Biology subjects

Gillespie, S. H.

Publications and source records attributed to Gillespie, S. H..

7 recordsLinked to original sources

bioRxiv2024

Integrated population clustering and genomic epidemiology with PopPIPE

Genetic distances between bacterial DNA sequences can be used to cluster populations into closely related subpopulations, and as an additional source of information when detecting possible transmission events. Due to their variable gene content and order, reference-free methods offer more sensitive detection of genetic differences, especially among closely related samples found in outbreaks. However, across longer genetic distances, frequent recombination can make calculation and interpretation of these differences more challenging, requiring significant bioinformatic expertise and manual intervention during the analysis process. Here we present a Population analysis PIPEline (PopPIPE) which combines rapid reference-free genome analysis methods to analyse bacterial genomes across these two scales, splitting whole populations into subclusters and detecting plausible transmission events within closely related clusters. We use k-mer sketching to split populations into strains, followed by split k-mer analysis and recombination removal to create alignments and subclusters within these strains. We first show that this approach creates high quality subclusters on a population-wide dataset of Streptococcus pneumoniae. When applied to nosocomial vancomycin resistant Enterococcus faecium samples, PopPIPE finds transmission clusters which are more epidemiologically plausible than core genome or MLST-based approaches. Our pipeline is rapid and reproducible, creates interactive visualisations, and can easily be reconfigured and re-run on new datasets. Therefore PopPIPE provides a user-friendly pipeline for analyses spanning species-wide clustering to outbreak investigations. Impact statementAs time passes, bacterial genomes accumulate small changes in their sequence due to mutations, or larger changes in their content due to horizontal gene transfer. Using their genome sequences, it is possible to use phylogenetics to work out the most likely order in which these changes happened, and how long they took to happen. Then, one can estimate the time that separates any two bacterial samples - if it is short then they may have been directly transmitted or acquired from the same source; but if it is long they must have been acquired separately. This information can be used to determine transmission chains, in conjunction with dates and locations of infections. Understanding transmission chains enables targeted infection control measures. However, correctly calculating the genetic evidence for transmission is made difficult by correctly distinguishing different types of sequence changes, dealing with large amounts of genome data, and the need to use multiple complex bioinformatic tools. We addressed this gap by creating a computational workflow, PopPIPE, which automates the process of detecting possible transmissions using genome sequences. PopPIPE applies state-of-the-art tools and is fast and easy to run - making this technology will be available to a wider audience of researchers. Data summaryThe code for this pipeline is available at https://github.com/bacpop/PopPIPE and as a docker image https://hub.docker.com/r/poppunk/poppipe. Raw sequencing reads for Enterococcus faecium isolates have been deposited at the NCBI under BioProject accession number PRJNA997588.

bioinformatics↗

bioRxiv2024

The human pathogen Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum trigger the same core set of late innate immune response genes in zebrafish larvae

Zebrafish is a natural host of various Mycobacterium species and a surrogate model organism for tuberculosis research. Mycobacterium marinum is evolutionarily one of the closest non-tuberculous species related to M. tuberculosis and shares the majority of virulence genes. Although zebrafish is not a natural host of the human pathogen, we have previously demonstrated successful robotic infection of zebrafish embryos with M. tuberculosis and performed drug treatment of the infected larvae. In the present study we examined for how long M. tuberculosis can be propagated in zebrafish larvae and tested a time series of infected larvae to study the transcriptional response via Illumina RNA deep sequencing (RNAseq). Granuloma-like structures carrying fluorescently labeled M. tuberculosis could be detected up to 9 days post infection. The continued presence of viable M. tuberculosis in the zebrafish larvae was further confirmed using the molecular bacterial load (MBL) assay. The infected larvae showed a clear and specific transcriptional immune response with a high similarity to the response of zebrafish larvae infected with the surrogate species M. marinum. We conclude that M. tuberculosis can be propagated in zebrafish larvae for at least one week after infection and provide further evidence that M. marinum is a good surrogate model for M. tuberculosis.

immunology↗

bioRxiv2022

Modelling the within-host spread of SARS-CoV-2 infection, and the subsequent immune response, using a hybrid, multiscale, individual-based model. Part I: Macrophages.

