Diversity of the Genomes and Neurotoxins of Strains of Clostridium botulinum Group I and Clostridium sporogenes Associated with Foodborne, Infant and Wound Botulism.

Clostridium botulinum Group I and Clostridium sporogenes are closely related bacteria responsible for foodborne, infant and wound botulism. A comparative genomic study with 556 highly diverse strains of C. botulinum Group I and C. sporogenes (including 417 newly sequenced strains) has been carried out to characterise the genetic diversity and spread of these bacteria and their neurotoxin genes. Core genome single-nucleotide polymorphism (SNP) analysis revealed two major lineages; C. botulinum Group I (most strains possessed botulinum neurotoxin gene(s) of types A, B and/or F) and C. sporogenes (some strains possessed a type B botulinum neurotoxin gene). Both lineages contained strains responsible for foodborne, infant and wound botulism. A new C. sporogenes cluster was identified that included five strains with a gene encoding botulinum neurotoxin sub-type B1. There was significant evidence of horizontal transfer of botulinum neurotoxin genes between distantly related bacteria. Population structure/diversity have been characterised, and novel associations discovered between whole genome lineage, botulinum neurotoxin sub-type variant, epidemiological links to foodborne, infant and wound botulism, and geographic origin. The impact of genomic and physiological variability on the botulism risk has been assessed. The genome sequences are a valuable resource for future research (e.g., pathogen biology, evolution of C. botulinum and its neurotoxin genes, improved pathogen detection and discrimination), and support enhanced risk assessments and the prevention of botulism.

Impact of whole genome sequencing on the investigation of food-borne outbreaks of Shiga toxin-producing Escherichia coli serogroup O157:H7, England, 2013 to 2017.

We aim to provide insight and guidance on the utility of whole genome sequencing (WGS) data for investigating food-borne outbreaks of Shiga toxin-producing Escherichia coli (STEC) O157:H7 in England between 2013 and 2017. Analysis of WGS data delivered an unprecedented level of strain discrimination when compared with multilocus variable number tandem repeat analysis. The robustness of the WGS method ensured confidence in the microbiological identification of linked cases, even when epidemiological links were obscured. There was evidence that phylogeny derived from WGS data can be used to trace the geographical origin of an isolate. Further analysis of the phylogenetic data provided insight on the evolutionary context of emerging pathogenic strains. Publically available WGS data linked to the clinical, epidemiological and environmental context of the sequenced strain has improved trace back investigations during outbreaks. Expanding the use of WGS-based typing analysis globally will ensure the rapid implementation of interventions to protect public health, inform risk assessment and facilitate the management of national and international food-borne outbreaks of STEC O157:H7.

Whole genome sequencing reveals an outbreak of Salmonella Enteritidis associated with reptile feeder mice in the United Kingdom, 2012-2015.

Analysis of whole genome sequencing data uncovered a previously undetected outbreak of Salmonella Enteritidis that had been on-going for four years. Cases were resident in all countries of the United Kingdom and 40% of the cases were aged less than 11 years old. Initial investigations revealed that 30% of cases reported exposure to pet snakes. A case-control study was designed to test the hypothesis that exposure to reptiles or their feed were risk factors. A robust case-definition, based on the single nucleotide polymorphism (SNP) profile, increased the power of the analytical study. Following univariable and multivariable analysis, exposure to snakes was the only variable independently associated with infection (Odds ratio 810 95% CI (85-7715) p < 0.001). Isolates of S. Enteritidis belonging to the outbreak profile were recovered from reptile feeder mice sampled at the retail and wholesale level. Control measures included improved public health messaging at point of sale, press releases and engagement with public health and veterinary counterparts across Europe. Mice destined to be fed to reptiles are not regarded as pet food and are not routinely tested for pathogenic bacteria. Routine microbiological testing to ensure feeder mice are free from Salmonella is recommended.

Identification of Salmonella for public health surveillance using whole genome sequencing.

