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Shiga-toxin-producing Escherichia coli is a foodborne pathogen commonly associated with human disease characterized by mild or bloody diarrhea hemorrhagic colitis and hemolytic uremic syndrome. This study investigated the occurrence of STEC in fecal samples of 289 goats in South Africa using microbiological culture and PCR. Furthermore, 628 goat STEC isolates were characterized by serotype (O:H) and major virulence factors by PCR. STEC was found in 80.2% (232/289) of goat fecal samples. Serotyping of 628 STEC isolates revealed 63 distinct serotypes including four of the major top seven STEC serogroups which were detected in 12.1% (35/289) of goats: O157:H7, 2.7% (8/289); O157:H8, 0.3%, (1/289); O157:H29, 0.3% (1/289); O103:H8, 7.6% (22/289); O103:H56, 0.3% (1/289); O26:H2, 0.3% (1/289); O111:H8, 0.3% (1/289) and 59 non-O157 STEC serotypes. Twenty-four of the sixty-three serotypes were previously associated with human disease. Virulence genes were distributed as follows: stx1, 60.6% (381/628); stx2, 72.7% (457/628); eaeA, 22.1% (139/628) and hlyA, 78.0% (490/628). Both stx1 and stx2 were found in 33.4% (210/628) of isolates. In conclusion, goats in South Africa are a reservoir and potential source of diverse STEC serotypes that are potentially virulent for humans. Further molecular characterization will be needed to fully assess the virulence potential of goat STEC isolates and their capacity to cause disease in humans.

The aim of this study was to compare the effect of a commercial formulation consisting of natural ingredients and dextrose on the production of salami. We analysed the effect on microbiological, physico-chemical and sensorial properties in the production of nitrite-free swine and roe deer (Capreolus capreolus) salami. Six batches of salami were prepared: four with the addition of starter cultures and diverse substrates (no substrate, skimmed milk, commercial formulation and dextrose) and two without starter cultures as controls (one without substrate and one with skimmed milk). Samples for microbiological and physico-chemical analysis were taken on day 0, day 5 and day 25. A sensory evaluation was performed at the end of the ripening process. The best results regarding the changes in the microbiota were observed in the batch produced with the addition of skimmed milk (final concentration for Pseudomonas spp. was 3.07 ± 1.11 log cfu g−1, for Enterobacteriaceae was 1.57 ± 0.51 log cfu g−1 and the total coliforms were undetectable). As regards the sensory evaluation, the best scores (in particular colour uniformity, fat/lean distribution and mould flavour) were assigned to the salami produced with the addition of dextrose. According to the results obtained in this study, the addition of the commercial formulation to manufacture nitrite and nitrate-free, dry-cured swine and roe deer salami led to no significant effects on their microbiological and sensory characteristics. Overall, the results obtained with the addition of the commercial formulation were very similar to those obtained by adding dextrose, which is the main component of the formulation itself.HIGHLIGHTS The effect of a commercial formulation on microbiological, chemical and sensory characteristics of nitrite-free swine and roe deer (Capreolus capreolus) salami was analysed. The results obtained with the addition of the commercial formulation were very similar to those obtained by simply adding dextrose or just skimmed milk. The use of commercial preparation seems therefore not worthwhile.

The term ‘packaging’ refers to the technological intervention aimed at the protection of food from a variety of factors, which provokes the product detriment. Packaging is considered as one of the most interesting technological aspects and a constantly evolving issue in food production. This paper aims at the evaluation of the properties of packaging currently used in the meat industry and analyses the advantages, the disadvantages and the microbiota involved. Packaging is a coordinated system, which prepares the products for transportation, distribution, storage, marketing and consumption. Even if several packaging alternatives are proposed, the common purpose is to guarantee high standards, yet maintaining the required characteristics as long as possible. Meat is a dynamic system with a limited shelf-life and the nutritional and sensory properties may change during storage due to microbial activity and physical or chemical changes. Microbial spoilage, for instance, determines an impact in meat, producing unattractive odours, flavours, discolouration, gas and slime.

Packaging is considered one of the most interesting technological aspects of food production and is a constantly evolving subject in food production. The type of packaging is important for the quality and safety of the product and for the visual appearance of the product to be immediately evaluated by consumers. The purpose of this study was to investigate the effect of four different types of modified atmosphere packaging (ATM) and vacuum packaging (VP) currently used by a company in central Italy, on the main qualitative characteristics of beef. For these two traditional and two new solutions with reduced environmental impact and compostable were evaluated. For each type of packaging, two different products were analyzed: steaks and hamburgers. The samples, immediately after production, were transported to the laboratory in refrigerated containers. Several parameters (color, pH, water holding capacity, drip loss, and microbiological characteristics) were evaluated at time 0 and after 7 (T7), 14 (T14) and 21 days (T21) of storage in the dark and at refrigeration temperature (+4 degrees C +/- 2 degrees C). The results showed that the two types of packaging have very similar effects on the water-retaining capacity of the steaks. More noticeable differences were recorded by the colorimetric analysis: for both steaks and hamburgers, the products packaged in the traditional packaging appeared brighter and redder than those packaged in the new alternatives. The microbiological analysis of the steaks showed higher values in the "new" packaging. The formation of abundant ropy slime was observed in one of the samples in the "new" modified atmosphere package at T21. The results of this study showed that the technological characteristics (in particular, the color) and the microbiological characteristics of the steaks and hamburgers were better in "old" packaging, with a better appearance and a longer shelf life. The results obtained show how the research for eco-sustainable products for packaging must be addressed, taking into account the effect of the materials on the qualitative and hygienic-sanitary characteristics of the meat.

