%0 Journal Article %J Microbiol Spectr %D 2023 %T Targeted Chromosomal Barcoding Establishes Direct Genotype-Phenotype Associations for Antibiotic Resistance in Mycobacterium abscessus. %A Juan Calvet-Seral %A Estefanía Crespo-Yuste %A Vanessa Mathys %A Rodriguez-Villalobos, Hector %A Pieter-Jan Ceyssens %A Martin, Anandi %A Jesús Gonzalo-Asensio %X

A bedaquiline-resistant Mycobacterium abscessus isolate was sequenced, and a candidate mutation in the 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 A64P mutation in analysis performed in independent M. abscessus strains and by independent researchers. 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 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 , 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 .

%B Microbiol Spectr %8 2023 Mar 29 %G eng %R 10.1128/spectrum.05344-22 %0 Journal Article %J Microorganisms %D 2023 %T Telacebec Interferes with Virulence Lipid Biosynthesis Protein Expression and Sensitizes to Other Antibiotics %A Zhou, Zhiyu %A Wattiez, Ruddy %A Patricia Constant %A Hedia Marrakchi %A Karine Soetaert %A Vanessa Mathys %A Véronique Fontaine %A Zeng, Sheng %B Microorganisms %V 11 %8 Jan-10-2023 %G eng %N 10 %R 10.3390/microorganisms11102469 %0 Journal Article %J Lancet Microbe %D 2022 %T The 2021 WHO catalogue of complex mutations associated with drug resistance: A genotypic analysis. %A Timothy M Walker %A Paolo Miotto %A Claudio U Köser %A Philip W Fowler %A Jeff Knaggs %A Iqbal, Zamin %A Martin Hunt %A Leonid Chindelevitch %A Maha Farhat %A Daniela Maria Cirillo %A Iñaki Comas %A James Posey %A Shaheed V Omar %A Timothy EA Peto %A Anita Suresh %A Swapna Uplekar %A Sacha Laurent %A Rebecca E Colman %A Carl-Michael Nathanson %A Matteo Zignol %A Ann Sarah Walker %A Derrick W Crook %A Nazir Ismail %A Timothy C Rodwell %A Vanessa Mathys %K Drug Resistance %K mutations %K Tuberculosis %X

Background: Molecular diagnostics are considered the most promising route to achieving rapid, universal drug susceptibility testing for complex (MTBC). We aimed to generate a WHO endorsed catalogue of mutations to serve as a global standard for interpreting molecular information for drug resistance prediction.

Methods: A candidate gene approach was used to identify mutations as associated with resistance, or consistent with susceptibility, for 13 WHO endorsed anti-tuberculosis drugs. 38,215 MTBC isolates with paired whole-genome sequencing and phenotypic drug susceptibility testing data were amassed from 45 countries. For each mutation, a contingency table of binary phenotypes and presence or absence of the mutation computed positive predictive value, and Fisher's exact tests generated odds ratios and Benjamini-Hochberg corrected p-values. Mutations were graded as Associated with Resistance if present in at least 5 isolates, if the odds ratio was >1 with a statistically significant corrected p-value, and if the lower bound of the 95% confidence interval on the positive predictive value for phenotypic resistance was >25%. A series of expert rules were applied for final confidence grading of each mutation.

Findings: 15,667 associations were computed for 13,211 unique mutations linked to one or more drugs. 1,149/15,667 (7·3%) mutations were classified as associated with phenotypic resistance and 107/15,667 (0·7%) were deemed consistent with susceptibility. For rifampicin, isoniazid, ethambutol, fluoroquinolones, and streptomycin, the mutations' pooled sensitivity was >80%. Specificity was over 95% for all drugs except ethionamide (91·4%), moxifloxacin (91·6%) and ethambutol (93·3%). Only two resistance mutations were classified for bedaquiline, delamanid, clofazimine, and linezolid as prevalence of phenotypic resistance was low for these drugs.

Interpretation: This first WHO endorsed catalogue of molecular targets for MTBC drug susceptibility testing provides a global standard for resistance interpretation. Its existence should encourage the implementation of molecular diagnostics by National Tuberculosis Programmes.

Funding: UNITAID, Wellcome, MRC, BMGF.

%B Lancet Microbe %V 3 %8 2022 Apr %G eng %N 4 %R 10.1016/S2666-5247(21)00301-3 %0 Journal Article %J Sci Transl Med %D 2022 %T The small-molecule SMARt751 reverses resistance to ethionamide in acute and chronic mouse models of tuberculosis. %A Flipo, Marion %A Frita, Rosangela %A Marilyne Bourotte %A Maria S Martínez-Martínez %A Markus Boesche %A Gary W Boyle %A Geo Derimanov %A Gerard Drewes %A Pablo Gamallo %A Sonja Ghidelli-Disse %A Stephanie Gresham %A Elena Jiménez %A Jaime de Mercado %A Esther Pérez-Herrán %A Esther Porras-De Francisco %A Joaquín Rullas %A Patricia Casado %A Florence Leroux %A Catherine Piveteau %A Kiass, Mehdi %A Vanessa Mathys %A Karine Soetaert %A Véronique Megalizzi %A Tanina, Abdalkarim %A Wintjens, René %A Antoine, Rudy %A Brodin, Priscille %A Delorme, Vincent %A Moune, Martin %A Djaout, Kamel %A Stéphanie Slupek %A Kemmer, Christian %A Gitzinger, Marc %A Lluis Ballell %A Alfonso Mendoza-Losana %A Sergio Lociuro %A Déprez, Benoit %A David Barros-Aguirre %A Modesto J Remuiñán %A Willand, Nicolas %A Baulard, Alain R %K Animals %K Antitubercular Agents %K Ethionamide %K mice %K Mycobacterium tuberculosis %K Prodrugs %K Tuberculosis %X

The sensitivity of , 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 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 , which regulates the 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 strains carrying mutations in the 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.

%B Sci Transl Med %V 14 %8 2022 05 04 %G eng %N 643 %R 10.1126/scitranslmed.aaz6280 %0 Journal Article %J J Clin Microbiol %D 2021 %T 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 %A Bert Bogaerts %A Thomas Delcourt %A Karine Soetaert %A Samira Boarbi %A Pieter-Jan Ceyssens %A Raf Winand %A Julien Van Braekel %A Sigrid C.J. De Keersmaecker %A Nancy Roosens %A Marchal, Kathleen %A Vanessa Mathys %A Kevin Vanneste %K Antimicrobial resistance %K Mycobacterium tuberculosis %K national reference center %K public health %K single nucleotide polymorphism %K VALIDATION %K whole genome sequencing %X

The use of whole genome sequencing (WGS) for routine typing of bacterial isolates has increased substantially in recent years. For (MTB), in particular, WGS has the benefit of drastically reducing the time 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, /RD, , 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 modified datasets allowing to greatly increase the set of evaluated mutations. The workflow demonstrated very high performance with performance metrics >99% for all assays, except for spoligotyping where sensitivity dropped to ∼90%. The validation framework for our WGS-based bioinformatics workflow can aid 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 non-profit usage through the Galaxy instance of our institute at https://galaxy.sciensano.be.

%B J Clin Microbiol %8 2021 Mar 31 %G eng %R 10.1128/JCM.00202-21 %0 Journal Article %J J Travel Med %D 2021 %T Integrative transnational analysis to dissect tuberculosis transmission events along the migratory route from Africa to Europe. %A Miguel Martínez-Lirola %A Rana Jajou %A Vanessa Mathys %A Martin, Anandi %A Andrea Maurizio Cabibbe %A Ana Valera %A Pedro J Sola-Campoy %A Estefanía Abascal %A Sandra Rodríguez-Maus %A Jose Antonio Garrido-Cárdenas %A Magdalena Bonillo %A Álvaro Chiner-Oms %A Begoña López %A Silvia Vallejo-Godoy %A Iñaki Comas %A Patricia Muñoz %A Daniela Maria Cirillo %A Dick van Soolingen %A Laura Pérez-Lago %A Darío García de Viedma %K Africa %K Cluster Analysis %K Europe %K Genotype %K Humans %K Mycobacterium tuberculosis %K Tuberculosis %X

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.

%B J Travel Med %V 28 %8 2021 06 01 %G eng %N 4 %R 10.1093/jtm/taab054 %0 Journal Article %J Acta Clin Belg %D 2021 %T Retrospective evaluation of routine whole genome sequencing of Mycobacterium tuberculosis at the Belgian National Reference Center, 2019. %A Karine Soetaert %A Pieter-Jan Ceyssens %A Samira Boarbi %A Bert Bogaerts %A Thomas Delcourt %A Kevin Vanneste %A Sigrid C.J. De Keersmaecker %A Nancy Roosens %A Alexandra Vodolazkaia %A Marina Mukovnikova %A Vanessa Mathys %X

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 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 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.

%B Acta Clin Belg %8 2021 Nov 09 %G eng %R 10.1080/17843286.2021.1999588 %0 Journal Article %J Int J Tuberc Lung Dis %D 2021 %T Whole-genome sequencing for TB source investigations: principles of ethical precision public health. %A A Van Rie %A D G de Viedma %A C Meehan %A I Comas %A T H Heupink %A E De Vos %A W A de Oñate %A Vanessa Mathys %A Pieter-Jan Ceyssens %A Groenen, G %A F González-Candelas %A A Forier %A E Juengst %X

Whole-genome sequencing (WGS) of allows rapid, accurate inferences about the sources, location and timing of transmission. However, in an era of heightened concern for personal privacy and science distrust, such inferences could result in unintended harm and undermine the public´s trust. We held interdisciplinary stakeholder discussions and performed ethical analyses of real-world illustrative cases to identify principles that optimise benefit and mitigate harm of WGS-driven TB source investigations. The speed and precision with which real-time WGS can be used to associate strains with sensitive information has raised important concerns. While detailed understanding of transmission events could mitigate harm to vulnerable patients and communities when otherwise unfairly blamed for TB outbreaks, the precision of WGS can also identify transmission events resulting in social blame, fear, discrimination, individual or location stigma, and the use of defaming language by the public, politicians and scientists. Public health programmes should balance the need to safeguard privacy with public health goals, transparency and individual rights, including the right to know who infects whom or where. Ethical challenges raised by real-time WGS-driven TB source investigation requires public health authorities to move beyond their current legal mandate and embrace transparency, privacy and community engagement.

%B Int J Tuberc Lung Dis %V 25 %8 2021 Mar 01 %G eng %N 3 %R 10.5588/ijtld.20.0886 %0 Journal Article %J Eur Respir J %D 2020 %T Deep amplicon sequencing for culture-free prediction of susceptibility or resistance to 13 anti-tuberculous drugs. %A Agathe Jouet %A Cyril Gaudin %A Nelly Badalato %A Allix-Béguec, Caroline %A Stéphanie Duthoy %A Alice Ferré %A Maren Diels %A Yannick Laurent %A Sandy Contreras %A Silke Feuerriegel %A Stefan Niemann %A Emmanuel André %A Michel K Kaswa %A Elisa Tagliani %A Andrea Cabibbe %A Vanessa Mathys %A Daniela Cirillo %A Bouke C de Jong %A Rigouts, Leen %A Supply, Philip %X

Conventional molecular tests for detecting 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 specificity of 97.4%. Fifty-six 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 2 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 specificity of 98.5/97.2/95.3%. Most residual discordances involved gene deletions/indels and 3-12% heteroresistant calls undetected by WGS analysis, or natural pyrazinamide resistance of globally rare " strains then unreported by Deeplex Myc-TB. On 1494 arduous sputa from a Democratic Republic of the Congo survey, 14 902 of 19 422 (76.7%) possible susceptible or resistance phenotypes could be predicted culture-free. Deeplex Myc-TB may enable fast, tailored tuberculosis treatment.

%B Eur Respir J %8 2020 Sep 17 %G eng %R 10.1183/13993003.02338-2020 %0 Journal Article %J Pediatr Infect Dis J %D 2020 %T Recurrent tuberculosis in a young child %A Alexandra Dreesman %A Oriane Stévart %A Catherine Adler %A Vanessa Mathys %A Françoise Mouchet %X

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.

%B Pediatr Infect Dis J %8 2020 Apr 13 %G eng %R 10.1097/INF.0000000000002685 %0 Journal Article %J Wellcome Open Res %D 2019 %T Antibiotic resistance prediction for Mycobacterium tuberculosis from genome sequence data with Mykrobe. %A Martin Hunt %A Phelim Bradley %A Lapierre, Simon Grandjean %A Simon Heys %A Mark Thomsit %A Michael B Hall %A Kerri M Malone %A Penelope Wintringer %A Timothy M Walker %A Daniela M Cirillo %A Iñaki Comas %A Maha R Farhat %A Phillip Fowler %A Jennifer Gardy %A Nazir Ismail %A Thomas A Kohl %A Vanessa Mathys %A Matthias Merker %A Stefan Niemann %A Shaheed Vally Omar %A Vitali Sintchenko %A Grace Smith %A Dick van Soolingen %A Supply, Philip %A Sabira Tahseen %A Mark Wilcox %A Irena Arandjelovic %A Tim E A Peto %A Derrick W Crook %A Zamin Iqbal %X

Two billion people are infected with , 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, , which provided offline species identification and drug resistance predictions for 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, , 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. 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 Illumina sequences. To give estimates of error rates, we use a prospectively collected dataset of 4362 . Using culture based DST as the reference, we estimate 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 gives concordant results with nanopore data.  We measure the ability of -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.

%B Wellcome Open Res %V 4 %8 2019 %G eng %R 10.12688/wellcomeopenres.15603.1 %0 Journal Article %J J Med Microbiol %D 2019 %T In vitro activity of bedaquiline against slow-growing nontuberculous mycobacteria. %A Martin, Anandi %A Godino, Isabel Torres %A Diana Angelica Aguilar-Ayala %A Vanessa Mathys %A Nacer Lounis %A Villalobos, Hector Rodriguez %K Bedaquiline %K Mycobacteria %K nontuberculous %X

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 and surprisingly a good bactericidal activity on the majority of the isolates tested with an MBC of 1-2 µg ml . Based on these preliminary results BDQ seems to be very promising for treatment of diseases caused by SGM.

%B J Med Microbiol %8 2019 Jun 18 %G eng %R 10.1099/jmm.0.001025 %0 Journal Article %J Antimicrob Agents Chemother %D 2019 %T Isoniazid Bactericidal Activity Involves Electron Transport Chain Perturbation. %A Zeng, Sheng %A Karine Soetaert %A Faustine Ravon %A Marie Vandeput %A Dirk Bald %A Kauffmann, Jean-Michel %A Vanessa Mathys %A Wattiez, Ruddy %A Véronique Fontaine %X

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 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 and FF ATP synthase, respectively. Moreover, the antioxidant -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 oxidase significantly reduced cell recovery during INH challenge in a culture settling model, suggesting that the respiratory reprogramming to the cytochrome oxidase contributes to the escape of INH killing. This study implicates mycobacterial ETC perturbation through NDHs, SDHs, cytochrome , and FF ATP synthase in INH's bactericidal activity and pinpoints the participation of the cytochrome oxidase in protection against this drug under stress conditions.

%B Antimicrob Agents Chemother %V 63 %8 2019 Mar %G eng %N 3 %R 10.1128/AAC.01841-18 %0 Generic %D 2019 %T RNA-Based susceptibility testing of Mycobacterium tuberculosis %A An Van den Bossche %A Roby Bhattacharyya %A Jean-Yves Coppee %A Alain Baulard %A Deborah Hung %A Vanessa Mathys %A Pieter-Jan Ceyssens %B 29th ECCMID %C Amsterdam, The Netherlands %8 1316 April, 2019 %G eng %N ESCMID %0 Journal Article %J Euro Surveill %D 2019 %T Strong increase of true and false positive mycobacterial cultures sent to the National Reference Centre in Belgium, 2007 to 2016. %A Karine Soetaert %A Lorenzo Subissi %A Pieter-Jan Ceyssens %A Vanfleteren, Brigitte %A Marianne Chantrenne %A Tommi Asikainen %A Els Duysburgh %A Vanessa Mathys %X

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 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. predominated (ca 20% of NTM cultures), but 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.

%B Euro Surveill %V 24 %8 2019 Mar %G eng %N 11 %R 10.2807/1560-7917.ES.2019.24.11.1800205 %0 Journal Article %J Tuberculosis %D 2019 %T Transcriptional profiling of a laboratory and clinical Mycobacterium tuberculosis strain suggests respiratory poisoning upon exposure to delamanid %A An Van den Bossche %A Hugo Varet %A Amandine Sury %A Odile Sismeiro %A Rachel Legendre %A Jean-Yves Coppee %A Vanessa Mathys %A Pieter-Jan Ceyssens %K Delamanid %K Mycobacterium tuberculosis %K RNA sequencing %K Transciptomics %X

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.

%B Tuberculosis %V 117 %8 Jan-07-2019 %G eng %R 10.1016/j.tube.2019.05.002 %0 Journal Article %J Tuberculosis (Edinb) %D 2019 %T Transcriptional profiling of a laboratory and clinical Mycobacterium tuberculosis strain suggests respiratory poisoning upon exposure to delamanid. %A An Van den Bossche %A Hugo Varet %A Amandine Sury %A Odile Sismeiro %A Rachel Legendre %A Jean-Yves Coppee %A Vanessa Mathys %A Pieter-Jan Ceyssens %K Mycobacterium tuberculosis %K Nitroimidazoles %K Transcriptome %X

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.

