Telacebec Interferes with Virulence Lipid Biosynthesis Protein Expression and Sensitizes to Other Antibiotics.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a public health issue, particularly due to multi-drug-resistant Mtb. The bacillus is wrapped in a waxy envelope containing lipids acting as essential virulence factors, accounting for the natural antibiotic resistance of mycobacteria. Telacebec (previously known as Q203) is a promising new anti-TB agent inhibiting the cytochrome bc1 complex of a mycobacterial electron transport chain (ETC). Here, we show that the telacebec-challenged M. bovis BCG exhibited a reduced expression of proteins involved in the synthesis of phthiocerol dimycocerosates (PDIMs)/phenolic glycolipids (PGLs), lipid virulence factors associated with cell envelope impermeability. Consistently, telacebec, at concentrations lower than its MIC, downregulated the transcription of a PDIM/PGL-synthesizing operon, suggesting a metabolic vulnerability triggered by the drug. The drug was able to synergize on BCG with rifampicin or vancomycin, the latter being a drug exerting a marginal effect on PDIM-bearing bacilli. Telacebec at a concentration higher than its MIC had no detectable effect on cell wall PDIMs, as shown by TLC analysis, a finding potentially explained by the retaining of previously synthesized PDIMs due to the inhibition of growth. The study extends the potential of telacebec, demonstrating an effect on mycobacterial virulence lipids, allowing for the development of new anti-TB strategies.

Targeted Chromosomal Barcoding Establishes Direct Genotype-Phenotype Associations for Antibiotic Resistance in Mycobacterium abscessus.

A bedaquiline-resistant Mycobacterium abscessus isolate was sequenced, and a candidate mutation in the atpE gene was identified as responsible for the antibiotic resistance phenotype. To establish a direct genotype-phenotype relationship of this mutation which results in a Asp-to-Ala change at position 29 (D29A), we developed a recombineering-based method consisting of the specific replacement of the desired mutation in the bacterial chromosome. As surrogate bacteria, we used two M. abscessus bedaquiline-susceptible strains: ATCC 19977 and the SL541 clinical isolate. The allelic exchange substrates used in recombineering carried either the sole D29A mutation or a genetic barcode of silent mutations in codons flanking the D29A mutation. After selection of bedaquiline-resistant M. abscessus colonies transformed with both substrates, we obtained equivalent numbers of recombinants. These resistant colonies were analyzed by allele-specific PCR and Sanger sequencing, and we demonstrated that the presence of the genetic barcode was linked to the targeted incorporation of the desired mutation in its chromosomal location. All recombinants displayed the same MIC to bedaquiline as the original isolate, from which the D29A mutation was identified. Finally, to demonstrate the broad applicability of this method, we confirmed the association of bedaquiline resistance with the atpE A64P mutation in analysis performed in independent M. abscessus strains and by independent researchers. IMPORTANCE Antimicrobial resistance (AMR) threatens the effective prevention and treatment of an ever-increasing range of infections caused by microorganisms. On the other hand, infections caused by Mycobacterium abscessus affect people with chronic lung diseases, and their incidence has grown alarmingly in recent years. Further, these bacteria are known to easily develop AMR to the few therapeutic options available, making their treatment long-lasting and challenging. The recent introduction of new antibiotics against M. abscessus, such as bedaquiline, makes us anticipate a future when a plethora of antibiotic-resistant strains will be isolated and sequenced. However, in the era of whole-genome sequencing, one of the challenges is to unequivocally assign a biological function to each identified polymorphism. Thus, in this study, we developed a fast, robust, and reliable method to assign genotype-phenotype associations for putative antibiotic-resistant polymorphisms in M. abscessus.

The small-molecule SMARt751 reverses Mycobacterium tuberculosis resistance to ethionamide in acute and chronic mouse models of tuberculosis.

