The few studies that have evaluated hygiene routines in farrowing accommodation to date have focu... more The few studies that have evaluated hygiene routines in farrowing accommodation to date have focused on pathogen elimination from pens, with little attention paid to pig growth and no information provided on pig health or medication usage. This study aimed to determine if implementation of an optimized farrowing accommodation hygiene routine could improve pig health and growth and reduce medication usage pre- and post-weaning (PW). Forty seven sows were blocked on parity, previous litter size and body weight and assigned to two treatments: T1) Basic hygiene: cold water washing only with minimal drying time; T2) Optimized hygiene: use of detergent and a chlorocresol-based disinfectant with a 6-d drying time. Total bacterial counts (TBC), Enterobacteriaceae counts and adenosine triphosphate (ATP) swabs were obtained from different areas within the farrowing pens. Pig growth and medication usage were monitored from birth to slaughter and carcass data were obtained at slaughter. On entry of sows to the farrowing pens, TBC and Enterobacteriaceae counts and ATP concentrations were lower on pen surfaces subjected to the optimized compared to the basic hygiene routine (P < 0.05). Pre-weaning diarrhea prevalence was lower in pigs born into optimal compared to basic hygiene pens (0 vs. 22%; P < 0.001). The number of clinical cases of disease and injections administered to piglets per litter was 75% and 79% less for the optimized compared to the basic hygiene routine, respectively (P < 0.001). This led to reductions of 77% (P < 0.001) and 75% (P < 0.01), respectively in the volume of antibiotics and anti-inflammatories administered per litter in the optimized hygiene group. Pigs from the optimized hygiene treatment were also heavier at weaning (P < 0.01) and their average daily gain (ADG) was higher from day 21 to weaning and days 22 to 49 PW (P < 0.05). However, these growth improvements did not carry through to the finisher period. In conclusion, implementation of an optimized hygiene routine reduced the bacterial load in farrowing pens, leading to a reduction in diarrhea and clinical cases of disease and therefore, medication usage, in suckling pigs. Pig growth was also improved during the suckling and early PW periods. Based on the results, an easily implementable farrowing room hygiene protocol with demonstrable benefits for pig health, growth, and welfare can be provided to farmers.
Nineteen Bacillus licheniformis strains and four strains of the closely related species Bacillus ... more 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.
Chlorate is a residue of concern in the dairy industry and is linked to cleaning-in-place strateg... more Chlorate is a residue of concern in the dairy industry and is linked to cleaning-in-place strategies that utilise chlorinated chemicals. Chlorine-free chemicals were adopted to minimise chlorate occurrence, but little is known about their performance. To address this, samples from single batches of commercially produced rennet casein powder and salted sweet cream butter were taken throughout their manufacturing processes. Manufacturing equipment was cleaned using chlorine-free chemicals. Samples were analysed for chlorate and total bacteria, thermoduric and spore former counts with 16S sequencing conducted on spore formers. Chlorate levels were 0.026 mg kg−1 in powder but were undetectable (<0.01 mg kg−1) in butter. Bacterial counts from butter and powder processes were ≤2.00 log10 cfu mL−1 and ≤4.00 log10 cfu mL−1, respectively. Spore formers were predominantly identified as Bacillus. This study demonstrates that satisfactory bacterial levels are achievable where chlorine-free chemicals are used for cleaning.
Sequential heating processes are commonly applied to milk by the dairy industry as part of their ... more Sequential heating processes are commonly applied to milk by the dairy industry as part of their microbiological control strategy. Often pasteurisation at 72 C is followed by a sequential high heat treatment step of up to 125 C; however, such severe heat treatment can lead to reduced protein quality. Nine temperature combinations (80e90 C) were evaluated to assess microbial reduction and whey protein nitrogen index values during pilot scale milk processing. A total of 110 bacterial isolates were identified to species level by 16S rDNA sequencing, with Bacillus licheniformis identified as the dominant species. While the experimental treatments did not achieve microbial reductions comparable with the control heating process, the results of this study provide a benchmark for milk processors relative to the effects of sequential heat treatments on milk and their impact on the survival of both thermally resistant microbial populations and thermally labile milk components during processing.
The different customer and regulatory specifications for mesophilic and thermophilic aerobic and ... more The different customer and regulatory specifications for mesophilic and thermophilic aerobic and anaerobic spore numbers in skim-milk powder, in addition to some specifications on specific sporeforming bacteria, such as Bacillus cereus, can be challenging for the industry to meet. Twenty-two samples of medium-heat skim-milk spray-dried powder from eight sources were analysed in triplicate with 16 bacterial and spore enumeration tests to understand the variety of spore-forming bacteria population. Using 16S rDNA sequencing, the species were identified for 269 isolates that were representative of the various tests. Of the isolates identified, 68% were Bacillus licheniformis, a facultative anaerobe that can survive and grow at mesophilic and thermophilic temperatures, making it difficult to eliminate in manufacturing environments. Using whole genome sequencing, 16 of 23 isolates identified as B. licheniformis by 16S sequencing were confirmed as B. licheniformis, four were identified as Bacillus paralicheniformis and three were identified as Bacillus sp. H15-1.
In the ‘One Health’ concept, animal health is linked to human health, through the matrix of food ... more In the ‘One Health’ concept, animal health is linked to human health, through the matrix of food (including the food-processing environment). Application of molecular methods for pathogen analysis of food is, therefore, important in the ‘One Health’ approach. This analysis can involve direct analysis of food for pathogens, using a combination of traditional and molecular methods, or application of molecular methods to characterisation of pathogenic bacteria isolated. Molecular methods offer many advantages in terms of specificity, sensitivity (under certain conditions), time-to-result and in characterisation of isolates, for example with whole genome sequencing. However, it is important to be aware of the current limitations of molecular methods used in food analysis. In this review, the advantages and disadvantages of molecular methods for analysis of food and of pathogens isolated from food are discussed with particular focus on the opportunities and current limitations of such methods.
