Mycobacterium Tuberculosis Complex Bacteria Research Articles

Mycobacterium tuberculosis complex in domestic goats in Southern Spain

Tuberculosis (TB) is a zoonotic infectious disease caused by bacteria belonging to the Mycobacterium tuberculosis complex (MTC), which can affect a wide variety of domestic and wild animal species. Although the role of goats as a reservoir of MTC bacteria has been evidenced, information about the circulation of MTC strains in this species is still very scarce. The aim of the present study was to determine the seroprevalence, spatial distribution, risk factors and MTC spoligotypes circulating in goats from Andalusia (Southern Spain), the Spanish region with the largest goat census and a hotspot area of TB in both cattle and wild ungulates. A total of 2155 serum samples from 80 goat flocks were analyzed by an in-house ELISA using the P22 protein complex as a coating antigen. Antibodies against MTC were detected in 473 goats (21.9%, 95% CI: 20.2–23.7) and the true seroprevalence was 22.3% (95% CI: 20.6–24.1). Seropositivity was found in 72 (90.0%) of the 80 flocks analyzed. The generalized estimating equation model showed that the management system (higher seroprevalence on intensive and semi-intensive farms), and the presence of hospital pens inside the regular stables, were risk factors potentially associated with MTC exposure in goats in Southern Spain. The spatial analysis identified a significant spatial cluster (p < 0.001) in Eastern Andalusia. A total of 16 different MTC spoligotypes, including five of M. caprae and eleven of M. bovis, were identified in goats between 2015 and 2022 in the study area, with SB0157 as the most frequently isolated. The results obtained indicate widespread and non-homogeneous spatial distribution of MTC in goat herds from Southern Spain. The high individual and herd-level seroprevalence values found suggest that goats could play a significant role in the maintenance and transmission of MTC in the study area. Our results highlight the importance of implementing control measures in this species.

Comparative pathology of experimental pulmonary tuberculosis in animal models.

Research in human tuberculosis (TB) is limited by the availability of human tissues from patients, which is often altered by therapy and treatment. Thus, the use of animal models is a key tool in increasing our understanding of the pathogenesis, disease progression and preclinical evaluation of new therapies and vaccines. The granuloma is the hallmark lesion of pulmonary tuberculosis, regardless of the species or animal model used. Although animal models may not fully replicate all the histopathological characteristics observed in natural, human TB disease, each one brings its own attributes which enable researchers to answer specific questions regarding TB immunopathogenesis. This review delves into the pulmonary pathology induced by Mycobacterium tuberculosis complex (MTBC) bacteria in different animal models (non-human primates, rodents, guinea pigs, rabbits, cattle, goats, and others) and compares how they relate to the pulmonary disease described in humans. Although the described models have demonstrated some histopathological features in common with human pulmonary TB, these data should be considered carefully in the context of this disease. Further research is necessary to establish the most appropriate model for the study of TB, and to carry out a standard characterisation and score of pulmonary lesions.

Comparative Functional Genomics Studies for Understanding the Hypothetical Proteins in Mycobacterium Tuberculosis Variant Microti 12

Comparative Functional Genomics Studies for Understanding the Hypothetical Proteins in Mycobacterium Tuberculosis Variant Microti 12

To Evaluate the Clinical and Laboratory Profiles of Patients with Abdominal Tuberculosis in Western Odisha

