Verocytotoxigenic Escherichia Coli Research Articles

Spatiotemporal dynamics of Escherichia coli presence and magnitude across a national groundwater monitoring network, Republic of Ireland, 2011–2020

Groundwater is a vital drinking water resource and its protection from microbiological contamination is paramount to safeguard public health. The Republic of Ireland (RoI) is characterised by the highest incidence of verocytotoxigenic Escherichia coli (VTEC) enteritis in the European Union (EU), linked to high reliance on unregulated groundwater sources (~16% of the population). Yet, the spatio-temporal factors influencing the frequency and magnitude of microbial contamination remain largely unknown, with past studies typically constrained to spatio-temporally ‘limited’ sampling campaigns. Accordingly, the current investigation sought to analyse an extensive spatially distributed time-series (2011−2020) of groundwater monitoring data in the RoI. The dataset, compiled by the Environmental Protection Agency (EPA), showed ‘high’ contamination rates, with 66.7% (88/132) of supplies testing positive for E. coli, and 29.5% (39/132) exceeding concentrations of 10MPN/100 ml (i.e. gross contamination) at least once during the 10-year monitoring period. Seasonal decomposition analyses indicate that E. coli detection rates peak during late autumn/early winter, coinciding with increases in annual rainfall, while gross contamination peaks in spring (May) and late-summer (August), likely reflecting seasonal shifts in agricultural practices. Mixed effects logistic regression modelling indicates that monitoring sources located in karst limestone are statistically associated with E. coli presence (OR = 2.76, p = 0.03) and gross contamination (OR = 2.54, p = 0.037) when compared to poorly productive aquifers (i.e., transmissivity below 10m2/d). Moreover, 5-day and 30-day antecedent rainfall increased the likelihood of E. coli contamination (OR = 1.027, p < 0.001 and OR = 1.005, p = 0.016, respectively), with the former also being associated with gross contamination (OR = 1.042, p < 0.001). As such, it is inferred that preferential flow and direct ingress of surface runoff are the most likely ingress mechanisms associated with E. coli groundwater supply contamination. The results presented are expected to inform policy change around groundwater source protection and provide insight for the development of groundwater monitoring programmes in geologically heterogeneous regions.

Terminal Ileum Perforation

Intestinal perforation is an uncommon complication and presentation of verocytotoxigenic Escherichia coli (VTEC) infection in individuals with Prader-Willi syndrome (PWS). The common site of perforation from VTEC infection is in the colon (and almost exclusively in the pediatric population), whereas PWS is in the stomach. Terminal ileum perforation is uncommon and is not reported in either these 2 conditions. We report a death from terminal ileum perforations in an adult who had PWS and was infected with VTEC. Potential reasons why the perforation occurred at this rare location, rather than in other more common location, in an adult are discussed.

Microbiological Quality of Slovak Traditional Cheese

Abstract The aim of this study was to investigate the micro-biological quality of traditional Slovak “bryndza” cheese made in Slovakia. Besides the common pathogenic bacteria, we focused on the analyses of verocytotoxigenic Escherichia coli (VTEC), the occurrence of which has been analysed only occasionally in a few products. As we chose food of the highest risk which contained raw milk, we expected several positive findings. The presence of Salmonella spp., Listeria monocytogenes and Campylobacter spp. was not confirmed. The enumeration of Staphylococcus aureus was more successful. In the case of VTEC stx and eae screening, the presence of genes producing verocytotoxins vtx1, vtx2 and the gene encoding virulent factor intimin—eae in nine samples by molecular-biological methods were revealed. Only one isolate, which carried genes vtx1 a vtx2 and did not belong to these serogroups: O157, O111, O26, O103, O145, or O104, was detected by confirmation assays.

