Microbiological Quality of Ready-to-Eat Salad Products Collected from Retail and Catering Settings in England during 2020 to 2021.

ABSTRACT: Salad and other fresh produce were collected in England from retail and catering businesses during 2020 to 2021 and were tested for Salmonella, Shiga toxin-producing Escherichia coli (STEC), Listeria, Bacillus cereus, and E. coli. Of the 604 samples collected, 57% were from retail settings and 43% were from catering settings; 61% were either salad leaves or salad leaves mixed with other products. Equal numbers of samples were prepacked or loose, and 50% were refrigerated at the time of sampling. Combining results for all microbiological parameters, 84% were interpreted as satisfactory, 12% were interpreted as borderline, and 4% were interpreted as unsatisfactory. One sample (prepacked leaves, cucumber, and tomato from a caterer) was categorized as unacceptable and potentially injurious because of detection of STEC O76; no STEC from human infections in the United Kingdom matched this isolate. No Salmonella enterica was detected, but Listeria monocytogenes was recovered from 11 samples: 1 at 20 CFU/g and the remainder at <20 CFU/g. B. cereus was detected at borderline levels (103 to ≤105 CFU/g) in 9% of samples and at an unsatisfactory level (>105 CFU/g) in one sample. E. coli was detected in 3% of samples at borderline levels (20 to ≤102 CFU/g) and in 4% at unsatisfactory levels (>102 CFU/g). There was a significant association between detection of L. monocytogenes and borderline or unsatisfactory levels of E. coli. There were no specific risk profiles associated with products with the higher levels of B. cereus, STEC, or Listeria, but elevated levels of E. coli were predominantly confined to loose products from the United Kingdom collected from caterers in summer or autumn 2021 and may have resulted from relaxation of COVID-19 restrictions. Among the L. monocytogenes isolates, only one matched those from human cases and was recovered from a prepacked mixed salad from a catering business in 2021. This isolate was the same strain as that responsible for a multicountry outbreak (2015 to 2018) associated with Hungarian-produced frozen sweet corn; no link to the outbreak food chain was established.

Human foodborne listeriosis in England and Wales, 1981 to 2015.

Almost all cases of human listeriosis are foodborne, however the proportion where specific exposures are identified is small. Between 1981 and 2015, 5252 human listeriosis cases were reported in England and Wales. The purpose of this study was to summarise data where consumption of specific foods was identified with transmission and these comprised 11 sporadic cases and 17 outbreaks. There was a single outbreak in the community of 378 cases (7% of the total) which was associated with pâté consumption and 112 cases (2% of the total) attributed to specific foods in all the other incidents. The proportion of food-attributed cases increased during this study with improvements in typing methods for Listeria monocytogenes. Ten incidents (one sporadic case and nine outbreaks of 2-9 cases over 4 days to 32 months) occurred in hospitals: all were associated with the consumption of pre-prepared sandwiches. The 18 community incidents comprised eight outbreaks (seven of between 3 and 17 cases) and 10 sporadic cases: food of animal origin was implicated in 16 of the incidents (sliced or potted meats, pork pies, pâté, liver, chicken, crab-meat, butter and soft cheese) and food of non-animal origin in the remaining two (olives and vegetable rennet).

An assessment of the microbiological quality of liver-based pâté in England 2012-13: comparison of samples collected at retail and from catering businesses.

