Microbiological challenge testing for Listeria monocytogenes in ready-to-eat food: a practical approach

Food business operators (FBOs) are the primary responsible for the safety of food they place on the market. The definition and validation of the product’s shelf-life is an essential part for ensuring microbiological safety of food and health of consumers. In the frame of the Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs, FBOs shall conduct shelf-life studies in order to assure that their food does not exceed the food safety criteria throughout the defined shelf-life. In particular this is required for ready-to-eat (RTE) food that supports the growth of Listeria monocytogenes. Among other studies, FBOs can rely on the conclusion drawn by microbiological challenge tests. A microbiological challenge test consists in the artificial contamination of a food with a pathogen microorganism and aims at simulating its behaviour during processing and distribution under the foreseen storage and handling conditions. A number of documents published by international health authorities and research institutions describes how to conduct challenge studies. The authors reviewed the existing literature and described the methodology for implementing such laboratory studies. All the main aspects for the conduction of L. monocytogenes microbiological challenge tests were considered, from the selection of the strains, preparation and choice of the inoculum level and method of contamination, to the experimental design and data interpretation. The objective of the present document is to provide an exhaustive and practical guideline for laboratories that want to implement L. monocytogenes challenge testing on RTE food

Hygienic and sensory quality factors affecting the shelf-life of Fruhe (Casu axedu) traditional Sardinian fresh cheese

A study was conducted to evaluate the dura- bility of the traditional fresh soft cheese Fruhe manufactured in Sardinia either from goats’ or sheep’s milk. Four farmstead cheese-making plants were visited three times during the Fruhe cheese-making season. During each visit environmental samples were collected from food contact and non-food contact sur- faces in order to evaluate the presence of Enterobacteriaceae, Escherichia coli, Pseudomonas spp. and Listeria spp. In a total of 60 environmental samples, Escherichia coli and Listeria spp. were never detected, while contamination with Enterobacteriaceae and Pseudomonas spp. was observed respectively in 48% and 43% of samples. The microbiological profile of 48 Fruhe cheese samples was assessed at different time points during the product shelf-life. Aerobic mesophilic bacteria, Enterobacteriaceae, E. coli, Pseudomonas spp., Bacillus cereus and Listeria monocytogenes were investigated at 0, 7, 14 and 21 days after production. E. coli, L. monocytogenes and B. cereus were never detected in the product. Enterobacteriaceae contamination was observed, showing decreasing levels over time. Pseudomonas spp. was recovered in only two Fruhe samples (3.3%) at day 0. Sensory analysis was also conducted using a triangle test to determine whether a difference between Fruhe samples at 14 and 21 days of shelf-life exists. Based on the evolution of the microbiological profile and the sensory attributes observed in the present study, it is reasonable to assume that the product shelf-life can be feasibly extended up to 21 days

Listeria monocytogenes contamination in dairy plants: evaluation of Listeria monocytogenes environmental contamination in two cheese-making plants using sheeps milk

Listeria monocytogenes harbouring niches established in the processing plant support post-process contamination of dairy products made from pasteurised or thermised milk. The present study investigated L. monocytogenes environmental contamination in two sheep’s milk cheese-making plants. Persistence of contamination in the area at higher risk was also investigated. During a one-year survey 7 samplings were carried out in each dairy plant, along the production lines of Pecorino Romano and ricotta salata cheese. A total of 613 environmental samples collected from food contact and non-food contact surfaces were analysed according to ISO 11290-1:2005 standard method. Identification of the isolated strains was carried out by polymerase chain reaction. L. monocytogenes prevalence was 23.2% in dairy A and 13.1% in dairy B, respectively. The higher prevalence rate was found in the following areas: salting, products washing, packaging, ricotta salata storage and Pecorino Romano ripening rooms. L. monocytogenes was never found in the cheese-making area. The probability of observing samples positive for the presence of L. monocytogenes was asso- ciated with dairy plant, sampling area and the period of cheese-making (P<0.001). The greater persistence of contamination over time was observed in the washing, salting, and Pecorino Romano ripening areas. The control of persistent environmental contamination relies on the identification of L. monocytogenes niches within the processing environment and the prevention of harborage sites formation. The importance of strict cleaning and sanitising procedure in controlling L. monocytogenes environmental contamination is confirmed by the lower level of contamination observed after these procedures were correctly implemented

Listeria monocytogenes contamination in dairy plants: evaluation of Listeria monocytogenes environmental contamination in two cheese-making plants using sheeps milk

