Electrochemical nucleic acid-based sensors for detection of Escherichia coli and Shiga toxin-producing E. coli-Review of the recent developments.

Escherichia coli are a group of bacteria that are a natural part of the intestinal flora of warm-blooded animals, including humans. Most E. coli are nonpathogenic and essential for the normal function of a healthy intestine. However, certain types, such as Shiga toxin-producing E. coli (STEC), which is a foodborne pathogen, can cause a life-threatening illness. The development of point-of-care devices for the rapid detection of E. coli is of significant interest with regard to ensuring food safety. The most suitable way to distinguish between generic E. coli and STEC is by using nucleic acid-based detection, focusing on the virulence factors. Electrochemical sensors based on nucleic acid recognition have attracted much attention in recent years for use in pathogenic bacteria detection. This review has summarized nucleic acid-based sensors for the detection of generic E. coli and STEC since 2015. First, the sequences of the genes used as recognition probes are discussed and compared to the most recent research regarding the specific detection of general E. coli and STEC. Subsequently, the collected literature regarding nucleic acid-based sensors is described and discussed. The traditional sensors were divided into four categories such as gold, indium tin oxide, carbon-based electrodes, and those using magnetic particles. Finally, we summarized the future trends in nucleic acid-based sensor development for E. coli and STEC including some examples of fully integrated devices.

A sensor-based system for rapid on-site testing of microbial contamination in meat samples and carcasses.

AIMS: To develop an oxygen sensor-based method for testing total aerobic viable counts (TVC) in raw meat samples and cattle carcass swabs, which is rapid, simple, affordable, provides good sensitivity and analytical performance and allows on-site use. METHODS AND RESULTS: The test uses the same sample preparation procedure as the established plate counting TVC method for meat samples and carcasses, ISO4833-1:2013. After this liquid samples are transferred into standard 25-ml vials with built-in phosphorescent O2  sensors and incubated on a block heater with hourly readings of sensor signals with a handheld reader, to determine signal threshold time (TT, hours) for each sample. The method is demonstrated with the quantification of TVC in industrial cuts of raw beef meat (CFU per g) and carcass swabs (CFU per cm2 ). Calibration curves were generated, which give the following analytical equations for calculating the TVC load in unknown samples from measured TT values: TVC [Log(CFU per cm2 )] = 7.83-0.73*TT(h) and TVC [Log(CFU per g)] = 8.74-0.70*TT(h) for the carcass swabs and meat samples respectively. The new tests show good correlation with the ISO methods, with correlation coefficients 0.85 and 0.83 respectively. The testing requires no dilutions, covers the ranges 2-7 Log(CFU per g) for the meat samples and 1-7 Log(CFU per cm2 ) for carcass swabs, and has time to result 1-10 h with faster detection of more contaminated samples. CONCLUSIONS: The sensor-based testing demonstrates simplicity, high speed, sample throughput and automation. It can provide a straightforward replacement for the conventional TVC tests, which are time consuming, laborious and have time to result of 48-72 h. SIGNIFICANCE AND IMPACT OF THE STUDY: The method(s) can be adopted by the meat industry and research labs, and used to improve microbial quality and safety of meat products and processes.

Prevalence and Whole-Genome Sequence-Based Analysis of Shiga Toxin-Producing Escherichia coli Isolates from the Recto-Anal Junction of Slaughter-Age Irish Sheep.