Background: Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tuberculosis) complex bacteria, is one of the oldest diseases known to affect humans and the leading cause of mortality worldwide. Milk contaminated with Mycobacterium bovis (reservoir cattle) can transmit tuberculosis to the gastrointestinal tract. TB can affect any portion of the digestive tract, from the buccal cavity to the anus, pancreato-biliary tree, and peritoneum. The peritoneum and ileocecal regions were most frequently affected. Aim: This study aimed to evaluate the clinical and laboratory profiles of patients with abdominal tuberculosis. Materials &amp; Methods: The current study was conducted at the VSS Institute of Medical Sciences and Research (VIMSAR), Burla, located at 21.5027 o N and 83.8865 o E in western Odisha, India, after receiving approval from the Institutional Ethics Committee. Outdoor (OPD) and indoor (IPD) patients of the Division of General Medicine, VIMSAR, Burla The duration of the research was from November 2019 to October 2021 (24 months). This was a cross-sectional study. All patients were admitted to the Department of General Medicine with suspected abdominal tuberculosis. Results: The distribution of socioeconomic status in the study population according to the modified Kuppuswamy scale was predominated by the upper lower class IV population (60.3%), followed by the lower middle group (26%); only 13.7% belonged to class II of the upper middle group. The residential patterns showed that 74% of the cases belonged to the rural population, whereas only 26% lived in urban areas. Anemia was the major finding in comments on peripheral smear examination in 74.2% of the population and pancytopenia in 10.6% of cases. A minimum number of patients (4.5%) had a normal peripheral smear. Erythrocyte sedimentation rate by the Westergren method was increased in 87.7% of cases, and the rest of the population was within the normal range. Conclusion: This study has once again shown how common tuberculosis is in our country, particularly in underdeveloped Western Odisha, due to lack of hygiene and poverty. Abdominal tuberculosis has undergone modification over time because of the rampant and improper use of antibiotics and other drugs, thus creating a clinical dilemma. The irregularity of daily drug intake is another factor leading to the emergence of drug-resistant organisms.

The Use of Xpert MTB/RIF Ultra Testing for Early Diagnosis of Tuberculosis: A Retrospective Study from a Single-Center Database.

Tuberculosis (TB) is a multisystemic contagious disease produced by Mycobacterium tuberculosis complex bacteria (MTBC), with a prevalence of 65:100,000 inhabitants in Romania (six times higher than the European average). The diagnosis usually relies on the detection of MTBC in culture. Although this is a sensitive method of detection and remains the "gold standard", the results are obtained after several weeks. Nucleic acid amplification tests (NAATs), being a quick and sensitive method, represent progress in the diagnosis of TB. The aim of this study is to assess the assumption that NAAT using Xpert MTB/RIF is an efficient method of TB diagnosis and has the capacity to reduce false-positive results. Pathological samples from 862 patients with TB suspicion were tested using microscopic examination, molecular testing and bacterial culture. The results show that the Xpert MTB/RIF Ultra test has a sensitivity of 95% and a specificity of 96.4% compared with 54.8% sensitivity and 99.5% specificity for Ziehl-Neelsen stain microscopy, and an average of 30 days gained in the diagnosis of TB compared with bacterial culture. The implementation of molecular testing in TB laboratories leads to an important increase in early diagnostics of the disease and the prompter isolation and treatment of infected patients.

Transmission Dynamics of a Mycobacterium tuberculosis Complex Outbreak in an Indigenous Population in the Colombian Amazon Region.

Whole genome sequencing (WGS) has become the main tool for studying the transmission of Mycobacterium tuberculosis complex (MTBC) strains; however, the clonal expansion of one strain often limits its application in local MTBC outbreaks. The use of an alternative reference genome and the inclusion of repetitive regions in the analysis could potentially increase the resolution, but the added value has not yet been defined. Here, we leveraged short and long WGS read data of a previously reported MTBC outbreak in the Colombian Amazon Region to analyze possible transmission chains among 74 patients in the indigenous setting of Puerto Nariño (March to October 2016). In total, 90.5% (67/74) of the patients were infected with one distinct MTBC strain belonging to lineage 4.3.3. Employing a reference genome from an outbreak strain and highly confident single nucleotide polymorphisms (SNPs) in repetitive genomic regions, e.g., the proline-glutamic acid/proline-proline-glutamic-acid (PE/PPE) gene family, increased the phylogenetic resolution compared to a classical H37Rv reference mapping approach. Specifically, the number of differentiating SNPs increased from 890 to 1,094, which resulted in a more granular transmission network as judged by an increasing number of individual nodes in a maximum parsimony tree, i.e., 5 versus 9 nodes. We also found in 29.9% (20/67) of the outbreak isolates, heterogenous alleles at phylogenetically informative sites, suggesting that these patients are infected with more than one clone. In conclusion, customized SNP calling thresholds and employment of a local reference genome for a mapping approach can improve the phylogenetic resolution in highly clonal MTBC populations and help elucidate within-host MTBC diversity. IMPORTANCE The Colombian Amazon around Puerto Nariño has a high tuberculosis burden with a prevalence of 1,267/100,000 people in 2016. Recently, an outbreak of Mycobacterium tuberculosis complex (MTBC) bacteria among the indigenous populations was identified with classical MTBC genotyping methods. Here, we employed a whole-genome sequencing-based outbreak investigation in order to improve the phylogenetic resolution and gain new insights into the transmission dynamics in this remote Colombian Amazon Region. The inclusion of well-supported single nucleotide polymorphisms in repetitive regions and a de novo-assembled local reference genome provided a more granular picture of the circulating outbreak strain and revealed new transmission chains. Multiple patients from different settlements were possibly infected with at least two different clones in this high-incidence setting. Thus, our results have the potential to improve molecular surveillance studies in other high-burden settings, especially regions with few clonal multidrug-resistant (MDR) MTBC lineages/clades.