Individual responses to SARS-CoV-2 infection vary significantly, ranging from mild courses of infection that do not require hospitalisation to the development of disease which not only requires hospitalisation but can be fatal. Whilst many immunological studies have revealed fundamental insights into SARS-CoV-2 infection and COVID-19, mathematical and computational modelling can offer an additional perspective and enhance understanding. The majority of mathematical models for the within-host spread of SARS-CoV-2 infection are ordinary differential equations, which neglect spatial variation. In this article, we present a hybrid, multiscale, individual-based model to study the within-host spread of SARS-CoV-2 infection. The model incorporates epithelial cells (each containing a dynamical model for viral entry and replication), macrophages and a subset of cytokines. We investigate the role of increasing initial viral deposition, increasing delay in type I interferon secretion from epithelial cells (as well as the magnitude of secretion), increasing macrophage virus internalisation rate and macrophage activation, on the spread of infection.

systems biology↗

bioRxiv2020

Deploying a novel tuberculosis molecular bacterial load assay to assess the elimination rate of Mycobacterium tuberculosis in patients with multidrug-resistant tuberculosis in Tanzania

BackgroundRifampin or multidrug-resistant-tuberculosis (RR/MDR-TB) treatment has transitioned to injectable-free regimens. We tested whether M. tuberculosis (Mtb) elimination rates measured by molecular bacterial load assay (TB-MBLA) in sputa correlate with composition of the RR/MDR-TB antibiotic regimen. MethodsSerial sputa were collected from patients with RR/MDR- and drug-sensitive TB at day 0, 3, 7, 14, and then monthly for 4 months of anti-TB treatment. TB-MBLA was used to quantify viable Mtb 16S rRNA in sputum for estimation of colony-forming-unit per mL (eCFU/mL). Mtb elimination rates were compared among regimens using nonlinear-mixed-effects modeling of repeated measures. ResultsAmong 37 patients with a total of 296 serial sputa; 7 patients received rifampin/isoniazid/pyrazinamide/ethambutol (RHZE), 8 an all-oral bedaquiline-based regimen, 9 an injectable and bedaquiline-containing regimen, and 13 an injectable-containing but bedaquiline-free regimen. The overall mean daily Mtb elimination was -0.24 [95% Confidence-Interval (CI); -0.39 to -0.08)] log10 eCFU/mL, and it varied with treatment-regimen (p < 0.001). Compared to the adjusted Mtb elimination of -0.17 (95% CI; -0.23 to -0.12) for the injectable-containing but bedaquiline-free reference regimen, the elimination rates were -0.62 (95% CI; -1.05 to -0.20) log10 eCFU/mL for the injectable and bedaquiline-containing regimen (p = 0.019), -0.35 (95% CI; -0.65 to -0.13) log10 eCFU/mL for the all-oral bedaquiline-based regimen (p = 0.054), and -0.29 (95% CI; -0.78 to +0.22) log10 eCFU/mL for RHZE (p = 0.332) ConclusionTB-MBLA distinguished Mtb elimination rates in sputa from patients receiving different treatment regimens, suggesting a reliable monitoring tool for RR/MDR-TB, that does not require mycobacterial culture.

microbiology↗

bioRxiv2020

Emergence of optrA-mediated linezolid resistance in multiple lineages and plasmids of Enterococcus faecalis revealed by long read sequencing