Listeria monocytogenes harbouring niches established in the processing plant support post-process contamination of dairy products made from pasteurised or thermised milk. The present study investigated L. monocytogenes environmental contamination in two sheep’s milk cheese-making plants. Persistence of contamination in the area at higher risk was also investigated. During a one-year survey 7 samplings were carried out in each dairy plant, along the production lines of Pecorino Romano and ricotta salata cheese. A total of 613 environmental samples collected from food contact and non-food contact surfaces were analysed according to ISO 11290-1:2005 standard method. Identification of the isolated strains was carried out by polymerase chain reaction. L. monocytogenes prevalence was 23.2% in dairy A and 13.1% in dairy B, respectively. The higher prevalence rate was found in the following areas: salting, products washing, packaging, ricotta salata storage and Pecorino Romano ripening rooms. L. monocytogenes was never found in the cheese-making area. The probability of observing samples positive for the presence of L. monocytogenes was asso- ciated with dairy plant, sampling area and the period of cheese-making (P<0.001). The greater persistence of contamination over time was observed in the washing, salting, and Pecorino Romano ripening areas. The control of persistent environmental contamination relies on the identification of L. monocytogenes niches within the processing environment and the prevention of harborage sites formation. The importance of strict cleaning and sanitising procedure in controlling L. monocytogenes environmental contamination is confirmed by the lower level of contamination observed after these procedures were correctly implemented

Hygienic and sensory quality factors affecting the shelf-life of Fruhe (Casu axedu) traditional Sardinian fresh cheese

A study was conducted to evaluate the dura- bility of the traditional fresh soft cheese Fruhe manufactured in Sardinia either from goats’ or sheep’s milk. Four farmstead cheese-making plants were visited three times during the Fruhe cheese-making season. During each visit environmental samples were collected from food contact and non-food contact sur- faces in order to evaluate the presence of Enterobacteriaceae, Escherichia coli, Pseudomonas spp. and Listeria spp. In a total of 60 environmental samples, Escherichia coli and Listeria spp. were never detected, while contamination with Enterobacteriaceae and Pseudomonas spp. was observed respectively in 48% and 43% of samples. The microbiological profile of 48 Fruhe cheese samples was assessed at different time points during the product shelf-life. Aerobic mesophilic bacteria, Enterobacteriaceae, E. coli, Pseudomonas spp., Bacillus cereus and Listeria monocytogenes were investigated at 0, 7, 14 and 21 days after production. E. coli, L. monocytogenes and B. cereus were never detected in the product. Enterobacteriaceae contamination was observed, showing decreasing levels over time. Pseudomonas spp. was recovered in only two Fruhe samples (3.3%) at day 0. Sensory analysis was also conducted using a triangle test to determine whether a difference between Fruhe samples at 14 and 21 days of shelf-life exists. Based on the evolution of the microbiological profile and the sensory attributes observed in the present study, it is reasonable to assume that the product shelf-life can be feasibly extended up to 21 days

Prevalence of Type 1 diabetes autoantibodies (GAD and IA2) in Sardinian children and adolescents with autoimmune thyroiditis

AIMS: Type 1 diabetes and autoimmune thyroiditis are common autoimmune diseases characterized by the presence of autoantibodies against tissue-specific components. Non-thyroid-specific autoantibodies are frequent in patients with autoimmune thyroiditis. The prevalence of Type 1 diabetes autoantibodies in patients with autoimmune thyroiditis is unknown. METHODS: The prevalence of Type 1 diabetes autoantibodies (GAD and IA2) was analysed in 236 Sardinian children and adolescents with autoimmune thyroiditis. GAD and IA2 antibodies were measured at the time of the diagnosis of autoimmune thyroiditis and re-evaluated after 1 year in the children who were shown to be positive. Autoantibody prevalence was evaluated in 949 healthy age-matched controls. RESULTS: The prevalence of GAD and/or IA2 antibodies was 8% in the children and adolescents with autoimmune thyroiditis and 4.1% in control subjects (P = 0.017). When Type 1 diabetes autoantibodies were separately analysed, the difference remained significant for IA2 (3.39% in autoimmune thyroiditis vs. 1.16% in control subjects, P = 0.012), but not for GAD (5.1% in autoimmune thyroiditis vs. 3.79% in control subjects, P = 0.367). Seven of 10 children with autoimmune thyroiditis and detectable Type 1 diabetes autoantibodies at the diagnosis remained positive after 1 year. In the course of 2 years of follow-up, two patients who were positive for Type 1 diabetes autoantibodies at the time of diagnosis of autoimmune thyroiditis developed diabetes. CONCLUSIONS: This is the first study reporting the prevalence of Type 1 diabetes autoantibodies in a selected cohort of genetically homogeneous children and adolescents with autoimmune thyroiditis. The main finding was that the prevalence of Type 1 diabetes autoantibodies and of newly diagnosed Type 1 diabetes in patients with autoimmune thyroiditis was significantly higher than that observed in the general paediatric population, suggesting that children with autoimmune thyroiditis are at increased risk of developing Type 1 diabetes