Antibiogram of Diarrhoeagenic Escherichia coli Patients under Five Years Attending Selected Hospitals in Kaduna State, Nigeria

Escherichia coli infections and poor nutritional status have implications on the growth and development of children under five years, physically, mentally and health wise with consequences such as diarrhoea, stunting, wasting, underweight and often times leading to death, depending on their severity. This study evaluated the antibiogram of Escherichia coli O157 and Verocytotoxigenic Escherichia coli (VTEC)and the nutritional status of diarrhoeic children under five years in Kaduna State, Nigeria, using Conventional isolation methods, latex agglutination tests, VTEC-ELISA tests, Chi-square (SPSS Version 19) and WHO Antro (Version 3.2.2). Purposive sampling was used to select 350 children presenting with diarrhoea in six government hospitals within the three senatorial zones of Kaduna State. The results obtained revealed that 76(21.7%) of the 350 stool samples were positive for E. coli and 28(36.8%) were positive for E. coli O157:H7serotype and 1(1.3%) verocytotoxigenic E. coli (VTEC) serotype. High susceptibility to ciprofloxacin, chloramphenicol and high resistance to sulphamethoxazole, cefotaxime, amoxicillin, gentamicin and tetracycline by the isolates were observed. The study concluded that antibiotics have not been very effective in the treatment of E. coli-related diarrhoea, with VTEC now emerging in this part of the world, making it a serious public health issue. The study therefore recommends the implementation of programmes geared towards good hygiene, good nutrition and good health.

The effect of antimicrobials on verocytotoxin bacteriophage transduction under bovine rumen fluid and broth conditions

AbstractThe verocytotoxin genes in verocytotoxigenicEscherichia coli(VTEC) are carried by bacteriophages, incorporated into the bacterial genome (prophage). Antibiotics may promote phage replication and release to infect other cells (transduction), thus leading to the emergence of new VTEC strains. This study investigated transduction of a verocytotoxin2-encoding bacteriophage (3538(vtx2::cat)) under laboratory conditions, including the effect of antibiotic treatments. Luria-Bertani Miller broth and rumen fluid (raw and sterilised by irradiation) were inoculated with the donor (C600φ3538(Δvtx2::cat)) and recipient (E. coli C600::kanamycinR) strains (4 log10cfu/mL) and incubated at 38°C. Antibiotic treatments (minimal inhibitory and sub-inhibitory concentrations of ampicillin, cefquinome, oxytetracycline and sodium sulfamethazine) were applied after 3 h. Samples were tested for donor, recipient, cell-free phage and transductants at times t = 0, 3, 4, 6, 27 (24 h post-antibiotic treatment) and 51 h. Free phage was detected in the untreated broth and rumen samples, as were the transductants confirmed by polymerase chain reaction. The antibiotic treatments did not significantly (P &gt; 0.01) increase the concentrations of free phage or transductants detected. It was therefore concluded that, under laboratory conditions, the antibiotics tested did not induce bacteriophage lysis, release and infection of new bacterial cells beyond that constitutively found in the phage population.

Molecular characterization of diarrheagenic Escherichia coli isolated from vegetables in Argentina

The aim of this study was to investigate the prevalence of diarrheagenic E. coli strains in vegetables from the humid Pampa region, Argentina, and to determine the occurrence of serotypes and virulence genes in the isolates. A total of 373 fresh vegetable samples obtained from 41 different geographical points were examined. E. coli was detected in 38.6% of the samples. Ten isolates could be obtained from 14 samples presumptively positive for diarrheagenic E. coli: 8 were identified as atypical Enteropathogenic E. coli (aEPEC) and 2 as Verocytotoxigenic E. coli (VTEC). Lettuce and beet were the vegetables most frequently contaminated with pathogenic E. coli. The isolates belonged to serotypes O1:H7, O28:H19, O39:H40, O86:H31, O132:H8, O139:H20, O178:H7 and O178:H19, some of which reportedly have caused human illness, and one isolate resulted non typeable. Taking into account the distribution of 16 nle genes, 7 profiles were detected. On the other hand, all tested isolates harbored the gene encoding for the adhesin HcpA. Other adhesion related genes were also identified: ecpA and elfA were detected in 90%, lpfA0113 in 60%, and ehaA in 50% of the isolates meanwhile ihaA was only observed in O178:H19 isolate. This VTEC isolate harbored, also, Cdt-V toxin and megaplasmid encoding genes such as espP, subA and epeA and exhibited a strong cytotoxic effect. These data is the first molecular E. coli report that confirms the presence of E. coli pathotypes circulating among vegetables in Argentina. Genetic characterization showed that in addition to eae or vtx genes, isolates obtained from vegetables harbored genes encoding other toxins, adhesins, and components related to the type III secretion system that could contribute to their virulence. In conclusion, this research shows that vegetables in Argentina may be the source of VTEC and EPEC infections in the community and therefore, they should be considered as vehicles for transmission of these potentially pathogenic bacteria.

