Genetic Determinants of Intrinsic Antibiotic Tolerance in Mycobacterium avium.

The Mycobacterium avium complex (MAC) is one of the most prevalent causes of nontuberculous mycobacteria pulmonary infection in the United States, and yet it remains understudied. Current MAC treatment requires more than a year of intermittent to daily combination antibiotic therapy, depending on disease severity. In order to shorten and simplify curative regimens, it is important to identify the innate bacterial factors contributing to reduced antibiotic susceptibility, namely, antibiotic tolerance genes. In this study, we performed a genome-wide transposon screen to elucidate M. avium genes that play a role in the bacterium's tolerance to first- and second-line antibiotics. We identified a total of 193 unique M. avium mutants with significantly altered susceptibility to at least one of the four clinically used antibiotics we tested, including two mutants (in DFS55_00905 and DFS55_12730) with panhypersusceptibility. The products of the antibiotic tolerance genes we have identified may represent novel targets for future drug development studies aimed at shortening the duration of therapy for MAC infections. IMPORTANCE The prolonged treatment required to eradicate Mycobacterium avium complex (MAC) infection is likely due to the presence of subpopulations of antibiotic-tolerant bacteria with reduced susceptibility to currently available drugs. However, little is known about the genes and pathways responsible for antibiotic tolerance in MAC. In this study, we performed a forward genetic screen to identify M. avium antibiotic tolerance genes, whose products may represent attractive targets for the development of novel adjunctive drugs capable of shortening the curative treatment for MAC infections.

In Vitro Biofilm Formation in Mycobacterium avium-intracellulare Complex / Formación in vitro de biopelículas en el complejo Mycobacterium avium-intracellulare

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Development of an in vivo-mimic silkworm infection model with Mycobacterium avium complex.

In vivo-mimic silkworm infection models with Mycobacterium avium and Mycobacterium intracellulare were newly established to evaluate the therapeutic effects of anti-M. avium complex (MAC) antibiotics. Silkworms raised at 37°C died within 72 hours of an injection of M. avium or M.intracellulare (2.5 × 107 colony-forming unit (CFU)/larva·g) into the hemolymph. Clinical anti-mycobacterial (tuberculosis) antibiotics were evaluated under these conditions. Clarithromycin, kanamycin, streptomycin, amikacin, and ciprofloxacin exerted therapeutic effects in a dose-dependent manner, which was consistent with those in the mouse model. Furthermore, three effective actinomycete culture broths were selected in the screening program of our microbial broth library using the silkworm model, and four active metabolites, ohmyungsamycins A and B (1 and 2), chartreusin (3), and griseoviridin (4), were identified. Among these compounds, 1 showed the lowest 50% effective dose (ED50) value (8.5 µg/larva·g), while 3 had the best ED50/minimum inhibitory concentration (MIC) ratio (7.4). These results indicate that silkworm models are a useful tool for identifying anti-MAC antibiotics candidates with veritable therapeutic effects.

Genome-wide identification of essential genes in Mycobacterium intracellulare by transposon sequencing - Implication for metabolic remodeling.

The global incidence of the human nontuberculous mycobacteria (NTM) disease is rapidly increasing. However, knowledge of gene essentiality under optimal growth conditions and conditions relevant to the natural ecology of NTM, such as hypoxia, is lacking. In this study, we utilized transposon sequencing to comprehensively identify genes essential for growth in Mycobacterium intracellulare. Of 5126 genes of M. intracellulare ATCC13950, 506 genes were identified as essential genes, of which 280 and 158 genes were shared with essential genes of M. tuberculosis and M. marinum, respectively. The shared genes included target genes of existing antituberculous drugs including SQ109, which targets the trehalose monomycolate transporter MmpL3. From 175 genes showing decreased fitness as conditionally essential under hypoxia, preferential carbohydrate metabolism including gluconeogenesis, glyoxylate cycle and succinate production was suggested under hypoxia. Virulence-associated genes including proteasome system and mycothiol redox system were also identified as conditionally essential under hypoxia, which was further supported by the higher effective suppression of bacterial growth under hypoxia compared to aerobic conditions in the presence of these inhibitors. This study has comprehensively identified functions essential for growth of M. intracellulare under conditions relevant to the host environment. These findings provide critical functional genomic information for drug discovery.

Clinical characteristics and treatment outcomes of patients with macrolide-resistant Mycobacterium avium complex pulmonary disease: a systematic review and meta-analysis.