Shiga toxin-producing Escherichia coli (STEC) organisms are a diverse group of pathogenic bacteria capable of causing serious human illness, and serogroups O157 and O26 are frequently implicated in human disease. Ruminant hosts are the primary STEC reservoir, and small ruminants are important contributors to STEC transmission. This study investigated the prevalence, serotypes, and shedding dynamics of STEC, including the supershedding of serogroups O157 and O26, in Irish sheep. Recto-anal mucosal swab samples (n = 840) were collected over 24 months from two ovine slaughtering facilities. Samples were plated on selective agars and were quantitatively and qualitatively assessed via real-time PCR (RT-PCR) for Shiga toxin prevalence and serogroup. A subset of STEC isolates (n = 199) were selected for whole-genome sequencing and analyzed in silico. In total, 704/840 (83.8%) swab samples were Shiga toxin positive following RT-PCR screening, and 363/704 (51.6%) animals were subsequently culture positive for STEC. Five animals were shedding STEC O157, and three of these were identified as supershedders. No STEC O26 was isolated. Post hoc statistical analysis showed that younger animals are more likely to harbor STEC and that STEC carriage is most prevalent during the summer months. Following sequencing, 178/199 genomes were confirmed as STEC. Thirty-five different serotypes were identified, 15 of which were not yet reported for sheep. Serotype O91:H14 was the most frequently reported. Eight Shiga toxin gene variants were reported, two stx1 and six stx2, and three novel Shiga-toxin subunit combinations were observed. Variant stx1c was the most prevalent, while many strains also harbored stx2b. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) bacteria are foodborne, zoonotic pathogens of significant public health concern. All STEC organisms harbor stx, a critical virulence determinant, but it is not expressed in most serotypes. Sheep shed the pathogen via fecal excretion and are increasingly recognized as important contributors to the dissemination of STEC. In this study, we have found that there is high prevalence of STEC circulating within sheep and that prevalence is related to animal age and seasonality. Further, sheep harbor a variety of non-O157 STEC, whose prevalence and contribution to human disease have been underinvestigated for many years. A variety of Stx variants were also observed, some of which are of high clinical importance.

An Overview of Shiga-Toxin Producing Escherichia coli Carriage and Prevalence in the Ovine Meat Production Chain.

Shiga-toxin producing Escherichia coli (STEC) are zoonotic foodborne pathogens that are capable of causing serious human illness. Ovine ruminants are recognized as an important source of STEC and a notable contributor to contamination within the food industry. This review examined the prevalence of STEC in the ovine food production chain from farm-to-fork, reporting carriage in sheep herds, during abattoir processing, and in raw and ready-to-eat meats and meat products. Factors affecting the prevalence of STEC, including seasonality and animal age, were also examined. A relative prevalence can be obtained by calculating the mean prevalence observed over multiple surveys, weighted by sample number. A relative mean prevalence was obtained for STEC O157 and all STEC serogroups at multiple points along the ovine production chain by using suitable published surveys. A relative mean prevalence (and range) for STEC O157 was calculated: for feces 4.4% (0.2-28.1%), fleece 7.6% (0.8-12.8%), carcass 2.1% (0.2-9.8%), and raw ovine meat 1.9% (0.2-6.3%). For all STEC independent of serotype, a relative mean prevalence was calculated: for feces 33.3% (0.9-90.0%), carcass 58.7% (2.0-81.6%), and raw ovine meat 15.4% (2.7-35.5%). The prevalence of STEC in ovine fleece was reported in only one earlier survey, which recorded a prevalence of 86.2%. Animal age was reported to affect shedding in many surveys, with younger animals typically reported as having a higher prevalence of the pathogen. The prevalence of STEC decreases significantly along the ovine production chain after the application of postharvest interventions. Ovine products pose a small risk of potential STEC contamination to the food supply chain.

A Simple Sensor System for Onsite Monitoring of O2 in Vacuum-Packed Meats during the Shelf Life.

Vacuum packaging (VP) is used to reduce exposure of retail meat samples to ambient oxygen (O2) and preserve their quality. A simple sensor system produced from commercial components is described, which allows for non-destructive monitoring of the O2 concentration in VP raw meat samples. Disposable O2 sensor inserts were produced by spotting small aliquots of the cocktail of the Pt-benzoporphyrin dye and polystyrene in ethyl acetate onto pieces of a PVDF membrane and allowing them to air-dry. These sensor dots were placed on top of the beef cuts and vacuum-packed. A handheld reader, FirestinGO2, was used to read nondestructively the sensor phase shift signals (dphi°) and relate them to the O2 levels in packs (kPa or %). The system was validated under industrial settings at a meat processing plant to monitor O2 in VP meat over nine weeks of shelf life storage. The dphi° readings from individual batch-calibrated sensors were converted into the O2 concentration by applying the following calibration equation: O2 (%) = 0.034 * dphi°2 - 3.413 * dphi° + 85.02. In the VP meat samples, the O2 levels were seen to range between 0.12% and 0.27%, with the sensor dphi signals ranging from 44.03° to 56.02°. The DIY sensor system demonstrated ease of use on-site, fast measurement time, high sample throughput, low cost and flexibility.