A loop-mediated isothermal amplification method for rapid direct detection and differentiation of nonpathogenic and verocytotoxigenic Escherichia coli in beef and bovine faeces.

To develop a multiplex loop-mediated isothermal amplification (LAMP) assay capable of quantifying Escherichia coli and differentiating verocytotoxigenic E.coli (VTEC). Primer sets were selected to amplify the phoA gene (all E.coli strains) and stx1 and/or stx2 genes (VTEC strains only). LAMP calibration curves demonstrated good quantification capability compared with conventional culture. The limits of detection 50% (LOD50 ) of the multiplex LAMP assay were 2·8 (95% CI 2·4-3·3), 3·2 (95% CI 2·5-3·9) and 2·8-3·2 (95% CI 2·1-3·5) log CFU per g for the phoA, stx1 and stx2 genes, respectively. When validated by testing retail beef and bovine faeces samples, good correlation between E.coli counts indicated by the LAMP assay and culture was observed; however, false-negative LAMP assay results were obtained for 12·5-14·7% of samples. A rapid, multiplex LAMP assay for direct quantification of E.coli and specific detection of VTEC in beef and faeces was successfully developed. Further optimisation of the assay would be needed to improve detection sensitivity. The multiplex LAMP assay represents a rapid alternative to culture for monitoring E.coli levels on beef for hygiene monitoring purposes, and, potentially, a method for detection of VTEC in beef and faeces.

English

Chicken meat is one of the predominantly consumed foods of animal origin in Nigeria with constant increase in demand normally met by local retail market. Processed chicken at the retail chicken meat markets in Abuja were screened for the presence of verocytotoxigenic Escherichia coli (VTEC) strains. A total of 273 faecal samples were collected using moistened sterile swabs and processed for E. coli isolation following standard cultural and biochemical procedures. Isolated E. coli samples were cultured on sorbitol McConkey (SMAC) and cefiximetellurite sorbitol McConkey (CT-SMAC) agar to assess their ability to ferment sorbitol. Samples were further characterized using commercially procured dry spot polyvalent serocheck and specific seroscreen agglutination test kits. Two (0.73%) of the samples tested positive to O157VTEC, while 5 (1.83%) tested positive to non-O157VTEC. There was no significant association (p&gt;0.05) between VTEC infection and season. The study indicated that processed chicken meat sold at the retail chicken market may serve as a potential vehicle for the spread of VTEC infection and other food borne pathogens. Consumer food safety education is important in control programmes. Key words: Occurrence, verocytotoxigenic Escherichia coli (VTEC), processed chicken, retail market, strain.

Genotypic study of verocytotoxic Escherichia coli isolates from deer by multiplex polymerase chain reaction.

Aim:This study was planned to study the genotypes of verocytotoxigenic Escherichia coli (VTEC) in fecal samples of deer due to its public health significance.Materials and Methods:A total of 160 fecal samples of deer were taken from Mathura district and Kanpur Zoo and screened for VTEC genes by polymerase chain reaction (PCR).Results:All fecal samples were positive for E. coli. All the E. coli isolates were screened by PCR to detect virulence genes stx1, stx2, eaeA, and hlyA. Of these, 15 isolates were found positive for VTEC having one or more genes in different combinations.Conclusion:Genes such as stx1, stx2, eaeA, and hlyA were prevalent in VTEC isolates from feces of deer. The presence of VTEC isolates having virulent genes may pose a threat to public health.

