Machine learning and structural analysis of Mycobacterium tuberculosis pan-genome identifies genetic signatures of antibiotic resistance.

Mycobacterium tuberculosis is a serious human pathogen threat exhibiting complex evolution of antimicrobial resistance (AMR). Accordingly, the many publicly available datasets describing its AMR characteristics demand disparate data-type analyses. Here, we develop a reference strain-agnostic computational platform that uses machine learning approaches, complemented by both genetic interaction analysis and 3D structural mutation-mapping, to identify signatures of AMR evolution to 13 antibiotics. This platform is applied to 1595 sequenced strains to yield four key results. First, a pan-genome analysis shows that M. tuberculosis is highly conserved with sequenced variation concentrated in PE/PPE/PGRS genes. Second, the platform corroborates 33 genes known to confer resistance and identifies 24 new genetic signatures of AMR. Third, 97 epistatic interactions across 10 resistance classes are revealed. Fourth, detailed structural analysis of these genes yields mechanistic bases for their selection. The platform can be used to study other human pathogens

Drug-Resistant Tuberculosis--Current Dilemmas, Unanswered Questions, Challenges and Priority Needs

Tuberculosis was declared a global emergency by the World Health Organization (WHO) in 1993. Following the declaration and the promotion in 1995 of directly observed treatment short course (DOTS), a cost-effective strategy to contain the tuberculosis epidemic, nearly 7 million lives have been saved compared with the pre-DOTS era, high cure rates have been achieved in most countries worldwide, and the global incidence of tuberculosis has been in a slow decline since the early 2000s. However, the emergence and spread of multidrug-resistant (MDR) tuberculosis, extensively drug-resistant (XDR) tuberculosis, and more recently, totally drug-resistant tuberculosis pose a threat to global tuberculosis control. Multidrug-resistant tuberculosis is a man-made problem. Laboratory facilities for drug susceptibility testing are inadequate in most tuberculosis-endemic countries, especially in Africa; thus diagnosis is missed, routine surveillance is not implemented, and the actual numbers of global drug-resistant tuberculosis cases have yet to be estimated. This exposes an ominous situation and reveals an urgent need for commitment by national programs to health system improvement because the response to MDR tuberculosis requires strong health services in general. Multidrug-resistant tuberculosis and XDR tuberculosis greatly complicate patient management within resource-poor national tuberculosis programs, reducing treatment efficacy and increasing the cost of treatment to the extent that it could bankrupt healthcare financing in tuberculosis-endemic areas. Why, despite nearly 20 years of WHO-promoted activity and >12 years of MDR tuberculosis–specific activity, has the country response to the drug-resistant tuberculosis epidemic been so ineffectual? The current dilemmas, unanswered questions, operational issues, challenges, and priority needs for global drug resistance screening and surveillance, improved treatment regimens, and management of outcomes and prevention of DR tuberculosis are discussed

Treatment outcomes and medication management of tuberculosis

Tuberculosis (TB) is a bacterial infectious disease. The majority of TB patients in the Netherlands are successfully treated but in some patientsextension of treatment duration is necessary due to complications. Only in a small number of patients this treatment fails. This thesis focuses on the multifaceted medical approach to optimize the treatment of TB and improve treatment outcomes.In the first part of this thesis, we used long-term data from the national tuberculosis registry to determine the risk factors of prolonged and unsuccessful TB treatment in different patients. By identifying risk factors at an early stage, appropriate measures can be taken to reduce their impact and improve TB treatment outcomes. In the second part of this thesis, we evaluated the efficacy of a well-known antibiotic called levofloxacin. We investigated the relationship between the amount of antibiotic in the blood and the effectiveness of the treatment, and side effects by combining and analyzing data from previous studies. We also investigated in patients with a highly resistant form of TB whether they had enough of the antibiotic in their blood to kill the bacteria.Based on this research, we conclude that the amount of antibiotic in the blood differs per patient, and monitoring of this value combined with measuring the sensitivity of the bacteria to the antibiotic is necessary to adjust the dosage and thereby optimize treatment

