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

Somatic cell count control strategies in dairy ewes

The consumption of milk products, especially made from raw milk, have been reported to be associated with food borne diseases. Since most sheep’s milk products are made from raw milk, it is clear how udder health is an important prerequisite to produce hygienic milk. Ewes with mastitis, particularly in their subclinical form, serve as reservoir of pathogens that can be shed into the milk and constitute a potential risk for human health. Milk somatic cell count (SCC) is not a public health concern itself but it is an indicator of the general state of udder health in a dairy sheep flock and can be used as an indication of hygienic milk and to improve safety of dairy products. This thesis presents three different strategies, within a comprehensive SCC control program in dairy ewes. All the studies were carried out at the University of Wisconsin, Madison dairy sheep research facility, which is the only University dairy sheep research station in North America. The study presented in Chapter 2 describes an automated method to assess SCC on farm. In Chapter 3 is presented a study aimed to determine the effect of intramammary antibiotic dry treatment on intramammary infection and somatic cell count in the subsequent lactation. Chapter 4 presents a study carried out to assess the impact of premilking teat sanitation on somatic cell count in dairy ewes. In Chapter 5 are presented the results of the combined effect of dry treatment and teat sanitation on SCC in dairy ewes

Antibiotic Resistance in Staphylococcus aureus and Coagulase Negative Staphylococci Isolated from Goats with Subclinical Mastitis

Antimicrobial resistance patterns and gene coding for methicillin resistance (mecA) were determined in 25 S. aureus and 75 Coagulase Negative Staphylococci (CNS) strains isolates from half-udder milk samples collected from goats with subclinical mastitis. Fourteen (56.0%) S. aureus and thirty-one (41.3%) CNS isolates were resistant to one or more antimicrobial agents. S. aureus showed the highest resistance rate against kanamycin (28.0%), oxytetracycline (16.0%), and ampicillin (12.0%). The CNS tested were more frequently resistant to ampicillin (36.0%) and kanamycin (6.7%). Multiple antimicrobial resistance was observed in eight isolates, and one Staphylococcus epidermidis was found to be resistant to six antibiotics. The mecA gene was not found in any of the tested isolates. Single resistance against β-lactamics or aminoglicosides is the most common trait observed while multiresistance is less frequent

A Survey on aflatoxin M₁ content in sheep and goat milk produced in Sardinia region, Italy (2005-2013)

In the present work the results of a survey conducted in Sardinia Region on Aflatoxin M1 (AFM1) contamination in milk of small ruminants from 2005 to 2013 are reported. A total of 517 sheep and 88 goat milk samples from bulk tank, tank trucks and silo tank milk were collected. Analyses were performed by the Regional Farmers Association laboratory using high-performance liquid chromatography following the ISO 14501:1998 standard. None of the sheep milk samples analysed during 2005- 2012 showed AFM1 contamination. In sheep milk samples collected in 2013, 8 out of 172 (4.6%) were contaminated by AFM1 with a concentration (mean±SD) of 12.59±14.05 ng/L. In one bulk tank milk sample 58.82 ng/L AFM1 was detected, exceeding the EU limit. In none of goat milk samples analysed from 2010 to 2012 AFM1 was detected. In 2013, 9 out of 66 goat milk samples (13.6%) showed an AFM1 concentration of 47.21±19.58 ng/L. Two of these samples exceeded the EU limit, with concentrations of 62.09 and 138.6 ng/L. Higher contamination frequency and concentration rates were detected in bulk tank milk samples collected at farm than in bulk milk truck or silo samples, showing a dilution effect on AFM1 milk content along small ruminants supply chain. The rate and levels of AFM1 contamination in sheep and goat milk samples were lower than other countries. However, the small number of milk samples analysed for AFM1 in Sardinia Region in 2005-2013 give evidence that food business operators check programmes should be improved to ensure an adequate monitoring of AFM1 contamination in small ruminant dairy chain

