Presence of phage-plasmids in multiple serovars of Salmonella enterica.

Evidence is accumulating in the literature that the horizontal spread of antimicrobial resistance (AMR) genes mediated by bacteriophages and bacteriophage-like plasmid (phage-plasmid) elements is much more common than previously envisioned. For instance, we recently identified and characterized a circular P1-like phage-plasmid harbouring a bla CTX-M-15 gene conferring extended-spectrum beta-lactamase (ESBL) resistance in Salmonella enterica serovar Typhi. As the prevalence and epidemiological relevance of such mechanisms has never been systematically assessed in Enterobacterales, in this study we carried out a follow-up retrospective analysis of UK Salmonella isolates previously sequenced as part of routine surveillance protocols between 2016 and 2021. Using a high-throughput bioinformatics pipeline we screened 47 784 isolates for the presence of the P1 lytic replication gene repL, identifying 226 positive isolates from 25 serovars and demonstrating that phage-plasmid elements are more frequent than previously thought. The affinity for phage-plasmids appears highly serovar-dependent, with several serovars being more likely hosts than others; most of the positive isolates (170/226) belonged to S. Typhimurium ST34 and ST19. The phage-plasmids ranged between 85.8 and 98.2 kb in size, with an average length of 92.1 kb; detailed analysis indicated a high amount of diversity in gene content and genomic architecture. In total, 132 phage-plasmids had the p0111 plasmid replication type, and 94 the IncY type; phylogenetic analysis indicated that both horizontal and vertical gene transmission mechanisms are likely to be involved in phage-plasmid propagation. Finally, phage-plasmids were present in isolates that were resistant and non-resistant to antimicrobials. In addition to providing a first comprehensive view of the presence of phage-plasmids in Salmonella, our work highlights the need for a better surveillance and understanding of phage-plasmids as AMR carriers, especially through their characterization with long-read sequencing.

Re-analysis of an outbreak of Shiga toxin-producing Escherichia coli O157:H7 associated with raw drinking milk using Nanopore sequencing.

The aim of this study was to compare Illumina and Oxford Nanopore Technology (ONT) sequencing data to quantify genetic variation to assess within-outbreak strain relatedness and characterise microevolutionary events in the accessory genomes of a cluster of 23 genetically and epidemiologically linked isolates related to an outbreak of Shiga toxin-producing Escherichia coli O157:H7 caused by the consumption of raw drinking milk. There were seven discrepant variants called between the two technologies, five were false-negative or false-positive variants in the Illumina data and two were false-negative calls in ONT data. After masking horizontally acquired sequences such as prophages, analysis of both short and long-read sequences revealed the 20 isolates linked to the outbreak in 2017 had a maximum SNP distance of one SNP between each other, and a maximum of five SNPs when including three additional strains identified in 2019. Analysis of the ONT data revealed a 47 kbp deletion event in a terminal compound prophage within one sample relative to the remaining samples, and a 0.65 Mbp large chromosomal rearrangement (inversion), within one sample relative to the remaining samples. Furthermore, we detected two bacteriophages encoding the highly pathogenic Shiga toxin (Stx) subtype, Stx2a. One was typical of Stx2a-phage in this sub-lineage (Ic), the other was atypical and inserted into a site usually occupied by Stx2c-encoding phage. Finally, we observed an increase in the size of the pO157 IncFIB plasmid (1.6 kbp) in isolates from 2019 compared to those from 2017, due to the duplication of insertion elements within the plasmids from the more recently isolated strains. The ability to characterize the accessory genome in this way is the first step to understanding the significance of these microevolutionary events and their impact on the genome plasticity and virulence between strains of this zoonotic, foodborne pathogen.

Genomic epidemiology of the clinically dominant clonal complex 1 in the Listeria monocytogenes population in the UK.