Evolution of the microbiological profile of vacuum-packed ricotta salata cheese during shelf-life

Ricotta salata cheese is a salted variety of ricotta traditionally made in Sardinia (Italy) from the whey remaining after the production of Pecorino Romano protected designation of origin or other sheep milk cheeses. Ricotta salata cheese is very critical for the possible growth of pathogenic and spoilage microorganisms. Sporadic cases of listeriosis associated with ricotta salata cheese have been reported over recent years. The objective of the present study was to assess the evolution of spoilage and pathogen microorganism of vacuum-packed ricotta salata cheese during the entire product shelf-life. The durability study was conducted on 18 vacuum-packed ricotta salata cheese samples analysed at the beginning of the shelf-life and after 60 and 90 days of refrigerated storage. Pathogens as Listeria monocytogenes and Bacillus cereus were never detected. During shelf-life total bacterial counts ranged between 7.90±0.64 and 9.19±0.58 CFU g-1 on the rind and between 2.95±0.68 and 4.27±1.10 CFU g-1 in the inner paste, while Enterobacteriaceae ranged between 4.22±0.66 and 5.30±0.73 CFU g-1 on the rind and 3.13±1.80 and 2.80±0.88 CFU g-1 in the inner paste. By considering the technology used, the intrinsic properties and the almost total absence of competing microflora, ricotta salata cheese can support the growth of spoilage and pathogen microorganisms originating from the processing environment. The high level of total bacterial counts and Enterobacteriaceae observed both on the rind and in the inner paste suggests contamination of the product from the processing environment. Therefore, a strict implementation of hygiene during processing is essential in order to reduce the load of environmental contaminants that may grow during refrigerated storage

Comparison of post-lethality thermal treatment conditions on the reduction of <i>Listeria monocytogenes</i> and sensory properties of vacuum packed ricotta salata cheese

Ricotta salata is a whey protein cheese produced in Sardinia that in the last decades has been linked to several recalls and in 2012 to a severe human listeriosis outbreak. Contamination of ricotta salata with Listeria monocytogenes mainly occurs during post-process handling and generally origins from the processing environment. The application of water bath heat treatment in vacuum packed ricotta salata is a possible strategy to control L. monocytogenes superficial contamination. The objective of the present study was to select a heat treatment able to inactivate L. monocytogenes count of at least 5 log. Nine temperature time combinations, 75 °C, 85 °C and 90 °C applied for 15 min, 25 min and 40 min each were tested in ricotta wheels artificially contaminated with a mixture of 5 L. monocytogenes strains. Inactivation was assessed respectively one day and 30 days after heat treatment. The efficacy of treatments was evaluated based on the reduction in L. monocytogenes counts, on the impact on sensory properties and on the cost of the treatment. Two out of nine treatment combinations, i.e. 85 °C for 40 min and 90 °C for 40 min, were effective in reducing L. monocytogenes contamination level of 5 log. No significant difference was observed in sensory properties after the heat treatments. Therefore both combinations are eligible to conduct a successive study aimed to extend the shelf-life of ricotta salata up to several months

Listeria spp. and Listeria monocytogenes contamination in ready-to-eat sandwiches collected from vending machines

Ready-to-eat (RTE) food is characterised by a long shelf-life at refrigerated temperature and can be consumed as such, without any treatment. The aim of the work was to evaluate the presence of Listeria spp. and Listeria monocytogenes in RTEs collected from refrigerated vending machines placed in hospital environment and accessible to the hospitalised patients. In 4 different sampling, 55 RTEs were collected from vending machines of six hospitals located in different areas of Sardinia region. All the samples were characterised by similar manufacturing process, such as the use of modified atmosphere packaging and belonged to 5 different producers. Listeria spp. was not countable using the enumeration method in all of the analysed samples. Using the detection method, Listeria spp. was recovered from 9 sandwich samples. Interestingly, 3 of these samples (5.5%) made by the manufacturer, were positive for L. monocytogenes contamination. The risk related to the L. monocytogenes presence in RTEs proportionally increases when food is introduced in susceptible environments, such as hospitals and consumed by susceptible people. Although the RTEs analysed showed values that complied with the European microbiological criteria for foodstuffs, the availability of these products in a susceptible environment should be carefully checked. Therefore, in order to limit the possible exposition to L. monocytogenes, more information on the risk related to RTE consumption should be provided to the hospitalised patients