The sensitivity of Mycobacterium tuberculosis, the pathogen that causes tuberculosis (TB), to antibiotic prodrugs is dependent on the efficacy of the activation process that transforms the prodrugs into their active antibacterial moieties. Various oxidases of M. tuberculosis have the potential to activate the prodrug ethionamide. Here, we used medicinal chemistry coupled with a phenotypic assay to select the N-acylated 4-phenylpiperidine compound series. The lead compound, SMARt751, interacted with the transcriptional regulator VirS of M. tuberculosis, which regulates the mymA operon encoding a monooxygenase that activates ethionamide. SMARt751 boosted the efficacy of ethionamide in vitro and in mouse models of acute and chronic TB. SMARt751 also restored full efficacy of ethionamide in mice infected with M. tuberculosis strains carrying mutations in the ethA gene, which cause ethionamide resistance in the clinic. SMARt751 was shown to be safe in tests conducted in vitro and in vivo. A model extrapolating animal pharmacokinetic and pharmacodynamic parameters to humans predicted that as little as 25 mg of SMARt751 daily would allow a fourfold reduction in the dose of ethionamide administered while retaining the same efficacy and reducing side effects.

Retrospective evaluation of routine whole genome sequencing of Mycobacterium tuberculosis at the Belgian National Reference Center, 2019.

OBJECTIVES: Since January 2019, the Belgian National Reference Center for Mycobacteria (NRC) has switched from conventional typing to prospective whole-genome sequencing (WGS) of all submitted Mycobacterium tuberculosis complex (MTB) isolates. The ISO17025 validated procedure starts with semi-automated extraction and purification of gDNA directly from the submitted MGIT tubes, without preceding subculturing. All samples are then sequenced on an Illumina MiSeq sequencer and analyzed using an in-house developed and validated bioinformatics workflow to determine the species and antimicrobial resistance. In this study, we retrospectively compare results obtained via WGS to conventional phenotypic and genotypic testing, for all Belgian MTB strains analyzed in 2019 (n = 306). RESULTS: In all cases, the WGS-based procedure was able to identify correctly the MTB species. Compared to MGIT drug susceptibility testing (DST), the sensitivity and specificity of genetic prediction of resistance to first-line antibiotics were respectively 100 and 99% (rifampicin, RIF), 90.5 and 100% (isoniazid, INH), 100 and 98% (ethambutol, EMB) and 61.1 and 100% (pyrazinamide, PZA). The negative predictive value was above 95% for these four first-line drugs. A positive predictive value of 100% was calculated for INH and PZA, 80% for RIF and 45% for EMB. CONCLUSIONS: Our study confirms the effectiveness of WGS for the rapid detection of M. tuberculosis complex and its drug resistance profiles for first-line drugs even when working directly on MGIT tubes, and supports the introduction of this test into the routine workflow of laboratories performing tuberculosis diagnosis.

A Bioinformatics Whole-Genome Sequencing Workflow for Clinical Mycobacterium tuberculosis Complex Isolate Analysis, Validated Using a Reference Collection Extensively Characterized with Conventional Methods and In Silico Approaches.

The use of whole-genome sequencing (WGS) for routine typing of bacterial isolates has increased substantially in recent years. For Mycobacterium tuberculosis (MTB), in particular, WGS has the benefit of drastically reducing the time required to generate results compared to most conventional phenotypic methods. Consequently, a multitude of solutions for analyzing WGS MTB data have been developed, but their successful integration in clinical and national reference laboratories is hindered by the requirement for their validation, for which a consensus framework is still largely absent. We developed a bioinformatics workflow for (Illumina) WGS-based routine typing of MTB complex (MTBC) member isolates allowing complete characterization, including (sub)species confirmation and identification (16S, csb/RD, hsp65), single nucleotide polymorphism (SNP)-based antimicrobial resistance (AMR) prediction, and pathogen typing (spoligotyping, SNP barcoding, and core genome multilocus sequence typing). Workflow performance was validated on a per-assay basis using a collection of 238 in-house-sequenced MTBC isolates, extensively characterized with conventional molecular biology-based approaches supplemented with public data. For SNP-based AMR prediction, results from molecular genotyping methods were supplemented with in silico modified data sets, allowing us to greatly increase the set of evaluated mutations. The workflow demonstrated very high performance with performance metrics of >99% for all assays, except for spoligotyping, where sensitivity dropped to ∼90%. The validation framework for our WGS-based bioinformatics workflow can aid in the standardization of bioinformatics tools by the MTB community and other SNP-based applications regardless of the targeted pathogen(s). The bioinformatics workflow is available for academic and nonprofit use through the Galaxy instance of our institute at https://galaxy.sciensano.be.

Integrative transnational analysis to dissect tuberculosis transmission events along the migratory route from Africa to Europe.