The problem of assessing the occurrence of the food-borne pathogen Listeria monocytogenes in the ... more The problem of assessing the occurrence of the food-borne pathogen Listeria monocytogenes in the food chain, and therefore the risk of exposure of the human population, is often challenging because of the limited scope of some studies. In this study the occurrence of L. monocytogenes in food from four major food groups, dairy products , meats, seafood and vegetables, and associated food processing environments in Ireland was studied over a three-year period. Fifty-four small food businesses participated in the study and sent both food and environmental samples every 2 months between 2013 and 2015. L. monocytogenes was isolated using the ISO11290 standard method. Confirmation of L. monocytogenes and identification of serogroups were achieved using a multiplex PCR assay, and for some isolates serotype was determined using commercial antisera. Pulsed-field gel electrophoresis (PFGE) analysis was performed on all isolates allowing the relatedness of isolates from different food businesses to be compared nationwide. In total, 86 distinct pulsotypes were identified. The overall occurrence of L. monocytogenes in food samples was 4.2%, while in environmental samples it was 3.8%. In general, the occurrence of L. monocytogenes in food businesses decreased over the course of the study, presumably reflecting increased awareness and vigilance. The majority of the pulsotypes detected were unique to a particular food group (63/ 86), while only three pulsotypes were found in all four food groups investigated. The highest occurrence in food was found in the meat category (7.5%) while seafood had the lowest rate of occurrence (1.8%). Seventeen of the pulsotypes detected in the study were persistent, where persistence was defined as repeated isolation from a single facility with a minimum time interval of 6 months. Using PFGE, 11 of the pulsotypes identified in this study were indistinguishable from those of 11 clinical isolates obtained from patients in Ireland over the last 4 years, highlighting the fact that these pulsotypes are capable of causing disease. Overall, the study shows the diversity of L. monocytogenes strains in the Irish food chain and highlights the ability of many of these strains to persist in food processing environments. The finding that a significant proportion of these pulsotypes are also found in clinical settings highlights the need for continued vigilance by food producers, including frequent sampling and typing of isolates detected.
Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a relatively rare gastroi... more Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a relatively rare gastrointestinal disease with a high mortality rate. As L. monocytogenes is ubiquitous in the environment, control of the organism in food processing environments is necessary to prevent cross-contamination of the food from the environment. In this study, the outcomes of a survey of management practices at 32 food processing facilities were statistically correlated with the occurrence and persistence of L. monocytogenes using multiple regression analyses and logistic regression analysis. No management practices correlated with persistence, while training of staff by management or external trainers, and separation of hygiene control areas correlated with a reduction in L. monocytogenes occurrence. The importance of management practices in reduction of L. monocytogenes in food processing environments was identified. Wider implementation of these practices could contribute to reduction in the occurrence of L. monocytogenes in food processing environments, facilitating production of safer food.
Purpose of Review Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a relat... more Purpose of Review Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a relatively rare but potentially fatal disease with a 19% mortality rate and a 99% hospitalisation rate. It affects mainly elderly and immunocompromised individuals. Ready-to-eat (RTE) foods are particularly dangerous with regard to L. monocytogenes as there is no further anti-microbial step between production and consumption. The purpose of this work is to review the importance of Listeria monocytogenes in the food processing environment. Recent Findings Cross-contamination from the processing environment to the food at production or at retail level is the most common route of RTE food contamination. If present on a food matrix, L. monocytogenes has a remarkable ability to survive and can grow during refrigeration to sufficient numbers to cause disease. Summary While hygiene processes and awareness can help control of L. monocytogenes in food processing environments, new methods such as bacteriophages and bacteriocins are being applied to control it in food, reducing public health issues.
Abstract: Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a relatively ra... more Abstract: Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a relatively rare, but potentially fatal, disease, with a mortality rate of 20–30%. In general, European Regulations require the absence of L. monocytogenes in five samples of 25 g before the food has left the producer, but if the food has been demonstrated not to support the growth of L. monocytogenes, up to 100 cfu g-1 are allowed in the food (except for foods for infants or medical purposes) during its shelf-life under reasonably foreseeable storage conditions. It is important for food producers to determine if their food supports the growth of L. monocytogenes. The European Union Reference Laboratory for L. monocytogenes published a Technical Guidance document for conducting shelf-life studies on L. monocytogenes in ready-to-eat foods in June 2014. Primarily based on the EURL guidance document for conducting challenge studies, the ability of cheese (feta and soft goat’s milk cheese), cold-smoked salmon, coleslaw, and pork pate to support the growth of L. monocytogenes was determined using a starting inoculum of approximately 100 cfu g−1. The cheese and pork pate were incubated at 8 °C for 14 days; the smoked salmon was incubated at 6 °C for 5 days and 8°C for 9 days; and the coleslaw was incubated at 8 °C for 7 days and 12 °C for 14 days. The results showed that the smoked salmon and pork pate supported growth, while coleslaw and cheese did not. From this study, it is evident that there are factors in food other than pH, water activity, and total bacterial count (TBC) that can inhibit the ability of L. monocytogenes to grow in food.
Listeria monocytogenes is a virulent pathogen that is the causative agent of listeriosis, a rare ... more Listeria monocytogenes is a virulent pathogen that is the causative agent of listeriosis, a rare and potentially fatal foodborne disease. The aim of this study was to assess the behaviour of L. monocytogenes in milk obtained from a cow with a sub-clinical infection, representing naturally contaminated milk. Within four hours of milking, the inhibitors Nisin V and pediocin from Pediococcus acidilactici, were added (at a concentration previously found to be inhibitory) to this naturally contaminated milk. The naturally contaminated milk was also pre-chilled at 4 C for 24 h and then the inhibitors added. All the milk samples were incubated at 30 C for 10 h. Samples were taken at hourly intervals for pH measurement and L. monocytogenes enumeration. The naturally contaminated milk was used to manufacture a laboratory-scale semi-soft cheese, in triplicate, using fresh and frozen starter cultures, and ripened at 15 C for 3 weeks. Using an isolated strain from the naturally contaminated milk, growth in milk and during cheesemaking and ripening was also studied. Increasing numbers of L. monocytogenes were measured in the naturally contaminated milk, even in the presence of the bacteriocins. The chilled naturally contaminated milk had a higher initial number of L. monocytogenes than the freshly analysed milk. This implies that in the naturally contaminated milk used in this study, 'increasing numbers' of L. monocytogenes was uncoupled from 'growth'; the increasing numbers may be due to breaking of clumps of cells in the naturally contaminated milk directly from the cow into individual cells, implying growth. Liquid starter culture of direct vat starter culture had little impact on the behaviour of L. monocytogenes during cheesemaking or ripening, the final numbers being the same in the ripened cheese as in the starting milk. The study contributes to risk assessment of naturally contaminated milk with respect to L. monocytogenes.