This study describes a simple method for the large‐scale isolation of pure Toxoplasma gondii tachyzoites and bradyzoites. T. gondii tachyzoites were obtained from infected human foreskin fibroblasts (HFFs) and peritoneal exudates of mice, while tissue cysts containing bradyzoites were collected from chronically infected mice. Harvested cells and brain tissues were incubated in Hanks balanced salt solution (HBSS), containing 0.25% trypsin and 0.5% taurodeoxycholic acid (TDC) for 5 min. Subsequent washes in phosphate buffered saline (PBS) were conducted, and the cell viability of the preparations was good, as determined by flow cytometry and ability to reinfect HFF cells and propagate in mice. The purification procedure allowed for a rapid preparation of pure T. gondii tachyzoites and bradyzoites in sufficient quantity that can be used for downstream procedures. The advantage of the new method is that it is convenient and inexpensive., We developed a rapid and simple method for purifying tachyzoites and bradyzoites with optimal yield and purity. The new proposed method has the following advantages: (1) zoites can be obtained quickly and easily; (2) the technique can be performed in low resource settings and capable of handling larger numbers of cells and tissues; (3) zoites obtained in this way are free of cellular debris; and (4) the viability and infectivity of tachyzoites, and bradyzoites are maintained. A more detailed study is required to explore the effect of trypsin on surface antigens of parasites.

International audience; This study investigated occurrence and antimicrobial resistance profiles of Campylobacter spp. isolates in beef cattle on five cow-calf operations in South Africa. A total of 537 fecal samples from adult beef cattle (n = 435) and rectal swabs from calves (n = 102) were screened for Campylobacter jejuni, Campylobacter coli, and Campylobacter upsaliensis by culture and polymerase chain reaction. Furthermore, 86 Campylobacter spp. isolates including 46 C. jejuni, 24 C. coli, and 16 C. upsaliensis were tested for antimicrobial resistance against a panel of 9 antimicrobials. Overall, Campylobacter spp. was detected in 29.7% of cattle. Among the 158 Campylobacter spp.-positive cattle, 61.8% carried C. jejuni, 25% carried C. coli, and 10% carried C. upsaliensis. Five animals (3.1%) had mixed infections: three cows carried C. jejuni and C. coli concurrently, one cow had both C. jejuni and C. upsaliensis, and one cow harbored C. coli and C. upsaliensis. Antimicrobial resistance profiling among 86 Campylobacter spp. isolates revealed that 52.3% of the isolates were resistant to one or more antimicrobials. Antimicrobial resistance was observed in 46.7% of C. jejuni isolates, 35.6% of C. coli, and 17.8% of C. upsaliensis. Thirty-six percent of isolates were resistant to clindamycin, 19.7% to nalidixic acid, 18.6% to tetracycline, and 17.4% to erythromycin. Lower resistance rates were recorded for azithromycin (8.1%), florfenicol (3.4%), gentamicin (4.8%), and telithromycin and ciprofloxacin (5.8%). Multidrug resistance (MDR) was observed in 32.5% of isolates. Significantly higher levels of MDR were detected among C. jejuni (36.9%) and C. coli (33.3%) isolates in comparison to C. upsaliensis (18.7%). Two main multiresistance patterns were detected: nalidixic acid/clindamycin (17.8%) and tetracycline/clindamycin (14.2%). To the best of our knowledge, this is the first study which has shown that beef cattle on cow-calf operations in South Africa constitute an important reservoir and a potential source of clinically relevant and antimicrobial resistant Campylobacter spp. strains.

The presence of lactic acid bacteria can be detrimental when the abundant growth of slime-producing strains (Lactobacillus spp. and Leuconostoc spp.) causes spoilage of meat products. Two strains of lactic acid bacteria were isolated from vacuum-packed cooked hams that had been withdrawn from the market for the so-called ropy slime defect and identified as Leuconostoc mesenteroides. In an attempt to define the behavior of ropy slime-producing bacteria, two strains of L. mesenteroides were incubated in de Man Rogosa Sharpe broth at different storage temperatures and conditions of thermal abuse (4, 12, 20, 30, 37, and 44°C). Both strains showed a lack of growth at 44°C, a good level of development at 30 and 37°C, and evident growth ability at low temperatures, with a long stationary phase. In particular, the bacterial concentration at 4°C was >105 CFU mL-1 after more than 120 days of incubation. This study demonstrates that the refrigeration temperature for cooked meat products does not constitute a hurdle for ropy slime producers and their subsequent ability to spoil. HIGHLIGHTS

The use antimicrobials for therapeutic and metaphylactic purpose in humans and agriculture exerts selective pressure on animal and environmental microbiota resulting in the survival and spread of antimicrobial resistance genes among bacteria and subsequent development of resistance in bacteria. Previous studies have shown that honey bees' microbiota (Apis mellifera) can accumulate antimicrobial resistance genes in their microbiome and act as collectors and disseminators of resistance genes. The aim of this study was to investigate to what extent honey bees act as reservoir of select antimicrobial resistance genes. This study was conducted on 35 groups of bees. Bees were collected from 35 sites in Umbria, Italy. PCR was used to screen pooled ground bees' specimens for genes that code for resistance against antimicrobials that are commonly used in humans and in veterinary medicine including aminoglycosides (aph), beta-lactams (blaZ), tetracycline (tetM) and sulphonamides (sul1 and sul2). Twenty-four samples out of 35 (68.57%) were positive for at least one antimicrobial resistance gene. Two samples were positive for the aph, 5.71%; eight for blaZ, 22.86%; three for tetM, 8.57%; ten for sul1, 28.57% and eighteen for sul2, 51.43%. Positivity to more than one antimicrobial resistance gene was observed in nine samples, 25.71%. The multivariate analysis identified "presence of farms nearby" as the factor most closely related to PCR positivity. Honey bees (Apis mellifera) from Umbria, Italy, carry antimicrobial resistance genes and can be used as indicators of the presence of resistance genes in the environment.