The purpose of this study was to investigate the microbiological quality of liver pâté. During 2012-13, a total of 870 samples, unrelated to the investigation of food-poisoning outbreaks, were collected either at retail (46%), catering (53%) or the point of manufacture (1%) and were tested using standard methods to detect Salmonella spp. or Campylobacter spp., and to enumerate for Listeria spp., including Listeria monocytogenes, Clostridium perfringens, coagulase-positive staphylococci including Staphylococcus aureus, Bacillus spp., including Bacillus cereus, Escherichia coli, Enterobacteriaceae, and aerobic colony counts (ACCs). Seventy-three percent of samples were of satisfactory microbiological quality, 18% were borderline and 9% unsatisfactory. Salmonella spp. or Campylobacter spp. was not recovered from any sample. The most common causes of unsatisfactory results were elevated ACCs (6% of the samples) and high Enterobacteriaceae counts (4% of samples). The remaining unsatisfactory results were due to elevated counts of: E. coli (three samples); B. cereus (one sample at 2·6 × 105 cfu/g); or L. monocytogenes (one sample at 2·9 × 103 cfu/g). Pâté from retail was less likely to be contaminated with L. monocytogenes than samples collected from catering and samples from supermarkets were of significantly better microbiological quality than those from catering establishments.

Investigation of an outbreak of vomiting in nurseries in South East England, May 2012.

On 30 May 2012, Surrey and Sussex Health Protection Unit was called by five nurseries reporting children and staff with sudden onset vomiting approximately an hour after finishing their lunch that day. Over the following 24 h 50 further nurseries supplied by the same company reported cases of vomiting (182 children, 18 staff affected). Epidemiological investigations were undertaken in order to identify the cause of the outbreak and prevent further cases. Investigations demonstrated a nursery-level attack rate of 55 out of 87 nurseries (63·2%, 95% confidence interval 52·2-73·3). Microbiological tests confirmed the presence of Bacillus cereus in food and environmental samples from the catering company and one nursery. This was considered microbiologically and epidemiologically consistent with toxin from this bacterium causing the outbreak. Laboratory investigations showed that the conditions used by the caterer for soaking of pearl haricot beans (known as navy bean in the USA) used in one of the foods supplied to the nurseries prior to cooking, was likely to have provided sufficient growth and toxin production of B. cereus to cause illness. This large outbreak demonstrates the need for careful temperature control in food preparation.

Infant botulism due to C. butyricum type E toxin: a novel environmental association with pet terrapins.

We describe two cases of infant botulism due to Clostridium butyricum producing botulinum type E neurotoxin (BoNT/E) and a previously unreported environmental source. The infants presented at age 11 days with poor feeding and lethargy, hypotonia, dilated pupils and absent reflexes. Faecal samples were positive for C. butyricum BoNT/E. The infants recovered after treatment including botulism immune globulin intravenous (BIG-IV). C. butyricum BoNT/E was isolated from water from tanks housing pet 'yellow-bellied' terrapins (Trachemys scripta scripta): in case A the terrapins were in the infant's home; in case B a relative fed the terrapin prior to holding and feeding the infant when both visited another relative. C. butyricum isolates from the infants and the respective terrapin tank waters were indistinguishable by molecular typing. Review of a case of C. butyricum BoNT/E botulism in the UK found that there was a pet terrapin where the infant was living. It is concluded that the C. butyricum-producing BoNT type E in these cases of infant botulism most likely originated from pet terrapins. These findings reinforce public health advice that reptiles, including terrapins, are not suitable pets for children aged <5 years, and highlight the importance of hand washing after handling these pets.

Identification of six Listeria species by real-time PCR assay.

UNLABELLED: The Listeria genus comprises 10 recognized species. Listeria monocytogenes causes listeriosis in humans and other animals primarily via contaminated food or animal feed. Listeria ivanovii causes listeriosis in animals and on rare occasions in humans. The identification of nonpathogenic species of Listeria in foods indicates that conditions exist that support the growth of pathogenic strains and is used to facilitate the implementation of control and prevention measures. This study shows the development and evaluation of a 5'exonuclease real-time PCR assay for the rapid identification of Listeria seeligeri, Listeria welshimeri, L. monocytogenes, L. ivanovii, Listeria grayi and Listeria innocua. The assay consists of two triplexes that were evaluated using 53 cultures of Gram-positive bacteria, including 49 Listeria spp. from human, animal, food or food-processing environments. The assay was rapid, specific and reproducible and could identify each of the six species from a mixture of strains. The developed assay proved to be a powerful means of rapidly identifying Listeria species and could be usefully implemented in busy specialist reference laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of species of Listeria from foods is important to monitor pathogenic strains and facilitates the implementation of control measures. This study shows the development and evaluation of a 5'exonuclease real-time PCR assay for the rapid identification of L. seeligeri, L. welshimeri, L. monocytogenes and L. ivanovii, L. grayi, L. innocua. The developed assay proved to be specific, rapid and reproducible and therefore could be implemented in busy specialist reference laboratories.