Irish Journal of Agricultural and Food Research, 2017
Listeria monocytogenes is a foodborne pathogen that causes a relatively rare foodborne disease ca... more Listeria monocytogenes is a foodborne pathogen that causes a relatively rare foodborne disease called listeriosis, with a high mortality rate of 20%-30% and an undefined dose response. Current European Union regulations permit up to 100 colony-forming units (cfu)/g in food at the end of its shelf life, where the food has been shown not to support the growth of this pathogenic bacterium. Therefore, enumeration of L. monocytogenes at low numbers in food is important. The objective of this study was to reduce the detection limit of L. monocytogenes in food by a factor of 10. The International Organisation for Standardisation (ISO) 11290-2 method for enumeration of L. monocytogenes in food recommends spreading 0.1 mL of a 1:10 dilution of the food on the surface of an agar plate (detection limit 100 cfu/g), or 1.0 mL spread in equal parts on the surface of three agar plates (detection limit: 10 cfu/g). The pour-plate method (using 1 or 10 mL of an appropriate dilution) was compared to the spread-plate method using the ISO-approved chromogenic medium Agar Listeria according to Ottaviani and Agosti (ALOA). Using the pour-plate method, the colony morphology and halo formation were similar to the spread-plate method from pure cultures and inoculated foods. Using the pour-plate method in a 140 mm Petri dish, 10 mL of a 1:10 dilution of food allowed determination of numbers as low as 1 cfu/g. Applying this method, L. monocytogenes in naturally contaminated food samples were enumerated at numbers as low as 1-9 cfu/g. Keywords ALOA agar • Listeria monocytogenes • low bacterial numbers • microbial detection limits in food • pour plate
Listeria monocytogenes is a foodborne pathogen and is the causative agent of listeriosis among hu... more Listeria monocytogenes is a foodborne pathogen and is the causative agent of listeriosis among humans and animals. The draft genome sequence of L. monocytogenes DPC6895, a serotype 1/2b strain isolated from the raw milk of a cow with subclinical bovine mastitis, is reported.
During routine sampling of bulk raw milk on a dairy farm, the pathogenic bacteria Listeria monocy... more During routine sampling of bulk raw milk on a dairy farm, the pathogenic bacteria Listeria monocytogenes was found to be a contaminant, at numbers < 100 cfu/ml. A strain with an indistinguishable pulsed-field gel electrophoresis pattern was isolated from the bulk milk two months later. Environmental swabs taken at the dairy environment were negative for the presence of L. monocytogenes, indicating a possible case of excretion of the L. monocytogenes directly into the milk. Milk samples were collected from the individual cows and analysed, resulting in the identification of L. monocytogenes excretion (at 280 cfu/ml) from one of the 4 mammary quarters of one dairy cow out of 180. When the infected cow was isolated from the herd, no L. monocytogenes was detected from the remaining herd. The pulsed-field gel electrophoresis pattern of the strain from the individual cow was indistinguishable from that originally isolated from the bulk milk. The infected cow did not show any clinical signs of disease, nor did the appearance of the milk have any physical abnormalities. Antibiotic treatment of the infected mammary quarter was found to be ineffective. This study shows that there can be risks associated with direct contamination of raw milk with L. monocytogenes.
Staphylococcal enterotoxin type C (SEC) is one of the classical enterotoxins implicated in staphy... more Staphylococcal enterotoxin type C (SEC) is one of the classical enterotoxins implicated in staphylococcal food poisoning. Staphylococcus aureus strains isolated from bovine unpasteurised milk and associated cheese samples produced the SECbovine sub-type. The objectives of this study were to determine the cell numbers required for SECbovine production in milk and to determine if SECbovine was produced by S. aureus during cheesemaking. To predict the point at which toxin production begins, SEC production was modelled against cell numbers of S. aureus (at different controlled pH/temperature combinations) in sterile reconstituted milk using a biphasic model fitted using the Solver routine in Excel. Under the conditions tested, the average cell number required for SECbovine production was log 8.43 ± 0.39 in sterile reconstituted milk. A semi-soft cheese was manufactured using milk inoculated with either washed (to remove any pre-formed toxin) or unwashed (containing toxin) S. aureus at 105 and 107 cfu.mL−1. An enzyme-linked immunosorbent assay was used to quantify toxin production and Baird-Parker agar to quantify S. aureus. Cheese made with washed and unwashed cells showed a one log increase in S. aureus cell numbers during cheesemaking. In cheese made with washed cells, no SEC was detected. With unwashed cells, the SEC concentration remained constant throughout cheesemaking. The results show that SECbovine is not formed in milk or cheese at <108 cfu.mL−1, and therefore, the risk-associated with SECbovine in cheese is low.
Toxin-producing Staphylococcus aureus can be present in raw milk and therefore in cheese made fro... more Toxin-producing Staphylococcus aureus can be present in raw milk and therefore in cheese made from raw milk. To determine the number and type of toxin producers in raw milk used for raw milk cheese production in Ireland, 117 samples of raw milk and related products from five raw milk suppliers, to four raw milk cheesemakers in the South of Ireland, were analysed for coagulase positive S. aureus . Enumeration, using ISO 688-2 and plating on Baird Parker Rabbit Plasma Fibrinogen selective agar showed samples were within limits set by EC regulations. Isolates (151 from 81 positive samples) were characterised for production of staphylococcal enterotoxins (SEs) SEA, SEB, SEC and SED by reverse passive latex agglutination (SET-RPLA) and by multiplex polymerase chain reaction for the sea , seb , sec , sed and see genes. The results showed 83.2% of the isolates did not contain the se genes or the toxin producing capability tested for. From only one supplier, 26 isolates contained the sec gene and produced SEC. Within these 26 isolates, there were only two PFGE types. One SEC-producing isolate showed no toxin production when grown in sterile 10% reconstituted skim milk at 10 °C and 12 °C for 96 and 74 h, respectively. Low concentrations of SEC were produced at 14 °C and 16 °C after 74 and 55 h, respectively. The results of this survey indicate that milk used for raw milk cheese production in Ireland poses a limited risk to public health, although further studies on occurrence of toxin producing S. aureus should be undertaken.
