Biology subjects

Rigouts, L.

Publications and source records attributed to Rigouts, L..

15 recordsLinked to original sources

bioRxiv2024

Direct thin-layer agar for bedaquiline-susceptibility testing of Mycobacterium tuberculosis at BSL2 level yields high accuracy in 15 days from sputum processing.

This study evaluated thin-layer agar (TLA) as a faster alternative for both indirect MIC determination of bedaquiline (BDQ) from culture isolates and direct drug-susceptibility testing (DST) from sputum samples. Indirect BDQ-MIC results from TLA were compared to the established 7H11 solid DST. Direct-TLA-DST performance was assessed using 143 baseline sputum samples from rifampicin-resistant TB cases. Direct-TLA tested susceptibility to rifampicin, isoniazid, levofloxacin, and BDQ, with results compared to Lowenstein-Jensen (LJ) and MGIT. For indirect BDQ-MIC determination, TLA demonstrated high accuracy, with MIC values for H37Rv falling within the WHO control range (0.015-0.12g/ml). The incubation conditions (5-10% CO2, standard) and reading time (day 7, 14) significantly affected results, with D7 readings in standard incubator providing optimal accuracy. Direct-TLA-DST detected MTB in 53.8% of samples, compared to 55.9% on LJ and 69.4% in MGIT. Uninterpretable results due to contamination or medium drying were low (4.9%). Median time to result was 15 days for smear-positive and 22 days for smear-negative samples, faster than WHO endorsed methods. Sensitivity was 100% for rifampicin and 87.8% for isoniazid, with a specificity of 100% for all drugs except isoniazid (96.2%). No BDQ nor levofloxacin resistance was detected, thus direct TLA sensitivity could not be assessed. In conclusion, direct-TLA-DST offers a reliable and faster alternative to conventional DST methods for BDQ and other anti-TB drugs. Essentially, this technique can be operated at BioSafety Level 2, allowing decentralization of pDST for managing drug-resistant TB in settings with limited laboratory infrastructure. ImportanceThis paper addresses the critical need for faster direct drug-susceptibility testing (DST) on sputum, especially for bedaquiline (BDQ), which is a key drug in treating drug-resistant TB. Currently, there is a lack of rapid, reliable methods for direct BDQ testing from sputum samples, limiting timely and accurate treatment decisions and monitoring. By demonstrating the potential of thin-layer agar (TLA) for direct BDQ-MIC determination, this study offers a promising solution that could significantly improve patient care.

microbiology↗

bioRxiv2024

Phylogenomic and genomic analysis reveals unique and shared genetic signatures of Mycobacterium kansasii complex species

Species belonging to the Mycobacterium kansasii complex (MKC) are frequently isolated from humans and the environment and can cause serious diseases. The most common MKC infections are caused by the species M. kansasii (stricto sensu), leading to tuberculosis-like disease. However, a broad spectrum of virulence, antimicrobial resistance and pathogenicity of these non-tuberculous mycobacteria (NTM) are observed across the MKC. Many genomic aspects of the MKC that relate to these broad phenotypes are not well elucidated. Here, we performed genomic analyses from a collection of 665 MKC strains, isolated from environmental, animal and human sources. We inferred the MKC pangenome, mobilome, resistome, virulome and defense systems and show that the MKC species harbors unique and shared genomic signatures. High frequency of presence of prophages and different types of defense systems was observed. We found that the M. kansasii species splits into four lineages, of which three are lowly represented and mainly in Brazil, while one lineage is dominant and globally spread. Moreover, we show that four sub-lineages of this most distributed M. kansasii lineage emerged during the 20th century. Further analysis of the M. kansasii genomes revealed almost 300 regions of difference contributing to genomic diversity, as well as fixed mutations that may explain the M. kansasiis increased virulence and drug resistance. RepositoriesBioProject PRJNA1048499. Impact statementMycobacterium kansasii complex (MKC) is a group of closely related non-tuberculous mycobacteria species, recognized as a significant source of human infection. Species belonging to the MKC may present a broad spectrum of virulence, antimicrobial resistance and pathogenicity and there is a lack of knowledge about their genomic content related to these broad phenotypes. We have provided whole genomic sequencing DNA for 342 MKC isolates and, together with public data, investigated the MKC pangenome, mobilome, resistome, virulome and defense systems and show unique and shared genetic signatures within MKC species. Furthermore, with phylogenomic and bayesian population analysis, we inferred the distribution and emergence of the Mycobacterium kansasii species lineages and sub-lineages. This study has considerably expanded the MKC available data, by providing genomic sequences of isolates from countries and global regions with unknown or poorly MKC data until now. Data summaryNGS data generated in the study are available in the NCBI Sequence Read Archive (SRA) repository under the accession number PRJNA1048499 and the accession numbers for all data sets used are provided in Supplementary Table 1.

