to tackle infectious diseases.

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.

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.