Four decades of transmission of a multidrug-resistant Mycobacterium tuberculosis outbreak strain

The rise of drug-resistant strains is a major challenge to containing the tuberculosis (TB) pandemic. Yet, little is known about the extent of resistance in early years of chemotherapy and when transmission of resistant strains on a larger scale became a major public health issue. Here we reconstruct the timeline of the acquisition of antimicrobial resistance during a major ongoing outbreak of multidrug-resistant TB in Argentina. We estimate that the progenitor of the outbreak strain acquired resistance to isoniazid, streptomycin and rifampicin by around 1973, indicating continuous circulation of a multidrug-resistant TB strain for four decades. By around 1979 the strain had acquired additional resistance to three more drugs. Our results indicate that Mycobacterium tuberculosis (Mtb) with extensive resistance profiles circulated 15 years before the outbreak was detected, and about one decade before the earliest documented transmission of Mtb strains with such extensive resistance profiles globally.Fil: Eldholm, Vegard. Norwegian Institute of Public Health; NoruegaFil: Monteserin, Johana. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rieux, Adrien. Colegio Universitario de Londres; Reino UnidoFil: Lopez, Beatriz. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; ArgentinaFil: Sobkowiak, Benjamin. Colegio Universitario de Londres; Reino UnidoFil: Ritacco, Gloria Viviana. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Balloux, Francois. Colegio Universitario de Londres; Reino Unid

The genetics and epidemiology of Shigella sonnei and Shigella flexneri in Vietnam

Shigella sonnei is rapidly emerging as the primary agent of bacillary dysentery, or shigellosis, in many developing countries, replacing the historically more prevalent species, S. flexneri, in these regions. There have been various theories proposed to explain this phenomenon, including environmental changes and increased antimicrobial use, though the precise reasons for this shift are still uncertain. Here I present four studies investigating key ecological and genetic differences between S. sonnei and S. flexneri from a region that has undergone this pattern of species replacement, Vietnam. This work combines experimental and bioinformatics techniques with the aim of identifying the extent that differences in disinfectant sensitivity, chromosomal antimicrobial resistance profiles and gene content will contribute to the successful spread of S. sonnei over S. flexneri. Firstly, I conducted in vitro experimental work to characterise differences between species with respect to resistance to chlorine disinfection and tolerance to the detergent SDS. The mechanisms by which the bacteria respond to this treatment, in particular the role of efflux pumps, were then explored to determine whether any informative variation in these systems will explain any differences in disinfectant sensitivity. The availability of high quality whole genome sequences for ~150 of each Shigella species allowed for robust bioinformatics work to describe genomic variation between species. These sequences are used to detect key resistance mutations in each species and look for associated fitness compensating mutations. Finally, the complete genome sequence of all coding regions in each strain was de novo assembled to look for species-level gene content variation that might contribute to functional differences between S. sonnei and S. flexneri. The results of these studies show that there are clear biological differences between S. sonnei and S. flexneri, though more work is necessary to fully elucidate the reasons for the species replacement in developing countries

Genome-Wide Association with Uncertainty in the Genetic Similarity Matrix

Genome-wide association studies (GWASs) are often confounded by population stratification and structure. Linear mixed models (LMMs) are a powerful class of methods for uncovering genetic effects, while controlling for such confounding. LMMs include random effects for a genetic similarity matrix, and they assume that a true genetic similarity matrix is known. However, uncertainty about the phylogenetic structure of a study population may degrade the quality of LMM results. This may happen in bacterial studies in which the number of samples or loci is small, or in studies with low-quality genotyping. In this study, we develop methods for linear mixed models in which the genetic similarity matrix is unknown and is derived from Markov chain Monte Carlo estimates of the phylogeny. We apply our model to a GWAS of multidrug resistance in tuberculosis, and illustrate our methods on simulated data

Bayesian reconstruction of Mycobacterium tuberculosis transmission networks in a high incidence area over two decades in Malawi reveals associated risk factors and genomic variants.

Understanding host and pathogen factors that influence tuberculosis (TB) transmission can inform strategies to eliminate the spread of Mycobacterium tuberculosis (Mtb). Determining transmission links between cases of TB is complicated by a long and variable latency period and undiagnosed cases, although methods are improving through the application of probabilistic modelling and whole-genome sequence analysis. Using a large dataset of 1857 whole-genome sequences and comprehensive metadata from Karonga District, Malawi, over 19 years, we reconstructed Mtb transmission networks using a two-step Bayesian approach that identified likely infector and recipient cases, whilst robustly allowing for incomplete case sampling. We investigated demographic and pathogen genomic variation associated with transmission and clustering in our networks. We found that whilst there was a significant decrease in the proportion of infectors over time, we found higher transmissibility and large transmission clusters for lineage 2 (Beijing) strains. By performing evolutionary convergence testing (phyC) and genome-wide association analysis (GWAS) on transmitting versus non-transmitting cases, we identified six loci, PPE54, accD2, PE_PGRS62, rplI, Rv3751 and Rv2077c, that were associated with transmission. This study provides a framework for reconstructing large-scale Mtb transmission networks. We have highlighted potential host and pathogen characteristics that were linked to increased transmission in a high-burden setting and identified genomic variants that, with validation, could inform further studies into transmissibility and TB eradication

