Biology subjects

Oravcova, K.

Publications and source records attributed to Oravcova, K..

6 recordsLinked to original sources

bioRxiv2026

Scale- and genotype-dependent spatial concordance of Mycobacterium bovis in cattle and badgers

Mycobacterium bovis causes tuberculosis (TB) in cattle, badgers (Meles meles), other wildlife, and humans, posing major animal and public health challenges in Britain and Ireland. Shared genotypes between cattle and badgers have been reported at regional scales, but whether this concordance holds at finer spatial scales, where control interventions operate, and whether it varies by genotype combination, remains unknown. Resolving this is critical for interpreting cross-species transmission risk and designing targeted control measures. We analyzed 1,248 M. bovis isolates from cattle and badgers (Northern Ireland, 1986-2018), including samples from a small, densely sampled intervention research area, using Multi-Locus Variable Number of Tandem Repeats Analysis (MLVA) and kernel discriminant analysis (KDA) across multiple spatial scales (25-95% density contours). We identified 36 MLVA types with four shared between species and one (MLV06) endemic to the area. Spatial concordance varied by type combination and scale: MLVA004-MLVA297 showed strong, bidirectional, scale-robust concordance; MLVA006-MLVA297 was concordant only at fine spatial scales; MLVA004-MLVA006 showed weak, asymmetric concordance; and no concordance was detected when all three types were analysed together. Our findings reveal spatial concordance between shared M. bovis genotypes varied by genotype combination and scale, indicating genotype-specific dynamics within this multi-host system.

ecology↗

bioRxiv2025

Nutrient-limited conditions reveal the activity of a minor groove binder, MGB-BP-3, against Escherichia coli

BackgroundAntimicrobial resistance demands new antibiotics, but conventional screening in nutrient-rich media often fails to predict in vivo efficacy, especially against Gram-negative bacteria. Minor groove binders (MGB), such as MGB-BP-3, are a promising new antibacterial with potent activity against Gram-positive bacteria but thought to have limited potency against Gram-negative bacteria. ObjectivesThis study evaluated the antibacterial activity of MGB-BP-3 under nutrient-limited, physiologically relevant conditions to better mimic in vivo environments. MethodsMinimum inhibitory concentrations (MICs) were determined against a panel of ESKAPEE pathogens in both rich (Cation Adjusted Mueller-Hinton Broth, CA-MHB) and nutrient-limited media (Roswell Park Memorial Institute, RPMI-1640 + 10% Luria-Bertani, LB). Synergy with efflux pump inhibitors and membrane permeabilizers was evaluated via checkerboard assays. Permeability was assessed using a fluorescent DNA-binding cell permeant (Hoechst 33342) accumulation assay. MICs were determined in the presence of different concentrations of ions, using both wild-type and efflux-compromised strains. In vivo efficacy was assessed using the Galleria mellonella infection model. ResultsThe activity of MGB-BP-3 against Gram-negative bacteria was improved by shifting the medium from rich to nutrient-limited conditions. Specifically, it showed potent activity against E. coli in nutrient-limited media (MIC 3.1 {micro}M) but was inactive in rich media (MIC 700 {micro}M). Synergy with efflux inhibitors occurred mainly in rich media, indicating barriers to intracellular accumulation were not present in nutrient-limited conditions. Increased permeability correlated with enhanced susceptibility and efflux-deficient strains were more susceptible, confirming the role of efflux pumps in resistance. MGB-BP-3 provided dose-dependent protection in G. mellonella larvae infected with E. coli or S. aureus. For example, at concentrations of 12.5 {micro}M and 100 {micro}M, MGB-BP-3 was able to protect the larvae by more than 70% over 5 days of treatment, respectively. ConclusionsNutrient-limited media better reveal MGB-BP-3s activity against Gram-negative bacteria and align with in vivo efficacy, highlighting the need for host-mimicking conditions in antibiotic screening.

