Genome sequencing of Mycobacterium abscessus isolates from patients in the united states and comparisons to globally diverse clinical strains.

Nontuberculous mycobacterial infections caused by Mycobacterium abscessus are responsible for a range of disease manifestations from pulmonary to skin infections and are notoriously difficult to treat, due to innate resistance to many antibiotics. Previous population studies of clinical M. abscessus isolates utilized multilocus sequence typing or pulsed-field gel electrophoresis, but high-resolution examinations of genetic diversity at the whole-genome level have not been well characterized, particularly among clinical isolates derived in the United States. We performed whole-genome sequencing of 11 clinical M. abscessus isolates derived from eight U.S. patients with pulmonary nontuberculous mycobacterial infections, compared them to 30 globally diverse clinical isolates, and investigated intrapatient genomic diversity and evolution. Phylogenomic analyses revealed a cluster of closely related U.S. and Western European M. abscessus subsp. abscessus isolates that are genetically distinct from other European isolates and all Asian isolates. Large-scale variation analyses suggested genome content differences of 0.3 to 8.3%, relative to the reference strain ATCC 19977(T). Longitudinally sampled isolates showed very few single-nucleotide polymorphisms and correlated genomic deletion patterns, suggesting homogeneous infection populations. Our study explores the genomic diversity of clinical M. abscessus strains from multiple continents and provides insight into the genome plasticity of an opportunistic pathogen.

In vitro synergy between clofazimine and amikacin in treatment of nontuberculous mycobacterial disease.

Disease caused by nontuberculous mycobacteria (NTM) is increasing in frequency. The outcome of treatment for NTM lung disease is poor, particularly lung disease caused by Mycobacterium simiae and M. abscessus. Exploring synergy between active available drugs is a sensible way forward given the lack of new active drugs. We tested for synergy between amikacin and clofazimine, using standardized methods, in 564 consecutive clinical isolates identified as 21 species of rapidly growing mycobacteria, 16 clinical M. avium complex isolates, and 10 M. simiae isolates. Clofazimine and amikacin are each active in vitro against NTM; 97% (n = 548) of the rapid growers revealed MICs of clofazimine of ≤1 µg/ml, and 93% (n = 524) proved susceptible to amikacin. The combination showed significant synergistic activity in 56 of 68 (82%) eligible M. abscessus isolates, 4 of 5 M. chelonae isolates, and 1 M. fortuitum and 1 M. cosmeticum isolate, with 4- to 8-fold decreases in MICs to both drugs. Significant synergy could also be demonstrated against all M. avium complex and M. simiae isolates, with fractional inhibitory concentrations of <0.5. Clofazimine and amikacin show significant synergistic activity against both rapidly and slowly growing nontuberculous mycobacteria. The safety and tolerability of adding clofazimine to amikacin-containing regimens should be tested in clinical trials, and the results of susceptibility tests for these two compounds and their combination merit clinical validation. Synergy between clofazimine and other antibiotics with intracellular targets should be explored.

The pharmacokinetics and pharmacodynamics of pulmonary Mycobacterium avium complex disease treatment.

RATIONALE: Currently recommended multidrug treatment regimens for Mycobacterium avium complex (MAC) lung disease yield limited cure rates. This results, in part, from incomplete understanding of the pharmacokinetics and pharmacodynamics of the drugs. OBJECTIVES: To study pharmacokinetics, pharmacodynamics, and drug interactions of multidrug treatment regimens in a large cohort of patients with MAC lung disease. METHODS: We retrospectively collected pharmacokinetic data of all patients treated for MAC lung disease in the Adult Care Unit at National Jewish Health, Denver, Colorado, in the January 2006 to January 2010 period; we retrospectively calculated areas under the time-concentration curve (AUC). Minimum inhibitory concentrations (MIC) of their MAC isolates were retrieved for pharmacodynamic calculations. MEASUREMENTS AND MAIN RESULTS: We included 531 pharmacokinetic analyses, performed for 481 patients (84% females; mean age, 63 yr; mean body mass index, 21.6). Peak serum concentrations (C(max)) below target range were frequent for ethambutol (48% of patients); clarithromycin (56%); and azithromycin (35%). Concurrent administration of rifampicin led to 68%, 23%, and 10% decreases in C(max) of clarithromycin, azithromycin, and moxifloxacin. C(max)/MIC or AUC/MIC ratios associated with bactericidal activity were seldom met; 57% of patients achieved target ratios for ethambutol, versus 42% for clarithromycin, 19% for amikacin, 18% for rifampicin, and 11% for moxifloxacin. CONCLUSIONS: Currently recommended regimens for MAC lung disease yield important pharmacologic interactions and low concentrations of key drugs including macrolides. Pharmacodynamic indices for rifampicin, clarithromycin, amikacin, and moxifloxacin are seldom met. This may partly explain the poor outcomes of currently recommended treatment regimens. Trials of new drugs and new dosing strategies are needed.

