• Antimicrobial activity of natural compounds against listeria spp. and their effects on sensory attributes in salmon (Salmo salar) and cod (Gadus morhua)

      Pedrós-Garrido, Selene; Clemente, Isabel; Calanche, J. B.; Condón-Abanto, Santiago; Beltrán, J. A.; Lyng, J. G.; Brunton, N.; Bolton, Declan; Whyte, Paul; Department of Agriculture, Food and the Marine; et al. (Elsevier, 2019-07-15)
      The application of natural preservatives on fresh fish has potential to extend shelf-life. In the present study, 8 essential oils (EOs) (lemon, lemongrass, lime, garlic, onion, oregano, thyme and rosemary) and 3 organic acids (OAs) (ascorbic, citric and lactic) were evaluated. The antimicrobial activity of these compounds was tested in-vitro against four confirmed Listeria spp. isolated from retail skin-packed salmon and cod. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were established for each compound. Then, a sensory evaluation was performed by a panel of ‘expert assessors’ on cooked fish treated with all of the OAs and any 4 EOs with a MIC <0.8%. A series of descriptors were assigned to characterize the combination of each compound with cooked salmon or cod. The highest antimicrobial effect against all Listeria spp. was observed for lactic acid (0.31–2.5%), but treatment with this compound resulted in the development of organoleptically unacceptable changes in salmon or cod. The most acceptable OAs for salmon and cod were ascorbic acid (1.25%) and citric acid (0.63%) respectively, which were shown to enhance certain organoleptic characteristics. The most effective EO against all Listeria strains evaluated was oregano oil (0.2%) and it was considered suitable as a treatment for salmon. In contrast, none of the EOs tested was organoleptically acceptable in combination with cod because of their strong odours and flavours that masked the fresh attributes associated with this fish.
    • Antimicrobial Resistance Determinants Circulating among Thermophilic Campylobacter Isolates Recovered from Broilers in Ireland Over a One-Year Period

      Lynch, Caoimhe T.; Lynch, Helen; Burke, Sarah; Hawkins, Kayleigh; Buttimer, Colin; McCarthy, Conor; Egan, John; Whyte, Paul; Bolton, Declan; Coffey, Aidan; et al. (MDPI AG, 2020-06-08)
      Campylobacteriosis is the leading cause of human bacterial gastroenteritis, very often associated with poultry consumption. Thermophilic Campylobacter (Campylobacter jejuni and Campylobacter coli) isolates (n = 158) recovered from broiler neck skin and caecal contents in Ireland over a one-year period, resistant to at least one of three clinically relevant antimicrobial classes, were screened for resistance determinants. All ciprofloxacin-resistant isolates (n = 99) harboured the C257T nucleotide mutation (conferring the Thr-86-Ile substitution) in conjunction with other synonymous and nonsynonymous mutations, which may have epidemiological value. The A2075G nucleotide mutation and amino acid substitutions in L4 and L22 were detected in all erythromycin-resistant isolates (n = 5). The tetO gene was detected in 100% (n = 119) of tetracycline-resistant isolates and three of which were found to harbour the mosaic tetracycline resistance gene tetO/32/O. Two streptomycin-resistant C. jejuni isolates (isolated from the same flock) harboured ant(6)-Ib, located in a multidrug resistance genomic island, containing aminoglycoside, streptothricin (satA) and tetracycline resistance genes (truncated tetO and mosaic tetO/32/O). The ant(6)-Ie gene was identified in two streptomycin-resistant C. coli isolates. This study highlights the widespread acquisition of antimicrobial resistance determinants among chicken-associated Campylobacter isolates, through horizontal gene transfer or clonal expansion of resistant lineages. The stability of such resistance determinants is compounded by the fluidity of mobile genetic element.
    • Controlling Blown Pack Spoilage Using Anti-Microbial Packaging

      Reid, Rachael; Bolton, Declan; Tiuftin, Andrey; Kerry, Joseph P.; Fanning, Seamus; Whyte, Paul (MDPI, 12/08/2017)
      Active (anti-microbial) packaging was prepared using three different formulations; Auranta FV; Inbac-MDA and sodium octanoate at two concentrations (2.5 and 3.5 times their minimum inhibitory concentration (MIC, the lowest concentration that will inhibit the visible growth of the organisms) against Clostridium estertheticum, DSMZ 8809). Inoculated beef samples were packaged using the active packaging and monitored for 100 days storage at 2 °C for blown pack spoilage. The time to the onset of blown pack spoilage was significantly (p < 0.01) increased using Auranta FV and sodium octanoate (caprylic acid sodium salt) at both concentrations. Moreover, sodium octanoate packs had significantly (p < 0.01) delayed blown pack spoilage as compared to Auranta FV. It was therefore concluded that Auranta FV or sodium octanoate, incorporated into the packaging materials used for vacuum packaged beef, would inhibit blown pack spoilage and in the case of the latter, well beyond the 42 days storage period currently required for beef primals
    • Development of a real-time multiplex PCR assay for the detection of multiple Salmonella serotypes in chicken samples.