Evaluation of an Alternative Experimental Infection Method, Which Closely Mimics the Natural Route of Transmission of Monophasic Salmonella Typhimurium in Pigs.

Salmonella carriage in pigs is a significant food safety issue. This study describes a new protocol of Salmonella infection based on exposure to an artificially contaminated environment that closely mimics natural exposure to the organism. The aim of the study was to develop and evaluate the effectiveness of this protocol, which could then be used as a tool in the investigation of control measures. In addition, Salmonella shedding pattern and growth performance of the pigs were examined. Trial pigs (n = 10) were placed in a pen that had been previously contaminated by housing two pigs experimentally challenged with a monophasic Salmonella Typhimurium (mST). A further 10 pigs were placed in a Salmonella-free pen. Pigs were weighed on days 0 and 28. Feces was collected on days 0, 2, 3, 5, 7, 14, 21, and 28 and examined for the presence and quantity of Salmonella. The trial was replicated once. All pigs in the contaminated pens shed Salmonella within the first 2 days of exposure with values ranging from 100 to 104 CFU/g. The noninfected pigs had significantly higher final body weights on day 28 than those exposed to the Salmonella contaminated environment in both replicates. The pigs in the Salmonella-free pen had significantly higher average daily weight gain over the 28-day period compared to the infected animals (p < 0.001). Although not significant, numerical improvements in average daily feed intake and feed conversion efficiency were observed in the Salmonella-free pigs when compared to the contaminated pigs. The approach used was successful in infecting pigs with Salmonella without the need for direct inoculation or exposure to seeder pigs. This "natural" method of infection in which pigs are exposed to low levels of environmental contamination with Salmonella may be an effective tool that could be utilized when investigating control measures.

Farm to Fork Quantitative Risk Assessment of Listeria monocytogenes Contamination in Raw and Pasteurized Milk Cheese in Ireland.

The objective of this study was to model and quantify the level of Listeria monocytogenes in raw milk cheese (RMc) and pasteurized milk cheese (PMc) from farm to fork using a Bayesian inference approach combined with a quantitative risk assessment. The modeling approach included a prediction of contamination arising from the farm environment as well from cross-contamination within the cheese-processing facility through storage and subsequent human exposure. The model predicted a high concentration of L. monocytogenes in contaminated RMc (mean 2.19 log10 CFU/g) compared to PMc (mean -1.73 log10 CFU/g). The mean probability of illness (P1 for low-risk population, LR) and (P2 for high-risk population, HR, e.g., immunocompromised) adult Irish consumers following exposure to contaminated cheese was 7 × 10(-8) (P1 ) and 9 × 10(-4) (P2 ) for RMc and 7 × 10(-10) (P1 ) and 8 × 10(-6) (P2 ) for PMc, respectively. In addition, the model was used to evaluate performance objectives at various stages, namely, the cheese making and ripening stages, and to set a food safety objective at the time of consumption. A scenario analysis predicted various probabilities of L. monocytogenes contamination along the cheese-processing chain for both RMc and PMc. The sensitivity analysis showed the critical factors for both cheeses were the serving size of the cheese, storage time, and temperature at the distribution stage. The developed model will allow food processors and policymakers to identify the possible routes of contamination along the cheese-processing chain and to reduce the risk posed to human health.

Evidence of metabolic switching and implications for food safety from the phenome(s) of Salmonella enterica serovar Typhimurium DT104 cultured at selected points across the pork production food chain.