ObjectivesTo characterise the genetic environment of optrA in linezolid-resistant Enterococcus faecalis isolates from Scotland. MethodsLinezolid-resistant E. faecalis were identified in three Scottish Health Boards and confirmed to carry the optrA gene at the national reference laboratory. WGS was performed with short read (Illumina MiSeq) and long read (Oxford Nanopore MinION) technologies to generate complete genome assemblies. Illumina reads for 94 E. faecalis bloodstream isolates were used to place the optrA-positive isolates in a larger UK phylogeny. ResultsSix optrA-positive linezolid-resistant E. faecalis were isolated from urogenital samples in three Scottish Health Boards (2014-2017). No epidemiological links were identified between the patients, four were community-based, and only one had recent linezolid exposure. Reference-based mapping confirmed the isolates were genetically distinct (>13,900 core SNPs). optrA was located on a plasmid in each isolate and these plasmids showed limited nucleotide similarity. There was variable presence of transposable elements surrounding optrA, (including IS1216, IS3, and Tn3) and not always as a recognisable gene cassette. OptrA amino acid sequences were also divergent, resulting in four protein variants differing in 1-20 residues. One isolate belonged to ST16 and clustered with three other isolates in the UK collection (76-182 SNPs), otherwise the optrA-positive isolates were genetically distinct from the bloodstream isolates (>6,000 SNPs). ConclusionsWe report multiple variants of the linezolid resistance gene optrA in diverse E. faecalis strain and plasmid backgrounds, suggesting multiple introductions of the gene into the E. faecalis population and selection driving recent emergence.

microbiology↗

bioRxiv2019

Modelling the effects of environmental heterogeneity within the lung on the tuberculosis life-cycle

Progress in shortening the duration of tuberculosis (TB) treatment is hampered by the lack of a predictive model that accurately reflects the diverse environment within the lung. This is important as TB has been shown to produce distinct localisations to different areas of the lung during different disease stages, with the environmental heterogeneity within the lung of factors such as air ventilation, blood perfusion and oxygen tension believed to contribute to the apical localisation witnessed during the post-primary form of the disease. Building upon our previous model of environmental lung heterogeneity, we present a networked metapopulation model that simulates TB across the whole lung, incorporating these notions of environmental heterogeneity across the whole TB life-cycle to show how different stages of the disease are influenced by different environmental and immunological factors. The alveolar tissue in the lung is divided into distinct patches, with each patch representing a portion of the total tissue and containing environmental attributes that reflect the internal conditions at that location. We include populations of bacteria and immune cells in various states, and events are included which determine how the members of the model interact with each other and the environment. By allowing some of these events to be dependent on environmental attributes, we create a set of heterogeneous dynamics, whereby the location of the tissue within the lung determines the disease pathological events that occur there. Our results show that the environmental heterogeneity within the lung is a plausible driving force behind the apical localisation during post-primary disease. After initial infection, bacterial levels will grow in the initial infection location at the base of the lung until an adaptive immune response is initiated. During this period, bacteria are able to disseminate and create new lesions throughout the lung. During the latent stage, the lesions that are situated towards the apex are the largest in size, and once a post-primary immune-suppressing event occurs, it is the uppermost lesions that reach the highest levels of bacterial proliferation. Our sensitivity analysis also shows that it is the differential in blood perfusion, causing reduced immune activity towards the apex, which has the biggest influence of disease outputs.

systems biology↗

bioRxiv2019

Improving diagnosis and monitoring of treatment response in pulmonary tuberculosis using the molecular bacterial load assay (MBLA)

ObjectivesBetter outcomes in tuberculosis require new diagnostic and treatment monitoring tools. In this paper we evaluated the utility of a marker of M. tuberculosis viable count, the Molecular Bacterial Load assay (MBLA) for diagnosis and treatment monitoring of tuberculosis in a high burden setting. MethodsPatients with smear positive pulmonary tuberculosis from two sites in Tanzania and one each in Malawi and Mozambique. Sputum samples were taken weekly for the first 12 weeks of treatment and evaluated by MBLA and mycobacterial growth indicator tube method (MGIT). ResultsThe results of high and low positive control samples confirmed inter site reproducibility. Over the 12 weeks of treatment there was a steady decline in the viable bacterial load as measured by the MBLA that corresponds to rise in time to a positive result (TTP) in the Mycobacterial Growth Indicator Tube. Both MBLA and MGIT provided similar time to test negativity. Importantly, as treatment progressed samples in MGIT were increasingly likely to be contaminated, which compromised the acquisition of results but did not affect MBLA samples. ConclusionsMBLA produces a reproducible measure of Mtb viable count comparable to that of MGIT that is not compromised by contamination in a real-world setting. As a molecular test, the results can be available in as little as four hours and could allow health care professionals to identify rapidly patients who are failing therapy.

microbiology↗