Tuberculosis Epidemiology and Spatial Ecology at the Cattle-Wild Boar Interface in Northern Spain

Tuberculosis (TB) is a contagious chronic disease due to infection with Mycobacterium tuberculosis complex (MTC) bacteria. Monitoring of wildlife, especially potential reservoirs, is important for detecting changes in disease occurrence and assessing the impact of interventions. Here, we examined whether wild boar (Sus scrofa) may contribute to the re-emergence of TB in Asturias (10,604 km2), northern Spain. Although this province was declared free of TB in cattle in November 2021, MTC bacteria remain prevalent in several “hotspots,” with the European badger (Meles meles) suggested as a TB potential wild reservoir. Drawing on data from the Spanish National Bovine Tuberculosis Eradication Program and the Government of the Principality of Asturias covering the period 2014–2020, we analyzed the prevalence of TB in cattle and wild boar in this region. In hotspots (592 km2), we also investigated the ranging behavior and habitat use of five cows that belonged to farms with a history of TB and six trapped sympatric wild boar. During the observation period, TB prevalence was 0.14% among cattle overall and 0.13–0.41% in hotspots, which was much lower than the prevalence in wild boar, which was 3.15% overall and 5.23–5.96% in hotspots. Infected cattle and infected wild boar in hotspots shared the same strains of M. bovis, and GPS tracking showed spatiotemporal overlap between the species, mainly around pastures during sunrise (06:00–07:00 h) and sunset (19:00–20:00 h). Our results suggest that in addition to cattle and badgers, wild boar possibly help maintain TB in northern Spain, increasing the host richness that influences TB transmission risk in the area, which should be taken into account in monitoring and eradication efforts.

Spoligotyping of the Mycobacterium tuberculosis complex using on-Chip PCR.

The aim of this study was to develop a rapid PCR-based method for spoligotyping of mycobacteria in the microarray format and to compare it to conventional spoligotyping by hybridization. The method employs the On-Chip PCR technique with primers specific for 43 spacers that separate direct repeats (DRs) in the DR region of mycobacterial DNA. The primers were immobilized on gel-based microarrays, and PCR was performed directly on the chips. The PCR fluorescence images were acquired and processed using a portable fluorescence analyzer equipped with dedicated software. Analysis takes 1.5-2 hours and can be carried out on clinical samples without additional handling. The analytical sensitivity of the method was 103 copies of target DNA. The spoligotyping results of 51 samples produced by the proposed method and by conventional reverse hybridization approach were in full concordance. High throughput capacity, computerized data analysis, compact equipment, and reliable results make the On-Chip PCR an attractive alternative to intra- and interspecific spoligotyping of Mycobacterium tuberculosis complex bacteria.

Optimizing DNA Extraction from Pediatric Stool for Diagnosis of Tuberculosis and Use in Next-Generation Sequencing Applications.

