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 phylogeographic diversity modulates the outcome of TB infection and disease. For decades, TB research and development has focused on the two canonical MTBC laboratory 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 laboratory 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 broadly effective

Comparative genomics of Mycobacterium africanum Lineage 5 and Lineage 6 from Ghana suggests distinct ecological niches.

Mycobacterium africanum (Maf) causes a substantial proportion of human tuberculosis in some countries of West Africa, but little is known on this pathogen. We compared the genomes of 253 Maf clinical isolates from Ghana, including N = 175 Lineage 5 (L5) and N = 78 Lineage 6 (L6). We found that the genomic diversity of L6 was higher than in L5 despite the smaller sample size. Regulatory proteins appeared to evolve neutrally in L5 but under purifying selection in L6. Even though over 90% of the human T cell epitopes were conserved in both lineages, L6 showed a higher ratio of non-synonymous to synonymous single nucleotide variation in these epitopes overall compared to L5. Of the 10% human T cell epitopes that were variable, most carried mutations that were lineage-specific. Our findings indicate that Maf L5 and L6 differ in some of their population genomic characteristics, possibly reflecting different selection pressures linked to distinct ecological niches

Transition bias influences the evolution of antibiotic resistance in Mycobacterium tuberculosis

Transition bias, an overabundance of transitions relative to transversions, has been widely reported among studies of the rates and spectra of spontaneous mutations. However, demonstrating the role of transition bias in adaptive evolution remains challenging. In particular, it is unclear whether such biases direct the evolution of bacterial pathogens adapting to treatment. We addressed this challenge by analyzing adaptive antibiotic-resistance mutations in the major human pathogen Mycobacterium tuberculosis (MTB). We found strong evidence for transition bias in two independently curated data sets comprising 152 and 208 antibiotic-resistance mutations. This was true at the level of mutational paths (distinct adaptive DNA sequence changes) and events (individual instances of the adaptive DNA sequence changes) and across different genes and gene promoters conferring resistance to a diversity of antibiotics. It was also true for mutations that do not code for amino acid changes (in gene promoters and the 16S ribosomal RNA gene rrs) and for mutations that are synonymous to each other and are therefore likely to have similar fitness effects, suggesting that transition bias can be caused by a bias in mutation supply. These results point to a central role for transition bias in determining which mutations drive adaptive antibiotic resistance evolution in a key pathogen

Is Holothuria tubulosa the golden goose of ecological aquaculture in the Mediterranean Sea?

The use of detritivores under sea farms is a promising avenue to mitigate the benthic impacts of marine fish farms. Sea cucumbers are interesting candidates for integrated multi-trophic aquaculture (IMTA) due to their prevalence in the marine environment, their diversified diet and their economic value. Yet limited information is available regarding their capacities to be stocked and reared underneath aquaculture cages and the associated effects on their survival, growth rate and body composition. This study focused on Holothuria tubulosa, a Mediterranean sea cucumber species candidate for rearing in the vicinity to marine fish cages. We investigated its potential for co-culture on the seabed more or less influenced by marine fish cages. The farm's waste footprint was predicted using a dispersion model (NewDEPOMOD) to estimate the farm's influence along a transect where we also sampled sediment at four distances from the cages (0 m, 25 m, 100 m from the cages, plus a reference site at 150 m). Organic composition of the sediment was analysed (TOC, TON, TOP, OM, stable isotope signature) and linked to the results from the dispersion model. Based on the model simulation, the maximum flux of matter reached almost 17 kg solids.m−2.year−1 below the cages, and gradually decreased with distance from the cages. An isotopic gradient was also found in the sediments according to the distance from the farm, with an enrichment in δN15 and a depletion in δC13 with increasing proximity to the farm. In parallel we investigated the response of adult sea cucumbers placed at varying distances from the fish cages for a period of one month, measuring their proximate composition, isotopic concentration, and fatty acid and protein composition. We found that despite good survival, growth was null over the experiment. While the isotope signature of the sea cucumbers was significantly affected by distance from the cage, this did not follow the pattern found in sediment. There was a clear difference in fatty acid composition between sites, with sea cucumbers closer to the cages having lower levels of short-chain fatty acids. The protein content was also lower in sea cucumbers reared right below the cages. These results suggest that while adult H. tubulosa can survive the environmental conditions below marine aquaculture cages, they do not nutritionally benefit from fish waste over short periods in the stocking conditions we tested. Their use in IMTA requires further investigation to find optimal stocking conditions

