Added insult to injury? The response of meat-associated pathogens to proposed antimicrobial interventions.

Modern requirements for 'green label' meat products have led to the design of novel antimicrobial innovations which prioritise quality, safety and longevity. Plasma-functionalised water (PFW), ultraviolet light and natural antimicrobial compositions have been investigated and optimised for control of foodborne pathogens like Campylobacter jejuni and Salmonella enterica serovar Typhimurium. However, given the adaptive mechanisms present in bacteria under external stresses, it is imperative to understand the effect that sublethal treatment may have on the bacterial transcriptome. In this study, Salmonella Typhimurium and C. jejuni were treated with sublethal doses of ultraviolet light, a citrus juice/essential oil marinade, and 'spark' or 'glow' cold plasma generation system-produced PFW. Immediately after treatment, cells were lysed and RNA was extracted and purified. mRNA was converted to cDNA by reverse transcription-PCR and sequenced by an Illumina MiSeq® system. Sequences were filtered and analysed using the Tuxedo workflow. Sublethal treatment of Campylobacter jejuni and Salmonella Typhimurium led to increased immediate cellular and metabolic activity, as well as diversification in protein and metabolic functioning. There was further expression of pathogenesis and virulence-associated traits associated with spark PFW and marinade treatment of Salmonella Typhimurium. However, similar concerns were not raised with glow PFW or UV-treated samples. This study provides science-based evidence of the efficacy of multi-hurdle antimicrobial system using green-label marinades and PFW or UV to inactivate pathogens without upregulating virulence traits in surviving cells. This study will inform policymakers and food industry stakeholders and reinforces the need to incorporate in-line novel technologies to ensure consumer safety. KEY POINTS: • Salmonella and C. jejuni showed increased cell activity in immediate response to stress. • Virulence genes showed increased expression when treated with natural antimicrobials and sPFW. • Reduced immediate transcriptomic response to gPFW and UV treatment indicates lower risk.

The efficiency of UV light-emitting diodes (UV-LED) in decontaminating Campylobacter and Salmonella and natural microbiota in chicken breast, compared to a UV pilot-plant scale device.

This study investigated the combined effect of Ultraviolet (UV) light-emitting diode (LED) technology treatment with refrigerated storage of chicken breast meat over 7 days on Campylobacter jejuni, Salmonella enterica serovar Typhimurium, total viable counts (TVC) and total Enterobacteriaceae counts (TEC). An optimised UV-LED treatment at 280 nm for 6 min decreased inoculated S. Typhimurium and C. jejuni populations by 0.6-0.64 log CFU/g, and TVC and TEC population by 1-1.2 log CFU/g in chicken samples. During a 7-day storage at 4 °C, a 0.73 log reduction in C. jejuni was achieved compared with non-treated samples. Moreover, the UV-LED effectiveness to reduce TVC and TEC during refrigerated storage was compared with a conventional UV lamp and a similar efficiency was observed. The impact of UV-LED and UV lamp devices on the microbial community composition of chicken meat during storage was further examined using 16 S rRNA gene amplicon sequencing. Although similar bacterial reductions were observed for both technologies, the microbial communities were impacted differently. Treatment with the UV conventional lamp increased the proportion of Brochothrix spp. In meat samples, whilst Photobacterium spp. Levels were reduced.

Green label marinades: A solution to salmonella and campylobacter in chicken products?

Introduction: The presence of meat-borne pathogens entering the home remains a concern for consumers, despite advances made in improving antimicrobial interventions and systems within the processing line. Naturally antibacterial food ingredients including citrus juice and essential oils have been proven to inhibit the proliferation of microbial growth with varying success. Aims: This study aims to investigate the antimicrobial and sensory effects of mixtures of essential oils, fruit juices and herbs at established Minimum Inhibitory Concentrations (MICs) for their biopreservative effect on general microbiota of chicken and against chicken challenged with selected pathogenic/surrogate microorganisms. Materials and methods: Three marinade compositions were designed for use on chicken meat; lemon juice, thyme oil and black pepper (M1), lime juice, lemongrass oil and chilli paste (M2), and olive oil, oregano oil, basil oil and garlic paste (M3). These marinades were assessed for antibacterial effects against Salmonella enterica, Campylobacter jejuni and Listeria innocua on marinaded chicken drumsticks stored in aerobic conditions at 4 °C. Consumer tasting sessions were also conducted with a small focus group using selected final marinades. Results: M1 and M2 were effective at significantly reducing initial pathogen carriage from 6 Log CFU/g to 2 Log CFU/g on refrigerated chicken meat as well as increasing the shelf-life of the product during cold-storage from 2 days to 7 days. However, consumer studies indicate that the flavours these marinades impart to treated products can be strong. Conclusion: These findings indicate that these designed marinades have shown excellent potential to improve food safety as well as shelf-life for the consumer, particularly in settings where food safety is often compromised such as barbecuing or in care settings. However, further recipe optimisation is required to make these marinades acceptable to consumers.

