Listeria monocytogenes: the silent assassin.

Listeriosis is a foodborne infection in humans caused by Listeria monocytogenes. Consumption of contaminated food can lead to severe infection in vulnerable patients, that can be fatal. Clinical manifestations include sepsis and meningitis, and in pregnancy-associated infection, miscarriage and stillbirth. Diagnosis is confirmed by culture and identification of the pathogen from blood, cerebrospinal fluid, vaginal swab, placenta or amniotic fluid. Treatment regimens recommend amoxicillin, ampicillin or an aminoglycoside. Virulence factors mediate bacterial adhesion and invasion of gut epithelial cells. Other factors mediate biofilm formation and tolerance to low temperatures and high salt concentrations facilitating persistence and survival in the environment.

An outbreak of human listeriosis associated with frozen sweet corn consumption: Investigations in the UK.

The use of Whole genome sequencing (WGS) identified a multi-country outbreak of human listeriosis associated with consumption of frozen sweet corn produced in Hungary. The purpose of this report was to summarise information on the cases occurring in the UK which were part of this outbreak and outline investigations on the presence of Listeria monocytogenes in the affected food chain. Prior to the international recall of this product in 2018, 12 UK cases of listeriosis were identified as infected by the outbreak strain between 2015 and 18. Epidemiological and microbiological investigations confirmed these cases as belonging to the outbreak. A further case occurred in 2019 and a contaminated frozen pack from one of the implicated batches of sweet corn was recovered from the patient's domestic freezer. The outbreak strain was also detected in products from a sandwich manufacturer in 2018 which added frozen sweet corn directly to sandwich fillings. The sandwich manufacturer's sweet corn was supplied by a distributor in England which obtained frozen products from the Hungarian manufacturer implicated in the outbreak. Within the distributor's premises, 208 food and environmental samples were taken: L. monocytogenes was detected in 44% of 70 samples of frozen sweet corn and 5% of 79 other foods. The outbreak strain was detected in the frozen sweet corn, in one other frozen food (mixed vegetables) and in the factory environment. The outbreak strain was also recovered from frozen beans on retail sale in the first four months of 2019. Five other L. monocytogenes strains together with two other Listeria species were detected in samples from the importer's premises. One of the L. monocytogenes strains in the importer's factory, which was distinct from the outbreak strain, was also recovered from sweet corn collected from the sandwich manufacturer, sweet corn tested in England in 2013 and 2016 and the blood of two cases of human listeriosis which occurred in England in 2014. This report shows how analysis by WGS provides evidence to understand complex food chains. This report also highlights risks for transmission of human listeriosis from frozen sweet corn and the potential for misuse of this food as a ready-to-eat product.

Occurrence of Listeria and Escherichia coli in frozen fruit and vegetables collected from retail and catering premises in England 2018-2019.

Frozen vegetables have previously been associated with outbreaks of listeriosis in both the USA and Europe. An outbreak of Listeria monocytogenes serogroup 4 caused 53 cases in five European countries between 2015 and 2018. Whole genome sequencing (WGS) indicated that frozen sweet corn from a producer in Hungary was the source of illness. However, limited data is available on the prevalence of Listeria in frozen produce. A study of frozen fruit and vegetables from catering and retail premises in England was therefore carried out to assess their microbiological quality with respect to Listeria and Escherichia coli. Between December 2018 and April 2019, 1050 frozen fruit and vegetable samples were collected. Of these, 99% were of a satisfactory or borderline microbiological quality. Eleven samples (1%) contained ≥100 cfu/g of Escherichia coli (considered unsatisfactory in products labelled as ready-to-eat). Listeria monocytogenes or other Listeria species were detected in six samples (2%) of fruit compared to 167 samples (24%) of vegetables and six samples (26%) of fruit and vegetable mixes, but none at a level of ≥100 cfu/g. Characterisation by WGS of 74 L. monocytogenes isolates identified ten genetic clusters indicating a common source. For 8 of the 10 clusters, the isolates came from homogenous food types: four were sweet corn, and there was one cluster each for beans, peas, peppers and broccoli. There were five genetic associations between isolates from frozen vegetables and from clinical cases of listeriosis, including two cultures from frozen beans that were indistinguishable from the 2015-2018 sweet corn outbreak strain. This study indicates that L. monocytogenes was present in 10% of frozen vegetables and even though products are generally not ready-to-eat and are intended to be cooked prior to consumption, these have the potential to cause illness. Clear cooking and handling instructions are therefore required on these products to ensure that the health of consumers is not put at risk, and appropriate Good Manufacturing Practice measures should be followed by all fruit and vegetable freezing plants in order to reduce contamination with Listeria during processing.