Sixteen cheesemaking facilities were sampled during the production season at monthly intervals ov... more Sixteen cheesemaking facilities were sampled during the production season at monthly intervals over a twoyear period. Thirteen facilities were found to have samples positive for Listeria monocytogenes. Samples were divided into 4 categories; cheese, raw milk, processing environment and external to the processing environment (samples from the farm such as silage, bedding, and pooled water). In order to attempt to identify the source, persistence and putative transfer routes of contamination with the L. monocytogenes isolates, they were differentiated using PFGE and serotyping. Of the 250 isolates, there were 52 different pulsotypes. No pulsotype was found at more than one facility. Two facilities had persistent pulsotypes that were isolated on sampling occasions at least 6 months apart. Of the samples tested, 6.3% of milk, 13.1% of processing environment and 12.3% of samples external to the processing environment, respectively, were positive for L. monocytogenes. Pulsotypes found in raw milk were also found in the processing environment, however, one of the pulsotypes from raw milk was found in cheese on only one occasion. One of the pulsotypes isolated from the environment external to the processing facility was found on the surface of cheese, however, a number of them were found in the processing environment. The results suggest that the farm environment external to the processing environment may in some cases be the source of processing environment contamination with L. monocytogenes.
Food safety is a critical factor in the production of farmhouse cheese. In Ireland the varieties ... more Food safety is a critical factor in the production of farmhouse cheese. In Ireland the varieties of farmhouse cheese produced reflect a much broader range than those produced commercially and some of these cheese varieties are associated with greater microbiological risk. These include cheese produced from unpasteurised milk and soft ripened cheese such as mould or smear-ripened cheeses which have high pH and relatively short ripening times. The aim of this study was to determine the microbiological quality of farmhouse cheeses in Ireland.
The few studies that have evaluated hygiene routines in farrowing accommodation to date have focu... more The few studies that have evaluated hygiene routines in farrowing accommodation to date have focused on pathogen elimination from pens, with little attention paid to pig growth and no information provided on pig health or medication usage. This study aimed to determine if implementation of an optimized farrowing accommodation hygiene routine could improve pig health and growth and reduce medication usage pre- and post-weaning (PW). Forty seven sows were blocked on parity, previous litter size and body weight and assigned to two treatments: T1) Basic hygiene: cold water washing only with minimal drying time; T2) Optimized hygiene: use of detergent and a chlorocresol-based disinfectant with a 6-d drying time. Total bacterial counts (TBC), Enterobacteriaceae counts and adenosine triphosphate (ATP) swabs were obtained from different areas within the farrowing pens. Pig growth and medication usage were monitored from birth to slaughter and carcass data were obtained at slaughter. On entry of sows to the farrowing pens, TBC and Enterobacteriaceae counts and ATP concentrations were lower on pen surfaces subjected to the optimized compared to the basic hygiene routine (P < 0.05). Pre-weaning diarrhea prevalence was lower in pigs born into optimal compared to basic hygiene pens (0 vs. 22%; P < 0.001). The number of clinical cases of disease and injections administered to piglets per litter was 75% and 79% less for the optimized compared to the basic hygiene routine, respectively (P < 0.001). This led to reductions of 77% (P < 0.001) and 75% (P < 0.01), respectively in the volume of antibiotics and anti-inflammatories administered per litter in the optimized hygiene group. Pigs from the optimized hygiene treatment were also heavier at weaning (P < 0.01) and their average daily gain (ADG) was higher from day 21 to weaning and days 22 to 49 PW (P < 0.05). However, these growth improvements did not carry through to the finisher period. In conclusion, implementation of an optimized hygiene routine reduced the bacterial load in farrowing pens, leading to a reduction in diarrhea and clinical cases of disease and therefore, medication usage, in suckling pigs. Pig growth was also improved during the suckling and early PW periods. Based on the results, an easily implementable farrowing room hygiene protocol with demonstrable benefits for pig health, growth, and welfare can be provided to farmers.
Nineteen Bacillus licheniformis strains and four strains of the closely related species Bacillus ... more 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.
Chlorate is a residue of concern in the dairy industry and is linked to cleaning-in-place strateg... more Chlorate is a residue of concern in the dairy industry and is linked to cleaning-in-place strategies that utilise chlorinated chemicals. Chlorine-free chemicals were adopted to minimise chlorate occurrence, but little is known about their performance. To address this, samples from single batches of commercially produced rennet casein powder and salted sweet cream butter were taken throughout their manufacturing processes. Manufacturing equipment was cleaned using chlorine-free chemicals. Samples were analysed for chlorate and total bacteria, thermoduric and spore former counts with 16S sequencing conducted on spore formers. Chlorate levels were 0.026 mg kg−1 in powder but were undetectable (<0.01 mg kg−1) in butter. Bacterial counts from butter and powder processes were ≤2.00 log10 cfu mL−1 and ≤4.00 log10 cfu mL−1, respectively. Spore formers were predominantly identified as Bacillus. This study demonstrates that satisfactory bacterial levels are achievable where chlorine-free chemicals are used for cleaning.
Sequential heating processes are commonly applied to milk by the dairy industry as part of their ... more Sequential heating processes are commonly applied to milk by the dairy industry as part of their microbiological control strategy. Often pasteurisation at 72 C is followed by a sequential high heat treatment step of up to 125 C; however, such severe heat treatment can lead to reduced protein quality. Nine temperature combinations (80e90 C) were evaluated to assess microbial reduction and whey protein nitrogen index values during pilot scale milk processing. A total of 110 bacterial isolates were identified to species level by 16S rDNA sequencing, with Bacillus licheniformis identified as the dominant species. While the experimental treatments did not achieve microbial reductions comparable with the control heating process, the results of this study provide a benchmark for milk processors relative to the effects of sequential heat treatments on milk and their impact on the survival of both thermally resistant microbial populations and thermally labile milk components during processing.
