Short-term genome stability of serial Clostridium difficile ribotype 027 isolates in an experimental gut model and recurrent human disease

Copyright: © 2013 Eyre et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedClostridium difficile whole genome sequencing has the potential to identify related isolates, even among otherwise indistinguishable strains, but interpretation depends on understanding genomic variation within isolates and individuals.Serial isolates from two scenarios were whole genome sequenced. Firstly, 62 isolates from 29 timepoints from three in vitro gut models, inoculated with a NAP1/027 strain. Secondly, 122 isolates from 44 patients (2–8 samples/patient) with mostly recurrent/on-going symptomatic NAP-1/027 C. difficile infection. Reference-based mapping was used to identify single nucleotide variants (SNVs).Across three gut model inductions, two with antibiotic treatment, total 137 days, only two new SNVs became established. Pre-existing minority SNVs became dominant in two models. Several SNVs were detected, only present in the minority of colonies at one/two timepoints. The median (inter-quartile range) [range] time between patients’ first and last samples was 60 (29.5–118.5) [0–561] days. Within-patient C. difficile evolution was 0.45 SNVs/called genome/year (95%CI 0.00–1.28) and within-host diversity was 0.28 SNVs/called genome (0.05–0.53). 26/28 gut model and patient SNVs were non-synonymous, affecting a range of gene targets.The consistency of whole genome sequencing data from gut model C. difficile isolates, and the high stability of genomic sequences in isolates from patients, supports the use of whole genome sequencing in detailed transmission investigations.Peer reviewe

Comparison of Control of Clostridium difficile Infection in Six English Hospitals Using Whole-Genome Sequencing

Background: Variation in Clostridium difficile infection (CDI) rates between healthcare institutions suggests overall incidence could be reduced if the lowest rates could be achieved more widely. Methods: We investigated whether whole-genome sequencing (WGS) of consecutive C. difficile isolates from six English hospitals over one year (2013-14) could be used to assess infection control performance. Fecal samples with a positive initial screen for C. difficile (GDH or toxin-PCR) were cultured and sequenced. Within each hospital, we estimated the proportion of cases plausibly acquired from previous cases, defined by an isolate ≤2 single nucleotide polymorphisms different from a previous isolate in the last 90-days. Results: 851/971(87.6%) sequenced culture-positive samples were toxigenic, and 451(46.4%) were fecal-toxin-positive. 128/652(20%,95%CI 17-23%) toxigenic isolates >90-days after the study started were genetically-linked to a prior patient’s isolate from the previous 90-days. Hospital-2 had the fewest linked isolates, 7/105(7%,3-13%), hospital-1 an intermediate proportion, 9/70(13%,6-23%), while hospitals 3-6 had similar proportions of linked isolates (22-26%) (p≤0.002 comparing hospital-2 vs 3-6). Results were similar adjusting for locally-circulating ribotypes. Adjusting for hospital, ribotype-027 had the highest proportion of linked isolates (57%, 95%CI 29-81%). Fecal-toxin-positive and toxin-negative patients were similarly infectious in terms of being a potential transmission donor, OR=1.01(0.68-1.49,p=0.97). There was no association between the estimated proportion of cases linked to a previous case within 90-days and testing rates (p=0.60). Conclusions: WGS can be used to identify varying rates of C. difficile transmission in different locations, and offers the potential to allow targeted efforts to reduce CDI incidence

Ethnically diverse urban transmission networks of Neisseria gonorrhoeae without evidence of HIV serosorting