Oral Phage Therapy of Acute Bacterial Diarrhea With Two Coliphage Preparations: A Randomized Trial in Children From Bangladesh

Background: Antibiotic resistance is rising in important bacterial pathogens. Phage therapy (PT), the use of bacterial viruses infecting the pathogen in a species-specific way, is a potential alternative. Method: T4-like coliphages or a commercial Russian coliphage product or placebo was orally given over 4 days to Bangladeshi children hospitalized with acute bacterial diarrhea. Safety of oral phage was assessed clinically and by functional tests; coliphage and Escherichia coli titers and enteropathogens were determined in stool and quantitative diarrhea parameters (stool output, stool frequency) were measured. Stool microbiota was studied by 16S rRNA gene sequencing; the genomes of four fecal Streptococcus isolates were sequenced. Findings: No adverse events attributable to oral phage application were observed (primary safety outcome). Fecal coliphage was increased in treated over control children, but the titers did not show substantial intestinal phage replication (secondary microbiology outcome). 60% of the children suffered from a microbiologically proven E. coli diarrhea; the most frequent diagnosis was ETEC infections. Bacterial co-pathogens were also detected. Half of the patients contained phage-susceptible E. coli colonies in the stool. E. coli represented less than 5% of fecal bacteria. Stool ETEC titers showed only a short-lived peak and were otherwise close to the replication threshold determined for T4 phage in vitro. An interim analysis after the enrollment of 120 patients showed no amelioration in quantitative diarrhea parameter by PT over standard care (tertiary clinical outcome). Stool microbiota was characterized by an overgrowth with Streptococcus belonging to the Streptococcus gallolyticus and Streptococcus salivarius species groups, their abundance correlated with quantitative diarrhea outcome, but genome sequencing did not identify virulence genes. Interpretation: Oral coliphages showed a safe gut transit in children, but failed to achieve intestinal amplification and to improve diarrhea outcome, possibly due to insufficient phage coverage and too low E. coli pathogen titers requiring higher oral phage doses. More knowledge is needed on in vivo phage–bacterium interaction and the role of E. coli in childhood diarrhea for successful PT. Funding: The study was supported by a grant from Nestlé Nutrition and Nestlé Health Science. The trial was registered with Identifier NCT00937274 at ClinicalTrials.gov

Whole genome sequencing distinguishes between relapse and reinfection in recurrent leprosy cases

Background Since leprosy is both treated and controlled by multidrug therapy (MDT) it is important to monitor recurrent cases for drug resistance and to distinguish between relapse and reinfection as a means of assessing therapeutic efficacy. All three objectives can be reached with single nucleotide resolution using next generation sequencing and bioinformatics analysis of Mycobacterium leprae DNA present in human skin. Methodology DNA was isolated by means of optimized extraction and enrichment methods from samples from three recurrent cases in leprosy patients participating in an open-label, randomized, controlled clinical trial of uniform MDT in Brazil (U-MDT/CT-BR). Genome-wide sequencing of M. leprae was performed and the resultant sequence assemblies analyzed in silico. Principal findings In all three cases, no mutations responsible for resistance to rifampicin, dapsone and ofloxacin were found, thus eliminating drug resistance as a possible cause of disease recurrence. However, sequence differences were detected between the strains from the first and second disease episodes in all three patients. In one case, clear evidence was obtained for reinfection with an unrelated strain whereas in the other two cases, relapse appeared more probable. Conclusions/Significance This is the first report of using M. leprae whole genome sequencing to reveal that treated and cured leprosy patients who remain in endemic areas can be reinfected by another strain. Next generation sequencing can be applied reliably to M. leprae DNA extracted from biopsies to discriminate between cases of relapse and reinfection, thereby providing a powerful tool for evaluating different outcomes of therapeutic regimens and for following disease transmission

Local adaptation in populations of Mycobacterium tuberculosis endemic to the Indian Ocean Rim

