The epistatic landscape of antibiotic resistance of different clades of Mycobacterium tuberculosis

Drug resistance (DR) remains a global challenge in tuberculosis (TB) control. In order to develop molecular-based diagnostic methods to replace the traditional culture-based diagnostics, there is a need for a thorough understanding of the processes that govern TB drug resistance. The use of whole-genome sequencing coupled with statistical and computational methods has shown great potential in unraveling the complexity of the evolution of DR-TB. In this study, we took an innovative approach that sought to determine nonrandom associations between polymorphic sites in Mycobacterium tuberculosis (Mtb) genomes. Attributable risk statistics were applied to identify the epistatic determinants of DR in different clades of Mtb and the possible evolutionary pathways of DR development. It was found that different lineages of Mtb exploited different evolutionary trajectories towards multidrug resistance and compensatory evolution to reduce the DR-associated fitness cost. Epistasis of DR acquisition is a new area of research that will aid in the better understanding of evolutionary biological processes and allow predicting upcoming multidrug-resistant pathogens before a new outbreak strikes humanity.The South African National Research Foundation (NRF)https://www.mdpi.com/journal/antibioticsam2022BiochemistryGeneticsMicrobiology and Plant Patholog

Resistance sniffer : an online tool for prediction of drug resistance patterns of Mycobacterium tuberculosis isolates using next generation sequencing data

The effective control of multidrug resistant tuberculosis (MDR-TB) relies upon the timely diagnosis and correct treatment of all tuberculosis cases. Whole genome sequencing (WGS) has great potential as a method for the rapid diagnosis of drug resistant Mycobacterium tuberculosis (Mtb) isolates. This method overcomes most of the problems that are associated with current phenotypic drug susceptibility testing. However, the application of WGS in the clinical setting has been deterred by data complexities and skill requirements for implementing the technologies as well as clinical interpretation of the next generation sequencing (NGS) data. The proposed diagnostic application was drawn upon recent discoveries of patterns of Mtb clade-specific genetic polymorphisms associated with antibiotic resistance. A catalogue of genetic determinants of resistance to thirteen anti-TB drugs for each phylogenetic clade was created. A computational algorithm for the identification of states of diagnostic polymorphisms was implemented as an online software tool, Resistance Sniffer (http://resistance-sniffer.bi.up. ac.za/), and as a stand-alone software tool to predict drug resistance in Mtb isolates using complete or partial genome datasets in different file formats including raw Illumina fastq read files. The program was validated on sequenced Mtb isolates with data on antibiotic resistance trials available from GMTV database and from the TB Platform of South African Medical Research Council (SAMRC), Pretoria. The program proved to be suitable for probabilistic prediction of drug resistance profiles of individual strains and large sequence data sets.The South African National Research Foundation (NRF)https://www.elsevier.com/locate/ijmmam2020BiochemistryGeneticsMicrobiology and Plant Patholog

The Epistatic Landscape of Antibiotic Resistance of Different Clades of Mycobacterium tuberculosis

Drug resistance (DR) remains a global challenge in tuberculosis (TB) control. In order to develop molecular-based diagnostic methods to replace the traditional culture-based diagnostics, there is a need for a thorough understanding of the processes that govern TB drug resistance. The use of whole-genome sequencing coupled with statistical and computational methods has shown great potential in unraveling the complexity of the evolution of DR-TB. In this study, we took an innovative approach that sought to determine nonrandom associations between polymorphic sites in Mycobacterium tuberculosis (Mtb) genomes. Attributable risk statistics were applied to identify the epistatic determinants of DR in different clades of Mtb and the possible evolutionary pathways of DR development. It was found that different lineages of Mtb exploited different evolutionary trajectories towards multidrug resistance and compensatory evolution to reduce the DR-associated fitness cost. Epistasis of DR acquisition is a new area of research that will aid in the better understanding of evolutionary biological processes and allow predicting upcoming multidrug-resistant pathogens before a new outbreak strikes humanity

Exploring the evolution of drug resistance in mycobacterium using whole genome sequencing data