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.

Phylogenomic analysis of gastroenteritis-associated Clostridium perfringens in England and Wales over a 7-year period indicates distribution of clonal toxigenic strains in multiple outbreaks and extensive involvement of enterotoxin-encoding (CPE) plasmids.

Clostridium perfringens is a major enteric pathogen known to cause gastroenteritis in human adults. Although major outbreak cases are frequently reported, only limited whole-genome sequencing (WGS) based studies have been performed to understand the genomic epidemiology and virulence gene content of outbreak-associated C. perfringens strains. We performed phylogenomic analysis on 109 C. perfringens isolates (human and food) obtained from disease cases in England and Wales between 2011 and 2017. Initial findings highlighted the enhanced discriminatory power of WGS in profiling outbreak C. perfringens strains, when compared to the current Public Health England referencing laboratory technique of fluorescent amplified fragment length polymorphism analysis. Further analysis identified that isogenic C. perfringens strains were associated with nine distinct care-home-associated outbreaks over the course of a 5-year interval, indicating a potential common source linked to these outbreaks or transmission over time and space. As expected, the enterotoxin cpe gene was encoded in all but 4 isolates (96.3 %; 105/109), with virulence plasmids encoding cpe (particularly pCPF5603 and pCPF4969 plasmids) extensively distributed (82.6 %; 90/109). Genes encoding accessory virulence factors, such as beta-2 toxin, were commonly detected (46.7 %; 51/109), and genes encoding phage proteins were also frequently identified. Overall, this large-scale genomic study of gastroenteritis-associated C. perfringens suggested that three major cpe-encoding (toxinotype F) genotypes underlie these outbreaks: strains carrying (1) pCPF5603 plasmid, (2) pCPF4969 plasmid and (3) chromosomal-cpe strains. Our findings substantially expanded our knowledge on type F C. perfringens involved in human-associated gastroenteritis, with further studies required to fully probe the dissemination and regional reservoirs of this enteric pathogen, which may help devise effective prevention strategies to reduce the food-poisoning disease burden in vulnerable patients, such as the elderly.

LISTERIA MONOCYTOGENES INFECTION OF FREE-LIVING WESTERN EUROPEAN HEDGEHOGS (ERINACEUS EUROPAEUS).

Listeria monocytogenes is a ubiquitous environmental bacterium that causes disease in a wide range of species. Infection with this pathogen is most frequently diagnosed in ruminant livestock, but is also known to infect people and occasionally wildlife. Postmortem examinations of Western European hedgehogs (Erinaceus europaeus) in Great Britain (2011-2017) identified five (5/266, 2%, 95% confidence interval: 0.8-4.3%) animals with L. monocytogenes infection. The L. monocytogenes isolates comprised three serogroup 1/2a and two serogroup 4 from three multilocus sequence types (2, 37, and 121), all of which were different by single-nucleotide polymorphism analysis, indicating they were distinct and epidemiologically unrelated. These findings are consistent with hedgehogs contracting sporadic infection from the environment, perhaps through eating soil-dwelling invertebrates. Examination of data from scanning surveillance programs focused on other British wildlife species indicates that the hedgehog is one of the wildlife species from which L. monocytogenes has been most frequently identified to date in Great Britain. However, further studies of multiple taxa with comparable sampling efforts are required to assess the relative frequency of L. monocytogenes infection in different wildlife species. The bacterium was isolated from extraintestinal sites in multiple hedgehogs, which may indicate septicemia. However, histological examination was limited and could not discriminate subclinical infection from disease (i.e., listeriosis). Although L. monocytogenes is a zoonotic pathogen, disease in people is typically contracted from the ingestion of contaminated foods. The risk to immunocompetent people of contracting listeriosis from hedgehogs is considered very low to negligible.

Utility of Whole Genome Sequencing To Describe the Persistence and Evolution of Listeria monocytogenes Strains within Crabmeat Processing Environments Linked to Two Outbreaks of Listeriosis.

