Quantitative Detection of Viable but Nonculturable Vibrio parahaemolyticus in Frozen Bivalve Molluscs.

Vibrio parahaemolyticus is a foodborne pathogen diffusely distributed in the marine environment and often isolated from raw seafood belonging to different species, mostly shellfish. Ingestion of under- or uncooked seafood contaminated by V. parahaemolyticus can cause severe gastrointestinal symptoms in humans. Due to its ability to withstand low temperatures, Vibrio spp. could survive in frozen seafoods for long periods by entering the viable but nonculturable state (VBNC) and may constitute an unrecognized source of food contamination and infection. In the present study, seventy-seven frozen bivalve molluscs (35 mussels; 42 clams) were subjected to the detection and enumeration of viable V. parahaemolyticus using standard culture methods. VBNC forms were detected and quantified by applying an optimized protocol based on Propidium Monoazide (PMA) and Quantitative PCR (qPCR). All samples were negative for both the detection and enumeration of V. parahaemolyticus by the standard culture methods. VBNC forms were detected in 11.7% of the samples (9/77), with values ranging from 1.67 to 2.29 Log CFU/g. Only clam samples were positive for the detection of VBNC forms. The results of this study highlighted that VBNC V. parahaemolyticus may be present in frozen bivalve molluscs. Further data on the prevalence of VBNC V. parahaemolyticus in frozen seafood are needed in order to perform a robust risk assessment.

Macroscopic sarcocystosis in a pig carcass from an Italian abattoir.

Different food-safety institutions, including the European Food Safety Authority, encourage monitoring and characterising Sarcocystis spp. in animals and foodstuffs; among meat-producing animals, domestic pigs (Sus scrofa domesticus) can host two different Sarcocystis spp., that is Sarcocystis miescheriana and the zoonotic Sarcocystis suihominis. Herein, we report for the first time the presence of macrocysts of Sarcocystis miescheriana in a domestic pig resulting in carcass condemnation. In North-West Italy, in June 2022 the carcass of a clinically healthy sow was condemned due to the detection of multifocal macroscopic whitish fusiform lesions. Affected muscle samples were submitted to histological and molecular analyses targeting the mtDNA cox1 and 18S rRNA genes. At gross examination and histology, well demarcated, oval or elongated macrocysts up to 8 mm in length characterized by a calcified central core surrounded by fibrosis were detected. The molecular amplification and sequencing of the cox1 mtDNA and 18S rRNA genes revealed the presence of Sarcocystis miescheriana DNA in all sampled macrocysts. Our study provides the first molecularly confirmed case of Sarcocystis miescheriana infection in a domestic pig in Italy. The present report highlights the need to increase data related to the occurrence and the prevalence of Sarcocystis spp. in meat-producing animals, and in wild and domestic pigs in particular, taking into account the zoonotic potential of Sarcocystis suihominis and the possible financial losses related to carcass discard due to macroscopic Sarcocystis spp. cysts.

Understanding the Effect of Ozone on Listeria monocytogenes and Resident Microbiota of Gorgonzola Cheese Surface: A Culturomic Approach.

The occurrence of Listeria monocytogenes on Gorgonzola cheese surface was reported by many authors, with risks arising from the translocation of the pathogen inside the product during cutting procedures. Among the novel antimicrobial strategies, ozone may represent a useful tool against L. monocytogenes contamination on Gorgonzola cheese rind. In this study, the effect of gaseous ozone (2 and 4 ppm for 10 min) on L. monocytogenes and resident microbiota of Gorgonzola cheese rind stored at 4 °C for 63 days was evaluated. A culturomic approach, based on the use of six media and identification of colonies by MALDI-TOF MS, was used to analyse variations of resident populations. The decrease of L. monocytogenes was less pronounced in ozonised rinds with final loads of ~1 log CFU/g higher than controls. This behaviour coincided with a lower maximum population density of lactobacilli in treated samples at day 28. No significant differences were detected for the other microbial determinations and resident microbiota composition among treated and control samples. The dominant genera were Candida, Carnobacterium, Staphylococcus, Penicillium, Saccharomyces, Aerococcus, Yarrowia, and Enterococcus. Based on our results, ozone was ineffective against L. monocytogenes contamination on Gorgonzola rinds. The higher final L. monocytogenes loads in treated samples could be associated with a suppressive effect of ozone on lactobacilli, since these are antagonists of L. monocytogenes. Our outcomes suggest the potential use of culturomics to study the ecosystems of complex matrices, such as the surface of mould and blue-veined cheeses.

