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

Decline of meticillin-resistant Staphylococcus aureus in Oxfordshire hospitals is strain-specific and preceded infection-control intensification

Background In the past, strains of Staphylococcus aureus have evolved, expanded, made a marked clinical impact and then disappeared over several years. Faced with rising meticillin-resistant S aureus (MRSA) rates, UK government-supported infection control interventions were rolled out in Oxford Radcliffe Hospitals NHS Trust from 2006 onwards. Methods Using an electronic Database, the authors identified isolation of MRS among 611 434 hospital inpatients admitted to acute hospitals in Oxford, UK, 1 April 1998 to 30 June 2010. Isolation rates were modelled using segmented negative binomial regression for three groups of isolates: from blood cultures, from samples suggesting invasion (eg, cerebrospinal fluid, joint fluid, pus samples) and from surface swabs (eg, from wounds). Findings MRSA isolation rates rose rapidly from 1998 to the end of 2003 (annual increase from blood cultures 23%, 95% CI 16% to 30%), and then declined. The decline accelerated from mid-2006 onwards (annual decrease post-2006 38% from blood cultures, 95% CI 29% to 45%, p=0.003 vs previous decline). Rates of meticillin-sensitive S aureus changed little by comparison, with no evidence for declines 2006 onward (p=0.40); by 2010, sensitive S aureus was far more common than MRSA (blood cultures: 2.9 vs 0.25; invasive samples 14.7 vs 2.0 per 10 000 bedstays). Interestingly, trends in isolation of erythromycin-sensitive and resistant MRSA differed. Erythromycin-sensitive strains rose significantly faster (eg, from blood cultures p=0.002), and declined significantly more slowly (p=0.002), than erythromycin-resistant strains (global p<0.0001). Bacterial typing suggests this reflects differential spread of two major UK MRSA strains (ST22/36), ST36 having declined markedly 2006-2010, with ST22 becoming the dominant MRSA strain. Conclusions MRSA isolation rates were falling before recent intensification of infection-control measures. This, together with strain-specific changes in MRSA isolation, strongly suggests that incompletely understood biological factors are responsible for the much recent variation in MRSA isolation. A major, mainly meticillin-sensitive, S aureus burden remains

The impact of sequencing depth on the inferred taxonomic composition and AMR gene content of metagenomic samples

Shotgun metagenomics is increasingly used to characterise microbial communities, particularly for the investigation of antimicrobial resistance (AMR) in different animal and environmental contexts. There are many different approaches for inferring the taxonomic composition and AMR gene content of complex community samples from shotgun metagenomic data, but there has been little work establishing the optimum sequencing depth, data processing and analysis methods for these samples. In this study we used shotgun metagenomics and sequencing of cultured isolates from the same samples to address these issues. We sampled three potential environmental AMR gene reservoirs (pig caeca, river sediment, effluent) and sequenced samples with shotgun metagenomics at high depth (~ 200 million reads per sample). Alongside this, we cultured single-colony isolates of Enterobacteriaceae from the same samples and used hybrid sequencing (short- and long-reads) to create high- quality assemblies for comparison to the metagenomic data. To automate data processing, we developed an open- source software pipeline, ‘ResPipe’

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

Extended Sequence Typing of Campylobacter spp., United Kingdom

Supplementing Campylobacter spp. multilocus sequence typing with nucleotide sequence typing of 3 antigen genes increased the discriminatory index achieved from 0.975 to 0.992 among 620 clinical isolates from Oxfordshire, United Kingdom. This enhanced typing scheme enabled identification of clusters and retained data required for long-range epidemiologic comparisons of isolates

Self-swabbing versus assisted swabbing for viral detection by qRT-PCR: the experience from SARS-CoV-2 based on a meta-analysis of six prospectively designed evaluations conducted in a UK setting