Survival studies of a temperate and lytic bacteriophage in bovine faeces and slurry.

Cattle are the main reservoir of verocytotoxigenic Escherichia coli (VTEC), food-borne pathogens that express verocytotoxins (vtx) encoded by temperate bacteriophage. Bovine faeces and unturned manure heaps can support the survival of VTEC and may propagate and transmit VTEC. This study investigated the survival of a vtx2 bacteriophage, φ24B ::Kan, in bovine faeces and slurry. The survival of an anti-Escherichia coli O157:H7 lytic bacteriophage, e11/2, was examined in the same matrices, as a possible bio-control option for VTEC. Samples were inoculated with φ24B ::Kan and/or e11/2 bacteriophage at a concentration of 7-8log10 PFUg(-1) (faeces) or ml(-1) (slurry), stored at 4 and 14°C and examined every 2 days for 36days. The ability of φ24B ::Kan to transduce E.coli cells was examined. Moreover, E.coli concentrations in the faeces and slurry were monitored throughout the experiment as were the pH and aw (faeces only). Both bacteriophages survived well in faeces and slurry. In addition, φ24B ::Kan was able to form lysogens. φ24B ::Kan and e11/2 phage can survive and remain infective in bovine faeces and slurry for at least 30days under representative Irish temperatures. Bovine faeces and slurry may act as a reservoir for vtx bacteriophages. The survival of the anti-O157 phage suggests it may be a suitable bio-control option in these matrices.

Influence of orally fed a select mixture of Bacillus probiotics on intestinal T-cell migration in weaned MUC4 resistant pigs following Escherichia coli challenge

Efficient strategies for treating enteritis caused by F4+ enterotoxigenic Escherichia coli (ETEC)/verocytotoxigenic Escherichia coli (VTEC)/enteropathogenic E. coli (EPEC) in mucin 4 resistant (MUC4 RR; supposed to be F4ab/ac receptor–negative [F4ab/acR−]) pigs remain elusive. A low (3.9 × 108 CFU/day) or high (7.8 × 108 CFU/day) dose of Bacillus licheniformis and Bacillus subtilis spore mixture (BLS-mix) was orally administered to MUC4 RR piglets for 1 week before F4+ ETEC/VTEC/EPEC challenge. Orally fed BLS-mix upregulated the expression of TLR4, NOD2, iNOS, IL-8, and IL-22 mRNAs in the small intestine of pigs challenged with E. coli. Expression of chemokine CCL28 and its receptor CCR10 mRNAs was upregulated in the jejunum of pigs pretreated with high-dose BLS-mix. Low-dose BLS-mix pretreatment induced an increase in the proportion of peripheral blood CD4−CD8− T-cell subpopulations and high-dose BLS-mix induced the expansion of CD4−CD8− T cells in the inflamed intestine. Immunostaining revealed that considerable IL-7Rα–expressing cells accumulated at the lamina propria of the inflamed intestines after E. coli challenge, even in pigs pretreated with either low- or high-dose BLS-mix, although Western blot analysis of IL-7Rα expression in the intestinal mucosa did not show any change. Our data indicate that oral administration of the probiotic BLS-mix partially ameliorates E. coli-induced enteritis through facilitating upregulation of intestinal IL-22 and IκBα expression, and preventing loss of intestinal epithelial barrier integrity via elevating ZO-1 expression. However, IL-22 also elicits an inflammatory response in inflamed intestines as a result of infection with enteropathogenic bacteria.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0355-8) contains supplementary material, which is available to authorized users.

