The microbiological quality of flour products in the UK with respect to Salmonella and Shiga-toxin-producing E. coli (STEC).

AIM: To investigate the possible contamination of raw flour and raw flour-based products, such as pancake/batter mixes, with Salmonella, generic E. coli and STEC. Samples included flours available for sale in the UK over a period of four months (January to April 2020). The Bread and Flour regulations, 1998 state the permitted ingredients in flour and bread but it does not specify the regular monitoring of the microbiological quality of flour and flour-based products. METHODS AND RESULTS: Samples of raw flour were collected by local authority sampling officers in accordance with current guidance on microbiological food sampling then transported to the laboratory for examination. Microbiological testing was performed to detect Salmonella spp., generic E. coli and Shiga-toxin-producing Escherichia coli (STEC) characterised for the presence of STEC virulence genes: stx1, stx2 and subtypes, eae, ipah, aggR, lt, sth and stp, using molecular methods (PCR). Of the 882 flours sampled, the incidence of Salmonella was 0.1% (a single positive sample that contained multiple ingredients such as flour, dried egg and dried milk, milled in the UK), and 68 samples (7.7%) contained generic E. coli at a level of >20 CFU/g. Molecular characterisation of flour samples revealed the presence of the Shiga-toxin (stx) gene in 10 samples (5 imported and 5 from the UK) (1.1%), from which STEC was isolated from 7 samples (0.8%). Salmonella and STEC isolates were sequenced to provide further characterisation of genotypes and to compare to sequences of human clinical isolates held in the UKHSA archive. Using our interpretive criteria based on genetic similarity, none of the STEC flour isolates correlated with previously observed human cases, while the singular Salmonella serotype Newport isolate from the mixed ingredient product was similar to a human case in 2019, from the UK, of S. Newport. Although there have been no reported human cases of STEC matching the isolates from these flour samples, some of the same serotypes and stx subtypes detected are known to have caused illness in other contexts. CONCLUSION: Results indicate that while the incidence was low, there is a potential for the presence of Salmonella and STEC in flour, and a genetic link was demonstrated between a Salmonella isolate from a flour-based product and a human case of salmonellosis.

Public health risks associated with Salmonella contamination of imported edible betel leaves: Analysis of results from England, 2011-2017.

Fresh betel leaves (Piper betle L.), imported into the UK are a traditional ready-to-eat food consumed by Asian populations. We report here the consolidation of routinely collected data to model the public health risks from consumption of this food. Amongst 2110 samples collected at Border Inspection, wholesale, catering or retail, Salmonella was detected in 488 (23%) of samples tested between 2011 and 2017 and was the most commonly Salmonella-contaminated ready-to-eat food examined by Public Health England during this period. Using data from multiple samples (usually 5) tested per consignment sampled at Border Inspection, contamination levels were calculated by most probable number: seasonal, temporal and country specific differences were detected. Quantitative contamination data was used to estimate the levels present at retail, and a ß-Poisson dose response model the probability of illness was calculated. Using data for products imported from India, the probability of acquiring infection following a single exposure (comprising of a single leaf) was estimated to be between 0.00003 (January-March) and 0.0001 (July-September). Using British Asian population data for individuals over 30 years of age in England in 2011, two estimates of consumption were modelled as 2.1 and 12.8 million servings per annum. Results from the model estimated 160 cases (range 102 to 242) and 960 cases (range 612 to 1456) per year in England for the two consumption estimates and equated to 34 (range 22 to 51) and 204 (range 130 to 310) salmonellosis cases per year reported to national surveillance. Salmonella from 475 of the contaminated samples were further characterised which showed a heterogeneous population structure with 46 S. enterica subsp. Enterica serovars, together with S. enterica subs diarizonae and salamae identified. Isolates from individual consignments were diverse and close genetic relationships between independent isolates were very rare except from within an individual consignment. There were no outbreaks detected as associated with betel leaf consumption. However analysis by whole genome sequencing of the 2014-17 data identified two cases where the clinical isolate had <5 single nucleotide polymorphism differences to isolates from betel leaves which is indicative of a likely epidemiological link and common source of contamination. Due to the diversity of the Salmonella contaminating this product, associations between salmonellosis cases and betel leaf consumption will appear sporadic and unlikely to be detected by current surveillance strategies based on outbreak detection.

Imported edible leaves collected at retail sale in England during 2017 with an emphasis on betel and curry leaves: microbiological quality with respect to Salmonella, Shiga-toxin-producing E. coli (STEC) and levels of Escherichia coli.