A case of foodborne listeriosis linked to a contaminated food-production process.

We report a case of listeriosis linked to consumption of contaminated ox tongue. A public health investigation identified intermittent contamination at a meat-production process and ox-tongue production was discontinued. Sensitive molecular subtyping methods are improving our ability to track sources of Listeria monocytogenes contamination through the food chain. Detailed investigation of sporadic cases of listeriosis can provide important public health information and its wider use is encouraged.

Hospital-acquired listeriosis associated with sandwiches in the UK: a cause for concern.

BACKGROUND: Hospital-acquired outbreaks of listeriosis are not commonly reported but remain a significant public health problem. AIM: To raise awareness of listeriosis outbreaks that have occurred in hospitals and describe actions that can be taken to minimize the risk of foodborne listeriosis to vulnerable patients. METHODS: Foodborne outbreaks and incidents of Listeria monocytogenes reported to the Health Protection Agency national surveillance systems were investigated and those linked to hospitals were extracted. The data were analysed to identify the outbreak/incident setting, the food vehicle, outbreak contributory factors and origin of problem. FINDINGS: Most (8/11, 73%) foodborne outbreaks of listeriosis that occurred in the UK between 1999 and 2011 were associated with sandwiches purchased from or provided in hospitals. Recurrently in the outbreaks the infecting subtype of L. monocytogenes was detected in supplied prepacked sandwiches and sandwich manufacturing environments. In five of the outbreaks breaches in cold chain controls of food also occurred at hospital level. CONCLUSIONS: The outbreaks highlight the potential for sandwiches contaminated with L. monocytogenes to cause severe infection in vulnerable people. Control of L. monocytogenes in sandwich manufacturing and within hospitals is essential to minimize the potential for consumption of this bacterium at levels hazardous to health. Manufacturers supplying sandwiches to hospitals should aim to ensure absence of L. monocytogenes in sandwiches at the point of production and hospital-documented food safety management systems should ensure the integrity of the food cold chain.

Detection by PCR of eight groups of enteric pathogens in 4,627 faecal samples: re-examination of the English case-control Infectious Intestinal Disease Study (1993-1996).

The English case-control Infectious Intestinal Disease Study (1993-1996) failed to detect an enteric pathogen or toxin in 49% of cases of gastroenteritis. In the present study, polymerase chain reaction (PCR) assays were applied to DNA and cDNA generated from 4,627 faecal samples from cases and controls archived during the original study for the detection of norovirus, rotavirus, sapovirus, Campylobacter spp., Salmonella spp., enteroaggregative Escherichia coli, Cryptosporidium spp., and Giardia spp. The percentage of archived samples from cases and from controls in which at least one agent (or toxin) was detected increased from 53% in the original study to 75% and from 19 to 42%, respectively, after the application of PCR assays. Among cases, the following percentages of enteric pathogens were detected: norovirus 36%, rotavirus A 31%, sapovirus 4%, Salmonella spp. 6%, Campylobacter jejuni 13%, Campylobacter coli 2%, other Campylobacter spp. 8%, enteroaggregative E. coli 6%, Giardia spp. 2%, and Cryptosporidium spp. 2%. The present study provides additional insight into the aetiology of infectious intestinal disease in England and highlights the occurrence of viral infections in cases as well as in asymptomatic individuals. Other notable findings include the frequent presence of Campylobacter spp. other than C. jejuni or C. coli, the high frequency of multiple agents in 41% of cases and in 13% of controls, and the variation in the aetiology and rate of infection found for different age groups. The results demonstrate the greater sensitivity of PCR-based methods compared to current conventional methods.