Listeria monocytogenes is a food-borne pathogen, typically affecting the elderly, immunocompromised patients and pregnant women. The aim of this study was to determine the population structure of L. monocytogenes clonal complex 1 (CC1) in the UK and describe the genomic epidemiology of this clinically significant CC. We interrogated a working dataset of 4073 sequences of L. monocytogenes isolated between January 2015 and December 2020 from human clinical specimens, food and/or food-production environments. A minimum spanning tree was reconstructed to determine the population structure of L. monocytogenes in the UK. Subsequent analysis focused on L. monocytogenes CC1, as the cause of the highest proportion of invasive listeriosis in humans. Sequencing data was integrated with metadata on food and environmental isolates, and information from patient questionnaires, including age, sex and clinical outcomes. All isolates either belonged to lineage I (n=1299/4073, 32%) or lineage II (n=2774/4073, 68%), with clinical isolates from human cases more likely to belong to lineage I (n=546/928, 59%) and food isolates more likely to belong to lineage II (n=2352/3067, 77%). Of the four largest CCs, CC1 (n=237) had the highest proportion of isolates from human cases of disease (CC1 n=160/237, 67.5 %; CC121 n=13/843, 2 %; CC9 n=53/360, 15 %; CC2 n=69/339, 20%). Within CC1, most cases were female (n=95/160, 59%, P=0.01771) and the highest proportion of cases were in people >60 years old (39/95, 41%, P=1.314×10-6) with a high number of them aged 20-39 years old (n=35/95, 37%) most linked to pregnancy-related listeriosis (n=29/35, 83%). Most of the male cases were in men aged over 60 years old (40/65, 62%), and most of the fatal cases in both males and females were identified in this age group (42/55, 76%). Phylogenetic analysis revealed 23 5 SNP single linkage clusters comprising 80/237 (34 %) isolates with cluster sizes ranging from 2 to 19. Five 5 SNP clusters comprised isolates from human cases and an implicated food item. Expanding the analysis to 25 SNP single linkage clusters resolved an additional two clusters linking human cases to a potential food vehicle. Analysis of demographic and clinical outcome data identified CC1 as a clinically significant cause of invasive listeriosis in the elderly population and in women of child-bearing age. Phylogenetic analysis revealed the population structure of CC1 in the UK comprised small, sparsely populated genomic clusters. Only clusters containing isolates from an implicated food vehicle, or food processing or farming environments, were resolved, emphasizing the need for clinical, food and animal-health agencies to share sequencing data in real time, and the importance of a One Health approach to public-health surveillance of listeriosis.

Understanding the impact of the COVID-19 pandemic response on GI infection surveillance trends in England, January 2020-April 2022.

Stepwise non-pharmaceutical interventions and health system changes implemented as part of the COVID-19 response have had implications on the incidence, diagnosis, and reporting of other communicable diseases. Here, we established the impact of the COVID-19 outbreak response on gastrointestinal (GI) infection trends using routinely collected surveillance data from six national English laboratory, outbreak, and syndromic surveillance systems using key dates of governmental policy to assign phases for comparison between pandemic and historic data. Following decreases across all indicators during the first lockdown (March-May 2020), bacterial and parasitic pathogens associated with foodborne or environmental transmission routes recovered rapidly between June and September 2020, while those associated with travel and/or person-to-person transmission remained lower than expected for 2021. High out-of-season norovirus activity was observed with the easing of lockdown measures between June and October 2021, with this trend reflected in laboratory and outbreak systems and syndromic surveillance indicators. Above expected increases in emergency department (ED) attendances may have reflected changes in health-seeking behaviour and provision. Differential reductions across specific GI pathogens are indicative of the underlying routes of transmission. These results provide further insight into the drivers for transmission, which can help inform control measures for GI infections.

Prevalence and Persistence of Antibiotic Resistance Determinants in the Gut of Travelers Returning to the United Kingdom is Associated with Colonization by Pathogenic Escherichia coli.