      O'Regan, Edel; McCabe, Evonne; Burgess, Catherine; McGuinness, Sheila; Barry, Thomas; Duffy, Geraldine; Whyte, Paul; Fanning, Seamus; Department of Agriculture, Food and the Marine, Ireland (Biomed Central, 21/09/2008)
      Background: A real-time multiplex PCR assay was developed for the detection of multiple Salmonella serotypes in chicken samples. Poultry-associated serotypes detected in the assay include Enteritidis, Gallinarum, Typhimurium, Kentucky and Dublin. The traditional cultural method according to EN ISO 6579:2002 for the detection of Salmonella in food was performed in parallel. The real-time PCR based method comprised a pre-enrichment step in Buffered Peptone Water (BPW) overnight, followed by a shortened selective enrichment in Rappaport Vasilliadis Soya Broth (RVS) for 6 hours and subsequent DNA extraction. Results: The real-time multiplex PCR assay and traditional cultural method showed 100% inclusivity and 100% exclusivity on all strains tested. The real-time multiplex PCR assay was as sensitive as the traditional cultural method in detecting Salmonella in artificially contaminated chicken samples and correctly identified the serotype. Artificially contaminated chicken samples resulted in a detection limit of between 1 and 10 CFU per 25 g sample for both methods. A total of sixty-three naturally contaminated chicken samples were investigated by both methods and relative accuracy, relative sensitivity and relative specificity of the real-time PCR method were determined to be 89, 94 and 87%, respectively. Thirty cultures blind tested were correctly identified by the real-time multiplex PCR method. Conclusion: Real-time PCR methodology can contribute to meet the need for rapid identification and detection methods in food testing laboratories.
    • The effect of antimicrobials on verocytotoxin bacteriophage transduction under bovine rumen fluid and broth conditions

      Nyambe, Sepa; Burgess, Catherine; Whyte, Paul; Bolton, Declan; Department of Agriculture, Food and the Marine, Ireland; 11/F/051 (Teagasc (Agriculture and Food Development Authority), Ireland, 15/11/2017)
      The verocytotoxin genes in verocytotoxigenic Escherichia 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 log10 cfu/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 > 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.
    • The effect of organic acid and sodium chloride dips on the shelf-life of refrigerated Irish brown crab (Cancer pagurus) meat

      McDermott, A.; Whyte, Paul; Brunton, Nigel; Lyng, L.; Fagan, John; Bolton, Declan; Department of Agriculture, Food and the Marine; 13F529 (Elsevier, 2018-08-18)
      Crab (Cancer pagurus) meat (white and brown) has a short shelf-life. Chemical treatments may inhibit microbial spoilage and extend shelf-life. The effect of 5% organic acids (lactic acid (LA), acetic acid (AA) and citric acid (CA)) and 5% sodium chloride (NaCl) on TVC (mesophiles and psychrophiles), Enterobacteriaceae, Pseudomonas spp. and lactic acid bacteria (LAB) were investigated during storage (2 °C for 12 days). AA was the most effective treatment for white meat, reducing the initial TVCm and TVCp by 1.6 and 1.8 log10 cfu/g, respectively, and extended the shelf life to 8–11.5 days, compared to 5 days for untreated control samples. LA treatment also significantly (P < 0.05) reduced the initial TVC, but the shelf life was only increased by 3 days. CA and NaCl treatments had no significant effect (P > 0.05). A similar pattern was observed for brown meat samples, although the shelf life was increased by a maximum of 1–3 days. The growth of Enterobacteriaceae, Pseudomonas spp. and LAB was significantly (P < 0.05) reduced on AA treated samples only. It was concluded that the shelf-life of crab meat may be extended by up to 3 days using lactic acid and more than doubled using acetic acid.
    • Efficacy of ultraviolet light (UV-C) and pulsed light (PL) for the microbiological decontamination of raw salmon (Salmo salar) and food contact surface materials