Whole-Transcriptome Analysis of Verocytotoxigenic Escherichia coli O157:H7 (Sakai) Suggests Plant-Species-Specific Metabolic Responses on Exposure to Spinach and Lettuce Extracts.

Verocytotoxigenic Escherichia coli (VTEC) can contaminate crop plants, potentially using them as secondary hosts, which can lead to food-borne infection. Currently, little is known about the influence of the specific plant species on the success of bacterial colonization. As such, we compared the ability of the VTEC strain, E. coli O157:H7 ‘Sakai,’ to colonize the roots and leaves of four leafy vegetables: spinach (Spinacia oleracea), lettuce (Lactuca sativa), vining green pea (Pisum sativum), and prickly lettuce (Lactuca serriola), a wild relative of domesticated lettuce. Also, to determine the drivers of the initial response on interaction with plant tissue, the whole transcriptome of E. coli O157:H7 Sakai was analyzed following exposure to plant extracts of varying complexity (spinach leaf lysates or root exudates, and leaf cell wall polysaccharides from spinach or lettuce). Plant extracts were used to reduce heterogeneity inherent in plant–microbe interactions and remove the effect of plant immunity. This dual approach provided information on the initial adaptive response of E. coli O157:H7 Sakai to the plant environment together with the influence of the living plant during bacterial establishment and colonization. Results showed that both the plant tissue type and the plant species strongly influence the short-term (1 h) transcriptional response to extracts as well as longer-term (10 days) plant colonization or persistence. We show that propagation temperature (37 vs. 18°C) has a major impact on the expression profile and therefore pre-adaptation of bacteria to a plant-relevant temperature is necessary to avoid misleading temperature-dependent wholescale gene-expression changes in response to plant material. For each of the plant extracts tested, the largest group of (annotated) differentially regulated genes were associated with metabolism. However, large-scale differences in the metabolic and biosynthetic pathways between treatment types indicate specificity in substrate utilization. Induction of stress-response genes reflected the apparent physiological status of the bacterial genes in each extract, as a result of glutamate-dependent acid resistance, nutrient stress, or translational stalling. A large proportion of differentially regulated genes are uncharacterized (annotated as hypothetical), which could indicate yet to be described functional roles associated with plant interaction for E. coli O157:H7 Sakai.

CapE (capture, amplify, extract): A rapid method for detection of low level contamination of water with Verocytotoxigenic Escherichia coli (VTEC)

Verocytotoxigenic Escherichia coli (VTEC) is associated with a wide spectrum of disease from mild self-limiting diarrhoea to haemolytic uremic syndrome. Contaminated drinking water is accepted as an important route of transmission in Ireland as elsewhere however established methods for detection of VTEC in drinking water have limitations. We describe a sensitive and rapid method for detection of VTEC from large volumes (20 to 30L) of drinking water based on filtration, enrichment culture of filters and real-time PCR detection of VTEC virulence and O antigen determinants from enrichments. The method has potential applications for other waterborne pathogens.

A Cross Sectional Sero-Study of Verocytotoxigenic &amp;lt;i&amp;gt;Escherichia coli&amp;lt;/i&amp;gt; (VTEC) Serotypes in Apparently Healthy and Diarrhoeic Cattle in Abuja, Federal Capital Territory (FCT), Nigeria

