Validating the Utility of Multilocus Variable Number Tandem-repeat Analysis (MLVA) as a Subtyping Strategy to Monitor Listeria monocytogenes In-built Food Processing Environments.

Listeria monocytogenes is a serious human pathogen and an enduring challenge to control for the ready-to-eat food processing industry. Cost-effective tools that can be deployed by commercial or in-house laboratories to rapidly investigate and resolve contamination events in the built food processing environment are of value to the food industry. Multilocus variable number tandem-repeat analysis (MLVA) is a molecular subtyping method, which along with other same-generation methods such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) is being superseded in disease tracking and outbreak investigations by whole-genome sequencing (WGS). In this paper, it is demonstrated that MLVA can continue to play a valuable role as a valid, fast, simple, and cost-effective method to identify and track Listeria monocytogenes subtypes in factory environments, with the method being highly congruent with MLST. Although MLVA does not have the discriminatory power of WGS to identify truly persistent clones, with careful interpretation of results alongside isolate metadata, it remains a powerful tool in situations and locations where WGS may not be readily available to food business operators.

Comparative genomic insights into Yersinia hibernica - a commonly misidentified Yersinia enterocolitica-like organism.

Food-associated outbreaks linked to enteropathogenic Yersinia enterocolitica are of concern to public health. Pigs and their meat are recognized risk factors for transmission of Y. enterocolitica. This study aimed to describe the comparative genomics of Y. enterocolitica along with a number of misclassified Yersinia isolates, now constituting the recently described Yersinia hibernica. The latter was originally cultured from an environmental sample taken at a pig slaughterhouse. Unique features were identified in the genome of Y. hibernica, including a novel integrative conjugative element (ICE), denoted as ICEYh-1 contained within a 255 kbp region of plasticity. In addition, a zebrafish embryo infection model was adapted and applied to assess the virulence potential among Yersinia isolates including Y. hibernica.

Longitudinal Study of Two Irish Dairy Herds: Low Numbers of Shiga Toxin-Producing Escherichia coli O157 and O26 Super-Shedders Identified.

A 12-month longitudinal study was undertaken on two dairy herds to ascertain the Shiga-toxin producing Escherichia coli (STEC) O157 and O26 shedding status of the animals and its impact (if any) on raw milk. Cattle are a recognized reservoir for these organisms with associated public health and environmental implications. Animals shedding E. coli O157 at >10,000 CFU/g of feces have been deemed super-shedders. There is a gap in the knowledge regarding super-shedding of other STEC serogroups. A cohort of 40 lactating cows from herds previously identified as positive for STEC in a national surveillance project were sampled every second month between August, 2013 and July, 2014. Metadata on any potential super-shedders was documented including, e.g., age of the animal, number of lactations and days in lactation, nutritional condition, somatic cell count and content of protein in milk to assess if any were associated with risk factors for super-shedding. Recto-anal mucosal swabs (RAMS), raw milk, milk filters, and water samples were procured for each herd. The swabs were examined for E. coli O157 and O26 using a quantitative real time PCR method. Counts (CFU swab-1) were obtained from a standard calibration curve that related real-time PCR cycle threshold (Ct) values against the initial concentration of O157 or O26 in the samples. Results from Farm A: 305 animals were analyzed; 15 E. coli O157 (5%) were recovered, 13 were denoted STEC encoding either stx1 and/or stx2 virulence genes and 5 (2%) STEC O26 were recovered. One super-shedder was identified shedding STEC O26 (stx1&2). Farm B: 224 animals were analyzed; eight E. coli O157 (3.5%) were recovered (seven were STEC) and 9 (4%) STEC O26 were recovered. Three super-shedders were identified, one was shedding STEC O157 (stx2) and two STEC O26 (stx2). Three encoded the adhering and effacement gene (eae) and one isolate additionally encoded the haemolysin gene (hlyA). All four super-shedders were only super-shedding once during the 1-year sampling period. The results of this study show, low numbers of super-shedders in the herds examined, with high numbers of low and medium shedding. Although four super-shedding animals were identified, no STEC O157 or O26 were recovered from any of the raw milk, milk filter, or water samples. The authors conclude that this study highlights the need for further surveillance to assess the potential for environmental contamination and food chain security.