Shotgun metagenomic sequencing of bulk tank milk filters reveals the role of Moraxellaceae and Enterobacteriaceae as carriers of antimicrobial resistance genes.

In the present context of growing antimicrobial resistance (AMR) concern, understanding the distribution of AMR determinants in food matrices such as milk is crucial to protect consumers and maintain high food safety standards. Herein, the resistome of different dairy farms was investigated through a shotgun metagenomic sequencing approach, taking advantage of in-line milk filters as promising tools. The application of both the reads-based and the assembly-based approaches has allowed the identification of numerous AMR determinants, enabling a comprehensive resolution of the resistome. Notably most of the species harboring AMR genes were predicted to be Gram-negative genera, namely Enterobacter, Acinetobacter, Escherichia, and Pseudomonas, pointing out the role of these bacteria as reservoirs of AMR determinants. In this context, the use of de novo assembly has allowed a more holistic AMR detection strategy, while the reads-based approach has enabled the detection of AMR genes from low abundance bacteria, usually undetectable by assembly-based methods. The application of both reads-based and assembly-based approaches, despite being computationally demanding, has facilitated the comprehensive characterization of a food chain resistome, while also allowing the construction of complete metagenome assembled genomes and the investigation of mobile genetic elements. Our findings suggest that milk filters can successfully be used to investigate the resistome of bulk tank milk through the application of the shotgun metagenomic sequencing. In accordance with our results, raw milk can be considered a source of AMR bacteria and genes; this points out the importance of properly informing food business operators about the risk associated with poor hygiene practices in the dairy production environment and consumers of the potential microbial food safety risks derived from raw milk products consumption. Translating these findings as risk assessment outputs heralds the next generation of food safety controls.

Effect of gaseous ozone treatment on biofilm of dairy-isolated Pseudomonas spp. strains.

Microbial biofilms existing in food industries have been implicated as important contamination sources of spoilage and pathogenic microorganisms in the finished products. Among the innovative strategies proposed to contrast biofilms in food environments, ozone is recognised as an environmentally friendly technology but there are few studies about its effect against bacterial biofilms. The objective of this study was to evaluate the effect of gaseous ozone (50 ppm for 6 h) in inhibition and eradication of biofilm formed by twenty-one dairyisolated Pseudomonas spp. strains. Before ozone treatments, all isolates were screened for biofilm formation according to a previously described method. Strains were then divided in four groups: weak, weak/moderate, moderate/strong, and strong biofilm producers based on the biofilm biomass value of each isolate determined using the optical density (OD - 595 nm). Inhibition treatment was effective on the strain (C1) belonging to the weak producers' group, on all strains classified as weak/moderate producers, on two strains (C8 and C12) belonging to the group of moderate/strong producers and on one strain (C13) classified as strong producer. Conversely, eradication treatments were ineffective on all strains tested, except for the strain C4 which reduced its biofilm-forming abilities after exposure to ozone gas. In conclusion, gaseous ozone may be used to enhance existing sanitation protocols in food processing environments, but its application alone not seems sufficient to contrast Pseudomonas spp. established biofilms.

Biofilm formation and genomic features of Listeria monocytogenes strains isolated from meat and dairy industries located in Piedmont (Italy).