%B Tuberculosis (Edinb) %V 117 %8 2019 07 %G eng %R 10.1016/j.tube.2019.05.002 %0 Generic %D 2019 %T Transcriptional profiling of Mycobacterium tuberculosis suggests respiratory poisoning upon exposure to delamanid %A An Van den Bossche %A Hugo Varet %A Amandine Sury %A Odile Sismeiro %A Rachel Legendre %A Jean-Yves Coppee %A Vanessa Mathys %A Pieter-Jan Ceyssens %X

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.

%B 40th ESM conference %C Valencia, Spain %8 30/6-3/7/2019 %G eng %N European Society of Mycobacteria %0 Journal Article %J Indian Journal of Microbiology %D 2018 %T Aloe Emodin Reduces Phthiodiolone Dimycocerosate Potentiating Vancomycin Susceptibility on Mycobacteria %A Céline Rens %A Pieter-Jan Ceyssens %A Françoise Laval %A Lefèvre, Philippe %A Vanessa Mathys %A Daffé, Mamadou %A Véronique Fontaine %B Indian Journal of Microbiology %8 Apr-05-2018 %G eng %R 10.1007/s12088-018-0734-0 %0 Journal Article %J Lancet Infect Dis %D 2018 %T A cluster of multidrug-resistant Mycobacterium tuberculosis among patients arriving in Europe from the Horn of Africa: a molecular epidemiological study. %A Timothy M Walker %A Matthias Merker %A Astrid M Knoblauch %A Peter Helbling %A Otto D Schoch %A J van der Werf %A Katharina Kranzer %A Lena Fiebig %A Stefan Kröger %A Walter Haas %A Harald Hoffmann %A Indra, Alexander %A Adrian Egli %A Daniela M Cirillo %A Jérôme Robert %A Thomas R Rogers %A Ramona Groenheit %A Anne T Mengshoel %A Vanessa Mathys %A Marjo Haanperä %A Dick van Soolingen %A Stefan Niemann %A Erik C Böttger %A Peter M Keller %K Cluster %K Tuberculosis %X

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).

%B Lancet Infect Dis %8 08-jan-2018 %G eng %R 10.1016/S1473-3099(18)30004-5 %0 Journal Article %J Int J Tuberc Lung Dis %D 2018 %T Meropenem-clavulanate for drug-resistant tuberculosis: a follow-up of relapse-free cases. %A M C Payen %A I Muylle %A Vandenberg, O %A Vanessa Mathys %A M Delforge %A S Van den Wijngaert %A N Clumeck %A De Wit, S %K Carbapenems %K repurposed drugs %K XDR-TB %X

BACKGROUND: Extensively drug-resistant tuberculosis (XDR-TB), defined as TB caused by a Mycobacterium strain resistant to at least rifampicin, isoniazid, any fluoroquinolone and one of the injectable anti-tuberculosis drugs, remains a worldwide public health threat. Among repurposed drugs empirically used for XDR-TB cases, carbapenems have been studied in vitro and in animal models, with encouraging results. However, only short-term follow-up data from clinical studies are currently available.

OBJECTIVES: To study the long-term follow-up of XDR-TB cases treated with a regimen containing meropenem-clavulanate (M/Clav).

DESIGN: Retrospective observational case series study at a single hospital.

METHODS: All hospitalised drug-resistant TB patients who received M/Clav as part of their treatment from 2009 to 2016 were included. Demographic and clinical data were extracted from medical records.

RESULTS: Eighteen XDR-TB patients were included in the analysis. The successful outcome and mortality rates were respectively 83.3% and 11.1%. No relapses were observed in cured patients after a median follow-up of 4 years. No specific adverse events were attributed to treatment with M/Clav.

CONCLUSION: The rate of sustained successful treatment outcome observed here is far higher than the 26% observed in the 2014 World Health Organization XDR-TB cohort, suggesting that carbapenems may be beneficial for the treatment of difficult-to-treat TB cases.

%B Int J Tuberc Lung Dis %V 22 %8 2018 Jan 01 %G eng %N 1 %R 10.5588/ijtld.17.0352 %0 Journal Article %J J Antimicrob Chemother %D 2018 %T The potential use of rifabutin for treatment of patients diagnosed with rifampicin-resistant tuberculosis. %A Michael G Whitfield %A Robin M Warren %A Vanessa Mathys %A Lesley Scott %A Elise De Vos %A Wendy Stevens %A Elizabeth M Streicher %A Groenen, Guido %A Frederick A Sirgel %A Annelies Van Rie %K rifabutin %K Tuberculosis %X

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.

%B J Antimicrob Chemother %8 2018 Jul 05 %G eng %R 10.1093/jac/dky248 %0 Journal Article %J New England Journal of MedicineNew England Journal of MedicineN Engl J Med %D 2018 %T Prediction of Susceptibility to First-Line Tuberculosis Drugs by DNA Sequencing %A The CRyPTIC Consortium and the 100000 Genomes Project %A Allix-Béguec, Caroline %A Irena Arandjelovic %A Lijun Bi %A Patrick Beckert %A Maryline Bonnet %A Phelim Bradley %A Andrea Cabibbe %A Irving Cancino-Muñoz %A Mark J. Caulfield %A Angkana Chaiprasert %A Daniela Cirillo %A David Clifton %A Iñaki Comas %A Derrick W Crook %A Maria R De Filippo %A Han De Neeling %A Roland Diel %A Francis A. Drobniewski %A Kiatichai Faksri %A Maha R. Farhat %A Joy Fleming %A Philip Fowler %A Tom A. Fowler %A Gao, Qian %A Jennifer Gardy %A Deborah Gascoyne-Binzi %A Ana-Luiza Gibertoni-Cruz %A Ana Gil-Brusola %A Tanya Golubchik %A Ximena Gonzalo %A Louis Grandjean %A Guangxue, He %A Jennifer L. Guthrie %A Sarah Hoosdally %A Martin Hunt %A Zamin Iqbal %A Nazir Ismail %A James Johnston %A Faisal Khanzada %A Chiea Khor %A Thomas A. Kohl %A Clare Kong %A Sam Lipworth %A Liu, Qingyun %A Gugu Maphalala %A Elena Martinez %A Vanessa Mathys %A Matthias Merker %A Paolo Miotto %A Mistry, Nerges %A David A.J. Moore %A Megan Murray %A Stefan Niemann %A Rick T.-H. Ong %A Tim E.A. Peto %A James E. Posey %A Prammananan, Therdsak %A Alexander Pym %A Camilla Rodrigues %A Mabel Rodrigues %A Timothy Rodwell %A Gian M. Rossolini %A Elisabeth Sánchez Padilla %A Marco Schito %A Xin Shen %A Jay Shendure %A Vitali Sintchenko %A Alex Sloutsky %A Grace E. Smith %A Matthew Snyder %A Karine Soetaert %A Angela M. Starks %A Supply, Philip %A Suriyapol, Prapat %A Sabira Tahseen %A Patrick Tang %A Teo, Yik-Ying %A Thuong N.T. Thuong %A Guy Thwaites %A Tortoli, Enrico %A Shaheed V. Omar %A Dick van Soolingen %A Sarah A. Walker %A Timothy M. Walker %A Mark Wilcox %A Daniel J. Wilson %A David Wyllie %A Yang, Yang %A Hongtai Zhang %A Zhao, Yanlin %A Zhu, Baoli %X

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.).

%B New England Journal of MedicineNew England Journal of MedicineN Engl J Med %8 2018/09/26 %@ 0028-4793 %G eng %R doi: 10.1056/NEJMoa1800474 %0 Generic %D 2018 %T RNA-based drug susceptibility testing of Mycobacterium tuberculosis %A An Van den Bossche %A Roby Bhattacharyya %A Jean-Yves Coppee %A Leen Rigouts %A Alain Baulard %A Alexandra Vodolazkaia %A Deborah Hung %A Vanessa Mathys %A Pieter-Jan Ceyssens %X

Background

Multidrug resistance of Tuberculosis strains (MDR-TB) are one of the major WHO health concerns. One of the challenges that hampers the effective response to MDR-TB is the long turnaround time of phenotypic Drug Susceptibility Testing (DST). To counter this, new fast and sensitive DNA-based methods were successfully introduced over the last years. However, these (a) are based on the knowledge on resistance mutations, (b) do not distinguish living from dead cells, (c) ignore all intrinsic resistance mechanisms, and (d) ignore the influence of compensatory mutations.

Objectives

We introduce a next-generation diagnostic test based on quantification of drug-specific RNA biomarkers. The basic principle is that a brief antibiotic exposure triggers specific transcriptional responses in susceptible, but not in resistant, microbes within a few hours. This has the advantage that long culture-dependent steps are avoided, yet the resistance phenotype is detected independent of the specific cause of resistance.

Materials & Methods

First, the global transcriptional response of two TB strains to 10 anti-TB drugs was determined using RNAtaq-Seq. A set of highly responsive genes was selected for each drug and RNA-targeting probes were designed.

Next, the RNA-based DST was developed in 96 well format. In short, 200 µl of a positively flagged MGITTM (BD) culture is spiked with a drug, while a replicate is incubated in absence of the drug. Multiplex mRNA quantification is performed directly on crude cell lysates using a combination of the bead-based MagPixTM (Luminex) and QuantigeneTM Plex (Thermo Fisher) technology. The normalized expression levels are combined to one numeric value which determines the drug susceptibility of the investigated strain.

Results

We successfully developed 8 primary sets of RNA biomarkers for ten 1st-line, 2nd-line and new drugs. Taking isoniazid as proof of principle, we present a biomarker set of 5 responsive genes and 3 normalizing genes, which enables to distinguish susceptible, low- and high resistant TB strains after 6 hours incubation. Next, preliminary results demonstrate that the biomarker sets can successfully discriminate between susceptible and resistance strains for the selected drugs.

Conclusion

We present a robust, RNA-based DST without the need for RNA extraction. The assay was proven to be efficient for isoniazid. With a total of 8 biomarker sets under optimization, the drug resistance profile of up to 14 drugs can be determined.

%B 2nd St. Petersburg Symposium on Tuberculosis and Mycobacteria: Molecular Approach %C Sint Petersburg %G eng %N Institut Pasteur Sint Petersburg %0 Generic %D 2018 %T RNA-based drug susceptibility testing of Mycobacterium tuberculosis %A An Van den Bossche %A Leen Rigouts %A Jean-Yves Coppee %A Vanessa Mathys %A Pieter-Jan Ceyssens %K Mycobacterium tuberculosis; antimicrobial resistance; diagnostics %X

To counter the long turnaround time of standard phenotypic Drug Susceptibility Testing (DST) of M. tuberculosis (Mtb), multiple DNA-based methods were successfully introduced over the last years. Although these are fast and sensitive, they (a) are based on the knowledge on resistance mutations which is limited for especially 2nd-line and new drugs, (b) do not distinguish living from death cells, (c) ignore all intrinsic resistance mechanisms like efflux pump overexpression, and (d) ignore the multifactorial influence of compensatory mutations. Here, we introduce a next-generation diagnostic test based on quantification of antibiotic-specific RNA biomarkers. The basic principle is that a brief antibiotic exposure triggers specific transcriptional responses in susceptible, but not in resistant, microbes within minutes to a few hours. A major advantage of this method is that it avoids a long culture-dependent step, yet detects the resistance phenotype, independent of the specific cause of resistance.

First, the global transcriptional response of H37Rv and clinical Mtb strains on ten anti-TB drugs including Bedaquiline, Pyrazinamide and Delamanid was determined, to identify RNA Biomarkers. In a next phase, the RNA-based DST was developed in 96 well format. In short, 200 µl of a positively flagged MGIT culture is spiked with a specific concentration of a drug, while a control sample is incubated in absence of the drug. Multiplex mRNA quantification is performed directly on crude cell lysate using a combination of the bead-based MagPixTM (Luminex) and QuantigeneTM Plex (Thermo Fischer) technology. Normalized, relative genes expression levels (control vs drug) are combined to one numeric value which determines the drug susceptibility of the investigated strain.

The assay was optimized for parameters as cell density, incubation time and lysis method. I will present the results, showing that following a biomarker set of 5 responsive genes and 3 normalizing genes enables to distinguish low- and high resistant Mtb strains after an incubation step of 6 hours. With a total of 8 biomarker sets under optimization, the phenotypic drug resistance profile of up to 14 drugs can be determined for any combination of antibiotics in 96 well format.

 

%B Combating resistance : microbes and vectors %C Paris, France %8 16/11/2018 %G eng %N Institute Pasteur %0 Generic %D 2018 %T RNA-based drug susceptibility testing of Mycobacterium tuberculosis %A An Van den Bossche %A Roby Bhattacharyya %A Jean-Yves Coppee %A Leen Rigouts %A Alain Baulard %A Deborah Hung %A Vanessa Mathys %A Pieter-Jan Ceyssens %K drug susceptibility testing %K Mycobacterium tuberculosis %B 39th Annual Congress of the European Society of Mycobacteriology %C Dresden, Germany %8 07/2018 %G eng %N ESM %0 Journal Article %J Int J Tuberc Lung Dis %D 2018 %T Time-and-motion tool for the assessment of working time in tuberculosis laboratories: a multicentre study. %A Vanessa Mathys %A E Roycroft %A P Raftery %A R Groenheit %A B D Folkvardsen %A D Homorodean %A E Vasiliauskiene %A L Vasiliauskaite %A C Kodmon %A J van der Werf %A F Drobniewski %A V Nikolayevskyy %K hands-on time %K laboratory diagnosis %K Workload %X

SETTING: Implementation of novel diagnostic assays in tuberculosis (TB) laboratory diagnosis requires effective management of time and resources.

OBJECTIVE: To further develop and assess at multiple centres a time-and-motion (T&M) tool as an objective means for recording the actual time spent on running laboratory assays.

DESIGN: Multicentre prospective study conducted in six European Union (EU) reference TB laboratories.

RESULTS: A total of 1060 specimens were tested using four laboratory assays. The number of specimens per batch varied from one to 60; a total of 64 recordings were performed. Theoretical hands-on times per specimen (TTPS) in h:min:s for Xpert® MTB/RIF, mycobacterial interspersed repetitive unit-variable number of tandem repeats genotyping, Ziehl-Neelsen staining and manual fluorescence microscopy were respectively 00:33:02 ± 00:12:32, 00:13:34 ± 00:03:11, 00:09:54 ± 00:00:53 and 00:06:23 ± 00:01:36. Variations between laboratories were predominantly linked to the time spent on reporting and administrative procedures. Processing specimens in batches could help save time in highly automated assays (e.g., line-probe) (TTPS 00:14:00 vs. 00:09:45 for batches comprising 7 and 31 specimens, respectively).

CONCLUSIONS: The T&M tool can be considered a universal and objective methodology contributing to workload assessment in TB diagnostic laboratories. Comparison of workload between laboratories could help laboratory managers justify their resource and personnel needs for the implementation of novel, time-saving, cost-effective technologies, as well as identify areas for improvement.

%B Int J Tuberc Lung Dis %V 22 %8 2018 Apr 01 %G eng %N 4 %R 10.5588/ijtld.17.0564 %0 Journal Article %J Acta Clin Belg %D 2017 %T Case report of a false positive result of the Xpert(®) MTB/RIF assay for rifampicin resistance in Mycobacterium tuberculosis complex. %A Claessens, Jolien %A Vanessa Mathys %A Derdelinckx, Inge %A Saegeman, Veroniek %X

In the present case, we report a false positive result for the detection of rifampicin (RIF) resistance by the Xpert(®) MTB/RIF assay, version G4.Miliary Mycobacterium tuberculosis infection (miliary TB) was suspected in a 50-year old Angolan woman. Imaging of the thorax and abdomen displayed diffuse lesions. The Xpert(®) MTB/RIF assay conducted on the broncho-alveolar lavage (BAL) fluid was positive for TB and positive for RIF resistance. Confirmatory molecular tests and the phenotypic drug susceptibility determination supported the diagnosis of TB but not RIF resistance. The patient was treated successfully with a conventional therapeutic scheme. Because, the Xpert(®) MTB/RIF assay allows the simultaneous detection of TB and RIF resistance, the World Health Organisation (WHO) recommends its use as initial diagnostic test, over microscopy, culture and phenotypic drug susceptibility testing. Even though specificity of the Xpert(®) MTB/RIF assay version G4 is high, false positive test results remain possible and have to be considered for the interpretation of the RIF resistance detection by Xpert(®) MTB/RIF assay.