Anaplasma phagocytophilum is a tick-borne pathogen of sheep and other ruminants, humans, horses and dogs. Current data on the prevalence and diffusion of A. phagocytophilum among domestic animals in Italy are fragmentary, especially those related to sheep, considered a natural domestic reservoir. We investigated the prevalence of A. phagocytophilum infection in dairy sheep from Tuscany (Italy), by testing 624 sera with the immunofluorescent antibody (IFA) test. The sera belonged to 33 different flocks distributed throughout the province of Grosseto (Tuscany). Positive results were found in 21 out of the 33 flocks tested (63.63%, 95% IC 47.22–80.05%) with a prevalence rate within flocks ranging from 3.33% to 59.26%. The seroprevalence against A. phagocytophilum within the sheep population was 8.01% (95% IC 6.13–10.41%), and most of the positive cases showed low antibody titres (1/40-1/80). The multivariate analysis identified "small flock" ( Our results reveal a moderate circulation of A. phagocytophilum among sheep flocks in central Italy.

The objective of this study was to investigate the effect of the antimicrobial drugs (AMD) on the shedding of resistant Enterobacteriaceae in feces of pre-weaned dairy calves. The AMD considered were ceftiofur, administered parenterally, and neomycin sulfate added in milk replacer and fed to calves during the first 20 days of life. Fifty-five calves, aged one to three days, were enrolled and followed to 64 days. Fecal samples were collected three times/week and treatments recorded daily. Enterobacteriaceae were quantified for a subset of 33 calves using spiral plating on plain, ceftiofur supplemented, and neomycin supplemented MacConkey agar. Negative binomial models were used to predict the association between treatment with AMD and the gain and loss of Enterobacteriaceae resistance over time. Acquisition of resistance by the Enterobacteriaceae occurred during treatment and peaked between days three to four post-treatment before decreasing to below treatment levels at days seven to eight post-treatment. Acquisition of neomycin resistance was observed on the first sampling day (day four from the start of feeding medicated milk replacer) to day eight, followed by cyclical peaks until day 29, when the Enterobacteriaceae counts decreased below pre-treatment. Enterobacteriaceae resistance against both AMD increased after AMD administration and didn’t return to pre-therapeutic status until seven or more days after therapy had been discontinued. The study findings provide valuable insights into the dynamics of Enterobacteriaceae under routine AMD use in calves.

Simple Summary The population of Procambarus clarkii was surveyed twenty years after its introduction into Lake Trasimeno (Central Italy), investigating both ecological and genetic aspects. Our results confirmed that P. clarkii is well acclimatized with a stable population structure, mainly characterized by a unique mitochondrial lineage, suggesting that a single introduction event may have occurred in the lake, complemented by secondary events. Abstract The deliberate or accidental introduction of invasive alien species (IAS) causes negative ecological and economic impacts altering ecosystem processes, imperiling native species and causing damage to human endeavors. A monthly monitoring program was performed in Lake Trasimeno (Central Italy) from July 2018 to July 2019 in order to provide an upgrade of the population ecology of Procambarus clarkii and to assess the genetic diversity by analyzing the relationships among mitochondrial DNA diversity. Our results confirmed that P. clarkii is well acclimatized in the lake, revealing a stable population structure favored by the resources and conditions typical of this ecosystem, which seem to be optimal for the maintenance of the species. Four distinct mitochondrial haplotypes were detected, but one of them was clearly overrepresented (76%), suggesting that a single predominant introduction event may have occurred in this area, likely followed by secondary events. The identification of the typical genetic variants provides a better understanding of the evolutionary scenarios of P. clarkii in this biotope and it can be helpful in management plans concerning the expanding populations of this invasive alien species.

The latest EU regulation on geographical indications (EU Regulation No. 1151/2012) has intro-duced a set of new tools for the protection and enhancement of food products in rural areas, under the group name of optional quality term (OQT). The Commission Delegated EU Regulation, No. 665/2014, regulated the conditions for the use of the optional quality term «mountain product» (MP), to support the implementation of a mountain value chain. This new tool is aimed at pro-moting local development, maintaining the economic activities in mountain areas and redistrib-uting wealth, whilst, at the same time, promoting the territory. Pecorino and goat cheeses are typ-ical Italian cheeses made usually with whole raw ewe's or raw goat's milk, without starter cul-ture addition. In an attempt to characterize these productions, the aim of this study was to inves-tigate the evolution of enterococci during the production and ripening of Pecorino cheese made in three different farms, located in Umbria, Italy in areas facing natural or other specific constraints as stipulated by Regulation 1305/2013 on support for rural development by the European Agri-cultural Fund for Rural Development (EAFRD). Enterococci are enteric organisms which are commonly isolated from ewe and goat's milk production in Umbria, Italy. Counts of enterococci in raw milk ranged from 1.75 for ovine milk to 3.62 for ewe milk and a marked reduction was observed after thermization especially in ovine milk. Out of 100 isolates, 69 were E. faecium, 23 E. durans, 8 E. faecalis and 2 E. casseliflavus and the distribution of species between farms and be-tween samples showed a prevalence of E. faecium in ovine farms and E. durans in ewes farms, with an equal distribution between samples. High percentages of susceptible isolates were found for amoxicillin/clavulanic acid, ampicillin, chloramphenicol, sulphamethoxazole, sulphameth-oxazole/trimethoprim, ticarcillin, vancomycin. A high prevalence of resistant strains (> 30%) was observed for amikacin, ciprofloxacin, ceftriaxone, kanamycin, tetracycline. A comparison of this results with those of previous works on similar dairy products revealed high levels of resistance to antimicrobials which needs to be addressed.

The objective of this study was to investigate the effect of the antimicrobial drugs (AMD) on the shedding of resistant Enterobacteriaceae in feces of pre-weaned dairy calves. The AMD considered were ceftiofur, administered parenterally, and neomycin sulfate added in milk replacer and fed to calves during the first 20 days of life. Fifty-five calves, aged one to three days, were enrolled and followed to 64 days. Fecal samples were collected three times/week and treatments recorded daily. Enterobacteriaceae were quantified for a subset of 33 calves using spiral plating on plain, ceftiofur supplemented, and neomycin supplemented MacConkey agar. Negative binomial models were used to predict the association between treatment with AMD and the gain and loss of Enterobacteriaceae resistance over time. Acquisition of resistance by the Enterobacteriaceae occurred during treatment and peaked between days three to four post-treatment before decreasing to below treatment levels at days seven to eight post-treatment. Acquisition of neomycin resistance was observed on the first sampling day (day four from the start of feeding medicated milk replacer) to day eight, followed by cyclical peaks until day 29, when the Enterobacteriaceae counts decreased below pre-treatment. Enterobacteriaceae resistance against both AMD increased after AMD administration and didn’t return to pre-therapeutic status until seven or more days after therapy had been discontinued. The study findings provide valuable insights into the dynamics of Enterobacteriaceae under routine AMD use in calves.