evolutionary biology↗

bioRxiv2023

Borderline rpoB mutations transmit at the same rate as common rpoB mutations in a tuberculosis cohort in Bangladesh

The spread of multidrug-resistant tuberculosis (MDR-TB) is a growing problem in many countries worldwide. Resistance to one of the primary first-line drugs, rifampicin, is caused by mutations in the Mycobacterium tuberculosis rpoB gene. While some of these infrequent mutations show lower fitness in vitro than more common mutations, their in vivo fitness is currently unknown. We used a dataset of 394 whole genome sequenced MDR-TB isolates from Bangladesh, representing around 44% of notified MDR-TB cases over 6 years, to look at differences in transmission clustering between isolates with borderline rpoB mutations and those with common rpoB mutations. We found a relatively low percentage of transmission clustering in the dataset (34.8%) but no difference in clustering between different types of rpoB mutations. Compensatory mutations in rpoA, rpoB, and rpoC were associated with higher levels of transmission clustering as were lineages 2, 3, and 4 relative to lineage 1. Young people as well as patients with high sputum smear positive TB were more likely to be in a transmission cluster. Our findings show that although borderline rpoB mutations have lower in vitro growth potential this does not translate into lower transmission potential or in vivo fitness. Proper detection of these mutations is crucial to ensure they do not go unnoticed and spread MDR-TB within communities. Data summaryWGS reads are available in the European Nucleotide Archive (PRJEB39569). In addition, WGS reads, as well as pDST and clinical data, are included in the ReSeqTB data platform and are accessible on registration at https://platform.reseqtb.org/. Custom scripts for clustering are available at https://github.com/conmeehan/pathophy.

genomics↗

bioRxiv2022

The GEN-ERA toolbox: unified and reproducible workflows for research in microbial genomics .

BackgroundMicrobial culture collections play a key role in taxonomy by studying the diversity of their accessions and providing well characterized strains to the scientific community for fundamental and applied research. These microbial resource centers thus need to implement new standards in species delineation, including whole-genome sequencing and phylogenomics. In this context, the genomic needs of the Belgian Coordinated Collections of Microorganisms (BCCM) were studied, resulting in the GEN-ERA toolbox. The latter is a unified cluster of bioinformatic workflows dedicated to both bacteria and small eukaryotes (i.e. yeasts). FindingsThis public toolbox allows researchers without a specific training in bioinformatics to perform robust phylogenetic analyses. Hence, it facilitates all steps from genome downloading and quality assessment, including genomic contamination estimation, to tree reconstruction. It also offers workflows for average nucleotide identity comparisons and metabolic modeling. Technical detailsNextflow workflows are launched by a single command and are available on the GEN-ERA GitHub repository (https://github.com/Lcornet/GENERA). All the workflows are based on Singularity containers to increase reproducibility. TestingThe toolbox was developed for a diversity of microorganisms, including bacteria and fungi. It was further tested on an empirical dataset of 18 (meta)genomes of early-branching Cyanobacteria, providing the most up-to-date phylogenomic analysis of the Gloeobacterales order, the first group to diverge in the evolutionary tree of Cyanobacteria. ConclusionThe GEN-ERA toolbox can be used to infer completely reproducible comparative genomic and metabolic analyses on prokaryotes and small eukaryotes. Although designed for routine bioinformatics of culture collections, it can also be useful for other applications, as shown by our case study on Gloeobacterales.

bioinformatics↗

bioRxiv2021

Rare alternative second line injectable drug resistance markers identified by gene-wise genome wide association in M. tuberculosis with unexplained resistance