%B Acta Clin Belg %V 72 %P 195-197 %8 2017 Jun %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/26400761?dopt=Abstract %R 10.1179/2295333715Y.0000000072 %0 Journal Article %J Clin Microbiol Infect %D 2017 %T Consensus numbering system for the rifampicin resistance-associated rpoB gene mutations in pathogenic mycobacteria. %A E André %A Goeminne, L %A Cabibbe, A %A Beckert, P %A B Kabamba Mukadi %A Vanessa Mathys %A Gagneux, S %A Niemann, S %A Van Ingen, J %A Cambau, E %K antibiotics %K Antitubercular %K Bacterial Proteins %K Consensus %K DNA-Directed RNA Polymerases %K Escherichia coli %K Escherichia coli Proteins %K Genotype %K Genotyping Techniques %K Humans %K Microbial Sensitivity Tests %K Mutant Proteins %K Mutation %K Mycobacterium %K Mycobacterium tuberculosis %K Rifampin %K Terminology as Topic %X

The rpoB gene codes for the RNA polymerase β subunit, which is the target of rifampicin, an essential drug in the treatment of tuberculosis and other mycobacterial infections. This gene is present in all bacteria, but its length and nucleotide sequence vary between bacterial species, including mycobacteria. Mutations in the rpoB gene alter the structure of this protein and cause drug resistance. To describe the resistance-associated mutations, the scientific and medical communities have been using, since 1993, a numbering system based on the Escherichia coli sequence annotation. Using E. coli reference for describing mutations in mycobacteria leads to misunderstandings, particularly with the increasing use of whole genome sequencing, which brought an alternative numbering system based on the Mycobacterium tuberculosis rpoB sequence. We propose using a consensus numbering system for the reporting of resistance mutations based on the reference genomes from the species interrogated (such as strain H37Rv for M. tuberculosis). This manuscript provides the necessary figures and tables allowing researchers, microbiologists and clinicians to easily convert other annotation systems into one common language.

%B Clin Microbiol Infect %V 23 %P 167-172 %8 2017 Mar %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/27664776?dopt=Abstract %& 167 %R 10.1016/j.cmi.2016.09.006 %0 Generic %D 2017 %T Global transcriptional analyses of Mycobacterium tuberculosis using old and new drugs %A An Van den Bossche %A Pieter-Jan Ceyssens %A Roby Bhattacharyya %A Deborah Hung %A Vanessa Mathys %K antibiotics %K Mycobacterium tuberculosis %K Transcriptomics %X

Background: With 1.5 million deaths in 2014, the most recent report of the WHO ranks tuberculosis (TB) alongside HIV as a leading cause of death by infectious diseases. In combination, the spread of (multi-)drug resistant Mycobacterium tuberculosis is rising (480 000 new cases), impeding the treatment of infected patients.

Current TB drug resistance research is mainly focused on the genomic level, using WGS to search for resistance-causing mutations. However, transcriptional analyses can be a powerful tool as well. Analyses on susceptible strains provide information on the response to the stress a drug is causing, showing which mechanisms are activated to counter the effect of the drug. This might lead to new strategies for the development of new/complimentary drugs. On the other hand, this response gives an view on the working mechanism of the drug. Moreover, comparing the response of sensitive and resistant strains can yield additional information, like the activation of specific efflux pumps.

However, due to the high cost of RNAseq analyses, genome wide transcriptional analyses of drug influences on M. tuberculosis remain rather limited. Here we present a global study of the response of two pansensitive M. tuberculosis strains to eight TB-drugs. By using the approach called RNAtag-Seq, multiple samples were combined in one run, reducing time and cost.

 

Material/methods: For each drug (isoniazid, rifampicin, ethambutol, capreomycin, amikacin, linezolid, moxifloxacin and bedaquilin), twice the critical concentration was added to the cultures of two pansensitive M. tuberculosis strains. Samples were taken 0h, 2h, 6h and 24h after the administration of the drug.  RNA was extracted combining bead-beating in TRIzol and the Direct-Zol purification kit. After quality control, the extracts were fragmented, barcoded and pooled, cDNA libraries were constructed and sequenced using the HiSeq technology (Illumina®). Reads were mapped to the reference genome and normalized read counts were calculated per gene.

 

Results: For each drug a specific transcriptional response was mapped, revealing lists of up- and down-regulated genes. As an example and in accordance with previous studies, the highest induced genes in the presence of isoniazid belong to a cluster of genes that encodes components of the FAS-II (fatty acid synthase II) complex, which is targeted by isoniazid. On the other hand, a remarkable down-regulation of the NADH-dehydrogenase (ndh) cluster genes was noted. This can be assigned to the dependence of isoniazid target inhA on NADH. Lowering the ndh-activity is also seen in resistant strains.

 

Conclusions: By using RNAtag-seq, a global study of the transcriptional response of M. tuberculosis to several drugs could be made. These analyses can reveal the working mechanisms of existing and new drugs and provide new insights on the mechanism of resistance of strains.

 

%B 38th Annual Congress of the European Society of Mycobacteriology %I ESM %C Sibenik, Craotia %8 06/2017 %G eng %N ESM %0 Generic %D 2017 %T Global transcriptional analyses of Mycobacterium tuberculosis using old and new drugs %A An Van den Bossche %A Pieter-Jan Ceyssens %A Roby Bhattacharyya %A Deborah Hung %A Vanessa Mathys %K antibiotics %K Mycobacterium tuberculosis %K Transcriptomics %X

Background: With 1.5 million deaths in 2014, the most recent report of the WHO ranks tuberculosis (TB) alongside HIV as a leading cause of death by infectious diseases. In combination, the spread of (multi-)drug resistant Mycobacterium tuberculosis is rising (480 000 new cases), impeding the treatment of infected patients.

Current TB drug resistance research is mainly focused on the genomic level, using WGS to search for resistance-causing mutations. However, transcriptional analyses can be a powerful tool as well. Analyses on susceptible strains provide information on the response to the stress a drug is causing, showing which mechanisms are activated to counter the effect of the drug. This might lead to new strategies for the development of new/complimentary drugs. On the other hand, this response gives an view on the working mechanism of the drug. Moreover, comparing the response of sensitive and resistant strains can yield additional information, like the activation of specific efflux pumps.

However, due to the high cost of RNAseq analyses, genome wide transcriptional analyses of drug influences on M. tuberculosis remain rather limited. Here we present a global study of the response of two pansensitive M. tuberculosis strains to eight TB-drugs. By using the approach called RNAtag-Seq, multiple samples were combined in one run, reducing time and cost.

 

Material/methods: For each drug (isoniazid, rifampicin, ethambutol, capreomycin, amikacin, linezolid, moxifloxacin and bedaquilin), twice the critical concentration was added to the cultures of two pansensitive M. tuberculosis strains. Samples were taken 0h, 2h, 6h and 24h after the administration of the drug.  RNA was extracted combining bead-beating in TRIzol and the Direct-Zol purification kit. After quality control, the extracts were fragmented, barcoded and pooled, cDNA libraries were constructed and sequenced using the HiSeq technology (Illumina®). Reads were mapped to the reference genome and normalized read counts were calculated per gene.

 

Results: For each drug a specific transcriptional response was mapped, revealing lists of up- and down-regulated genes. As an example and in accordance with previous studies, the highest induced genes in the presence of isoniazid belong to a cluster of genes that encodes components of the FAS-II (fatty acid synthase II) complex, which is targeted by isoniazid. On the other hand, a remarkable down-regulation of the NADH-dehydrogenase (ndh) cluster genes was noted. This can be assigned to the dependence of isoniazid target inhA on NADH. Lowering the ndh-activity is also seen in resistant strains.

 

Conclusions: By using RNAtag-seq, a global study of the transcriptional response of M. tuberculosis to several drugs could be made. These analyses can reveal the working mechanisms of existing and new drugs and provide new insights on the mechanism of resistance of strains.

 

%B ECCMID 2017 %I ECCMID %C Vienna, Austria %8 04/2017 %G eng %N ESCMID %0 Journal Article %J J Clin Microbiol %D 2017 %T Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Combined Species Identification and Drug Sensitivity Testing in Mycobacteria. %A Pieter-Jan Ceyssens %A Karine Soetaert %A Timke, Markus %A An Van den Bossche %A Sparbier, Katrin %A Koen De Cremer %A Kostrzewa, Markus %A Marijke Hendrickx %A Vanessa Mathys %K Antitubercular Agents %K Bacteriological Techniques %K Humans %K Mycobacterium tuberculosis %K Nontuberculous Mycobacteria %K Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization %X

Species identification and drug susceptibility testing (DST) of mycobacteria are important yet complex processes traditionally reserved for reference laboratories. Recent technical improvements in matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has started to facilitate routine mycobacterial identifications in clinical laboratories. In this paper, we investigate the possibility of performing phenotypic MALDI-based DST in mycobacteriology using the recently described MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA). We randomly selected 72 clinical Mycobacterium tuberculosis and nontuberculous mycobacterial (NTM) strains, subjected them to MBT-ASTRA methodology, and compared its results to current gold-standard methods. Drug susceptibility was tested for rifampin, isoniazid, linezolid, and ethambutol (M. tuberculosis, n = 39), and clarithromycin and rifabutin (NTM, n = 33). Combined species identification was performed using the Biotyper Mycobacteria Library 4.0. Mycobacterium-specific MBT-ASTRA parameters were derived (calculation window, m/z 5,000 to 13,000, area under the curve [AUC] of >0.015, relative growth [RG] of <0.5; see the text for details). Using these settings, MBT-ASTRA analyses returned 175/177 M. tuberculosis and 65/66 NTM drug resistance profiles which corresponded to standard testing results. Turnaround times were not significantly different in M. tuberculosis testing, but the MBT-ASTRA method delivered on average a week faster than routine DST in NTM. Databases searches returned 90.4% correct species-level identifications, which increased to 98.6% when score thresholds were lowered to 1.65. In conclusion, the MBT-ASTRA technology holds promise to facilitate and fasten mycobacterial DST and to combine it directly with high-confidence species-level identifications. Given the ease of interpretation, its application in NTM typing might be the first in finding its way to current diagnostic workflows. However, further validations and automation are required before routine implementation can be envisioned.

%B J Clin Microbiol %V 55 %P 624-634 %8 2017 Feb %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/28003422?dopt=Abstract %R 10.1128/JCM.02089-16 %0 Journal Article %J PLoS One %D 2017 %T Molecular epidemiology of Mycobacterium tuberculosis complex in Brussels, 2010-2013. %A Vluggen, Christelle %A Soetaert, Karine %A Groenen, Guido %A Wanlin, Maryse %A Spitaels, Martine %A Arrazola de Oñate, Wouter %A Fauville-Dufaux, Maryse %A Saegerman, Claude %A Vanessa Mathys %K ADOLESCENT %K Adult %K Bacterial Typing Techniques %K Belgium %K Child %K Cluster Analysis %K Contact Tracing %K Drug Resistance, Multiple, Bacterial %K Emigrants and Immigrants %K Family Health %K Female %K Genetic Variation %K Genotype %K Hospitals, Urban %K Humans %K incidence %K Male %K middle aged %K Mycobacterium tuberculosis %K Phylogeny %K Population Surveillance %K Tuberculosis %K Tuberculosis, Multidrug-Resistant %K Urban Health %K Young adult %X

The tuberculosis (TB) incidence rate in Brussels-Capital Region is 3-fold higher than in Belgium as a whole. Eight years after the realization of initial prospective population-based molecular epidemiology investigations in this Region, a similar study over the period 2010-2013 was conducted. TB strains isolated from 945 patients were submitted to genotyping by standardized 24-locus-MIRU-VNTR typing and spoligotyping. The phylogenetic analysis showed that the LAM (16.7%) and Haarlem (15.7%) branches are the two most prevalent TB lineages circulating in Brussels. Analysis of the MDR subgroup showed an association with Beijing strains (39.9%) and patients native of Eastern Europe (40.7%). Genotyping detected 113 clusters involving 321 patients, giving a recent transmission index of 22.9%. Molecular-guided epidemiological investigations and routine surveillance activities revealed family transmission or social contact for patients distributed over 34 clusters. Most of the patients were foreign-born (75.7%). However, cluster analysis revealed only limited trans-national transmission. Comparison with the previous study shows a stable epidemiological situation except for the mean age difference between Belgian-born and foreign-born patients which has disappeared. This study confirms that molecular epidemiology has become an important determinant for TB control programs. However, sufficient financial means need to be available to perform all required epidemiological investigations.

%B PLoS One %V 12 %P e0172554 %8 2017 %G eng %N 2 %1 https://www.ncbi.nlm.nih.gov/pubmed/28222189?dopt=Abstract %& e0172554 %R 10.1371/journal.pone.0172554 %0 Journal Article %J Acta Clin Belg %D 2017 %T Nontuberculous mycobacteria among pulmonary tuberculosis patients: a retrospective Belgian multicenter study. %A De Keukeleire, Steven %A Vanessa Mathys %A Van den Wijngaert, Sigi %A Van De Vyvere, Martine %A Jonckheere, Stijn %A De Beenhouwer, Hans %A de Bel, Annelies %A Arrazola de Oñate, Wouter %A Wanlin, Maryse %A Piérard, Denis %A Nulens, Eric %A Saegeman, Veroniek %K Adult %K Belgium %K Coinfection %K Female %K Humans %K Male %K middle aged %K Mycobacterium Infections, Nontuberculous %K Nontuberculous Mycobacteria %K Retrospective Studies %K Tuberculosis, Pulmonary %X

OBJECTIVES: Currently, there are no European data about the frequency and clinical significance of nontuberculous mycobacteria (NTM) grown from respiratory samples during the treatment of tuberculosis (TB). We determined the frequency and clinical significance of NTM isolated before or during pulmonary tuberculosis treatment in Belgian laboratories.

METHODS: We conducted a nationwide retrospective multicenter cohort study on the co-isolation of TB and NTM in Belgium. Starting from laboratory data between 2006 and 2013, possible TB-NTM co-isolations were searched for.

RESULTS: A total of 2569 unique culture-positive pulmonary tuberculosis cases were included in the study. Only 35 (1.4%) of these TB cases had an NTM co-isolated, and two of these 35 fulfilled the ATS criteria for NTM lung disease.

CONCLUSION: A very low prevalence of 1.4% NTM co-isolations was found in Belgian patients with culture-proven pulmonary TB.

%B Acta Clin Belg %V 72 %P 45-48 %8 2017 Feb %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/27345031?dopt=Abstract %R 10.1080/17843286.2016.1200823 %0 Journal Article %J Science %D 2017 %T Reversion of antibiotic resistance in Mycobacterium tuberculosis by spiroisoxazoline SMARt-420. %A Blondiaux, Nicolas %A Moune, Martin %A Desroses, Matthieu %A Frita, Rosangela %A Flipo, Marion %A Vanessa Mathys %A Karine Soetaert %A Kiass, Mehdi %A Delorme, Vincent %A Djaout, Kamel %A Trebosc, Vincent %A Kemmer, Christian %A Wintjens, René %A Wohlkönig, Alexandre %A Antoine, Rudy %A Huot, Ludovic %A Hot, David %A Coscolla, Mireia %A Feldmann, Julia %A Gagneux, Sebastien %A Locht, Camille %A Brodin, Priscille %A Gitzinger, Marc %A Déprez, Benoit %A Willand, Nicolas %A Baulard, Alain R %X

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.