Patterns of antimicrobial resistance in pathogenic Escherichia coli isolates from cases of calf enteritis during the spring-calving season.

Neonatal enteritis is a common condition of young calves and can be caused by pathogenic strains of Escherichia coli. We hypothesised that on-farm antimicrobial use would result in an increased frequency of resistance in these strains during the calving season. We also sought to determine if the frequency of resistance reflected on-farm antimicrobial use. Faecal samples were collected from cases of calf enteritis on 14 spring-calving dairy farms during two 3 week periods: Period 1 - February 11th through March 2nd 2008 and Period 2 - April 14th through May 5th 2008. E. coli were cultured from these samples, pathogenic strains were identified and antimicrobial susceptibility testing was carried out on these pathogenic isolates. Antimicrobial prescribing data were collected from each farm for the previous 12 months as an indicator of antimicrobial use. The correlation between antimicrobial use and resistance was assessed using Spearman's correlation coefficient. Logistic regression analysis was used to investigate the relationship between resistance, sampling period and pathotype. Penicillins and aminopenicillins, streptomycin, and tetracyclines were the most frequently prescribed antimicrobials and the greatest frequencies of resistance were detected to these 3 antimicrobial classes. A strong correlation (ρ=0.879) was observed between overall antimicrobial use and frequencies of antimicrobial resistance on farms. Sampling period was significant in the regression model for ampicillin resistance while pathotype was significant in the models for streptomycin, tetracycline and trimethoprim/sulphamethoxazole resistance. The frequencies of resistance observed have implications for veterinary therapeutics and prudent antimicrobial use. Resistance did not increase during the calving season and factors other than antimicrobial use, such as calf age and bacterial pathotype, may influence the occurrence of resistance in pathogenic E. coli.

Complete Genome Sequence of Salmonella enterica Serovar Agona Pulsed-Field Type SAGOXB.0066, Cause of a 2008 Pan-European Outbreak.

Salmonella enterica serovar Agona is in the top 10 most common nontyphoidal serovars reported in humans in the European Union. Here we report the complete genome sequence of an S. enterica serovar Agona isolate, designated 24249, that was the cause of a pan-European outbreak in 2008 with 163 confirmed cases reported.

Yersinia enterocolitica: a brief review of the issues relating to the zoonotic pathogen, public health challenges, and the pork production chain.

Yersinia enterocolitica is a zoonotic agent that causes gastrointestinal disease in humans, as well as reactive arthritis and erythema nodosum. Enteropathogenic Yersinia are the etiological agents for yersiniosis, which can be acquired through the consumption of contaminated foods. As porcine animals are the main carriers of Y. enterocolitica, food safety measures to minimize human infection are of increasing interest to the scientific and medical community. In this review, we examine why it is imperative that information on the reservoirs, prevalence, virulence, and ability of this pathogen to survive in different environments is further investigated to provide rational measures to prevent or decrease associated disease risks.

Molecular epidemiology of Campylobacter isolates from poultry production units in southern Ireland.

This study aimed to identify the sources and routes of transmission of Campylobacter in intensively reared poultry farms in the Republic of Ireland. Breeder flocks and their corresponding broilers housed in three growing facilities were screened for the presence of Campylobacter species from November 2006 through September 2007. All breeder flocks tested positive for Campylobacter species (with C. jejuni and C. coli being identified). Similarly, all broiler flocks also tested positive for Campylobacter by the end of the rearing period. Faecal and environmental samples were analyzed at regular intervals throughout the rearing period of each broiler flock. Campylobacter was not detected in the disinfected house, or in one-day old broiler chicks. Campylobacter jejuni was isolated from environmental samples including air, water puddles, adjacent broiler flocks and soil. A representative subset of isolates from each farm was selected for further characterization using flaA-SVR sub-typing and multi-locus sequence typing (MLST) to determine if same-species isolates from different sources were indistinguishable or not. Results obtained suggest that no evidence of vertical transmission existed and that adequate cleaning/disinfection of broiler houses contributed to the prevention of carryover and cross-contamination. Nonetheless, the environment appears to be a potential source of Campylobacter. The population structure of Campylobacter isolates from broiler farms in Southern Ireland was diverse and weakly clonal.