Listeria monocytogenes is considered a major challenge for the food industry as it can persist for long periods in food processing plants by forming biofilms. The aims of this study were: i) to assess the biofilm producing ability of 57 Listeria monocytogenes isolates previously subjected to whole-genome sequencing (WGS); ii) to compare the levels of biofilm formation with the presence or absence of biofilm associated genes. To determine the presence or absence of a known set of biofilm associated genes, a comparative genomic analysis was performed on each strain. Among Listeria monocytogenes isolates, 58 %, 38.5 % and 3.5 % exhibited weak, moderate or strong biofilm production, respectively. No difference in biofilm production was observed between food and environmental isolates. The percentage of Listeria monocytogenes strains isolated from meat products (57 %) classified as moderate or strong biofilm producers was higher than the percentage obtained for strains isolated from dairy products (28 %). The presence of the Stress Survival Islet 1, the arsD stress gene and the truncated inlA protein was significantly associated with increased levels of biofilm. Combining biofilm phenotype with molecular and genotyping data may provide the opportunity to better understand the relationship between genes linked to biofilm formation in Listeria monocytogenes.

Anisakicidal Effects of R (+) Limonene: An Alternative to Freezing Treatment in the Industrial Anchovy Marinating Process.

Anisakiasis is a fish-borne zoonotic disease caused by the ingestion of raw/undercooked fishes or cephalopods parasitized by members of the genus Anisakis. Freezing ensures the inactivation of viable Anisakis larvae; however, since it affects the organoleptic properties of food, essential oils and their compounds were proposed as an alternative. In this study, fresh anchovy fillets were experimentally parasitized with L3 Anisakis larvae to test the anisakicidal efficacy of R (+) limonene (LMN) in marinated fishery products. The anisakicidal effectiveness and organoleptic influence of several LMN concentrations (0.5%, 1%, and 5%) were tested during the marinating process (MS) and storage in sunflower seed oil (SO) of marinated anchovy fillets. Double treatment (DT) with 1% LMN was also performed both during marination and subsequent storage in oil. MS treatment resulted only in a reduction in larvae viability after 48 h, while a complete inactivation was observed in SO after 8, 10, and 20 days of treatment with 5%, 1%, and 0.5% LMN, respectively. DT was the most effective with complete larval inactivation after 7 days. Only 5% LMN influenced the sensory characteristics of the fillets, resulting, however, in a pleasant lemon-like odor and taste. Considering the results obtained, LMN might be a suitable natural alternative to manage Anisakis risk in the fishery industry.

Development of a predictive model for the shelf-life of Atlantic mackerel (Scomber scombrus).

Despite its commercial value, the shelflife of the Atlantic mackerel (Scomber scombrus) during refrigerated storage was poorly investigated. In this regard, the Quality Index Method (QIM) was proposed as a suitable scoring system for freshness and quality sensorial estimation of fishery products. This study aims to develop a deterministic mathematical model based on dynamic temperatures conditions and a successive statistical analysis of the results obtained. This model will be exploited to predict the shelf-life of the Atlantic mackerel based on specific storage temperatures. A total of 60 fresh fishes were subdivided into two groups and respectively stored in ice for 12 days at a constant temperature of 1±0.5°C (Group A) and a fluctuating temperature ranging between 1 and 7°C (Group B). Microbiological analysis and sensory evaluation through the QIM were performed on each fish at regular time intervals. A critical value of 6 Log cfu/g of spoilage bacteria (mainly psychotropic) associated with a significant decay of the sensorial characteristics was exceeded after 9 days of storage for Group A and 3 days for Group B. A reliable prediction of fish freshness was obtained by modelling the QIM as a function of the spoilage bacteria behaviour. A coefficient ß of correlation was determined to convert the spoilage bacteria load into a Quality Index score. The adoption of mathematical predictive models to assess microbial behaviour under different environmental conditions is an interesting tool for food industries to maximize production and reduce waste.

The Use of Ozone as an Eco-Friendly Strategy against Microbial Biofilm in Dairy Manufacturing Plants: A Review.

