In vitro activity of bedaquiline against rapidly growing nontuberculous mycobacteria

Bedaquiline (BDQ) has been proven to be effective in the treatment of multidrug-resistant tuberculosis. We hypothesized that BDQ could be a potential agent to treat nontuberculous mycobacterial (NTM) infection. The objective of this study was to evaluate the in vitro activity of BDQ against rapidly growing mycobacteria by assessing the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against 18 NTM strains. For MIC determination we performed the resazurin microtitre assay broth dilution, and for the MBC the c.f.u. was determined. BDQ exhibited a strong inhibitory effect against most NTM tested; however, for some NTM strains the MBC was significantly higher than the MIC. A new finding is that Mycobacterium flavescens has a mutation in the gene atpE associated with natural resistance to BDQ. These preliminary promising results demonstrate that BDQ could be potentially useful for the treatment of NTM

The transcriptome of Mycobacterium tuberculosis in a lipid-rich dormancy model through RNAseq analysis

Tuberculosis (TB) is currently the number one killer among infectious diseases worldwide. Lipids are abundant molecules during the infectious cycle of Mycobacterium tuberculosis (Mtb) and studies better mimicking its actual metabolic state during pathogenesis are needed. Though most studies have focused on the mycobacterial lipid metabolism under standard culture conditions, little is known about the transcriptome of Mtb in a lipid environment. Here we determined the transcriptome of Mtb H37Rv in a lipid-rich environment (cholesterol and fatty acid) under aerobic and hypoxic conditions, using RNAseq. Lipids significantly induced the expression of 368 genes. A main core lipid response was observed involving efflux systems, iron caption and sulfur reduction. In co-expression with ncRNAs and other genes discussed below, may act coordinately to prepare the machinery conferring drug tolerance and increasing a persistent population. Our findings could be useful to tag relevant pathways for the development of new drugs, vaccines and new strategies to control TB

Evaluation of phage assay for rapid phenotypic detection of rifampicin resistance in Mycobacterium tuberculosis

BACKGROUND: Conventional methods for susceptibility testing require several months before results can be reported. However, rapid methods to determine drug susceptibility have been developed recently. Phage assay have been reported as a rapid useful tools for antimicrobial susceptibility testing. The aim of this study was to apply the Phage assay for rapid detection of resistance on Mycobacterium tuberculosis strains in Cuba. METHODS: Phage D29 assay was performed on 102 M. tuberculosis strains to detect rifampicin resistance. The results were compared with the proportion method (gold standard) to evaluate the sensitivity and specificity of Phage assay. RESULTS: Phage assay results were available in 2 days whereas Proportion Methods results were obtain in 42 days. A total of 44 strains were detected as rifampicin resistant by both methods. However, one strains deemed resistant by Proportion Methods was susceptible by Phage assay. The sensitivity and specificity of Phage assay were 97.8 % and 100% respectively. CONCLUSION: Phage assay provides rapid and reliable results for susceptibility testing; it's easy to perform, requires no specialized equipment and is applicable to drug susceptibility testing in low income countries where tuberculosis is a major public health problem

Profiling Mycobacterium ulcerans with hsp65

Univ Fed Sao Paulo, Escola Paulista Med, Dept Microbiol, BR-04023062 Sao Paulo, BrazilInst Trop Med, B-2000 Antwerp, BelgiumUniv Fed Sao Paulo, Escola Paulista Med, Dept Microbiol, BR-04023062 Sao Paulo, BrazilWeb of Scienc

Fitness of Mycobacterium tuberculosis Strains of the W-Beijing and Non-W-Beijing Genotype

BACKGROUND: Multidrug resistant tuberculosis (MDR-TB) is a major threat for global tuberculosis control. The W-Beijing Mycobacterium tuberculosis genotype has been associated with drug resistance. Elucidation of the mechanisms underlying this epidemiological finding may have an important role in the control of MDR-TB. The aim of this study was to evaluate the fitness of drug-susceptible and MDR M. tuberculosis strains of the W-Beijing genotype compared with that of Non-W-Beijing strains. METHODOLOGY/PRINCIPAL FINDINGS: Fitness of M. tuberculosis strains was determined by evaluating the difference in the growth curves obtained in the MGIT960 automated system and assessing the competitive growth capacity between W-Beijing and non-W-Beijing strains. The W-Beijing MDR strains had a significant longer lag phase duration compared to the other strains but did not present a significant fitness cost. When grown in competition they had an advantage only in medium containing 0.1% Tween 80. CONCLUSIONS/SIGNIFICANCE: It was not possible to confirm a selective advantage of W-Beijing strains to grow, except for differences in their resistance to Tween 80. Further studies are needed to elucidate the putative advantage of W-Beijing strains compared to other genotypes

Characterization of Mycobacterium chelonae-like strains by comparative genomics

Isolates of the Mycobacterium chelonae-M. abscessus complex are subdivided into four clusters (CHI to CHIV) in the INNO-LiPA (R) Mycobacterium spp DNA strip assay. A considerable phenotypic variability was observed among isolates of the CHII cluster. In this study, we examined the diversity of 26 CHII cluster isolates by phenotypic analysis, drug susceptibility testing, whole genome sequencing and single-gene analysis. Pairwise genome comparisons were performed using several approaches, including average nucleotide identity (ANI) and genome-to-genome distance (GGD) among others. Based on ANI and GGD the isolates were identified as M. chelonae (14 isolates), M. franklinii (2 isolates) and M. salmoniphium (1 isolate). The remaining 9 isolates were subdivided into three novel putative genomospecies. Phenotypic analyses including drug susceptibility testing, as well as whole genome comparison by TETRA and delta differences, were not helpful in separating the groups revealed by ANI and GGD. The analysis of standard four conserved genomic regions showed that rpoB alone and the concatenated sequences clearly distinguished the taxonomic groups delimited by whole genome analyses. In conclusion, the CHII INNO-LiPa is not a homogeneous cluster; on the contrary, it is composed of closely related different species belonging to the M. chelonae-M. abscessus complex and also several unidentified isolates. The detection of these isolates, putatively novel species, indicates a wider inner variability than the presently known in this complex