It is reckoned worldwide that verocytotoxigenic Escherichia coli (VTEC) serotypes are important food borne pathogens causing severe health problems in humans. A cross sectional epidemiological study was carried out to determine the prevalence of VTEC serotypes (O157 and non O157) in both apparently healthy and diarrhoeic cattle in Abuja, FCT. A total of 718 faecal samples collected from abattoirs and cattle herds from Abuja, FCT representing 381 from apparently healthy and 337 from diarrhoeic cattle were analyzed. Primary isolation of typical E. coli was done using Eosin Methylene Blue (EMB) agar and performing biochemical tests. Samples were further analyzed using Cefixime, Tellurite-Sorbitol McConkey (CT-SMAC) agar to identify sorbitol and non sorbitol fermenting E. coli. Further characterization of both the sorbitol fermenting and non sorbitol fermenting E. coli was done using commercially procured latex agglutination test kits from Oxoid, United Kingdom. The prevalence of VTEC O157 in apparently healthy cattle was 1.84% and 2.96% for diarrhoeic cattle while the prevalence of non O157 VTEC was 3.67% and 7.12% for apparently healthy and diarrhoeic cattle respectively. There was no strong association (p > 0.05) between faecal consistency and infection with VTEC O157. A strong association (p < 0.05) however existed between faecal consistency and infection with non-O157 VTEC. Diarrhoeic cattle appear likely to be more affected. The implication of the study is that individuals in contact with cattle such as veterinarians, abattoir workers and cattle herdsmen are at risk of exposure to VTEC and proper hygienic control measures should be adopted.

Prevalence of vero toxic Escherichia coli in fecal samples of domestic as well as wild ruminants in Mathura districts and Kanpur zoo.

Aim:The present study was planned to reveal the prevalence of verocytotoxigenic Escherichia coli (VTEC) in fecal samples of domestic and wild ruminants in Mathura district and Kanpur zoo.Materials and Methods:A total of 240 fecal samples comprising 60 each of cattle, buffalo, sheep and deer from Mathura districts and Kanpur zoo were screened for the presence of E. coli and VTEC genes positive by polymerase chain reaction (PCR).Result:Out of 240 fecal samples, 212 E. coli strains were obtained. All the E. coli isolates were screened by PCR to detect virulence genes stx1, stx2, eaeA and hlyA. Of these, 25 isolates were identified as VTEC. The prevalence of VTEC in cattle, buffalo, sheep and deer was found 13.4% (8/60), 13.4% (8/60), 6.67% (4/60) and 8.33% (5/60), respectively.Conclusion:stx1, stx2, eaeA and hlyA genes were prevalent in VTEC isolates from feces of cattle, buffalo, sheep and deer population of Mathura districts and Kanpur zoo. The presence of VTEC isolates in this region may pose a threat to public health.

Lactobacillus plantarum LB95 impairs the virulence potential of Gram-positive and Gram-negative food-borne pathogens in HT-29 and Vero cell cultures.

Listeria monocytogenes, Salmonella enterica and verocytotoxigenic Escherichia coli (VTEC) are amongst the most important agents responsible for food outbreaks occurring worldwide. In this work, two Lactobacillus spp. strains (LABs), Lactobacillus plantarum (LB95) and Lactobacillus paraplantarum (LB13), previously isolated from spontaneously fermenting olive brines, and two reference probiotic strains, Lactobacillus casei Shirota and Lactobacillus rhamnosus GG, were investigated for their ability to attenuate the virulence of the aforementioned pathogens using animal cell culture assays. In competitive exclusion assays, the relative percentages of adhesion and invasion of S. enterica subsp. enterica serovar Enteritidis were significantly reduced when the human HT-29 cell line was previously exposed to LB95. The relative percentage of invasion by Listeria monocytogenes was significantly reduced when HT-29 cells were previously exposed to LB95. In the cytotoxicity assays, the cell-free supernatant of the co-culture (CFSC)of VTEC with LB95 accounted for the lowest value obtained amongst the co-cultures of VTEC with LABs, and was significantly lower than the value obtained with the co-culture of VTEC with the two probiotic reference strains. The cytotoxicity of CFSC of VTEC with both LB95 and LB13 exhibited values not significantly different from the cell-free supernatant of the nonpathogenic E. coli B strain. Our results suggested that LB95 may be able to attenuate the virulence of Gram-positive and Gram-negative food-borne pathogens; together with other reported features of these strains, our data reveal their possible use in probiotic foods due to their interesting potential in preventing enteric infections in humans.