Temporal trends in dioxin, furan and polychlorinated biphenyl concentrations in bovine milk from farms adjacent to industrial and chemical installations over a 15 year period.

The concentrations of polychlorinated dibenzo-p-dioxins, furans and polychlorinated biphenyls (PCBs) were measured in pooled bovine milk samples collected between 1991 and 2005 in County Cork, Ireland. The pooled samples were of bulk-tank milk collected from farms adjacent to industrial, chemical and pharmaceutical installations (target milk) or from rural farms distant from industrial activity (control milk). Comparing data between the first and last 3-year periods of the study revealed a 62% decrease in the mean total dioxin concentration in target milk from 1.58 to 0.60pg toxic equivalents (TEQ)/g fat. On the same basis the dioxin-like PCB concentration in target milk decreased by 80% over the study period (from 0.95pg to 0.19pg TEQ/g fat). The mean 'marker' PCB concentration in target milk from 1991 to 1993 inclusive was 3359pg/g fat. This value decreased by 75% to a mean of 849pg/g fat for the years 2003-2005 inclusive. The results of this study are consistent with low background dietary/environmental PCB contamination in both target and control herds. The total dioxin concentrations in all samples were well below the maximum tolerable limits permitted for marketable milk. The decrease in the total dioxin concentration in target and control milk samples over the study period was chiefly due to decreases in the concentration of dioxin-like PCBs, consistent with significant reductions in the concentration of PCBs in the dairy cow diet over the 15 year study period.

First report: Yersinia enterocolitica recovered from canine tonsils.

Yersinia enterocolitica (Y. enterocolitica) is a known zoonotic pathogen and is often found in pig tonsils as the primary site of colonisation. In this study we investigated whether or not Y. enterocolitica could be recovered from canine tonsils. During a study on the prevalence of Y. enterocolitica in animal populations in Ireland, 144 canine tonsils and 72 canine rectal swabs were procured over a ten-month period and subjected to microbiological examination for the presence of this human pathogen. Molecular methods were used to determine virulence and all strains were negative for the chromosomally mediated virulence factor (ail) and plasmid-encoded adhesion molecule (pYad). Y. enterocolitica was recovered from 25 of 216 (12%) samples. Twenty-four strains were from tonsils along with one from a rectal swab. All were biotype 1A. Antimicrobial resistance profiling showed two of 25 (8%) were resistant to cephalothin and the remaining strains were resistant to ampicillin and cephalothin with six of these additionally resistant to streptomycin. Our evidence that a human pathogen may be harboured in the oral cavity of dogs' adds a new dimension to the epidemiology of this organism, identifying a potential public health risk following exposure to dogs.

Sensitive and rapid molecular detection assays for Salmonella enterica serovars Typhimurium and Heidelberg.

Salmonella enterica is a significant cause of gastroenteritis worldwide, with serovars Typhimurium and Heidelberg being particularly prevalent, which have broad host ranges infecting poultry, dairy animals, and humans. Traditional methods used for the detection of Salmonella from contaminated food products are time-consuming and labor-intensive. The aim of this study was to develop a sensitive and rapid PCR-based detection method with optimized specificity for high-throughput screening of food and clinical samples. We used bioinformatics to identify potential serovar-specific regions from the available S. enterica sequenced genomes. We designed primer pairs to targeted regions unique to Typhimurium and Heidelberg. A primer pair targeting a putative cytoplasmic protein STM4492 amplified a 759-bp product specific to Typhimurium, and a primer pair targeting a putative inner membrane protein STM2745 amplified a 199-bp product from both Typhimurium and Heidelberg. A primer pair for the oriC locus was used to identify all Salmonella. We screened 217 isolates including the Salmonella reference collections A and B, validating the specificity of each primer set. Next, a multiplex PCR (mPCR) assay and quantitative real-time PCR assay were optimized for identification and differentiation of Typhimurium and Heidelberg. An mPCR assay was developed and successfully detected S. enterica isolates from inoculated Cheddar cheese, raw turkey, and cooked turkey at concentrations as low as 1 CFU/g of food. The reaction conditions for this mPCR have significantly reduced the time needed to identify S. enterica Typhimurium and Heidelberg, making this a rapid selective tool.