Objective We aimed to characterise gonorrhoea transmission patterns in a diverse urban population by linking genomic, epidemiological and antimicrobial susceptibility data. Methods Neisseria gonorrhoeae isolates from patients attending sexual health clinics at Barts Health NHS Trust, London, UK, during an eleven-month period underwent whole-genome sequencing and antimicrobial susceptibility testing. We combined laboratory and patient data to investigate the transmission network structure. Results One hundred and fifty-eight isolates from 158 patients were available with associated descriptive data. One hundred and twenty-nine (82%) patients identified as male and 25 (16%) as female; 4 (3%) records lacked gender information. Self-described ethnicities were: 51 (32%) English/Welsh/Scottish; 33 (21%) White, other; 23 (15%) Black British/Black African/Black, other; 12 (8%) Caribbean; 9 (6%) South Asian; 6 (4%) mixed ethnicity; 10 (6%) other; data were missing for 14 (9%). Self-reported sexual orientations were 82 (52%) men who have sex with men; 49 (31%) heterosexual; 2 (1%) bisexual; data missing for 25 individuals. Twenty-two (14%) patients were HIV-positive. Whole genome sequence data were generated for 151 isolates, which linked 75 (50%) patients to at least one other case. Using sequencing data, we found no evidence of transmission networks related to specific ethnic groups (p=0.64) or of HIV serosorting (p=0.35). Of 82 MSM/bisexual patients with sequencing data, 45 (55%) belonged to clusters of ≥2 cases, compared to 16/44 (36%) heterosexuals with sequencing data (p=0.06). Conclusion We demonstrate links between 50% of patients in transmission networks using a relatively small sample in a large cosmopolitan city. We found no evidence of HIV serosorting. Our results do not support assortative selectivity as an explanation for differences in gonorrhoea incidence between ethnic groups

Mortality risks associated with empirical antibiotic activity in E. coli bacteraemia: an analysis of electronic health records

Background: Reported bacteraemia outcomes following inactive empirical antibiotics (based on in vitro testing) are conflicting, potentially reflecting heterogeneity in causative species, MIC breakpoints defining resistance/susceptibility, and times to rescue therapy. Methods: We investigated adult inpatients with Escherichia coli bacteraemia at Oxford University Hospitals, UK, from 4 February 2014 to 30 June 2021 who were receiving empirical amoxicillin/clavulanate with/without other antibiotics. We used Cox regression to analyse 30 day all-cause mortality by in vitro amoxicillin/clavulanate susceptibility (activity) using the EUCAST resistance breakpoint (>8/2 mg/L), categorical MIC, and a higher resistance breakpoint (>32/2 mg/L), adjusting for other antibiotic activity and confounders including comorbidities, vital signs and blood tests. Results: A total of 1720 E. coli bacteraemias (1626 patients) were treated with empirical amoxicillin/clavulanate. Thirty-day mortality was 193/1400 (14%) for any active baseline therapy and 52/320 (16%) for inactive baseline therapy (P = 0.17). With EUCAST breakpoints, there was no evidence that mortality differed for inactive versus active amoxicillin/clavulanate [adjusted HR (aHR) = 1.27 (95% CI 0.83–1.93); P = 0.28], nor of an association with active aminoglycoside (P = 0.93) or other active antibiotics (P = 0.18). Considering categorical amoxicillin/clavulanate MIC, MICs > 32/2 mg/L were associated with mortality [aHR = 1.85 versus MIC = 2/2 mg/L (95% CI 0.99–3.73); P = 0.054]. A higher resistance breakpoint (>32/2 mg/L) was independently associated with higher mortality [aHR = 1.82 (95% CI 1.07–3.10); P = 0.027], as were MICs > 32/2 mg/L with active empirical aminoglycosides [aHR = 2.34 (95% CI 1.40–3.89); P = 0.001], but not MICs > 32/2 mg/L with active non-aminoglycoside antibiotic(s) [aHR = 0.87 (95% CI 0.40–1.89); P = 0.72]. Conclusions: We found no evidence that EUCAST-defined amoxicillin/clavulanate resistance was associated with increased mortality, but a higher resistance breakpoint (MIC > 32/2 mg/L) was. Additional active baseline non-aminoglycoside antibiotics attenuated amoxicillin/clavulanate resistance-associated mortality, but aminoglycosides did not. Granular phenotyping and comparison with clinical outcomes may improve AMR breakpoints

Genomic diversity affects the accuracy of bacterial single-nucleotide polymorphism-calling pipelines