      Pedrós-Garrido, S.; Condón-Abanto, S.; Clemente, I.; Beltrán, J.A.; Lyng, James G.; Bolton, Declan; Brunton, Nigel; Whyte, Paul; Department of Agriculture, Food and the Marine; 13F458 (Elsevier, 2018-10-03)
      The decontamination effect of two light-based technologies on salmon, polyethylene (PE) and stainless steel (SS) was evaluated. Optimization of treatment conditions for ultraviolet light (UV-C) and pulsed light (PL) was carried out on raw salmon, obtaining inactivation levels of 0.9 and 1.3 log CFU/g respectively. The effects of treatments on several microbial groups present in salmon were then evaluated. For both technologies, Pseudomonas spp. were found to be the most resistant group of microorganisms tested. Three different strains from within this group were isolated and speciated, including a P. fluorescens strain which was selected for subsequent studies. PE and SS surfaces were inoculated with a suspension of the P. fluorescens suspended in a ‘salmon juice’ solution, and treated with UV-C and PL at different doses (mJ/cm2). PE surfaces were effectively decontaminated a low doses for both technologies, with a reduction of >4 log cycles observed. Decontamination of SS was also effective when treated with PL, although at higher doses than for PE. When SS was treated with UV-C, the maximum reduction of P. fluorescens achieved was 2 log cycles, even at the highest dose.
    • Genetic characterisation of a subset of Campylobacter jejuni isolates from clinical and poultry sources in Ireland

      Truccollo, Brendha; Whyte, Paul; Burgess, Catherine; Bolton, Declan; Teagasc; 0028 (Public Library of Science (PLoS), 2021-03-09)
      Campylobacter spp. is a significant and prevalent public health hazard globally. Campylobacter jejuni is the most frequently recovered species from human cases and poultry are considered the most important reservoir for its transmission to humans. In this study, 30 Campylobacter jejuni isolates were selected from clinical (n = 15) and broiler (n = 15) sources from a larger cohort, based on source, virulence, and antimicrobial resistance profiles. The objective of this study was to further characterise the genomes of these isolates including MLST types, population structure, pan-genome, as well as virulence and antimicrobial resistance determinants. A total of 18 sequence types and 12 clonal complexes were identified. The most common clonal complex was ST-45, which was found in both clinical and broiler samples. We characterised the biological functions that were associated with the core and accessory genomes of the isolates in this study. No significant difference in the prevalence of virulence or antimicrobial resistance determinants was observed between clinical and broiler isolates, although genes associated with severe illness such as neuABC, wlaN and cstIII were only detected in clinical isolates. The ubiquity of virulence factors associated with motility, invasion and cytolethal distending toxin (CDT) synthesis in both clinical and broiler C. jejuni genomes and genetic similarities between groups of broiler and clinical C. jejuni reaffirm that C. jejuni from poultry remains a significant threat to public health.
    • Impact of direct and indirect heating systems in broiler units on environmental conditions and flock performance

      Smith, Shaun; Meade, Joseph; Gibbons, James; McGill, Kevina; Bolton, Declan; Whyte, Paul; Department of Agriculture, Food and Marine; 11SF328 (Elsevier, 2016-11-11)
      This study compared the impact of three indirect heating systems to direct gas flame heaters on a selection of flock performance and environmental indicators in commercial broiler units. No statistically significant differences (P≥0.05) were found in flock mortality rates, bird weight, water consumption, stress response, carbon dioxide, ammonia, temperature, relative humidity, litter quality, within-flock Campylobacter levels or mean Campylobacter counts when flock data from any of the three indirect heating systems were compared to flocks reared in houses with direct heating systems. Differences in litter quality were observed between upper and lower litter layers in all houses, regardless of heating type, which may have implications for bird health and welfare. Carbon dioxide concentrations in houses with direct heating systems were significantly higher than those in houses with indirect heating systems during the first 10 days of bird life (P≤0.05). This was due to the increased use of heating systems during this period of the flock cycle. Differences in CO2 concentrations had no effect on flock performance, possibly due to the fact that concentrations did not exceed known safe levels. A statistically significant increase in stress response was observed in birds as a result of partial depopulation (thinning) within houses, irrespective of heating system type used (P≤0.05). Stress associated with thinning may have consequences for bird welfare and food safety. In conclusion, the results of our study suggest that indirect heating systems do not appear to negatively impact on flock performance, stress response, within-flock Campylobacter levels or mean Campylobacter counts and do not appear to significantly alter environmental conditions within broiler houses when compared to houses equipped with direct heating systems. Indirect systems are a viable alternative for heating broiler houses in terms of flock performance, bird welfare and food safety.
    • An investigation of the ecological niches and seasonal nature of Clostridium estertheticum and Clostridium gasigenes in the Irish beef farm environment