BACKGROUND: Growing international migration has increased the complexity of tuberculosis transmission patterns. Italy's decision to close its borders in 2018 made of Spain the new European porte entrée for migration from the Horn of Africa (HA). In one of the first rescues of migrants from this region at the end of 2018, tuberculosis was diagnosed in eight subjects, mainly unaccompanied minors. METHODS: Mycobacterium tuberculosis isolates from these recently arrived migrants were analysed by Mycobacterial Interspersed Repetitive-Unit/Variable-Number of Tandem Repeat (MIRU-VNTR) and subsequent whole genome sequencing (WGS) analysis. Data were compared with those from collections from other European countries receiving migrants from the HA and a strain-specific PCR was applied for a fast searching of common strains. Infections in a cellular model were performed to assess strain virulence. RESULTS: MIRU-VNTR analysis allowed identifying an epidemiological cluster involving three of the eight cases from Somalia (0 single-nucleotide polymorphisms between isolates, HA cluster). Following detailed interviews revealed that two of these cases had shared the same migratory route in most of the trip and had spent a long time at a detention camp in Libya. To confirm potential en route transmission for the three cases, we searched the same strain in collections from other European countries receiving migrants from the HA. MIRU-VNTR, WGS and a strain-specific PCR for the HA strain were applied. The same strain was identified in 12 cases from Eritrea diagnosed soon after their arrival in 2018 to the Netherlands, Belgium and Italy. Intracellular replication rate of the strain did not reveal abnormal virulence. CONCLUSIONS: Our study suggests a potential en route transmission of a pan-susceptible strain, which caused at least 15 tuberculosis cases in Somalian and Eritrean migrants diagnosed in four different European countries.

Deep amplicon sequencing for culture-free prediction of susceptibility or resistance to 13 anti-tuberculous drugs.

Conventional molecular tests for detecting Mycobacterium tuberculosis complex (MTBC) drug resistance on clinical samples cover a limited set of mutations. Whole-genome sequencing (WGS) typically requires culture.Here, we evaluated the Deeplex Myc-TB targeted deep-sequencing assay for prediction of resistance to 13 anti-tuberculous drugs/drug classes, directly applicable on sputum.With MTBC DNA tests, the limit of detection was 100-1000 genome copies for fixed resistance mutations. Deeplex Myc-TB captured in silico 97.1-99.3% of resistance phenotypes correctly predicted by WGS from 3651 MTBC genomes. On 429 isolates, the assay predicted 92.2% of 2369 first- and second-line phenotypes, with a sensitivity of 95.3% and a specificity of 97.4%. 56 out of 69 (81.2%) residual discrepancies with phenotypic results involved pyrazinamide, ethambutol and ethionamide, and low-level rifampicin or isoniazid resistance mutations, all notoriously prone to phenotypic testing variability. Only two out of 91 (2.2%) resistance phenotypes undetected by Deeplex Myc-TB had known resistance-associated mutations by WGS analysis outside Deeplex Myc-TB targets. Phenotype predictions from Deeplex Myc-TB analysis directly on 109 sputa from a Djibouti survey matched those of MTBSeq/PhyResSE/Mykrobe, fed with WGS data from subsequent cultures, with a sensitivity of 93.5/98.5/93.1% and a specificity of 98.5/97.2/95.3%, respectively. Most residual discordances involved gene deletions/indels and 3-12% heteroresistant calls undetected by WGS analysis or natural pyrazinamide resistance of globally rare "Mycobacterium canettii" strains then unreported by Deeplex Myc-TB. On 1494 arduous sputa from a Democratic Republic of the Congo survey, 14 902 out of 19 422 (76.7%) possible susceptible or resistance phenotypes could be predicted culture-free.Deeplex Myc-TB may enable fast, tailored tuberculosis treatment.

Recurrent Tuberculosis in a Young Child.

A young child, 19 months of age, presented with a second episode of tuberculosis after full recovery from initial tuberculosis disease 6 months earlier. Mycobacterium tuberculosis strains isolated from both episodes were genotyped and differed from one another. We present the first case of proven tuberculosis reinfection in a likely immunocompetent child, living in a high-risk environment favorable for exposition to M. tuberculosis but in a low-incidence country.

Transcriptional profiling of a laboratory and clinical Mycobacterium tuberculosis strain suggests respiratory poisoning upon exposure to delamanid.