Drug susceptibility testing and pharmacokinetics question current treatment regimens in Mycobacterium simiae complex disease.

The Mycobacterium simiae complex bacteria can cause opportunistic infections in humans. In the case of definite disease, there are no evidence-based treatment regimens and outcomes are very disappointing. To increase the evidence base underpinning treatment regimens for M. simiae complex disease, drug susceptibility patterns and rifampicin/ethambutol synergy were assessed retrospectively in 69 clinical M. simiae complex isolates from 60 patients (22 patients with M. simiae, 24 with Mycobacterium lentiflavum, 8 with Mycobacterium triplex, 5 with Mycobacterium parascrofulaceum and 1 with Mycobacterium stomatepiae) submitted to the mycobacteriology laboratory at National Jewish Health (Denver, CO). Quantitative drug susceptibility testing (DST) was performed using the radiometric BacTec 460 macrodilution method. Results were related to pharmacokinetic (PK) measurements, where available. All M. simiae complex species proved susceptible to clarithromycin and, to a lesser extent, rifabutin, clofazimine, streptomycin and moxifloxacin. Synergy or additive action between rifampicin and ethambutol was observed for all species except M. simiae. Mycobacterium simiae is poorly susceptible in vitro to rifampicin and ethambutol alone as well as in combination; PK measurements support the limited efficacy of these drugs against M. simiae. The triple-drug regimen of a rifamycin, ethambutol and a macrolide may be advised to treat disease caused by M. lentiflavum, M. triplex, M. parascrofulaceum and M. stomatepiae; for M. simiae, this regimen appears less active. These findings may partly explain the limited treatment results in M. simiae disease. A treatment regimen including a macrolide, moxifloxacin and one or two additional drugs based on DST results may be advisable; clofazimine and amikacin or streptomycin are potential candidates.

Are phylogenetic position, virulence, drug susceptibility and in vivo response to treatment in mycobacteria interrelated?

Phylogenetic analyses on the basis of multiple house-keeping genes and whole genome sequences have offered new insights in the phylogeny of the genus Mycobacterium. This genus yields obligate pathogens, the M. tuberculosis complex and M. leprae, as well as opportunistic pathogens (e.g. M. avium, M. intracellulare, M. kansasii, M. marinum, M. malmoense) and saprophytes (e.g. M. phlei, M. sphagni, M. gordonae). The most virulent mycobacteria, the M. tuberculosis complex, M. leprae and the M. kansasii-M. szulgai-M. marinum-M. ulcerans group are phylogenetically related and infections by these organisms are better treatable than those caused by less virulent and phylogenetically more distantly related Mycobacterium species. The most virulent Mycobacterium species are also characterized by high levels of natural drug susceptibility. In this paper, we review studies of phylogeny, drug susceptibility, and clinical significance to support our hypothesis that drug susceptibility in mycobacteria is acquired and reflects the low level of competition in -and adaptation to- a closer-to-human (environmental) niche. In turn, mycobacteria that inhabit the most competitive environmental niches are the least adapted to humans, thus of low clinical significance, but most tolerant to antibiotics derived from microbes with which they share their habitat, lowering the chances of cure in case of infection.

Mycobacterium tuberculosis complex differentiation by genomic deletion patterns with multiplex polymerase chain reaction and melting analysis.

Differentiation of Mycobacterium tuberculosis complex (MTC) species is important for patient management. We developed a genomic deletion assay based on multiplex polymerase chain reaction with melting temperature analysis that correctly identified 124 (96%) of 129 MTC isolates. This assay is a fast single-tube method to differentiate members of MTC.

Inhibition of M. tuberculosis in vitro in monocytes and in mice by aminomethylene pyrazinamide analogs.

Pyrazinamide is unusual among anti-tuberculous agents in its ability to promote a durable cure and shorten the duration of therapy. Yet the basis for this effect is not well understood. A particularly effective strategy for the development of new drugs can be to synthetically manipulate the well-established structures to improve either the spectrum of activity or some pharmacological properties. Similar to previously described aminomethylene amides such as morphazinamide, it was found that novel aminomethylene amides can have in vitro activity at higher than the very acidic pH conditions where pyrazinamide is inactive as well as retaining activity against pyrazinamide-resistant M. tuberculosis. These new compounds have shown an improved anti-tuberculous activity in infected human macrophages relative to pyrazinamide. Compound 1, in combination with rifamycin, was especially effective in both infected human macrophages and in a murine model of infection. The activity of these analogs against pyrazinamide-resistant strains suggests that the development of second generation pyrazinamide analogs may be especially fruitful.