The different customer and regulatory specifications for mesophilic and thermophilic aerobic and ... more The different customer and regulatory specifications for mesophilic and thermophilic aerobic and anaerobic spore numbers in skim-milk powder, in addition to some specifications on specific sporeforming bacteria, such as Bacillus cereus, can be challenging for the industry to meet. Twenty-two samples of medium-heat skim-milk spray-dried powder from eight sources were analysed in triplicate with 16 bacterial and spore enumeration tests to understand the variety of spore-forming bacteria population. Using 16S rDNA sequencing, the species were identified for 269 isolates that were representative of the various tests. Of the isolates identified, 68% were Bacillus licheniformis, a facultative anaerobe that can survive and grow at mesophilic and thermophilic temperatures, making it difficult to eliminate in manufacturing environments. Using whole genome sequencing, 16 of 23 isolates identified as B. licheniformis by 16S sequencing were confirmed as B. licheniformis, four were identified as Bacillus paralicheniformis and three were identified as Bacillus sp. H15-1.
In the ‘One Health’ concept, animal health is linked to human health, through the matrix of food ... more In the ‘One Health’ concept, animal health is linked to human health, through the matrix of food (including the food-processing environment). Application of molecular methods for pathogen analysis of food is, therefore, important in the ‘One Health’ approach. This analysis can involve direct analysis of food for pathogens, using a combination of traditional and molecular methods, or application of molecular methods to characterisation of pathogenic bacteria isolated. Molecular methods offer many advantages in terms of specificity, sensitivity (under certain conditions), time-to-result and in characterisation of isolates, for example with whole genome sequencing. However, it is important to be aware of the current limitations of molecular methods used in food analysis. In this review, the advantages and disadvantages of molecular methods for analysis of food and of pathogens isolated from food are discussed with particular focus on the opportunities and current limitations of such methods.
The problem of assessing the occurrence of the food-borne pathogen Listeria monocytogenes in the ... more The problem of assessing the occurrence of the food-borne pathogen Listeria monocytogenes in the food chain, and therefore the risk of exposure of the human population, is often challenging because of the limited scope of some studies. In this study the occurrence of L. monocytogenes in food from four major food groups, dairy products , meats, seafood and vegetables, and associated food processing environments in Ireland was studied over a three-year period. Fifty-four small food businesses participated in the study and sent both food and environmental samples every 2 months between 2013 and 2015. L. monocytogenes was isolated using the ISO11290 standard method. Confirmation of L. monocytogenes and identification of serogroups were achieved using a multiplex PCR assay, and for some isolates serotype was determined using commercial antisera. Pulsed-field gel electrophoresis (PFGE) analysis was performed on all isolates allowing the relatedness of isolates from different food businesses to be compared nationwide. In total, 86 distinct pulsotypes were identified. The overall occurrence of L. monocytogenes in food samples was 4.2%, while in environmental samples it was 3.8%. In general, the occurrence of L. monocytogenes in food businesses decreased over the course of the study, presumably reflecting increased awareness and vigilance. The majority of the pulsotypes detected were unique to a particular food group (63/ 86), while only three pulsotypes were found in all four food groups investigated. The highest occurrence in food was found in the meat category (7.5%) while seafood had the lowest rate of occurrence (1.8%). Seventeen of the pulsotypes detected in the study were persistent, where persistence was defined as repeated isolation from a single facility with a minimum time interval of 6 months. Using PFGE, 11 of the pulsotypes identified in this study were indistinguishable from those of 11 clinical isolates obtained from patients in Ireland over the last 4 years, highlighting the fact that these pulsotypes are capable of causing disease. Overall, the study shows the diversity of L. monocytogenes strains in the Irish food chain and highlights the ability of many of these strains to persist in food processing environments. The finding that a significant proportion of these pulsotypes are also found in clinical settings highlights the need for continued vigilance by food producers, including frequent sampling and typing of isolates detected.
Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a relatively rare gastroi... more Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a relatively rare gastrointestinal disease with a high mortality rate. As L. monocytogenes is ubiquitous in the environment, control of the organism in food processing environments is necessary to prevent cross-contamination of the food from the environment. In this study, the outcomes of a survey of management practices at 32 food processing facilities were statistically correlated with the occurrence and persistence of L. monocytogenes using multiple regression analyses and logistic regression analysis. No management practices correlated with persistence, while training of staff by management or external trainers, and separation of hygiene control areas correlated with a reduction in L. monocytogenes occurrence. The importance of management practices in reduction of L. monocytogenes in food processing environments was identified. Wider implementation of these practices could contribute to reduction in the occurrence of L. monocytogenes in food processing environments, facilitating production of safer food.
Purpose of Review Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a relat... more Purpose of Review Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a relatively rare but potentially fatal disease with a 19% mortality rate and a 99% hospitalisation rate. It affects mainly elderly and immunocompromised individuals. Ready-to-eat (RTE) foods are particularly dangerous with regard to L. monocytogenes as there is no further anti-microbial step between production and consumption. The purpose of this work is to review the importance of Listeria monocytogenes in the food processing environment. Recent Findings Cross-contamination from the processing environment to the food at production or at retail level is the most common route of RTE food contamination. If present on a food matrix, L. monocytogenes has a remarkable ability to survive and can grow during refrigeration to sufficient numbers to cause disease. Summary While hygiene processes and awareness can help control of L. monocytogenes in food processing environments, new methods such as bacteriophages and bacteriocins are being applied to control it in food, reducing public health issues.
Abstract: Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a relatively ra... more Abstract: Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a relatively rare, but potentially fatal, disease, with a mortality rate of 20–30%. In general, European Regulations require the absence of L. monocytogenes in five samples of 25 g before the food has left the producer, but if the food has been demonstrated not to support the growth of L. monocytogenes, up to 100 cfu g-1 are allowed in the food (except for foods for infants or medical purposes) during its shelf-life under reasonably foreseeable storage conditions. It is important for food producers to determine if their food supports the growth of L. monocytogenes. The European Union Reference Laboratory for L. monocytogenes published a Technical Guidance document for conducting shelf-life studies on L. monocytogenes in ready-to-eat foods in June 2014. Primarily based on the EURL guidance document for conducting challenge studies, the ability of cheese (feta and soft goat’s milk cheese), cold-smoked salmon, coleslaw, and pork pate to support the growth of L. monocytogenes was determined using a starting inoculum of approximately 100 cfu g−1. The cheese and pork pate were incubated at 8 °C for 14 days; the smoked salmon was incubated at 6 °C for 5 days and 8°C for 9 days; and the coleslaw was incubated at 8 °C for 7 days and 12 °C for 14 days. The results showed that the smoked salmon and pork pate supported growth, while coleslaw and cheese did not. From this study, it is evident that there are factors in food other than pH, water activity, and total bacterial count (TBC) that can inhibit the ability of L. monocytogenes to grow in food.