Genetic Diversity of norA, Coding for a Main Efflux Pump of Staphylococcus aureus

Funding Information: This work was partially supported by Fundação para a Ciência e a Tecnologia (FCT, Portugal), through funds to GHTM – UID/Multi/04413/2013. SC was supported by grant SFRH/BPD/97508/2013 from FCT, Portugal. TC was funded by the Medical Research Council United Kingdom (Grant Nos. MR/K000551/1, MR/M01360X/1, MR/N010469/1, and MR/R020973/1) and BBSRC United Kingdom (BB/R013063/1). BS was funded by the Medical Research Council United Kingdom (Grant No. MR/N010469/1). Publisher Copyright: © 2007 - 2019 Frontiers Media S.A. All Rights Reserved.NorA is the best studied efflux system of Staphylococcus aureus and therefore frequently used as a model for investigating efflux-mediated resistance in this pathogen. NorA activity is associated with resistance to fluoroquinolones, several antiseptics and disinfectants and several reports have pointed out the role of efflux systems, including NorA, as a first-line response to antimicrobials in S. aureus. Genetic diversity studies of the gene norA have described three alleles; norAI, norAII and norAIII. However, the epidemiology of these alleles and their impact on NorA activity remains unclear. Additionally, increasing studies do not account for norA variability when establishing relations between resistance phenotypes and norA presence or reported absence, which actually corresponds, as we now demonstrate, to different norA alleles. In the present study we assessed the variability of the norA gene present in the genome of over 1,000 S. aureus isolates, corresponding to 112 S. aureus strains with whole genome sequences publicly available; 917 MRSA strains sourced from a London-based study and nine MRSA isolates collected in a major Hospital in Lisbon, Portugal. Our analyses show that norA is part of the core genome of S. aureus. It also suggests that occurrence of norA variants reflects the population structure of this major pathogen. Overall, this work highlights the ubiquitous nature of norA in S. aureus which must be taken into account when studying the role played by this important determinant on S. aureus resistance to antimicrobials.publishersversionpublishe

Do Global Diversity Patterns of Vertebrates Reflect Those of Monocots?

Few studies of global diversity gradients in plants exist, largely because the data are not available for all species involved. Instead, most global studies have focussed on vertebrates, as these taxa have historically been associated with the most complete data. Here, we address this shortfall by first investigating global diversity gradients in monocots, a morphologically and functionally diverse clade representing a quarter of flowering plant diversity, and then assessing congruence between monocot and vertebrate diversity patterns. To do this, we create a new dataset that merges biome-level associations for all monocot genera with country-level associations for almost all ∼70,000 species. We then assess the evidence for direct versus indirect effects of this plant diversity on vertebrate diversity using a combination of linear regression and structural equation modelling (SEM). Finally, we also calculate overlap of diversity hotspots for monocots and each vertebrate taxon. Monocots follow a latitudinal gradient although with pockets of extra-tropical diversity, mirroring patterns in vertebrates. Monocot diversity is positively associated with vertebrate diversity, but the strength of correlation varies depending on the clades being compared. Monocot diversity explains marginal amounts of variance (<10%) after environmental factors have been accounted for. However, correlations remain among model residuals, and SEMs apparently reveal some direct effects of monocot richness. Our results suggest that collinear responses to environmental gradients are behind much of the congruence observed, but that there is some evidence for direct effects of producer diversity on consumer diversity. Much remains to be done before broad-scale diversity gradients among taxa are fully explained. Our dataset of monocot distributions will aid in this endeavour

Identifying mixed Mycobacterium tuberculosis infections from whole genome sequence data.

BACKGROUND: Mixed, polyclonal Mycobacterium tuberculosis infection occurs in natural populations. Developing an effective method for detecting such cases is important in measuring the success of treatment and reconstruction of transmission between patients. Using whole genome sequence (WGS) data, we assess two methods for detecting mixed infection: (i) a combination of the number of heterozygous sites and the proportion of heterozygous sites to total SNPs, and (ii) Bayesian model-based clustering of allele frequencies from sequencing reads at heterozygous sites. RESULTS: In silico and in vitro artificially mixed and known pure M. tuberculosis samples were analysed to determine the specificity and sensitivity of each method. We found that both approaches were effective in distinguishing between pure strains and mixed infection where there was relatively high (> 10%) proportion of a minor strain in the mixture. A large dataset of clinical isolates (n = 1963) from the Karonga Prevention Study in Northern Malawi was tested to examine correlations with patient characteristics and outcomes with mixed infection. The frequency of mixed infection in the population was found to be around 10%, with an association with year of diagnosis, but no association with age, sex, HIV status or previous tuberculosis. CONCLUSIONS: Mixed Mycobacterium tuberculosis infection was identified in silico using whole genome sequence data. The methods presented here can be applied to population-wide analyses of tuberculosis to estimate the frequency of mixed infection, and to identify individual cases of mixed infections. These cases are important when considering the evolution and transmission of the disease, and in patient treatment