Background: Accurately identifying SNPs from bacterial sequencing data is an essential requirement for using genomics to track transmission and predict important phenotypes such as antimicrobial resistance. However, most previous performance evaluations of SNP calling have been restricted to eukaryotic (human) data. Additionally, bacterial SNP calling requires choosing an appropriate reference genome to align reads to, which, together with the bioinformatic pipeline, affects the accuracy and completeness of a set of SNP calls obtained. This study evaluates the performance of 209 SNP calling pipelines using a combination of simulated data from 254 strains of 10 clinically common bacteria and real data from environmentally-sourced and genomically diverse isolates within the genera Citrobacter, Enterobacter, Escherichia and Klebsiella. Results: We evaluated the performance of 209 SNP calling pipelines, aligning reads to genomes of the same or a divergent strain. Irrespective of pipeline, a principal determinant of reliable SNP calling was reference genome selection. Across multiple taxa, there was a strong inverse relationship between pipeline sensitivity and precision, and the Mash distance (a proxy for average nucleotide divergence) between reads and reference genome. The effect was especially pronounced for diverse, recombinogenic, bacteria such as Escherichia coli, but less dominant for clonal species such as Mycobacterium tuberculosis. Conclusions: The accuracy of SNP calling for a given species is compromised by increasing intra-species diversity. When reads were aligned to the same genome from which they were sequenced, among the highest performing pipelines was Novoalign/GATK. By contrast, when reads were aligned to particularly divergent genomes, the highest-performing pipelines often employed the aligners NextGenMap or SMALT, and/or the variant callers LoFreq, mpileup or Strelka

Constraining the Milky Way potential with a 6-D phase-space map of the GD-1 stellar stream

The narrow GD-1 stream of stars, spanning 60 deg on the sky at a distance of ~10 kpc from the Sun and ~15 kpc from the Galactic center, is presumed to be debris from a tidally disrupted star cluster that traces out a test-particle orbit in the Milky Way halo. We combine SDSS photometry, USNO-B astrometry, and SDSS and Calar Alto spectroscopy to construct a complete, empirical 6-dimensional phase-space map of the stream. We find that an eccentric orbit in a flattened isothermal potential describes this phase-space map well. Even after marginalizing over the stream orbital parameters and the distance from the Sun to the Galactic center, the orbital fit to GD-1 places strong constraints on the circular velocity at the Sun's radius V_c=224 \pm 13 km/s and total potential flattening q_\Phi=0.87^{+0.07}_{-0.04}. When we drop any informative priors on V_c the GD-1 constraint becomes V_c=221 \pm 18 km/s. Our 6-D map of GD-1 therefore yields the best current constraint on V_c and the only strong constraint on q_\Phi at Galactocentric radii near R~15 kpc. Much, if not all, of the total potential flattening may be attributed to the mass in the stellar disk, so the GD-1 constraints on the flattening of the halo itself are weak: q_{\Phi,halo}>0.89 at 90% confidence. The greatest uncertainty in the 6-D map and the orbital analysis stems from the photometric distances, which will be obviated by Gaia.Comment: 16 pages, 18 figures; accepted to ApJ; full resolution version is available at http://www.ast.cam.ac.uk/~koposov/files/gd1_fullres.pd

Relationship between bacterial strain type, host biomarkers, and mortality in clostridium difficile infection

Background: Despite substantial interest in biomarkers, their impact on clinical outcomes and variation with bacterial strain has rarely been explored using integrated databases. Methods: From September 2006 to May 2011, strains isolated from Clostridium difficile toxin enzyme immunoassay (EIA)-positive fecal samples from Oxfordshire, United Kingdom (approximately 600 000 people) underwent multilocus sequence typing. Fourteen-day mortality and levels of 15 baseline biomarkers were compared between consecutive C. difficile infections (CDIs) from different clades/sequence types (STs) and EIA-negative controls using Cox and normal regression adjusted for demographic/clinical factors. Results: Fourteen-day mortality was 13% in 2222 adults with 2745 EIA-positive samples (median, 78 years) vs 5% in 20 722 adults with 27 550 EIA-negative samples (median, 74 years) (absolute attributable mortality, 7.7%; 95% CI, 6.4%-9.0%). Mortality was highest in clade 5 CDIs (25% [16 of 63]; polymerase chain reaction (PCR) ribotype 078/ST 11), then clade 2 (20% [111 of 560]; 99% PCR ribotype 027/ST 1) versus clade 1 (12% [137 of 1168]; adjusted P <. 0001). Within clade 1, 14-day mortality was only 4% (3 of 84) in ST 44 (PCR ribotype 015) (adjusted P =. 05 vs other clade 1). Mean baseline neutrophil counts also varied significantly by genotype: 12.4, 11.6, and 9.5 × 109 neutrophils/L for clades 5, 2 and 1, respectively, vs 7.0 × 109 neutrophils/L in EIA-negative controls (P <. 0001) and 7.9 × 109 neutrophils/L in ST 44 (P =. 08). There were strong associations between C. difficile-type-specific effects on mortality and neutrophil/white cell counts (rho = 0.48), C-reactive-protein (rho = 0.43), eosinophil counts (rho =-0.45), and serum albumin (rho =-0.47). Biomarkers predicted 30%-40% of clade-specific mortality differences. Conclusions: C. difficile genotype predicts mortality, and excess mortality correlates with genotype-specific changes in biomarkers, strongly implicating inflammatory pathways as a major influence on poor outcome after CDI. PCR ribotype 078/ST 11 (clade 5) leads to severe CDI; thus ongoing surveillance remains essential