The gut microbiota constitutes an ideal environment for the selection, exchange, and carriage of antibiotic resistance determinants (ARDs), and international travel has been identified as a risk factor for acquisition of resistant organisms. Here, we present a longitudinal metagenomic analysis of the gut resistome in travellers to "high-risk" countries (Gutback). Fifty volunteers, recruited at a travel clinic in London, United Kingdom, provided stool samples before (pre-travel), immediately after (post-travel), and 6 months after their return (follow-up) from a high-risk destination. Fecal DNA was extracted, metagenomic sequencing performed and the resistome profiled. An increase in abundance and diversity of resistome was observed after travel. Significant increases in abundance were seen in antimicrobial genes conferring resistance to macrolides, third-generation cephalosporins, aminoglycosides, and sulfonamides. There was a significant association with increased resistome abundance if the participant experienced diarrhea during travel or took antibiotics, but these two variables were co-correlated. The resistome abundance returned to pre-travel levels by the 6-month sample point but there was evidence of persistence of several ARDs. The post-travel samples had an increase in abundance Escherichia coli which was positively associated with many acquired resistant determinants. Virulence and phylogenetic profiling revealed pathogenic E. coli significantly contributed to this increase abundance. In summary, in this study, foreign travel remains a significant risk factor for acquisition of microbes conferring resistance to multiple classes of antibiotics, often associated with symptomatic exposure to diarrhoeagenic E. coli. IMPORTANCE A future where antimicrobial therapy is severely compromised by the increase in resistant organisms is of grave concern. Given the variability in prevalence and diversity of antimicrobial resistance determinants in different geographical settings, international travel is a known risk factor for acquisition of resistant organisms into the gut microbiota. In this study, we show the utility of metagenomic approaches to quantify the levels of acquisition and carriage of resistance determinants after travel to a "high-risk" setting. Significant modulation to the resistome was seen after travel that is largely resolved within 6 months, although evidence of persistence of several ARDs was observed. Risk factors for acquisition included experiencing a diarrheal episode and the use of antibiotics. Colonization by pathogenic Escherichia coli was correlated with an increase in acquisition of antimicrobial resistance determinants, and as such established public health guidance to travelers on food and water safety remain an important message to reduce the spread of antibiotic resistance.

Evaluation of Genomic Typing Methods in the Salmonella Reference Laboratory in Public Health, England, 2012-2020.

We aim to provide an evidence-based evaluation of whole genome sequence (WGS) methods, employed at the Salmonella reference laboratory in England, in terms of its impact on public health and whether these methods remain a fit for purpose test under UKAS ISO 15189. The evaluation of the genomic methods were mapped against the value of detecting microbiological clusters to support the investigation of food-borne outbreaks of Salmonella in England between 2012-2020. The analysis of WGS with both SNP- and allelic-based methods provided an unprecedented level of strain discrimination and detection of additional clusters when comparing to all of the previous typing methods. The robustness of the routine genomic sequencing at the reference laboratory ensured confidence in the microbiological identifications, even in large outbreaks with complex international food distribution networks. There was evidence that the phylogeny derived from the WGS data can be used to inform the provenance of strains and support discrimination between domestic and non-domestic transmission events. Further insight on the evolutionary context of the emerging pathogenic strains was enabled with a deep dive of the phylogenetic data, including the detection of nested clusters. The public availability of the WGS data linked to the clinical, epidemiological and environmental context of the sequenced strains has improved the trace-back investigations during outbreaks. The global expansion in the use of WGS-based typing in reference laboratories has shown that the WGS methods are a fit for purpose test in public health as it has ensured the rapid implementation of interventions to protect public health, informed risk assessment and has facilitated the management of national and international food-borne outbreaks of Salmonella.

Identification of domestic reservoirs and common exposures in an emerging lineage of Shiga toxin-producing Escherichia coli O157:H7 in England: a genomic epidemiological analysis.

BACKGROUND: The zoonotic pathogen Shiga toxin-producing Escherichia coli (STEC) O157:H7 emerged during the 1980s as a causative agent of foodborne outbreaks associated with haemorrhagic colitis and haemolytic uraemic syndrome, which can be fatal. We investigated the emerging lineage IIc that was causing outbreaks of STEC O157:H7, identified and quantified the domestic and non-domestic reservoirs, and quantified patient exposures across the population of England. METHODS: In this genomic epidemiological analysis study, all human STEC O157:H7 lineage IIc (n=925) isolates cultured from faecal specimens submitted to the UK Health Security Agency between June 1, 2015, and Dec 31, 2020, from patients in England in the community or in hospital, were whole-genome sequenced and the genomic population structure was described. Explanatory variables were obtained from microbiological surveillance data and STEC Enhanced Surveillance Questionnaire responses. Ancestral-state reconstruction using patient travel information was used to define domestic and non-domestic clades and transmission dynamics. Exposures for patients infected with isolates from domestic clades were assessed using mixed-effects multinomial univariable and multivariable regression. FINDINGS: Lineage IIc emerged 50 years ago, and subsequent clonal expansions have resolved into six major extant clades. We defined two English domestic clades that emerged during the past 30 years, and four non-domestic clades comprising isolates that infected or were transmitted to patients in England via international travel or the consumption or handling of imported food. Throughout the study period, non-domestic clades contributed approximately twice the number of infections as domestic clades did. Patients infected with domestic IIc clade strains reported more frequent exposure to fresh produce (raw vegetables p=0·012; prepackaged salad p=0·0009), contact with animals (cattle p=0·021), and visits to farms (p=0·0053) than patients infected with strains from other STEC O157:H7 lineages. A multivariable mixed-effects multinomial model confirmed that within the domestic clades, the major risk factors for infection were prepackaged salad (clade 2.3.3, relative risk ratio [RRR] 1·72, 95% CI 1·09-2·72; p=0·019) and visits to farms (clade 2.5.2, RRR 1·98, 1·12-3·52; p=0·020) as fixed effects. Local authority district as a random variable had a strong but variable effect for clades 2.3.3 and 2.5.2. INTERPRETATION: Lineage IIc has emerged as the most prevalent lineage of STEC O157:H7 in England, with a sizeable domestic reservoir. Human infection is associated with the consumption of contaminated fresh produce and contact with domestic livestock. The collection of routine, detailed exposure data on patients who are infected, integrated with high-resolution microbiological typing, enables powerful reframing of our understanding of foodborne disease risk within a One Health context. FUNDING: National Institute for Health and Care Research Health and UK Health Security Agency.