Salmonella enterica serovar Typhimurium DT104 is a recognized food-borne pathogen that displays a multidrug-resistant phenotype and that is associated with systemic infections. At one extreme of the food chain, this bacterium can infect humans, limiting the treatment options available and thereby contributing to increased morbidity and mortality. Although the antibiotic resistance profile is well defined, little is known about other phenotypes that may be expressed by this pathogen at key points across the pork production food chain. In this study, 172 Salmonella enterica serovar Typhimurium DT104/DT104b isolated from an extensive "farm-to-fork" surveillance study, focusing on the pork food chain, were characterized in detail. Isolates were cultured from environmental, processing, retail, and clinical sources, and the study focused on phenotypes that may have contributed to persistence/survival in these different niches. Molecular subtypes, along with antibiotic resistance profiles, tolerance to biocides, motility, and biofilm formation, were determined. As a basis for human infection, acid survival and the ability to utilize a range of energy sources and to adhere to and/or invade Caco-2 cells were also studied. Comparative alterations to biocide tolerance were observed in isolates from retail. l-Tartaric acid and d-mannose-1-phosphate induced the formation of biofilms in a preselected subset of strains, independent of their origin. All clinical isolates were motile and demonstrated an enhanced ability to survive in acidic conditions. Our data report on a diverse phenotype, expressed by S. Typhimurium isolates cultured from the pork production food chain. Extending our understanding of the means by which this pathogen adapts to environmental niches along the "farm-to-fork" continuum will facilitate the protection of vulnerable consumers through targeted improvements in food safety measures.

Hybrid genome assemblies of four Shiga toxin-producing Escherichia coli strains containing a complete locus of enterocyte effacement and an O-Island 122.

This study describes the hybrid genome assemblies of four Shiga toxin-producing Escherichia coli strains isolated from the recto-anal junction of slaughter-age Irish sheep. In silico serotyping and genome analysis determined that each of the strains harbored a Shiga-toxin subtype, a complete locus of enterocyte effacement, and a rare O-island 122.

Draft Genome Sequences of Bacillus licheniformis and Bacillus paralicheniformis Strains Isolated from Irish Skim Milk Powder.

Nineteen Bacillus licheniformis strains and four strains of the closely related species Bacillus paralicheniformis were isolated from a variety of Irish medium-heat skim milk powders. The draft genome sequences of these 23 isolates provide valuable genetic data for research work relevant to dairy products and process development. The isolates are available at Teagasc.

Evaluation of carvacrol for the control of Escherichia coli O157 on cattle hide and carcass cuts.

The antimicrobial activity of carvacrol against Escherichia coli O157 was assessed on cattle hide and beef carcass cuts. Carvacrol (0, 10, 20, and 30 mg/mL) was applied using a spray bottle to cattle hide and beef carcass cuts, inoculated with a cocktail of E. coli O157 (5-6 log(10) CFU/cm(2)) and left in contact for 10 min. Following treatment, hide and carcass cuts were sampled using a swab method, and E. coli O157 were enumerated by plate counts on selective media. Carvacrol (30 mg/mL) significantly (p < 0.05) reduced inoculated E. coli O157 (1.4 and 1.58 log(10) CFU/cm(2)) compared to the no wash and water wash (0 mg/mL) controls on both carcass cuts and hide respectively. This preliminary study shows that carvacrol has the potential to control E. coli O157 on bovine hide and carcass cuts, but further research with larger scale trials is needed.

Impact of Industrial Practices on the Microbial and Quality Attributes of Fresh Vacuum-Packed Lamb Joints.

The impact of different industrial practices at lamb export abattoirs in Ireland on the microbial and quality attributes of fresh vacuum-packed (VP) lamb leg joints, including Clean Livestock Policy (CLP), fleece clipping, carcass chilling times and vacuum pack storage, at typical chill and retail display temperatures was investigated. Five separate slaughter batches of lamb (ranging in size from 38 to 60 lambs) were followed at two lamb export plants over a two-year period, accounting for seasonal variation. In general, fleece clipping resulted in significantly lower microbial contamination on the fleece than the use of CLP alone. Lamb from carcasses chilled for 24 h had significantly lower psychrophilic total viable counts and Brochothrix thermosphacta and pseudomonad counts than carcasses chilled for 72 h. Following vacuum-packed (VP) storage of meat from these carcasses at 1.7 ± 1.6 °C for 23 days in the meat plant followed by retail display at 3.9 ± 1.7 °C (up to day 50), the dominant microorganisms were lactic acid bacteria, Br. thermosphacta, Enterobacteriaceae and pseudomonads, and all had reached maximum population density by storage day 34. Aligned with this, after day 34, the quality of the raw meat samples also continued to deteriorate, with off-odours and colour changes developing. While the mean values for cooked meat eating quality attributes did not change significantly over the VP storage period, high variability in many attributes, including off-flavours and off-odours, were noted for lamb meat from all storage times, highlighting inconsistences in lamb quality within and between slaughter batches.