In April 2015, Public Health England implemented whole genome sequencing (WGS) as a routine typing tool for public health surveillance of Salmonella, adopting a multilocus sequence typing (MLST) approach as a replacement for traditional serotyping. The WGS derived sequence type (ST) was compared to the phenotypic serotype for 6,887 isolates of S. enterica subspecies I, and of these, 6,616 (96%) were concordant. Of the 4% (n = 271) of isolates of subspecies I exhibiting a mismatch, 119 were due to a process error in the laboratory, 26 were likely caused by the serotype designation in the MLST database being incorrect and 126 occurred when two different serovars belonged to the same ST. The population structure of S. enterica subspecies II-IV differs markedly from that of subspecies I and, based on current data, defining the serovar from the clonal complex may be less appropriate for the classification of this group. Novel sequence types that were not present in the MLST database were identified in 8.6% of the total number of samples tested (including S. enterica subspecies I-IV and S. bongori) and these 654 isolates belonged to 326 novel STs. For S. enterica subspecies I, WGS MLST derived serotyping is a high throughput, accurate, robust, reliable typing method, well suited to routine public health surveillance. The combined output of ST and serovar supports the maintenance of traditional serovar nomenclature while providing additional insight on the true phylogenetic relationship between isolates.

Public Health Investigation of Two Outbreaks of Shiga Toxin-Producing Escherichia coli O157 Associated with Consumption of Watercress.

An increase in the number of cases of Shiga toxin-producing Escherichia coli (STEC) O157 phage type 2 (PT2) in England in September 2013 was epidemiologically linked to watercress consumption. Whole-genome sequencing (WGS) identified a phylogenetically related cluster of 22 cases (outbreak 1). The isolates comprising this cluster were not closely related to any other United Kingdom strain in the Public Health England WGS database, suggesting a possible imported source. A second outbreak of STEC O157 PT2 (outbreak 2) was identified epidemiologically following the detection of outbreak 1. Isolates associated with outbreak 2 were phylogenetically distinct from those in outbreak 1. Epidemiologically unrelated isolates on the same branch as the outbreak 2 cluster included those from human cases in England with domestically acquired infection and United Kingdom domestic cattle. Environmental sampling using PCR resulted in the isolation of STEC O157 PT2 from irrigation water at one implicated watercress farm, and WGS showed this isolate belonged to the same phylogenetic cluster as outbreak 2 isolates. Cattle were in close proximity to the watercress bed and were potentially the source of the second outbreak. Transfer of STEC from the field to the watercress bed may have occurred through wildlife entering the watercress farm or via runoff water. During this complex outbreak investigation, epidemiological studies, comprehensive testing of environmental samples, and the use of novel molecular methods proved invaluable in demonstrating that two simultaneous outbreaks of STEC O157 PT2 were both linked to the consumption of watercress but were associated with different sources of contamination.

Insight into Shiga toxin genes encoded by Escherichia coli O157 from whole genome sequencing.

The ability of Shiga toxin-producing Escherichia coli (STEC) to cause severe illness in humans is determined by multiple host factors and bacterial characteristics, including Shiga toxin (Stx) subtype. Given the link between Stx2a subtype and disease severity, we sought to identify the stx subtypes present in whole genome sequences (WGS) of 444 isolates of STEC O157. Difficulties in assembling the stx genes in some strains were overcome by using two complementary bioinformatics methods: mapping and de novo assembly. We compared the WGS analysis with the results obtained using a PCR approach and investigated the diversity within and between the subtypes. All strains of STEC O157 in this study had stx1a, stx2a or stx2c or a combination of these three genes. There was over 99% (442/444) concordance between PCR and WGS. When common source strains were excluded, 236/349 strains of STEC O157 had multiple copies of different Stx subtypes and 54 had multiple copies of the same Stx subtype. Of those strains harbouring multiple copies of the same Stx subtype, 33 had variants between the alleles while 21 had identical copies. Strains harbouring Stx2a only were most commonly found to have multiple alleles of the same subtype (42%). Both the PCR and WGS approach to stx subtyping provided a good level of sensitivity and specificity. In addition, the WGS data also showed there were a significant proportion of strains harbouring multiple alleles of the same Stx subtype associated with clinical disease in England.

Applying phylogenomics to understand the emergence of Shiga-toxin-producing Escherichia coli O157:H7 strains causing severe human disease in the UK.