This article describes the identification and investigation of two extended outbreaks of listeriosis in which crabmeat was identified as the vehicle of infection. Comparing contemporary and retrospective typing data of Listeria monocytogenes isolates from clinical cases and from food and food processing environments allowed the detection of cases going back several years. This information, combined with the analysis of routinely collected enhanced surveillance data, helped to direct the investigation and identify the vehicle of infection. Retrospective whole genome sequencing (WGS) analysis of isolates provided robust microbiological evidence of links between cases, foods, and the environments in which they were produced and demonstrated that for some cases and foods, identified by fluorescent amplified fragment length polymorphism, the molecular typing method in routine use at the time, were not part of the outbreak. WGS analysis also showed that the strains causing illness had persisted in two food production environments for many years and in one producer had evolved into two strains over a period of around 8 years. This article demonstrates the value of reviewing L. monocytogenes typing data from clinical cases together with that from foods as a means of identifying potential vehicles and sources of infection in outbreaks of listeriosis. It illustrates the importance of reviewing retrospective L. monocytogenes typing alongside enhanced surveillance data to characterize extended outbreaks and inform control measures. Also, this article highlights the advantages of WGS analysis for strain discrimination and clarification of evolutionary relationships that refine outbreak investigations and improve our understanding of L. monocytogenes in the food chain.

LiSEQ - whole-genome sequencing of a cross-sectional survey of Listeria monocytogenes in ready-to-eat foods and human clinical cases in Europe.

We present the LiSEQ (Listeria SEQuencing) project, funded by the European Food Safety Agency (EFSA) to compare Listeria monocytogenes isolates collected in the European Union from ready-to-eat foods, compartments along the food chain (e.g. food-producing animals, food-processing environments) and humans. In this article, we report the molecular characterization of a selection of this data set employing whole-genome sequencing analysis. We present an overview of the strain diversity observed in different sampled sources, and characterize the isolates based on their virulence and resistance profile. We integrate into our analysis the global L. monocytogenes genome collection described by Moura and colleagues in 2016 to assess the representativeness of the LiSEQ collection in the context of known L. monocytogenes strain diversity.

Mortality risk factors for listeriosis - A 10 year review of non-pregnancy associated cases in England 2006-2015.

LISTERIOSIS: is a foodborne illness that can result in septicaemia, Central Nervous System (CNS) disease, foetal loss and death in high risk patients. OBJECTIVES: To analyse the demographic trends, clinical features and treatment of non-perinatal listeriosis cases over a ten year period and identify mortality-associated risk factors. METHODS: Reported laboratory-confirmed non-pregnancy associated cases of listeriosis between 2006 and 2015 in England were included and retrospectively analysed. Multivariate logistic regression analysis was performed to determine independent risk factors for mortality. RESULTS: 1357/1683 reported cases met the inclusion criteria. Overall all-cause mortality was 28.7%; however, mortality rates declined from 42.1% to 20.2%. Septicaemia was the most common presentation 69.5%, followed by CNS involvement 22.4%. CNS presentations were significantly associated with age < 50 years, and septicaemia with older age. Age > 80 years (OR 3.32 95% CI 1.92-5.74), solid-organ malignancy (OR 3.42 95% CI 2.29-5.11), cardiovascular disease (OR 3.30 95% CI 1.64-6.63), liver disease (OR 4.61 95% CI 2.47-8.61), immunosuppression (OR 2.12 95% CI 1.40-3.21) and septicaemia (OR 1.60 95% CI 1.17-2.20) were identified as independent mortality risk factors. CONCLUSIONS: High risk groups identified in this study should be the priority focus of future public health strategies aimed at reducing listeriosis incidence and mortality.

A pragmatic harm reduction approach to manage a large outbreak of wound botulism in people who inject drugs, Scotland 2015.

BACKGROUND: People who inject drugs (PWID) are at an increased risk of wound botulism, a potentially fatal acute paralytic illness. During the first 6 months of 2015, a large outbreak of wound botulism was confirmed among PWID in Scotland, which resulted in the largest outbreak in Europe to date. METHODS: A multidisciplinary Incident Management Team (IMT) was convened to conduct an outbreak investigation, which consisted of enhanced surveillance of cases in order to characterise risk factors and identify potential sources of infection. RESULTS: Between the 24th of December 2014 and the 30th of May 2015, a total of 40 cases were reported across six regions in Scotland. The majority of the cases were male, over 30 and residents in Glasgow. All epidemiological evidence suggested a contaminated batch of heroin or cutting agent as the source of the outbreak. There are significant challenges associated with managing an outbreak among PWID, given their vulnerability and complex addiction needs. Thus, a pragmatic harm reduction approach was adopted which focused on reducing the risk of infection for those who continued to inject and limited consequences for those who got infected. CONCLUSIONS: The management of this outbreak highlighted the importance and need for pragmatic harm reduction interventions which support the addiction needs of PWID during an outbreak of spore-forming bacteria. Given the scale of this outbreak, the experimental learning gained during this and similar outbreaks involving spore-forming bacteria in the UK was collated into national guidance to improve the management and investigation of future outbreaks among PWID.