AIMS: To investigate the microbiological quality of imported fresh leaves on retail sale during 2017 with respect to Salmonella, Shiga-toxin-producing Escherichia coli (STEC) and levels of E. coli. METHODS AND RESULTS: Two hundred and seventy-nine samples of imported edible leaves (69 banana, 77 betel, 118 curry and 15 other types) were tested. Salmonella spp. were confirmed by whole-genome sequencing and isolated from 44 samples, 26% from curry leaves, 14% from betel and 2·4% from all other leaf types: 80% of all samples contained ≥102 , 44% ≥103 and 22% ≥104 CFU of E. coli CFU per g. All samples where Salmonella were detected also yielded ≥20 CFU of E. coli/g. 54 samples were tested for STEC which was detected in six samples and isolated from three: one was identified as STEC O157:H7. CONCLUSIONS: This report further highlights an ongoing problem of Salmonella contamination of imported fresh edible leaves. SIGNIFICANCE AND IMPACT OF THE STUDY: Among all food tested by Public Health England (approximately 11 000 per annum), curry leaves were the herb most commonly contaminated with Salmonella, and betel leaves were the most commonly contaminated ready-to-eat food. The high proportion with unsatisfactory E. coli levels and the detection of STEC suggests risks of contamination by multiple enteric pathogens.

Whole genome sequencing improved case ascertainment in an outbreak of Shiga toxin-producing Escherichia coli O157 associated with raw drinking milk.

Five cases of STEC O157 phage type (PT) 21/28 reported consumption of raw cows' drinking milk (RDM) produced at a dairy farm in the South West of England. STEC O157 PT21/28 was isolated from faecal specimens from milking cows on the implicated farm. Whole genome sequencing (WGS) showed that human and cattle isolates were the same strain. Further analysis of WGS data confirmed that sequences of isolates from an additional four cases (who did not report consumption of RDM when first questioned) fell within the same five single nucleotide polymorphism cluster as the initial five cases epidemiologically linked to the consumption of RDM. These four additional cases identified by WGS were investigated further and were, ultimately, associated with the implicated farm. The RDM outbreak strain encoded stx2a, which is associated with increased pathogenicity and severity of symptoms. Further epidemiological analysis showed that 70% of isolates within a wider cluster containing the outbreak strain were from cases residing in, or linked to, the same geographical region of England. During this RDM outbreak, use of WGS improved case ascertainment and provided insights into the evolution of a highly pathogenic clade of STEC O157 PT21/28 stx2a associated with the South West of England.

Trends in ExPEC serogroups in the UK and their significance.

Extra-intestinal pathogenic Escherichia coli are a significant cause of urinary tract infection and bacteraemia within the UK. We sought to identify the serogroups of 658 E. coli isolates collected in the UK between January 2011 and March 2012, to better understand the ExPEC population and understand the relevance of serogroups in this pathotype. Isolates were typed and serogroup identified using established phenotypic and molecular methods. Sixty-two serogroups were identified; 54 among urinary isolates and 35 among bloodstream isolates. However, serogroups O25, O6, and O2 dominated both infection types. These serogroups were linked to the major ExPEC STs as follows: ST131-O25, ST73-O6, ST127-O6, and ST95-O2. The serogroup data from this study have increased our understanding of the ExPEC population in the UK, but also highlighted key ST-serogroup relationships within the major ExPEC clones. These data can be used to guide vaccine design and in the development of laboratory diagnostic tests targeting the ExPEC population.

Comparative study of all Salmonella enterica serovar Enteritidis strains isolated from food and food animals in Greece from 2008 to 2010 with clinical isolates.

The aim of the present work was to study the epidemiology of Salmonella enterica serovar Enteritidis (S. Enteritidis) in Greece, comparing all the food and food animal isolates during a 3-year period with clinical isolates. Submission of the generated data to the PulseNet Europe database was carried out in order to study the population structure of this particular serovar and indicate possible connections with European strains. One hundred and sixty-eight (168) S. Enteritidis strains of human, animal, and food origin, isolated during the period 2008-2010 in Greece, were studied. Strains were characterized by phenotypic (antibiotic resistance) and molecular [pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)] methods. PFGE revealed 39 XbaI, 48 BlnI, and 80 XbaI-BlnI distinct pulsotypes, suggesting several clones circulating through the food chain and multiple sources of transmission. Submission to the PulseNet Europe database indicated that PFGE profile SENTXB.0001, the most common PFGE profile in Europe, was also predominant in Greece (33.3 %). MLST showed that all the strains studied shared the same sequence type (ST11), representing the most common ST in Europe. High rates of resistance to nalidixic acid were observed among human and poultry isolates (~25 %), indicating the potential fluoroquinolone treatment failure. Our data suggest that strains originating from multiple reservoirs circulated in Greece through the food chain during the study period. Predominant profiles in Greece were common to PulseNet Europe profiles, indicating similarities between the S. Enteritidis populations in Greece and Europe.

Chromosomal location of blaCTX-M genes in clinical isolates of Escherichia coli from Germany, The Netherlands and the UK.