Point mutations in the rrs gene and eis promoter are known to confer resistance to second-line injectable drugs (SLIDs) amikacin (AMK), capreomycin (CAP), and kanamycin (KAN). While mutations in these canonical genes confer a majority of SLID-resistance, alternative mechanisms of resistance are not uncommon and threaten effective treatment decisions when using conventional molecular diagnostics. In total, 1184 clinical M. tuberculosis isolates from 7 countries were studied for genomic markers associated with phenotypic resistance. The markers rrs:A1401G and rrs:G1484T were associated with resistance to all three SLIDs, and three known markers in the eis promoter (eis:G-10A, eis:C-12T, and eis:C-14T) were similarly associated with kanamycin resistance (KAN-R). Among 325, 324, 270 AMK-R, CAP-R, and KAN-R isolates, 264 (81.2%), 250 (77.2%), and 249 (92.3%) harbored canonical mutations, respectively. Thirteen isolates harbored more than one canonical mutation. Canonical mutations did not account for 111 of the phenotypically resistant isolates. A gene-wise method identified three genes and promoters with mutations that, on aggregate, associated with unexplained resistance to at least one SLID. Our analysis associated whiB7 promoter mutations with KAN resistance, supporting clinical relevance for the previously demonstrated role of whiB7 overexpression in KAN resistance. We also provide evidence for the novel association of ppe51 (a gene previously associated with various antimicrobial compounds) with AMK resistance, and for the novel association of thrB with AMK and CAP resistance. The use of gene-wise association can provide additional insight, and therefore is recommended for identification of rare mechanisms of resistance when individual mutations carry insufficient statistical power.

genomics↗

bioRxiv2021

Reconstituting the genus Mycobacterium

The definition of a genus has wide ranging implications both in terms of binomial species names and also evolutionary relationships. In recent years, the definition of the genus Mycobacterium has been debated due to the proposed split of this genus into five new genera (Mycolicibacterium, Mycolicibacter, Mycolicibacillus, Mycobacteroides and an emended Mycobacterium). Since this group of species contains many important obligate and opportunistic pathogens, it is important that any renaming of species is does not cause confusion in clinical treatment as outlined by the nomen periculosum rule (56a) of the Prokaryotic Code. In this study, we evaluated the proposed and original genus boundaries for the mycobacteria, to determine if the split into five genera was warranted. By combining multiple approaches for defining genus boundaries (16S rRNA gene similarity, amino acid identity index, average nucleotide identity, alignment fraction and percentage of conserved proteins) we show that the original genus Mycobacterium is strongly supported over the proposed five-way split. Thus, we propose that the original genus label be reapplied to all species within this group, with the proposed five genera used as sub-genus complex names.

microbiology↗

bioRxiv2020

Mycobacterium tuberculosis complex lineage 5 exhibits high levels of within-lineage genomic diversity

Pathogens of the Mycobacterium tuberculosis complex (MTBC) are considered monomorphic, with little gene content variation between strains. Nevertheless, several genotypic and phenotypic factors separate the different MTBC lineages (L), especially L5 and L6 (traditionally termed Mycobacterium africanum), from each other. However, genome variability and gene content especially of L5 and L6 strains have not been fully explored and may be potentially important for pathobiology and current approaches for genomic analysis of MTBC isolates, including transmission studies. We compared the genomes of 358 L5 clinical isolates (including 3 completed genomes and 355 Illumina WGS (whole genome sequenced) isolates) to the L5 complete genomes and H37Rv, and identified multiple genes differentially present or absent between H37Rv and L5 strains. Additionally, considerable gene content variability was found across L5 strains, including a split in the L5.3 sublineage into L5.3.1 and L5.3.2. These gene content differences had a small knock on effect on transmission cluster estimation, with clustering rates influenced by the selection of reference genome, and with potential over-estimation of recent transmission when using H37Rv as the reference genome. Our data show that the use of H37Rv as reference genome results in missing SNPs in genes unique for L5 strains. This potentially leads to an underestimation of the diversity present in the genome of L5 strains and in turn affects the transmission clustering rates. As such, a full capture of the gene diversity, especially for high resolution outbreak analysis, requires a variation of the single H37Rv-centric reference genome mapping approach currently used in most WGS data analysis pipelines. Moreover, the high within-lineage gene content variability suggests that the pan-genome of M. tuberculosis is at least several kilobases larger than previously thought, implying a concatenated or reference-free genome assembly (de novo) approach may be needed for particular questions. Data summarySequence data for the Illumina dataset are available at European Genome-phenome Archive (EGA; https://www.ebi.ac.uk/ega/) under the study accession numbers PRJEB38317 and PRJEB38656. Individual runs accession numbers are indicated in Table S8. PacBio raw reads for the L5 Benin genome are available on the ENA accession SAME3170744. The assembled L5 Benin genome is available on NCBI with accession PRJNA641267. To ensure naming conventions of the genes in the three L5 genomes can be followed, we have uploaded these annotated GFF files to figshare at https://doi.org/10.6084/m9.figshare.12911849.v1. Custom python scripts used in this analysis can be found at https://github.com/conmeehan/pathophy.