A Small Study on Clostridioides difficile in Spinach Field Soil and the Chemical and Microbial Factors that may Influence Prevalence.

Clostridioides difficile is a human pathogen that is ubiquitous in soil. Despite increasing infection rates and evidence of foodborne transmission, there is limited data on prevalence in soil or which factors influence persistence. The aim of this study was to investigate the prevalence of these bacteria in soil from three different spinach fields and to examine the chemical composition (carbon, organic carbon, nitrogen, organic matter, minerals and pH) and microbiota to gain insight into the factors that may promote/inhibit C. difficile. The overall C. difficile prevalence (10%) was lower than expected (based on international studies) and a significantly (P < 0.05) higher prevalence was obtained in Field 3 (20%) as compared to Fields 1 and 2 (5% each). Analysis of the soil suggested that the pH as well as organic matter, calcium and phosphorus content directly and indirectly (via the microbiota) influenced the prevalence of C. difficile in adjacent fields, where other factors (eg. climate) are similar. Although further studies are required to validate our findings, the data provides the first step in developing potential soil based control strategies.

Investigation of differences in susceptibility of Campylobacter jejuni strains to UV light-emitting diode (UV-LED) technology.

Campylobacter jejuni remains a high priority in public health worldwide. Ultraviolet light emitting-diode technology (UV-LED) is currently being explored to reduce Campylobacter levels in foods. However, challenges such as differences in species and strain susceptibilities, effects of repeated UV-treatments on the bacterial genome and the potential to promote antimicrobial cross-protection or induce biofilm formation have arisen. We investigated the susceptibility of eight C. jejuni clinical and farm isolates to UV-LED exposure. UV light at 280 nm induced different inactivation kinetics among strains, of which three showed reductions greater than 1.62 log CFU/mL, while one strain was particularly resistant to UV light with a maximum reduction of 0.39 log CFU/mL. However, inactivation was reduced by 0.46-1.03 log CFU/mL in these three strains and increased to 1.20 log CFU/mL in the resistant isolate after two repeated-UV cycles. Genomic changes related to UV light exposure were analysed using WGS. C. jejuni strains with altered phenotypic responses following UV exposure were also found to have changes in biofilm formation and susceptibility to ethanol and surface cleaners.

Characterization of Food Chain Clostridioides difficile Isolates in Terms of Ribotype and Antimicrobial Resistance.

The aim of this study was to characterize C. difficile isolates from the farm, abattoir, and retail outlets in Ireland in terms of ribotype and antibiotic resistance (vancomycin, erythromycin, metronidazole, moxifloxacin, clindamycin, and rifampicin) using PCR and E-test methods, respectively. The most common ribotype in all stages of the food chain (including retail foods) was 078 and a variant (RT078/4). Less commonly reported (014/0, 002/1, 049, and 205) and novel (RT530, 547, and 683) ribotypes were also detected, but at lower frequencies. Approximately 72% (26/36 tested) of the isolates tested were resistant to at least one antibiotic, with the majority of these (65%; 17/26) displaying a multi-drug (three to five antibiotics) resistant phenotype. It was concluded that ribotype 078, a hypervirulent strain commonly associated with C. difficile infection (CDI) in Ireland, was the most frequent ribotype along the food chain, resistance to clinically important antibiotics was common in C. difficile food chain isolates, and there was no relationship between ribotype and antibiotic resistance profile.

The Gut Microbiota of Broilers Reared with and without Antibiotic Treatment.