String test: a potentially useful tool in the diagnosis of pulmonary tuberculosis in Brazilian children and adolescents

This study investigated the potential use of the String Test (ST) for the diagnosis of pulmonary tuberculosis (PTB) in children and adolescents. This is a case series of patients aged 4-15 years presenting with clinically presumed PTB and submitted to ST in three pediatric TB referral centers in Brazil, between November 2017 and July 2020. The ST was performed in the morning, after 4-12 h of fasting, followed by ingestion of the capsule by the patient, which was attached to the patient’s malar region. The material was collected for simultaneous smear microscopy (acid-fast bacilli - AFB), culture and the molecular investigation by the GeneXpert MTB/RIF®. Thirty-three patients with presumed PTB were included and ST was performed in 26 (78.8%) of them and 7 (21.2%) patients could not swallow the cord. The diagnosis of PTB was established in 11 (42.3%) of the 26 patients who underwent the ST. The diagnosis of PTB was confirmed (by culture or GeneXpert MTB/RIF®) in 5 patients, 4 of whom were also positive by the ST. Two of them showed positivity by the GeneXpert MTB/RIF® only in the ST sample. Two other patients had a positive ST following the induced sputum test (AFB, GeneXpert MTB/RIF®, and positive culture in both specimens). Thus, ST was positive in 36.4% of the patients in whom PTB was diagnosed. ST could be a useful test for diagnosing PTB in children and adolescents

Rapid culture-based methods for drug-resistance detection in Mycobacterium tuberculosis.

Tuberculosis still represents a major public health problem, especially in low-resource countries where the burden of the disease is more important. Multidrug-resistant and extensively drug drug-resistant tuberculosis constitute serious problems for the efficient control of the disease stressing the need to investigate resistance to first- and second-line drugs. Conventional methods for detecting drug-resistance in Mycobacterium tuberculosis are slow and cumbersome. The most commonly used proportion method on Löwenstein-Jensen medium or Middlebrook agar requires a minimum of 3-4 weeks to produce results. Several new approaches have been proposed in the last years for the rapid and timely detection of drug-resistance in tuberculosis. This review will address phenotypic culture-based methods for rapid drug susceptibility testing in M. tuberculosis

Rifampin-Isoniazid Oligonucleotide Typing: an Alternative Format for Rapid Detection of Multidrug-Resistant Mycobacterium tuberculosis

Fil: Hernandez-Neuta, Iván. Corporacion CorpoGen; Colombia.Fil: Varela, Andres. Corporacion CorpoGen; Colombia.Fil: Martin, Anandi. Institute of Tropical Medicine. Mycobacteriology Unit; Bélgica.Fil: von Groll, Andrea. Institute of Tropical Medicine. Mycobacteriology Unit; Bélgica.Fil: Jureen, Pontus. Swedish Institute for Infectious Disease Control; Suecia.Fil: López, Beatriz. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Imperiale, Belen. Hospital Dr. Cetrángolo; Argentina.Fil: Skenders, Girts. State Agency of Tuberculosis and Lung Diseases; Letonia.Fil: Ritacco, Viviana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Hoffner, Sven. Swedish Institute for Infectious Disease Control; Suecia.Fil: Morcillo, Nora. Hospital Dr. Cetrángolo; Argentina.Fil: Palomino, Juan Carlos. Institute of Tropical Medicine. Mycobacteriology Unit; Bélgica.Fil: Del Portillo, Patricia. Corporacion CorpoGen; Colombia.on of rifampin and isoniazid resistance in clinical isolates of Mycobacterium tuberculosis was based on the same amplification/reverse hybridization principle of the widely used spoligotyping. The test involved probing nine DNA regions that are targets of common drug resistance-associated mutations in the genes rpoB, katG, and inhA. Addition of quaternary amine tetramethyl ammonium chloride to the hybridization buffer promoted multiple hybrid formations at a single annealing temperature irrespective of the different GC contents of probes. The assay was standardized using 20 well-documented strains from the Institute of Tropical Medicine (Belgium) and evaluated blindly in a central laboratory with 100 DNA samples that were obtained from cultured clinical isolates and shipped dried from three other countries. Compared with drug susceptibility testing, both sensitivity and specificity for rifampin resistance detection were 93.0% while for isoniazid the values were 87.7% and 97.7%, respectively. Compared with sequencing and GenoType MTBDRplus methods, sensitivity and specificity reached 96.4% and 95.5% for rifampin and 92.7% and 100% for isoniazid. Altogether, 40/45 (89%) multidrug-resistant isolates were correctly identified. Advantages of this in-house development include versatility, capacity to run up to 41 samples by triplicate in a single run, and reuse of the membrane at least 10 times. These features substantially reduce cost per reaction and make the assay an attractive tool for use in reference laboratories of countries that have a high burden of multidrug-resistant tuberculosis but that cannot afford expensive commercial tests because of limited resources