Managing spoilage and pathogenic bacteria contaminations represents a major challenge for the food industry, especially for the dairy sector. Biofilms formed by these microorganisms in food processing environment continue to pose concerns to food manufacturers as they may impact both the safety and quality of processed foods. Bacteria inside biofilm can survive in harsh environmental conditions and represent a source of repeated food contamination in dairy manufacturing plants. Among the novel approaches proposed to control biofilm in food processing plants, the ozone treatment, in aqueous or gaseous form, may represent one of the most promising techniques due to its antimicrobial action and low environmental impact. The antimicrobial effectiveness of ozone has been well documented on a wide variety of microorganisms in planktonic forms, whereas little data on the efficacy of ozone treatment against microbial biofilms are available. In addition, ozone is recognized as an eco-friendly technology since it does not leave harmful residuals in food products or on contact surfaces. Thus, this review intends to present an overview of the current state of knowledge on the possible use of ozone as an antimicrobial agent against the most common spoilage and pathogenic microorganisms, usually organized in biofilm, in dairy manufacturing plants.

Effect of Gaseous Ozone on Listeria monocytogenes Planktonic Cells and Biofilm: An In Vitro Study.

Among food-borne pathogens, Listeria monocytogenes continues to pose concerns to food business operators due to its capacity to form biofilm in processing environments. Ozone may be an eco-friendly technology to control microbial contaminations, but data concerning its effect on Listeria monocytogenes biofilm are still limited. In this study, the effect of gaseous ozone at 50 ppm on planktonic cells and biofilm of reference and food-related Listeria monocytogenes strains was evaluated. Ozone caused a reduction in microbial loads of 3.7 ± 0.4 and 3.9 ± 0.4 Log10 CFU/mL after 10 and 30 min, respectively. A complete inactivation of planktonic cells after 6 h of treatment was observed. Biofilm inhibition and eradication treatments (50 ppm, 6 h) resulted in a significant decrease of the biofilm biomass for 59% of the strains tested, whilst a slight dampening of live cell loads in the biofilm state was observed. In conclusion, gaseous ozone is not sufficient to completely counteract Listeria monocytogenes biofilm, but it may be useful as an additional tool to contrast Listeria monocytogenes free-living cells and to improve the existing sanitization procedures in food processing environments.

Molecular detection of cattle Sarcocystis spp. in North-West Italy highlights their association with bovine eosinophilic myositis.

BACKGROUND: Cattle are intermediate hosts of six Sarcocystis species, among which Sarcocystis hominis and Sarcocystis heydorni can infect humans through the consumption of raw or undercooked meat. In addition to the zoonotic potential, there is increasing interest in these protozoa because of the evidence supporting the role of Sarcocystis spp. in the occurrence of bovine eosinophilic myositis (BEM), a specific inflammatory myopathy which leads to carcass condemnation and considerable economic losses. Actually, all the prevalence studies carried out on cattle in Italy have been based on either morphological or 18S rDNA-based molecular techniques, most likely leading to misidentification of closely related species. Therefore, there is a strong need for new data on the prevalence of the different Sarcocystis spp. in cattle in Italy and their association with bovine eosinophilic myositis. METHODS: To reach our aim, individual striated muscle samples from BEM condemned carcasses (N = 54) and diaphragm muscle samples from randomly sampled carcasses (N = 59) were obtained from Northwest Italy slaughterhouses. Genomic DNA was extracted and analyzed by multiplex-PCR targeting 18S rDNA and cox1 genes. PCR products amplified using the genus-specific primer set in absence of the specific fragment for S. hirsuta, S. cruzi, S. hominis or S. bovifelis were sequenced to achieve species identification. RESULTS: Sarcocystis DNA was detected in 67.8% of the samples from slaughter cattle and in 90.7% of the samples from BEM condemned carcasses. S. cruzi was identified as the most prevalent species in slaughter cattle (61%), followed by S. bovifelis (10.2%), S. hominis (8.5%) and S. hirsuta (1.7%). Notably, among the different Sarcocystis spp. detected, the presence of S. bovifelis and S. hominis was significantly higher in samples isolated from BEM condemned carcasses (46.3% and 40.7% respectively), while there was no statistically significant difference between the presence of S. cruzi or S. hirsuta in BEM condemned carcasses (42.6% and 1.8%, respectively) and randomly sampled carcasses. Furthermore, DNA sequence analysis revealed the presence of a putative new species in two carcasses. CONCLUSIONS: Our study contributes to updating the data on the prevalence of the different Sarcocystis spp. in cattle in Italy, highlighting the presence of three Sarcocystis spp., S. cruzi, S. hominis and S. bovifelis, in BEM lesions and allowing us to speculate on the possible role of S. hominis and S. bovifelis as the major sarcosporidian species involved in bovine eosinophilic myositis.