microbiology↗

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

Target-enrichment sequencing yields valuable genomic data for difficult-to-culture bacteria of public health importance

Genomic data contribute invaluable information to the epidemiological investigation of pathogens of public health importance. However, whole genome sequencing (WGS) of bacteria typically relies on culture, which represents a major hurdle for generating such data for a wide range of species for which culture is challenging. In this study, we assessed the use of culture-free target-enrichment sequencing as a method for generating genomic data for two bacterial species: 1) Bacillus anthracis, which causes anthrax in both people and animals and whose culture requires high level containment facilities; and 2) Mycoplasma amphoriforme, a fastidious emerging human respiratory pathogen. We obtained high quality genomic data for both species directly from clinical samples, with sufficient coverage (>15X) for confident variant calling over at least 80% of the baited genomes for over two thirds of the samples tested. Higher qPCR cycle threshold (Ct) values (indicative of lower pathogen concentrations in the samples), pooling libraries prior to capture, and lower captured library concentration were all statistically associated with lower capture efficiency. The Ct value had the highest predictive value, explaining 52% of the variation in capture efficiency. Samples with Ct values [≤] 30 were over 6 times more likely to achieve the threshold coverage than those with a Ct > 30. We conclude that target-enrichment sequencing provides a valuable alternative to standard WGS following bacterial culture and creates opportunities for an improved understanding of the epidemiology and evolution of many clinically important pathogens for which culture is challenging. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. Scripts used in this study can be accessed on GitHub at https://github.com/tristanpwdennis/bactocap. All sequence data generated during this study have been deposited in the European Nucleotide Archive (ENA) Sequence Read Archive (SRA) under project accession numbers PRJEB46822 (B. anthracis) and PRJEB50216 (M. amphoriforme). Accession numbers for individual samples, along with metadata, laboratory parameters and sequence quality metrics, are available at the University of Glasgows data repository, Enlighten, at http://dx.doi.org/10.5525/gla.researchdata.1249.

molecular biology↗

bioRxiv2021

Genomic epidemiology of Mycobacterium bovis infection in sympatric badger and cattle populations in Northern Ireland.

BackgroundBovine tuberculosis (bTB) is a costly, epidemiologically complex, multi-host, endemic disease. Pathogen whole genome sequencing can improve the resolution of epidemiological tracing. We genome sequenced an exceptional data set of 619 Mycobacterium bovis isolates from badgers and cattle in a 100km2 bTB hotspot. Historical molecular subtyping data permitted the targeting of an endemic pathogen lineage, whose long-term persistence provided an opportunity to study genome epidemiology in detail. To assess whether badger population genetic structure was associated with the spatial distribution of pathogen genetic diversity, we microsatellite genotyped hair samples from 769 badgers trapped in this area. ResultsEight lineages of M. bovis were circulating in the study area, seven of which were likely non-endemic, and imported by animal movement. The endemic lineage exhibited low genetic diversity with an average inter-isolate genetic distance of 7.6 SNPs (s.d. {+/-} 4.0), consistent with contemporary transmission. Bayesian phylogenetic methods determined an evolutionary rate of 0.30 substitutions per genome per year for this lineage, estimating its emergence 40-50 years before present, while Bayesian Skyline analysis identified significant population expansion of the endemic lineage in the 1990s and again in 2011-2012. The phylogeny revealed distinct sub-lineages, all of which contained isolates from both cattle and badger hosts, indicative of the sharing of closely related strains and inter-species transmission. However, the presence of significant badger population genetic structure was not associated with the spatial distribution of M. bovis genetic diversity. ConclusionsOur data provided unparalleled detail on the evolutionary history of an endemic M. bovis lineage. Findings are consistent with ongoing interspecies transmission in the study area but suggest that badger intra-species transmission may not be a major driver of persistence in this area. In addition, the data collected permitted the tracking of incursions of novel pathogen lineages into the study area and means to determine if they were involved in disease transmission.

evolutionary 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↗