Phylogenetically informative mutations in genes implicated in antibiotic resistance in Mycobacterium tuberculosis complex

Funder: Joachim Herz Stiftung; doi: http://dx.doi.org/10.13039/100008662Abstract: Background: A comprehensive understanding of the pre-existing genetic variation in genes associated with antibiotic resistance in the Mycobacterium tuberculosis complex (MTBC) is needed to accurately interpret whole-genome sequencing data for genotypic drug susceptibility testing (DST). Methods: We investigated mutations in 92 genes implicated in resistance to 21 anti-tuberculosis drugs using the genomes of 405 phylogenetically diverse MTBC strains. The role of phylogenetically informative mutations was assessed by routine phenotypic DST data for the first-line drugs isoniazid, rifampicin, ethambutol, and pyrazinamide from a separate collection of over 7000 clinical strains. Selected mutations/strains were further investigated by minimum inhibitory concentration (MIC) testing. Results: Out of 547 phylogenetically informative mutations identified, 138 were classified as not correlating with resistance to first-line drugs. MIC testing did not reveal a discernible impact of a Rv1979c deletion shared by M. africanum lineage 5 strains on resistance to clofazimine. Finally, we found molecular evidence that some MTBC subgroups may be hyper-susceptible to bedaquiline and clofazimine by different loss-of-function mutations affecting a drug efflux pump subunit (MmpL5). Conclusions: Our findings underline that the genetic diversity in MTBC has to be studied more systematically to inform the design of clinical trials and to define sound epidemiologic cut-off values (ECOFFs) for new and repurposed anti-tuberculosis drugs. In that regard, our comprehensive variant catalogue provides a solid basis for the interpretation of mutations in genotypic as well as in phenotypic DST assays

Resistance to First-Line Anti-TB Drugs Is Associated with Reduced Nitric Oxide Susceptibility in Mycobacterium tuberculosis

Background and objective: The relative contribution of nitric oxide (NO) to the killing of Mycobacterium tuberculosis in human tuberculosis (TB) is controversial, although this has been firmly established in rodents. Studies have demonstrated that clinical strains of M. tuberculosis differ in susceptibility to NO, but how this correlates to drug resistance and clinical outcome is not known. Methods: In this study, 50 sputum smear- and culture-positive patients with pulmonary TB in Gondar, Ethiopia were included. Clinical parameters were recorded and drug susceptibility profile and spoligotyping patterns were investigated. NO susceptibility was studied by exposing the strains to the NO donor DETA/NO. Results: Clinical isolates of M. tuberculosis showed a dose- and time-dependent response when exposed to NO. The most frequent spoligotypes found were CAS1-Delhi and T3_ETH in a total of nine known spoligotypes and four orphan patterns. There was a significant association between reduced susceptibility to NO (>10% survival after exposure to 1mM DETA/NO) and resistance against first-line anti-TB drugs, in particular isoniazid (INH). Patients infected with strains of M. tuberculosis with reduced susceptibility to NO showed no difference in cure rate or other clinical parameters, but a tendency towards lower rate of weight gain after two months of treatment. Conclusion: There is a correlation between resistance to first-line anti-TB drugs and reduced NO susceptibility in clinical strains of M. tuberculosis. Further studies including the mechanisms of reduced NO susceptibility are warranted and could identify targets for new therapeutic interventions

Antioxidants Protect Keratinocytes against M. ulcerans Mycolactone Cytotoxicity

BACKGROUND: Mycobacterium ulcerans is the causative agent of necrotizing skin ulcerations in distinctive geographical areas. M. ulcerans produces a macrolide toxin, mycolactone, which has been identified as an important virulence factor in ulcer formation. Mycolactone is cytotoxic to fibroblasts and adipocytes in vitro and has modulating activity on immune cell functions. The effect of mycolactone on keratinocytes has not been reported previously and the mechanism of mycolactone toxicity is presently unknown. Many other macrolide substances have cytotoxic and immunosuppressive activities and mediate some of their effects via production of reactive oxygen species (ROS). We have studied the effect of mycolactone in vitro on human keratinocytes--key cells in wound healing--and tested the hypothesis that the cytotoxic effect of mycolactone is mediated by ROS. METHODOLOGY/PRINCIPAL FINDINGS: The effect of mycolactone on primary skin keratinocyte growth and cell numbers was investigated in serum free growth medium in the presence of different antioxidants. A concentration and time dependent reduction in keratinocyte cell numbers was observed after exposure to mycolactone. Several different antioxidants inhibited this effect partly. The ROS inhibiting substance deferoxamine, which acts via chelation of Fe(2+), completely prevented mycolactone mediated cytotoxicity. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that mycolactone mediated cytotoxicity can be inhibited by deferoxamine, suggesting a role of iron and ROS in mycolactone induced cytotoxicity of keratinocytes. The data provide a basis for the understanding of Buruli ulcer pathology and the development of improved therapies for this disease

Tuberculosis : Diagnosis and drug susceptibility testing where resources are scarce