BACKGROUND: Macrolide is a key drug in the treatment of Mycobacterium avium complex pulmonary disease (MAC-PD). Macrolide-resistant MAC is gaining importance, but there are little data in clinical characteristics and treatment outcomes of macrolide-resistant MAC-PD (MR-MAC-PD). METHODS: We performed a systematic review and meta-analysis of published studies reporting clinical characteristics and treatment outcomes of patients with MR-MAC-PD. Risk of bias was assessed using the modified Newcastle-Ottawa Scale. RESULTS: Nine studies (seven retrospective and two prospective) comprising 319 patients were identified through a database search. Around 73% were women, and 52% had the fibrocavitary form. Pooled sputum culture conversion rate after combined multiple antibiotics or surgical resection was 21% (95% confidence interval [CI], 14-30%), and the one-year all-cause mortality was 10% (95% CI, 5-20%). There was no significant difference in treatment outcomes between nodular bronchiectatic and fibrocavitary types. CONCLUSIONS: Even combination therapy with fluoroquinolone, aminoglycoside, and surgical resection, the treatment outcomes of MR-MAC-PD were poor. The investigation of new treatment modalities is urgent.

JVA, an isoniazid analogue, is a bioactive compound against a clinical isolate of the Mycobacterium avium complex.

Bacteria belonging to Mycobacterium avium complex are organisms of low pathogenicity that infect immunosuppressed individuals. Infection is treated with an antimicrobial macrolide, Clarithromycin (CAM) or Azitromycin, associated with Ethambutol and Rifabutin during 12 months. Regimen long duration and side effects hinder patient's commitment to treatment favoring emergence of antibiotic resistance. In this present study, we evaluated the activity of JVA, an Isoniazid (INH) derivative, against M. avium 2447, a clinical isolate. We demonstrated that JVA reduces M. avium 2447 growth in macrophages, more efficiently than CAM and INH. In order to explore JVA mechanism of action, we investigated compound properties and performed pH-dependent stability studies. Our results suggest an enhanced ability of JVA to cross biological membranes. Furthermore, we suggest that in acidic conditions of macrophages' phagosomes, where mycobacteria replicate, JVA would be promptly hydrolyzed to INH, delivering the adduct INH-nicotinamide adenine dinucleotide and thus inhibiting M. avium 2447 growth.

Identification of Clinically Relevant Mycobacterial Species After Extended Incubation Times in the BACTEC MGIT System.

Objectives: Traditionally, for mycobacterial culture both solid and broth media are used and routinely held for 6 weeks minimum to optimize yield. We retrospectively reviewed all positive mycobacterial cultures over a 12-month period to assess growth kinetics of clinically relevant isolates. Methods: From January to December 2015, 658 positive mycobacteria cultures by solid (7H11 and 7H10 plates) and/or broth (BACTEC MGIT) media were identified and reviewed. Results: In broth-only cultures, 21 of 153 (13.7%) from 21 patients were positive after 28 days' incubation. Subsequent chart review revealed the following species: 11 Mycobacterium avium intracellulare complex (MAI), five Mycobacterium tuberculosis (MTB), and five other non-MTB/MAI mycobacteria. Two of the cases of MTB were first-time isolates, and 11.4% of MTB-positive cultures became positive after 4 weeks' incubation. Conclusions: These data provide strong evidence reaffirming that clinically meaningful results are frequently detected after extended incubation times by broth-only methods, including several MTB isolates.

Do infections with disseminated Mycobacterium avium complex precede sweet's syndrome? A case report and literature review.

Sweet's syndrome is reportedly associated with preceding nontuberculous mycobacterial infections (NTMIs). Here, we report on a systemic Mycobacterium intracellulare infection in a patient on corticoid therapy for Sweet's syndrome. Literature searches show that 69.1% of patients with Sweet's syndrome and NTMIs developed this syndrome later than NTMIs and 89.3% of them developed during the clinical course of a rapidly growing mycobacterial infection. The residual cases were associated with slow-growing mycobacteria (14.3%), but only three cases of Mycobacterium avium complex (MAC) infections before the onset of Sweet's syndrome have been reported, and all of them were caused by disseminated MAC disease. One of these cases developed during corticoid therapy for Sweet's syndrome, while another case had underlying diabetes mellitus. Hence, the occurrence of systemic MAC disease may be an inevitable consequence of long-term steroid use and underlying diseases. Literature searches also show that cervical lymphadenitis was a predominant symptom in NTMIs (90.5%). The present case did not have cervical lymphadenitis although the previously reported MAC cases did experience it. Therefore, lymphadenitis from NTMIs may be related to the pathogenesis of Sweet's syndrome. Hence, should a patient have systemic infection without lymphadenitis, it will be more difficult to clinically confirm that MAC disease is a predisposing factor for Sweet's syndrome.