This study aimed to detect and characterise clinical Escherichia coli isolates suspected of carrying chromosomally encoded CTX-M enzymes. Escherichia coli (n=356) obtained in Germany, The Netherlands and the UK (2005-2009) and resistant to third-generation cephalosporins were analysed for the presence of ESBL-/AmpC-encoding genes within the European SAFEFOODERA-ESBL project. ß-Lactamases and their association with IS26 and ISEcp1 were investigated by PCR. Isolates were typed by phylogenetic grouping, MLST and PFGE. Plasmids were visualised by S1 nuclease PFGE, and the location of blaCTX-M genes was determined by Southern hybridisation of XbaI-, S1- and I-CeuI-digested DNA. ESBL enzymes could not be located on plasmids in 17/356 isolates (4.8%). These 17 isolates, from different countries and years, were ascribed to phylogenetic groups D (9), B2 (6) and B1 (2), and to seven sequence types, with ST38 being the most frequent (7 phylogroup D isolates). Eleven isolates produced CTX-M-15. blaCTX-M-15 genes were associated with ISEcp1. The remaining isolates expressed the CTX-M group 9 ß-lactamases CTX-M-14 (4), CTX-M-9 (1) and CTX-M-51 (1). blaCTX-M probes hybridised with I-CeuI- and/or XbaI-digested DNA, but not with S1-digested DNA, corroborating their chromosomal location. To summarise, only 4.8% of a large collection of ESBL-producing E. coli isolates harboured chromosomal blaCTX-M genes. These isolates were of human origin and belonged predominantly to ST38 and ST131, which possibly indicates the role of these sequence types in this phenomenon. However, heterogeneity among isolates was found, suggesting that their spread is not only due to the dispersion of successful E. coli clones.

Investigating the link between the presence of enteroaggregative Escherichia coli and infectious intestinal disease in the United Kingdom, 1993 to 1996 and 2008 to 2009.

There are an estimated 17 million human diarrhoea cases annually in the United Kingdom. In 2008 and 2009, enteroaggregative E. coli (EAEC) were identified in 1.9% of stools. However, it remains unclear whether there is a causal link between presence of EAEC and disease. This study used bacterial load, the presence of co-infections and demographic data to assess if EAEC was independently associated with intestinal infectious disease. Quantitative real-time PCR data (Ct values) generated directly from stool specimens for several pathogen targets were analysed to identify multiple pathogens, including EAEC, in the stools of cases and healthy controls. Sensitivity and specificity using Ct value (60% and 60%) was not useful for identifying cases or controls, but an independent association between disease and EAEC presence was demonstrated: multivariate logistic regression for EAEC presence (odds ratio: 2.41; 95% confidence interval: 1.78­3.26; p<0.001). The population-attributable fraction was 3.3%. The group of bacteria known as EAEC are associated with gastrointestinal disease in at least half of the cases with EAEC positive stools. We conclude that the current definition of EAEC, by plasmid gene detection, includes true pathogens as well as non-pathogenic variants.

Characterisation of Exiguobacterium aurantiacum isolates from blood cultures of six patients.

Exiguobacterium spp. are alkaliphilic, halotolerant, non-spore-forming Gram-positive bacilli, hitherto uncharacterized from human infections. Six isolates of Exiguobacterium aurantiacum were obtained from patients with bacteraemia, three of whom had myeloma. All isolates formed orange-yellow pigmented colonies on blood agar, were catalase- and DNase-positive, and grew on nutrient agar at pH 10 and in the presence of NaCl 6% w/v. The six isolates were susceptible to all antimicrobial agents tested and were uniform in their fatty acid and mass spectrum profiles.

Comparative analysis of virulence determinants and mass spectral profiles of Finnish and Lithuanian endodontic Enterococcus faecalis isolates.

INTRODUCTION: Putative virulence factors of Enterococcus faecalis have been proposed by several workers and, by analogy, these have been linked to strains of endodontic origin. However, their distribution within the cell population is unknown. In the present study, isolates were taken from the dental root canals of two defined human populations, Lithuanian and Finnish, and examined for a range of virulence properties. In addition, surface-associated molecules and intracellular proteins were compared using matrix-assisted laser desorption-ionization/mass spectrometry (MALDI-TOF-MS) and ProteinChip capture/MS (SELDI-TOF-MS), respectively. METHODS: Twenty-three Lithuanian and 35 Finnish dental root canal isolates were included. The esp, gelE, ace and efaA genes were detected by polymerase chain reaction, and cytolysin and gelatinase phenotypes were determined by hydrolysis of horse blood agar and gelatine agar, respectively. Protein extracts and surface-associated molecules of whole cells were analysed by SELDI-TOF-MS and MALDI-TOF-MS, respectively. RESULTS: Presence of esp (n = 15), cytolysin (n = 9), ace (n = 55) and efaA (n = 58) was not statistically different in the two samples, whereas gelE and gelatinase production was detected more frequently in the Finnish material (chi-squared, P < 0.01). Analysis of protein profiles by SELDI-TOF-MS showed clustering of cytolysin-producing strains, whereas MALDI-TOF-MS generated profiles that clustered according to the samples' origin and, furthermore, to atypical quinupristin-dalfopristin susceptibility. CONCLUSION: A high prevalence of virulence factors was demonstrated in both population types. SELDI-TOF-MS and MALDI-TOF-MS proved useful in distinguishing between different E. faecalis phenotypes and they may be useful technologies for elucidating the eco-distribution of E. faecalis in humans.