The aim of this study was to examine the microbiota in broilers reared with and without antibiotics and to investigate differences between the upper, middle and lower sections of the gastrointestinal tract (GIT). One of two commercial flocks was treated with an antibiotic (T) (20 mg trimethoprim and 100 mg sulfamethoxazole per ml in the drinking water for 3 days) and the other was left untreated (UT). The GIT contents of 51 treated and untreated birds were aseptically removed from the upper (U), middle (M) and lower (L) sections. These were pooled in triplicate (n = 17 per section per flock), the DNA extracted and purified, 16S amplicon metagenomic sequencing performed and the resultant data analysed using a range of bioinformatics software. There were significant differences in the microbiota of the upper, middle and lower GIT, and treatment with the antibiotic significantly affected the microbiota in each of these sections. This study provides new data on broiler GIT microbiota and suggests that GIT location is a more important determinant of the constituent bacterial flora rather than the use or otherwise of antimicrobial treatments, at least when applied early in the production cycle.

The effect of cold storage and cooking on the viability of Clostridioides difficile spores in consumer foods.

The increased detection of clinical cases of Clostridioides difficile coupled with the persistence of clostridial spores at various stages along the food chain suggest that this pathogen may be foodborne. This study examined C. difficile (ribotypes 078 and 126) spore viability in chicken breast, beef steak, spinach leaves and cottage cheese during refrigerated (4 °C) and frozen (-20 °C) storage with and without a subsequent sous vide mild cooking (60 °C, 1 h). Spore inactivation at 80 °C in phosphate buffer solution, beef and chicken were also investigated to provide D80°C values and determine if PBS was a suitable model system for real food matrices. There was no decrease in spore concentration after chilled or frozen storage and/or sous vide cooking at 60 °C. Non-log-linear thermal inactivation was observed for both C. difficile ribotypes at 80 °C in phosphate buffer solution (PBS), beef and chicken. The predicted PBS D80°C values of 5.72±[2.90, 8.55] min and 7.50±[6.61, 8.39] min for RT078 and RT126, respectively, were in agreement with the food matrices D80°C values of 5.65 min (95% CI range from 4.29 to 8.89 min) for RT078 and 7.35 min (95% CI range from 6.81 to 7.01 min) for RT126. It was concluded that C. difficile spores survive chilled and frozen storage and mild cooking at 60 °C but may be inactivated at 80 °C. Moreover thermal inactivation in PBS was representative of that observed in real food matrices (beef and chicken).

Detection and Genomic Characterisation of Clostridioides difficile from Spinach Fields.

Despite an increased incidence of Clostridioides difficile infections, data on the reservoirs and dissemination routes of this bacterium are limited. This study examined the prevalence and characteristics of C. difficile isolates in spinach fields. C. difficile was detected in 2/60 (3.3%) of spinach and 6/60 (10%) of soil samples using culture-based techniques. Whole genome sequencing (WGS) analysis identified the spinach isolates as belonging to the hypervirulent clade 5, sequence type (ST) 11, ribotypes (RT) 078 and 126 and carried the genes encoding toxins A, B and CDT. The soil isolates belonged to clade 1 with different toxigenic ST/RT (ST19/RT614, ST12/RT003, ST46/RT087, ST16/RT050, ST49/RT014/0) strains and one non-toxigenic ST79/RT511 strain. Antimicrobial resistance to erythromycin (one spinach isolate), rifampicin (two soil isolates), clindamycin (one soil isolate), both moxifloxacin and rifampicin (one soil isolate), and multi-drug resistance to erythromycin, vancomycin and rifampicin (two soil isolates) were observed using the E test, although a broader range of resistance genes were detected using WGS. Although the sample size was limited, our results demonstrate the presence of C. difficile in horticulture and provide further evidence that there are multiple sources and dissemination routes for these bacteria.

Genomic diversity, virulence and source of Campylobacter jejuni contamination in Irish poultry slaughterhouses by whole genome sequencing.