Listeria monocytogenes is a virulent pathogen that is the causative agent of listeriosis, a rare ... more Listeria monocytogenes is a virulent pathogen that is the causative agent of listeriosis, a rare and potentially fatal foodborne disease. The aim of this study was to assess the behaviour of L. monocytogenes in milk obtained from a cow with a sub-clinical infection, representing naturally contaminated milk. Within four hours of milking, the inhibitors Nisin V and pediocin from Pediococcus acidilactici, were added (at a concentration previously found to be inhibitory) to this naturally contaminated milk. The naturally contaminated milk was also pre-chilled at 4 C for 24 h and then the inhibitors added. All the milk samples were incubated at 30 C for 10 h. Samples were taken at hourly intervals for pH measurement and L. monocytogenes enumeration. The naturally contaminated milk was used to manufacture a laboratory-scale semi-soft cheese, in triplicate, using fresh and frozen starter cultures, and ripened at 15 C for 3 weeks. Using an isolated strain from the naturally contaminated milk, growth in milk and during cheesemaking and ripening was also studied. Increasing numbers of L. monocytogenes were measured in the naturally contaminated milk, even in the presence of the bacteriocins. The chilled naturally contaminated milk had a higher initial number of L. monocytogenes than the freshly analysed milk. This implies that in the naturally contaminated milk used in this study, 'increasing numbers' of L. monocytogenes was uncoupled from 'growth'; the increasing numbers may be due to breaking of clumps of cells in the naturally contaminated milk directly from the cow into individual cells, implying growth. Liquid starter culture of direct vat starter culture had little impact on the behaviour of L. monocytogenes during cheesemaking or ripening, the final numbers being the same in the ripened cheese as in the starting milk. The study contributes to risk assessment of naturally contaminated milk with respect to L. monocytogenes.
Irish Journal of Agricultural and Food Research, 2017
Listeria monocytogenes is a foodborne pathogen that causes a relatively rare foodborne disease ca... more Listeria monocytogenes is a foodborne pathogen that causes a relatively rare foodborne disease called listeriosis, with a high mortality rate of 20%-30% and an undefined dose response. Current European Union regulations permit up to 100 colony-forming units (cfu)/g in food at the end of its shelf life, where the food has been shown not to support the growth of this pathogenic bacterium. Therefore, enumeration of L. monocytogenes at low numbers in food is important. The objective of this study was to reduce the detection limit of L. monocytogenes in food by a factor of 10. The International Organisation for Standardisation (ISO) 11290-2 method for enumeration of L. monocytogenes in food recommends spreading 0.1 mL of a 1:10 dilution of the food on the surface of an agar plate (detection limit 100 cfu/g), or 1.0 mL spread in equal parts on the surface of three agar plates (detection limit: 10 cfu/g). The pour-plate method (using 1 or 10 mL of an appropriate dilution) was compared to the spread-plate method using the ISO-approved chromogenic medium Agar Listeria according to Ottaviani and Agosti (ALOA). Using the pour-plate method, the colony morphology and halo formation were similar to the spread-plate method from pure cultures and inoculated foods. Using the pour-plate method in a 140 mm Petri dish, 10 mL of a 1:10 dilution of food allowed determination of numbers as low as 1 cfu/g. Applying this method, L. monocytogenes in naturally contaminated food samples were enumerated at numbers as low as 1-9 cfu/g. Keywords ALOA agar • Listeria monocytogenes • low bacterial numbers • microbial detection limits in food • pour plate
Listeria monocytogenes is a foodborne pathogen and is the causative agent of listeriosis among hu... more Listeria monocytogenes is a foodborne pathogen and is the causative agent of listeriosis among humans and animals. The draft genome sequence of L. monocytogenes DPC6895, a serotype 1/2b strain isolated from the raw milk of a cow with subclinical bovine mastitis, is reported.
During routine sampling of bulk raw milk on a dairy farm, the pathogenic bacteria Listeria monocy... more During routine sampling of bulk raw milk on a dairy farm, the pathogenic bacteria Listeria monocytogenes was found to be a contaminant, at numbers < 100 cfu/ml. A strain with an indistinguishable pulsed-field gel electrophoresis pattern was isolated from the bulk milk two months later. Environmental swabs taken at the dairy environment were negative for the presence of L. monocytogenes, indicating a possible case of excretion of the L. monocytogenes directly into the milk. Milk samples were collected from the individual cows and analysed, resulting in the identification of L. monocytogenes excretion (at 280 cfu/ml) from one of the 4 mammary quarters of one dairy cow out of 180. When the infected cow was isolated from the herd, no L. monocytogenes was detected from the remaining herd. The pulsed-field gel electrophoresis pattern of the strain from the individual cow was indistinguishable from that originally isolated from the bulk milk. The infected cow did not show any clinical signs of disease, nor did the appearance of the milk have any physical abnormalities. Antibiotic treatment of the infected mammary quarter was found to be ineffective. This study shows that there can be risks associated with direct contamination of raw milk with L. monocytogenes.