Tuberculosis (TB) is the most deadly infectious disease worldwide. To reduce TB incidence and counter the spread of multidrug resistant TB, the discovery and characterization of new drugs is essential. In this study, the transcriptional response of two Mycobacterium tuberculosis strains to a pressure of the recently approved delamanid is investigated. Total RNA sequencing revealed that the response to this bicyclic nitroimidazole shows many similarities with pretomanid, an anti-tuberculous drug from the same class. Although delamanid is found to inhibit cell wall synthesis, the expression of genes involved in this process were only mildly affected. In contrast, a clear parallel was found with components that affect aerobic respiration. This demonstrates that, besides the inhibition of cell wall synthesis, respiratory poisoning plays a fundamental role in the bactericidal effect of delamanid. Remarkably, the most highly induced genes comprise poorly characterized genes for which functional characterization might hint to the target molecule(s) of delamanid and its exact mode(s) of action.

In vitro activity of bedaquiline against slow-growing nontuberculous mycobacteria.

Bedaquiline (BDQ) is a recently approved antibiotic for the treatment of multidrug-resistant tuberculosis, but its potential against slow-growing mycobacteria (SGM) is still unknown. The objective of this study was to determine the in vitro activity of BDQ on SGM by assessing their MIC and minimal bactericidal concentration (MBC). The MIC of BDQ against 17 clinical isolates including Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium kansasii and Mycobacterium simiae species was determined by the resazurin microtitre assay and the MBC by the c.f.u. determination on 7H10 agar plates. BDQ has a bacteriostatic activity on all SGM tested with a MIC range from 0.03 to 0.007 µg ml-1 and surprisingly a good bactericidal activity on the majority of the isolates tested with an MBC of 1-2 µg ml-1 . Based on these preliminary results BDQ seems to be very promising for treatment of diseases caused by SGM.

Strong increase of true and false positive mycobacterial cultures sent to the National Reference Centre in Belgium, 2007 to 2016.

IntroductionIn 2007, a new federal legislation in Belgium prohibited non-biosafety level 3 laboratories to process culture tubes suspected of containing mycobacteria.AimTo present mycobacterial surveillance/diagnosis data from the Belgian National Reference Centre for mycobacteria (NRC) from 2007 to 2016.MethodsThis retrospective observational study investigated the numbers of analyses at the NRC and false positive cultures (interpreted as containing mycobacteria at referring clinical laboratories, but with no mycobacterial DNA detected by PCR in the NRC). We reviewed mycobacterial species identified and assessed trends over time of proportions of nontuberculous mycobacteria (NTM) vs Mycobacterium tuberculosis complex (MTBc), and false positive cultures vs NTM.ResultsFrom 2007 to 2016, analyses requests to the NRC doubled from 12.6 to 25.3 per 100,000 inhabitants. A small but significant increase occurred in NTM vs MTBc proportions, from 57.9% (587/1,014) to 60.3% (867/1,437) (p < 0.001). Although NTM infection notification is not mandatory in Belgium, we annually received up to 8.6 NTM per 100,000 inhabitants. M. avium predominated (ca 20% of NTM cultures), but M. intracellulare culture numbers rose significantly, from 13.0% (74/587) of NTM cultures in 2007 to 21.0% (178/867) in 2016 (RR: 1.05; 95% CI: 1.03-1.07). The number of false positive cultures also increased, reaching 43.3% (1,097/2,534) of all samples in 2016.ConclusionWe recommend inclusion of NTM in sentinel programmes. The large increase of false positive cultures is hypothesised to result from processing issues prior to arrival at the NRC, highlighting the importance of sample decontamination/transport and equipment calibration in peripheral laboratories.

Isoniazid Bactericidal Activity Involves Electron Transport Chain Perturbation.