Characterisation of a Cryptosporidium isolate from water buffalo (Bubalus bubalis) by sequencing of a fragment of the Cryptosporidium oocyst wall protein gene (COWP).

Cryptosporidium oocysts were detected using a direct immunofluorescence antibody test in the faeces of an asymptomatic water buffalo (Bubalus bubalis) heifer from a dairy farm close to Santiago de Compostela (NW Spain). Oocysts were morphologically indistinguishable from Cryptosporidium parvum. Using DNA extracted from this sample and a PCR-RFLP analysis of a 341 base pairs fragment of the Cryptosporidium oocyst wall protein (COWP) gene, a previously undescribed fragment pattern was generated. The COWP gene fragment was cloned and sequencing analyses revealed it to be similar to the C. parvum 'pig' genotype but with four base pairs substitutions.

Blinded application of microscopy, bacteriological culture, immunoassays and PCR to detect gastrointestinal pathogens from faecal samples of patients with community-acquired diarrhoea.

A blinded trial in two different laboratories was performed to compare the detection of selected enteric pathogens in 92 unselected faecal samples collected from patients with community-acquired diarrhoea by conventional and PCR-based techniques. Conventional techniques detected a single potential etiological agent in 15% of the samples, whereas results of PCR detected evidence of at least one agent in 41% of the samples. Overall, the detection rates for the different pathogens were as follows: adenovirus serogroup F, 1%; Campylobacter spp., 7.6%; Salmonella spp., 4%; enteroaggregative Escherichia coli, 9.8%; enteropathogenic E. coli, 6.5%; enterotoxigenic Clostridium perfringens, 3%; Cryptosporidium spp., 13%; and Giardia spp., 11%. Results for the detection of Salmonella spp., Campylobacter spp. and C. perfringens were similar by both techniques, whereas Cryptosporidium and Giardia spp. were detected 22 times more often by PCR than by conventional microscopy. It was not possible to compare the results for detection of enteroaggregative E. coli and enteropathogenic E. coli since these were only investigated by PCR. The results of this small study clearly demonstrate the advantages of PCR-based methods compared to conventional techniques for the detection of gastrointestinal pathogens.

Detection and identification by real time PCR/RFLP analyses of Cryptosporidium species from human faeces.

AIMS: To detect a wide range of Cryptosporidium species from human faeces by analysis of the Cryptosporidium oocyst wall protein gene by PCR. METHODS AND RESULTS: The nested-assay comprised an initial amplification using a conventional thermocycler followed by real time PCR using a LightCycler with SYBR Green I for the characterization of the amplicons. The technique uses four sets of primers composed of five to six oligonucleotides with one to six base differences corresponding to the inter-species sequence differences of the gene fragment. Restriction fragment length polymorphism analysis identified Cryptosporidium hominis and C. parvum. The assay was evaluated using DNA extracted from purified material and faecal specimens containing a range of potential pathogens (including Cryptosporidium). The assay was specific, sensitive, reproducible and rapid. CONCLUSIONS: This unique technique enables the rapid detection of a range of polymorphic COWP gene sequences directly from faeces using real time PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates a novel approach to identification of Cryptosporidium species and the identification of C. hominis and C. parvum. The technique may be especially useful for the analysis of environmental samples which are likely to contain heterogeneous mixtures of Cryptosporidium species.

Detection of Cryptosporidium and Giardia in molluscan shellfish by multiplexed nested-PCR.

