Biology subjects

Peto, T. E.

Publications and source records attributed to Peto, T. E..

7 recordsLinked to original sources

bioRxiv2018

A role for tetracycline selection in the evolution of Clostridium difficile PCR-ribotype 078

Farm animals have been identified as reservoirs of Clostridium difficile PCR-ribotype 078 (RT078). Since 2005, the incidence of human clinical cases (frequently severe), with this genotype has increased. We aimed to understand this change, by studying the recent evolutionary history of RT078. Phylogenetic analysis of international genomes (isolates from 2006-2014) revealed several recent clonal expansions. A common ancestor of each expansion had independently acquired different alleles of the tetracycline resistance gene tetM. Consequently, an unusually high proportion of RT078 genomes were tetM positive (76.5%). Additional tetracycline resistance determinants were also identified, some for the first time in C. difficile (efflux pump tet40). Each tetM-clonal expansion lacked geographic structure, indicating rapid international spread. Resistance determinants for C. difficile-infection-triggering antimicrobials including fluoroquinolones and clindamycin were comparatively rare in RT078. Tetracyclines are used intensively in agriculture; this selective pressure, plus rapid spread via the food-chain may explain the increased RT078 prevalence in humans.

microbiology↗

bioRxiv2018

Control of artefactual variation in reported inter-sample relatedness during clinical use of a Mycobacterium tuberculosis sequencing pipeline

Contact tracing requires reliable identification of closely related bacterial isolates. When we noticed the reporting of artefactual variation between M. tuberculosis isolates during routine next generation sequencing of Mycobacterium spp, we investigated its basis in 2,018 consecutive M. tuberculosis isolates. In the routine process used, clinical samples were decontaminated and inoculated into broth cultures; from positive broth cultures DNA was extracted, sequenced, reads mapped, and consensus sequences determined. We investigated the process of consensus sequence determination, which selects the most common nucleotide at each position. Having determined the high-quality read depth and depth of minor variants across 8,006 M. tuberculosis genomic regions, we quantified the relationship between the minor variant depth and the amount of non-Mycobacterial bacterial DNA, which originates from commensal microbes killed during sample decontamination. In the presence of non-Mycobacterial bacterial DNA, we found significant increases in minor variant frequencies of more than 1.5 fold in 242 regions covering 5.1% of the M. tuberculosis genome. Included within these were four high variation regions strongly influenced by the amount of non-Mycobacterial bacterial DNA. Excluding these four regions from pairwise distance comparisons reduced biologically implausible variation from 5.2% to 0% in an independent validation set derived from 226 individuals. Thus, we have demonstrated an approach identifying critical genomic regions contributing to clinically relevant artefactual variation in bacterial similarity searches. The approach described monitors the outputs of the complex multi-step laboratory and bioinformatics process, allows periodic process adjustments, and will have application to quality control of routine bacterial genomics.

microbiology↗

bioRxiv2018

Validation of a 14-drug microtitre plate that includes delamanid and bedaquiline for susceptibility testing of M. tuberculosis

UKMYC5 is a 96-well microtitre plate designed by the Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) to enable the measurement of minimum inhibitory concentrations (MICs) of 14 different anti-TB compounds for >30,000 clinical tuberculosis isolates. Unlike the MYCOTB plate, on which UKMYC5 is based, the plate included two new (bedaquiline and delamanid) and two repurposed (clofazimine and linezolid) compounds. UKMYC5 plates were tested by seven laboratories on four continents using a panel of 19 external quality assessment (EQA) strains, including H37Rv. To assess the optimal combination of reading method and incubation time, MICs were measured from each plate by two readers using three methods (mirrored-box, microscope and Vizion Digital viewing system) after 7, 10, 14 and 21 days incubation. In addition, all EQA strains were whole-genome sequenced and phenotypically characterized by 7H10/7H11 agar proportion method (APM) and MGIT960. We conclude that the UKMYC5 plate is optimally read using the Vizion system after 14 days incubation, achieving an inter-reader agreement of 97.9% and intra- and inter-laboratory reproducibilities of 95.6% and 93.1%, respectively. The mirrored-box had similar reproducibilities. Strains classified as resistant by APM, MGIT960 or the presence of mutations known to confer resistance consistently record elevated MICs compared with those strains classified as susceptible. Finally, the UKMYC5 plate records intermediate MICs for one strain which the APM measured MICs close the applied critical concentration, providing early evidence that the UKMYC5 plate can quantitatively measure the magnitude of resistance to anti-TB compounds due to specific genetic variation.

microbiology↗

bioRxiv2017

Automated detection of Mycobacterial growth on 96-well plates for rapid and accurate Tuberculosis drug susceptibility testing