Use of Nanopore Sequencing to Characterise the Genomic Architecture of Mobile Genetic Elements Encoding bla CTX-M-15 in Escherichia coli Causing Travellers' Diarrhoea.

Increasing levels of antimicrobial resistance (AMR) have been documented in Escherichia coli causing travellers' diarrhoea, particularly to the third-generation cephalosporins. Diarrhoeagenic E. coli (DEC) can act as a reservoir for the exchange of AMR genes between bacteria residing in the human gut, enabling them to survive and flourish through the selective pressures of antibiotic treatments. Using Oxford Nanopore Technology (ONT), we sequenced eight isolates of DEC from four patients' specimens who had all recently returned to the United Kingdome from Pakistan. Sequencing yielded two DEC harbouring bla CTX-M-15 per patient, all with different sequence types (ST) and belonging to five different pathotypes. The study aimed to determine whether bla CTX-M-15 was located on the chromosome or plasmid and to characterise the drug-resistant regions to better understand the mechanisms of onward transmission of AMR determinants. Patients A and C both had one isolate where bla CTX-M-15 was located on the plasmid (899037 & 623213, respectively) and one chromosomally encoded (899091 & 623214, respectively). In patient B, bla CTX-M-15 was plasmid-encoded in both DEC isolates (786605 & 7883090), whereas in patient D, bla CTX-M-15 was located on the chromosome in both DEC isolates (542093 & 542099). The two bla CTX-M-15-encoding plasmids associated with patient B were different although the bla CTX-M-15-encoding plasmid isolated from 788309 (IncFIB) exhibited high nucleotide similarity to the bla CTX-M-15-encoding plasmid isolated from 899037 (patient A). In the four isolates where bla CTX-M-15 was chromosomally encoded, two isolates (899091 & 542099) shared the same insertion site. The bla CTX-M-15 insertion site in isolate 623214 was described previously, whereas that of isolate 542093 was unique to this study. Analysis of Nanopore sequencing data enables us to characterise the genomic architecture of mobile genetic elements encoding AMR determinants. These data may contribute to a better understanding of persistence and onward transmission of AMR determinants in multidrug-resistant (MDR) E. coli causing gastrointestinal and extra-intestinal infections.

Impact of the COVID-19 pandemic on gastrointestinal infection trends in England, February-July 2020.