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

Survey on the fatty acids profile of fluid goat milk

Fluid goat milk submitted to thermal treatment has interesting nutritional properties and a potential expanding market. The present study was aimed to conduct fatty acids profile characterisation of goat milk placed on market. Forty-nine fluid milk samples were collected: 12 pasteurised, 12 pasteurised at high temperature, 11 ultrahigh temperature (UHT) whole milk and 14 UHT semi-skimmed milk. Milk samples were collected at retail level from 7 different companies and from different production batches. After extraction and methilation, fatty acids (FAs) profile was determined on each sample using a gas chromatograph with flame ionisation detector (GC-FID) with high-polarity capillary column. The concentration (g/100mL) of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs), trans fatty acids (t-FAs), and isomers of conjugated linoleic acid (CLA) was determined. N-6/n-3 ratio, atherogenic index (AI) and thrombogenic index (TI) were also assessed. Fluid goat milk lipid profile was characterised by SFAs (68.4% of total FAs), PUFAs (5.3%), MUFAs (21.3%), t-FAs (3.6%) and CLA (0.8%). The most represented fatty acids were: 16:0 (24.5%), 9cis-18:1 (18.2%), 18:0 (9.6%), 14:0 (9.5%), 10:0 (9.3%) and 12:0 (4.5%). Nutritional indices were 2.8-6.8 for n-6/n-3 ratio; 2.3-2.9 for AI; and 2.7-3.2 for TI. Milk produced by small scale plants, with no milk fat standardisation, showed greater differences in fatty acid profile as compared to industrial plants milk. Large scale production is characterised by commingled bulk tank milk of different origins and then is more homogeneous. The whole goat milk supply chain should be controlled to obtain milk with fatty acids of high nutritional value

Evaluation of vacuum packaging for extending the shelf life of Sardinian fermented sausage

Salsiccia sarda or Sardinian fermented sausage is a traditional dry-fermented sausage included in the list of traditional food products of Sardinia (Italy). At the request of some producing plants, the possibility of extending the shelf life of the vacuum-packed product up to 120 days was evaluated. Manufacturing of 90 samples, representing 3 different batches of Sardinian fermented sausage was carried out in two producing plants (A and B). In the packaged product and subsequently every 30 days for four months (T0, T30, T60, T120), the following analyses were conducted on all samples: physicochemical characteristics, total aerobic mesophilic count, Enterobacteriaceae count, detection of Listeria monocytogenes, Salmonella spp., mesophilic lactic acid bacteria, and coagulase-positive Staphylococci. Moreover, surfaces in contact and surfaces not in contact with food were sampled in both producing plants. Sensory profile analysis was also performed for every analysis time. At the end of the extended shelf life, pH values were equal to 5.90±0.11 (producing plant A) and 5.61±0.29 (producing plant B). Water activity mean values at T120 were 0.894±0.02 (producing plant A) and 0.875±0.01 (producing plant B). L. monocytogenes was detected in 73.3% (33/45) of the samples from producing plant A, with mean levels of 1.12±0.76 log10 CFU/g. In producing plant B, L. monocytogenes was never detected. Enterobacteriaceae were detected in 91.1% (41/45) of samples in producing plant A with mean values of 3.15±1.21 log10 CFU/g, and in 35.5% (16/45) samples in producing plant B samples with mean values of 0.72±0.86 log10 CFU/g. Salmonella and Staphylococcus aureus were never detected. Regarding environmental samples, the sites that were most contaminated by L. monocytogenes were the bagging table (contact surface) and processing room floor drains (non-contact surface) with a prevalence of 50% each (8/16 positive samples for both sampling sites). Sensory analysis results showed that at T30 the overall sensory quality was at its highest; moreover, the visual-tactile aspect, the olfactory characteristics, the gustatory aspects, and the texture showed significant differences in samples throughout the shelf life, with a decreased intensity at 120 days of storage. Overall, the quality and sensory acceptance of the vacuum-packed Sardinian fermented sausage were not affected until 120 days of shelf-life. However, the possible contamination by L. monocytogenes calls attention to the hygienic management of the entire technological process. The environmental sampling was confirmed as a useful verification tool during control

Occurrence and traceability of Salmonella spp. in five Sardinian fermented sausage facilities