The Effect of Transposable Element Insertions on Gene Expression Evolution in Rodents

Background:Many genomes contain a substantial number of transposable elements (TEs), a few of which are known to be involved in regulating gene expression. However, recent observations suggest that TEs may have played a very important role in the evolution of gene expression because many conserved non-genic sequences, some of which are know to be involved in gene regulation, resemble TEs. Results:Here we investigate whether new TE insertions affect gene expression profiles by testing whether gene expression divergence between mouse and rat is correlated to the numbers of new transposable elements inserted near genes. We show that expression divergence is significantly correlated to the number of new LTR and SINE elements, but not to the numbers of LINEs. We also show that expression divergence is not significantly correlated to the numbers of ancestral TEs in most cases, which suggests that the correlations between expression divergence and the numbers of new TEs are causal in nature. We quantify the effect and estimate that TE insertion has accounted for ~20% (95% confidence interval: 12% to 26%) of all expression profile divergence in rodents. Conclusions:We conclude that TE insertions may have had a major impact on the evolution of gene expression levels in rodents

Identifying Verticillium dahliae resistance in strawberry through disease screening of multiple populations and image based phenotyping

© 2019 Cockerton, Li, Vickerstaff, Eyre, Sargent, Armitage, Marina-Montes, Garcia-Cruz, Passey, Simpson and Harrison. Verticillium dahliae is a highly detrimental pathogen of soil cultivated strawberry (Fragaria x ananassa). Breeding of Verticillium wilt resistance into commercially viable strawberry cultivars can help mitigate the impact of the disease. In this study we describe novel sources of resistance identified in multiple strawberry populations, creating a wealth of data for breeders to exploit. Pathogen-informed experiments have allowed the differentiation of subclade-specific resistance responses, through studying V. dahliae subclade II-1 specific resistance in the cultivar “Redgauntlet” and subclade II-2 specific resistance in “Fenella” and “Chandler.” A large-scale low-cost phenotyping platform was developed utilizing automated unmanned vehicles and near infrared imaging cameras to assess field-based disease trials. The images were used to calculate disease susceptibility for infected plants through the normalized difference vegetation index score. The automated disease scores showed a strong correlation with the manual scores. A co-dominant resistant QTL; FaRVd3D, present in both “Redgauntlet” and “Hapil” cultivars exhibited a major effect of 18.3% when the two resistance alleles were combined. Another allele, FaRVd5D, identified in the “Emily” cultivar was associated with an increase in Verticillium wilt susceptibility of 17.2%, though whether this allele truly represents a susceptibility factor requires further research, due to the nature of the F1 mapping population. Markers identified in populations were validated across a set of 92 accessions to determine whether they remained closely linked to resistance genes in the wider germplasm. The resistant markers FaRVd2B from “Redgauntlet” and FaRVd6D from “Chandler” were associated with resistance across the wider germplasm. Furthermore, comparison of imaging versus manual phenotyping revealed the automated platform could identify three out of four disease resistance markers. As such, this automated wilt disease phenotyping platform is considered to be a good, time saving, substitute for manual assessment