Purpose: In April 2020, the UK Government implemented NHS Test and Trace to provide SARS-CoV-2 quantitative reverse transcription polymerase chain reaction (qRT-PCR) testing for the public, with nose-and-throat swabbing for samples performed by trained staff. Self-swabbing (SS) would allow rapid scale-up of testing capacity and access. Six studies were undertaken to determine whether SS was as effective for detecting SARS-CoV-2 as swabbing performed by trained staff. Methods: Six prospective studies were conducted between April–October 2020, using six swab/media combinations. Differences between assisted swabbing (AS) and SS were evaluated for concordance, positivity, sensitivity, cycle threshold (Ct) values and void rates. Statistical analysis was performed using 95% confidence intervals (CIs), paired t-tests and model-based methods. Results: Overall, 3,253 individuals were recruited (median age 37 years, 49% female), with 2,933 having valid paired qRT-PCR results. Pooled concordance rate was 98% (95% CI: 96%, 99%). Positivity rate differences for SS (8.1%) and AS (8.4%) and differences in pooled sensitivities between SS (86%; 95% CI: 78%, 92%) and AS (91%; 95% CI: 78%, 96%) were nonsignificant. Both types of swabbing led to pooled void rates below 2% and strongly correlated Ct values. Age, sex and previous swabbing experience did not have a significant impact on concordance or sensitivity. Conclusion: The UK adopted a policy to promote self-testing for SARS-CoV-2 based on data demonstrating equivalence of SS versus AS. Positive outcomes with SS are likely generalisable to testing for other respiratory pathogens, and we consider self-sampling and self-testing essential for future pandemic preparedness

Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae

Metagenomic sequencing of fecal DNA can usefully characterise an individual’s intestinal resistome but is limited by its inability to detect important pathogens that may be present at low abundance, such as carbapenemase or extended-spectrum beta-lactamase producing Enterobacteriaceae. Here we aimed to develop a hybrid protocol to improve detection of resistance genes in Enterobacteriaceae by using a short period of culture enrichment prior to sequencing of DNA extracted directly from the enriched sample. Volunteer feces were spiked with carbapenemase-producing Enterobacteriaceae and incubated in selective broth culture for 6 hours before sequencing. Different DNA extraction methods were compared, including a plasmid extraction protocol to increase the detection of plasmid-associated resistance genes. Although enrichment prior to sequencing increased the detection of carbapenemase genes, the differing growth characteristics of the spike organisms precluded accurate quantification of their concentration prior to culture. Plasmid extraction increased detection of resistance genes present on plasmids, but the effects were heterogeneous and dependent on plasmid size. Our results demonstrate methods of improving the limit of detection of selected resistance mechanisms in a fecal resistome assay, but they also highlight the difficulties in using these techniques for accurate quantification and should inform future efforts to achieve this goa

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. We 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 70 Illumina MiSeq/MiniSeq the workflow from patient sample to results can be completed in 44/16 hours at a reagent cost of £96/£198 per sample. We 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 flowcell version R9.4, the estimated turnaround time from patient to identification of BCG, detection of pyrazinamide resistance, and phylogenetic placement was 7.5 hours, with full susceptibility results 5 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

Protection against SARS-CoV-2 Omicron BA.4/5 variant following booster vaccination or breakthrough infection in the UK

Following primary SARS-CoV-2 vaccination, whether boosters or breakthrough infections provide greater protection against SARS-CoV-2 infection is incompletely understood. Here we investigated SARS-CoV-2 antibody correlates of protection against new Omicron BA.4/5 (re-)infections and anti-spike IgG antibody trajectories after a third/booster vaccination or breakthrough infection following second vaccination in 154,149 adults ≥18 y from the United Kingdom general population. Higher antibody levels were associated with increased protection against Omicron BA.4/5 infection and breakthrough infections were associated with higher levels of protection at any given antibody level than boosters. Breakthrough infections generated similar antibody levels to boosters, and the subsequent antibody declines were slightly slower than after boosters. Together our findings show breakthrough infection provides longer-lasting protection against further infections than booster vaccinations. Our findings, considered alongside the risks of severe infection and long-term consequences of infection, have important implications for vaccine policy

Improving the representativeness of UK’s national COVID-19 Infection Survey through spatio-temporal regression and post-stratification

Population-representative estimates of SARS-CoV-2 infection prevalence and antibody levels in specific geographic areas at different time points are needed to optimise policy responses. However, even population-wide surveys are potentially impacted by biases arising from differences in participation rates across key groups. Here, we used spatio-temporal regression and post-stratification models to UK’s national COVID-19 Infection Survey (CIS) to obtain representative estimates of PCR positivity (6,496,052 tests) and antibody prevalence (1,941,333 tests) for different regions, ages and ethnicities (7-December-2020 to 4-May-2022). Not accounting for vaccination status through post-stratification led to small underestimation of PCR positivity, but more substantial overestimations of antibody levels in the population (up to 21 percentage points), particularly in groups with low vaccine uptake in the general population. There was marked variation in the relative contribution of different areas and age-groups to each wave. Future analyses of infectious disease surveys should take into account major drivers of outcomes of interest that may also influence participation, with vaccination being an important factor to consider