Accumulating evidence suggests that the bactericidal activity of some antibiotics may not be directly initiated by target inhibition. The activity of isoniazid (INH), a key first-line bactericidal antituberculosis drug currently known to inhibit mycolic acid synthesis, becomes extremely poor under stress conditions, such as hypoxia and starvation. This suggests that the target inhibition may not fully explain the bactericidal activity of the drug. Here, we report that INH rapidly increased Mycobacterium bovis BCG cellular ATP levels and enhanced oxygen consumption. The INH-triggered ATP increase and bactericidal activity were strongly compromised by Q203 and bedaquiline, which inhibit mycobacterial cytochrome bc1 and FoF1 ATP synthase, respectively. Moreover, the antioxidant N-acetylcysteine (NAC) but not 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) abrogated the INH-triggered ATP increase and killing. These results reveal a link between the energetic (ATP) perturbation and INH's killing. Furthermore, the INH-induced energetic perturbation and killing were also abrogated by chemical inhibition of NADH dehydrogenases (NDHs) and succinate dehydrogenases (SDHs), linking INH's bactericidal activity further to the electron transport chain (ETC) perturbation. This notion was also supported by the observation that INH dissipated mycobacterial membrane potential. Importantly, inhibition of cytochrome bd oxidase significantly reduced cell recovery during INH challenge in a culture settling model, suggesting that the respiratory reprogramming to the cytochrome bd oxidase contributes to the escape of INH killing. This study implicates mycobacterial ETC perturbation through NDHs, SDHs, cytochrome bc1, and FoF1 ATP synthase in INH's bactericidal activity and pinpoints the participation of the cytochrome bd oxidase in protection against this drug under stress conditions.

Antibiotic resistance prediction for Mycobacterium tuberculosis from genome sequence data with Mykrobe.

Two billion people are infected with Mycobacterium tuberculosis, leading to 10 million new cases of active tuberculosis and 1.5 million deaths annually. Universal access to drug susceptibility testing (DST) has become a World Health Organization priority. We previously developed a software tool, Mykrobe predictor, which provided offline species identification and drug resistance predictions for M. tuberculosis from whole genome sequencing (WGS) data. Performance was insufficient to support the use of WGS as an alternative to conventional phenotype-based DST, due to mutation catalogue limitations.  Here we present a new tool, Mykrobe, which provides the same functionality based on a new software implementation. Improvements include i) an updated mutation catalogue giving greater sensitivity to detect pyrazinamide resistance, ii) support for user-defined resistance catalogues, iii) improved identification of non-tuberculous mycobacterial species, and iv) an updated statistical model for Oxford Nanopore Technologies sequencing data. Mykrobe is released under MIT license at https://github.com/mykrobe-tools/mykrobe. We incorporate mutation catalogues from the CRyPTIC consortium et al. (2018) and from Walker et al. (2015), and make improvements based on performance on an initial set of 3206 and an independent set of 5845 M. tuberculosis Illumina sequences. To give estimates of error rates, we use a prospectively collected dataset of 4362 M. tuberculosis isolates. Using culture based DST as the reference, we estimate Mykrobe to be 100%, 95%, 82%, 99% sensitive and 99%, 100%, 99%, 99% specific for rifampicin, isoniazid, pyrazinamide and ethambutol resistance prediction respectively. We benchmark against four other tools on 10207 (=5845+4362) samples, and also show that Mykrobe gives concordant results with nanopore data.  We measure the ability of Mykrobe-based DST to guide personalized therapeutic regimen design in the context of complex drug susceptibility profiles, showing 94% concordance of implied regimen with that driven by phenotypic DST, higher than all other benchmarked tools.

Prediction of Susceptibility to First-Line Tuberculosis Drugs by DNA Sequencing.