The WHO has endorsed the use of stool samples for diagnosis of tuberculosis (TB) in children, and targeted next-generation sequencing (tNGS) of stool has been shown to support diagnosis and provide information about drug susceptibility (DS). Optimizing extraction of DNA from stool for sequencing is critical to ensure high diagnostic sensitivity and accurate DS information. Human stool samples were spiked with various concentrations of Mycobacterium bovis bacillus Calmette-Guérin (BCG), and DNA was extracted from the samples using four different DNA extraction kits. Each sample was subjected to quantitative PCR for identifying Mycobacterium tuberculosis complex bacteria and underwent further analysis to assess the overall DNA yield, fragment length, and purity. This same process was performed with 10 pediatric participants diagnosed with pulmonary TB, and the samples underwent tNGS. The FastDNA spin kit for soil showed the best results on model samples spiked with known quantities of BCG, compared to the other extraction methods evaluated. For clinical samples, the FastDNA and PowerFecal Pro DNA (PowerFecal) kits both showed an increase in the overall DNA quantity, M. tuberculosis-specific DNA quantity, and successful targeted sequencing when testing was performed on stool samples, compared to the two other kits. Three samples extracted via PowerFecal and three samples extracted via FastDNA (from different patients) provided successful sequencing data, with an average depth of coverage of the rpoB region for FastDNA of 298 (range, 107 to 550) and for PowerFecal of 310 (range, 182 to 474), results that were comparable to one another (P = 0.946). The PowerFecal Pro and FastDNA spin kits were superior for extracting DNA from pediatric stool samples for tNGS. IMPORTANCE This is the first study to compare Mycobacterium tuberculosis DNA extraction techniques from pediatric stool samples for use with sequencing technologies. It provides an important starting point for other researchers to isolate quality DNA for this purpose to further the field and to continue to optimize protocols and approaches.

Era, a GTPase-like protein of the Ras family, does not control ribosome assembly in Mycobacterium tuberculosis.

Era GTPase is universally present in microbes including Mycobacterium tuberculosis (Mtb) complex bacteria. While Era is known to regulate ribosomal assembly in Escherichia coli and predicted to be essential for in vitro growth, its function in mycobacteria remains obscured. Herein, we show that Era ortholog in the attenuated Mtb H37Ra strain, MRA_2388 (annotated as EraMT) is a cell envelope localized protein harbouring critical GTP-binding domains, which interacts with several envelope proteins of Mtb. The purified Era from M. smegmatis (annotated as EraMS) exhibiting ~90 % sequence similarity with EraMT, exists in monomeric conformation. While it is co-purified with RNA upon overexpression in E. coli, the presence of RNA does not modulate the GTPase activity of the EraMS as against its counterpart from other organisms. CRISPRi silencing of eraMT does not show any substantial effect on the in vitro growth of Mtb H37Ra, which suggests a redundant function of Era in mycobacteria. Notably, no effect on ribosome assembly, protein synthesis or bacterial susceptibility to protein synthesis inhibitors was observed upon depletion of EraMT in Mtb H37Ra, further indicating a divergent role of Era GTPase in mycobacteria.

Prevalence of active tuberculosis and associated factors among people with chronic psychotic disorders at St. Amanuel Mental Specialized Hospital and Gergesenon Mental Rehabilitation center, Addis Ababa, Ethiopia

BackgroundTuberculosis (TB) is an airborne chronic infectious disease mainly caused by Mycobacterium tuberculosis complex bacteria. Currently, about 1.7 billion (26%) of the world’s population are considered to be infected with M. tuberculosis. The risk of acquiring tuberculosis is higher on some segments of societies including people with severe mental illness. As a result, World health organization (WHO) strongly recommends screening for tuberculosis in such risk groups and setting.MethodsA cross-sectional study was conducted to assess the prevalence of active tuberculosis and associated factors among patients with chronic psychotic disorders admitted at St. Amanuel Mental Specialized Hospital and Gergesenon Mental rehabilitation center from February to June, 2020. All admitted patients were screened for any sign of TB as recommended by WHO. Presumptive TB cases were identified. Sputum samples were collected and tested by Xpert MTB/RIF assay. Data analysis was performed using SPSS version 25.0 statistical software and Chi square analysis was used to test the statistical association.ResultsFrom a total 3600 pschotic patients screened for TB, 250 (6.94%) presumptive tuberculosis cases were detected. From these, 27 (10.8%) were positive by Xpert MTB/RIF assay. Most of the patients were males (68.4%). The mean ± SD age of the participant was 36.5 ± 9.7 years. The overall prevalence of tuberculosis was found to be 750 per 100,000 population. The number of patients per room (p = 0.039) was associated with Xpert MTB/RIF positive active tuberculosis.ConclusionThe prevalence of active tuberculosis among chronic psychotic patients was high. Number of admitted patients per room was identified as risk factors for Xpert MTB/RIF positive active tuberculosis. Therefore, to control TB transmission in chronic mental health treatment facilities, efforts should be directed to periodic screening for early case detection and improving the number of patients per room.