      Esteves, Eden; Whyte, Paul; Gupta, Tanushree B; Bolton, Declan; Teagasc Walsh Fellowship Programme; 6910NF (Wiley, 2020-07)
      Blown pack spoilage (BPS) of vacuum packaged beef is caused by psychrotolerant and psychrophilic Clostridium species (PPC), primarily Clostridium estertheticum and Clostridium gasigenes. The aim of this study was to investigate the environmental niches and impact of season on these BPS Clostridium spp. on Irish beef farms. On each of 5 different beef farms, faecal (10), soil (5), silage (5), bedding straw (5), drinking water (5), puddle/ditch water (5) and air (5) samples were collected during Spring, Summer, Autumn and Winter and tested for C. estertheticum and C. gasigenes using culture (direct plating and enrichment) and molecular, (conventional PCR and quantitative PCR (qPCR)), based techniques. C. estertheticum and C. gasigenes were detected in all sample types, with qPCR detection rates ranging from 4% to 50% and at concentrations of up to 1.5 log10 cfu g‐1 and 3.5 log10 cfu g‐1, respectively. The impact of season was not clear as the results were mixed depending on the detection method used. It was concluded that BPS causing C. estertheticum and C. gasigenes are widely distributed in the beef farm environment.
    • Molecular Epidemiology of Campylobacter Isolates from Poultry Production Units in Southern Ireland

      O'Mahony, Emer; Buckley, James F.; Bolton, Declan; Whyte, Paul; Fanning, Seamus; Safefood; 04-RESR-04 (PLOS, 2011-12-06)
      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.
    • Ranking hazards pertaining to human health concerns from land application of anaerobic digestate

      Nag, Rajat; Whyte, Paul; Markey, Bryan K.; O'Flaherty, Vincent; Bolton, Declan; Fenton, Owen; Richards, Karl G.; Cummins, Enda; Department of Agriculture, Food and the Marine; 14/SF/847 (Elsevier BV, 2020-03)
      Anaerobic digestion (AD) has been identified as one of the cleanest producers of green energy. AD typically uses organic materials as feedstock and, through a series of biological processes, produces methane. Farmyard manure and slurry (FYM&S) are important AD feedstock and are typically mixed with agricultural waste, grass and/or food wastes. The feedstock may contain many different pathogens which can survive the AD process and hence also possibly be present in the final digestate. In this study, a semi-quantitative screening tool was developed to rank pathogens of potential health concern emerging from AD digestate. A scoring system was used to categorise likely inactivation during AD, hazard pathways and finally, severity as determined from reported human mortality rates, number of global human-deaths and infections per 100,000 populations. Five different conditions including mesophilic and thermophilic AD and three different pasteurisation conditions were assessed in terms of specific pathogen inactivation. In addition, a number of scenarios were assessed to consider foodborne incidence data from Ireland and Europe and to investigate the impact of raw FYM&S application (without AD and pasteurisation). A sensitivity analysis revealed that the score for the mortality rate (S3) was the most sensitive parameter (rank coefficient 0.49) to influence the final score S; followed by thermal inactivation score (S1, 0.25) and potential contamination pathways (S2, 0.16). Across all the scenarios considered, the screening tool prioritised Cryptosporidium parvum, Salmonella spp., norovirus, Streptococcus pyogenes, enteropathogenic E. coli (EPEC), Mycobacterium spp., Salmonella typhi (followed by S. paratyphi), Clostridium spp., Listeria monocytogenes and Campylobacter coli as the highest-ranking pathogens of human health concern resulting from AD digestate in Ireland. This tool prioritises potentially harmful pathogens which can emerge from AD digestate and highlights where regulation and intervention may be required.
    • Spoilage indicator bacteria in farmed Atlantic salmon (Salmo salar) stored on ice for 10 days

      Fogarty, Colin; Whyte, Paul; Brunton, Nigel; Lyng, James; Smyth, Conor; Fagan, John; Bolton, Declan; Department of Agriculture, Food and the Marine; 13F458 (Elsevier, 2018-08-02)
      This study investigated the growth of indicator and spoilage bacteria on whole Atlantic salmon (Salmo salar) stored aerobically at 2 °C. On days 0, 2, 3, 6, 8 and 10 microbiological analysis was carried out on inner flesh and outer skin samples as well as outer skin swabs (25 cm2 surface areas). Mesophilic total viable counts (TVCm) on skin, flesh and swab samples increased from 1.9, 1.1 and 2.7 log10 CFUcm2 to 6.0, 5.1 and 5.7 log10 CFU/cm2 after 10 days, respectively. Psychrotrophic counts (TVCp), increased from 2.2, 1.8 and 3.1 log10 CFU/cm2 to 6.2, 5.3 and 5.9 log10 CFU/cm2, for skin, flesh and swab samples respectively. Hydrogen sulphide producing bacteria (HSPB), lactic acid bacteria (LAB), Pseudomonas spp., Brochothrix thermosphacta and Photobacterium spp. grew well with similar growth rates (mean generation times of 17.2–26 h). It was concluded that the shelf-life of salmon at 2 °C was approximately 10 days and that HSPB, LAB, Pseudomonas spp., Br. thermosphacta and Photobacterium spp. may be a better indicator of fish spoilage rather than TVC growth, with a count of 5–6 log10 CFU/cm2 indicating the end of shelf-life.