During the last ten years, the worldwide interest in using insects as food and feed has surged. Edible insects fall within the category of novel foods, i.e., the category of food not consumed in significant amounts in the European Union before 15 May 1997 (the date of entry into force of Regulation (EC) No. 258/1997, later repealed by Regulation (EU) No. 2283/2015). One of the most promising insect species to be raised for food is the house cricket (Acheta domesticus). In this study, the rearing of a stock of house crickets was studied over a period of four months. The microbiological quality of the farm was studied using swabs on the surface of the rearing boxes to analyze the trend over time of different populations of microorganisms (total aerobic mesophilic microbiota, Lactobacillus spp., enterococci, Staphylococcus spp., Enterobacteriaceae, total coliforms, Pseudomonas spp. and molds). The presence of four antimicrobial resistance genes (aph, blaZ, sul1, and tetM) was investigated by polymerase chain reaction. A production scheme was also developed in order to obtain a cricket-based flour, which was analyzed for its microbiological and chemical-centesimal profile. The results obtained in this study demonstrate that the contamination increases with time and that a proper management of the farming system for insects is of the utmost importance, as it is for conventional farm animals such as ungulates, poultry, and rabbits. The old-fashioned adage “all full, all empty” for the farming system summarizes the need for proper cleaning and disinfection of the structures at the end of each production cycle.

Food security, as part as public health protection, constitutes one of the main objectives for countries aiming to ensure the health of all their citizens. However, food security is compromised worldwide by conflict, political instability, or economic crises, both in developed and developing countries. Conversely, because of the importance of agriculture to the economies of rural areas both in developed and developing countries, this sector can contribute to improving food stability, as well as to furthering food security. Thus, livestock and traditional meat products represent a key factor in ensuring food availability. Overall, biosecurity measures improve animal welfare by decreasing the occurrence of diseases that compromise the stability by causing fluctuations in the availability of meat and animal-derived food products such as milk, eggs, or traditional fermented products. As a consequence, an absence of biosecurity measures affects food security (in its quantitative definition, as described above) as well as the productive, sanitary, and environmental sustainability of the rural environment. Products of animal origin support local trade and the regional economy, while contributing to the availability of foods without great external dependence. The manufacture of foods of animal origin aims to create products that are durable and that maintain food availability for long periods of time, even during seasons with scarce resources. Thus, dry-cured or fermented meat products play an important role in food availability. Food security also refers to food access under healthy economic conditions; therefore, knowledge of the main tools that guarantee the safety of these kinds of food products is essential to achieving food stability and further food security.

The immediate refrigeration of meat after slaughter is a key issue for the proper storage and aging of meat. The industry standard cold chain relies on low temperatures and ventilation to lower the internal carcass temperature to 0–4 °C within the first 48 h, i.e., within four times the so-called semi-cooling time. On the other hand, for games, once bled and eviscerated, the carcass must be sent to a point where it can be sectioned or kept on air for maturation at refrigeration temperature. The precautions to observe are few and simple but essential: protect the meat and start the cooling process quickly. After preparing the animal (bleeding and evisceration), it may be necessary to face a period of transport that is sometimes long and not very easy, while small animals can be easily transported in a backpack, larger ones must necessarily be carried by several people or sometimes dragged to the vehicle capable of transporting them. It is obvious that a wild boar opened from the jaws to the pelvis and dragged for hundreds of meters will tend to be contaminated, although these contaminations are to be considered secondary for the preservation of the meat, compared to contamination by the intestinal contents. In an attempt to investigate the effect of delayed refrigeration on wild boar carcass contamination, the aim of this work was to determine a correlation between several hunting and logistic parameters (age, sex, animal weight, shooting distance, number of shots, weather and temperature and time from shot to refrigeration and to analysis) and bacterial contamination of the carcass. The correlation coefficient, r, was found to be 0.038 for the eviscerated body weight (p &lt, 0.05), 0.091 for the external temperature on the day of hunting (p &lt, 0.05), 0.027 for the time from shot to refrigeration (p = 0.081), 0.038 for the time from refrigeration to analysis (p &lt, 0.05) and 0.043 for the time from shot to analysis (p &lt, 0.05). These results stand for a negative correlation between the bacterial population and eviscerated carcass weight and between the bacterial population and external temperature and for a positive correlation between the time from shot to analysis and from refrigeration to analysis. No association was demonstrated between the bacterial population and the time from shot to refrigeration.

Enterococci are enteric organisms which are commonly isolated from ewe and goat's milk production in Umbria, Italy. For years enterococci have been considered as microorganisms only indicative of inadequate hygienic practices or exposure of the food to conditions that would permit multiplication of other undesirable bacteria. However, enterococci largely occur in many cheeses, and are now considered to be usual components of their typical microflora. They play a major role in cheese ripening due to lipolytic, proteolytic and caseinolytic activities. Enterococci have been also shown to be involved in food poisoning outbreaks although only E. faecalis has been demonstrated to cause changes in dairy products, thus being the only species of concern in dairy production. The aim of this study was to was to investigate the evolution of enterococci during the production and ripening of Pecorino cheese made with two different cheesemaking processes and characterize Enterococcus spp. isolates all along the cheesemaking and ripening process.