Nontuberculous mycobacteria in aerosol droplets and bulk water samples from therapy pools and hot tubs.

Hot tub exposure has been causally associated with a steroid-responsive, granulomatous lung disease featuring nontuberculous mycobacterial (NTM) growth in both clinical and environmental samples. Little is known regarding prevalence of and risk factors for NTM-contamination and associated illness in these settings. In this study, the frequency of NTM growth and aerosolization in 18 public hot tubs and warm water therapy pools and the factors associated with mycobacterial growth were analyzed. Each site was characterized by water chemistry analysis; a questionnaire on maintenance, disinfection, and water quality; and air and water sampling for quantitative NTM culture. NTM were detected in air or water from 13/18 (72%) sites; a strong correlation was found between the maximum air and water NTM concentrations (rho 0.49, p = 0.04). Use of halogen (chlorine or bromine) disinfection was associated with significantly lower air and water concentrations of NTM compared with disinfection using ultraviolet light and hydrogen peroxide (p = 0.01-0.04). Higher water turnover rates were also associated with lower air and water NTM concentrations (p = 0.02-0.03). These findings suggest that NTM are frequently detectable in the air and water of spas and therapy pools and that particular maintenance and disinfection approaches affect NTM bioaerosol concentrations in these settings.

Pulmonary infection with Mycobacterium neoaurum identified by 16S ribosomal DNA sequence.

Mycobacterium neoaurum infection has rarely been found in humans, and only a limited number of cases have been reported. We describe the first case of pulmonary infection with M. neoaurum. We speculate that unrecognized aspiration and long-term corticosteroid therapy predisposed our patient to this rare mycobacterial infection.

The potential advantages of nanoparticle drug delivery systems in chemotherapy of tuberculosis.

Nanoparticle-based drug delivery systems have considerable potential for treatment of tuberculosis (TB). The important technological advantages of nanoparticles used as drug carriers are high stability, high carrier capacity, feasibility of incorporation of both hydrophilic and hydrophobic substances, and feasibility of variable routes of administration, including oral application and inhalation. Nanoparticles can also be designed to allow controlled (sustained) drug release from the matrix. These properties of nanoparticles enable improvement of drug bioavailability and reduction of the dosing frequency, and may resolve the problem of nonadherence to prescribed therapy, which is one of the major obstacles in the control of TB epidemics. This article highlights some of the issues of nanotechnology relevant to the anti-TB drugs.

Ancestral antibiotic resistance in Mycobacterium tuberculosis.

Chemotherapeutic options to treat tuberculosis are severely restricted by the intrinsic resistance of Mycobacterium tuberculosis to the majority of clinically applied antibiotics. Such resistance is partially provided by the low permeability of their unique cell envelope. Here we describe a complementary system that coordinates resistance to drugs that have penetrated the envelope, allowing mycobacteria to tolerate diverse classes of antibiotics that inhibit cytoplasmic targets. This system depends on whiB7, a gene that pathogenic Mycobacterium shares with Streptomyces, a phylogenetically related genus known as the source of diverse antibiotics. In M. tuberculosis, whiB7 is induced by subinhibitory concentrations of antibiotics (erythromycin, tetracycline, and streptomycin) and whiB7 null mutants (Streptomyces and Mycobacterium) are hypersusceptible to antibiotics in vitro. M. tuberculosis is also antibiotic sensitive within a monocyte model system. In addition to antibiotics, whiB7 is induced by exposure to fatty acids that pathogenic Mycobacterium species may accumulate internally or encounter within eukaryotic hosts during infection. Gene expression profiling analyses demonstrate that whiB7 transcription determines drug resistance by activating expression of a regulon including genes involved in ribosomal protection and antibiotic efflux. Components of the whiB7 system may serve as attractive targets for the identification of inhibitors that render M. tuberculosis or multidrug-resistant derivatives more antibiotic-sensitive.

Capreomycin is active against non-replicating M. tuberculosis.

BACKGROUND: Latent tuberculosis infection (LTBI) is affecting one-third of the world population, and activation of LTBI is a substantial source of new cases of tuberculosis. LTBI is caused by tubercle bacilli in a state of non-replicating persistence (NRP), and the goal of this study was to evaluate the activity in vitro of various antimicrobial agents against non-replicating M. tuberculosis. METHODS: To achieve a state of NRP we placed broth cultures of M. tuberculosis (three strains) in anaerobic conditions, and in this model tested all known anti-TB drugs and some other antimicrobial agents (a total of 32 drugs). The potential effect was evaluated by plating samples from broth cultures for determining the number of viable bacteria (CFU/ml) during a prolonged period of cultivation. Besides drug-free controls we used metronidazole for positive controls, the only drug known so far to be effective against tubercle bacilli in anaerobic setting. RESULTS: On a background of non-replicating conditions in drug-free cultures and clear bactericidal effect of metronidazole none of the antimicrobial agents tested produced effect similar to that of metronidazole except capreomycin, which was as bactericidal at the same level as metronidazole. CONCLUSION: The unique ability of capreomycin to be bactericidal in vitro among the anti-TB drugs against non-replicating tubercle bacilli may justify the search for other drugs among peptide antibiotics with similar activity. This phenomenon requires further studies on the mechanism of action of capreomycin, and evaluation of its activity in appropriate animal models.