Shiga-toxin-producing Escherichia coli (STEC) O157:H7 is a recently emerged zoonotic pathogen with considerable morbidity. Since the emergence of this serotype in the 1980s, research has focussed on unravelling the evolutionary events from the E. coli O55:H7 ancestor to the contemporaneous globally dispersed strains observed today. In this study, the genomes of over 1000 isolates from both human clinical cases and cattle, spanning the history of STEC O157:H7 in the UK, were sequenced. Phylogenetic analysis revealed the ancestry, key acquisition events and global context of the strains. Dated phylogenies estimated the time to evolution of the most recent common ancestor of the current circulating global clone to be 175 years ago. This event was followed by rapid diversification. We show the acquisition of specific virulence determinates has occurred relatively recently and coincides with its recent detection in the human population. We used clinical outcome data from 493 cases of STEC O157:H7 to assess the relative risk of severe disease including haemolytic uraemic syndrome from each of the defined clades in the population and show the dramatic effect Shiga toxin repertoire has on virulence. We describe two strain replacement events that have occurred in the cattle population in the UK over the last 30 years, one resulting in a highly virulent strain that has accounted for the majority of clinical cases in the UK over the last decade. There is a need to understand the selection pressures maintaining Shiga-toxin-encoding bacteriophages in the ruminant reservoir and the study affirms the requirement for close surveillance of this pathogen in both ruminant and human populations.

Salmonella enterica serovar Enteritidis, England and Wales, 1945-2011.

In England and Wales, the emergence of Salmonella enterica serovar Enteritidis resulted in the largest and most persistent epidemic of foodborne infection attributable to a single subtype of any pathogen since systematic national microbiological surveillance was established. We reviewed 67 years of surveillance data to examine the features, underlying causes, and overall effects of S. enterica ser. Enteritidis. The epidemic was associated with the consumption of contaminated chicken meat and eggs, and a decline in the number of infections began after the adoption of vaccination and other measures in production and distribution of chicken meat and eggs. We estimate that >525,000 persons became ill during the course of the epidemic, which caused a total of 6,750,000 days of illness, 27,000 hospitalizations, and 2,000 deaths. Measures undertaken to control the epidemic have resulted in a major reduction in foodborne disease in England and Wales.

Epidemiology and microbiology of Shiga toxin-producing Escherichia coli other than serogroup O157 in England, 2009-2013.

The implementation of direct testing of clinical faecal specimens for gastrointestinal (GI) pathogens by PCR offers a sensitive and comprehensive approach for the detection of Shiga toxin-producing Escherichia coli (STEC). The introduction of a commercial PCR assay, known as GI PCR, for the detection of GI pathogens at three frontline hospital laboratories in England between December 2012 and December 2013 led to a significant increase in detection of STEC other than serogroup O157 (non-O157 STEC). In 2013, 47 isolates were detected in England, compared with 57 in the preceding 4 years (2009-2012). The most common non-O157 STEC serogroup detected was O26 (23.2 %). A total of 47 (47.5 %) STEC isolates had stx2 only, 28 (28.3 %) carried stx1 and stx2, and the remaining 24 (24.2 %) had stx1 only. Stx2a (64.0 %) was the most frequently detected Stx2 subtype. The eae (intimin) gene was detected in 52 (52.5 %) non-O157 STEC isolates. Six strains of STEC O104 had aggR, but this gene was not detected in any other STEC serogroups in this study. Haemolytic ureamic syndrome was significantly associated with STEC strains possessing eae [odds ratio (OR) 5.845, P = 0.0235] and/or stx2a (OR 9.56, P = 0.0034) subtypes. A matched case-control analysis indicated an association between non-O157 STEC cases and contact with farm animals. Widespread implementation of the PCR approach in England will determine the true incidence of non-O157 STEC infection, highlight the burden in terms of morbidity and mortality, and facilitate the examination of risk factors to indicate whether there are niche risk exposures for particular strains.

An investigation of the diversity of strains of enteroaggregative Escherichia coli isolated from cases associated with a large multi-pathogen foodborne outbreak in the UK.

Following a large outbreak of foodborne gastrointestinal (GI) disease, a multiplex PCR approach was used retrospectively to investigate faecal specimens from 88 of the 413 reported cases. Gene targets from a range of bacterial GI pathogens were detected, including Salmonella species, Shigella species and Shiga toxin-producing Escherichia coli, with the majority (75%) of faecal specimens being PCR positive for aggR associated with the Enteroaggregative E. coli (EAEC) group. The 20 isolates of EAEC recovered from the outbreak specimens exhibited a range of serotypes, the most frequent being O104:H4 and O131:H27. None of the EAEC isolates had the Shiga toxin (stx) genes. Multilocus sequence typing and single nucleotide polymorphism analysis of the core genome confirmed the diverse phylogeny of the strains. The analysis also revealed a close phylogenetic relationship between the EAEC O104:H4 strains in this outbreak and the strain of E. coli O104:H4 associated with a large outbreak of haemolytic ureamic syndrome in Germany in 2011. Further analysis of the EAEC plasmids, encoding the key enteroaggregative virulence genes, showed diversity with respect to FIB/FII type, gene content and genomic architecture. Known EAEC virulence genes, such as aggR, aat and aap, were present in all but one of the strains. A variety of fimbrial genes were observed, including genes encoding all five known fimbrial types, AAF/1 to AAF/V. The AAI operon was present in its entirety in 15 of the EAEC strains, absent in three and present, but incomplete, in two isolates. EAEC is known to be a diverse pathotype and this study demonstrates that a high level of diversity in strains recovered from cases associated with a single outbreak. Although the EAEC in this study did not carry the stx genes, this outbreak provides further evidence of the pathogenic potential of the EAEC O104:H4 serotype.