A 'shock and awe' thioridazine and moxifloxacin combination-based regimen for pulmonary Mycobacterium avium-intracellulare complex disease.

OBJECTIVES: To develop a thioridazine/moxifloxacin-based combination regimen for treatment of pulmonary infection due to Mycobacterium avium-intracellulare complex (MAC) that kills bacteria faster than the standard treatment regimen. METHODS: Monocytes were infected with MAC and inoculated into the hollow-fibre system model for pulmonary MAC disease (HFS-MAC). We co-administered ethambutol plus azithromycin daily for 28 days, to achieve the same human concentration-time profiles that result from standard doses, in three HFS-MAC systems. Two experimental regimens consisted of thioridazine at an exposure associated with optimal kill, given intermittently on days 0, 3, 7 and 10. Regimen A consisted of thioridazine in combination with standard dose azithromycin for the entire study duration. Regimen B was thioridazine plus moxifloxacin at concentration-time profiles achieved by the standard daily dose administered for 14 days, followed by daily azithromycin. Each HFS-MAC was sampled for bacterial burden every 7 days. RESULTS: The bacteria in the non-treated HFS-MAC grew at a rate of 0.11 ±âŸ0.01 log10 cfu/mL/day. The azithromycin/ethambutol regimen decreased bacterial burden by 1.21 ±âŸ0.74 log10 cfu/mL below baseline during the first 7 days, after which it failed. Regimen A killed 3.28 ±âŸ0.32 log10 cfu/mL below baseline up to day 14, after which regrowth occurred once thioridazine treatment stopped. Regimen B killed bacteria to below the limits of detection in 7 days (≥5.0 log10 cfu/mL kill), with rebound in the azithromycin continuation phase. CONCLUSIONS: The thioridazine/moxifloxacin regimen demonstrated that rapid microbial kill could be achieved within 7 days. This is a proof of principle that short-course chemotherapy for pulmonary MAC is possible.

Outcomes of Mycobacterium avium complex lung disease based on clinical phenotype.

The effect of the clinical phenotype of Mycobacterium avium complex (MAC) lung disease on treatment outcome and redevelopment of nontuberculous mycobacterial (NTM) lung disease after treatment completion has not been studied systematically.We evaluated 481 treatment-naïve patients with MAC lung disease who underwent antibiotic treatment for ≥12 months between January 2002 and December 2013.Out of 481 patients, 278 (58%) had noncavitary nodular bronchiectatic (NB) disease, 80 (17%) had cavitary NB disease and 123 (25%) had fibrocavitary disease. Favourable outcome was higher in patients with noncavitary disease (88%) than in patients with cavitary disease (76% for fibrocavitary and 78% for cavitary NB disease; p<0.05). Cavitary disease was independently associated with unfavourable outcomes (p<0.05). Out of 402 patients with favourable outcomes, 118 (29%) experienced redevelopment of NTM lung disease, with the same MAC species recurring in 65 (55%) patients. The NB form was an independent risk factor for redevelopment of NTM lung disease (p<0.05). In patients with recurrent MAC lung disease due to the same species, bacterial genotyping revealed that 74% of cases were attributable to reinfection and 26% to relapse.Treatment outcomes and redevelopment of NTM lung disease after treatment completion differed by clinical phenotype of MAC lung disease.

The characteristics of patients with pulmonary Mycobacterium avium-intracellulare complex disease diagnosed by bronchial lavage culture compared to those diagnosed by sputum culture.

BACKGROUND AND OBJECTIVE: The utility of bronchoscopy for the diagnosis of pulmonary Mycobacterium avium-intracellulare complex (MAC) disease has been reported; however, which patients require bronchoscopy remains unclear. Our objective was to identify the characteristics of the patients in whom bronchoscopy is needed for the diagnosis of MAC disease. METHODS: Fifty-four patients with pulmonary MAC disease were divided into two groups according to established diagnostic criteria: 39 patients were diagnosed by sputum culture and 15 patients were diagnosed by bronchial lavage culture. We analysed the differences in demographic and clinical characteristics as well as microbiological and radiological data between the two groups. RESULTS: There were no significant differences in age, sex, smoking status, MAC species, underlying diseases, or steroid use. Significantly more patients diagnosed by sputum culture than bronchial lavage culture had a positive sputum smear for acid-fast bacilli (79.5% vs. 0.0%, respectively; p < 0.001) and any symptoms (75.3% vs. 46.2%, respectively; p = 0.0059). No significant differences were found in the prevalence of each computed tomography finding, including nodules, air-space disease, bronchiectasis, and cavities. However, more patients diagnosed by sputum culture than bronchial lavage culture had abnormalities in the left upper division (48.7% vs. 13.3%, respectively; p = 0.017) and higher numbers of affected lobes (4.3 ± 1.4 vs. 3.3 ± 1.6, respectively; p = 0.034). CONCLUSION: If patients suspected of having pulmonary MAC disease have a negative sputum smear, no symptoms, no abnormal findings in the left upper division, or fewer affected lobes on computed tomography, bronchoscopy might be needed for the diagnosis.