%B Science %V 355 %P 1206-1211 %8 2017 03 17 %G eng %N 6330 %1 http://www.ncbi.nlm.nih.gov/pubmed/28302858?dopt=Abstract %R 10.1126/science.aag1006 %0 Journal Article %J Veterinary Record %D 2017 %T Trend analysis suggested a change in subspecies among Mycobacterium avium isolated from pigs in Belgium, 1967–2013 %A Karine Soetaert %A C. Vluggen %A L Duytschaever %A J. Denoël %A Virginie Roupie %A F. Smeets %A Nicolas Bruffaerts %A Huygen, K. %A David Fretin %A M. Diels %A L. Rigouts %A C. Saegerman %A Vanessa Mathys %B Veterinary Record %V 180 %8 Jun-05-2017 %G eng %N 18 %R 10.1136/vr.103951 %0 Journal Article %J Vet Rec %D 2017 %T Trend analysis suggested a change in subspecies among isolated from pigs in Belgium, 1967-2013. %A Karine Soetaert %A Duytschaever, L %A Denoël, J %A Virginie Roupie %A Smeets, F %A Nicolas Bruffaerts %A Huygen, K %A David Fretin %A Diels, M %A Rigouts, L %A Saegerman, C %A Vanessa Mathys %K Animals %K Belgium %K Genotyping Techniques %K Mycobacterium avium %K Swine %K Swine Diseases %K Tuberculosis %B Vet Rec %V 180 %P 449 %8 2017 May 06 %G eng %N 18 %1 https://www.ncbi.nlm.nih.gov/pubmed/28283669?dopt=Abstract %R 10.1136/vr.103951 %0 Conference Proceedings %B WIV-ISP website %D 2017 %T Tuberculose en antibioticaresistentie: het WIV draagt bij tot de ontwikkeling van een nieuw prototype van een geneesmiddel %A Vanessa Mathys %A Karine Soetaert %K antibioresistentie %K geneesmiddelen %K tuberculose %B WIV-ISP website %I WIV-ISP %C Brussels, Belgium %P 1 %8 20/03/2017 %U https://www.wiv-isp.be/nl/pershoek/tuberculose-en-antibioticaresistentie-het-wiv-draagt-bij-tot-de-ontwikkeling-van-een-nieuw %0 Conference Proceedings %B WIV-ISP website %D 2017 %T Tuberculose et antibiorésistance : l’ISP contribue au développement d’un nouveau prototype de médicament %A Vanessa Mathys %A Karine Soetaert %K antibiorésistance %K médicament %K tuberculose %B WIV-ISP website %I WIV-ISP %C Brussels, Belgium %P 1 %8 20/03/2017 %U https://www.wiv-isp.be/fr/coin-presse/tuberculose-et-antibioresistance-lisp-contribue-au-developpement-dun-nouveau-prototype %0 Journal Article %J PLoS One %D 2017 %T Virulence and immunogenicity of genetically defined human and porcine isolates of M. avium subsp. hominissuis in an experimental mouse infection. %A Bruffaerts, Nicolas %A Vluggen, Christelle %A Roupie, Virginie %A Duytschaever, Lucille %A Van den Poel, Christophe %A Denoël, Joseph %A Wattiez, Ruddy %A Letesson, Jean-Jacques %A Fretin, David %A Rigouts, Leen %A Chapeira, Ophélie %A Vanessa Mathys %A Saegerman, Claude %A Huygen, Kris %K Adult %K Animals %K Child, Preschool %K Female %K Genome, Bacterial %K Genotype %K Humans %K Interferon-gamma %K Interleukins %K Liver %K Lung %K Male %K mice %K Mice, Inbred BALB C %K Mycobacterium avium %K Spleen %K Swine %K Tuberculosis %K virulence %X

Mycobacterium avium subsp. hominissuis (Mah) represents a health concern for humans and to a lesser extent for pigs, but its zoonotic potential remains elusive. Using multispacer sequence typing (MST) we previously identified 49 different genotypes of Mah among Belgian clinical and porcine isolates, with 5 MSTs shared by both hosts. Using experimental intranasal infection of BALB/c mice, we compared the virulence and immunogenicity of porcine and clinical human isolates with shared genotype or with a genotype only found in humans or pigs. Bacterial replication was monitored for 20 weeks in lungs, spleen and liver and mycobacteria specific spleen cell IFN-γ, IL-10 and IL-17 production as well as serum antibody responses were analyzed. Isolates varied in virulence, with human and porcine isolates sharing MST22 genotype showing a thousand fold higher bacterial replication in lungs and more dissemination to spleen and liver than the human and porcine MST91 isolates. Virulent MST22 type was also associated with progressive suppression of IFN-γ and IL-17 responses, and increased IL-10 production. Whole genome sequencing of the two virulent isolates with MST22 genotype and two avirulent isolates of genotype MST91 and comparison with two well-studied M. avium subsp. hominissuis reference strains i.e. Mah 104 and Mah TH135, identified in the two MST22 isolates nine specific virulence factors of the mammalian cell entry family, that were identical with Mah 104 strain. Despite the obvious limitations of the mouse model, a striking link of virulence and identity at the genome level of porcine and human isolates with the same multisequence type, for which no correlation of place of residence (humans) or farm of origin (pigs) was observed, seems to point to the existence in the environment of certain genotypes of Mah which may be more infectious both for humans and pigs than other genotypes.

%B PLoS One %V 12 %P e0171895 %8 2017 %G eng %N 2 %1 https://www.ncbi.nlm.nih.gov/pubmed/28182785?dopt=Abstract %& e0171895 %R 10.1371/journal.pone.0171895 %0 Journal Article %J PLOS ONE %D 2017 %T Virulence and immunogenicity of genetically defined human and porcine isolates of M. avium subsp. hominissuis in an experimental mouse infection %A Nicolas Bruffaerts %A Vluggen, Christelle %A Virginie Roupie %A Duytschaever, Lucille %A Christophe Van den Poel %A Joseph Noël %A Wattiez, Ruddy %A Letesson, Jean-Jacques %A David Fretin %A Rigouts, Leen %A Elie Chapeira %A Vanessa Mathys %A Saegerman, Claude %A Huygen, Kris %E Cloeckaert, Axel %K Paratuberculosis %X

Mycobacterium avium subsp. hominissuis (Mah) represents a health concern for humans and to a lesser extent for pigs, but its zoonotic potential remains elusive. Using multispacer sequence typing (MST) we previously identified 49 different genotypes of Mah among Belgian clinical and porcine isolates, with 5 MSTs shared by both hosts. Using experimental intranasal infection of BALB/c mice, we compared the virulence and immunogenicity of porcine and clinical human isolates with shared genotype or with a genotype only found in humans or pigs. Bacterial replication was monitored for 20 weeks in lungs, spleen and liver and mycobacteria specific spleen cell IFN-γ, IL-10 and IL-17 production as well as serum antibody responses were analyzed. Isolates varied in virulence, with human and porcine isolates sharing MST22 genotype showing a thousand fold higher bacterial replication in lungs and more dissemination to spleen and liver than the human and porcine MST91 isolates. Virulent MST22 type was also associated with progressive suppression of IFN-γ and IL-17 responses, and increased IL-10 production. Whole genome sequencing of the two virulent isolates with MST22 genotype and two avirulent isolates of genotype MST91 and comparison with two well-studied M. avium subsp. hominissuis reference strains i.e. Mah 104 and Mah TH135, identified in the two MST22 isolates nine specific virulence factors of the mammalian cell entry family, that were identical with Mah 104 strain. Despite the obvious limitations of the mouse model, a striking link of virulence and identity at the genome level of porcine and human isolates with the same multisequence type, for which no correlation of place of residence (humans) or farm of origin (pigs) was observed, seems to point to the existence in the environment of certain genotypes of Mah which may be more infectious both for humans and pigs than other genotypes.

%B PLOS ONE %V 12 %8 Sep-02-2017 %G eng %N 2 %R 10.1371/journal.pone.017189510.1371/ %0 Generic %D 2016 %T Détermination des sous-espèces et génotypage des souches humaines et porcines de Mycobacterium avium isolées en Belgique %A Christelle Vluggen %A Karine Soetaert %A Dutschaever,L. %A J. Denoel %A Smeets,F. %A Nicolas Bruffaerts %A K Huygen %A David Fretin %A Saegerman,C. %A Vanessa Mathys %K Belgique %K de %K EN %K ET %K Mycobacterium %B Belspo RT-project %I NA %C NA %8 0/0/2016 %G eng %N Belspo %1 37202 %2 16/03/2016 %0 Generic %D 2016 %T Evolution des espèces mycobactériennes identifiées au CNR au cours du temps. %A Vanessa Mathys %E FARES-VRGT %K de %K espèces %K Evolution %K Surveillance %B Réunion des laboratoires du réseau de surveillance de la (multi)résistance. %8 0/0/2016 %G eng %N FARES %1 37196 %2 18/02/2016 %0 Journal Article %J J Med Microbiol %D 2016 %T Frequency of Mycobacterium chimaera among Belgian patients, 2015. %A Karine Soetaert %A Vluggen, Christelle %A Emmanuel André %A Vanhoof, Raymond %A Vanfleteren, Brigitte %A Vanessa Mathys %K Adult %K Aged %K Aged, 80 and over %K Belgium %K DNA, Bacterial %K Female %K Humans %K Male %K middle aged %K Mycobacterium %K Mycobacterium Infections %K Phylogeny %K RNA, Ribosomal, 16S %K Young adult %X

Mycobacterium chimaera arouses an increasing public health concern, as this non-tuberculous mycobacterium (NTM) has recently been associated with life-threatening cardiac infections. M. chimaera and Mycobacteriumintracellulare are genetically very close but recently appeared to present different epidemiological and clinical significance. Therefore, it has become important for laboratories to use adequate techniques allowing precise species identification. To date, most commercially available laboratory assays cannot distinguish them and erroneously identify M. chimaera as M. intracellulare. We performed a re-analysis of the 149 M. intracellulare strains received by the Belgian National Reference Laboratory using 16S rRNA gene sequencing, representing 25 % of all NTM collected in 2015. We found that M. chimaera represents the majority (n=94, 63 %) of the previous M. intracellulare. This study reports the large presence of M. intracellulare/chimaera among Belgian patients infected by an NTM and the predominance of the species M. chimaera among this group. This study also stresses the public health importance of M. chimaera and demonstrates the inability of commonly used laboratory techniques to correctly diagnose these infections.

%B J Med Microbiol %V 65 %P 1307-1310 %8 2016 Nov %G eng %N 11 %1 http://www.ncbi.nlm.nih.gov/pubmed/27902393?dopt=Abstract %R 10.1099/jmm.0.000359 %0 Journal Article %J Genome Announc %D 2016 %T Genome Sequences of Four Strains of Mycobacterium avium subsp. hominissuis, Isolated from Swine and Humans, Differing in Virulence in a Murine Intranasal Infection Model. %A Nicolas Bruffaerts %A Duytschaever, L %A Vanessa Mathys %A Saegerman, C %A Chapeira, O %A Huygen, K %K genome sequence %K hominissuis %K Humans %K intranasal infection model %K murine %K Mycobacterium avium %K Swine %K virulence %X

This paper announces the genome sequences of four strains of Mycobacterium avium subsp. hominissuis, isolated from cases of lymphadenopathy in swine and humans, differing in virulence in a murine intranasal infection model.

%B Genome Announc %V 4 %8 2016 Jun 16 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/27313293?dopt=Abstract %R 10.1128/genomeA.00533-16 %0 Journal Article %J Euro Surveill %D 2016 %T Genotyping and strain distribution of Mycobacterium avium subspecies hominissuis isolated from humans and pigs in Belgium, 2011-2013. %A Vluggen, Christelle %A Karine Soetaert %A Duytschaever, Lucille %A Denoël, Joseph %A Fauville-Dufaux, Maryse %A Smeets, François %A Nicolas Bruffaerts %A Huygen, Kris %A David Fretin %A Rigouts, Leen %A Saegerman, Claude %A Vanessa Mathys %K Animals %K Belgium %K Genetic Variation %K Genotype %K Humans %K Minisatellite Repeats %K Mycobacterium avium %K Phylogeny %K polymerase chain reaction %K Polymorphism, Restriction Fragment Length %K Retrospective Studies %K Sequence Analysis, DNA %K Swine %K Swine Diseases %K Tuberculosis %X

Mycobacterium avium represents a health concern for both humans and pigs. The characterisation of its subspecies is an important step improving the understanding of the epidemiology and the control of this pathogen. Ninety-two human M. avium strains were selected for a retrospective study. Subspecies determination by rpoB sequencing and IS1245/IS901 analysis showed that 98.9% of Belgian human M. avium strains belong to the subspecies hominissuis (MAH). Some of these MAH strains present particular IS1245/IS901 profiles (absence of IS1245 and false IS901 detection provoked by the presence of ISMav6). In addition, 54 MAH strains isolated from submandibular lymph nodes of Belgian pigs with lymphadenitis were included in this study. Genotyping of human and porcine isolates was performed using multispacer sequence typing (MST). In total, 49 different MST types were identified among pig (n = 11) and human (n = 43) MA isolates, with only five shared by both hosts. Among these MST types, 34 were newly identified. Our findings demonstrate the extensive genetic diversity among MAH isolates. Some genotypes were more prevalent in human or pigs but no correlation was observed between MST type and place of residence or the farm of origin for human and porcine isolates respectively, suggesting an environmental source of infection.

%B Euro Surveill %V 21 %P 30111 %8 2016 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/26835872?dopt=Abstract %R 10.2807/1560-7917.ES.2016.21.3.30111 %0 Generic %D 2016 %T Infections mycobactériennes chez l'homme et l'animal %A Vanessa Mathys %A David Fretin %K ET %K health %K INFECTION %K infections %B One Health Seminar %I NA %C NA %8 0/0/2016 %G eng %N WIV-ISP %1 37199 %2 25/05/2016 %0 Generic %D 2016 %T MALDI-TOF for drug resistance testing in mycobacterium tuberculosis %A Pieter-Jan Ceyssens %A Karine Soetaert %A Koen De Cremer %A Sophie Bertrand %A Marijke Hendrickx %A Vanessa Mathys %K DRUG %K Drug Resistance %K MALDI-TOF %K Mycobacterium %K resistance %K TESTING %B ECCMID 2016 %I NA %C NA %8 9/4/2016 %G eng %N ECCMID %1 39257 %2 09/04/2016 %0 Generic %D 2016 %T MALDI-TOF for the identification of Mycobacteria and their drug resistance profiles %A An Van den Bossche %A Pieter-Jan Ceyssens %A Marijke Hendrickx %A Vanessa Mathys %K drug susceptibility testing %K identification %K MALDI-TOF %K Mycobacterium tuberculosis %X

In the last years, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been increasingly introduced as a valuable method for the identification of bacteria and yeast1. This technique, which generates mass spectra that are compared to reference spectra in databases, is accurate, fast and cost-effective compared to traditional biochemical and molecular techniques.

For the identification of Mycobacteria, this method is more challenging due to the special need for inactivation and protein extraction. Although several studies have been published, there is no clear consensus on the processing of Mycobacteria for MALDI-TOF MS and outcomes vary from 55% correct identifications to 97%2-4. In this study, we optimized an extraction protocol and evaluated its efficiency using the Brüker Biotyper system v3.0 for 194 cultures. In total 88.6% (172/194) of the samples were correctly identified on the species-level using a cut-off score of 1.8 and 91.75% (178/194) using a cut-off of 1.65. All samples of the Mycobacterium tuberculosis complex were correctly classified (52/52), while 126 of the 142 Non-tuberculosis Mycobacteria were accurately identified at a 1.65 cut-off.

Recently, it was described that MALDI-TOF MS can also be used for drug-susceptibility testing (DST) in bacteria. The MS-ASTRA technology is based on the parallel incubation of a culture in presence or absence of an antibiotic. By adding an internal standard during the protein extraction, semi-quantitative measurements of the bacterial biomass can be derived from the MALDI-TOF spectra7.

In this study, we show that this technology can be applied on Mycobacteria. 34 clinical M. tuberculosis isolates were investigated for their resistance profile to four different drugs (rifampicin, isoniazid, ethambutol and linezolid), yielding a 100% concordance to the BACTEC MGIT results. Moreover, incubation with serial dilutions allowed a correct determination of the Minimal Inhibitory Concentrations of isoniazid and rifampicin. Therefore, this method has the potential to provide a cost-effective and fast alternative for classical phenotypic DST, independent of the mechanism of drug resistance. However, a current lack of automation hinders implementation in diagnostic laboratories.

%B 37th Annual Congress of the European Society of Mycobacteriology %I ESM %C Catania, Sicily, Italy %8 06/2017 %G eng %0 Generic %D 2016 %T Mycobacteria: interactions between human, animals and environment %A Vanessa Mathys %K Animal %K Animals %K environment %K Human %K interaction %K interactions %K Mycobacterium %K study %B FSVEE study day %I NA %C NA %8 0/0/2016 %G eng %N KULeuven %1 37197 %2 28/10/2016 %0 Journal Article %J Acta Clinica Belgica %D 2016 %T Nontuberculous mycobacteria among pulmonary tuberculosis patients: a retrospective Belgian multicentre study %A De Keukeleire,S. %A Vanessa Mathys %A Van Den Wijngaert,S. %A Van de Vyvere,M. %A S. Jonckheere %A De Beenhouwer,H. %A A. De Bel %A W. Arrazola de Onate %A Wanlin,M. %A Denis Piérard %A Nulens,E. %A Saegeman,V. %K a %K Belgian %K Mycobacterium %K Nontuberculous Mycobacteria %K Patient %K patients %K Pulmonary %K Tuberculosis %X . %B Acta Clinica Belgica %V . %8 24/6/2016 %G eng %1 37111 %R http://dx.doi.org/10.1080/17843286.2016.1200823 %0 Journal Article %J Org Biomol Chem %D 2016 %T Synthesis and anti-tubercular activity of N(2)-arylbenzo[g]isoquinoline-5,10-dione-3-iminium bromides. %A Rotthier, G %A Davie Cappoen %A Nguyen, Quang Trung %A Dang Thi, Tuyet Anh %A Vanessa Mathys %A Nguyen, Van Tuyen %A Huygen, K %A Maes, L %A Cos, P %A Abbaspour Tehrani, K %K Antitubercular Agents %K Dose-Response Relationship, Drug %K Hydrocarbons, Brominated %K Isoquinolines %K Macrophages %K Microbial Sensitivity Tests %K Molecular Conformation %K Mycobacterium tuberculosis %K Structure-Activity Relationship %K Tuberculosis, Multidrug-Resistant %X

Tuberculosis has remained a challenge for medicinal chemists worldwide. In the framework of a collaborative program to identify and evaluate novel antitubercular candidate compounds, the biological properties of benzo[g]isoquinoline-5,10-diones have been found to be very promising. In this paper we have further expanded the library by incorporation of an amidinium moiety into the benzo[g]isoquinoline-5,10-dione scaffold. The presence of this functional group also increased the solubility of the quinones in polar solvents. To this purpose N(2)-arylbenzo[g]isoquinoline-5,10-dione-3-iminium bromides were synthesized in a straightforward way by means of a reaction of anilines with 2-(bromomethyl)-3-(cyanomethyl)-1,4-dimethoxynaphthalene. Following the biological evaluation, N(2)-(4-chlorophenyl)-5,10-dioxobenzo[g]isoquinoline-3(2H)-iminium bromide (MIC = 1.16 μM, CC50 = 28.51 μM, SI = 24.58) was selected as the most promising representative. Apart from the nano-molar anti-mycobacterial activity, the compound was able to target intracellular residing Mycobacterium tuberculosis and the susceptibility of a multi-drug-resistant strain towards the compound was confirmed.

