Biology subjects

James, R. S.

Publications and source records attributed to James, R. S..

5 recordsLinked to original sources

bioRxiv2020

Metabolic potential of uncultured Antarctic soil bacteria revealed through long-read metagenomic sequencing

The growing problem of antibiotic resistance has led to the exploration of uncultured bacteria as potential sources of new antimicrobials. PCR amplicon analyses and short-read sequencing studies of samples from different environments have reported evidence of high biosynthetic gene cluster (BGC) diversity in metagenomes. However, few complete BGCs from uncultivated bacteria have been recovered, making assessment of BGC diversity difficult. Here, long-read sequencing and genome mining were used to recover >1400 mostly complete BGCs that demonstrate the rich diversity of BGCs from uncultivated lineages present in soil from Mars Oasis, Antarctica. The phyla Acidobacteriota, Verrucomicrobiota and Gemmatimonadota, but also the actinobacterial classes Acidimicrobiia, Thermoleophilia, and the gammaproteobacterial order UBA7966, were found to encode a large number of highly divergent BGCs. Our findings underline the biosynthetic potential of underexplored phyla as well as unexplored lineages within seemingly well-studied producer phyla. They also showcase long-read metagenomic sequencing as a promising way to access the untapped reservoir of specialised metabolites of the uncultured majority of microbes.

microbiology↗

bioRxiv2020

Metagenomics Strain Resolution on Assembly Graphs

We introduce a novel bioinformatics pipeline, STrain Resolution ON assembly Graphs (STRONG), which identifies strains de novo, when multiple metagenome samples from the same community are available. STRONG performs coassembly, followed by binning into metagenome assembled genomes (MAGs), but uniquely it stores the coassembly graph prior to simplification of variants. This enables the subgraphs for individual single-copy core genes (SCGs) in each MAG to be extracted. It can then thread back reads from the samples to compute per sample coverages for the unitigs in these graphs. These graphs and their unitig coverages are then used in a Bayesian algorithm, BayesPaths, that determines the number of strains present, their sequences or haplotypes on the SCGs and their abundances in each of the samples. Our approach both avoids the ambiguities of read mapping and allows more of the information on co-occurrence of variants in reads to be utilised than if variants were treated independently, whilst at the same time exploiting the correlation of variants across samples that occurs when they are linked in the same strain. We compare STRONG to the current state of the art on synthetic communities and demonstrate that we can recover more strains, more accurately, and with a realistic estimate of uncertainty deriving from the variational Bayesian algorithm employed for the strain resolution. On a real anaerobic digestor time series we obtained strain-resolved SCGs for over 300 MAGs that for abundant community members match those observed from long Nanopore reads.

bioinformatics↗

bioRxiv2020

Genetic characterisation of a large antibiotic resistant environmental ST131 E. coli plasmid using a long read hybrid assembly approach

Escherichia coli Strain Type 131 are a globally disseminated environmental E. coli that has been linked to the capture and spread of plasmid mediated blaCTX-M type extended spectrum beta-lactamase (ESBLs). Accurately identifying such resistance genes in their wider genetic context provides a greater understanding of the mechanisms of selection and persistence in the environment. In this study we use a novel DNA extraction and enrichment method in combination with a custom long-read scaffold hybrid-assembly and polishing pipe line to identify the genetic context of the plasmid borne blaCTX-M gene previously identified in an ST131 environmental E. coli isolate. This has allowed us to discern the complete structure of a ~100kb environmental plasmid and further resolve the blaCTX-M variant to the group 9 blaCTX-M-27 gene. The upstream IS26 insertion element associated with the global capture and dissemination of blaCTX-M-15 was also identified in proximity to blaCTX-M-27. Furthermore, the lack of conjugative machinery identified on this plasmid, in combination with a toxin-antitoxin and plasmid partitioning system, indicates a mechanism of vertical transmission to maintain persistence in a population.

molecular biology↗

bioRxiv2020

Impact of trimethoprim on the river microbiome and antimicrobial resistance.

Recent evidence suggests that anthropogenic activity can increase the levels of antimicrobial resistance (AMR) in the environment. Rivers and waterways are significant examples of environmental settings that have become repositories of antibiotics and antibiotic resistance genes (ARGs). Our recent study quantified drug concentrations in freshwater samples taken at a range of sites located on the Thames catchment; the highest levels of antibiotics and other drugs were recorded downstream of waste water treatment plants (WWTPs). One specific antibiotic: Trimethoprim (TMP) was shown at elevated concentrations reaching 2000ng/L at particular sites. We have also shown a correlative relationship between the residue of TMP and the prevalence of sulfonamide antibiotic resistance genes such as sul1. Despite this, there is still no evidence of a causative relationship between TMP concentrations and the prevalence of the ARGs at river sites. The aim of the current study was to conduct in-depth analysis using a combination of large metagenomic, geospatial and chemical datasets, in order to conduct a comparison between those sites with the highest TMP and lowest TMP levels across the Thames catchment. We aimed to establish the proximity of these sites to WWTPs, their population equivalence (PE) and land coverage. A secondary aim was to investigate seasonal variation in TMP and ARGs. Exploring these factors will help to decipher the clinical relevance of ARG accumulation at river sites. A significant correlation was shown between TMP levels at river sites and their distance downstream from a WWTP. Three sites located on the Rivers Cut and Ray showed significantly higher TMP concentrations in winter compared to summer. The population equivalence (PE) for sites with the highest TMP levels was significantly higher than those with the lowest levels. The land coverage of sites with the highest TMP levels was significantly more urban/suburban than sites with the lowest TMP concentrations, which were found to be significantly more arable. Five ARGs relevant to TMP and sulfonamides were identified across the Thames catchment. The most prevalent ARG was sul1, which was significantly more prevalent in winter compared to summer. By contrast sul2 was found to be significantly more prevalent in summer compared to winter at a site on the River Coln. The prevalence of the class 1 integron marker gene (inti1) did not differ significantly by season or between sites with the highest/lowest TMP levels.

microbiology↗

bioRxiv2019

LAMBDR: Long-range amplification and Nanopore sequencing of Mycobacterium bovis direct-repeat region. A novel method for in-silico spoligotyping of M. bovis directly from badger faeces.

The environment is an overlooked source of Mycobacterium bovis, the causative agent of bovine TB. Long read, end to end sequencing of variable repeat regions across the M. bovis genome was evaluated as a method of acquiring rapid strain level resolution directly from environmental samples. Eight samples of M. bovis, two BCG strains (Danish and Pasteur), and a single M. tuberculosis type culture (NCTC 13144) were used to generate data for this method. Long range PCR amplification of the direct repeat region was used to synthesize [~]5kb template DNA for onward sequence analysis. This has permitted culture independent identification of M. bovis spoligotypes present in the environment. Sequence level analysis of the direct repeat region showed that spoligotyping may underestimate strain diversity due to the inability to identify both SNPs and primer binding mutations using a biotinylated hybridisation approach.

molecular biology↗