M. tuberculosis grows slowly and is challenging to work with experimentally compared with many other bacteria. Although microtitre plates have the potential to enable high-throughput phenotypic testing of M. tuberculosis, they can be difficult to read and interpret. Here we present a software package, the Automated Mycobacterial Growth Detection Algorithm (AMyGDA), that measures how much M. tuberculosis is growing in each well of a 96-well microtitre plate. The plate used here has serial dilutions of 14 anti-tuberculosis drugs, thereby permitting the minimum inhibitory concentrations (MICs) to be elucidated. The two participating laboratories each inoculated ten 96-well plates with the standard H37Rv reference strain and, after two weeks incubation, measured the MICs for all 14 drugs on each plate and took a photograph. By analysing the images, we demonstrate that AMyGDA is reproducible, and that the MICs measured are comparable to those measured by a laboratory scientist. AMyGDA software will be used by the Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) to measure the drug susceptibility profile of a large number (> 30,000) of samples of M. tuberculosis from patients over the next few years.

microbiology↗

bioRxiv2017

Severe infections emerge from the microbiome by adaptive evolution

Bacteria responsible for the greatest global mortality colonize the human microbiome far more frequently than they cause severe infections. Whether mutation and selection within the microbiome accompany infection is unknown. We investigated de novo mutation in 1163 Staphylococcus aureus genomes from 105 infected patients with nose-colonization. We report that 72% of infections emerged from the microbiome, with infecting and nose-colonizing bacteria showing parallel adaptive differences. We found 2.8-to-3.6-fold enrichments of protein-altering variants in genes responding to rsp, which regulates surface antigens and toxicity; agr, which regulates quorum-sensing, toxicity and abscess formation; and host-derived antimicrobial peptides. Adaptive mutations in pathogenesis-associated genes were 3.1-fold enriched in infecting but not nose-colonizing bacteria. None of these signatures were observed in healthy carriers nor at the species-level, suggesting disease-associated, short-term, within-host selection pressures. Our results show that infection, like a cancer of the microbiome, emerges through spontaneous adaptive evolution, raising new possibilities for diagnosis and treatment.\n\nOne Sentence SummaryLife-threatening S. aureus infections emerge from nose microbiome bacteria in association with repeatable adaptive evolution.

genomics↗

bioRxiv2017

Genomic epidemiology of global Klebsiella pneumoniae carbapenemase (KPC)-producing E. coli

The dissemination of carbapenem resistance in Escherichia coli has major implications for the management of common human infections. blaKPC, encoding a transmissible carbapenemase (KPC), has historically largely been associated with Klebsiella pneumoniae, a predominant plasmid (pKpQIL), and a specific transposable element (Tn4401, ~10kb). Here we characterize the genetic features of the emergence of blaKPC in global E. coli, 2008-2013, using both long-and short-read whole genome sequencing.\n\nAmongst 43/45 successfully sequenced blaKPC-E. coli strains, we identified high strain (n=21 sequence types, 18% of annotated genes in the core genome); plasmid ([≥]9 replicon types); and blaKPC-associated, mobile genetic element (MGE) diversity (50% not within complete Tn4401 elements). We also found evidence of interspecies, regional and international plasmid spread. In several cases blaKPC was found on high copy number, small Col-like plasmids, previously associated with horizontal transmission of resistance genes in the absence of antimicrobial selection pressures.\n\nE. coli is a common human pathogen, but also a commensal in a multiple environmental and animal reservoirs, and easily transmissible. The association of blaKPC with a range of MGEs previously linked to the successful spread of widely endemic resistance mechanisms (e.g. blaTEM, blaCTX-M) suggests that it is likely to become similarly prevalent.

microbiology↗

bioRxiv2016

Same-day diagnostic and surveillance data for tuberculosis via whole genome sequencing of direct respiratory samples.

Routine full characterization of Mycobacterium tuberculosis (TB) is culture-based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near point of care.\n\nWe demonstrate a low-cost DNA extraction method for TB WGS direct from patient samples. We initially evaluated the method using the Illumina MiSeq sequencer (40 smear-positive respiratory samples, obtained after routine clinical testing, and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction was obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. Using an Illumina MiSeq/MiniSeq the workflow from patient sample to results can be completed in 44/16 hours at a cost of {pound}96/{pound}198 per sample.\n\nWe then employed a non-specific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis BCG strain (BCG), and to combined culture-negative sputum DNA and BCG DNA. For the latest flowcell, the estimated turnaround time from patient to identification of BCG was 6 hours, with full susceptibility and surveillance results 2 hours later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of the MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis in direct samples.

microbiology↗