BACKGROUND: The World Health Organization recommends drug-susceptibility testing of Mycobacterium tuberculosis complex for all patients with tuberculosis to guide treatment decisions and improve outcomes. Whether DNA sequencing can be used to accurately predict profiles of susceptibility to first-line antituberculosis drugs has not been clear. METHODS: We obtained whole-genome sequences and associated phenotypes of resistance or susceptibility to the first-line antituberculosis drugs isoniazid, rifampin, ethambutol, and pyrazinamide for isolates from 16 countries across six continents. For each isolate, mutations associated with drug resistance and drug susceptibility were identified across nine genes, and individual phenotypes were predicted unless mutations of unknown association were also present. To identify how whole-genome sequencing might direct first-line drug therapy, complete susceptibility profiles were predicted. These profiles were predicted to be susceptible to all four drugs (i.e., pansusceptible) if they were predicted to be susceptible to isoniazid and to the other drugs or if they contained mutations of unknown association in genes that affect susceptibility to the other drugs. We simulated the way in which the negative predictive value changed with the prevalence of drug resistance. RESULTS: A total of 10,209 isolates were analyzed. The largest proportion of phenotypes was predicted for rifampin (9660 [95.4%] of 10,130) and the smallest was predicted for ethambutol (8794 [89.8%] of 9794). Resistance to isoniazid, rifampin, ethambutol, and pyrazinamide was correctly predicted with 97.1%, 97.5%, 94.6%, and 91.3% sensitivity, respectively, and susceptibility to these drugs was correctly predicted with 99.0%, 98.8%, 93.6%, and 96.8% specificity. Of the 7516 isolates with complete phenotypic drug-susceptibility profiles, 5865 (78.0%) had complete genotypic predictions, among which 5250 profiles (89.5%) were correctly predicted. Among the 4037 phenotypic profiles that were predicted to be pansusceptible, 3952 (97.9%) were correctly predicted. CONCLUSIONS: Genotypic predictions of the susceptibility of M. tuberculosis to first-line drugs were found to be correlated with phenotypic susceptibility to these drugs. (Funded by the Bill and Melinda Gates Foundation and others.).

Aloe Emodin Reduces Phthiodiolone Dimycocerosate Potentiating Vancomycin Susceptibility on Mycobacteria.

Treatment of tuberculosis still represent a major public health issue. The emergence of multi-and extensively-drug resistant (MDR and XDR) Mycobacterium tuberculosis clinical strains further pinpoint the urgent need for new anti-tuberculous drugs. We previously showed that vancomycin can target mycobacteria lacking cell wall integrity, especially those lacking related phthiocerol and phthiodolone dimycocerosates, PDIM A and PDIM B, respectively. As aloe emodin was previously hypothesized to be able to target the synthesis of mycobacterial cell wall lipids, we tested its ability to potentiate glycopeptides antimycobacterial activity. The aloe emodin with the vancomycin induced a combination effect beyond simple addition, close to synergism, at a concentration lower to reported IC50 cytotoxic value, on M. bovis BCG and on H37Rv M. tuberculosis. Interestingly, out of six MDR and pre-XDR clinical strains, one showed a strong synergic susceptibility to the drug combination. Mycobacterial cell wall lipid analyses highlighted a selective reduction of PDIM B by aloe emodin.

The potential use of rifabutin for treatment of patients diagnosed with rifampicin-resistant tuberculosis.

Background: Use of the Xpert MTB/RIF assay has increased the number of people diagnosed with rifampicin-resistant tuberculosis (RR-TB), especially in South Africa where Xpert is now the initial diagnostic for individuals with TB symptoms. We hypothesized that a proportion of RR-TB patients determined by Xpert can be treated with a rifabutin-containing regimen. Methods: Rifabutin susceptibility by rpoB mutation was assessed in 349 individuals from South Africa and 172 from Belgium. rpoB polymorphisms were identified by Sanger sequencing. Rifampicin and rifabutin susceptibility was assessed phenotypically. A systematic review was performed to comprehensively collate information on rifabutin susceptibility by rpoB polymorphism. Rifabutin susceptibility was assigned to rpoB polymorphisms based on their positive likelihood ratios and ORs. Results: One hundred and twelve rpoB polymorphisms (67.9% from literature) were identified from all 2045 RR-TB patients, of which 17 polymorphisms could be classified as susceptible/resistant to rifabutin. Eleven polymorphisms were associated with rifabutin susceptibility. The 516GTC mutation was the most common, representing 70% (South Africa) and 76% (Belgium) of all rifabutin-susceptible isolates. At a population level, the 11 polymorphisms associated with rifabutin susceptibility occurred in 33.2% and 16.6% of all South African and Belgian patients diagnosed with RR-TB, respectively. Conclusions: Identification of the exact rpoB polymorphism leading to the diagnosis of RR-TB has the potential to allow inclusion of rifabutin in the treatment regimen of a substantial proportion of RR-TB patients. A randomized controlled trial evaluating the efficacy of a rifabutin-containing TB treatment regimen in these selected patients is needed to provide the evidence required for a change in policy.

A cluster of multidrug-resistant Mycobacterium tuberculosis among patients arriving in Europe from the Horn of Africa: a molecular epidemiological study.