Background: Tuberculosis remains a major global public health problem. Not surprisingly, most cases of this disease occur in poor countries and an increasing number of patients harbor drug - resistant bacteria. The cornerstone of bacteriological diagnosis of tuberculosis is direct sputum smear microscopy. This method is rapid, inexpensive and specific, however, sensitivity is discouragingly low. Regarding drug susceptibility testing, the methods available today are either cheap and slow or fast but too costly to be applicable in most high incidence areas. The aim of this investigation was to improve detection and drug susceptibility testing of Mycobacterium. tuberculosis with special emphasis on settings with scarce resources. Methods: We evaluated an improved method for sputum microscopy, which is based on liquefaction of the sample with household bleach, followed by its concentration by centrifugation prior to staining and microscopy. The bleach microscopy method was compared to standard direct microscopy in Honduras. Then, we performed a literature review in search for all studies that have compared the bleach method to the direct method in low- and middle-income countries. We further sent out questionnaires to key persons in national tuberculosis control programs in order to investigate the knowledge and opinions about this alternative method. We also developed and evaluated a new method for rapid and inexpensive drug susceptibility testing, which is based on the ability of M. tuberculosis to reduce nitrate to nitrite. The performance of this method, the Nitrate Reductase Assay, for drug susceptibility testing was compared to the standard BACTEC 460 liquid culture system. Moreover, we evaluated the performance of a commercial automatic culture system called BacT/ALERT 3D for primary detection of M. tuberculosis in clinical samples and for drug susceptibility testing. Results: We found that the bleach method could improve sensitivity with 3 7 %, Arith unchanged specificity, in Honduras. In the scientific literature, we found 19 studies that had compared the bleach microscopy method with the direct smear. In 15 out of these there was a significant improvement ranging from 7-253 % of the proportion of positive tests using the new method. We received answers from 84 key persons in 69/85 included countries (81 %). Thirty-six key persons thought that the bleach method could increase case detection in their countries, 40 did not know and five thought it could not. Furthermore, recommendations from the World Health Organization or the International Union Against Tuberculosis and Lung Disease, as well as studies in their own countries, were factors that would make the key persons promote the bleach microscopy method for routine use. When tested in 57 M. tuberculosis strains, the Nitrate Reductase Assay showed equivalent susceptibility results to the BACTEC method for isoniazid and rifampicin, and had a similar turn-around time. The BacT/ALERT technique, which was tested on 2659 clinical specimens, detected M. tuberculosis at a similar rate as when cultured on standard Löwenstein-Jensen medium. Concerning drug susceptibility testing, this new technique showed a lesser sensitivity in detecting drug resistance compared to the BACTEC method in 50 M. tuberculosis strains. Conclusions: The bleach microscopy method can clearly improve case detection of tuberculosis and key persons in national tuberculosis control programs are interested in this technique. I my own opinion, the World Health Organization should recommend its evaluation and introduction for routine use. The Nitrate Reductase Assay might become a suitable option for rapid and inexpensive drug susceptibility testing of rifampicin and isoniazid (the two most important antituberculosis drugs) if it proves successful in field studies. The BacT/ALERT 3D system is a valid alternative for primary isolation but should be further developed before it can be used for drug susceptibility testing. The heavy cost of the apparatus and substrates limits, however, the applicability where resources are scarce

Rapid and Inexpensive Drug Susceptibility Testing of Mycobacterium tuberculosis with a Nitrate Reductase Assay

Multidrug-resistant tuberculosis is an increasing public health concern in many parts of the world, especially in low-income countries, where most cases occur. Traditional drug susceptibility testing is either time-consuming, such as the proportion method on solid media, or expensive, such as the BACTEC 460 system. We have evaluated a new nitrate reductase assay (NRA) that depends on the ability of Mycobacterium tuberculosis to reduce nitrate to nitrite. The reduction can be detected using specific reagents, which produce a color change. We tested a panel of 57 M. tuberculosis strains with various resistance patterns. The bacteria were inoculated on Löwenstein-Jensen medium, either without drugs or with rifampin, isoniazid, streptomycin, or ethambutol and with potassium nitrate (KNO(3)) incorporated. After incubation for 7, 10, or 14 days, the reagents were added and nitrate reduction, indicating growth, could be detected by a color change. Sensitivities to and specificities for drugs as determined by the NRA method compared to those determined by the BACTEC 460 method were 100 and 100% for rifampin, 97 and 96% for isoniazid, 95 and 83% for streptomycin, and 75 and 98% for ethambutol, respectively. The results were in the majority of the cases available in 7 days. The evaluated method is rapid and inexpensive and could correctly identify most resistant and sensitive M. tuberculosis strains. It has the potential to become an interesting alternative to existing methods, such as the proportion and BACTEC methods, particularly in resource-poor settings

Challenging a dogma: antimicrobial susceptibility testing breakpoints for Mycobacterium tuberculosis