A multiplexed nested-PCR procedure (ABC-PCR) previously developed to detect Cryptosporidium spp. and Giardia duodenalis assemblages A and B in whole human faeces was applied to DNA extracted from filter-feeding molluscs. Species of Cryptosporidium and G. duodenalis were identified by restriction fragment analysis of the PCR products and by DNA sequencing. The extraction and ABC-PCR procedures were shown to be suitable for application to shellfish by amplification of specific target sequences using DNA from Cryptosporidium parvum genotype 2 and G. duodenalis assemblages A and B which were spiked into DNA extracted from mussels. Using 49 molluscan shellfish specimens (18 clam, 22 mussel and 9 oyster samples) from Spain, cryptosporidial oocysts were detected in 56% by immunofluorescence microscopy, and in 44% by ABC-PCR. For detection of Cryptosporidium, there was a significant association, but not total agreement, between the results of microscopy and PCR. G. duodenalis assemblage B was detected from one oyster sample by PCR. Amongst 38 specimens (20 mussel and 18 cockle samples) collected in the UK and tested by the ABC-PCR, G. duodenalis was not detected, and Cryptosporidium was detected in 11% of the samples. Overall, the 26 samples where Cryptosporidium was detected, C. hominis/C. parvum genotype 1 was detected in 1, C. parvum genotype 2 in 22, and the remaining three samples contained either sequences similar to C. parvum genotype 2 or heterogeneous mixtures of Cryptosporidium species. There was no significant association between the level of Escherichia coli detected by conventional microbiological methods and the presence of Cryptosporidium detected by ABC-PCR.

Blinded evaluation of DNA extraction and genotyping of stained Cryptosporidium on glass slides.

AIMS: A blinded trial was performed on Cryptosporidium genotyping using polymerase chain reaction (PCR)/restriction fragment length polymorphism analysis of the Cryptosporidium oocyst wall protein (COWP) gene between DNA extracted from oocyst suspensions as compared with DNA from fixed and stained faecal smears on glass microscope slides. METHODS AND RESULTS: Sixty-five faecal smears on slides were stained by one of three different methods comprising 50 positives and 15 negatives as determined by the observation of Cryptosporidium oocysts by microscopy. The expected result in terms of detection and the COWP genotype detected was achieved using DNA extracted from 94% of the slides tested. CONCLUSIONS: This study shows that DNA, which can be amplified by PCR, is present in stained smears on glass microscope slides. SIGNIFICANCE AND IMPACT OF THE STUDY: The method may be useful for molecular epidemiological studies on a range of gastrointestinal pathogens where samples are collected from locations remote from the testing laboratory.

Sensitive PCR-restriction fragment length polymorphism assay for detection and genotyping of Giardia duodenalis in human feces.

An assay that uses heminested PCR-restriction fragment length polymorphism analysis for the detection and genotyping of Giardia duodenalis on the basis of polymorphism in the triose phosphate isomerase (tpi) gene was developed. This assay was evaluated with DNA extracted from purified parasite material, bacterial cultures, whole human feces containing G. duodenalis and other parasites, and their corresponding immunofluorescence-stained fecal smears on glass microscope slides. The assay was specific and discriminated between G. duodenalis assemblages A and B. RFLP analysis further distinguished two groups (designated groups I and II) within assemblage A. Among 35 DNA samples extracted from whole feces from patients with confirmed sporadic giardiasis, the tpi gene was amplified from 33 (94%). Of these, nine (27%) samples contained assemblage A group II, 21 (64%) contained assemblage B, and 3 (9%) contained a mixture of assemblage A group II and assemblage B. The tpi gene of G. duodenalis assemblage B was amplified from 21 of 24 (88%) DNA samples extracted from whole feces from patients with confirmed cases of infection in a nursery outbreak. No amplification was detected from the remaining three DNA samples. Overall, analysis of DNA extracted from material recovered from stained microscope slides identified identical G. duodenalis genotypes in 35 (65%) of the 54 samples for which a genotype was established with DNA from whole feces. The heminested PCR method developed is sensitive, simple, and rapid to perform and is applicable for the analysis of other intestinal pathogens.