Staphylococcal enterotoxin type C (SEC) is one of the classical enterotoxins implicated in staphy... more Staphylococcal enterotoxin type C (SEC) is one of the classical enterotoxins implicated in staphylococcal food poisoning. Staphylococcus aureus strains isolated from bovine unpasteurised milk and associated cheese samples produced the SECbovine sub-type. The objectives of this study were to determine the cell numbers required for SECbovine production in milk and to determine if SECbovine was produced by S. aureus during cheesemaking. To predict the point at which toxin production begins, SEC production was modelled against cell numbers of S. aureus (at different controlled pH/temperature combinations) in sterile reconstituted milk using a biphasic model fitted using the Solver routine in Excel. Under the conditions tested, the average cell number required for SECbovine production was log 8.43 ± 0.39 in sterile reconstituted milk. A semi-soft cheese was manufactured using milk inoculated with either washed (to remove any pre-formed toxin) or unwashed (containing toxin) S. aureus at 105 and 107 cfu.mL−1. An enzyme-linked immunosorbent assay was used to quantify toxin production and Baird-Parker agar to quantify S. aureus. Cheese made with washed and unwashed cells showed a one log increase in S. aureus cell numbers during cheesemaking. In cheese made with washed cells, no SEC was detected. With unwashed cells, the SEC concentration remained constant throughout cheesemaking. The results show that SECbovine is not formed in milk or cheese at <108 cfu.mL−1, and therefore, the risk-associated with SECbovine in cheese is low.
Toxin-producing Staphylococcus aureus can be present in raw milk and therefore in cheese made fro... more Toxin-producing Staphylococcus aureus can be present in raw milk and therefore in cheese made from raw milk. To determine the number and type of toxin producers in raw milk used for raw milk cheese production in Ireland, 117 samples of raw milk and related products from five raw milk suppliers, to four raw milk cheesemakers in the South of Ireland, were analysed for coagulase positive S. aureus . Enumeration, using ISO 688-2 and plating on Baird Parker Rabbit Plasma Fibrinogen selective agar showed samples were within limits set by EC regulations. Isolates (151 from 81 positive samples) were characterised for production of staphylococcal enterotoxins (SEs) SEA, SEB, SEC and SED by reverse passive latex agglutination (SET-RPLA) and by multiplex polymerase chain reaction for the sea , seb , sec , sed and see genes. The results showed 83.2% of the isolates did not contain the se genes or the toxin producing capability tested for. From only one supplier, 26 isolates contained the sec gene and produced SEC. Within these 26 isolates, there were only two PFGE types. One SEC-producing isolate showed no toxin production when grown in sterile 10% reconstituted skim milk at 10 °C and 12 °C for 96 and 74 h, respectively. Low concentrations of SEC were produced at 14 °C and 16 °C after 74 and 55 h, respectively. The results of this survey indicate that milk used for raw milk cheese production in Ireland poses a limited risk to public health, although further studies on occurrence of toxin producing S. aureus should be undertaken.
Sixteen cheesemaking facilities were sampled during the production season at monthly intervals ov... more Sixteen cheesemaking facilities were sampled during the production season at monthly intervals over a twoyear period. Thirteen facilities were found to have samples positive for Listeria monocytogenes. Samples were divided into 4 categories; cheese, raw milk, processing environment and external to the processing environment (samples from the farm such as silage, bedding, and pooled water). In order to attempt to identify the source, persistence and putative transfer routes of contamination with the L. monocytogenes isolates, they were differentiated using PFGE and serotyping. Of the 250 isolates, there were 52 different pulsotypes. No pulsotype was found at more than one facility. Two facilities had persistent pulsotypes that were isolated on sampling occasions at least 6 months apart. Of the samples tested, 6.3% of milk, 13.1% of processing environment and 12.3% of samples external to the processing environment, respectively, were positive for L. monocytogenes. Pulsotypes found in raw milk were also found in the processing environment, however, one of the pulsotypes from raw milk was found in cheese on only one occasion. One of the pulsotypes isolated from the environment external to the processing facility was found on the surface of cheese, however, a number of them were found in the processing environment. The results suggest that the farm environment external to the processing environment may in some cases be the source of processing environment contamination with L. monocytogenes.
Food safety is a critical factor in the production of farmhouse cheese. In Ireland the varieties ... more Food safety is a critical factor in the production of farmhouse cheese. In Ireland the varieties of farmhouse cheese produced reflect a much broader range than those produced commercially and some of these cheese varieties are associated with greater microbiological risk. These include cheese produced from unpasteurised milk and soft ripened cheese such as mould or smear-ripened cheeses which have high pH and relatively short ripening times. The aim of this study was to determine the microbiological quality of farmhouse cheeses in Ireland.
PFGE is a valuable tool for assessing L. monocytogenes strain interrelatedness. It is based on th... more PFGE is a valuable tool for assessing L. monocytogenes strain interrelatedness. It is based on the study of total bacterial DNA restriction patterns. Cells are embedded in agarose plugs before being lysed. The released DNA is then digested into large fragments by restriction enzymes. As DNA fragments are too large to be separated by traditional electrophoresis in an agarose gel, changes in the direction of the electrical current are periodically applied in order to allow the proper migration of large DNA fragments. Strains are characterized by the obtained DNA fragment patterns or pulsotypes which vary depending on the number and size of bands.
n book: Case studies in food safety and authenticity: lessons from real-life situations 2012, Edi... more n book: Case studies in food safety and authenticity: lessons from real-life situations 2012, Edition: 2012, Chapter: Troubleshooting the environmental source of contamination with Listeria monocytogenes in a typical small food manufacturing plant in Ireland., Publisher: Woodhead Publishing Ltd, Editors: Hoorfar, J, pp.pp. 95-101
In book: Case studies in food safety and authenticity: lessons from real-life situations, Edition... more In book: Case studies in food safety and authenticity: lessons from real-life situations, Edition: 2012, Chapter: The continuing debate over increasing consumption of raw (unpasteurised) milk: is it safe?, Publisher: Woodhead Publishing Ltd, Editors: Hoorfar, pp.215-221
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Papers by Triona O'Brien
with little attention paid to pig growth and no information provided on pig health or medication usage. This study aimed to determine if implementation of an optimized farrowing accommodation hygiene routine could improve pig health and growth and reduce medication usage pre- and
post-weaning (PW). Forty seven sows were blocked on parity, previous litter size and body weight and assigned to two treatments: T1) Basic
hygiene: cold water washing only with minimal drying time; T2) Optimized hygiene: use of detergent and a chlorocresol-based disinfectant with
a 6-d drying time. Total bacterial counts (TBC), Enterobacteriaceae counts and adenosine triphosphate (ATP) swabs were obtained from different
areas within the farrowing pens. Pig growth and medication usage were monitored from birth to slaughter and carcass data were obtained at
slaughter. On entry of sows to the farrowing pens, TBC and Enterobacteriaceae counts and ATP concentrations were lower on pen surfaces
subjected to the optimized compared to the basic hygiene routine (P < 0.05). Pre-weaning diarrhea prevalence was lower in pigs born into optimal compared to basic hygiene pens (0 vs. 22%; P < 0.001). The number of clinical cases of disease and injections administered to piglets
per litter was 75% and 79% less for the optimized compared to the basic hygiene routine, respectively (P < 0.001). This led to reductions of
77% (P < 0.001) and 75% (P < 0.01), respectively in the volume of antibiotics and anti-inflammatories administered per litter in the optimized
hygiene group. Pigs from the optimized hygiene treatment were also heavier at weaning (P < 0.01) and their average daily gain (ADG) was higher
from day 21 to weaning and days 22 to 49 PW (P < 0.05). However, these growth improvements did not carry through to the finisher period.