The rise in multidrug-resistant tuberculosis makes it increasingly important that antimicrobial susceptibility testing of Mycobacterium tuberculosis produce clinically meaningful and technically reproducible results. Unfortunately, this is not always the case because mycobacteriology specialists have not followed generally accepted modern principles for the establishment of susceptibility breakpoints for bacterial and fungal pathogens. These principles specifically call for a definition of the minimum inhibitory concentrations (MICs) applicable to organisms without resistance mechanisms (also known as wild-type MIC distributions), to be used in combination with data on clinical outcomes, pharmacokinetics and pharmacodynamics. In a series of papers the authors have defined tentative wild-type MIC distributions for M. tuberculosis and hope that other researchers will follow their example and provide confirmatory data. They suggest that some breakpoints are in need of revision because they either (i) bisect the wild-type distribution, which leads to poor reproducibility in antimicrobial susceptibility testing, or (ii) are substantially higher than the MICs of wild-type organisms without supporting clinical evidence, which may result in some strains being falsely reported as susceptible. The authors recommend, in short, that susceptibility breakpoints for antituberculosis agents be systematically reviewed and revised, if necessary, using the same modern tools now accepted for all other bacteria and fungi by the scientific community and by the European Medicines Agency and the European Centre for Disease Prevention and Control. For several agents this would greatly improve the accuracy and reproducibility of antimicrobial susceptibility testing of M. tuberculosi

Drug Susceptibility Testing of Mycobacterium tuberculosis by a Nitrate Reductase Assay Applied Directly on Microscopy-Positive Sputum Samples

Current methods for drug susceptibility testing of Mycobacterium tuberculosis are either costly or slow. As the prevalence of multidrug-resistant strains increases, the need for fast, reliable, and inexpensive methods that can also be applied in settings with scarce resources is obvious. We evaluated a rapid colorimetric nitrate reductase assay (NRA) for direct drug susceptibility testing of M. tuberculosis directly from clinical sputum samples with positive microscopy results for acid-fast bacilli with more than 10 acid-fast bacilli per high-power field. We have saved valuable time by omitting the preisolation step. The sensitivity (ability to detect true drug resistance) and specificity (ability to detect true drug susceptibility) of the direct NRA, using the direct proportion method as the reference, were 100 and 100%, 93 and 100%, 76 and 100%, and 55 and 99% for rifampin, isoniazid, streptomycin, and ethambutol, respectively, when tested on M. tuberculosis strains present in 121 samples. The results were in most cases available in 14 days. The direct NRA could be used as a rapid, inexpensive, and accurate method to determine rifampin and isoniazid susceptibility directly from sputum. The technique might become a valid alternative to traditional methods, especially in low-income countries

Phylogenetically informative mutations in genes implicated in antibiotic resistance in Mycobacterium tuberculosis complex.

BACKGROUND:A comprehensive understanding of the pre-existing genetic variation in genes associated with antibiotic resistance in the Mycobacterium tuberculosis complex (MTBC) is needed to accurately interpret whole-genome sequencing data for genotypic drug susceptibility testing (DST). METHODS:We investigated mutations in 92 genes implicated in resistance to 21 anti-tuberculosis drugs using the genomes of 405 phylogenetically diverse MTBC strains. The role of phylogenetically informative mutations was assessed by routine phenotypic DST data for the first-line drugs isoniazid, rifampicin, ethambutol, and pyrazinamide from a separate collection of over 7000 clinical strains. Selected mutations/strains were further investigated by minimum inhibitory concentration (MIC) testing. RESULTS:Out of 547 phylogenetically informative mutations identified, 138 were classified as not correlating with resistance to first-line drugs. MIC testing did not reveal a discernible impact of a Rv1979c deletion shared by M. africanum lineage 5 strains on resistance to clofazimine. Finally, we found molecular evidence that some MTBC subgroups may be hyper-susceptible to bedaquiline and clofazimine by different loss-of-function mutations affecting a drug efflux pump subunit (MmpL5). CONCLUSIONS:Our findings underline that the genetic diversity in MTBC has to be studied more systematically to inform the design of clinical trials and to define sound epidemiologic cut-off values (ECOFFs) for new and repurposed anti-tuberculosis drugs. In that regard, our comprehensive variant catalogue provides a solid basis for the interpretation of mutations in genotypic as well as in phenotypic DST assays

Antioxidant protection against mycolactone cytotoxicity.

<p>(A) Antioxidants were added 30 min before mycolactone (300 ng/ml) and the incubation was continued for 48 h when cell numbers were determined by measuring WST-1color at 450 nm. Data represent means of triplicate determinations and standard deviations from one representative experiment out of two performed (*, p<0.05, one way ANOVA and students t-test). (B) Deferoxamine (D) and TEMPOL were added at different concentrations alone or in combination 30 min before mycolactone (300 ng/ml) and the incubation was continued for 48 h when cell numbers were determined by measuring neutral red uptake Concentrations are in µM for all substances except catalase which is in U/ml. Data shown are means and SEM of four experiments (*, p<0.05, ANOVA and students t-test). TEMPOL 1600 µM was not included in the statistical analysis (N = 2).</p