Characterization of the temperature fluctuation effect on shelf life of an octopus semi-preserved product.

The aim of this work is to study the effect of temperature fluctuations on spoilage microbial flora behaviour of a semi-preserved seafood product in modified atmosphere packaging (MAP) as well as to find correct interpretation criteria for simulating temperature fluctuations during storage tests. The study concerned 54 packages of "Octopus carpaccio" that were grouped in three batches and stored at 3 different temperature profiles: the first (16 packages - Group 4°C) was stored at 4±0.5°C; the second (16 packages - Group 8°C) was stored at 8±0.5°C; the third (16 packages - Group F) was stored under a fluctuating temperature regime between 2°C and 14°C. Spoilage microflora, pH and AW has been monitored, at regular intervals, along the storage period (44 days). A predictive model was constructed according to the accredited scientific literature and validated against the observed growth curves of the above three groups. Afterwards, the predictive model has been used setting the temperature at the mean value of fluctuations (6.72°C), at the kinetic mean value of fluctuations (7.80°C) and at the 75th percentile value of fluctuations (11.14°C). The best fitting to the observed data was obtained with the kinetic mean temperature value and this result shows that this parameter can be proposed to reproduce the temperature fluctuation along the distribution and the domestic storage when a storage test has to be carried out.

Activity of R(+) Limonene Against Anisakis Larvae.

The aim of this work is to evaluate the activity of R(+) limonene of against Anisakidae larvae. Its effectiveness was tested in vitro. The results obtained showed a significant activity of the compound against Anisakis larvae, suggesting further investigation on its potential use in the industrial marinating process. In this regard, the use of R(+) limonene in seafood products could be interesting, also due the sensory attributes resulting from its use and its relatively safe status.

Effect of allyl isothiocyanate against Anisakis larvae during the anchovy marinating process.

Allyl isothiocyanate (AITC), is a natural compound found in plants belonging to the family Cruciferae and has strong antimicrobial activity and a biocidal activity against plants parasites. Anisakidosis is a zoonotic disease caused by the ingestion of larval nematodes in raw, almost raw, and marinated and/or salted seafood dishes. The aim of this work was to evaluate the effect of AITC against Anisakis larvae and to study its potential use during the marinating process. The effects of AITC against Anisakis larvae were tested in three experiment: in vitro with three liquid media, in semisolid media with a homogenate of anchovy muscle, and in a simulation of two kinds of anchovy fillets marinating processes. For all tests, the concentrations of AITC were 0, 0.01, 0.05, and 0.1%. Significant activity of AITC against Anisakis larvae was observed in liquid media, whereas in the semisolid media, AITC was effective only at higher concentrations. In anchovy fillets, prior treatment in phosphate buffer solution (1.5% NaCl, pH 6.8) with 0.1% AITC and then marination under standard conditions resulted in a high level of larval inactivation. AITC is a good candidate for further investigation as a biocidal agent against Anisakis larvae during the industrial marinating process.