OBJECTIVE: To establish the impact of the first 6 months of the COVID-19 outbreak response on gastrointestinal (GI) infection trends in England. DESIGN: Retrospective ecological study using routinely collected national and regional surveillance data from seven UK Health Security Agency coordinated laboratory, outbreak and syndromic surveillance systems using key dates of UK governmental policy change to assign phases for comparison between 2020 and historic data. RESULTS: Decreases in GI illness activity were observed across all surveillance indicators as COVID-19 cases began to peak. Compared with the 5-year average (2015-2019), during the first 6 months of the COVID-19 response, there was a 52% decrease in GI outbreaks reported (1544 vs 3208 (95% CI 2938 to 3478)) and a 34% decrease in laboratory confirmed cases (27 859 vs 42 495 (95% CI 40 068 to 44 922)). GI indicators began to rise during the first lockdown and lockdown easing, although all remained substantially lower than historic figures. Reductions in laboratory confirmed cases were observed across all age groups and both sexes, with geographical heterogeneity observed in diagnosis trends. Health seeking behaviour changed substantially, with attendances decreasing prior to lockdown across all indicators. CONCLUSIONS: There has been a marked change in trends of GI infections in the context of the COVID-19 pandemic. The drivers of this change are likely to be multifactorial; while changes in health seeking behaviour, pressure on diagnostic services and surveillance system ascertainment have undoubtably played a role, there has likely been a true decrease in the incidence for some pathogens resulting from the control measures and restrictions implemented. This suggests that if some of these changes in behaviour such as improved hand hygiene were maintained, then we could potentially see sustained reductions in the burden of GI illness.

Covid-19 Vaccine Effectiveness against the Omicron (B.1.1.529) Variant.

BACKGROUND: A rapid increase in coronavirus disease 2019 (Covid-19) cases due to the omicron (B.1.1.529) variant of severe acute respiratory syndrome coronavirus 2 in highly vaccinated populations has aroused concerns about the effectiveness of current vaccines. METHODS: We used a test-negative case-control design to estimate vaccine effectiveness against symptomatic disease caused by the omicron and delta (B.1.617.2) variants in England. Vaccine effectiveness was calculated after primary immunization with two doses of BNT162b2 (Pfizer-BioNTech), ChAdOx1 nCoV-19 (AstraZeneca), or mRNA-1273 (Moderna) vaccine and after a booster dose of BNT162b2, ChAdOx1 nCoV-19, or mRNA-1273. RESULTS: Between November 27, 2021, and January 12, 2022, a total of 886,774 eligible persons infected with the omicron variant, 204,154 eligible persons infected with the delta variant, and 1,572,621 eligible test-negative controls were identified. At all time points investigated and for all combinations of primary course and booster vaccines, vaccine effectiveness against symptomatic disease was higher for the delta variant than for the omicron variant. No effect against the omicron variant was noted from 20 weeks after two ChAdOx1 nCoV-19 doses, whereas vaccine effectiveness after two BNT162b2 doses was 65.5% (95% confidence interval [CI], 63.9 to 67.0) at 2 to 4 weeks, dropping to 8.8% (95% CI, 7.0 to 10.5) at 25 or more weeks. Among ChAdOx1 nCoV-19 primary course recipients, vaccine effectiveness increased to 62.4% (95% CI, 61.8 to 63.0) at 2 to 4 weeks after a BNT162b2 booster before decreasing to 39.6% (95% CI, 38.0 to 41.1) at 10 or more weeks. Among BNT162b2 primary course recipients, vaccine effectiveness increased to 67.2% (95% CI, 66.5 to 67.8) at 2 to 4 weeks after a BNT162b2 booster before declining to 45.7% (95% CI, 44.7 to 46.7) at 10 or more weeks. Vaccine effectiveness after a ChAdOx1 nCoV-19 primary course increased to 70.1% (95% CI, 69.5 to 70.7) at 2 to 4 weeks after an mRNA-1273 booster and decreased to 60.9% (95% CI, 59.7 to 62.1) at 5 to 9 weeks. After a BNT162b2 primary course, the mRNA-1273 booster increased vaccine effectiveness to 73.9% (95% CI, 73.1 to 74.6) at 2 to 4 weeks; vaccine effectiveness fell to 64.4% (95% CI, 62.6 to 66.1) at 5 to 9 weeks. CONCLUSIONS: Primary immunization with two doses of ChAdOx1 nCoV-19 or BNT162b2 vaccine provided limited protection against symptomatic disease caused by the omicron variant. A BNT162b2 or mRNA-1273 booster after either the ChAdOx1 nCoV-19 or BNT162b2 primary course substantially increased protection, but that protection waned over time. (Funded by the U.K. Health Security Agency.).

Hospitalization and Mortality Risk for COVID-19 Cases With SARS-CoV-2 AY.4.2 (VUI-21OCT-01) Compared to Non-AY.4.2 Delta Variant Sublineages.