AIMS: The aim was to exploit whole genome sequencing (WGS) to assess genomic diversity, identify virulence genes and deduce the proportion of Campylobacter colonized broilers that directly contaminate their carcasses. METHODS AND RESULTS: Campylobacter jejuni isolates (107) from caeca and carcass neck skin samples (50 pairs from the same batch plus 7 individual caeca) sampled at three poultry slaughterhouses over a one-year period were selected for sequencing (MiSeq; Illumina). FastQ files were submitted to BioNumerics for analysis using the wgMLST scheme for allele calling. Campylobacter cgMLST and hierarchical clustering was performed by applying the single linkage algorithm. Sequence types (STs) were determined in silico from the WGS data and isolates were assigned into clonal complexes (CCs) using the Campylobacter PubMLST.org database. Virulence genes were determined by downloading core sequences from the virulence factor database (VFDB) and the National Center for Biotechnology Information (NCBI). A high degree of diversity was observed with 23 different STs identified. ST257 and CC-21 were the most common STs and CCs, respectively. cgMLST analysis suggested that 56% of carcass contamination was a direct result of contamination from caeca from the same batch. Virulence genes known to play a role in human C. jejuni infection were identified such as the wlaN gene and the genes associated with lipooligosaccharide synthesis, which were identified in 30% of isolates. CONCLUSIONS: Caecal colonization was the more plausible occurring source of C. jejuni contamination of broiler carcasses, compared with cross-contamination from another batch or the environment. The high rate of genetic diversity observed amongst caecal isolates is consistent with a wide variety of Campylobacter strains circulating in poultry flocks in Ireland. SIGNIFICANCE AND IMPACT OF STUDY: The results will further inform broiler processors and regulators about the influence and importance of on-farm colonization versus slaughterhouse cross-contamination and the relationship between C. jejuni in caeca and carcasses during processing.

An Investigation of the Effect of Water Additives on Broiler Growth and the Caecal Microbiota at Harvest.

Campylobacter is the most common foodborne pathogen in developed countries and most cases are associated with poultry. This study investigated the effect of three anti-Campylobacter water additives on broiler growth and on the caecal microbiota at harvest using 16S rRNA amplicon sequencing. Mixtures of organic acids (OA) and essential oils (EO) were administered to broilers for the entirety of the production cycle (35 d) and medium-chain fatty acids (MCFA) for 5 d immediately before harvest, under commercial conditions. Bird weight gain was significantly (p < 0.001) reduced in broilers receiving the OA and EO treatments. While this was most likely due to reduced water intake and corresponding lower feed consumption, changes to the caecal microbiota may also have contributed. Firmicutes made up over 75% of the bacteria regardless of sample type, while the minor phyla included Bacteroidetes, Actinobacteria, Melainabacteria, and Proteobacteria. There were no significant (p > 0.05) differences in the alpha diversity as measured using ACE, Chao1, and Shannon indices, except for control (water) versus MCFA and OA versus MCFA, using the Wilcox test. In contrast, there was a significant (p < 0.05) difference in beta diversity when the treated were compared to the untreated control and main flock samples, while linear discriminant analysis effect size (LeFSe) identified three OTUs that were present in the control but absent in the treated birds. It was concluded that the water additives tested adversely affected broiler performance, which may, at least in part, be due to changes in the caecal microbiota, assuming that the altered microbiota at day 35 is indicative of a change throughout the production cycle.

Effect of Doxycycline Use in the Early Broiler Production Cycle on the Microbiome.

16S rRNA amplicon sequencing was used to investigate changes in the broiler gastrointestinal tract (GIT) microbiota throughout the rearing period and in combination with antibiotic treatment. Thirty birds (from a commercial flock) were removed at multiple points throughout the rearing period on days 13, 27, and 33, euthanised, and their GIT aseptically removed and divided into upper (the crop, proventriculus, and the gizzard), middle (the duodenum, jejunum, and ileum) and lower (the large intestine, the caeca, and the cloaca) sections. In a separate commercial flock, on the same farm with similar husbandry practices and feed, doxycycline (100 mg/ml per kg body weight) was administered in drinking water between day 8 and 12 (inclusive) of the production cycle. Birds were removed on days, 13, 27, and 33 and GIT samples prepared as above. The contents of three merged samples from each GIT section were pooled (n = 60), the DNA extracted and analysed by 16S rRNA amplicon metagenomic sequencing and analysed. Major changes in the broiler microbiota were observed as the birds aged particularly with the Firmicutes/Bacteroidetes ratio (F:B) of the lower GIT. Moreover, Chao1, ACE, and Shannon indices showed the antibiotic treatment significantly altered the microbiota, and this change persisted throughout the rearing period. Further research is required to investigate the effect of these changes on bird performance, susceptibility to infections and Campylobacter carriage.

Prevalence and levels of Campylobacter in broiler chicken batches and carcasses in Ireland in 2017-2018.