%B Org Biomol Chem %V 14 %P 2041-51 %8 2016 Feb 14 %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/26763748?dopt=Abstract %R 10.1039/c5ob02138c %0 Generic %D 2016 %T TB-BRU-NET 2010-2013 %A Vanessa Mathys %A Christelle Vluggen %A Karine Soetaert %E FARES-VRGT %K meeting %K TB-BRU-NET %B MDR meeting %8 0/0/2016 %G eng %N FARES %1 37200 %2 9/06/2016 %0 Generic %D 2016 %T Time and motion exercise %A Vanessa Mathys %E ERL-TB %K Exercise %K meeting %K time %B ERL-TB annual meeting %8 0/0/2016 %G eng %N ERL-TB %1 37198 %2 27/04/2016 %0 Generic %D 2015 %T Amélioration de l'activité thérapeutique de l'ethionamide. %A Vanessa Mathys %E FARES-VRGT %K de %B FARES annual meeting %8 0/0/2015 %G eng %N FARES %1 39186 %2 26/02/2015 %0 Generic %D 2015 %T Analysis of the virulence and immunogenicity of porcine and human M. avium ssp. hominissuis isolates in an experimental mouse model. %A Nicolas Bruffaerts %A Christelle Vluggen %A L Duytschaever %A J. Denoel %A Wattiez,R. %A J.J. Letesson %A Vanessa Mathys %A Virginie Roupie %A David Fretin %A Saegerman,C. %A K Huygen %E Research Center Borstel %K an %K analysi %K analysis %K European %K Human %K M %K MODEL %K ON %K symposium %K virulence %B European Symposium on Non-tuberculous Mycobacteria %8 0/0/2015 %G eng %N Research Center Borstel %1 37032 %2 24/06/2015;27/06/2015 %0 Generic %D 2015 %T Antimicrobial resistance testing in 2020: Will we still be looking at the phenotype? %A Pieter-Jan Ceyssens %A Wesley Mattheus %A Vanessa Mathys %A R. Vanhoof %A Michael Kalai %A Sophie Bertrand %E Institut Pasteur %K Antimicrobial %K Antimicrobial resistance %K at %K Phenotype %K resistance %K Still %K TESTING %B RIIP Meeting %8 0/0/2015 %G eng %N Institut Pasteur %1 37023 %2 04/2015 %0 Journal Article %J Acta Clin.Belg. %D 2015 %T Case report of a false positive result of the Xpert MTB/RIF assay for rifampicin resistance in mycobacterium tuberculosis complex %A Claessens,J. %A Vanessa Mathys %A Derdelinckx,I. %A Saegeman,V. %K a %K Abdomen %K article %K AS %K Case %K conventional %K culture %K detection %K Diagnosis %K DRUG %K health %K INFECTION %K IS %K IT %K journal %K Microscopy %K Molecular %K Mycobacterium %K Mycobacterium tuberculosis %K old %K ON %K organisation %K Patient %K present %K Print %K report %K resistance %K result %K results %K specificity %K Test %K TESTING %K tests %K Tuberculosis %K use %K Version %K WHO %K world %K World Health %X In the present case, we report a false positive result for the detection of rifampicin (RIF) resistance by the Xpert(R) MTB/RIF assay, version G4.Miliary Mycobacterium tuberculosis infection (miliary TB) was suspected in a 50-year old Angolan woman. Imaging of the thorax and abdomen displayed diffuse lesions. The Xpert(R) MTB/RIF assay conducted on the broncho-alveolar lavage (BAL) fluid was positive for TB and positive for RIF resistance. Confirmatory molecular tests and the phenotypic drug susceptibility determination supported the diagnosis of TB but not RIF resistance. The patient was treated successfully with a conventional therapeutic scheme. Because, the Xpert(R) MTB/RIF assay allows the simultaneous detection of TB and RIF resistance, the World Health Organisation (WHO) recommends its use as initial diagnostic test, over microscopy, culture and phenotypic drug susceptibility testing. Even though specificity of the Xpert(R) MTB/RIF assay version G4 is high, false positive test results remain possible and have to be considered for the interpretation of the RIF resistance detection by Xpert(R) MTB/RIF assay %B Acta Clin.Belg. %V . %P 2295333715Y0000000072 %8 24/9/2015 %G eng %1 37103 %& 2295333715Y0000000072 %R http://dx.doi.org/10.1179/2295333715Y.0000000072 %0 Journal Article %J J Med Microbiol %D 2015 %T Evaluation of the Speed-Oligo Mycobacteria assay for the identification of nontuberculous mycobacteria. %A Ivy Bastos Ramis %A Cnockaert, Margo %A Von Groll, Andrea %A Vanessa Mathys %A Simon, Anne %A Tortoli, Enrico %A Palomino, Juan Carlos %A Almeida da Silva, Pedro Eduardo %A Vandamme, Peter %A Emmanuel André %A Martin, Anandi %K DNA Probes %K DNA, Ribosomal %K DNA, Ribosomal Spacer %K Humans %K Mycobacterium Infections, Nontuberculous %K Nontuberculous Mycobacteria %K Nucleic Acid Hybridization %K Oligonucleotides %K polymerase chain reaction %K Reagent Kits, Diagnostic %K RNA, Ribosomal, 16S %K Sensitivity and Specificity %K Sequence Analysis, DNA %K Species Specificity %K Time Factors %X

Nontuberculous mycobacteria (NTM) causing human infectious disease have become increasingly common. Rapid and accurate identification to the species level is, therefore, critical. The Speed-Oligo Mycobacteria assay is an oligochromatographic method that was made available recently for the identification and differentiation of mycobacteria. The present study aimed to evaluate the performance of the Speed-Oligo Mycobacteria assay for the identification of NTM. We examined a total of 62 strains (9 type strains, 19 reference strains and 34 clinical isolates) belonging to 13 different species (Mycobacterium intracellulare, M. fortuitum, M. gordonae, M. kansasii, M. marinum, M. peregrinum, M. scrofulaceum, M. abscessus, M. bovis BCG, M. chelonae, M. avium, M. malmoense and M. xenopi). The Speed-Oligo Mycobacteria assay was performed according to the manufacturer's instructions. Discrepant results between Speed-Oligo Mycobacteria and the original identification were reassessed by the Speed-Oligo Mycobacteria assay and resolved by the GenoType Mycobacterium CM assay and by sequencing of 16S rRNA and protein-encoding genes. We found 93.5 % (58/62) concordance for the identification of NTM as compared with the original identification. Three strains were erroneously identified by Speed-Oligo Mycobacteria: one M. kansasii strain was identified as Mycobacterium tuberculosis complex, and one M. chelonae strain and one M. peregrinum strain were both identified as Mycobacterium abscessus. Moreover, one M. chelonae strain was not identified by Speed-Oligo Mycobacteria since it did not react with any species-specific probe. For these strains, sequencing of the genes hsp65, 16S rRNA and rpoB and the GenoType Mycobacterium CM assay were performed. The Speed-Oligo Mycobacteria assay can be a useful tool for the rapid and easy identification of the most common NTM. If applied in clinical practice it could reduce diagnostic delays and contribute to correct clinical and better management of infections caused by NTM.

%B J Med Microbiol %V 64 %P 283-7 %8 2015 Mar %G eng %N Pt 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/25596120?dopt=Abstract %R 10.1099/jmm.0.000025 %0 Generic %D 2015 %T Improvement of the ETH therapeutic activity. %A Vanessa Mathys %K Activity %K Improvement %B MDR meeting %I NA %C NA %8 0/0/2015 %G eng %N MDR-TB comity %1 39185 %2 11/06/2015 %0 Generic %D 2015 %T Mycobacteria : current molecular laboratory techniques. %A Vanessa Mathys %K Diagnosis %K future %K Laboratories %K Molecular %K Molecular diagnosis %K Mycobacterium %K Technique %B The future of the molecular diagnosis. %I NA %C NA %8 0/0/2015 %G eng %N VAKB UZ Leuven %1 39187 %2 05/02/2015 %0 Generic %D 2015 %T Mycobacterium tuberculosis & les mycobactéries atypiques: techniques de laboratoire. %A Vanessa Mathys %E FARES-VRGT %K de %K LE %K Mycobactéries %K Mycobacterium %K Mycobacterium tuberculosis %K Technique %K Tuberculosis %B Formation infirmière %8 0/0/2015 %G eng %N FARES %1 39183 %2 25/09/2015 %0 Journal Article %J J.Antimicrob.Chemother. %D 2015 %T Revisiting susceptibility testing in MDR-TB by a standardized quantitative phenotypic assessment in a European multicentre study9 %A Cambau,E. %A Viveiros,M. %A Machado,D. %A Raskine,L. %A Ritter,C. %A Tortoli,E. %A Vanessa Mathys %A S Hoffner %A Richter,E. %A Perez Del Molino,M.L. %A D M. Cirillo %A D. van Soolingen %A Bottger,E.C. %K 0 %K a %K acid %K Agent %K Agents %K Amikacin %K an %K Antitubercular Agents %K article %K AS %K assessment %K at %K Belgium %K Biology %K Brussels %K care %K Cell %K Clinical %K Concordance %K de %K detection %K disease %K Diseases %K DRUG %K drug effects %K Drug Resistance %K drugs %K e %K electronic %K environment %K Establish %K estimation %K ET %K Ethionamide %K Europe %K European %K EVALUATION %K France %K gene %K genetics %K Genotype %K Genotyping Techniques %K Germany %K growth & development %K health %K Help %K Humans %K im %K Impact %K Infectious %K Infectious diseases %K Institute %K IS %K isolation & purification %K ITALY %K journal %K Laboratories %K LEVEL %K levels %K medical %K method %K methods %K Microbial Sensitivity Tests %K microbiology %K Mutation %K Mycobacterium %K Mycobacterium tuberculosis %K national %K Netherlands %K objectives %K observed %K ON %K outcome %K paris %K pathogen %K Patient %K Patient Care %K patients %K pharmacology %K portugal %K proportion %K protocol %K public %K public health %K Public-health %K Pulmonary %K Pyrazinamide %K region %K resistance %K result %K results %K SB - IM %K SCREENING %K Service %K Short %K SOFTWARE %K Spain %K standards %K Streptomycin %K study %K Sweden %K Switzerland %K Technique %K TESTING %K The Netherlands %K therapeutic use %K time %K treatment %K Treatment Outcome %K Tuberculosis %K Tuberculosis,Multidrug-Resistant %K tumor %K Universities %K university %K use %K VALIDATION %K WIV-ISP %X OBJECTIVES: Treatment outcome of MDR-TB is critically dependent on the proper use of second-line drugs as per the result of in vitro drug susceptibility testing (DST). We aimed to establish a standardized DST procedure based on quantitative determination of drug resistance and compared the results with those of genotypes associated with drug resistance. METHODS: The protocol, based on MGIT 960 and the TB eXiST software, was evaluated in nine European reference laboratories. Resistance detection at a screening drug concentration was followed by determination of resistance levels and estimation of the resistance proportion. Mutations in 14 gene regions were investigated using established techniques. RESULTS: A total of 139 Mycobacterium tuberculosis isolates from patients with MDR-TB and resistance beyond MDR-TB were tested for 13 antituberculous drugs: isoniazid, rifampicin, rifabutin, ethambutol, pyrazinamide, streptomycin, para-aminosalicylic acid, ethionamide, amikacin, capreomycin, ofloxacin, moxifloxacin and linezolid. Concordance between phenotypic and genotypic resistance was >80%, except for ethambutol. Time to results was short (median 10 days). High-level resistance, which precludes the therapeutic use of an antituberculous drug, was observed in 49% of the isolates. The finding of a low or intermediate resistance level in 16% and 35% of the isolates, respectively, may help in designing an efficient personalized regimen for the treatment of MDR-TB patients. CONCLUSIONS: The automated DST procedure permits accurate and rapid quantitative resistance profiling of first- and second-line antituberculous drugs. Prospective validation is warranted to determine the impact on patient care %B J.Antimicrob.Chemother. %V 70 %P 686 - 696 %8 0/3/2015 %G eng %N 3 %1 9 %& 686 %R dku438 [pii];10.1093/jac/dku438 [doi] %0 Generic %D 2015 %T Time and motion exercise: MIRU-VNTR typing. %A Vanessa Mathys %E ERL-TB %K Exercise %K time %B ERL-TB net annual meeting %8 0/0/2015 %G eng %N ERL-TB %1 38293 %2 12/05/2015 %0 Generic %D 2015 %T Tuberculose humaine %A Vanessa Mathys %E Scicom %E FAVV-AFSCA %K tuberculose %B Scicom %8 0/0/2015 %G eng %N Scicom, AFSCA %1 37015 %2 26/08/2015 %0 Journal Article %J J.Med.Chem. %D 2014 %T 1,2,3,4,8,9,10,11-Octahydrobenzo[j]phenanthridine-7,12-diones as New Leads against Mycobacterium tuberculosis %A Davie Cappoen %A Claes,P. %A J. Jacobs %A R. Anthonissen %A Vanessa Mathys %A Luc Verschaeve %A K Huygen %A Kimpe,N.D. %K Activity %K acute %K Agent %K article %K AS %K at %K Belgium %K continue %K Death %K Design %K Diagnostics %K disease %K Diseases %K DRUG %K Drug Resistance %K drugs %K effective %K electronic %K Exploration %K genotoxicity %K global %K health %K HIV %K immunology %K Infectious %K Infectious diseases %K Institute %K IS %K journal %K Lead %K Mycobacterium %K Mycobacterium tuberculosis %K pandemic %K PROGRAM %K public %K public health %K Public-health %K Research %K resistance %K Service %K strain %K study %K Target %K toxicity %K Tuberculosis %K vaccine %K vaccines %X Tuberculosis (TB) continues to be a worldwide health problem with over 1.4 million deaths each year. Despite efforts to develop more effective vaccines, more reliable diagnostics, and chemotherapeutics, tuberculosis remains a threat to global health, fueled by the HIV pandemic and the rapid generation of drug resistance. The exploration of novel drugs to serve as a companion drug for existing drugs is of paramount importance. As part of our program to design new 2-aza-anthraquinones with antimycobacterial activity, various tetrahydro- and octahydrobenzo[j]phenanthridinediones were synthesized. These compounds showed high in vitro potency against Mycobacterium tuberculosis, the etiological agent of TB and against other clinically relevant mycobacterial species at submicromolar concentrations. The susceptibility of a multidrug resistant strain toward these compounds and their ability to target intracellular replicating Mycobacterium tuberculosis was demonstrated. Next to the acute toxicity, the genotoxicity of these compounds was investigated. Often overlooked in studies, genotoxicity could be dismissed for the investigated compounds, making them a promising scaffold in TB drug research %B J.Med.Chem. %V 57 %P 2895 - 2907 %8 10/4/2014 %G eng %N 7 %1 37497 %& 2895 %R http://dx.doi.org/10.1021/jm401735w %0 Journal Article %J Eur J Med Chem %D 2014 %T 2,4-Dialkyl-8,9,10,11-tetrahydrobenzo[g]pyrimido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraones as new leads against Mycobacterium tuberculosis. %A Claes, Pieter %A Davie Cappoen %A Uythethofken, Cynthia %A Jacobs, Jan %A Birgit Mertens %A Vanessa Mathys %A Luc Verschaeve %A Huygen, Kris %A De Kimpe, Norbert %K Animals %K Antitubercular Agents %K Cell Line %K Dose-Response Relationship, Drug %K Isoquinolines %K Macrophages %K mice %K Microbial Sensitivity Tests %K Molecular Structure %K Mycobacterium tuberculosis %K Pyrimidinones %K Quinones %K Structure-Activity Relationship %K Tuberculosis, Multidrug-Resistant %X

Given the re-emergence of tuberculosis in Europe and beyond, the search for novel bio-active compound classes against this disease is of utmost importance. As a result of a high intrinsic tolerance of the etiological agent, Mycobacterium tuberculosis, towards most antibiotics and xenobiotics, the search for such new compounds is far from trivial. Further exacerbated by the rapid generation and spread of drug resistant M. tuberculosis and fuelled by the HIV/AIDS pandemic, halting the tuberculosis epidemic is of paramount importance. As part of our program to design new 2-aza-anthraquinones with anti-mycobacterial activity, various dialkyltetrahydrobenzo[g]pyrimido[4,5-c]isoquinolinetetraones were designed and synthesised. The compounds were submitted to a biological evaluation in which the activity against M.tb H37Rv(lux) was observed, as well as the acute toxicity towards J774 A.1 macrophages. From these results, the selectivity index was calculated. Furthermore, the activity of the most promising compounds was further studied against a multi-drug resistant LAM-1 strain and against intracellular replicating M.tb. The study was further extended with a comet assay and a VITOTOX™ assay to investigate the possibility of observable genotoxic effects caused by these compounds.