Evaluating the use of multilocus variable number tandem repeat analysis of Shiga toxin-producing Escherichia coli O157 as a routine public health tool in England.

Multilocus variable number tandem repeat analysis (MLVA) provides microbiological support for investigations of clusters of cases of infection with Shiga toxin-producing E. coli (STEC) O157. All confirmed STEC O157 isolated in England and submitted to the Gastrointestinal Bacteria Reference Unit (GBRU) during a six month period were typed using MLVA, with the aim of assessing the impact of this approach on epidemiological investigations. Of 539 cases investigated, 341 (76%) had unique (>2 single locus variants) MLVA profiles, 12% of profiles occurred more than once due to known household transmission and 12% of profiles occurred as part of 41 clusters, 21 of which were previously identified through routine public health investigation of cases. The remaining 20 clusters were not previously detected and STEC enhanced surveillance data for associated cases were retrospectively reviewed for epidemiological links including shared exposures, geography and/or time. Additional evidence of a link between cases was found in twelve clusters. Compared to phage typing, the number of sporadic cases was reduced from 69% to 41% and the diversity index for MLVA was 0.996 versus 0.782 for phage typing. Using MLVA generates more data on the spatial and temporal dispersion of cases, better defining the epidemiology of STEC infection than phage typing. The increased detection of clusters through MLVA typing highlights the challenges to health protection practices, providing a forerunner to the advent of whole genome sequencing as a diagnostic tool.

Infant botulism: advice on avoiding feeding honey to babies and other possible risk factors.

Botulism is a rare, but extremely serious, disease and Public Health England is responsible for its diagnosis and surveillance in the UK. Over the past five years (2008-2013), the most common form of the disease recognised in the UK has been infant botulism. The aim of this article is to raise awareness of infant botulism and highlight advice for parents and carers of infants that honey should not be fed to infants under 12 months old. Other possible risk factors for infant botulism are also discussed in this article, including household pet reptiles and herbal teas.

Whole genome sequencing of an unusual serotype of Shiga toxin-producing Escherichia coli.

Shiga toxin-producing Escherichia coli serotype O117:K1:H7 is a cause of persistent diarrhea in travelers to tropical locations. Whole genome sequencing identified genetic mechanisms involved in the pathoadaptive phenotype. Sequencing also identified toxin and putative adherence genes flanked by sequences indicating horizontal gene transfer from Shigella dysenteriae and Salmonella spp., respectively.

Infections with spore-forming bacteria in persons who inject drugs, 2000-2009.

Since 2000 in the United Kingdom, infections caused by spore-forming bacteria have been associated with increasing illness and death among persons who inject drugs (PWID). To assess temporal and geographic trends in these illnesses (botulism, tetanus, Clostridium novyi infection, and anthrax), we compared rates across England and Scotland for 2000-2009. Overall, 295 infections were reported: 1.45 per 1,000 PWID in England and 4.01 per 1,000 PWID in Scotland. The higher rate in Scotland was mainly attributable to C. novyi infection and anthrax; rates of botulism and tetanus were comparable in both countries. The temporal and geographic clustering of cases of C. novyi and anthrax into outbreaks suggests possible contamination of specific heroin batches; in contrast, the more sporadic nature of tetanus and botulism cases suggests that these spores might more commonly exist in the drug supply or local environment although at varying levels. PWID should be advised about treatment programs, injecting hygiene, risks, and vaccinations.

Characterization of a verocytotoxin-producing enteroaggregative Escherichia coli serogroup O111:H21 strain associated with a household outbreak in Northern Ireland.

A strain of Escherichia coli O111:H21 recently isolated in the United Kingdom harbored the phage-encoded vtx2c gene and the aggregative adherence plasmid. Although exhibiting the same pathogenic profile as the E. coli O104:H4 strain linked to the outbreak in Germany, there were important differences in strain characteristics and in the epidemiological setting.