Variability in resistance to Cold Atmospheric Plasma (CAP) and Ultraviolet light (UV) and multiple stress resistance analysis of pathogenic verocytotoxigenic Escherichia coli (VTEC)

This study assessed the resistance to Ultraviolet light (UV) and Cold Atmospheric Plasma (CAP) of ten verocytotoxigenic Escherichia coli (VTEC) isolates and two laboratory non-pathogenic E. coli strains and monitored the effects that UV and CAP have on VTEC molecular composition by using Fourier transform infrared (FTIR) spectroscopy. In addition, serotype-related resistance patterns to different stresses (acid, alkaline, heat, high hydrostatic pressure, UV, CAP and biofilm formation ability) were determined through multivariate analysis of stress resistance parameters. While UV treatments were very effective in inactivating VTEC, with >5log reductions at the lowest intensity tested (15mJ) and low intraspecies variability, the efficacy of CAP treatments was more limited, with in general <3log reductions after a treatment of up to 3min in the lab-scale CAP equipment and larger interstrain variability. Two VTEC strains with non-sense mutations in the global stress response regulator rpoS showed an increased sensitivity to CAP at short treatment times, what suggests that RpoS is important for the cellular defensive response to this technology of preservation. Stepwise Discriminant Analysis (SDA) of FTIR spectra allowed the differentiation of untreated and treated samples based on a subrange of the spectral region w4 (1200 to 900cm−1), which suggests that the damage inflicted to the cells is focused, both for UV and CAP treatments, in nucleic acids and the cell wall. Principal Component Analysis (PCA) of stress resistance parameters allowed the identification of significant associations between VTEC serotypes and their stress resistance patterns. Strains of the serotype O157, more commonly associated with human disease, were in general more resistant to food-related stresses than strains of other serotypes when they had a functional RpoS, emphasizing the influence that stressors may have in the epidemiology, ecology and disease-causing potential of VTEC.

The European Union summary report on trends and sources of zoonoses, zoonotic agents and food‐borne outbreaks in 2014

This report of the European Food Safety Authority and the European Centre for Disease Prevention and Control presents the results of the zoonoses monitoring activities carried out in 2014 in 32 European countries (28 Member States (MS) and four non-MS). Campylobacteriosis was the most commonly reported zoonosis with an increase in confirmed human cases in the European Union (EU) since 2008. In food the occurrence of Campylobacter remained high in broiler meat. The decreasing EU trend for confirmed human salmonellosis cases since 2008 continued. More human Salmonella Enteritidis cases were reported whereas the S. Stanley cases remained, as in 2013, at a higher level compared with 2011–2012. Most MS met their Salmonella reduction targets for poultry but isolates of S. Infantis increased at EU level. In foodstuffs, the EU-level Salmonella non-compliance in fresh and processed poultry meat was rare and low, respectively. The numbers of human listeriosis cases further increased, since 2008. In ready-to-eat foods Listeria seldom exceeded the EU food safety limit. The decreasing EU trend for confirmed yersiniosis cases since 2008 continued. Positive findings for Yersinia were mainly reported in pig meat and products thereof. The number of confirmed verocytotoxigenic Escherichia coli (VTEC) infections in humans slightly decreased compared with 2013. VTEC was reported from food and animals. A total of 5,251 food-borne outbreaks, including water-borne outbreaks, were reported. Most food-borne outbreaks were caused by viruses, followed by Salmonella, bacterial toxins and Campylobacter and with unknown causative agent in 29.1% of all outbreaks. Important food vehicles in strong-evidence food-borne outbreaks were ‘eggs and egg products’, followed by ‘mixed food’ and ‘crustaceans, shellfish, molluscs and products thereof’. The report further summarises trends and sources along the food chain of tuberculosis due to Mycobacterium bovis, Brucella, Trichinella, Echinococcus, Toxoplasma, rabies, Coxiella burnetii (Q fever), West Nile virus and tularaemia.

Diarrheagenic pathogens in adults attending a hospital in Singapore.