%B Eur J Med Chem %V 77 %P 409-21 %8 2014 Apr 22 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/24681334?dopt=Abstract %R 10.1016/j.ejmech.2014.03.024 %0 Journal Article %J Eur J Med Chem %D 2014 %T Anti-mycobacterial activity of 1,3-diaryltriazenes. %A Davie Cappoen %A Vajs, Jure %A Uythethofken, Cynthia %A Virag, Andrej %A Vanessa Mathys %A Kočevar, Marijan %A Luc Verschaeve %A Gazvoda, Martin %A Polanc, Slovenko %A Huygen, Kris %A Košmrlj, Janez %K Animals %K Antitubercular Agents %K Cell Line %K Cell Survival %K Dose-Response Relationship, Drug %K Macrophages %K mice %K Microbial Sensitivity Tests %K Molecular Structure %K Mycobacterium avium %K Mycobacterium bovis %K Mycobacterium ulcerans %K Structure-Activity Relationship %K Triazenes %X

The rapid generation and spread of the drug resistant tuberculosis has led to an ongoing demand for novel compounds for therapeutic use. Identification and study of compounds with the ability to inhibit Mycobacterium tuberculosis is of paramount importance. For this reason, a library of substituted 1,3-diaryltriazenes based on the acting component of the anti-trypanosomal drug, diminazene aceturate was created and evaluated for its potential as anti-tubercular agent. Several compounds were identified with sub-micro molar inhibitory concentrations against M. tuberculosis and other clinically relevant mycobacterial species such as Mycobacterium bovis, Mycobacterium avium and Mycobacterium ulcerans. Although the library of the compounds showed a considerable acute cytotoxicity, a genotoxicity could not be observed. Finally, the triazene 14 was selected with the best biological properties (IC50 = 3.26 μM, NI50 = 24.22 μM, SI = 7.44). The compound 14 showed the ability to inhibit the growth of intracellular replicating and multi-drug resistant M. tuberculosis. The results suggest the molecule to be an interesting scaffold for further study and optimization.

%B Eur J Med Chem %V 77 %P 193-203 %8 2014 Apr 22 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/24631899?dopt=Abstract %R 10.1016/j.ejmech.2014.02.065 %0 Journal Article %J Eur J Med Chem %D 2014 %T Biological evaluation of diazene derivatives as anti-tubercular compounds. %A Davie Cappoen %A Majce, Vita %A Uythethofken, Cynthia %A Urankar, Damijana %A Vanessa Mathys %A Kočevar, Marijan %A Luc Verschaeve %A Polanc, Slovenko %A Huygen, Kris %A Košmrlj, Janez %K Antitubercular Agents %K Drug Screening Assays, Antitumor %K Imides %X

Despite efforts made in chemotherapeutic research in the past and present, Mycobacterium tuberculosis (M.tb), the etiological agent of tuberculosis, still causes more than a million deadly casualties each year, second only to HIV. The rapid generation and spread of drug resistant strains, a problem exacerbated by co-infection with HIV demands further efforts in the investigation of novel classes of anti-tubercular compounds. A library of eight substituted diazenecarboxamides, three carbamoyldiazenecarboxylates and four diazene-1,2-dicarboxamides was synthesized in a straightforward manner followed by a biological evaluation of the compounds. We observed minimal inhibitory concentrations below 10 μg/mL against the H37Rv lab strain of M.tb. Three compounds that showed a potency of 90% growth inhibition of M.tb at a concentration lower than 10 μg/mL were further evaluated and showed potency against other clinically relevant mycobacterial species such as Mycobacterium bovis, Mycobacterium avium and Mycobacterium ulcerans. The selected compounds were examined for acute cell toxicity on a murine macrophage like monocyte cell line J774 A.1 in which the cell viability was reduced by 50% at concentrations ranging from 7.4 μg/mL to 20.7 μg/mL. Neither of the three compounds showed signs of genotoxicity by VITOTOX or by Comet assay. The study was complemented by demonstration of the inhibition of intracellular replication of M.tb H37Rv inside J774 A.1 cells at 2 μg/mL concentration and the susceptibility of a MDR LAM-1 strain at concentrations between 5 and 1 μg/mL of the most active compound.

%B Eur J Med Chem %V 74 %P 85-94 %8 2014 Mar 03 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/24448419?dopt=Abstract %R 10.1016/j.ejmech.2013.12.057 %0 Journal Article %J Int J Tuberc Lung Dis %D 2014 %T False-positive rifampicin resistance on Xpert® MTB/RIF caused by a silent mutation in the rpoB gene. %A Vanessa Mathys %A van de Vyvere, M %A de Droogh, E %A Groenen, G %K Aged %K Amikacin %K Amino Acid Sequence %K Antibiotics, Antitubercular %K Bacterial Proteins %K DNA-Directed RNA Polymerases %K Drug Resistance, Multiple, Bacterial %K Ethambutol %K Ethionamide %K Fluoroquinolones %K Genotype %K Humans %K Isoniazid %K Male %K Molecular Sequence Data %K Mutation %K Mycobacterium tuberculosis %K Phenotype %K Pyrazinamide %K Rifampin %K Sensitivity and Specificity %K sputum %K Tuberculosis, Pulmonary %X

The Xpert® MTB/RIF assay detects the presence of Mycobacterium tuberculosis and its resistance to rifampicin (RMP) directly in sputum samples. Discrepant results were observed in a case of smear-positive pulmonary tuberculosis that was Xpert-resistant but phenotypically susceptible to RMP. Complementary investigations (repeat Xpert, Genotype®MTBDRplus assay and sequencing of the rpoB gene) revealed the presence of a silent mutation in the rpoB gene, leading to the conclusion of a false-positive Xpert result. As misinterpretation of Xpert results may lead to inappropriate treatment, the presence of rpoB mutations should be confirmed by sequencing the rpoB gene.

%B Int J Tuberc Lung Dis %V 18 %P 1255-7 %8 2014 Oct %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/25216843?dopt=Abstract %R 10.5588/ijtld.14.0297 %0 Generic %D 2014 %T Simultaneous determination of anti-tuberculosis drug levels in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry for subsequent Therapeutic Drug Monitoring %A Tim Reyns %A Vanessa Mathys %A Groenen,G. %A Mirjana Andjelkovic %A Joris Van Loco %K alternative %K Anti tubercolosis drugs %K DRUG %K Drug Monitoring %K Human %K LC-MS/MS %K LEVEL %K levels %K Liquid chromatography-tandem mass spectrometry %K Mass %K Mass Spectrometry %K meeting %K Monitoring %K plasma %K Strategies %K Strategy %K TDM %K Therapeutic drug monitoring %K Toxicology %K ultra-performance liquid chromatography-tandem mass spectrometry %B Alternative Sampliong Strategies in Toxicology and Therapeutic Drug Monitoring - IATDMCT Satellite Meeting 2014 %I NA %C NA %8 18/9/2014 %G eng %N IATDMCT %1 2270 %2 18/09/2014 - 19/09/2014 %0 Journal Article %J Eur J Med Chem %D 2013 %T Biological evaluation of bisbenzaldehydes against four Mycobacterium species. %A Davie Cappoen %A Forge, Delphine %A Vercammen, Frank %A Vanessa Mathys %A Kiass, Mehdi %A Virginie Roupie %A Anthonissen, Roel %A Luc Verschaeve %A Vanden Eynde, Jean Jacques %A Huygen, Kris %K Animals %K Anti-Bacterial Agents %K Antitubercular Agents %K Benzaldehydes %K Cell Line %K Cell Survival %K Cells, Cultured %K Dose-Response Relationship, Drug %K Drug Resistance, Bacterial %K Hepatocytes %K Host-Pathogen Interactions %K Humans %K Macrophages %K mice %K Microbial Sensitivity Tests %K Molecular Structure %K Mycobacterium %K Mycobacterium avium %K Mycobacterium bovis %K Mycobacterium tuberculosis %K Mycobacterium ulcerans %K Species Specificity %X

A series of bisbenzaldehydes and structurally related analogs, conveniently synthesized via microwave-assisted reactions, were evaluated in vitro against drug susceptible and multi-drug resistant Mycobacterium tuberculosis, against virulent Mycobacterium bovis, against Mycobacterium ulcerans and against two Mycobacterium avium subspecies. Among the 33 substances that were tested, compound 12, i.e. 4,4'-[1,12-dodecanediyl(oxy)]bisbenzaldehyde, emerged as the most promising hit. Its activity was further confirmed in an intracellular growth inhibition assay of M. tb in murine J774 A.1 macrophages. None of the compounds showed significant cytotoxicity on human C3A hepatocytes in a neutral red dye uptake assay and no genotoxicity or mutagenicity was observed as demonstrated by a VITOTOX™ test and confirmed with a comet assay.

%B Eur J Med Chem %V 63 %P 731-8 %8 2013 May %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/23567963?dopt=Abstract %R 10.1016/j.ejmech.2013.03.023 %0 Report %D 2013 %T Evaluation externe de qualité 2013 en microbiologie. Identification de Mycobactéries et test de sensibilité de Mycobacterium tuberculosis à l'isoniazide (INH), la rifampicine (RMP), l'éthambutol (EMB) et facultatif la pyrazinamide (PZA). %A Vanessa Mathys %A Bernard China %A Kris Vernelen %K antibiotics %K bactériologie %K biologie clinique %K de %K EEQ %K EVALUATION %K identification %K microbiologie %K Mycobactéries %K Mycobacterium %K Pyrazinamide %K Test %K Tuberculosis %I WIV-ISP %C Bruxelles %P 11 %8 20/12/2013 %G eng %1 567 %0 Journal Article %J PLoS One %D 2013 %T From multidrug- to extensively drug-resistant tuberculosis: upward trends as seen from a 15-year nationwide study. %A Stoffels, Karolien %A Allix-Béguec, Caroline %A Groenen, Guido %A Wanlin, Maryse %A Berkvens, Dirk %A Vanessa Mathys %A Supply, Philip %A Fauville-Dufaux, Maryse %K ADOLESCENT %K Adult %K Antitubercular Agents %K Belgium %K Extensively Drug-Resistant Tuberculosis %K Female %K health surveys %K Humans %K incidence %K Male %K Mutation %K Mycobacterium tuberculosis %K REGISTRIES %K Treatment Outcome %K Tuberculosis, Multidrug-Resistant %K Young adult %X

BACKGROUND: Emergence of extensively drug-resistant tuberculosis (XDR-TB) represents an enormous challenge to Public Health globally.

METHODS: Progression towards XDR-TB was investigated in Belgium, a country with a typically low TB incidence, by analyzing the magnitude, characteristics, and treatment success of multidrug-resistant tuberculosis (MDR-TB) through a population-based study from 1994 to 2008.

RESULTS: Among the 174 MDR-TB patients, 81% were foreign-born, 48% of these being asylum seekers. Although the number of MDR-TB patients remained stable through the study period at around 15 new cases annually, frequencies of resistance of the patients' first MDR-TB isolate to second-line drugs increased, as well as the total number of antibiotics it was resistant to (p<0.001). XDR-TB cases were detected from 2002 onwards. For 24 patients, additional resistance to several second-line drugs was acquired during treatment. Molecular-guided investigations indicated little to no contribution of in-country clonal spread or exogenous re-infection. The increase of pre-XDR and XDR cases could be attributed to rising proportions of patients from Asia and Central and Eastern Europe (p<0.001) and an increase in the isolation of Beijing strains in these groups (p<0.001). Despite augmented resistance, the treatment success rate improved from 63.0% to 75.8% (p = 0.080) after implementation in 2005 of improved surveillance measures and therapeutic access.

CONCLUSIONS: Increasing severity in drug resistance patterns leading to more XDR- and "panresistant" TB cases in a country with a low TB incidence like Belgium represents a strong alert on worsening situations in other world regions and requires intense public health measures.

%B PLoS One %V 8 %P e63128 %8 2013 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/23671662?dopt=Abstract %R 10.1371/journal.pone.0063128 %0 Journal Article %J J.Biol.Chem. %D 2013 %T Para-Aminosalicylic acid is a prodrug targeting dihydrofolate reductase in Mycobacterium tuberculosis33991 %A Zheng,J. %A Rubin,E.J. %A Bifani,P. %A Vanessa Mathys %A Lim,V. %A Au,M. %A Jang,J. %A Nam,J. %A Dick,T. %A Walker,J.R. %A Pethe,K. %A L. Camacho %K 0 %K a %K acid %K Activity %K Agent %K Agents %K Aminosalicylic Acid %K an %K antagonists & inhibitors %K Antitubercular Agents %K article %K AS %K Bacterial Proteins %K Benzoic Acid %K biosynthesis %K Clinical %K Dihydropteroate Synthase %K disease %K Diseases %K DRUG %K drugs %K electronic %K evidence %K Folate %K Folic Acid %K Folic Acid Antagonists %K Folic-acid %K Functional %K Gene Knockdown Techniques %K genetics %K identification %K im %K Institute %K IS %K IT %K journal %K Laboratories %K mechanism %K metabolism %K Mutation %K Mycobacterium %K Mycobacterium tuberculosis %K Pa %K PAS %K pharmacokinetics %K pharmacology %K prevent %K Prodrugs %K protein %K Proteins %K report %K Research %K Research Support %K resistance %K SB - IM %K strain %K Tetrahydrofolate Dehydrogenase %K Tuberculosis %K use %X para-Aminosalicylic acid (PAS) is one of the antimycobacterial drugs currently used for multidrug-resistant tuberculosis. Although it has been in clinical use for over 60 years, its mechanism(s) of action remains elusive. Here we report that PAS is a prodrug targeting dihydrofolate reductase (DHFR) through an unusual and novel mechanism of action. We provide evidences that PAS is incorporated into the folate pathway by dihydropteroate synthase (DHPS) and dihydrofolate synthase (DHFS) to generate a hydroxyl dihydrofolate antimetabolite, which in turn inhibits DHFR enzymatic activity. Interestingly, PAS is recognized by DHPS as efficiently as its natural substrate para-amino benzoic acid. Chemical inhibition of DHPS or mutation in DHFS prevents the formation of the antimetabolite, thereby conferring resistance to PAS. In addition, we identified a bifunctional enzyme (riboflavin biosynthesis protein (RibD)), a putative functional analog of DHFR in a knock-out strain. This finding is further supported by the identification of PAS-resistant clinical isolates encoding a RibD overexpression mutation displaying cross-resistance to genuine DHFR inhibitors. Our findings reveal that a metabolite of PAS inhibits DHFR in the folate pathway. RibD was shown to act as a functional analog of DHFR, and as for DHFS, both were shown to be associated in PAS resistance in laboratory strains and clinical isolates %B J.Biol.Chem. %V 288 %P 23447 - 23456 %8 9/8/2013 %G eng %N 32 %1 38480 %& 23447 %R http://dx.doi.org/10.1074/jbc.M113.475798 %0 Journal Article %J Eur.J.Med.Chem. %D 2013 %T Synthesis and antimycobacterial activity of analogues of the bioactive natural products sampangine and cleistopholine36497 %A Claes,P. %A Davie Cappoen %A Mbala,B.M. %A J. Jacobs %A Birgit Mertens %A Vanessa Mathys %A Luc Verschaeve %A K Huygen %A N. De Kimpe %K acute toxicity %K antibiotics %K Cleistopholine %K Pyridinium ylids %K Sampangine %K Tuberculosis %X

Identification and investigation of novel classes and compounds for the treatment of tuberculosis remains of utmost importance in the fight against the disease. Despite many efforts, the weakly gram positive Mycobacterium tuberculosis keeps demanding its toll in human lives. For this reason a small library of substituted and unsubstituted aza analogues of cleistopholine and sampangine were synthesized in a short and straightforward manner and tested in vitro against M.tb. The compounds showed promising activity against the M.tb H37Rv strain and Minimal Inhibitory Concentrations (MIC) could be observed as low as 0.88 muM. Accompanied by moderate acute toxicity against C3A hepatocytes, the therapeutic index showed an acceptable range. Further tests confirmed the inhibition by up to 74% of intracellular growth of M.tb inside macrophages conferred by 1-hydroxybenzo[g]isoquinoline-5,10-diones. Activity of the library against other clinically relevant mycobacterial species such as Mycobacterium bovis, Mycobacterium avium and Mycobacterium ulcerans was confirmed. Furthermore the activity against a multi-drug-resistant MDR LAM-1 M.tb strain was tested and the MIC value situated around 1 muM. The lacking genotoxicity of a group of enamine substituted cleistopholine analogues indicates this group as a hit and encourages their use as a scaffold for further studies