genetics↗

bioRxiv2020

Characterization of genomic variants associated with resistance to bedaquiline and delamanid in naïve Mycobacterium tuberculosis clinical strains

The role of genetic mutations in genes associated to phenotypic resistance to bedaquiline (BDQ) and delamanid (DLM) in Mycobacterium tuberculosis complex (MTBc) strains is poorly understood. However, a clear understanding of the role of each genetic variant is crucial to guide the development of molecular-based drug susceptibility testing (DST). In this work, we analysed all mutations in candidate genomic regions associated with BDQ- and DLM-resistant phenotypes using a whole genome sequencing (WGS) dataset from a collection of 4795 MTBc clinical isolates from six high-burden countries of tuberculosis (TB). From WGS analysis, we identified 61 and 158 unique mutations in genomic regions potentially involved in BDQ- and DLM-resistant phenotypes, respectively. Importantly, all strains were isolated from patients who likely have never been exposed to the medicines. In order to characterize the role of mutations, we performed an energetic in silico analysis to evaluate their effect in the available protein structures Ddn (DLM), Fgd1 (DLM) and Rv0678 (BDQ), and minimum inhibitory concentration (MIC) assays on a subset of MTBc strains carrying mutations to assess their phenotypic effect. The combination of structural protein information and phenotypic data allowed for cataloging the mutations clearly associated with resistance to BDQ (n= 4) and DLM (n= 35), as well as about a hundred genetic variants without any correlation with resistance. Importantly, these results show that both BDQ and DLM resistance-related mutations are diverse and distributed across the entire region of each gene target, which is of critical importance to the development of comprehensive molecular diagnostic tools. ImportancePhenotypic drug susceptibility tests (DST) are too slow to provide an early indication of drug susceptibility status at the time of treatment initiation and very demanding in terms of specimens handling and biosafety. The development of molecular assays to detect resistance to bedaquiline (BDQ) and delamanid (DLM) requires accurate categorization of genetic variants according to their association with phenotypic resistance. We have evaluated a large multi-country set of clinical isolates to identify mutations associated with increased minimum inhibitory concentrations (MICs) and used an in silico protein structure analysis to further unravel the potential role of these mutations in drug resistance mechanisms. The results of this study are an important source of information for the development of molecular diagnostic tests to improve the provision of appropriate treatment and care to TB patients.

microbiology↗

bioRxiv2020

A sister lineage of the Mycobacterium tuberculosis complex discovered in the African Great Lakes region

The human- and animal-adapted lineages of the Mycobacterium tuberculosis complex (MTBC) are thought to have clonally expanded from a common progenitor in Africa. However, the molecular events that accompanied this emergence remain largely unknown. Here, we describe two MTBC strains isolated from patients with multidrug-resistant tuberculosis, representing an as-yet-unknown lineage, named Lineage 8 (L8), seemingly restricted to the African Great Lakes region. Using genome-based phylogenetic reconstruction, we show that L8 is a sister clade to the known MTBC lineages. Comparison with other complete mycobacterial genomes indicate that the divergence of L8 preceded the loss of the cobF genome region - involved in the cobalamin/vitamin B12 synthesis - and gene interruptions in a subsequent common ancestor shared by all other known MTBC lineages. This discovery further supports an East African origin for the MTBC and provides additional molecular clues on the ancestral genome reduction associated with adaptation to a pathogenic lifestyle.