Mycobacterium tuberculosis (Mtb) remains a global challenge that has been worsened by the emergence of drug resistant strains of Mtb. We used publicly available Next Generation Sequencing (NGS) and drug susceptibility (DST) data to develop “Resistance sniffer”, an online software program for the rapid prediction of lineage and Mtb drug resistance. Based on the distribution of polymorphisms in the genomes of Mtb, we calculated the power of association between the polymorphisms in different clades of Mtb and resistance to 13 anti-TB drugs. Our data suggests that the development of drug resistance in Mtb is a stepwise process that involves the accumulation of polymorphisms in the Mtb genome. We carefully curated the polymorphisms based on their association powers to create a diagnostic key that captures patterns of these polymorphisms that can be used to predict lineage and drug resistance in Mtb. This diagnosis key was incorporated into the Resistance Sniffer tool, an online software program that we developed for the rapid diagnosis of drug resistance in Mtb. The tool was tested using sequence data from the South Africa Medical Research Council (SA-MRC). Our data suggests that the majority of the strains in SA may have been brought by the arrival of European settlers while the more resistant strains may have been introduced in the region by Asian travellers later on. We next sought to determine non-random associations between polymorphic sites in genomes of Mtb. Using the attributable risk (Ra) statistical methods, we distinguished between functional associations and associations that may have been due to genetic drift events for different Mtb clades. We then integrated the (Ra) data with drug susceptibility and annotation data to generate networks in Cytoscape 3.71. These networks were then used to infer evolutionary trajectories that drive the emergence and fixation of the drug resistant phenotype in different clades of Mtb. We demonstrate that strains from the Lineage 1.2 are associated with less complex functional associations compared to the strains from other clades such as the Asian and Euro-American clades. Our data also shows that the predisposition of strains from the Asian clades to develop multi-drug resistance may be attributed to a complex network of functional interactions of mutations in genes that are involved in several aspects of Mtb physiology such as cell wall modelling, lipid metabolism, stress response and DNA repair.Dissertation (MSc)--University of Pretoria, 2019.BiochemistryMScUnrestricte

Genetic, Epigenetic and Phenotypic Diversity of Four Bacillus velezensis Strains Used for Plant Protection or as Probiotics

Bacillus velezensis strains are applied as ecologically safe biopesticides, plant growth promoting rhizobacteria (PGPR), and in veterinary probiotics. They are abundant in various environments including soil, plants, marine habitats, the intestinal micro-flora, etc. The mechanisms underlying this adaptive plasticity and bioactivity are not well understood, nor is it clear why several strains outperform other same species isolates by their bioactivities. The main objective of this work was to demonstrate versatility of bioactivities and lifestyle strategies of the selected B. velezensis strains suitable to serve as model organisms in future studies. Here, we performed a comparative study of newly sequenced genomes of four B. velezensis isolates with distinct phenotypes and isolation origin, which were assessed by RNA sequencing under the effect of root exudate stimuli and profiled by epigenetic modifications of chromosomal DNA. Among the selected strains, UCMB5044 is an oligotrophic PGPR strain adapted to nutrient poor desert soils. UCMB5113 and At1 are endophytes that colonize plants and require nutrient rich media. In contrast, the probiotic strain, UCMB5007, is a copiotroph, which shows no propensity to colonize plants. PacBio and Illumina sequencing approaches were used to generate complete genome assemblies, tracing epigenetic modifications, and determine gene expression profiles. All sequence data was deposited at NCBI. The strains, UCMB5113 and At1, show 99% sequence identity and similar phenotypes despite being isolated from geographically distant regions. UCMB5007 and UCMB5044 represent another group of organisms with almost identical genomes but dissimilar phenotypes and plant colonization propensity. The two plant associated strains, UCMB5044 and UCMB5113, share 398 genes putatively associated with root colonization, which are activated by exposure to maize root exudates. In contrast, UCMB5007 did not respond to root exudate stimuli. It was hypothesized that alterations in the global methylation pattern and some other epigenetic modifications enable adaptation of strains to different habitats and therefore may be of importance in terms of the biotechnological applicability of these bacteria. Contrary, the ability to grow on root exudates as a sole source of nutrients or a strong antagonism against phytopathogens showed by the strains in vitro cannot be considered as good predictors of PGPR activities.Peer Reviewe