Identification of a Mycobacterium tuberculosis strain with stable, low-level resistance to isoniazid.

We have identified a potential quality control strain of Mycobacterium tuberculosis to monitor isoniazid susceptibility testing. This strain (strain A) has a stable phenotypic low-level resistance to isoniazid, has a mutation of C (-15) --> T in the inhA promoter region, and gave consistent susceptibility test results in 141 laboratories.

Cough-generated aerosols of Mycobacterium tuberculosis: a new method to study infectiousness.

The concentration and size distribution of infectious aerosols produced by patients with pulmonary tuberculosis (TB) has never been directly measured. We aimed to assess the feasibility of a method that we developed to collect and quantify culturable cough-generated aerosols of Mycobacterium tuberculosis. Subjects were recruited from a referral hospital and most had multidrug-resistant TB. They coughed into a chamber containing microbial air samplers while cough frequency was measured during two 5-minute sessions. Cough-generated aerosol cultures were positive in 4 of 16 subjects (25%) with smear-positive pulmonary TB. There was a rapid decrease in the cough-generated aerosol cultures within the first 3 weeks of effective treatment. Culture-positive cough aerosols were associated with lack of treatment during the previous week (p = 0.007), and there was a trend in the association with cough frequency (p = 0.08). The size distributions of these aerosols were variable, but most particle sizes were in the respirable range. Quantification of viable cough-generated aerosols is feasible and offers a new approach to study infectiousness and transmission of M. tuberculosis and other airborne pathogens.

Mycobacterial infections caused by nontuberculous mycobacteria.

The purpose of this review is to familiarize a broad range of medical professionals with a relatively new and growing problem of infections caused by mycobacteria other than M. tuberculosis and M. leprae. There are at least 60 mycobacterial species that have been identified as causative agents of diseases in humans. They are all environmental bacteria, and they are not transmitted from person to person. The usual source of infection is water, soil, and aerosols developed from these sources. The probability of contracting such a disease depends not only on the closeness of interaction with an environment containing an enhanced concentration of bacteria but also and foremost on the sensitivity of an individual to these infections, which may depend on the state of immunity and other so-called predisposing conditions. Therefore, these infections are often referred to as opportunistic, and the group of organisms causing them are usually referred to as nontuberculous mycobacteria (NTM). This review addresses in a condensed form various aspects of these infections, including bacteriology, clinical manifestations, diagnosis, and therapy. The reader will be able to find more details on each of these topics in several reviews and some original papers cited in this article.

A multicentre comparison of a novel surrogate marker for determining the specific potency of anti-tuberculosis drugs.

A model for evaluating the potency of a new anti-tuberculosis drug or a drug combination, based on a decline in the number of viable tubercle bacilli in patient's sputum during 5 days mono-therapy has been reported. One popular measure is based on the analysis of the decline in bacterial counts during the first 48 h of therapy and has been called early bactericidal activity (EBA). Such analyses could detect EBA for only a few drugs and were subject to variations in results obtained in different sites. To address these problems we applied a reiterative exponential decay model to evaluate the data on bacterial counts during 5 days of mono-therapy. The validity of this approach was tested using data from three previously published studies. For patients treated with isoniazid 300 mg daily, the values for the time taken to reduce the viable count by 50% (vt50) measured in days were, from a Kenyan study 0.58 days S.E.M. 0.18, from a Tanzanian study 0.41 days S.E.M. 0.04, and from a United States study 0.55 days s.e.m. 0.12. These differences were not statistically significant (P = 0.77 Kruskal-Wallis non-parametric ANOVA). Mean values of vt50 for all of the major anti-tuberculosis agents showed that there was an overlapping spectrum of activity from isoniazid 300 mg (vt50 0.58 days) to para-amino-salicylic acid (vt50 2.9 days) The variation between column means was greater than could be expected by chance (P = 0.0002 Kruskal-Wallis non-parametric ANOVA). From this, we conclude that the reiterative exponential decay model permits comparison between the data obtained in different centres and would allow the activity of a new drug to be compared with that of the currently available agents.