DNA repair systems and the pathogenesis of Mycobacterium tuberculosis: varying activities at different stages of infection

Mycobacteria, including most of all MTB (Mycobacterium tuberculosis), cause pathogenic infections in humans and, during the infectious process, are exposed to a range of environmental insults, including the host's immune response. From the moment MTB is exhaled by infected individuals, through an active and latent phase in the body of the new host, until the time they reach the reactivation stage, MTB is exposed to many types of DNA-damaging agents. Like all cellular organisms, MTB has efficient DNA repair systems, and these are believed to play essential roles in mycobacterial pathogenesis. As different stages of infection have great variation in the conditions in which mycobacteria reside, it is possible that different repair systems are essential for progression to specific phases of infection. MTB possesses homologues of DNA repair systems that are found widely in other species of bacteria, such as nucleotide excision repair, base excision repair and repair by homologous recombination. MTB also possesses a system for non-homologous end-joining of DNA breaks, which appears to be widespread in prokaryotes, although its presence is sporadic within different species within a genus. However, MTB does not possess homologues of the typical mismatch repair system that is found in most bacteria. Recent studies have demonstrated that DNA repair genes are expressed differentially at each stage of infection. In the present review, we focus on different DNA repair systems from mycobacteria and identify questions that remain in our understanding of how these systems have an impact upon the infection processes of these important pathogens

Investigating the genomic basis of antimicrobial resistance in Mycobacterium tuberculosis (Mtb) using genome-wide methodologies

Characterizing the drug resistance mutations that have evolved in Mycobacterium tuberculosis (Mtb), has important implications for control of tuberculosis (TB) disease, through more accurate and timely use of therapy. Whole genome sequencing of Mtb can assist this characterization by providing insights into loci and specific mutations underlying drug resistance and the transmission success that enables their spread. We hypothesised that genetic variation outside of known resistance-conferring mutations might give additional information concerning drug resistance and fitness. Firstly, we explored the effect of lineage on the identification of drug resistance associations, applying novel lineage level genome-wide association study (GWAS) and convergence-based (PhyC) methods to drug resistance phenotypes of a global dataset of Mtb lineages 2 and 4. We identified known drug resistance variants and novel associations, uniquely identifying associations for lineage-specific GWAS analyses and reporting 17 novel associations between antimicrobial resistance phenotypes and Mtb genomic variants, demonstrating the utility of lineage-specific GWAS. To further examine the genomic basis of extensively drug resistant (XDR)-TB, we next applied the GWAS and PhyC techniques to a global dataset of 18,255 Mtb isolates. Through GWAS we identified 20 loci in novel associations within highly drug-resistant Mtb strains. Cluster-based GWAS and a lack of overlap with associations identified through convergent-evolution-based analyses confirmed that many such associations have been driven by transmission in outbreaks of XDR-TB. We then investigated the feasibility of applying a learning classifier system to this dataset to predict rifampicin resistance and discover candidate loci for novel involvement, finally enabling a sensitivity of 93.7% and a specificity of 94.8% of rifampicin resistance prediction. Finally, we applied this methodology to the XDR phenotype in lineages 2 and 4 of a global dataset (n=13,270), achieving high accuracy of prediction and identifying a number of candidate loci for involvement in XDR, including candidates for epistasis

Genomic analysis of globally diverse Mycobacterium tuberculosis strains provides insights into the emergence and spread of multidrug resistance

Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. Here we examined a data set of whole-genome sequences from 5,310 M. tuberculosis isolates from five continents. Despite the great diversity of these isolates with respect to geographical point of isolation, genetic background and drug resistance, the patterns for the emergence of drug resistance were conserved globally. We have identified harbinger mutations that often precede multidrug resistance. In particular, the katG mutation encoding p.Ser315Thr, which confers resistance to isoniazid, overwhelmingly arose before mutations that conferred rifampicin resistance across all of the lineages, geographical regions and time periods. Therefore, molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of polymorphisms that occur before the emergence of multidrug resistance, particularly katG p.Ser315Thr, into molecular diagnostics should enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.ope