BACKGROUND: The risk of tuberculosis outbreaks among people fleeing hardship for refuge in Europe is heightened. We describe the cross-border European response to an outbreak of multidrug-resistant tuberculosis among patients from the Horn of Africa and Sudan. METHODS: On April 29 and May 30, 2016, the Swiss and German National Mycobacterial Reference Laboratories independently triggered an outbreak investigation after four patients were diagnosed with multidrug-resistant tuberculosis. In this molecular epidemiological study, we prospectively defined outbreak cases with 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) profiles; phenotypic resistance to isoniazid, rifampicin, ethambutol, pyrazinamide, and capreomycin; and corresponding drug resistance mutations. We whole-genome sequenced all Mycobacterium tuberculosis isolates and clustered them using a threshold of five single nucleotide polymorphisms (SNPs). We collated epidemiological data from host countries from the European Centre for Disease Prevention and Control. FINDINGS: Between Feb 12, 2016, and April 19, 2017, 29 patients were diagnosed with multidrug-resistant tuberculosis in seven European countries. All originated from the Horn of Africa or Sudan, with all isolates two SNPs or fewer apart. 22 (76%) patients reported their travel routes, with clear spatiotemporal overlap between routes. We identified a further 29 MIRU-VNTR-linked cases from the Horn of Africa that predated the outbreak, but all were more than five SNPs from the outbreak. However all 58 isolates shared a capreomycin resistance-associated tlyA mutation. INTERPRETATION: Our data suggest that source cases are linked to an M tuberculosis clone circulating in northern Somalia or Djibouti and that transmission probably occurred en route before arrival in Europe. We hypothesise that the shared mutation of tlyA is a drug resistance mutation and phylogenetic marker, the first of its kind in M tuberculosis sensu stricto. FUNDING: The Swiss Federal Office of Public Health, the University of Zurich, the Wellcome Trust, National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC), the Medical Research Council, BELTA-TBnet, the European Union, the German Center for Infection Research, and Leibniz Science Campus Evolutionary Medicine of the Lung (EvoLUNG).

Comparison of the RGM medium and the mycobacterial growth indicator tube automated system for isolation of non-tuberculous mycobacteria from sputum samples of cystic fibrosis patients in Belgium.

PURPOSE: Pulmonary infections due to non-tuberculous mycobacteria (NTM) are an emerging issue in the cystic fibrosis (CF) population. Due to bacterial and fungal overgrowth, isolation of mycobacteria from the sputum samples of these patients remains challenging. RGM medium, a novel agar-based culture medium was evaluated for the isolation of NTM from sputum samples of CF patients. METHODOLOGY: Sputum samples were inoculated onto RGM medium and conventional Mycobacterial Growth Indicator Tube (MGIT™, Becton Dickinson, USA). Agar plates were incubated at 35 °C and growth was recorded once a week during 42 days. We compared the yield of the two media. RESULTS: 217 samples were obtained from 124 CF patients. 20 samples (13 patients) had a positive culture for NTM. 79/217 (36.4%) MGIT had to be discontinued due to contamination compared to 18/217 (8.3%) for RGM. We reported equivalent NTM detection performances for RGM and MGIT (P = 0.579): these media enabled the isolation of 15 and 12 NTM strains respectively. CONCLUSION: RGM medium increases the proportion of interpretable results and the number of NTM cultured. Taking into account the non-inferiority compared to conventional methods and ease of use of RGM medium, we estimate that this test can replace current approaches for the screening of NTM among people with CF. Additionally, RGM provides semi-quantitative results (number of colonies) and information on the morphology of colonies, which may be clinically relevant information.

Reversion of antibiotic resistance in Mycobacterium tuberculosis by spiroisoxazoline SMARt-420.

Antibiotic resistance is one of the biggest threats to human health globally. Alarmingly, multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis have now spread worldwide. Some key antituberculosis antibiotics are prodrugs, for which resistance mechanisms are mainly driven by mutations in the bacterial enzymatic pathway required for their bioactivation. We have developed drug-like molecules that activate a cryptic alternative bioactivation pathway of ethionamide in M. tuberculosis, circumventing the classic activation pathway in which resistance mutations have now been observed. The first-of-its-kind molecule, named SMARt-420 (Small Molecule Aborting Resistance), not only fully reverses ethionamide-acquired resistance and clears ethionamide-resistant infection in mice, it also increases the basal sensitivity of bacteria to ethionamide.