Development and evaluation of DNA and RNA real-time assays for food analysis using the hilA gene of Salmonella enterica subspecies enterica.

The objective of this study was the development of DNA and RNA real-time PCR methods for detection of food-borne Salmonella sp. as rapid alternatives to the traditional cultural method (ISO 6579, 2004) in fresh meat carcasses and processed meat samples. These PCR methods were based on the hilA sequence, with primers and hybridisation probes designed against this gene target. The primers and probes were evaluated for their efficiency and dynamic range and subsequently the specificity of the assay was tested using 106 Salmonella enterica subspecies enterica strains and 30 non-salmonellae strains. An internal amplification control (IAC) was also developed for incorporation. The optimum copy number of IAC was determined to be 500 copies per reaction. A complementary enrichment protocol was adapted from the existing standard ISO 6579:2004 and consisted of enrichment in Buffered Peptone Water (BPW) 22 ± 2 h and a second selective enrichment for 6 h in Rappaport Vassiliadis with Soya (RVS). The DNA and RNA-based real-time PCR protocols, were applied to meat samples inoculated with Salmonella enterica subspecies enterica strains, including swabs from meat carcasses and minced beef samples which were heat treated or frozen. The developed methods have the potential as useful alternatives to the standard ISO 6579:2004 method for the detection of Salmonella enterica subspecies enterica on carcass swabs and raw meat using hilA as a target. The DNA assay is a useful tool for the screening of meat samples in the abattoir within 3 days of slaughter or in a food production process and the RNA-based assay has the potential to detect viable Salmonella enterica subspecies enterica in ready-to-eat products.

Natural Anti-Microbials for Enhanced Microbial Safety and Shelf-Life of Processed Packaged Meat.

Microbial food contamination is a major concern for consumers and food industries. Consumers desire nutritious, safe and "clean label" products, free of synthetic preservatives and food industries and food scientists try to meet their demands by finding natural effective alternatives for food preservation. One of the alternatives to synthetic preservatives is the use of natural anti-microbial agents in the food products and/or in the packaging materials. Meat and processed meat products are characteristic examples of products that are highly perishable; hence natural anti-microbials can be used for extending their shelf-life and enhancing their safety. Despite several examples of the successful application of natural anti-microbial agents in meat products reported in research studies, their commercial use remains limited. This review objective is to present an extensive overview of recent research in the field of natural anti-microbials, covering essential oils, plant extracts, flavonoids, animal-derived compounds, organic acids, bacteriocins and nanoparticles. The anti-microbial mode of action of the agents, in situ studies involving meat products, regulations and, limitations for usage and future perspectives are described. The review concludes that naturally derived anti-microbials can potentially support the meat industry to provide "clean label", nutritious and safe meat products for consumers.

In vivo and ex vivo evaluations of bacteriophages e11/2 and e4/1c for use in the control of Escherichia coli O157:H7.