In conclusion, implementation of an optimized hygiene routine reduced the bacterial load in farrowing pens, leading to a reduction in diarrhea
and clinical cases of disease and therefore, medication usage, in suckling pigs. Pig growth was also improved during the suckling and early PW
periods. Based on the results, an easily implementable farrowing room hygiene protocol with demonstrable benefits for pig health, growth, and
welfare can be provided to farmers.
potentially fatal, disease, with a mortality rate of 20–30%. In general, European Regulations require
the absence of L. monocytogenes in five samples of 25 g before the food has left the producer, but if
the food has been demonstrated not to support the growth of L. monocytogenes, up to 100 cfu g-1 are
allowed in the food (except for foods for infants or medical purposes) during its shelf-life under
reasonably foreseeable storage conditions. It is important for food producers to determine if their
food supports the growth of L. monocytogenes. The European Union Reference Laboratory for L.
monocytogenes published a Technical Guidance document for conducting shelf-life studies on L.
monocytogenes in ready-to-eat foods in June 2014. Primarily based on the EURL guidance document
for conducting challenge studies, the ability of cheese (feta and soft goat’s milk cheese), cold-smoked
salmon, coleslaw, and pork pate to support the growth of L. monocytogenes was determined using a
starting inoculum of approximately 100 cfu g−1. The cheese and pork pate were incubated at 8 °C for
14 days; the smoked salmon was incubated at 6 °C for 5 days and 8°C for 9 days; and the coleslaw
was incubated at 8 °C for 7 days and 12 °C for 14 days. The results showed that the smoked salmon
and pork pate supported growth, while coleslaw and cheese did not. From this study, it is evident
that there are factors in food other than pH, water activity, and total bacterial count (TBC) that can
inhibit the ability of L. monocytogenes to grow in food.
with little attention paid to pig growth and no information provided on pig health or medication usage. This study aimed to determine if implementation of an optimized farrowing accommodation hygiene routine could improve pig health and growth and reduce medication usage pre- and
post-weaning (PW). Forty seven sows were blocked on parity, previous litter size and body weight and assigned to two treatments: T1) Basic
hygiene: cold water washing only with minimal drying time; T2) Optimized hygiene: use of detergent and a chlorocresol-based disinfectant with
a 6-d drying time. Total bacterial counts (TBC), Enterobacteriaceae counts and adenosine triphosphate (ATP) swabs were obtained from different
areas within the farrowing pens. Pig growth and medication usage were monitored from birth to slaughter and carcass data were obtained at
slaughter. On entry of sows to the farrowing pens, TBC and Enterobacteriaceae counts and ATP concentrations were lower on pen surfaces
subjected to the optimized compared to the basic hygiene routine (P < 0.05). Pre-weaning diarrhea prevalence was lower in pigs born into optimal compared to basic hygiene pens (0 vs. 22%; P < 0.001). The number of clinical cases of disease and injections administered to piglets
per litter was 75% and 79% less for the optimized compared to the basic hygiene routine, respectively (P < 0.001). This led to reductions of
77% (P < 0.001) and 75% (P < 0.01), respectively in the volume of antibiotics and anti-inflammatories administered per litter in the optimized
hygiene group. Pigs from the optimized hygiene treatment were also heavier at weaning (P < 0.01) and their average daily gain (ADG) was higher
from day 21 to weaning and days 22 to 49 PW (P < 0.05). However, these growth improvements did not carry through to the finisher period.
In conclusion, implementation of an optimized hygiene routine reduced the bacterial load in farrowing pens, leading to a reduction in diarrhea
and clinical cases of disease and therefore, medication usage, in suckling pigs. Pig growth was also improved during the suckling and early PW
periods. Based on the results, an easily implementable farrowing room hygiene protocol with demonstrable benefits for pig health, growth, and
welfare can be provided to farmers.
potentially fatal, disease, with a mortality rate of 20–30%. In general, European Regulations require
the absence of L. monocytogenes in five samples of 25 g before the food has left the producer, but if
the food has been demonstrated not to support the growth of L. monocytogenes, up to 100 cfu g-1 are
allowed in the food (except for foods for infants or medical purposes) during its shelf-life under
reasonably foreseeable storage conditions. It is important for food producers to determine if their
food supports the growth of L. monocytogenes. The European Union Reference Laboratory for L.
monocytogenes published a Technical Guidance document for conducting shelf-life studies on L.
monocytogenes in ready-to-eat foods in June 2014. Primarily based on the EURL guidance document
for conducting challenge studies, the ability of cheese (feta and soft goat’s milk cheese), cold-smoked
salmon, coleslaw, and pork pate to support the growth of L. monocytogenes was determined using a
starting inoculum of approximately 100 cfu g−1. The cheese and pork pate were incubated at 8 °C for
14 days; the smoked salmon was incubated at 6 °C for 5 days and 8°C for 9 days; and the coleslaw
was incubated at 8 °C for 7 days and 12 °C for 14 days. The results showed that the smoked salmon
and pork pate supported growth, while coleslaw and cheese did not. From this study, it is evident
that there are factors in food other than pH, water activity, and total bacterial count (TBC) that can
inhibit the ability of L. monocytogenes to grow in food.