Multiple-locus variable number of tandem repeat analysis (MLVA) of Irish verocytotoxigenic Escherichia coli O157 from feedlot cattle: uncovering strain dissemination routes.

BACKGROUND: The identification of the routes of dissemination of Escherichia coli (E. coli) O157 through a cohort of cattle is a critical step to control this pathogen at farm level. The aim of this study was to identify potential routes of dissemination of E. coli O157 using Multiple-Locus Variable number of tandem repeat Analysis (MLVA). RESULTS: Thirty-eight environmental and sixteen cattle faecal isolates, which were detected in four adjacent pens over a four-month period were sub-typed. MLVA could separate these isolates into broadly defined clusters consisting of twelve MLVA types. Strain diversity was observed within pens, individual cattle and the environment. CONCLUSION: Application of MLVA is a broadly useful and convenient tool when applied to uncover the dissemination of E. coli O157 in the environment and in supporting improved on-farm management of this important pathogen. These data identified diverse strain types based on amplification of VNTR markers in each case.

Comparison of Salmonella species recovered from Irish liquid milk production holdings with temporal clinical veterinary isolates.

The presence of microbiological hazards in foodstuffs including, Salmonella, form a major source of food-borne diseases in humans. In-line milk filters from 97 liquid milk production holdings in Cork, the largest dairy region in Ireland, were surveyed for the presence of Salmonella species at herd level over a 2-year period (September 2001-September 2003). Each dairy farm was visited 6 times at 4 monthly intervals (denoted by cycles A-F). Six of the 97 herds (6%) were positive. Ten isolates were detected based on culture methods. These included five (5%) Salmonella Typhimurium DT104, 4 (4%) Salmonella Dublin, and 1 (1%) Salmonella Agona from a total of 556 filters. During cycle C, in addition to the milk filters, a bulk tank milk (BTM) sample was procured from each dairy holding and analysed but no Salmonella were isolated. For comparison purposes a further 26 temporal veterinary clinical isolates (21 S. Typhimurium of varying phage type, and 5 S. Dublin) were procured from the Cork Veterinary Clinical Diagnostic Laboratory, Cork. The study collection showed resistance to one or more antimicrobial agents. During the study, Salmonella spp. were isolated from five of the herds prior to any clinical signs in the farm animals. Pulsed-field gel electrophoresis (PFGE) profiles indicated clonality among the isolates pre- and post-clinical illness. A phenotypic and genotypic database for Salmonella spp. has been developed and used for comparative purposes.

Investigation of a global collection of nontyphoidal Salmonella of various serotypes cultured between 1953 and 2004 for the presence of class 1 integrons.

In this study, antibiotic resistance profiles, and the presence of class 1 integrons were determined for 108 Salmonella isolates comprising 37 serotypes cultured from a variety of sources between 1953 and 2004. Antibiogram analyses showed that all isolates were resistant to streptomycin/spectinomycin. Molecular analysis revealed that 50% of the collection contained an integrase-encoding gene (int1) and 25% contained class 1 integrons. A Salmonella Wien isolate possessing a complete class 1 integron with a dfrA5-ereA2 gene arrangement within the variable region was characterized.

Development and application of Multiple-Locus Variable number of tandem repeat Analysis (MLVA) to subtype a collection of Listeria monocytogenes.

In this study we report the development and application of a Multiple-Locus Variable number of tandem repeat Analysis (MLVA) strategy for subtyping Listeria monocytogenes. Genome profiles of a collection of forty-five food-borne L. monocytogenes isolates were compared using MLVA. These isolates were obtained as part of an active surveillance programme of foods in the south-east region of Ireland. MLVA successfully discriminated amongst the isolates. The method was easy to perform, relatively fast and could be deployed in any molecular laboratory with basic laboratory equipment. This approach is a valuable tool, which has the capability to provide comparable results when compared with other more established typing methods, including pulsed-field gel electrophoresis (PFGE).