This study investigated the effect of bacteriophages (phages) e11/2 and e4/1c against Escherichia coli O157:H7 in an ex vivo rumen model and in cattle in vivo. In the ex vivo rumen model, samples were inoculated with either 10³ or 106 CFU/ml inoculum of E. coli O157:H7 and challenged separately with each bacteriophage. In the presence of phage e11/2, the numbers of E. coli O157:H7 bacteria were significantly (P < 0.05) reduced to below the limit of detection within 1 h. Phage e4/1c significantly (P < 0.05) reduced E. coli O157:H7 numbers within 2 h of incubation, but the number of surviving E. coli O157:H7 bacteria then remained unchanged over a further 22-h incubation period. The ability of a phage cocktail of e11/2 and e4/1c to reduce the fecal shedding of E. coli O157:H7 in experimentally inoculated cattle was then investigated in two cattle trials. Cattle (yearlings, n = 20 for trial one; adult fistulated cattle, n = 2 for trial two) were orally inoculated with 10(10) CFU of E. coli O157:H7. Animals (n = 10 for trial one; n = 1 for trial two) were dosed daily with a bacteriophage cocktail of 10(11) PFU for 3 days postinoculation. E. coli O157:H7 and phage numbers in fecal and/or rumen samples were determined over 7 days postinoculation. E. coli O157:H7 numbers rapidly declined in all animals within 24 to 48 h; however, there was no significant difference (P > 0.05) between the numbers of E. coli O157:H7 bacteria shed by the phage-treated or control animals. Phages were recovered from the rumen but not from the feces of the adult fistulated animal in trial two but were recovered from the feces of the yearling animals in trial one. While the results from the rumen model suggest that phages are effective in the rumen, further research is required to improve the antimicrobial effectiveness of phages for the elimination of E. coli O157:H7 in vivo.

Survival characteristics of Cronobacter spp. in model bovine gut and in the environment.

Cronobacter spp. (formally Enterobacter sakazakii) have been linked to illness in infants from contaminated powdered infant formula. The source of the pathogen remains unclear, and it is believed that farm environments and cattle could play a role in the transmission of Cronobacter spp. The aim of this study was to establish if the organism would survive passage through a model of the bovine rumen and abomasum and in bovine feces in the farm environment. Models of the bovine abomasum and rumen were inoculated with Cronobacter strains (final concentrations of 5.7 and 6.5 log(10) CFU/mL, respectively), and survival was examined over time in these environments using an adapted ISO/DTS 22964 culture protocol. Fecal samples were inoculated with Cronobacter (final concentration 6.0 log(10) CFU/mL), and survival on soil and in containers stored outdoors was examined over time. The results showed no significant changes in the number of Cronobacter in rumen fluid over a 24-h period. Cronobacter were undetectable after 30 min of incubation in the model abomasum. The pathogen survived 105 days in sealed containers and was detectable after 112 days in soil. This study indicated that Cronobacter spp. are unlikely to be shed in bovine feces as supported by previous surveillance studies; however, the study did show that the organism survives well in the farm environment.

Emerging Technologies for Aerial Decontamination of Food Storage Environments to Eliminate Microbial Cross-Contamination.

Air is recognized as an important source of microbial contamination in food production facilities and has the potential to contaminate the food product causing food safety and spoilage issues for the food industry. Potential for aerial microbial contamination of food can be a particular issue during storage in cold rooms when the food is not packaged and is exposed to contaminated air over a prolonged period. Thus, there are potential benefits for the food industry for an aerial decontamination in cold storage facilities. In this paper, aerial decontamination approaches are reviewed and challenges encountered for their applications are discussed. It is considered that current systems may not be completely effective and environmentally friendly, therefore, it is of great significance to consider the development of nonresidual and verified decontamination technologies for the food industry and, in particular, for the cold storage rooms.

Investigation of the Causes of Shigatoxigenic Escherichia coli PCR Positive and Culture Negative Samples.

Molecular methods may reveal the presence of pathogens in samples through the detection of specific target gene(s) associated with microorganisms, but often, the subsequent cultural isolation of the pathogen is not possible. This discrepancy may be related to low concentration of the cells, presence of dead cells, competitive microflora, injured cells and cells in a viable but non-culturable state, free DNA and the presence of free bacteriophages which can carry the target gene causing the PCR-positive/culture-negative results. Shiga-toxigenic Escherichia coli (STEC) was used as a model for studying this phenomenon, based on the phage-encoded cytotoxins genes (Stx family) as the detection target in samples through real-time qPCR. Stx phages can be integrated in the STEC chromosome or can be isolated as free particles in the environment. In this study, a combination of PCR with culturing was used for investigating the presence of the stx1 and stx2 genes in 155 ovine recto-anal junction swab samples (method (a)-PCR). Samples which were PCR-positive and culture-negative were subjected to additional analyses including detection of dead STEC cells (method (b)-PCR-PMA dye assay), presence of Stx phages (method (c)-plaque assays) and inducible integrated phages (method (d)-phage induction). Method (a) showed that even though 121 samples gave a PCR-positive result (78%), only 68 samples yielded a culturable isolate (43.9%). Among the 53 (34.2%) PCR-positive/culture-negative samples, 21 (39.6%) samples were shown to have STEC dead cells only, eight (15.1%) had a combination of dead cells and inducible stx phage, while two samples (3.8%) had a combination of dead cells, inducible phage and free stx phage, and a further two samples had Stx1 free phages only (3.8%). It was thus possible to reduce the samples with no explanation to 20 (37.7% of 53 samples), representing a further step towards an improved understanding of the STEC PCR-positive/culture-negative phenomenon.