In 2008, an EU wide baseline survey of broilers revealed a high Campylobacter prevalence. To assist with industry-wide controls, updated data were required. The primary objective of this study was to establish up-to-date data on Campylobacter carriage and carcass contamination in Irish broilers. Monthly samples were collected from the three largest broiler processing plants in Ireland over a twelve-month period. Samples were taken from both first and final thin birds (partial and full depopulation) from 358 batches of broilers. From each batch, a composite sample of 10 caecal contents (n = 358) and 5 neck skins (n = 1790) were collected and numbers of Campylobacter in each sample were determined. Of the 1790 neck skin samples tested, 53% were Campylobacter positive. Campylobacter was detected in the caecal contents of 66% of all batches tested. Depopulation and/or age had a significant effect on Campylobacter prevalence with 67% of final thin broilers yielding Campylobacter-positive neck skin samples in contrast to 38% of first thin broilers that yielded positive neck skin samples (P ≤ 0.002). A significant seasonal variation was observed in the rate of Campylobacter-positive caecal samples with higher prevalence seen in July (85%) than the colder months of November (61%), December (50%), January (61%) March (57%) and April (59%). Neck skin samples were 7 times more likely to be Campylobacter positive if the caecal contents from the same batch were positive (odds ratio = 7.1; P ≤ 0.0001). The decrease in Campylobacter prevalence observed in neck skin and caecal contents demonstrates the improvements and progress made in reducing prevalences of this important enteropathogen in the Irish poultry industry since the 2008 EU baseline survey. It also provides further supporting data on the impact of thinning, the processing environment and season on Campylobacter prevalence.

Stress response modulation: the key to survival of pathogenic and spoilage bacteria during poultry processing.

The control of bacterial contaminants on meat is a key area of interest in the food industry. Bacteria are exposed to a variety of stresses during broiler processing which challenge bacterial structures and metabolic pathways causing death or sublethal injury. To counter these stresses, bacteria possess robust response systems that can induce shifts in the transcriptome and proteome to enable survival. Effective adaptive responses, such as biofilm formation, shock protein production and metabolic flexibility, require rapid induction and implementation at a cellular and community level to facilitate bacterial survival in adverse conditions. This review aims to provide an overview of the scientific literature pertaining to the regulation of complex adaptive processes used by bacteria to survive the processing environment, with particular focus on species that impact the quality and safety of poultry products like Campylobacter spp., Salmonella enterica and Pseudomonas spp.

Quantitative microbial risk assessment associated with ready-to-eat salads following the application of farmyard manure and slurry or anaerobic digestate to arable lands.

Farmyard manure and slurry (FYM&S) and anaerobic digestate are potentially valuable soil conditioners providing important nutrients for plant development and growth. However, these organic fertilisers may pose a microbial health risk to humans. A quantitative microbial risk assessment (QMRA) model was developed to investigate the potential human exposure to pathogens following the application of FYM&S and digestate to agricultural land. The farm-to-fork probabilistic model investigated the fate of microbial indicators (total coliforms and enterococci) and foodborne pathogens in the soil with potential contamination of ready-to-eat salads (RTEs) at the point of human consumption. The processes examined included pathogen inactivation during mesophilic anaerobic digestion (M-AD), post-AD pasteurisation, storage, dilution while spreading, decay in soil, post-harvest washing processes, and finally, the potential growth of the pathogen during refrigeration/storage at the retail level in the Irish context. The QMRA highlighted a very low annual probability of risk (Pannual) due to Clostridium perfringens, norovirus, and Salmonella Newport across all scenarios. Mycobacterium avium may result in a very high mean Pannual for the application of raw FYM&S, while Cryptosporidium parvum and pathogenic E. coli showed high Pannual, and Listeria monocytogenes displayed moderate Pannual for raw FYM&S application. The use of AD reduces this risk; however, pasteurisation reduces the Pannual to an even greater extent posing a very low risk. An overall sensitivity analysis revealed that mesophilic-AD's inactivation effect is the most sensitive parameter of the QMRA, followed by storage and the decay on the field (all negatively correlated to risk estimate). The information generated from this model can help to inform guidelines for policymakers on the maximum permissible indicator or pathogen contamination levels in the digestate. The QMRA can also provide the AD industry with a safety assessment of pathogenic organisms resulting from the digestion of FYM&S.