microbiology↗

bioRxiv2019

Comparative Genomics Shows Differences in the Electron Transport and Carbon Metabolic Pathways of Mycobacterium africanum relative to Mycobacterium tuberculosis and suggests an adaptation to low oxygen tension

The geographically restricted Mycobacterium africanum lineages (MAF) are primarily found in West Africa, where they account for a significant proportion of tuberculosis. Despite this phenomenon, little is known about the co-evolution of these ancient lineages with West Africans. MAF and M. tuberculosis sensu stricto lineages (MTB) differ in their clinical, in vitro and in vivo characteristics for reasons not fully understood. Therefore, we compared genomes of 289 MAF and 205 MTB clinical isolates from the 6 main human-adapted M. tuberculosis complex lineages, for mutations in their Electron Transport Chain and Central Carbon Metabolic pathway in order to explain these metabolic differences. Furthermore, we determined, in silico, whether each mutation could affect the function of genes encoding enzymes in these pathways. We found more mutations with the potential to affect enzymes in these pathways in MAF lineages compared to MTB lineages. We also found that similar mutations occurred in these pathways between MAF and some MTB lineages. Generally, our findings show further differences between MAF and MTB lineages that may have contributed to the MAF clinical and growth phenotype and indicate potential adaptation of MAF lineages to a distinct ecological niche, which we suggest includes areas characterized by low oxygen tension.

microbiology↗

bioRxiv2019

An African origin for Mycobacterium bovis

Background and objectivesMycobacterium bovis and Mycobacterium caprae are two of the most important agents of tuberculosis (TB) in livestock and the most important causes of zoonotic TB in humans. However, little is known about the global population structure, phylogeography and evolutionary history of these pathogens. MethodologyWe compiled a global collection of 3364 whole-genome sequences from M. bovis and M. caprae originating from 35 countries and inferred their phylogenetic relationships, geographic origins and age. ResultsOur results resolved the phylogenetic relationship among the four previously defined clonal complexes of M. bovis, and another eight newly described here. Our phylogeographic analysis showed that M. bovis likely originated in East Africa. While some groups remained restricted to East- and West Africa, others have subsequently dispersed to different parts of the world. Conclusions and implicationsOur results allow a better understanding of the global population structure of M. bovis and its evolutionary history. This knowledge can be used to define better molecular markers for epidemiological investigations of M. bovis in settings where whole genome sequencing cannot easily be implemented.

evolutionary biology↗

bioRxiv2019

How well do routine molecular diagnostics detect rifampicin heteroresistance in Mycobacterium tuberculosis?

Rifampicin heteroresistance - where rifampicin-resistant and -susceptible tuberculosis (TB) bacilli co-exist - may result in failed standard TB treatment and potential spread of rifampicin-resistant strains. Detection of rifampicin heteroresistance in routine rapid diagnostic tests (RDTs) allows for patients to receive prompt and effective multidrug-resistant-TB treatment, and may improve rifampicin-resistant TB control.\n\nThe limit of detection (LOD) of rifampicin heteroresistance for phenotypic drug susceptibility testing by the proportion method is 1%, yet is insufficiently documented for RDTs. We therefore aimed to determine, for the four RDTs (XpertMTB/RIF, XpertMTB/RIF Ultra, GenoTypeMTBDRplusv2.0, and GenoscholarNTM+MDRTBII), the LOD per probe and mutation, validated by colony-forming-unit-counting and targeted deep sequencing (Deeplex-MycTB).\n\nWe selected one rifampicin-susceptible and four rifampicin-resistant strains, with mutation D435V, H445D, H445Y, and S450L respectively, mixed them in various proportions in triplicate, tested them with each RDT, and determined the LODs per mutation type. Deeplex-MycTB revealed concordant proportions of the minority resistant variants in the mixtures. The Deeplex-MycTB-validated-LODs ranged from 20-80% for XpertMTB/RIF, 20-70% for Xpert Ultra, 5-10% for GenoTypeMTBDRplusv2.0, and 1-10% for GenoscholarNTM+MTBII for the different mutations.\n\nDeeplex-MycTB, GenoTypeMTBDRplusv2.0, and GenoscholarNTM+MDRTBII, provide explicit information on rifampicin heteroresistance for the most frequently detected mutations. Classic Xpert and Ultra report rifampicin heteroresistance as rifampicin resistance, while Ultra may denote rifampicin heteroresistance through mixed patterns of wild-type and mutant melt probe melt peak temperatures.\n\nOverall, our findings inform end-users that the threshold for reporting resistance in case of rifampicin heteroresistance is the highest for Classic Xpert and Ultra, to resolve phenotypic and genotypic discordant rifampicin-resistant TB results.