Role of KATP channels in sepsis.

Sepsis is an infection-induced inflammatory syndrome responsible for approximately 10% of all deaths worldwide. While pathophysiological mechanisms remain to be fully unravelled, new insights and discoveries are yielding significant improvements in outcome, particularly in the high mortality conditions of shock and multi-organ failure. One potential target is the ATP-sensitive potassium (K(ATP)) channel, an ion channel critical to the cardiovascular stress response. Excessive activation of the vascular channel is now recognised as a major cause of hypotension and vascular hyporesponsiveness to catecholamines in septic shock. Some researchers advocate therapeutic blockade of these channels; however, outside the vasculature, channel opening may actually represent a protective mechanism against cellular damage. In this review we critically examine the role of the K(ATP) channel in sepsis.

In-line milk filter analysis: Escherichia coli O157 surveillance of milk production holdings.

Escherichia coli O157 is a major etiological agent of food-borne illness. Bovine animals are recognized reservoirs for this organism and represent a significant source from where these pathogens can enter the food chain. Food products derived from these animals are convenient vehicles, and are often the focal point(s) of infection. As a useful strategy to provide herd-level surveillance and to investigate for the presence of this pathogen in a population of Irish dairy cattle, milk filters from 97 farms were analysed by conventional culture and other methods. Five hundred and thirty-six milk filters were evaluated over a 2-year period. Filters from 12 of the 97 farms (12%) were found to contain E. coli O157, based on culture methods. Sixteen verocytotoxigenic E. coli O157 organisms were recovered and characterized in detail. The farm families in each case were consuming raw milk from their respective herds. The potential risk to public health associated with the detection of E. coli O157 and the local consumption of raw milk are discussed.

The pore-forming subunit of the K(ATP) channel is an important molecular target for LPS-induced vascular hyporeactivity in vitro.

ATP-sensitive K(+) (K(ATP)) channel activation is implicated in the vascular hyporeactivity occurring in septic shock. However, channel inhibition with the sulphonylurea receptor (SUR) antagonist, glibenclamide (Glib) fails to reverse lipopolysaccharide (LPS)-induced vascular hyporeactivity in vitro. We investigated whether inhibitors that act by binding to the K(ATP) channel pore could be effective. Ring segments of endothelium-intact rat mesenteric artery were incubated with LPS in culture media for either 6 or 20 h before contractile responses to phenylephrine were assessed in the absence or presence of K(ATP) channel inhibitors. The pore-forming subunit inhibitors barium chloride (BaCl(2); 300 microM) and PNU-37883A (1 microM) significantly reversed hyporeactivity at both time points, although less so at 20 h. In contrast, the SUR inhibitors, Glib (10 microM), tolbutamide (Tolb) (1 mM) and PNU-99963 (1 microM) were ineffective. In LPS-incubated tissues, Glib and Tolb antagonised contractions to the thromboxane A2 mimetic, U46619 (9,11-dideoxy-9alpha, 11alpha-methanoepoxy prostaglandin F(2alpha)) (10(-7) M), whereas the pinacidil-derived inhibitor, PNU-99963, did not. Contractions to 60 mM KCl were unaffected by LPS at 6 h, but were significantly depressed by LPS at 20 h, suggesting that K(+)-channel-independent pathways contribute to hyporeactivity at the later time point. The inducible nitric oxide synthase (iNOS) inhibitor, 1400 W (10 microM) and Tolb inhibited the production of nitrite induced by LPS, whereas BaCl(2) and PNU-37883A had no effect. In conclusion, K(ATP) channels contribute to LPS-induced vascular hyporeactivity via the iNOS pathway in rat mesenteric artery. The effectiveness of pore inhibitors over SUR inhibitors of the K(ATP) channel suggests altered SUR function following LPS administration, which cannot be explained by thromboxane receptor inhibition.