To investigate if the AY.4.2 sublineage of the SARS-CoV-2 delta variant is associated with hospitalization and mortality risks that differ from non-AY.4.2 delta risks, we performed a retrospective cohort study of sequencing-confirmed COVID-19 cases in England based on linkage of routine health care datasets. Using stratified Cox regression, we estimated adjusted hazard ratios (aHR) of hospital admission (aHR = 0.85; 95% confidence interval [CI], .77-.94), hospital admission or emergency care attendance (aHR = 0.87; 95% CI, .81-.94), and COVID-19 mortality (aHR = 0.85; 95% CI, .71-1.03). The results indicate that the risks of hospitalization and mortality are similar or lower for AY.4.2 compared to cases with other delta sublineages.

Premature neonatal gut microbial community patterns supporting an epithelial TLR-mediated pathway for necrotizing enterocolitis.

BACKGROUND: Necrotising enterocolitis (NEC) is a devastating bowel disease, primarily affecting premature infants, with a poorly understood aetiology. Prior studies have found associations in different cases with an overabundance of particular elements of the faecal microbiota (in particular Enterobacteriaceae or Clostridium perfringens), but there has been no explanation for the different results found in different cohorts. Immunological studies have indicated that stimulation of the TLR4 receptor is involved in development of NEC, with TLR4 signalling being antagonised by the activated TLR9 receptor. We speculated that differential stimulation of these two components of the signalling pathway by different microbiota might explain the dichotomous findings of microbiota-centered NEC studies. Here we used shotgun metagenomic sequencing and qPCR to characterise the faecal microbiota community of infants prior to NEC onset and in a set of matched controls. Bayesian regression was used to segregate cases from control samples using both microbial and clinical data. RESULTS: We found that the infants suffering from NEC fell into two groups based on their microbiota; one with low levels of CpG DNA in bacterial genomes and the other with high abundances of organisms expressing LPS. The identification of these characteristic communities was reproduced using an external metagenomic validation dataset. We propose that these two patterns represent the stimulation of a common pathway at extremes; the LPS-enriched microbiome suggesting overstimulation of TLR4, whilst a microbial community with low levels of CpG DNA suggests reduction of the counterbalance to TLR4 overstimulation. CONCLUSIONS: The identified microbial community patterns support the concept of NEC resulting from TLR-mediated pathways. Identification of these signals suggests characteristics of the gastrointestinal microbial community to be avoided to prevent NEC. Potential pre- or pro-biotic treatments may be designed to optimise TLR signalling.

Outbreak of Shiga toxin-producing Escherichia coli O157 linked with consumption of a fast-food product containing imported cucumbers, United Kingdom, August 2020.

BACKGROUND: In August 2020, an outbreak of Shiga toxin-producing Escherichia coli (STEC) O157:H7 occurred in the United Kingdom. Whole genome sequencing revealed that these cases formed a genetically distinct cluster. METHODS: Hypotheses generated from case interviews were tested in analytical studies, and results informed environmental sampling and food chain analysis. A case-case study used non-outbreak 'comparison' STEC cases; a case-control study used a market research panel to recruit controls. RESULTS: A total of 36 cases were identified; all cases reported symptom onset between August 3 and August 16, 2020. The majority of cases (83%) resided in the Midlands region of England and in Wales. A high proportion of cases reported eating out, with one fast-food restaurant chain mentioned by 64% (n = 23) of cases. Both the case-case study (adjusted odds ratio (aOR) 31.8, 95% confidence interval (CI) 1.6-624.9) and the case-control study (aOR 9.19, 95% CI 1.0-82.8) revealed statistically significant results, showing that the consumption of a specific fast-food product was independently associated with infection. CONCLUSIONS: Consumption of a specific fast-food product was a likely cause of this outbreak. The only ingredient specific to the product was cucumbers. The supply of cucumbers was immediately halted, and no further cases have been identified.

Phylogenetic structure of Shiga toxin-producing Escherichia coli O157:H7 from sub-lineage to SNPs.