Twenty years of global surveillance of antituberculosis-drug resistance

Antimicrobial resistance represents a major threat to global health and security. In 2014, the World Health Assembly called on all nations and the international community to take every necessary measure to control it, including surveillance of its emergence and spread.1 The development of drug resistance in Mycobacterium tuberculosis was first documented in the late 1940s, soon after antibiotic therapy was introduced for tuberculosis treatment.2 It quickly became obvious that combination chemotherapy could prevent the emergence of drug resistance3 and that patients infected with drug-resistant strains were less likely to be cured.4 Nevertheless, it was only in the early 1990s that drugresistant tuberculosis began to receive global attention as a public health threat. This coincided with the detection of outbreaks of multidrugresistant (MDR) tuberculosis (defined as resistance to at least rifampin and isoniazid) that were associated with high mortality among patients coinfected with the human immunodeficiency virus (HIV).5-8 The urgent need for a global mechanism to monitor the emergence and spread of resistance to antituberculosis drugs became clear. In 1994, the Global Tuberculosis Program of the World Health Organization (WHO), with the support of the International Union against Tuberculosis and Lung Disease (the Union), established the Global Project on Anti-Tuberculosis Drug Resistance Surveillance (hereafter referred to as \u201cthe project\u201d) to measure the magnitude of drug resistance and to monitor trends. This project remains the oldest and largest initiative on the surveillance of antimicrobial resistance in the world.9 In this article, we describe the history of global surveillance of drug resistance in tuberculosis and discuss methods for surveillance, the quality of available data, the key achievements and findings to date, the main challenges that remain, and future directions

Pseudomonas aeruginosa antibiotic susceptibility profiles, genomic epidemiology and resistance mechanisms: a nation-wide five-year time lapse analysis

Background: Pseudomonas aeruginosa healthcare-associated infections are one of the top antimicrobial resistance threats world-wide. In order to analyze the current trends, we performed a Spanish nation-wide high-resolution analysis of the susceptibility profiles, the genomic epidemiology and the resistome of P. aeruginosa over a five-year time lapse. Methods: A total of 3.180 nonduplicated P. aeruginosa clinical isolates from two Spanish nation-wide surveys performed in October 2017 and 2022 were analyzed. MICs of 13 antipseudomonals were determined by ISO-EUCAST. Multidrug resistance (MDR)/extensively drug resistance (XDR)/difficult to treat resistance (DTR)/pandrug resistance (PDR) profiles were defined following established criteria. All XDR/DTR isolates were subjected to whole genome sequencing (WGS). Findings: A decrease in resistance to all tested antibiotics, including older and newer antimicrobials, was observed in 2022 vs 2017. Likewise, a major reduction of XDR (15.2% vs 5.9%) and DTR (4.2 vs 2.1%) profiles was evidenced, and even more patent among ICU isolates [XDR (26.0% vs 6.0%) and DTR (8.9% vs 2.6%)] (p < 0.001). The prevalence of Extended-spectrum β-lactamase/carbapenemase production was slightly lower in 2022 (2.1%. vs 3.1%, p = 0.064). However, there was a significant increase in the proportion of carbapenemase production among carbapenem-resistant strains (29.4% vs 18.1%, p = 0.0246). While ST175 was still the most frequent clone among XDR, a slight reduction in its prevalence was noted (35.9% vs 45.5%, p = 0.106) as opposed to ST235 which increased significantly (24.3% vs 12.3%, p = 0.0062). Interpretation: While the generalized decrease in P. aeruginosa resistance, linked to a major reduction in the prevalence of XDR strains, is encouraging, the negative counterpart is the increase in the proportion of XDR strains producing carbapenemases, associated to the significant advance of the concerning world-wide disseminated hypervirulent high-risk clone ST235. Continued high-resolution surveillance, integrating phenotypic and genomic data, is necessary for understanding resistance trends and analyzing the impact of national plans on antimicrobial resistance.This work was supported by MSD and by the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea —NextGenerationEU through grants PI21/00017 and Personalized and precision medicine grant (MePRAM Project, PMP22/00092).S