Leonid Heifets

pncA Mutations as a Major Mechanism of Pyrazinamide Resistance in Mycobacterium tuberculosis: Spread of a Monoresistant Strain in Quebec, Canada

ABSTRACT Pyrazinamide (PZA) is an important first-line tuberculosis drug that is part of the currently used short-course tuberculosis chemotherapy. PZA is a prodrug that has to be converted to the active form pyrazinoic acid by pyrazinamidase (PZase) activity, encoded by thepncA gene of Mycobacterium tuberculosis, and loss of PZase activity is associated with PZA resistance. To further define the genetic basis of PZA resistance and determine the frequency of PZA-resistant strains having pncA mutations, we sequenced the pncA gene from a panel of 59 PZA-resistant clinical isolates from Canada, the United States, and Korea. Two strains that did not contain pncA mutations and had positive PZase turned out to be falsely resistant. Three PZase-negative strains (MIC, >900 μg of PZA per ml) and one PZase-positive strain (strain 9739) (MIC, >300 μg of PZA per ml) did not havepncA mutations. The remaining 53 of the 57 PZA-resistant isolates had pncA mutations, confirming thatpncA mutation is the major mechanism of PZA resistance. Various new and diverse mutations were found in the pncAgene. Interestingly, 20 PZA-monoresistant strains and 1 multidrug-resistant isolate from Quebec, Canada, all had the samepncA mutation profile, consisting of an 8-nucleotide deletion and an amino acid substitution of Arg140→Ser. Strain typing indicated that these strains are highly related and share almost identical IS6110 patterns. These data strongly suggest the spread of a PZA-monoresistant strain, which has not previously been described.

Mycobacterium avium strains resistant to clarithromycin and azithromycin.

Mycobacterium avium strains susceptible to clarithromycin and azithromycin contain mutants resistant to these macrolides with a frequency of 1.1 x 10(-10) to 1.2 x 10(-6). Cross-resistance between clarithromycin and azithromycin was demonstrated with mutants selected in the laboratory as well as with resistant strains isolated from patients. The susceptibility-resistance patterns of the macrolide-resistant strains with drugs other than macrolides were the same as those of the original susceptible strains. The emergence of clarithromycin resistance in patients was a result of multiplication of the preexisting resistant mutants that survived the elimination of bacteria during the initial period of treatment and was an exclusive cause of the relapse of bacteremia.

Comparison of activities of rifapentine and rifampin against Mycobacterium tuberculosis residing in human macrophages.

The activities of rifapentine and rifampin against Mycobacterium tuberculosis residing in human monocyte-derived macrophages were determined. The MICs and MBCs of rifapentine for intracellular bacteria were two- to fourfold lower than those of rifampin. For extracellular bacteria, this difference was less noticeable. Nevertheless, the more favorable pharmacokinetics of rifapentine over rifampin was addressed in other experimental models. These models showed substantial differences after short pulsed exposures of the infected macrophages to the drugs and when the infected macrophages were exposed to changing drug concentrations that imitated the pharmacokinetic curves observed in blood. Once-a-week exposures to rifapentine concentrations equivalent to those attained in blood after one 600-mg dose resulted during the first week in a dramatic decline in the number of bacteria, and this decline was maintained at a minimal level for a period of four weeks. The results of this study have shown the suitability of rifapentine for intermittent-treatment regimens. The prolonged effect of rifapentine found in this study may be associated with high ratios of intracellular accumulation, which were four- to fivefold higher than those found for rifampin. Further studies on the intracellular distribution of rifamycins and on the sites of actual interaction between the drugs and bacteria residing in macrophages are necessary.

Inhibitory and bactericidal activities of levofloxacin against Mycobacterium tuberculosis in vitro and in human macrophages.

Levofloxacin exhibited twofold greater inhibitory and bactericidal activities than ofloxacin against either extracellular or intracellular tubercle bacilli. The activities of both drugs against extracellular and intracellular bacteria were about the same, despite the accumulation of the drugs in macrophages at a level four- to fivefold greater than that in the extracellular medium. The activities of both drugs against intracellular bacteria were largely associated with the short, 2-h pulsed exposures of the infected macrophages to the concentrations which correspond to those attainable in blood during the period of the maximum concentration of drug in serum.

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.

Effects of clarithromycin and rifabutin alone and in combination on intracellular and extracellular replication of Mycobacterium avium.

The combined effect of clarithromycin and rifabutin against Mycobacterium avium multiplying either within human monocyte-derived macrophages or extracellularly in a liquid medium was additive: both MICs and MBCs were twofold lower for the combination than they were for each drug alone. Prolonged exposure for 4 weeks of M. avium-infected macrophages to combined concentrations that were only twofold greater than the MICs resulted in a 100-fold decrease in the number of viable bacteria, while in the drug-free controls a 100-fold or greater increase in comparison with the initial viable counts took place. Comparison of this effect with the results of the prolonged exposure to each drug alone suggested that under these experimental conditions rifabutin enhanced the antimicrobial activity of clarithromycin against intracellular bacteria. At the same time, inhibition of intracellular growth by a 2-h pulsed exposure of the infected macrophages to the combination of the two drugs was not different from the effect induced by clarithromycin alone. In conclusion, clarithromycin played the major role in the antimicrobial activity of the tested combination, while rifabutin may have enhanced this effect during a prolonged exposure of the intracellular bacteria to these two agents.

Genome Sequencing of Mycobacterium abscessus Isolates from Patients in the United States and Comparisons to Globally Diverse Clinical Strains

ABSTRACT 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 19977T. 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.

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.

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

ABSTRACT 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.

Expedited detection of drug resistance in tuberculosis patients

The increase in disease-causing organisms resistant to standard drug therapy has captured the attention of the medical community and the lay press. Drug resistance in rapidly growing bacteria can be detected within a short period. However, in the case of Mycobacterium tuberculosis, detection may take weeks. This paper examines the current methods available to determine drug susceptibility in M tuberculosis. These laboratory methods can be used as a model to assist the clinician in making rational decisions when managing patients with potentially resistant bacterial infections.