Sequence similarity of pathogen genomes can infer the relatedness between isolates as the fewer genetic differences identified between pairs of isolates, the less time since divergence from a common ancestor. Clustering based on hierarchical single linkage clustering of pairwise SNP distances has been employed to detect and investigate outbreaks. Here, we evaluated the evidence-base for the interpretation of phylogenetic clusters of Shiga toxin-producing Escherichia coli (STEC) O157:H7. Whole genome sequences of 1193 isolates of STEC O157:H7 submitted to Public Health England between July 2015 and December 2016 were mapped to the Sakai reference strain. Hierarchical single linkage clustering was performed on the pairwise SNP difference between all isolates at descending distance thresholds. Cases with known epidemiological links fell within 5-SNP single linkage clusters. Five-SNP single linkage community clusters where an epidemiological link was not identified were more likely to be temporally and/or geographically related than sporadic cases. Ten-SNP single linkage clusters occurred infrequently and were challenging to investigate as cases were few, and temporally and/or geographically dispersed. A single linkage cluster threshold of 5-SNPs has utility for the detection of outbreaks linked to both persistent and point sources. Deeper phylogenetic analysis revealed that the distinction between domestic UK and imported isolates could be inferred at the sub-lineage level. Cases associated with domestically acquired infection that fall within clusters that are predominantly travel associated are likely to be caused by contaminated imported food.

Analysis of a small outbreak of Shiga toxin-producing Escherichia coli O157:H7 using long-read sequencing.

Compared to short-read sequencing data, long-read sequencing facilitates single contiguous de novo assemblies and characterization of the prophage region of the genome. Here, we describe our methodological approach to using Oxford Nanopore Technology (ONT) sequencing data to quantify genetic relatedness and to look for microevolutionary events in the core and accessory genomes to assess the within-outbreak variation of four genetically and epidemiologically linked isolates. Analysis of both Illumina and ONT sequencing data detected one SNP between the four sequences of the outbreak isolates. The variant calling procedure highlighted the importance of masking homologous sequences in the reference genome regardless of the sequencing technology used. Variant calling also highlighted the systemic errors in ONT base-calling and ambiguous mapping of Illumina reads that results in variations in the genetic distance when comparing one technology to the other. The prophage component of the outbreak strain was analysed, and nine of the 16 prophages showed some similarity to the prophage in the Sakai reference genome, including the stx2a-encoding phage. Prophage comparison between the outbreak isolates identified minor genome rearrangements in one of the isolates, including an inversion and a deletion event. The ability to characterize the accessory genome in this way is the first step to understanding the significance of these microevolutionary events and their impact on the evolutionary history, virulence and potentially the likely source and transmission of this zoonotic, foodborne pathogen.

The airways microbiome of individuals with asthma treated with high and low doses of inhaled corticosteroids.

BACKGROUND: Inhaled corticosteroids (ICS) are the mainstay of asthma treatment, but evidence suggests a link between ICS usage and increased rates of respiratory infections. We assessed the composition of the asthmatic airways microbiome in asthma patients taking low and high dose ICS and the stability of the microbiome over a 2 week period. METHODS: We prospectively recruited 55 individuals with asthma. Of these, 22 were on low-dose ICS and 33 on high-dose ICS (16 on budesonide, 17 on fluticasone propionate). Sputum from each subject underwent DNA extraction, amplification and 16S rRNA gene sequencing of the bacterial component of the microbiome. 19 subjects returned for further sputum induction after 24 h and 2 weeks. RESULTS: A total of 5,615,037 sequencing reads revealed 167 bacterial taxa in the asthmatic airway samples, with the most abundant being Streptococcus spp. No significant differences in sputum bacterial load or overall community composition were seen between the low- and high-dose ICS groups. However, Streptococcus spp. showed significantly higher relative abundance in subjects taking low-dose ICS (p = 0.002). Haemophilus parainfluenzae was significantly more abundant in subjects on high-dose fluticasone propionate than those on high-dose budesonide (p = 0.047). There were no statistically significant changes in microbiota composition over a 2-week period. DISCUSSION: Whilst no significant differences were observed between the low- and high-dose ICS groups, increased abundance of the potential pathogen H. parainfluenzae was observed in patients taking high-dose fluticasone propionate compared to those taking high-dose budesonide. The microbiota were stable over fourteen days, providing novel evidence of the established community of bacteria in the asthmatic airways. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT02671773.

Evaluation of chromogenic selective agar (CHROMagar STEC) for the direct detection of Shiga toxin-producing Escherichia coli from faecal specimens.