Whole-genome epidemiology links phage-mediated acquisition of a virulence gene to the clonal expansion of a pandemic Salmonella enterica serovar Typhimurium clone.

Epidemic and pandemic clones of bacterial pathogens with distinct characteristics continually emerge, replacing those previously dominant through mechanisms that remain poorly characterized. Here, whole-genome-sequencing-powered epidemiology linked horizontal transfer of a virulence gene, sopE, to the emergence and clonal expansion of a new epidemic Salmonella enterica serovar Typhimurium (S. Typhimurium) clone. The sopE gene is sporadically distributed within the genus Salmonella and rare in S. enterica Typhimurium lineages, but was acquired multiple times during clonal expansion of the currently dominant pandemic monophasic S. Typhimurium sequence type (ST) 34 clone. Ancestral state reconstruction and time-scaled phylogenetic analysis indicated that sopE was not present in the common ancestor of the epidemic clade, but later acquisition resulted in increased clonal expansion of sopE-containing clones that was temporally associated with emergence of the epidemic, consistent with increased fitness. The sopE gene was mainly associated with a temperate bacteriophage mTmV, but recombination with other bacteriophage and apparent horizontal gene transfer of the sopE gene cassette resulted in distribution among at least four mobile genetic elements within the monophasic S. enterica Typhimurium ST34 epidemic clade. The mTmV prophage lysogenic transfer to other S. enterica serovars in vitro was limited, but included the common pig-associated S. enterica Derby (S. Derby). This may explain mTmV in S. Derby co-circulating on farms with monophasic S. Typhimurium ST34, highlighting the potential for further transfer of the sopE virulence gene in nature. We conclude that whole-genome epidemiology pinpoints potential drivers of evolutionary and epidemiological dynamics during pathogen emergence, and identifies targets for subsequent research in epidemiology and bacterial pathogenesis.

An appraisal of the use of meat-juice serology monitoring data for estimating prevalence of cecal Salmonella carriage of pigs at slaughter by means of herd-level and animal-level simulation.

Some attempts have been made to elucidate the association between positive serology and Salmonella detection by bacterial culture in individual pigs and pig herds. This study aimed to appraise whether the existing knowledge on such association provides grounds for the utilization of serology monitoring data for predicting Salmonella subclinical infection of pigs entering the abattoir. Serology test results of pig carcasses (taken at abattoirs) originating from 436 representative active herds in Ireland were utilized to estimate the overall cecal Salmonella carriage of Irish slaughter pigs. To this effect, two separate simulations were conducted using (i) herd-level regression data and (ii) animal-level sensitivity (0.2890) and specificity (0.8895) data, which were extracted from published articles. The herd-level approach estimated a moderate prevalence of cecal Salmonella carriage of 0.222 (sigma = 0.094; 95% confidence interval [CI]: 0.069 to 0.431), which matched closely the mean prevalence value from the surveys' validation data of Salmonella-positive cecal samples (n = 1,098) obtained at Irish abattoirs (0.215; 95% CI: 0.192 to 0.240). The animal-level simulation generated an output distribution with slightly more uncertainty (sigma = 0.102 and 95% CI: 0.146 to 0.537) and a higher estimate of cecal carriage (0.312), which was an effect of the low relative sensitivity of serology, common under field conditions. While the herd-level simulation appeared to be technically more appropriate, since its correlation is only moderate, further elucidation of other factors related to subclinical infection should be attained for their incorporation in prospective dynamic on-farm models, which would be useful in the ultimate goal of estimating the risk of carcass contamination during slaughter.