Transcriptomic Profile of Mycobacterium smegmatis in Response to an Imidazo[1,2-b][1,2,4,5]tetrazine Reveals Its Possible Impact on Iron Metabolism

Tuberculosis (TB), caused by the Mycobacterium tuberculosis complex bacteria, is one of the most pressing health problems. The development of new drugs and new therapeutic regimens effective against the pathogen is one of the greatest challenges in the way of tuberculosis control. Imidazo[1,2-b][1,2,4,5]tetrazines have shown promising activity against M. tuberculosis and M. smegmatis strains. Mutations in MSMEG_1380 lead to mmpS5–mmpL5 operon overexpression, which provides M. smegmatis with efflux-mediated resistance to imidazo[1,2-b][1,2,4,5]tetrazines, but the exact mechanism of action of these compounds remains unknown. To assess the mode of action of imidazo[1,2-b][1,2,4,5]tetrazines, we analyzed the transcriptomic response of M. smegmatis to three different concentrations of 3a compound: 1/8×, 1/4×, and 1/2× MIC. Six groups of genes responsible for siderophore synthesis and transport were upregulated in a dose-dependent manner, while virtual docking revealed proteins involved in siderophore synthesis as possible targets for 3a. Thus, we suggest that imidazo[1,2-b][1,2,4,5]tetrazines may affect mycobacterial iron metabolism.

Global trends of epidemiological research in livestock tuberculosis for the last four decades.

Animal tuberculosis (TB) caused by Mycobacterium tuberculosis complex (MTC) bacteria remains as one of the most significant infectious diseases of livestock, despite decades of eradication programmes and research efforts, in an era where the livestock sector is among the most important and rapidly expanding commercial agricultural segments worldwide. This work provides a global overview of the spatial and temporal trends of reported scientific knowledge of TB in livestock, aiming to gain insights into research subtopics within the animal TB epidemiology domain and to highlight territorial inequalities regarding data reporting and research outputs over the years. To deliver such information, peer-reviewed reports of TB studies in livestock were retrieved from the Web of Science and Google Scholar, systematized and dissected. The validated data set contained 443 occurrence observations, covering the 1981-2020 period (39years). We highlight a clear move towards transdisciplinary areas and the One Health approach, with a global temporal increase in publications combining livestock with wildlife and/or human components, which reflect the importance of non-prototypical hosts as key to understanding animal TB. It becomes evident that cattle is the main host across works from all continents; however, many regions remain poorly surveyed. TB research in livestock in low-/middle-income countries is markedly growing, reflecting changes in animal husbandry, but also mirroring the globalization era, with a marked increase in international collaboration and capacitation programmes for scientific and technological development. This review gives an overview of the most prolific continents, countries and research fields in animal TB epidemiology, clearly outlining knowledge gaps and key priority topics. The estimated growth trend of livestock production until 2050, particularly in Asia and Africa, in response to human population growth and animal-protein demand, will require further investment in early surveillance and adaptive research to accommodate the higher diversity of livestock species and MTC members and raising the possibility to fine-tune funding schemes.

Utility of xpert® MTB/RIF ultra assay in the rapid diagnosis of bovine tuberculosis in wildlife and livestock animals from South Africa

Members of the Mycobacterium tuberculosis complex (MTBC) bacteria, mainly Mycobacterium bovis (M. bovis), cause bovine tuberculosis (bTB) in livestock and wildlife animals. Confirmation of the disease is through culture and verification of the causative agent by molecular tests. In this study, we assessed the utility of the Xpert ® MTB/RIF Ultra assay, an automated molecular test originally designed to improve the detection of tuberculosis (TB) and rifampicin resistance in clinical sputum samples of human origin, by conducting a comparative evaluation with a culture based method routinely used at the Onderstepoort Veterinary Research (OVR). A total of 167 samples (tissue, n = 165; pus, n = 1; abscess, n = 1) from different wildlife and livestock animals (from 65 individual animals) were analyzed. Mycobacterium tuberculosis complex species was isolated in 63 (37.72 %) of the 167 samples, and was detected in 79 (47.3 %) of the samples by Xpert ® MTB/RIF Ultra assay. Based on the standard culture test, the diagnostic sensitivity and specificity of the Xpert ® MTB/RIF Ultra assay was found to be 95.24 % and 82 % respectively. All animals that were confirmed bTB positive by culture method were also found to be positive with the Xpert ® MTB/RIF Ultra assay in at least one sample (indicating a 100 % sensitivity of the method at the animal level). Non-tuberculous mycobacteria were isolated in 9 (3.4 %) of the samples analysed and none were detected by Xpert ® MTB/RIF Ultra assay, highlighting that this molecular test is highly specific. Xpert ® MTB/RIF Ultra assay was found to have great potential for the rapid diagnosis of the bTB in animals, hence allowing early intervention by regulatory authorities.