Wound botulism, its neurological manifestations, treatment and outcomes: A case series from the Glasgow outbreak, 2015.

Background and aims We examined the neurological manifestations, treatment and outcomes of a subset of 25 patients within the largest ever outbreak of wound botulism in Europe. Methods and results All 25 cases were intravenous drug users. The most common presenting symptom was dysarthria in 19/25 (76%), followed by dysphagia in 12/25 (48%), blurred vision in 10/25 (40%) and double vision in 8/25 (32%). Microbiological analysis confirmed the diagnosis in nine cases (36%). Duration of admission positively correlated with time to antitoxin, time to wound debridement and female sex. Conclusion As the outbreak continued, hospital stays shortened, reflecting growing awareness of the outbreak and quicker treatment initiation.

Factors influencing the time between onset of illness and specimen collection in the diagnosis of non-pregnancy associated listeriosis in England and Wales.

BACKGROUND: Listeriosis is an opportunistic bacterial infection caused by Listeria monocytogenes and predominantly affects people who are immunocompromised. Due to its severity and the population at risk, prompt clinical diagnosis and treatment of listeriosis is essential. A major step to making a clinical diagnosis is the collection of the appropriate specimen(s) for testing. This study explores factors that may influence the time between onset of illness and collection of specimen in order to inform clinical policy and develop necessary interventions. METHODS: Enhanced surveillance data on non-pregnancy associated listeriosis in England and Wales between 2004 and 2013 were collected and analysed. The difference in days between onset of symptoms and collection of specimen was calculated and factors influencing the time difference were identified using a gamma regression model. RESULTS: The median number of days between onset of symptoms and collection of specimen was two days with 27.1 % of cases reporting one day between onset of symptoms and collection of specimen and 18.8 % of cases reporting more than seven days before collection of specimen. The median number of days between onset of symptoms and collection of specimen was shorter for cases infected with Listeria monocytogenes serogroup 1/2b (one day) and cases with an underlying condition (one day) compared with cases infected with serotype 4 (two days) and cases without underlying conditions (two days). CONCLUSIONS: Our study has shown that Listeria monocytogenes serotype and the presence of an underlying condition may influence the time between onset of symptoms and collection of specimen.

Assessment of the Microbiological Safety of Precut Fruit from Retail and Catering Premises in the United Kingdom.

Fresh fruit has been associated with a number of foodborne outbreaks in recent years. In particular, a large outbreak of listeriosis in the United States in 2011 was associated with consumption of cantaloupe melon, and an outbreak of Salmonella Newport in the United Kingdom and Europe (also in 2011) was linked to watermelon consumption. A study of precut fruit products from catering and retail premises in the United Kingdom was, therefore, carried out to assess their microbiological safety. Between January and March 2012, samples (1,188) of ready-to-eat precut fruit were collected from retail and catering premises in the United Kingdom, and 99% were of satisfactory microbiological quality. However, four samples (0.3%) were of an unsatisfactory quality (one with 800 CFU/g Listeria monocytogenes and three with >100 CFU/g Escherichia coli), and five samples (0.4%) were of a borderline quality owing to the presence of E. coli (two samples with a level of 20 CFU/g), Staphylococcus aureus (two samples with levels of >50 CFU/g), or L. monocytogenes (one sample with a level of 80 CFU/g). L. monocytogenes or other Listeria species were detected in a further 54 samples (4.5%) at levels below the threshold considered to be borderline or unsatisfactory. A significantly larger proportion of samples from one national supermarket chain was contaminated with L. monocytogenes than other supermarkets, and two types were, in this study, unique to this supermarket. This study shows that overall, the microbiological quality of ready-to-eat precut fruit was good. However, the presence of Listeria species in 5% of samples highlights the need for good hygiene during preparation and satisfactory temperature and time control during storage of these food products.