BackgroundSingapore’s diarrhoeal notification system is based on specific pathogens. Official data may thus be skewed towards notifiable diseases. Limited information is available on the profiles of aetiological agents responsible for acute gastroenteritis (AGE) cases, especially among the adult population. To understand the frequency and distribution of potential causative agents of diarrheal disease in Singapore, we screened adults’ stool samples collected from a large public hospital.MethodsThe stool samples were screened for 18 diarrheagenic pathogens using a combination of commercial multiplex polymerase chain reaction (PCR), in-house singleplex PCR and immunochromatographic assays. One hundred adult faecal samples that were collected from October 2013 to January 2014 for routine diagnostic purposes and submitted for culture at Tan Tock Seng Hospital, Singapore were used.ResultsPathogens were detected in 32 % of the samples. The predominant organisms encountered were norovirus genogroup II (11 %), Aeromonas spp. (9 %) and Campylobacter spp. (5 %). One sample was positive for both verocytotoxigenic E. coli (VTEC) and E. coli O157:H7. Two other samples were positive for VTEC only, and one other sample was positive for E. coli O157:H7 only. Astrovirus, C. perfringens, Shigella spp. and toxigenic C. difficile were each detected in 2 % of the samples. Cryptosporidium parvum, Giardia lamblia, group A rotavirus, Salmonella spp. and Vibrio spp. were each detected in 1 % of the samples. No L. monocytogenes, Y. enterocolitica, enteric adenovirus, or norovirus genogroup I were detected.ConclusionOur preliminary findings suggest that pathogens causing non-notifiable diseases might have contributed considerably to the adult hospitalised AGE cases. However, as the samples were from an adult hospital, the data obtained may not be representative of the whole community. Thus, a larger study to collect clinical samples and risk exposure data from primary healthcare clinics and children hospital is planned for, to gain a more holistic perspective on the epidemiology of AGE in Singapore. A larger study may also offer valuable insights for improving the approach of microbiological surveillance of food, as well as strategizing inspection efforts along the food supply chain by public health authorities.

Development of a quantitative real time PCR assay to detect and enumerate Escherichia coli O157 and O26 serogroups in bovine recto-anal swabs

Escherichia coli O157 and O26 shedding patterns in cattle are known to vary widely. To address gaps in the understanding of the underlying factors which impact on shedding dynamics, sensitive and rapid quantitative methods which can be applied in surveillance studies on cattle are required. Current approaches for enumeration of verocytotoxigenic E. coli (VTEC) in cattle faeces are based on direct plating onto selective agars, most probable number (MPN) or real time PCR applied directly to faecal samples, all of which have limitations in terms of the labour involved or their sensitivity. The objective of this study was to develop a sensitive real time quantitative PCR assay, to quantify O157 and O26 in bovine recto-anal junction (RAJ) swabs. The approach was to target serogroup specific genes rfbE and wzx, and to couple a short enrichment, with the use of a standard calibration curve relating real time PCR cycle threshold (Ct) values against the initial concentration of the pathogen in the sample. Following initial experiments in broth culture, a 5h enrichment in modified tryptone soya broth with novobiocin (20mg/l) (mTSBn) was found to be optimal, and a linear correlation between inocula (Log10 1 to 6CFUml−1) and the PCR Ct values for both E. coli O157 (R2=0.99, rsd=0.58) and E. coli O26 (R2=0.99, rsd=0.44) was confirmed. The developed method was then applied to bovine RAJ swab samples (n=153), which were inoculated with E. coli O157 or O26 (Log10 1 to 7CFUswab−1). Calibration curves yielded correlations for E. coli O157 of R2=0.86, rsd=0.72 and for O26 (R2=0.88, rsd=0.69). In conclusion, a sensitive method for detection and enumeration of two significant VTEC serogroups in bovine RAJ samples has been developed and validated, and will support studies on the bovine shedding dynamics of these pathogens in cattle.