Staphylococcus aureus is not only a common cause of bovine mastitis, but also an agent of food poisoning in humans. In an attempt to determine whether staphylococci causing bovine mastitis could also cause food poisoning, 60 isolates of presumed S . aureus were isolated in the period between March and August 2017 from 3,384 routine, composite, quarter milk samples of individual cows raised on 12 dairy farms in central Italy. Seventeen out of 60 isolates were confirmed as S . aureus after coagulase, thermonuclease, and biochemical tests. These isolates were analyzed by PCR for the presence of the nuc , sea , seb , sec , sed , and see genes. The positive isolates were nuc , 100% (17); sea , 35.29% (6); seb , 5.88% (1); sec , 5.88% (1); sed , 29.41% (5); and see , 47.06% (8). The isolates were also tested with 2 enzyme immunoassay diagnostic kits, one for the screening detection of the production of staphylococcal enterotoxins (SEA, SEB, SEC, SED, SEE) and one for the detection of specific enterotoxin produced by each isolate. Seven out of 17 (41.18%) were enterotoxin producers: 7 produced SEA (41.18%), 1 SEB (5.88%), 1 SEC (5.88%), 5 SED (29.41%), and 6 SEE (35.29%). To further characterize the isolates, they were analyzed by the Kirby Bauer test for susceptibility to 13 antimicrobials (ampicillin, ciprofloxacin, kanamycin, tetracycline, gentamicin, methicillin, nalidixic acid, erythromycin, amoxicillin/clavulanic acid, streptomycin, vancomycin, neomycin, and enrofloxacin), and we detected resistance to ampicillin (52.94%), nalidixic acid (70.59%), erythromycin (5.88%), and amoxicillin/clavulanic acid (17.65%). The isolates were sensitive to the main classes of antimicrobials used for the treatment of bovine subclinical mastitis. The presence of enterotoxin-producing isolates of S . aureus in bovine milk means that a temperature abuse or a breakdown in the thermal treatment of the milk could present a food safety risk, particularly if all enterotoxigenic isolates could potentially produce SEA in milk.

Fermented sausages have been manufactured traditionally in the Mediterranean basin. Istrian sausages are one of the many regionally specific products distinguished by the specific traditional recipes and consequentially exceptional sensory characteristics. The aim of this study was to evaluate sensory, physico- chemical and microbiological changes occurring during production and ripening of Istrian sausages made at traditional households, and to compare the results obtained with the identical product produced in industrial conditions. Additional goal was to characterise the autochthonous lactic acid bacteria populations and to obtain the candidate starter strains. Production stages and product ripening parameters could be strictly defined in controlled industrial conditions of the production process, and the results of this study can serve to standardise the production of traditional Istrian sausages. The results from sensory and physico- chemical examination do not show differences in relation to sausages of a similar type investigated in other papers. Lactic acid bacteria were favoured during fermentation at rising salt concentrations, consisting mostly of Lactobacillus strains followed with the abundance of coagulase-negative cocci. Further, we targeted the autochthonous lactic LAB population to select the candidate starter strains for controlled sausage fermentation. Fifty Gram-positive strains with catalase-negative phenotype, rod-shape or cocci morphology, effective acidification kinetics and production of lactic acid identified by sequence analysis of 16S rRNA were considered LAB. The selected LAB strains were able to grow in a range of pH values and high salt concentration important for their metabolic activity during sausage fermentation. According to our results, Lactobacillus sakei, which is a prevalent strain during fermentation of various types of fermented sausages, was frequently detected. Lactobacillus plantarum and Lactobacillus curvatus were also detected in abundance, implying their possible competitiveness among the complex microbial population. Isolated autochthonous strains L. curvatus S30, L. sakei S32 and L. plantarum S50 produced high lactic acid concentrations, tolerated NaCl concentrations present during sausage fermentation, survived at high concentrations of hydrogen ions and inhibited six pathogens associated with contamination of meat products in a strain-specific manner.

In this research communication we report on the diversity of yeast and mould species in 69 samples of milk and different dairy products from three plants located in Umbria, central Italy. Isolates were characterised both macroscopically and microscopically and then identified by PCR and genome sequencing of the ITS region and the D1–D2 domain of the large-subunit rRNA gene for filamentous fungi and yeasts, respectively. Out of the 69 samples analysed, 51 (73.9%) tested positive for the presence of yeasts, whereas moulds were detected in 25 (36.2%) samples. A total of 9 yeast species belonging to 8 different genera and 13 mould species belonging to 6 different genera were isolated. The most common genera isolated were Debaryomyces and Kluyveromyces among the yeasts and Penicillium and Galactomyces among the moulds. Microbiota play a key role in the formation of flavour, aroma, texture and appearance of dairy products. This complex microbial ecosystem includes both cultured and external bacteria, yeasts and moulds. Some of them have an important role in the production of cheeses, whereas others are responsible for dairy product spoilage, resulting in significant food waste and economic losses. Some species can produce mycotoxins, representing a potential hazard for the consumer's safety. This study provides interesting information on the diversity of fungi species in dairy products from central Italy that can be of major importance to identify these products and to develop adequate strategies for fungal spoilage control and consumer safety.

Among various food sources, milk proteins remain the major vector for functional peptides endowed with several biological activities. Particularly, the proteolytic activity of lactic acid bacteria during milk fermentation has been one of the most followed strategies to produce bioactive peptides. In the present study, the exploration of the activity of several starter cultures, at different fermentation times, was firstly investigated by reversed phase-high performance liquid chromatography. Among the tested strains, Lactobacillus helveticus showed a higher proteolytic activity and it was submitted to further investigations by changing the fermentation substrate (skim milk, brain heart infusion, peptone water) as well as the extraction strategy (trichloroacetic acid vs. glass beads). The chromatographic analyses and the in vitro antioxidant and antihypertensive assays highlighted considerable differences for L. helveticus hydrolysates from different substrates, while a negligible impact by the two extraction protocols emerged. Furthermore, nano-high pressure liquid chromatography coupled with a high resolution mass spectrometry analyzer allowed the preliminary discrimination of fractions from fermented skim milk, likely responsible for the found activity. The obtained results suggest the possibility of varying the fermentation parameters in order to maximize the functional effects of the bioactive peptides.