Complete genome sequence of the proteolytic Clostridium botulinum type A5 (B3') strain H04402 065.

H04402 065 is one of a very small group of strains of proteolytic Clostridium botulinum that form type A5 neurotoxin. Here, we report the complete 3.9-Mb genome sequence and annotation of strain H04402 065, which was isolated from a botulism patient in the United Kingdom in 2004.

Report of two unlinked cases of infant botulism in the UK in October 2007.

Infant botulism is a rare disease in the UK, with the first case being recognized in 1978 and only five subsequent cases being reported before 2007. This study reports two unlinked cases of infant botulism, caused by two distinct strains of Clostridium botulinum (toxin types A and B, respectively), that occurred within a single month in the south-east of England in October 2007. The use of real-time PCR to detect C. botulinum neurotoxin genes in clinical specimens to improve the diagnostic procedure and to follow carriage of the causative organism in the infant gut is described. The laboratory investigation of these two cases demonstrated that a combination of the mouse bioassay, real-time PCR assays and conventional microbiological culture can provide rapid confirmation of a clinical diagnosis and affect patient management. Both infants (aged 4 and 8 months) were previously healthy prior to the onset of symptoms, and in both cases, a diagnosis of infant botulism was delayed for at least 10 days after initial admission to hospital. Once diagnosed, one of the infants was the first in the UK to be treated with human-derived botulism immunoglobulin. Real-time PCR was used to demonstrate that C. botulinum was excreted in the infants' faeces for up to 68 and 81 days, respectively. Despite the infrequency of infant botulism in the UK, clinicians should be aware of this rare but serious condition and should seek microbiological advice when presented with young infants with compatible symptomologies.

Association between a case study of asymptomatic ovine listerial mastitis and the contamination of soft cheese and cheese processing environment with Listeria monocytogenes in Portugal.

For 5 months, the udders of milking ewes, raw ewe's milk, cheese, and the plant and environment of a cheese manufacturer in Portugal were investigated using standard methods for the presence of Listeria spp. An association between subclinical mastitis and Listeria monocytogenes in a single lactating sheep was investigated by visual inspection of udders for signs of inflammation, application of somatic cell counts, the California mastitis test, pH measurement to milk, and culture of L. monocytogenes and Staphylococcus spp. To track the routes of contamination by L. monocytogenes, 103 isolates were characterized by molecular serotyping and amplified fragment length polymorphism, and a selection was further tested by pulsed-field gel electrophoresis. This study provides molecular and epidemiological evidence tracking the persistence of a single L. monocytogenes strain causing a subclinical udder infection without obvious inflammation in a single ewe. This infection was the likely source of contamination of raw milk that was subsequently used to produce unpasteurised milk cheese and resulted in a single strain of this bacterium colonizing the processing environment and the final cheese product.

The identification and characterization of Clostridium perfringens by real-time PCR, location of enterotoxin gene, and heat resistance.

Clostridium perfringens carrying the enterotoxin gene is an important cause of both foodborne and non-foodborne diarrheal disease. Rapid identification of isolates carrying the enterotoxin gene is invaluable for outbreak investigation whilst information on the genomic location of the enterotoxin (cpe) gene can improve our understanding of disease transmission. This paper describes the validation of a real-time polymerase chain reaction (PCR) assay for the identification of C. perfringens and assessment of the potential to cause diarrhea, together with an investigation into the genomic location of the cpe genes in isolates from confirmed incidents of C. perfringens diarrhea. The real-time assay was shown to be specific for the identification of 253 C. perfringens cultures and gave results concordant with those from motility nitrate and lactose gelatine media, the Nagler reaction, and a conventional block-based PCR assay. The cpe gene was detected in 223 of 253 C. perfringens cultures isolated in association with human gastrointestinal disease. A subset of cpe-positive C. perfringens isolates associated with separate incidents of diarrheal disease were investigated further for plasmid or chromosomal location of the cpe gene using a multiplex PCR assay. The cpe gene was plasmid encoded in two isolates from cases of sporadic diarrhea and six isolates from cases of food poisoning. The cpe gene from the remaining 11 isolates from different food poisoning outbreaks was found to be chromosomally encoded. One of the C. perfringens strains with a plasmid encoded cpe gene formed spores of high heat resistance and five formed spores that were sensitive to heating. Eight of the isolates with a chromosomal cpe gene formed heat-resistant spores, and two formed spores with an intermediate heat resistance.