Tiled Amplicon Sequencing Enables Culture-free Whole-Genome Sequencing of Pathogenic Bacteria From Clinical Specimens

Pathogen sequencing is an important tool for disease surveillance and demonstrated its high value during the COVID-19 pandemic. Viral sequencing during the pandemic allowed us to track disease spread, quickly identify new variants, and guide the development of vaccines. Tiled amplicon sequencing, in which a panel of primers is used for multiplex amplification of fragments across an entire genome, was the cornerstone of SARS-CoV-2 sequencing. The speed, reliability, and cost-effectiveness of this method led to its implementation in academic and public health laboratories across the world and adaptation to a broad range of viral pathogens. However, similar methods are not available for larger bacterial genomes, for which whole-genome sequencing typically requires in vitro culture. This increases costs, error rates and turnaround times. The need to culture poses particular problems for medically important bacteria such as Mycobacterium tuberculosis, which are slow to grow and challenging to culture. As a proof of concept, we developed two novel whole-genome amplicon panels for M. tuberculosis and Streptococcus pneumoniae. Applying our amplicon panels to clinical samples, we show the ability to classify pathogen subgroups and to reliably identify markers of drug resistance without culturing. Development of this work in clinical settings has the potential to dramatically reduce the time of diagnosis of drug resistance for multiple drugs in parallel, enabling earlier intervention for high priority pathogens

Distribution and transmission of M. tuberculosis in a high-HIV prevalence city in Malawi : a genomic and spatial analysis

Funding: Funding: MHC, ELC, VN, SB, CC, JRA, PGTC, PJD, DME, MF, YL, JL, NAM, JLW, JAS, TC acknowledge funding from the National Institute of Allergy and Infectious Diseases (NIAID) (R01AI147854) (https://www.niaid.nih.gov/). JWI-E acknowledges funding from the Bill & Melinda Gates Foundation (INV-006733, INV-002606) (https://www.gatesfoundation.org/) and the MRC Centre for Global Infectious Disease Analysis (reference MR/X020258/1), funded by the UK Medical Research Council (MRC)(https://www.ukri.org/councils/mrc/). This UK funded award is carried out in the frame of the Global Health EDCTP3 Joint Undertaking.Delays in identifying and treating individuals with infectious tuberculosis (TB) contribute to poor health outcomes and allow ongoing community transmission of M. tuberculosis (Mtb). Current recommendations for screening for tuberculosis specify community characteristics (e.g., areas with high local tuberculosis prevalence) that can be used to target screening within the general population. However, areas of higher tuberculosis burden are not necessarily areas with higher rates of transmission. We investigated the transmission of Mtb using high-resolution surveillance data in Blantyre, Malawi. We extracted and performed whole genome sequencing on mycobacterial DNA from cultured M. tuberculosis isolates obtained from culture-positive tuberculosis cases at the time of tuberculosis (TB) notification in Blantyre, Malawi between 2015-2019. We constructed putative transmission networks identified using TransPhylo and investigated individual and pair-wise demographic, clinical, and spatial factors associated with person-to-person transmission. We found that 56% of individuals with sequenced isolates had a probable transmission link to at least one other individual in the study. We identified thirteen putative transmission networks that included five or more individuals. Five of these networks had a single spatial focus of transmission in the city, and each focus centered in a distinct neighborhood in the city. We also found that approximately two-thirds of inferred transmission links occurred between individuals residing in different geographic zones of the city. While the majority of detected tuberculosis transmission events in Blantyre occurred between people living in different zones, there was evidence of distinct geographical concentration for five transmission networks. These findings suggest that targeted interventions in areas with evidence of localized transmission may be an effective local tactic, but will likely need to be augmented by city-wide interventions to improve case finding to have sustained impact.Peer reviewe

Phylogenetic inference of pneumococcal transmission from cross-sectional data, a pilot study.

Background: Inference on pneumococcal transmission has mostly relied on longitudinal studies which are costly and resource intensive. Therefore, we conducted a pilot study to test the ability to infer who infected whom from cross-sectional pneumococcal sequences using phylogenetic inference. Methods: Five suspected transmission pairs, for which there was epidemiological evidence of who infected whom, were selected from a household study. For each pair, Streptococcus pneumoniae full genomes were sequenced from nasopharyngeal swabs collected on the same day. The within-host genetic diversity of the pneumococcal population was used to infer the transmission direction and then cross-validated with the direction suggested by the epidemiological records. Results: The pneumococcal genomes clustered into the five households from which the samples were taken. The proportion of concordantly inferred transmission direction generally increased with increasing minimum genome fragment size and single nucleotide polymorphisms. We observed a larger proportion of unique polymorphic sites in the source bacterial population compared to that of the recipient in four of the five pairs, as expected in the case of a transmission bottleneck. The only pair that did not exhibit this effect was also the pair that had consistent discordant transmission direction compared to the epidemiological records suggesting potential misdirection as a result of false-negative sampling. Conclusions: This pilot provided support for further studies to test if the direction of pneumococcal transmission can be reliably inferred from cross-sectional samples if sequenced with sufficient depth and fragment length