The aim of this work was to study the effect of a selected lactic acid bacteria formulation on the microbiological characteristics and colour of beef hamburgers stored at different temperatures. All hamburgers were evaluated on day-0, day-1, day-3, and day-5 for the following microbiological parameters (Staphylococcusspp., enterococci, Lactococcusspp., Lactobacillusspp., total mesophilic aerobes, Pseudomonasspp., total coliforms) according to standard methods and for colorimetric measurements performed with Colorimeter - Digital Color Picker for iOS 10, under a 6500K light, with the CIELab system. All data (geometric mean for microbiological data) were elaborated with GraphPad InStat, 3.0b and GraphPad Prism 6.0d for Mac OS X. Two-way analysis of variance (ANOVA) followed by the Tukey's multiple comparisons test was performed. The analysis of the colour proved that the addition of LAB does not affect the natural colour of ground meat, avoiding the risk of hiding the spoilage or fastening it. The addition of the starter has preserved the colour stability throughout the preservation period, with the same behaviour both in the hamburgers stored at 4 °C and in those at 10 °C after thermal abuse or not. In conclusion, the application of the proposed LAB formulation maintains hamburgers quality standards and can be a potential tool to increase their shelf-life.

Probiotic viability is generally determined by quantifying its resistance to simulated gastric juice or to simulated intestinal fluid in in vitro tests, which measure microbial survival after given periods of contact. The use of a neutralizing agent is needed to avoid a carry-over of gastric or intestinal juice into the culture media of the subsequent analysis and to avoid any antimicrobial effect extended over the defined period of contact of the test. Neutralization of gastric juice and intestinal juice are of the utmost importance to present data accurately. Failing to do so determines a carry-over of bactericidal activity to the plates used for the enumeration, which further reduces the number of surviving cells. Examples of such incorrect adaptation of the test are available in literature. The purpose of this perspective stems from the discovery that many studies do not adhere to internationally recognized standards, e.g., EN 1040:2005 (European Committee for Standardization [ECS], 2005), to evaluate the basic, bactericidal activity of compounds, especially for the neutralization step.

This study investigated occurrence and antimicrobial resistance profiles of

The use of antioxidants in meat preparation and meat products is highly debated. Regulations define the use of antioxidants mostly in terms of the age-old subdivision between meat preparations and meat products. Best practices are not well represented in regulations. Antioxidants for foodstuffs during processing or before packing protect colour, aroma and nutrient content. As regards food safety regulations, long-term efforts have been made in terms of food standards, food control systems, food legislation and regulatory approaches. These have, however, generated several questions on how to apply the law to diverse food businesses. To answer these questions, a thorough examination of the EU legislator’s choices for food additives and definitions is provided and discussed in relation to factors affecting microbial growth. The paper highlights the regulatory aspects along with the correct application and interpretation of the norms

The CoViD-19 pandemic has caused a sudden spike in demand and production of hand sanitisers. Concerns are rising regarding the quality of such products, as the safeguard of consumers is a priority worldwide. We analyse here the ethanolic content of seven off-the-shelf hand sanitiser gels (two biocides and five cosmetics) from the Italian market, using gas chromatography. The WHO recommends that products containing ethanol should have 60–95% (v/v) alcohol. Four of the tested hand gels have ethanolic contents within the recommended range, while three products (all cosmetics) contain

Ground beef contamination with Escherichia coli is usually a result of carcass faecal contamination during the slaughter process. Carcasses are contaminated when they come into contact with soiled hides or intestinal leakage content during dressing and the evisceration processes. A more recent and compelling hypothesis is that, when lymph nodes are present in manufacturing beef trimmings, they can be a potential source of Enterobacteriaceae contamination of ground beef. The aim of this study was to investigate the occurrence of E. coli in lymph nodes from beef carcasses used for ground meat production, in six slaughter plants situated in central Italy A total of 597 subiliac (precrural) lymph nodes were obtained from 597 cattle carcasses and screened for E. coli by culture. Furthermore, E. coli isolates (one per positive carcass) were tested for stx1, stx2 eaeA and hlyA genes that are commonly used to identify and characterise shiga toxin-producing E. coli (STEC). In addition, the E. coli isolates were profiled for antimicrobial susceptibility. A proportion of 34.2% (204/597) carcasses were positive for E. coli. PCR revealed that 29% (59/204) of E. coli possessed stx1 or stx2 which corresponded to 9.9% of the cattle sampled. Moreover, a combination of stx1 or stx2 and eaeA was found in in 4 isolates (2% among E. coli positive samples and 1% among cattle sampled) and a combination of stx1 or stx2 and eaeA and hly in 1 isolate (0.5% and 0.2%). More than 95% of isolates were susceptible to gentamicin, ceftriaxone, cyprofloxacin and cefotaxime while high rates of resistance were recorded for cephalotin, ampicillin, tetracycline, tripe sulfa and streptomycin. The multivariate analysis identified "age" as the factor most closely related to E. coli positivity (either generic E. coli or STEC) in bovine lymph nodes. In conclusion, subiliac lymph nodes represent a source of E. coli for ground beef. These results are of major importance for risk assessment and improving good manufacturing practices during animal slaughter and ground meat production.