The efficacy of organic acid, medium chain fatty acid and essential oil based broiler treatments; in vitro anti-Campylobacter jejuni activity and the effect of these chemical-based treatments on broiler performance.

AIMS: This research tested the anti-Campylobacter properties of organic acids (OA), medium chain fatty acids (MCFA) and essential oils (EO) in vitro and commenced in vivo suitability testing focused on broiler performance. METHODS AND RESULTS: Nine active compounds were tested at different concentrations and times against Campylobacter jejuni in sterile distilled water, Mueller Hinton broth and grower feed digestate (GFD). Sodium caprate (1.5%, v/v), thymol (0.25% and 2.5%, v/v), carvacrol (1.25%, v/v) and potassium sorbate (1.5%, v/v) each achieved C. jejuni reductions of ≥4.5 log10  CFU per ml in GFD, the matrix most representative of the broiler gut, after 60 s. Similar reductions were achieved after 60 min with lactic acid (1.25%, v/v), formic acid (3.1%, v/v), sodium caprylate (1.5%, v/v) and carvacrol (1.25%, v/v). However, in vivo these compounds adversely affected broiler performance, resulting in dimished water intake and reduced weight. CONCLUSIONS: OA, MFCA and EO based compounds are effective anti-Campylobacter treatments in laboratory model studies but cannot be applied in vivo. SIGNIFICANCE AND IMPACT OF THE STUDY: This study illustrates that OAs, MCFAs and EOs can achieve significant reductions in Campylobacter in vitro but identifies a major issue, inhibition of broiler performance, preventing their use in practice.

Genomic Characterisation of Campylobacter jejuni Isolates Recovered During Commercial Broiler Production.

Background: Campylobacter is commonly transmitted to humans from chickens. Campylobacter jejuni is the species most frequently associated with human illness, and the most prevalent species recovered from poultry. Objective: The objective of this study was to analyse a sub-population of C. jejuni from two broiler flocks on the farm and at slaughter using whole-genome sequencing to gain insights into the changes in the Campylobacter population during broiler production, including changes in virulence and antimicrobial resistance profiles. Methods: In this study, ten composite faecal samples (n=10), obtained by pooling ten fresh faecal samples (n=10), were collected in the broiler house on two farms on days 14, 21, 28, and 34 (n=80) and ten composite (n=10) caecal samples were collected at the time of slaughter for each flock (n=20). These were tested for C. jejuni using the ISO 10272-2:2016 method. Seven isolates were randomly selected from each of the nine Campylobacter-positive sampling points (n=63) and were subjected to antimicrobial susceptibility tests. Their genomes were sequenced and the data obtained was used to characterise the population structure, virulence, antimicrobial resistance determinants and inter-strain variation. Results: The Farm 1 isolates had three MLST types (ST257-257, ST814-661 and ST48-48) while those on Farm 2 were ST6209-464 and ST9401. Interestingly, only the MLST types positive for most of the virulence genes tested in this study persisted throughout the production cycle, and the detection of antimicrobial resistance determinants (gyrA T86I and tetO) increased after thinning and at slaughter, with the detection of new strains. Conclusion: The persistence of the most virulent strains detected in this study throughout the production cycle has important implications for the risk to consumers and requires further investigation. The detection of new strains within the population corresponding with the time of thinning and transportation reflects previous reports and provides further evidence that these activities pose a risk of introducing new Campylobacter strains to broiler batches.

Quantitative microbial human exposure model for faecal indicator bacteria and risk assessment of pathogenic Escherichia coli in surface runoff following application of dairy cattle slurry and co-digestate to grassland.