%B Eur.J.Med.Chem. %V 67 %P 98 - 110 %8 0/9/2013 %G eng %1

36497

%& 98 %R http://dx.doi.org/10.1016/j.ejmech.2013.06.010 %0 Journal Article %J J.Med.Chem. %D 2012 %T Ethionamide boosters. 2. Combining bioisosteric replacement and structure-based drug design to solve pharmacokinetic issues in a series of potent 1,2,4-oxadiazole EthR inhibitors31058 %A Flipo,M. %A Desroses,M. %A Lecat-Guillet,N. %A Villemagne,B. %A Blondiaux,N. %A Leroux,F. %A Piveteau,C. %A Vanessa Mathys %A Flament,M.P. %A Siepmann,J. %A Villeret,V. %A Wohlkonig,A. %A Wintjens,R. %A Soror,S.H. %A Christophe,T. %A Jeon,H.K. %A Locht,C. %A Brodin,P. %A Deprez,B. %A Baulard,A.R. %A Willand,N. %K 0 %K a %K Administration,Oral %K Agent %K Agents %K Animals %K antagonists & inhibitors %K Antibiotic %K Antitubercular Agents %K article %K Cell Line %K chemical synthesis %K chemistry %K Combination %K Control %K Crystallography,X-Ray %K de %K Design %K DRUG %K Drug Design %K drug effects %K Drug Synergism %K electronic %K Ethionamide %K EVALUATION %K exposure %K expression %K France %K Human %K im %K IS %K journal %K Macrophages %K metabolism %K mice %K microbiology %K Microsomes,Liver %K Models,Molecular %K Mycobacterium %K Mycobacterium tuberculosis %K ON %K Oxadiazoles %K pathogen %K Pharmacokinetic %K pharmacokinetics %K Piperidines %K PROCESSES %K Prodrugs %K protein %K Proteins %K Repressor Proteins %K Research %K Research Support %K SB - IM %K SENSITIVITY %K series %K stability %K Stereoisomerism %K Structure-Activity Relationship %K study %K Tuberculosis %K work %X Mycobacterial transcriptional repressor EthR controls the expression of EthA, the bacterial monooxygenase activating ethionamide, and is thus largely responsible for the low sensitivity of the human pathogen Mycobacterium tuberculosis to this antibiotic. We recently reported structure-activity relationships of a series of 1,2,4-oxadiazole EthR inhibitors leading to the discovery of potent ethionamide boosters. Despite high metabolic stability, pharmacokinetic evaluation revealed poor mice exposure; therefore, a second phase of optimization was required. Herein a structure-property relationship study is reported according to the replacement of the two aromatic heterocycles: 2-thienyl and 1,2,4-oxadiazolyl moieties. This work was done using a combination of structure-based drug design and in vitro/ex vivo evaluations of ethionamide boosters on the targeted protein EthR and on the human pathogen Mycobacterium tuberculosis. Thanks to this process, we identified compound 42 (BDM41906), which displays improved efficacy in addition to high exposure to mice after oral administration %B J.Med.Chem. %V 55 %P 68 - 83 %8 12/1/2012 %G eng %N 1 %1 38193 %& 68 %R http://dx.doi.org/10.1021/jm200825u %0 Journal Article %J Antimicrob Agents Chemother %D 2012 %T Evolutionary changes in antimicrobial resistance of invasive Neisseria meningitidis isolates in Belgium from 2000 to 2010: increasing prevalence of penicillin nonsusceptibility. %A Sophie Bertrand %A Carion, Françoise %A Wintjens, René %A Vanessa Mathys %A Vanhoof, Raymond %K 3' Flanking Region %K alleles %K Anti-Bacterial Agents %K Bacterial Typing Techniques %K Belgium %K Biological Evolution %K Ceftriaxone %K Drug Resistance, Bacterial %K Gene Frequency %K Genes, Bacterial %K Humans %K Longitudinal Studies %K Meningococcal Infections %K Microbial Sensitivity Tests %K Neisseria meningitidis %K Penicillin G %K Penicillin-Binding Proteins %K Sequence Analysis, DNA %X

This study was conducted to evaluate the evolution of the antimicrobial susceptibility of Neisseria meningitidis causing invasive diseases in Belgium in the period of January 2000 to December 2010. A total of 1,933 cases of N. meningitidis from invasive infections were analyzed by antimicrobial susceptibility testing at the Belgian Meningococcal Reference Centre. The majority of strains were susceptible to antibiotics that are currently used for the treatment and prophylaxis of meningococcal disease, but the prevalence of clinical isolates with reduced susceptibility to penicillin increased over the years. The phenotyping, genotyping, and determination of MICs of penicillin G were performed. The systematic shift of the curves toward higher penicillin MICs in the susceptible population indicated that this population became less sensitive to penicillin in this period. A 402-bp DNA fragment in the 3' end of penA was sequenced for the 296 nonsusceptible meningococcal strains isolated between 2000 and 2010 to examine the genetic diversity and evolution of their penA gene. In conclusion, the data obtained in our study support the statement that the position of penicillin G as a first choice in the treatment of invasive meningococcal diseases in Belgium should be reexamined. Despite an important number of isolates displaying a reduced susceptibility to penicillin, at present the expanded-spectrum cephalosporins, such as ceftriaxone, are not affected. The follow-up of the evolutionary changes in antimicrobial resistance has also proved to be essential for the recommendation of an appropriate antimicrobial treatment for invasive meningococcal diseases.

%B Antimicrob Agents Chemother %V 56 %P 2268-72 %8 2012 May %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/22290951?dopt=Abstract %R 10.1128/AAC.06310-11 %0 Generic %D 2012 %T The fluoropyrimidine chemotherapeutic agent 5-fluorouracil displays strong anti-tubercular activity in vitro %A Vanessa Mathys %A Singhal,A. %A P. Lefevre %A Kiass,M. %A Wang,X.M. %A Mathema,B. %A Kurepina,N. %A B.N. Kreiswirth %A Bifani,P. %K Activity %K Agent %K general %K meeting %B 107th General Meeting %I NA %C NA %8 0/0/2012 %G eng %N American Society for Microbiology (ASM) %1 38291 %2 05/06/2008 %0 Generic %D 2012 %T Le rat Wistar : modèle pour la tuberculose latente? %A Vanessa Mathys %E WIV-ISP %K Antibiotic %K antibiotics %K journal %K latence %K LE %K tuberculose %K Wistar rat %B Journal Club Antibiotics (JCA) %I NA %C NA %8 0/0/2012 %G eng %N Scientific Institute of Public Health, Service Maladies Bactériennes %1 38292 %2 21/09/2011 %0 Journal Article %J Antimicrob Agents Chemother %D 2012 %T Systematic analysis of pyrazinamide-resistant spontaneous mutants and clinical isolates of Mycobacterium tuberculosis. %A Stoffels, Karolien %A Vanessa Mathys %A Fauville-Dufaux, Maryse %A Wintjens, René %A Bifani, Pablo %K Amidohydrolases %K Antitubercular Agents %K Crystallography, X-Ray %K Microbial Sensitivity Tests %K Mutation %K Mycobacterium tuberculosis %K Pyrazinamide %X

Pyrazinamide (PZA) is a first-line antitubercular drug known for its activity against persistent Mycobacterium tuberculosis bacilli. We set out to systematically determine the PZA susceptibility profiles and mutations in the pyrazinamidase (pncA) gene of a collection of multidrug-resistant tuberculosis (MDR-TB) clinical isolates and PZA-resistant (PZA(r)) spontaneous mutants. The frequency of acquired resistance to PZA was determined to be 10(-5) bacilli in vitro. Selection at a lower concentration of PZA yielded a significantly larger number of spontaneous mutants. The methodical approach employed allowed for determination of the frequency of the PZA(r) phenotype correlated with mutations in the pncA gene, which was 87.5% for the laboratory-selected spontaneous mutants examined in this study. As elucidated by structural analysis, most of the identified mutations were foreseen to affect protein activity through either alteration of an active site residue or destabilization of protein structure, indicating some preferential mutation site rather than random scattering. Twelve percent of the PZA(r) mutants did not have a pncA mutation, strongly indicating the presence of at least one other mechanism(s) of PZA(r).

%B Antimicrob Agents Chemother %V 56 %P 5186-93 %8 2012 Oct %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/22825123?dopt=Abstract %R 10.1128/AAC.05385-11 %0 Journal Article %J Infect.Immun. %D 2012 %T Urease activity represents an alternative pathway for Mycobacterium tuberculosis nitrogen metabolism31064 %A Lin,W. %A Vanessa Mathys %A Ang,E.L. %A Koh,V.H. %A J.M. Martinez Gomez %A Ang,M.L. %A S.Z. Zainul Rahim %A Tan,M.P. %A Pethe,K. %A Alonso,S. %K a %K Activity %K alternative %K an %K article %K AS %K bacteria %K Carbon %K data %K effect %K electronic %K environment %K Functional %K growth %K Help %K immunology %K INFECTION %K IS %K IT %K journal %K Liver %K Lung %K M %K Macrophage %K Macrophages %K Medicine %K metabolism %K mice %K microbiology %K Mycobacterium %K Mycobacterium tuberculosis %K national %K Nitrogen %K pathogen %K pathogenic %K profile %K Profiles %K programme %K Role %K School %K Spleen %K strain %K survival %K Tuberculosis %K Type %K Universities %K university %K Urea %K urease %K virulence %K work %X Urease represents a critical virulence factor for some bacteria species through its alkalizing effect that helps neutralize the acidic microenvironment of the pathogen. In addition, urease serves as a nitrogen source provider for bacterial growth. Pathogenic mycobacteria express a functional urease but its role during infection has yet to be characterized. Here, we constructed a urease-deficient M. tuberculosis (Mtb) strain and confirmed the alkalizing effect of the urease activity within the mycobacteria-containing vacuole in resting macrophages but not in the more acidic phagolysosomal compartment of activated macrophages. However, the urease-mediated alkalizing effect did not confer any growth advantage to Mtb in macrophage as evidenced by comparable growth profiles for the mutant, wild type (WT) and complemented strains. In contrast, the urease-deficient mutant exhibited impaired in vitro growth compared to the WT and complemented strains when urea is the sole source of nitrogen. Substantial amounts of ammonia were produced by the WT and complemented strains, but not with the urease-deficient mutant, which represents the actual nitrogen source for mycobacterial growth. However, the urease-deficient mutant displayed parental colonization profiles in the lungs, spleen and liver in mice. Together, our data demonstrate a role of the urease activity in Mtb nitrogen metabolism that could be crucial for the pathogen's survival in nutrient-limited microenvironments where urea is the sole nitrogen source. Our work supports the notion that Mtb virulence correlates with its unique metabolic versatility and ability to utilize virtually any carbon and nitrogen sources available in its environment %B Infect.Immun. %V 80 %P 2771 - 2779 %8 0/8/2012 %G eng %N 8 %1 31064 %& 2771 %R http://dx.doi.org/10.1128/IAI.06195-11 %0 Journal Article %J PLoS.One. %D 2011 %T BCG induces protection against Mycobacterium tuberculosis infection in the Wistar rat model36573 %A Singhal,A. %A Vanessa Mathys %A Kiass,M. %A Creusy,C. %A Delaire,B. %A M. El Aliouat %A Dartois,V. %A Kaplan,G. %A Bifani,P. %K a %K analysi %K analysis %K Animal %K Animals %K article %K at %K BALANCE %K BCG %K Biology %K Cell %K cells %K challenge %K Control %K Correlation %K data %K disease %K Diseases %K electronic %K EVALUATION %K expression %K gene %K Genes %K Granuloma %K growth %K i %K im %K immune response %K immunohistochemistry %K INFECTION %K Inflammation %K Institute %K IS %K journal %K Lung %K MODEL %K Mycobacterium %K Mycobacterium bovis %K Mycobacterium tuberculosis %K observed %K PCR %K present %K protection %K Pulmonary %K rats %K real time PCR %K regulation %K Research %K Research Support %K response %K Responses %K SB - IM %K specific %K Still %K study %K time %K Tuberculosis %K Type %K Vaccination %K Wistar rat %X Our understanding of the correlation of Mycobacterium bovis Bacille Calmette-Guerin (BCG)-mediated immune responses and protection against Mycobacterium tuberculosis (Mtb) infection is still limited. We have recently characterized a Wistar rat model of experimental tuberculosis (TB). In the present study, we evaluated the efficacy of BCG vaccination in this model. Upon Mtb challenge, BCG vaccinated rats controlled growth of the bacilli earlier than unvaccinated rats. Histopathology analysis of infected lungs demonstrated a reduced number of granulomatous lesions and lower parenchymal inflammation in vaccinated animals. Vaccine-mediated protection correlated with the rapid accumulation of antigen specific CD4(+) and CD8(+) T cells in the infected lungs. Immunohistochemistry further revealed higher number of CD8(+) cells in the pulmonary granulomas of vaccinated animals. Evaluation of pulmonary immune responses in vaccinated and Mtb infected rats by real time PCR at day 15 post-challenge showed reduced expression of genes responsible for negative regulation of Th1 immune responses. Thus, early protection observed in BCG vaccinated rats correlated with a similarly timed shift of immunity towards the Th1 type response. Our data support the importance of (i) the Th1-Th2 balance in the control of mycobacterial infection and (ii) the value of the Wistar rats in understanding the biology of TB %B PLoS.One. %V 6 %P e28082 %8 0/0/2011 %G eng %N 12 %1 38349 %& e28082 %R http://dx.doi.org/10.1371/journal.pone.0028082 %0 Journal Article %J PLoS.One. %D 2011 %T Experimental tuberculosis in the Wistar rat: a model for protective immunity and control of infection36574 %A Singhal,A. %A M. El Aliouat %A Herve,M. %A Vanessa Mathys %A Kiass,M. %A Creusy,C. %A Delaire,B. %A Tsenova,L. %A Fleurisse,L. %A Bertout,J. %A L. Camacho %A Foo,D. %A Tay,H.C. %A Siew,J.Y. %A Boukhouchi,W. %A Marta Romano %A Mathema,B. %A Dartois,V. %A Kaplan,G. %A Bifani,P. %K a %K Analyses %K analysi %K analysis %K Animal %K Animals %K article %K Cell %K cells %K Control %K Cost %K disease %K Disease Models,Animal %K Diseases %K DRUG %K electronic %K Granuloma %K growth %K Host-Pathogen Interactions %K Human %K Humans %K im %K immunology %K INFECTION %K Institute %K interaction %K interactions %K IS %K journal %K Kinetics %K Lung %K lymphocyte %K Lymphocytes %K Macrophage %K Macrophages %K MODEL %K models %K Mycobacterium %K Mycobacterium tuberculosis %K need %K observed %K ON %K pathology %K Pharmacokinetic %K prevention & control %K rats %K Rats,Wistar %K Research %K Research Support %K result %K SB - IM %K Size %K Still %K strain %K study %K Toxicology %K Tuberculosis %K Type %K use %K virulence %K Wistar rat %X BACKGROUND: Despite the availability of many animal models for tuberculosis (TB) research, there still exists a need for better understanding of the quiescent stage of disease observed in many humans. Here, we explored the use of the Wistar rat model for the study of protective immunity and control of Mycobacterium tuberculosis (Mtb) infection. METHODOLOGY/PRINCIPAL FINDINGS: The kinetics of bacillary growth, evaluated by the colony stimulating assay (CFU) and the extent of lung pathology in Mtb infected Wistar rats were dependent on the virulence of the strains and the size of the infecting inoculums. Bacillary growth control was associated with induction of T helper type 1 (Th1) activation, the magnitude of which was also Mtb strain and dose dependent. Histopathology analysis of the infected lungs demonstrated the formation of well organized granulomas comprising epithelioid cells, multinucleated giant cells and foamy macrophages surrounded by large numbers of lymphocytes. The late stage subclinical form of disease was reactivated by immunosuppression leading to increased lung CFU. CONCLUSION: The Wistar rat is a valuable model for better understanding host-pathogen interactions that result in control of Mtb infection and potentially establishment of latent TB. These properties together with the ease of manipulation, relatively low cost and well established use of rats in toxicology and pharmacokinetic analyses make the rat a good animal model for TB drug discovery %B PLoS.One. %V 6 %P e18632 %8 0/0/2011 %G eng %N 4 %1 38350 %& e18632 %R http://dx.doi.org/10.1371/journal.pone.0018632 %0 Journal Article %J Eur.J.Clin.Microbiol.Infect.Dis. %D 2011 %T Extremely high prevalence of multidrug resistant tuberculosis in Murmansk, Russia: a population-based study36555 %A Makinen,J. %A Marjamaki,M. %A Haanpera-Heikkinen,M. %A Marttila,H. %A Endourova,L.B. %A Presnova,S.E. %A Vanessa Mathys %A Bifani,P. %A Ruohonen,R. %A Viljanen,M.K. %A Soini,H. %K 0 %K 2004 %K a %K Agent %K Agents %K ALL %K Antimicrobial %K Antimicrobial resistance %K Antitubercular Agents %K article %K AS %K at %K Case %K classification %K DRUG %K drug effects %K Drug Resistance %K electronic %K epidemiology %K Finland %K gene %K Genetic %K Genotype %K health %K High prevalence %K Humans %K im %K Institute %K IS %K isolation & purification %K journal %K Laboratories %K Microbial Sensitivity Tests %K Molecular %K Molecular Epidemiology %K Molecular Typing %K Mutation %K Mycobacterium tuberculosis %K n %K national %K pharmacology %K Polymorphism,Genetic %K POPULATION %K population based %K population-based %K Population-based studies %K population-based study %K prevalence %K region %K resistance %K Rifampin %K Russia %K SB - IM %K strain %K study %K Surveillance %K Target %K Targets %K Tuberculosis %K Tuberculosis,Multidrug-Resistant %K welfare %X Drug resistance and molecular epidemiology of tuberculosis (TB) in the Murmansk region was investigated in a 2-year, population-based surveillance of the civilian population. During 2003 and 2004, isolates from all culture-positive cases were collected (n = 1,226). Prevalence of multi-drug resistance (MDR) was extremely high, as 114 out of 439 new cases (26.0%), and 574 out of 787 previously treated cases (72.9%) were resistant to at least isoniazid (INH) and rifampin (RIF). Spoligotyping of the primary MDR-TB isolates revealed that most isolates grouped to the Beijing SIT1 genotype (n = 91, 79.8%). Isolates of this genotype were further analyzed by IS6110 RFLP. Sequencing of gene targets associated with INH and RIF resistance further showed that the MDR-TB strains are highly homogeneous as 78% of the MDR, SIT1 strains had the same resistance-conferring mutations. The genetic homogeneity of the MDR-TB strains indicates that they are actively transmitted in Murmansk %B Eur.J.Clin.Microbiol.Infect.Dis. %V 30 %P 1119 - 1126 %8 0/9/2011 %G eng %N 9 %1 38273 %& 1119 %R http://dx.doi.org/10.1007/s10096-011-1200-7 %0 Generic %D 2011 %T Le rat Wistar: un modèle pour la tuberculose latente? %A Singhal,A. %A M. el Aliouat %A Herve,M. %A Vanessa Mathys %A Kiass,M. %A Creusy,C. %A Delaire,B. %A Tsenova,L. %A Fleurisse,L. %A Boukhouchi,W. %A Marta Romano %A Dartois,V. %A Kaplan,G. %A Bifani,P. %E Mycoclub %K latence %K LE %K tuberculose %K Wistar rat %B Mycoclub2 %8 0/0/2011 %G eng %N Mycoclub %1 38348 %2 26/09/2011 %0 Generic %D 2011 %T M. tuberculosis : traitement et résistance aux antibiotiques %A Vanessa Mathys %A M. Fauville %K Antibiotique %K de %K ET %K M %K resistance %K Surveillance %K traitement %K Tuberculosis %B Réunion des laboratoires du réseau de surveillance de la (multi)résistance %I NA %C NA %8 0/0/2011 %G eng %N FARES-VRGT %1 38289 %2 15/02/2011 %0 Generic %D 2011 %T Molecular epidemiology of tuberculosis in Brussels - 1 year results (2010) %A Vanessa Mathys %A Vanfleteren,B. %A M. Fauville %K 2010 %K Brussels %K epidemiology %K meeting %K Molecular %K Molecular Epidemiology %K result %K results %K Tuberculosis %B TB-PANNET Mid-Term Meeting %I NA %C NA %8 0/0/2011 %G eng %N TB-PANNET %1 38290 %2 30/05/2011 %0 Journal Article %J Tuberculosis.(Edinb.) %D 2011 %T Mycobacterium tuberculosis infection induces hypoxic lung lesions in the rat36548 %A Heng,Y. %A Seah,P.G. %A Siew,J.Y. %A Tay,H.C. %A Singhal,A. %A Vanessa Mathys %A Kiass,M. %A Bifani,P. %A Dartois,V. %A Herve,M. %K 0 %K Activity %K Animals %K Anoxia %K article %K cause %K disease %K Disease Models,Animal %K Diseases %K DRUG %K Drug Resistance %K electronic %K Evaluating %K Female %K Granuloma %K im %K immunohistochemistry %K in vivo %K INFECTION %K Institute %K IS %K journal %K Lung %K microbiology %K MODEL %K Mycobacterium %K Mycobacterium tuberculosis %K Nitroimidazoles %K Oxygen %K pathogenicity %K pathology %K pharmacology %K rats %K region %K Research %K Research Support %K resistance %K result %K results %K SB - IM %K State %K Tension %K Tuberculosis %K Tuberculosis,Pulmonary %K use %X Hypoxia is believed to influence the metabolic state of Mycobacterium tuberculosis and cause phenotypic drug resistance. Using pimonidazole adduct staining, we show that lung lesions of infected rats contain regions of low oxygen tension. Our results support the use of the rat model for evaluating anaerobic drug activity in vivo %B Tuberculosis.(Edinb.) %V 91 %P 339 - 341 %8 0/7/2011 %G eng %N 4 %1 38225 %& 339 %R http://dx.doi.org/10.1016/j.tube.2011.05.003 %0 Generic %D 2011 %T TB BRU-NET…une partie de TB-PAN-NET %A Vanessa Mathys %A M. Fauville %K de %K Surveillance %K TB-BRU-NET %B Réunion des laboratoires du réseau de surveillance de la (multi)résistance %I NA %C NA %8 0/0/2011 %G eng %N FARES-VRGT %1 38288 %2 15/02/2011 %0 Journal Article %J Tuberculosis (Edinb) %D 2010 %T Effect of PstS sub-units or PknD deficiency on the survival of Mycobacterium tuberculosis. %A Vanzembergh, Frederic %A Peirs, Priska %A Lefèvre, Philippe %A Celio, Nathalie %A Vanessa Mathys %A Content, Jean %A Kalai, Michael %K Animals %K ATP-Binding Cassette Transporters %K Bacterial Proteins %K Blotting, Western %K Cell Proliferation %K Gene Expression Regulation, Bacterial %K mice %K Molecular Sequence Data %K Mycobacterium tuberculosis %K Protein Kinases %K Reverse Transcriptase Polymerase Chain Reaction %K Tuberculosis %X