Gene expression in Listeria monocytogenes exposed to sublethal concentration of benzalkonium chloride.

In this study, tolerance at sublethal concentration of benzalkonium chloride and transcription levels of mdrL, ladR, lde, sigB and bcrABC genes in Listeria monocytogenes strains were evaluated. Viable cells reduction occurred in 45% of strains and clinical isolates showed lower sensitivity than isolates from foods. An increased transcription of an efflux system encoding gene was found in 60% of strains, and simultaneous mdrL overexpression and ladR underexpression occurred in 30% of isolates. A significant association between reduced benzalkonium chloride activity and both mdrL and sigB overexpression was observed; sigB expression also correlated with both mdrL and ladR genes. The bcrABC gene was only found in six strains, all isolated from foods and sensitive to benzalkonium chloride, and in four strains an underexpression was observed. Disinfection at sublethal concentration was less effective in clinical isolates, and mdrL and sigB expression was significantly affected by disinfection. Further insights are needed to understand the adaptation to benzalkonium chloride and to evaluate whether changes in gene expression could affect the L. monocytogenes virulence traits and persistence in the environment.

Fluorescent amplified fragment length polymorphism (fAFLP) analysis of Listeria monocytogenes.

Fluorescent amplified fragment length polymorphism (fAFLP) is based on the selective PCR amplification of restriction fragments from a digest of total genomic DNA. Genomic DNA extracted from a purified bacterial isolate is completely digested with two endonucleases generating fragments which are ligated to specific double-stranded adaptors. The ligated fragments are then amplified by PCR using fluorescently labelled primers. Fluorescent amplified fragments are separated by size on an automated sequencer with a size standard. fAFLP is a rapid, highly reproducible technique which can be used to discriminate and subtype Listeria monocytogenes strains.

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.

Fluorescence amplified fragment length polymorphism compared to pulsed field gel electrophoresis for Listeria monocytogenes subtyping.

BACKGROUND: Listeriosis is a severe infection which mainly affects pregnant women, neonates and immuno-compromised adults. ANSES's Laboratory for Food safety has been the European Union Reference Laboratory (EURL) for L. monocytogenes in the food chain since 2006. Pulsed Field Gel Electrophoresis (PFGE) is routinely used in the EURL for the surveillance of L. monocytogenes isolated from foods, animals and the environment. One of the main EURL activities is to evaluate alternative molecular subtyping methods to PFGE, and integrate their use within the National Reference Laboratories (NRL) network. Since 2008, the United Kingdom (UK)-NRL for L. monocytogenes at the Health Protection Agency (HPA), London, has used fluorescent Amplified Fragment Length Polymorphism (fAFLP) for the routine surveillance of L. monocytogenes isolated from human clinical cases, food and food processing environments in the UK. This study compares fAFLP with PFGE for subtyping L. monocytogenes. RESULTS: A panel of 109 L. monocytogenes isolates from either human cases of listeriosis, foods, food processing environments and animals were used for the comparative evaluation. Among these, 2 strains were tested from duplicate culture by both methods. The panel also included field isolates, isolates associated with outbreaks or sporadic cases and reference strains. The two strains tested in duplicate displayed the same fAFLP and PFGE types. Strains known to be epidemiologically associated with one another were found to have unique PFGE and fAFLP types. FAFLP and PFGE divided the strains into 76 and 82 distinct profiles, or types, respectively. The discriminatory index calculated was 0.993 and 0.996 for fAFLP and PFGE, respectively. CONCLUSIONS: The discriminatory ability of fAFLP was similar to that of PFGE for the subtyping of L. monocytogenes isolates. As a less labour intensive technique fAFLP may be a better method to use than PFGE in investigating outbreaks of human listeriosis and tracking the source of contamination in food processing facilities in real time.

Real-time PCRs assay for serogrouping Listeria monocytogenes and differentiation from other Listeria spp.

A 5'-exonuclease real-time triplex-PCR assay was developed for serogrouping Listeria monocytogenes, and differentiation from other Listeria spp. The assay was evaluated on 109 Listeria cultures, and results were compared with a previously validated gel-based multiplex-PCR procedure. All L. monocytogenes were correctly classified into four serogroups, including atypical serotype 4b strains, and differentiated from other Listeria species. The assay is a rapid method for categorisation of suspect L. monocytogenes.