Animal waste contains high numbers of microorganisms and therefore can present a potential biological threat to human health. During episodic rainfall events resulting in runoff, microorganisms in the waste and soil may migrate into surface runoff, contaminating surface water resources. A probabilistic human exposure (HE) model was created to determine exposure to faecal indicator bacteria (FIB): total coliforms (TC), E. coli and enterococci following application of bio-based fertiliser (dairy cattle slurry, digestate) to grassland; using a combination of experimental field results and literature-based data. This step was followed by a quantitative microbial risk assessment (QMRA) model for pathogenic E. coli based on a literature-based dose-response model. The results showed that the maximum daily HE (HEdaily) is associated with E. coli for unprocessed slurry (treatment T1) on day 1, the worst-case scenario where the simulated mean HEdaily was calculated as 2.84 CFU day -1. The results indicate that the overall annual probability of risk (Pannual) of illness from E. coli is very low or low based on the WHO safe-limit of Pannual as 10 -6. In the worst-case scenario, a moderate risk was estimated with simulated mean Pannual as 1.0 × 10 -5. Unpasteurised digestate application showed low risk on day 1 and 2 (1.651 × 10 -6, 1.167 × 10 -6, respectively). Pasteurised digestate showed very low risk in all scenarios. These results support the restriction imposed on applying bio-based fertiliser if there is any rain forecast within 48 h from the application time. This study proposes a future extension of the probabilistic model to include time, intensity, discharge, and distance-dependant dilution factor. The information generated from this model can help policymakers ensure the safety of surface water sources through the quality monitoring of FIB levels in bio-based fertiliser.

An investigation into the anaerobic spoilage microbiota of beef carcass and rump steak cuts using high- throughput sequencing.

The presence of anaerobic microflora on fresh beef carcass and rump steaks, which may contribute to meat spoilage, was explored in this study. A total of 120 carcass and 120 rump steak swabs were collected immediately after slaughtering and boning, respectively from five meat plants, anaerobically incubated and enriched at 4°C for 3 weeks. This was followed by DNA extraction and 16S rRNA amplicon sequencing using the Illumina MiSeq, with subsequent bioinformatics analysis. The enriched microbiota of the samples was classified and grouped into 149 operational taxonomic units (OTUs). The microbiota recovered from both sample types consisted mainly of Carnobacterium, with an average relative abundance of 28.4% and 32.8% in beef carcasses and beef rump steaks, respectively. This was followed by Streptococcus, Serratia, Lactococcus, Enterococcus, Escherichia-Shigella, Raoultella and Aeromonas ranging from 1.5 to 20% and 0.1 to 29.8% in enriched carcasses and rump steak swabs, respectively. Trichococcus, Bacteroides, Dysgomonas, Providencia, Paraclostridium and Proteus were also present ranging from 0 to 0.8% on carcass and 0 to 1.8% on rump steak swabs, respectively. Alpha and beta diversity measurements showed limited diversity between the two sample types, but some differences between samples from the beef plants investigated were evident. This study highlights the presence of potential spoilage bacteria, mainly anaerobic genera on and between carcass and rump steaks, as an indication of contamination on and between these samples.

Evaluation of pathogen concentration in anaerobic digestate using a predictive modelling approach (ADRISK).

Farmyard manure and slurry (FYM&S) is a valuable feedstock for anaerobic digestion (AD) plants. However, FYM&S may contain high concentrations of pathogens, and complete inactivation through the AD process is unlikely. Thus, following land application of digestate, pathogens may contaminate a range of environmental media posing a potential threat to public health. The present study aimed to combine primary laboratory data with literature-based secondary data to develop an Excel-based exposure assessment model (ADRISK) using a gamma generalised linear model to predict the final microorganism count in the digestate. This research examines the behaviour of a suite of pathogens (Cryptosporidium parvum, norovirus, Mycobacterium spp., Salmonella spp., Listeria monocytogenes, Clostridium spp., and pathogenic Escherichia coli) and indicators (total coliforms, E. coli, and enterococci) during mesophilic anaerobic digestion (M-AD) at 37 °C, pre/post-AD pasteurisation, and after a period of storage (with/without lime) for different feedstock proportions (slurry:food waste: 0:1, 1:3, 2:1, and 3:1). ADRISK tool simulations of faecal indicator bacteria levels across all scenarios show that the digestate can meet the EU standard without pasteurisation if the AD runs at 37 °C or a higher temperature with a higher C:N ratio (recipe 3) and a hydraulic retention time ≥ 7 days. The storage of digestate also reduced levels of microorganisms in the digestate. The Irish pasteurisation process (60 °C for 4 days), although more energy-intensive, is more effective than the EU pasteurisation (70 °C for 1 h) specification. Pre-AD pasteurisation was more effective for C. parvum, norovirus, Mycobacterium thermoresistibile. However, post-AD literature-based pasteurisation is most likely to assure the safety of the digestate. The information generated from this model can inform policy-makers regarding the optimal M-AD process parameters necessary to maximise the inactivation of microorganisms, ensuring adverse environmental impact is minimised, and public health is protected.