This work was supported by the Swiss National Science Foundation (grants 310030_188888, CRSII5_177163, IZRJZ3_164171 and IZLSZ3_170834) and the European Research Council (309540‑EVODRTB and 883582-ECOEVODRTB)Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Institute of Biomedicine of Valencia. Valencia, Spain.Universidade Federal do Rio de Janeiro. Instituto de Microbiologia. Laboratório de Micobactérias. Rio de Janeiro, RJ, Brazil / Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Programa de Pós-graduação em Pesquisa Clínica e Doenças Infecciosas. Rio de Janeiro, RJ, Brazil.University of Valencia- joint Unit. I2SysBio,Valencia, Spain.University of Cape Town. Wellcome Centre for Infectious Diseases Research in Africa. Institute of Infectious Diseases and Molecular Medicine. Cape Town, South Africa.Makerere University. Department of Medical Microbiology. Kampala, Uganda.National Health Research Institutes. National Institute of Infectious Diseases and Vaccinology. Zhunan, Taiwan.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland / University of Bern. Institute for Social and Preventive Medicine. Switzerland.Victorian Infectious Diseases Reference Laboratory. Victoria, Australia.Fudan University. School of Basic Medical Science. Institutes of Biomedical Sciences and Institute of Medical Microbiology. The Key Laboratory of Medical Molecular Virology of Ministries of Education and Health. Shanghai, China.Instituto de Investigación Sanitaria Gregorio Marañón. Hospital General Universitario Gregorio Marañón. Madrid, Spain / CIBER Enfermedades Respiratorias. Spain.Universitat de Barcelona. Hospital Clínic. Barcelona Institute for Global Health. Barcelona, Spain / Centro de Investigação em Saúde de Manhiça. Maputo, Mozambique.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland / United Republic of Tanzania. Ifakara Health Institute, Bagamoyo, Bagamoyo District Hospital. Bagamoyo, Tanzania.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland / United Republic of Tanzania. Ifakara Health Institute. Bagamoyo District Hospital. Bagamoyo, Tanzania.University of California. School of Medicine. San Francisco, USA.Fudan University. School of Basic Medical Science. Institutes of Biomedical Sciences and Institute of Medical Microbiology. The Key Laboratory of Medical Molecular Virology of Ministries of Education and Health. Shanghai, China.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland / Papua New Guinea Institute of Medical Research. Goroka, Papua New Guinea.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Mahidol University. Faculty of Science. Department of Microbiology. Pornchai Matangkasombut Center for Microbial Genomics / National Science and Technology Development Agency. Bangkok, Thailand.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Mahidol University. Faculty of Science. Department of Microbiology. Pornchai Matangkasombut Center for Microbial Genomics / National Science and Technology Development Agency. Bangkok, Thailand.Institut Pasteur de Madagascar. Mycobacteriology Unit. Antananarivo, Madagascar.Institut Pasteur de Madagascar. Mycobacteriology Unit. Antananarivo, Madagascar.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.University of Basel. Basel, Switzerland / Swiss Tropical and Public Health Institute. Department of Medicine. Basel, Switzerland.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland / United Republic of Tanzania. Ifakara Health Institute. Bagamoyo District Hospital. Bagamoyo, Tanzania.Universidade Federal do Rio de Janeiro. Instituto de Microbiologia. Laboratório de Micobactérias. Rio de Janeiro, RJ, Brazil.Université Paris-Saclay. Paris, France / Paris Diderot University. Sorbonne Paris Cité. Paris, France.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brazil.Universidade do Estado do Pará. Centro de Ciências Biológicas e da Saúde. Programa de Pós-graduação em Biologia Parasitária na Amazônia. Belém, PA, Brazil / Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.University of Ghana. Noguchi Memorial Institute for Medical Research. Accra, Ghana.ETH Zürich. Department of Biosystems Science and Engineering. Basel, Switzerland.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Lineage 1 (L1) and 3 (L3) are two lineages of the Mycobacterium tuberculosis complex (MTBC), causing tuberculosis (TB) in humans. L1 and L3 are endemic to the Rim of the Indian Ocean, the region that accounts for most of the world’s new TB cases. Despite their relevance for this region, L1 and L3 remain understudied. Here we analyzed 2,938 L1 and 2,030 L3 whole genome sequences originating from 69 countries. We show that South Asia played a central role in the dispersion of these two lineages to neighboring regions. Moreover, we found that L1 exhibits signatures of local adaptation at the esxH locus, a gene coding for a secreted effector that targets the human endosomal sorting complex, and is included in several vaccine candidates. Our study highlights the importance of genetic diversity in the MTBC, and sheds new light on two of the most important MTBC lineages affecting humans