Metabolic Changes of Mycobacterium tuberculosis during the Anti-Tuberculosis Therapy.

Tuberculosis, caused by Mycobacterium tuberculosis complex bacteria, remains one of the most pressing health problems. Despite the general trend towards reduction of the disease incidence rate, the situation remains extremely tense due to the distribution of the resistant forms. Most often, these strains emerge through the intra-host microevolution of the pathogen during treatment failure. In the present study, the focus was on three serial clinical isolates of Mycobacterium tuberculosis Beijing B0/W148 cluster from one patient with pulmonary tuberculosis, to evaluate their changes in metabolism during anti-tuberculosis therapy. Using whole genome sequencing (WGS), 9 polymorphisms were determined, which occurred in a stepwise or transient manner during treatment and were linked to the resistance (GyrA D94A; inhA t-8a) or virulence. The effect of the inhA t-8a mutation was confirmed on both proteomic and transcriptomic levels. Additionally, the amount of RpsL protein, which is a target of anti-tuberculosis drugs, was reduced. At the systemic level, profound changes in metabolism, linked to the evolution of the pathogen in the host and the effects of therapy, were documented. An overabundance of the FAS-II system proteins (HtdX, HtdY) and expression changes in the virulence factors have been observed at the RNA and protein levels.

Differential Sensitivity of Mycobacteria to Isoniazid Is Related to Differences in KatG-Mediated Enzymatic Activation of the Drug.

Isoniazid (INH) is a cornerstone of antitubercular therapy. Mycobacterium tuberculosis complex bacteria are the only mycobacteria sensitive to clinically relevant concentrations of INH. All other mycobacteria, including M. marinum and M. avium subsp. paratuberculosis are resistant. INH requires activation by bacterial KatG to inhibit mycobacterial growth. We tested the role of the differences between M. tuberculosis KatG and that of other mycobacteria in INH sensitivity. We cloned the M. boviskatG gene into M. marinum and M. avium subsp. paratuberculosis and measured the MIC of INH. We recombinantly expressed KatG of these mycobacteria and tested in vitro binding to, and activation of, INH. Introduction of katG from M. bovis into M. marinum and M. avium subsp. paratuberculosis rendered them 20 to 30 times more sensitive to INH. Analysis of different katG sequences across the genus found KatG evolution diverged from RNA polymerase-defined mycobacterial evolution. Biophysical and biochemical tests of M. bovis and nontuberculous mycobacteria (NTM) KatG proteins showed lower affinity to INH and substantially lower enzymatic capacity for the conversion of INH into the active form in NTM. The KatG proteins of M. marinum and M. avium subsp. paratuberculosis are substantially less effective in INH activation than that of M. tuberculosis, explaining the relative INH insensitivity of these microbes. These data indicate that the M. tuberculosis complex KatG is divergent from the KatG of NTM, with a reciprocal relationship between resistance to host defenses and INH resistance. Studies of bacteria where KatG is functionally active but does not activate INH may aid in understanding M. tuberculosis INH-resistance mechanisms, and suggest paths to overcome them.

Towards next-generation diagnostics for tuberculosis: identification of novel molecular targets by large-scale comparative genomics.