Shiga toxin-producing Escherichia coli (STEC) are zoonotic pathogens that cause symptoms of severe gastrointestinal disease, including haemolytic uraemic syndrome (HUS), in humans. Currently in England, STEC serotypes other than O157:H7 are not cultured at the local hospital laboratories. The aim of this study was to evaluate the utility of CHROMagar STEC for the direct detection of STEC from faecal specimens in a diagnostic setting, compared to the current reference laboratory method using PCR targeting the Shiga-toxin gene (stx) to test multiple colonies cultured on MacConkey agar. Of the 292 consecutive faecal specimens submitted to the Gastrointestinal Bacterial Reference Unit that tested positive for stx by PCR, STEC could not be cultured on MacConkey agar or CHROMagar STEC from 87/292 (29.8 %). Of the 205 that were cultured, 106 (51.7 %) were detected on both MacConkey agar and CHROMagar STEC and 99 (48.3 %) were detected on MacConkey agar only. All 106 (100 %) isolates that grew on CHROMagar STEC had the ter gene cassette, known to be associated with resistance to tellurite, compared to 13/99 (13.1 %) that were not detected on CHROMagar STEC. CHROMagar STEC supported the growth of 36/40 (90 %) isolates harbouring stx2a or stx2d, the subtypes most frequently associated with progression to HUS. Of the 92 isolates harbouring eae, an important STEC virulence marker, 77 (83.7 %) grew on CHROMagar STEC. CHROMagar STEC is a useful selective media for the rapid, near-patient detection of STEC that have the potential to cause HUS.

Comparison of single-nucleotide variants identified by Illumina and Oxford Nanopore technologies in the context of a potential outbreak of Shiga toxin-producing Escherichia coli.

BACKGROUND: We aimed to compare Illumina and Oxford Nanopore Technology sequencing data from the 2 isolates of Shiga toxin-producing Escherichia coli (STEC) O157:H7 to determine whether concordant single-nucleotide variants were identified and whether inference of relatedness was consistent with the 2 technologies. RESULTS: For the Illumina workflow, the time from DNA extraction to availability of results was ∼40 hours, whereas with the ONT workflow serotyping and Shiga toxin subtyping variant identification were available within 7 hours. After optimization of the ONT variant filtering, on average 95% of the discrepant positions between the technologies were accounted for by methylated positions found in the described 5-methylcytosine motif sequences, CC(A/T)GG. Of the few discrepant variants (6 and 7 difference for the 2 isolates) identified by the 2 technologies, it is likely that both methodologies contain false calls. CONCLUSIONS: Despite these discrepancies, Illumina and Oxford Nanopore Technology sequences from the same case were placed on the same phylogenetic location against a dense reference database of STEC O157:H7 genomes sequenced using the Illumina workflow. Robust single-nucleotide polymorphism typing using MinION-based variant calling is possible, and we provide evidence that the 2 technologies can be used interchangeably to type STEC O157:H7 in a public health setting.

Phylogenetic context of Shiga toxin-producing Escherichia coli serotype O26:H11 in England.

The increasing use of PCR for the detection of gastrointestinal pathogens in hospital laboratories in England has improved the detection of Shiga toxin-producing Escherichia coli (STEC), and the diagnosis of haemolytic uraemic syndrome (HUS). We aimed to analyse the microbiological characteristics and phylogenetic relationships of STEC O26:H11, clonal complex (CC) 29, in England to inform surveillance, and to assess the threat to public health. There were 502 STEC belonging to CC29 isolated between 2014 and 2019, of which 416 were from individual cases. The majority of isolates belonged to one of three major sequence types (STs), ST16 (n=37), ST21 (n=350) and ST29 (n=24). ST16 and ST29 were mainly isolated from cases reporting recent travel abroad. Within ST21, there were three main clades associated with domestic acquisition. All three domestic clades had Shiga toxin subtype gene (stx) profiles associated with causing severe clinical outcomes including STEC-HUS, specifically either stx1a, stx2a or stx1a/stx2a. Isolates from the same patient, same household or same outbreak with an established source for the most part fell within 5-SNP single linkage clusters. There were 19 5-SNP community clusters, of which six were travel-associated and one was an outbreak of 16 cases caused by the consumption of contaminated salad leaves. Of the remaining 12 clusters, 9/12 were either temporally or geographically related or both. Exposure to foodborne STEC O26:H11 ST21 capable of causing severe clinical outcomes, including STEC-HUS, is an emerging risk to public health in England. The lack of comprehensive surveillance of this STEC serotype is a concern, and there is a need to expand the implementation of methods capable of detecting STEC in local hospital settings.