LdtMav2, a nonclassical transpeptidase and susceptibility of Mycobacterium avium to carbapenems.

AIM: Mycobacterium avium infections, especially in immune-compromised individuals, present a significant challenge as therapeutic options are limited. In this study, we investigated if M. avium genome encodes nonclassical transpeptidases and if newer carbapenems are effective against this mycobacteria. MATERIALS & METHODS: Biochemical and microbiological approaches were used to identify and characterize a nonclassical transpeptidase, namely L,D-transpeptidase, in M. avium. RESULTS & CONCLUSION: We describe the biochemical and physiological attributes of a L,D-transpeptidase in M. avium, LdtMav2. Suggestive of a constitutive requirement, levels of LdtMav2, a L,D-transpeptidase in M. avium, remain constant during exponential and stationary phases of growth. Among ß-lactam antibacterials, only a subset of carbapenems inhibit LdtMav2 and tebipenem, a new oral carbapenem, inhibits growth of M. avium.

Clinical Characteristics, Treatment Outcomes, and Resistance Mutations Associated with Macrolide-Resistant Mycobacterium avium Complex Lung Disease.

Macrolide antibiotics are key components of the multidrug treatment regimen for treating lung disease (LD) due to Mycobacterium avium complex (MAC). Despite the emergence of macrolide resistance, limited data are available on macrolide-resistant MAC-LD. This study evaluated the clinical features and treatment outcomes of patients with macrolide-resistant MAC-LD and the molecular characteristics of the macrolide-resistant isolates. A retrospective review of the medical records of 34 patients with macrolide-resistant MAC-LD who were diagnosed between January 2002 and December 2014 was performed, along with genetic analysis of 28 clinical isolates. Nineteen (56%) patients had the fibrocavitary form of MAC-LD, and 15 (44%) had the nodular bronchiectatic form. M. intracellulare was the etiologic organism in 21 (62%) patients. Approximately two-thirds (22/34 [65%]) of the patients had been treated with currently recommended multidrug regimens that included macrolide, ethambutol, and rifamycin prior to the emergence of macrolide resistance, and none had been treated with macrolide monotherapy. The median duration of treatment after the detection of macrolide resistance was 23.0 months (interquartile range, 16.8 to 45.3 months). Treatment outcomes were poor after the development of macrolide resistance, with favorable treatment outcomes achieved in only five (15%) patients, including two patients who underwent surgical resection. One-, 3-, and 5-year mortality rates were 9, 24, and 47%, respectively. Molecular analysis of 28 clinical isolates revealed that 96% (27/28) had point mutations at position 2058 or 2059 of the 23S rRNA gene. Our analyses indicate that more effective therapy is needed to treat macrolide-resistant MAC-LD and prevent its development.

Peak Plasma Concentration of Azithromycin and Treatment Responses in Mycobacterium avium Complex Lung Disease.

Macrolides, such as azithromycin (AZM) and clarithromycin, are the cornerstones of treatment for Mycobacterium avium complex lung disease (MAC-LD). Current guidelines recommend daily therapy with AZM for cavitary MAC-LD and intermittent therapy for noncavitary MAC-LD, but the effectiveness of these regimens has not been thoroughly investigated. This study evaluated associations between microbiological response and estimated peak plasma concentrations (Cmax) of AZM. The AZM Cmax was measured in patients receiving daily therapy (250 mg of AZM daily, n = 77) or intermittent therapy (500 mg of AZM three times weekly, n = 89) for MAC-LD and daily therapy for Mycobacterium abscessus complex LD (MABC-LD) (250 mg of AZM daily, n = 55). The AZM Cmax was lower with the daily regimen for MAC-LD (median, 0.24 µg/ml) than with the intermittent regimen for MAC-LD (median, 0.65 µg/ml; P < 0.001) or daily therapy for MABC-LD (median, 0.53 µg/ml; P < 0.001). After adjusting for confounding factors, AZM Cmax was independently associated with favorable microbiological responses in MAC-LD patients receiving a daily regimen (adjusted odds ratio [aOR], 1.58; 95% confidence interval [CI], 1.01 to 2.48; P = 0.044) but not an intermittent regimen (aOR, 0.85; 95% CI, 0.58 to 1.23, P = 0.379). With the daily AZM-based multidrug regimen for MAC-LD, a low AZM Cmax was common, whereas a higher AZM Cmax was associated with favorable microbiologic responses. The results also suggested that the addition of rifampin may lower AZM Cmax When a daily AZM-based multidrug regimen is used for treating severe MAC-LD, such as cavitary disease, the currently recommended AZM dose might be suboptimal. (This study has been registered at ClinicalTrials.gov under identifier NCT00970801.).