microbiology↗

bioRxiv2018

Genome wide association with quantitative resistance phenotypes in Mycobacterium tuberculosis reveals novel resistance genes and regulatory regions

Drug resistance is threatening attempts at tuberculosis epidemic control. Molecular diagnostics for drug resistance that rely on the detection of resistance-related mutations could expedite patient care and accelerate progress in TB eradication. We performed minimum inhibitory concentration testing for 12 anti-TB drugs together with Illumina whole genome sequencing on 1452 clinical Mycobacterium tuberculosis (MTB) isolates. We then used a linear mixed model to evaluate genome wide associations between mutations in MTB genes or noncoding regions and drug resistance, followed by validation of our findings in an independent dataset of 792 patient isolates. Novel associations at 13 genomic loci were confirmed in the validation set, with 2 involving noncoding regions. We found promoter mutations to have smaller average effects on resistance levels than gene body mutations in genes where both can contribute to resistance. Enabled by a quantitative measure of resistance, we estimated the heritability of the resistance phenotype to 11 anti-TB drugs and identify a lower than expected contribution from known resistance genes. We also report the proportion of variation in resistance levels explained by the novel loci identified here. This study highlights the complexity of the genomic mechanisms associated with the MTB resistance phenotype, including the relatively large number of potentially causative or compensatory loci, and emphasizes the contribution of the noncoding portion of the genome.

evolutionary biology↗

bioRxiv2018

Reference Set of Mycobacterium tuberculosis Clinical Strains: A tool for research and product development

The Mycobacterium tuberculosis complex (MTBC) causes tuberculosis (TB) in humans and various other mammals. The human-adapted members of the MTBC comprise seven phylogenetic lineages that differ in their geographical distribution. There is growing evidence that this phylogenetic diversity modulates the outcome of TB infection and disease. For decades, TB research and development has focused on the two canonical MTBC reference strains H37Rv and Erdman, both of which belong to Lineage 4. Relying on only a few laboratory-adapted strains can be misleading as study results might not be directly transferrable to clinical settings where patients are infected with a diverse array of strains, including drug-resistant variants. Here, we argue for the need to expand TB research and development by incorporating the phylogenetic diversity of the MTBC. To facilitate such work, we have assembled a group of 20 genetically well-characterized clinical strains representing the seven known human-adapted MTBC lineages. With the \"MTBC clinical strains reference set\" we aim to provide a standardized resource for the TB community. We hope it will enable more direct comparisons between studies that explore the physiology of MTBC beyond the lab strains used thus far. We anticipate that detailed phenotypic analyses of this reference strain set will increase our understanding of TB biology and assist in the development of new control tools that are universally effective.

microbiology↗

bioRxiv2018

Xpert Ultra can unambiguously identify specific rifampicin resistance-conferring mutations

Introduction Introduction Methods Microorganisms Results Conclusions References The deluge of data produced by XpertMTB/RIF (Cepheid) can help improve global rifampicin-resistant tuberculosis (RR-TB) control strategies through molecular epidemiological surveillance (1, 2). Recently, a new version of the test - Xpert Ultra (hereinafter called Ultra) was released (3). Determining the relationship between RR-conferring rpoB mutations, Ultra probes, and melting temperature shifts ({Delta}Tm) - the difference between mutant and wildtype melting temperatures - allows Ultra results to be utilized for rapid detection of RR-TB strains and related underlying rpoB mutations. ...

microbiology↗