The aims of the present study were to evaluate the presence of Salmonella in five fermented sausage processing plants and their products during the production process, and to trace the possible sources of contamination. A total of 270 samples were collected: mixture of ground pork meat and fat, products at the end of acidification, sausages at the end of ripening and, during production stages, surfaces in contact with meat and surfaces not in contact with meat. For samples of ground meat, product at the end of acidification and sausages at the end of ripening, the pH and water activity (aw), were determined. All the samples were tested for the presence of Salmonella. Thirtytwo Salmonella isolates were obtained, subjected to serotyping and PFGE. The sausages at the end of ripening pH and aw mean values were 5.39±0.24 and 0.91±0.03, respectively. Salmonella was detected in three processing plants with an overall prevalence of 16.7% in food samples and 5.8% in environmental samples. Salmonella prevalence was 24% in ground meat and products at the end of acidification and was also detected in a sample of sausage at the end of ripening (2%). In environmental samples, Salmonella was detected in 6.6% of surfaces in contact with meat and 5% of surfaces not in contact with meat. Five serotypes were identified among 32 isolates: S. Derby (37.5%), S. Typhimurium and S. Rissen (both 25%), S. Give and monophasic S. Typhimurium (both 6.25%). Six different pulsotypes were obtained with PFGE. The serotypes and the PFGE pattern of the strains were specific for each facility with no overlapping between different processing plants. The same observation can be pointed out considering different sampling days for the same processing plants, thus presumably indicating the raw material (ground pork meat and fat) as the source of contamination. The detection of Salmonella in a sample of sausage at the end of ripening highlights the ability of the pathogen to survive during manufacturing process

Double carbapenem as a rescue strategy for the treatment of severe carbapenemase-producing Klebsiella pneumoniae infections: A two-center, matched case-control study

Background: Recent reports have suggested the efficacy of a double carbapenem (DC) combination, including ertapenem, for the treatment of carbapenem-resistant Klebsiella pneumoniae (CR-Kp) infections. We aimed to evaluate the clinical impact of such a regimen in critically ill patients. Methods: This case-control (1:2), observational, two-center study involved critically ill adults with a microbiologically documented CR-Kp invasive infection treated with the DC regimen matched with those receiving a standard treatment (ST) (i.e., colistin, tigecycline, or gentamicin). Results: The primary end point was 28-day mortality. Secondary outcomes were clinical cure, microbiological eradication, duration of mechanical ventilation and of vasopressors, and 90-day mortality. Forty-eight patients treated with DC were matched with 96 controls. Occurrence of septic shock at infection and high procalcitonin levels were significantly more frequent in patients receiving DC treatment (p < 0.01). The 28-day mortality was significantly higher in patients receiving ST compared with the DC group (47.9% vs 29.2%, p = 0.04). Similarly, clinical cure and microbiological eradication were significantly higher when DC was used in patients infected with CR-Kp strains resistant to colistin (13/20 (65%) vs 10/32 (31.3%), p = 0.03 and 11/19 (57.9%) vs 7/27 (25.9%), p = 0.04, respectively). In the logistic regression and multivariate Cox-regression models, the DC regimen was associated with a reduction in 28-day mortality (OR 0.33, 95% CI 0.13-0.87 and OR 0.43, 95% CI 0.23-0.79, respectively). Conclusions: Improved 28-day mortality was associated with the DC regimen compared with ST for severe CR-Kp infections. A randomized trial is needed to confirm these observational results. Trial registration: ClinicalTrials.gov NCT03094494. Registered 28 March 2017

Designing the Future: An Intelligent System for Zero-Mile Food Production by Upcycling Wastewater

The project deals with the environmental problem of water consumption. The aim of this work is to experiment the recycling of dishwasher wastewater through its reuse in growing edible vegetables or ornamental plants; this can also accomplish the valorization of nutrients present in the wastewater. This new process allows to ensure washing functions coupled with vegetables production and to affect users’ environmental awareness and habits, following a user-centered system design approach to understand the users and involve them actively in the system development. The presented work is also aimed to experiment a multidisciplinary approach in order to face environmental problems