Simple Summary Staphylococcus aureus is not only a common cause of bovine mastitis, but also an agent of food poisoning in humans. S. aureus has the ability to produce branched-chain amino acids (BCAAs) under certain nutritional conditions. We show that levels of two BCCA (leucine and isoleucine) are correlated to the load of S. aureus in composite milk samples. The application of the ANOVA and Tukey-Kramer analysis showed statistically significant differences in the content of the BCAAs, isoleucine and leucine, between the two groups based on S. aureus positivity (p < 0.001), while bivariate Pearson correlation analysis showed a strong relationship between S. aureus load and the content of these two BCAAs. Abstract The early diagnosis of mastitis is an essential factor for the prompt detection of the animal for further actions. In fact, if not culled, infected cows must be segregated from the milking herd and milked last, or milked with separate milking units. Besides microbiological analysis, the somatic cell count (SCC) commonly used as predictor of intramammary infection, frequently lead to a misclassification of milk samples. To overcome these limitations, more specific biomarkers are continuously evaluated. The total amino acid content increases significantly in mastitic milk compared to normal milk. S. aureus requires branched-chain amino acids (BCAAs—isoleucine, leucine, and valine) for protein synthesis, branched-chain fatty acids synthesis, and environmental adaptation by responding to their availability via transcriptional regulators. The increase of BCAAs in composite milk has been postulated to be linked to mammary infection by S. aureus. The aim of this work is to demonstrate, by a direct ion-pairing reversed-phase method, based on the use of the evaporative light-scattering detector (IP-RP-HPLC-ELSD), applied to 65 composite cow milk samples, a correlation between the concentration of isoleucine and leucine, and S. aureus load. The correlation coefficient, r, was found to be 0.102 for SCC (p = 0.096), 0.622 for isoleucine (p < 0.0001), 0.586 for leucine (p < 0.0001), 0.013 for valine (p = 0.381), and 0.07 for tyrosine (p = 0.034), standing for a positive correlation between S. aureus and isoleucine and leucine concentration. The link between the content of BCAAs, isoleucine and leucine, and udder infection by S. aureus demonstrated with our study has an important clinical value for the rapid diagnosis of S. aureus mastitis in cows.

Cattle are a major reservoir of Shiga toxin-producing Escherichia coli. This study investigated the occurrence of seven major STEC serogroups including O157, O145, O103, O121, O111, O45 and O26 among 578 STEC isolates previously recovered from 559 cattle. The isolates were characterized for serotype and major virulence genes. Polymerase chain reaction revealed that 41.7% (241/578) of isolates belonged to STEC O157, O145, O103, O121, O45 and O26, and 33 distinct serotypes. The 241 isolates corresponded to 16.5% (92/559) of cattle that were STEC positive. The prevalence of cattle that tested positive for at least one of the six serogroups across the five farms was variable ranging from 2.9% to 43.4%. Occurrence rates for individual serogroups were as follows: STEC O26 was found in 10.2% (57/559); O45 in 2.9% (16/559); O145 in 2.5% (14/559); O157 in 1.4% (8/559); O121 in 1.1% (6/559); and O103 in 0.4% (2/559). The following proportions of virulence genes were observed: stx1, 69.3% (167/241); stx2, 96.3% (232/241); eaeA, 7.1% (17/241); ehxA, 92.5% (223/241); and both stx1 and stx2, 62.2% (150/241) of isolates. These findings are evidence that cattle in South Africa carry STEC that belong to six major STEC serogroups commonly incriminated in human disease. However, only a subset of serotypes associated with these serogroups were clinically relevant in human disease. Most STEC isolates carried stx1, stx2 and ehxA but lacked eaeA, a major STEC virulence factor in human disease.

Listeria monocytogenes has been referred to as a concern microorganism in cheese making due to its ability to survive and grow in a wide range of environmental conditions, such as refrigeration temperatures, low pH and high salt concentration at the end of the production process. Since cheese may be a potential hazard for consumers, especially high-risk consumers (e.g., pregnant, young children, the elderly, people with medical conditions), efforts of the dairy industry have been aimed at investigating new conservation techniques based on natural additives to meet consumers’ demands on less processed foods without compromising the food safety. Thus, the aim of this study was to evaluate the efficacy of Myrtus communis L. (myrtle) and Rosmarinus officinalis L. (rosemary) essential oils (EO) against Listeria monocytogenes ATCC 679 spiked in sheep cheese before ripening. After the cheesemaking process, the samples were stored at 8 °C for 2 h, 1 d, 3 d, 14 d and 28 d. The composition of EO was identified by gas chromatography-mass spectrometry (GC-MS) analysis. Constituents such as 1,8-cineole, limonene, methyl-eugenol, α-pinene, α-terpineol, α-terpinolene and β-pinene were present in both EO, accounting for 44.61% and 39.76% from the total of chemical compounds identified for myrtle and rosemary EO, respectively. According to the chemical classification, both EO were mainly composed of monoterpenes. Minimum inhibitory concentration (MIC) against L. monocytogenes was obtained at 31.25 μL/mL to myrtle EO and at 0.40 μL/mL to rosemary EO. Then, cheeses were inoculated with L. monocytogenes (Ca. 6 log CFU/mL) and EO was added at MIC value. The addition of rosemary and myrtle EO displayed lower counts of L. monocytogenes (p &lt, 0.01) (about 1–2 log CFU/g) during the ripening period compared to control samples. Ripening only influences (p &lt, 0.001) the growth of L. monocytogenes in control samples. Since rosemary and myrtle EO do not exert any negative impact on the growth of native microflora (p &gt, 0.05), their use as natural antimicrobial additives in cheese demonstrated a potential for dairy processors to assure safety against L. monocytogenes.

California is the largest agricultural producer in the United States and local dairy industry produces 21.5% of the national milk supply. There are 1470 dairies, 1789 million dairy cows and a total annual milk production of 18 million metric tons. The amount of dead cows to be disposed of is remarkable in intensive farming and it increases in periods of extreme weather events, such as drought in California. Composting of bovine mortalities is prohibited in California as a means of disposal of carcases, and can only be done under an emergency declaration. Composting is an effective disposal method that can aid in carcase disposal, especially during an emergency. The objective of this study was to use screened dairy manure solids (SDMS) as the composting amendment for carcase decomposition. Our hypothesis was that temperatures would be sufficiently high and of sufficient duration to destroy most bacteria within the carcases and that the leachate from the carcases would penetrate less than one foot into the underlying soil. No significant amounts of leachate were noted in the collection pipes buried beneath either soil type. Total bacterial counts exceeded 1 × 106 CFU/ml in approximately 19% of the swab samples from the sample collection pipes. The sandy soil had higher bacterial counts than the clay soil. Results of these trials indicate that adult dairy cows can be successfully composted without significant impact on the nearby surrounding environment. The basic hypotheses have been verified by the simple and multiple regression and chi-square non-parametric test.