MotivationTuberculosis (TB) remains one of the main causes of death worldwide. The long and cumbersome process of culturing Mycobacterium tuberculosis complex (MTBC) bacteria has encouraged the development of specific molecular tools for detecting the pathogen. Most of these tools aim to become novel TB diagnostics, and big efforts and resources are invested in their development, looking for the endorsement of the main public health agencies. Surprisingly, no study has been conducted where the vast amount of genomic data available is used to identify the best MTBC diagnostic markers.ResultsIn this work, we used large-scale comparative genomics to identify 40 MTBC-specific loci. We assessed their genetic diversity and physiological features to select 30 that are good targets for diagnostic purposes. Some of these markers could be used to assess the physiological status of the bacilli. Remarkably, none of the most used MTBC markers is in our catalog. Illustrating the translational potential of our work, we develop a specific qPCR assay for quantification and identification of MTBC DNA. Our rational design of targeted molecular assays for TB could be used in many other fields of clinical and basic research.Availability and implementationThe database of non-tuberculous mycobacteria assemblies can be accessed at: 10.5281/zenodo.3374377.Supplementary information Supplementary data are available at Bioinformatics online.

Alphanumerical Visual Display Made of DNA Logic Gates for Drug Susceptibility Testing of Pathogens.

Molecular diagnostics of drug-resistant pathogens require the analysis of point mutations in bacterial or viral genomes, which is usually performed by trained professionals and/or by sophisticated computer algorithms. We have developed a DNA-based logic system that autonomously analyzes mutations found in the genome of Mycobacterium tuberculosis complex (MTC) bacteria and communicates the output to a human user as alphanumeric characters read by the naked eye. The five-gate system displays "O" ("no infection") for the absence of MTC infection and "P" or "F" for passing or failing a drug-susceptibility test, respectively.

Evidence of Mycobacterium tuberculosis complex bacteraemia in intradermal skin test positive cattle detected using phage-RPA

ABSTRACTBovine tuberculosis is a zoonotic infectious disease caused by Mycobacterium bovis that affects cattle and can cause tuberculosis in a range of wildlife animals. A bacteriophage-based method combined with PCR (phage-PCR) has been recently used to detect and identify viable pathogenic mycobacteria in the peripheral blood mononuclear cells (PBMCs) of animals suffering from paratuberculosis. To adapt this method for the detection of M. bovis in blood, a new isothermal DNA amplification protocol using Recombinase Polymerase Amplification (RPA) was developed and was found to be able to detect M. bovis BCG within 48 h, with a limit of detection of approximately 10 cells per ml of blood for artificially inoculated blood samples. When blood samples (2 ml) from a Single Comparative Cervical Intradermal Tuberculin (SCCIT)- negative beef herd were tested, Mycobacterium tuberculosis complex (MTC) cells were not detected from any (45) of the blood samples. However when blood samples from SCCIT-positive animals were tested, viable MTC bacteria were detected in 66 % (27/41) of samples. Of these 41 animals sampled, 32 % (13) had visible lesions. In the visible lesion (VL) group, 85 % (11/13) had detectable levels of MTC whereas only 57 % (16/28) of animals which had no visible lesions (NVL) were found to have detectable mycobacteraemia. These results indicated that this simple, rapid method can be applied for the study of M. bovis infections. The frequency with which viable mycobacteria were detected in the peripheral blood of SCCIT-positive animals changes the paradigm of this disease.

Environmental Presence of Mycobacterium tuberculosis Complex in Aggregation Points at the Wildlife/Livestock Interface.

The members of the Mycobacterium tuberculosis complex (MTC) cause tuberculosis (TB). Infection is transmitted within and between livestock and wildlife populations, thus hampering TB control. Indirect transmission might be facilitated if MTC bacteria persist in the environment long enough to represent a risk of exposure to different species sharing the same habitat. We have, for the first time, addressed the relationship between environmental MTC persistence and the use of water resources in two TB endemic areas in southern Spain with the objective of identifying the presence of environmental MTC and its driving factors at ungulates' water aggregation points. Camera-trap monitoring and MTC diagnosis (using a new MTC complex-specific PCR technique) were carried out at watering sites. Overall, 55.8% of the water points tested positive for MTC in mud samples on the shore, while 8.9% of them were positive in the case of water samples. A higher percentage of MTC-positive samples was found at those waterholes where cachectic animals were identified using camera-trap monitoring, and at the smallest waterholes. Our results help to understand the role of indirect routes of cross-species TB transmission and highlight the importance of certain environmental features in maintaining infection in multihost systems. This will help to better target actions and implement control strategies for TB at the wildlife/livestock interface.