Interaction between antimicrobial peptides and mycobacteria.

Mycobacteria can cause different severe health problems, including tuberculosis (TB). The treatment of TB with conventional antibiotics is successful, however, the number of multi-drug and extensively-drug resistant Mycobacterium tuberculosis strains increases. Moreover, many classical antimycobacterial antibiotics have severe side effects. Therefore, antimicrobial peptides (AMPs) seem to be good candidates for new therapeutic strategies. On the one hand AMPs can be used as a single drug or in combination with conventional antibiotics to directly kill mycobacteria, or on the other hand to act as immunstimulatory agents. This review summarizes the findings on the role of endogenous human AMPs being involved in TB, the antimycobacterial activity of various AMPs, and the molecular modes of action. Most active AMPs interact with the mycobacterial cell envelope and in particular with the mycomembrane and the plasma membrane. The mycomembrane is a very rigid membrane probably leading to a lower activity of the AMPs against mycobacteria as compared to other Gram-negative or Gram-positive bacteria. For some AMPs also other targets have been identified. Because of the complex environment of intracellular mycobacteria being trapped in the phagosome, within the macrophage, within the granuloma, within the lung, the external administration of AMPs in the latent phase of TB is a challenge. However, in the acute phase the AMPs can attack mycobacteria in a direct way. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Azithromycin Dose To Maximize Efficacy and Suppress Acquired Drug Resistance in Pulmonary Mycobacterium avium Disease.

Mycobacterium aviumcomplex is now the leading mycobacterial cause of chronic pneumonia in the United States. Macrolides and ethambutol form the backbone of the regimen used in the treatment of pulmonary disease. However, therapy outcomes remain poor, with microbial cure rates of 4% in cavitary disease. The treatment dose of azithromycin has mostly been borrowed from that used to treat other bacterial pneumonias; there are no formal dose-response studies in pulmonaryM. aviumdisease and the optimal dose is unclear. We utilized population pharmacokinetics and pharmacokinetics/pharmacodynamics-derived azithromycin exposures associated with optimal microbial kill or resistance suppression to perform 10,000 patient Monte Carlo simulations of dose effect studies for daily azithromycin doses of 0.5 to 10 g. The currently recommended dose of 500 mg per day achieved the target exposures in 0% of patients. Exposures associated with optimal kill and resistance suppression were achieved in 87 and 54% of patients, respectively, only by the very high dose of 8 g per day. The azithromycin susceptibility breakpoint above which patients failed therapy on the very high doses of 8 g per day was an MIC of 16 mg/liter, suggesting a critical concentration of 32 mg/liter, which is 8-fold lower than the currently used susceptibility breakpoint of 256 mg/liter. If the standard dose of 500 mg a day were used, then the critical concentration would fall to 2 mg/liter, 128-fold lower than 256 mg/liter. The misclassification of resistant isolates as susceptible could explain the high failure rates of current doses.

Direct detection of Mycobacterium avium in environmental water and scale samples by loop-mediated isothermal amplification.

We previously demonstrated the colonization of Mycobacterium avium complex in bathrooms by the conventional culture method. In the present study, we aimed to directly detect M. avium organisms in the environment using loop-mediated isothermal amplification (LAMP), and to demonstrate the efficacy of LAMP by comparing the results with those obtained by culture. Our data showed that LAMP analysis has detection limits of 100 fg DNA/reaction for M. avium. Using an FTA(®) elute card, DNA templates were extracted from environmental samples from bathrooms in the residences of 29 patients with pulmonary M. avium disease. Of the 162 environmental samples examined, 143 (88%) showed identical results by both methods; 20 (12%) and 123 (76%) samples were positive and negative, respectively, for M. avium. Of the remaining 19 samples (12%), seven (5%) and 12 (7%) samples were positive by the LAMP and culture methods, respectively. All samples that contained over 20 colony forming units/primary isolation plate, as measured by the culture method, were also positive by the LAMP method. Our data demonstrate that the combination of the FTA elute card and LAMP can facilitate prompt detection of M. avium in the environment.