The membrane-associated phosphate-specific transporter (Pst) complex is composed of four different proteins: PstS, PstC, PstA and PstB. The PstS component detects and binds Pi with high affinity; the PstA and PstC form transmembrane pores for Pi entry, while PstB provides energy through ATP hydrolysis. In the Mycobacterium tuberculosis genome, four different gene clusters encode three PstS, and two of each of the other sub-units. We used RT-PCR to show that these clusters represent at least three distinct operons. The pstS3-containing operon was the only one induced by lack of environmental Pi. To study the physiologic role of the different PstS sub-units and that of another potential Pi receptor, PknD, we constructed and complemented their knockout (KO) mutants. In Sauton medium, the PstS1-3 KO grew faster than the Wt or the PknD KO. Following 24 h of complete starvation, the PstS3 or PknD deficient strains died if exposed to Pi poor conditions while the PstS1 and PstS2 KO survived and still grew faster than the Wt strain. These results suggest that PstS1-3 may play a role in the regulation of M. tuberculosis growth or metabolism while PstS3 and PknD contribute to the survival of the bacteria in phosphate poor conditions.

%B Tuberculosis (Edinb) %V 90 %P 338-45 %8 2010 Nov %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/20933472?dopt=Abstract %R 10.1016/j.tube.2010.09.004 %0 Generic %D 2010 %T Etude des mécanismes de résistance de M. tuberculosis aux agents antituberculeux %A Vanessa Mathys %K Agent %K Agents %K Antibiotic %K antibiotics %K de %K journal %K M %K resistance %K Tuberculosis %B Journal Club Antibiotics %I NA %C NA %8 0/0/2010 %G eng %N WIV-ISP %1 36880 %2 23/09/2012 %0 Generic %D 2010 %T Identification d'espèce des mycobactéries non tuberculeuses: quel fragment génique séquencer et dans quelle(s) base(s) de séquences analyser le résultat? %A Vanessa Mathys %A Vanfleteren,B. %A Stoffels,K. %A M. Fauville %E Mycoclub %K de %K espèces %K ET %K gene %K identification %K LE %K Mycobactéries %K tuberculose %B Mycoclub %8 0/0/2010 %G eng %N Mycoclub %1 38286 %2 28/10/2010 %0 Generic %D 2010 %T Identification of non-tuberculous mycobacteria by sequencing a fragment of the 16S rRNA gene : which internet database use to analyze the sequence? %A Vanessa Mathys %A M. Fauville %K 16S rRNA %K a %K Database %K gene %K identification %K Internet %K meeting %K Mycobacterium %K Nontuberculous Mycobacteria %K use %B ESM annual Meeting %I NA %C NA %8 0/0/2010 %G eng %N European Society of Mycobacteriology (ESM) %1 36881 %2 30/06/2010 %0 Generic %D 2010 %T Molecular Genetics of para -Aminosalicylic Acid Resistance in Clinical Isolates and Spontaneous Mutants of Mycobacterium tuberculosis %A Vanessa Mathys %K acid %K Clinical %K Genetic %K genetics %K meeting %K Molecular %K Mycobacterium %K Mycobacterium tuberculosis %K resistance %K Tuberculosis %B TB-PAN-NET 1st Anual Meeting %I NA %C NA %8 0/0/2010 %G eng %N TB-PANNET %1 38285 %2 26/05/2010 %0 Generic %D 2010 %T Mycobacterium tuberculosis : treatment and resistance to antibiotics %A Vanessa Mathys %K Antibiotic %K antibiotics %K Mycobacterium %K Mycobacterium tuberculosis %K resistance %K treatment %K Tuberculosis %B Tuesday Seminar %I NA %C NA %8 0/0/2010 %G eng %N WIV-ISP %1 38287 %2 19/10/2010 %0 Report %D 2009 %T Contribution à la compréhension des mécanismes moléculaires de résistance de Mycobacterium tuberculosis aux agents anti-tuberculeux %A Vanessa Mathys %K Agent %K Agent anti-tuberculeux %K Agents %K contribution %K de %K Mycobacterium %K Mycobacterium tuberculosis %K resistance %K Tuberculosis %I Univeristé Libre de Bruxelles (ULB) %C Brussels, Belgium %P 225 %8 0/0/2009 %G eng %1 38283 %0 Journal Article %J Antimicrob Agents Chemother %D 2009 %T Molecular genetics of para-aminosalicylic acid resistance in clinical isolates and spontaneous mutants of Mycobacterium tuberculosis. %A Vanessa Mathys %A Wintjens, René %A Lefèvre, Philippe %A Bertout, Julie %A Singhal, Amit %A Kiass, Mehdi %A Kurepina, Natalia %A Wang, Xiao-Ming %A Mathema, Barun %A Baulard, Alain %A Kreiswirth, Barry N %A Bifani, Pablo %K Aminosalicylic Acid %K Antitubercular Agents %K Bacterial Proteins %K Drug Resistance, Bacterial %K Folic Acid %K Humans %K Microbial Sensitivity Tests %K Mutation %K Mycobacterium tuberculosis %K Structure-Activity Relationship %K Thymidylate Synthase %K Thymine %X

The emergence of Mycobacterium tuberculosis resistant to first-line antibiotics has renewed interest in second-line antitubercular agents. Here, we aimed to extend our understanding of the mechanisms underlying para-aminosalicylic acid (PAS) resistance by analysis of six genes of the folate metabolic pathway and biosynthesis of thymine nucleotides (thyA, dfrA, folC, folP1, folP2, and thyX) and three N-acetyltransferase genes [nhoA, aac(1), and aac(2)] among PAS-resistant clinical isolates and spontaneous mutants. Mutations in thyA were identified in only 37% of the clinical isolates and spontaneous mutants. Overall, 24 distinct mutations were identified in the thyA gene and 3 in the dfrA coding region. Based on structural bioinformatics techniques, the altered ThyA proteins were predicted to generate an unfolded or dysfunctional polypeptide. The MIC was determined by Bactec/Alert and dilution assay. Sixty-three percent of the PAS-resistant isolates had no mutations in the nine genes considered in this study, revealing that PAS resistance in M. tuberculosis involves mechanisms or targets other than those pertaining to the biosynthesis of thymine nucleotides. The alternative mechanism(s) or pathway(s) associated with PAS resistance appears to be PAS concentration dependent, in marked contrast to thyA-mutated PAS-resistant isolates.

%B Antimicrob Agents Chemother %V 53 %P 2100-9 %8 2009 May %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/19237648?dopt=Abstract %R 10.1128/AAC.01197-08 %0 Journal Article %J Nat.Med. %D 2009 %T Synthetic EthR inhibitors boost antituberculous activity of ethionamide36849 %A Willand,N. %A Dirie,B. %A Carette,X. %A Bifani,P. %A Singhal,A. %A Desroses,M. %A Leroux,F. %A Willery,E. %A Vanessa Mathys %A Deprez-Poulain,R. %A Delcroix,G. %A Frenois,F. %A Aumercier,M. %A Locht,C. %A Villeret,V. %A Deprez,B. %A Baulard,A.R. %K 0 %K a %K Activity %K Agent %K Agents %K Animals %K antagonists & inhibitors %K Antitubercular Agents %K article %K AS %K Binding Sites %K chemistry %K concept %K conventional %K culture %K de %K DNA-Binding Proteins %K DRUG %K Drug Resistance %K Drug Synergism %K drug therapy %K drugs %K effect %K effects %K electronic %K ET %K Ethionamide %K France %K Hydrogen Bonding %K identify %K im %K improve %K index %K interaction %K IS %K journal %K Ligands %K metabolism %K mice %K Models,Molecular %K Mycobacterium %K Mycobacterium tuberculosis %K national %K Oxadiazoles %K production %K protein %K Protein Conformation %K Proteins %K Repressor Proteins %K Research %K Research Support %K resistance %K risk %K SB - IM %K Side effects %K Side-effects %K structure %K Synthetic %K therapeutic use %K Therapy %K Thiophenes %K treatment %K Tuberculosis %K use %X The side effects associated with tuberculosis therapy bring with them the risk of noncompliance and subsequent drug resistance. Increasing the therapeutic index of antituberculosis drugs should thus improve treatment effectiveness. Several antituberculosis compounds require in situ metabolic activation to become inhibitory. Various thiocarbamide-containing drugs, including ethionamide, are activated by the mycobacterial monooxygenase EthA, the production of which is controlled by the transcriptional repressor EthR. Here we identify drug-like inhibitors of EthR that boost the bioactivation of ethionamide. Compounds designed and screened for their capacity to inhibit EthR-DNA interaction were co-crystallized with EthR. We exploited the three-dimensional structures of the complexes for the synthesis of improved analogs that boosted the ethionamide potency in culture more than tenfold. In Mycobacterium tuberculosis-infected mice, one of these analogs, BDM31343, enabled a substantially reduced dose of ethionamide to lessen the mycobacterial load as efficiently as the conventional higher-dose treatment. This provides proof of concept that inhibiting EthR improves the therapeutic index of thiocarbamide derivatives, which should prompt reconsideration of their use as first-line drugs %B Nat.Med. %V 15 %P 537 - 544 %8 0/5/2009 %G eng %N 5 %1 38473 %& 537 %R http://dx.doi.org/10.1038/nm.1950 %0 Generic %D 2008 %T The anti-cancer drug 5-fluorouracil is highly active against Mycobacterium tuberculosis in vitro but not in vivo %A Vanessa Mathys %A Singhal,A. %A Kiass,M. %A P. Lefevre %A Kurepina,N. %A Wang,X.M. %A Mathema,B. %A Baulard,A. %A Bifani,P. %K Control %K DRUG %K in vivo %K INFECTION %K infections %K IS %K Mycobacterium %K Mycobacterium tuberculosis %K Tuberculosis %B Pathogenesis and Control of Emerging Infections and Drug-Resistant Organisms %I NA %C NA %8 0/0/2008 %G eng %N Keystone Symposia %1 38282 %2 24/10/2008 %0 Generic %D 2007 %T Mutations associated with para-aminosalicylic acid (PAS) resistance in clinical isolates and spontaneous mutants of Mycobacterium tuberculosis %A Vanessa Mathys %A Quatannens,J. %A Wintjens,R. %A Kurepina,N. %A Singhal,A. %A Kiass,M. %A Mathema,B. %A Baulard,A. %A B.N. Kreiswirth %A Bifani,P. %K acid %K Clinical %K de %K Mutation %K Mycobacterium %K Mycobacterium tuberculosis %K Pa %K PAS %K resistance %K Tuberculosis %B 7ème journée des Doctorants %I NA %C NA %8 0/0/2007 %G eng %N Université Libre de Bruxelles (ULB) %1 36787 %2 18/12/2007 %0 Generic %D 2006 %T Molecular characterization of genes associated with ethionamide resistance in Mycobacterium tuberculosis clinical isolates and spontaneous mutants %A Vanessa Mathys %A Baulard,A. %A Kurepina,N. %A M. Fauville %A B.N. Kreiswirth %A Bifani,P. %K Clinical %K Ethionamide %K gene %K general %K Genes %K meeting %K Molecular %K Mycobacterium %K Mycobacterium tuberculosis %K resistance %K Tuberculosis %B 106th General Meeting %I NA %C NA %8 0/0/2006 %G eng %N American Society for Microbiology (ASM) %1 38281 %2 24/05/2006 %0 Generic %D 2006 %T Mutations associated with PAS resistance in clinical isolates and spontaneous mutants of Mycobaterium tuberculosis %A Vanessa Mathys %A Quatannens,J. %A Kurepina,N. %A Wintjens,R. %A Mathema,B. %A Baulard,A. %A B.N. Kreiswirth %A Bifani,P. %E Belgian Society for Microbiology %K Clinical %K meeting %K Mutation %K Pa %K PAS %K resistance %K Tuberculosis %B BSM meeting %8 0/0/2006 %G eng %N Belgian Society of Microbiology (BSM) %1 38280 %2 24/11/2006 %0 Government Document %D 0 %T Rapport annuel - CNR Mycobacterium %A Vanessa Mathys %G eng