The early diagnosis of mastitis is an essential factor for the prompt detection of the animal for further actions. In fact, if not culled, infected cows must be segregated from the milking herd and milked last, or milked with separate milking units. Besides microbiological analysis, the somatic cell count (SCC) commonly used as predictor of intramammary infection, frequently lead to a misclassification of milk samples. To overcome these limitations, more specific biomarkers are continuously evaluated. The total amino acid content increases significantly in mastitic milk compared to normal milk. S. aureus requires branched-chain amino acids (BCAAs&mdash, isoleucine, leucine, and valine) for protein synthesis, branched-chain fatty acids synthesis, and environmental adaptation by responding to their availability via transcriptional regulators. The increase of BCAAs in composite milk has been postulated to be linked to mammary infection by S. aureus. The aim of this work is to demonstrate, by a direct ion-pairing reversed-phase method, based on the use of the evaporative light-scattering detector (IP-RP-HPLC-ELSD), applied to 65 composite cow milk samples, a correlation between the concentration of isoleucine and leucine, and S. aureus load. The correlation coefficient, r, was found to be 0.102 for SCC (p = 0.096), 0.622 for isoleucine (p &lt, 0.0001), 0.586 for leucine (p &lt, 0.0001), 0.013 for valine (p = 0.381), and 0.07 for tyrosine (p = 0.034), standing for a positive correlation between S. aureus and isoleucine and leucine concentration. The link between the content of BCAAs, isoleucine and leucine, and udder infection by S. aureus demonstrated with our study has an important clinical value for the rapid diagnosis of S. aureus mastitis in cows.

In this study, 140 cattle STEC isolates belonging to serogroups O157, O26, O145, O121, O103 and O45 were characterized for 38 virulence-associated genes, antimicrobial resistance profiles and genotyped by PFGE. The majority of isolates carried both stx1 and stx2 concurrently, stx2c, and stx2d; plasmid-encoded genes ehxA, espP, subA and saa but lacked katP and etpD and eaeA. Possession of eaeA was significantly associated with the presence of nle genes, katP, etpD, ureC and terC. However, saa and subA, stx1c and stx1d were only detected in eaeA negative isolates. A complete OI-122 and most non-LEE effector genes were detected in only two eaeA positive serotypes, including STEC O157:H7 and O103:H2. The eaeA gene was detected in STEC serotypes that are commonly implicated in severe humans disease and outbreaks including STEC O157:H7, STEC O145:H28 and O103:H2. PFGE revealed that the isolates were highly diverse with very low rates of antimicrobial resistance. In conclusion, only a small number of cattle STEC serotypes that possessed eaeA, had the highest number of virulence-associated genes, indicative of their high virulence. Further characterization of STEC O157:H7, STEC O145:H28 and O103:H2 using whole genome sequencing will be needed to fully understand their virulence potential for humans.

Shiga toxin-producing Escherichia coli (STEC) isolates (N = 38) that were incriminated in human disease from 2006 to 2013 in South Africa were characterized by serotype, virulence-associated genes, antimicrobial resistance and pulsed-field gel electrophoresis (PFGE). The isolates belonged to 11 O:H serotypes. STEC O26:H11 (24%) was the most frequent serotype associated with human disease, followed by O111:H8 (16%), O157:H7 (13%) and O117:H7 (13%). The majority of isolates were positive for key virulence-associated genes including stx1 (84%), eaeA (61%), ehxA (68.4%) and espP (55%), but lacked stx2 (29%), katP (42%), etpD (16%), saa (16%) and subA (3%). stx2 positive isolates carried stx2c (26%) and/or stx2d (26%) subtypes. All pathogenicity island encoded virulence marker genes were detected in all (100%) isolates except nleA (47%), nleC (84%) and nleD (76%). Multidrug resistance was observed in 89% of isolates. PFGE revealed 34 profiles with eight distinct clusters that shared &ge, 80% intra-serotype similarity, regardless of the year of isolation. In conclusion, STEC isolates that were implicated in human disease between 2006 and 2013 in South Africa were mainly non-O157 strains which possessed virulence genes and markers commonly associated with STEC strains that have been incriminated in mild to severe human disease worldwide. Improved STEC monitoring and surveillance programs are needed in South Africa to control and prevent STEC disease in humans.

Possible contamination by Staphylococcus aureus of the production environment and of the meat of a canned meat production factory was analysed. A total of 108 samples were taken from nine critical control points, 13 of them were positive for S. aureus. None of the isolates produced enterotoxins. To determine how much time can elapse between can seaming and sterilisation in the autoclave without any risk of enterotoxin production by S. aureus, the growth and enterotoxin production of three enterotoxin A producing strains of S. aureus (one ATCC strain and two field strains) in canned meat before sterilisation was investigated at three different temperatures (37, 20 and 10 °C). Two types of meat were used, one with and one without sodium nitrite. In the canned products, the spiked bacteria spread throughout the meat and reached high levels. Enterotoxin production was shown to start 10 hours after incubation at 37 °C and after 48 h after incubation at 20 °C; the production of enterotoxin was always detected in the transition between the exponential and the stationary growth phase. At 10 °C, the enterotoxin was never detected. The statistical analysis of the data showed that the difference between the two different types of meat was not statistically significant (p value > 0.05). Since it is well known that following heat treatment, staphylococcal enterotoxins, although still active (in in vivo assays), can be undetectable (loss of serological recognition) depending on the food matrix and pH, it is quite difficult to foresee the impact of heat treatment on enterotoxin activity. Therefore, although the bacteria are eliminated, the toxins may remain and cause food poisoning. The significance of the results of this study towards implementing good manufacturing practices and hazard analysis critical control points in a canned meat factory are discussed with reference to the management of pre-retorting steps after seaming.