Impact of Fibrous Microplastic Pollution on Commercial Seafood and Consumer Health: A Review.

The omnipresence of microfibers in marine environments has raised concerns about their availability to aquatic biota, including commercial fish species. Due to their tiny size and wide distribution, microfibers may be ingested by wild-captured pelagic or benthic fish and farmed species. Humans are exposed via seafood consumption. Despite the fact that research on the impact of microfibers on marine biota is increasing, knowledge on their role in food security and safety is limited. The present review aims to examine the current knowledge about microfiber contamination in commercially relevant fish species, their impact on the marine food chain, and their probable threat to consumer health. The available information suggests that among the marine biota, edible species are also contaminated, but there is an urgent need to standardize data collection methods to assess the extent of microfiber occurrence in seafood. In this context, natural microfibers should also be investigated. A multidisciplinary approach to the microfiber issue that recognizes the interrelationship and connection of environmental health with that of animals and humans should be used, leading to the application of strategies to reduce microfiber pollution through the control of the sources and the development of remediation technologies.

Prevalence and Antibiotic Resistance Phenotypes of Pseudomonas spp. in Fresh Fish Fillets.

In fresh fish products, excessive loads of Pseudomonas can lead to their rapid spoilage. It is wise for Food Business Operators (FBOs) to consider its presence both in whole and prepared fish products. With the current study, we aimed to quantify Pseudomonas spp. in fresh fillets of Salmo salar, Gadus morhua and Pleuronectes platessa. For all three fish species, we detected loads of presumptive Pseudomonas no lower than 104-105 cfu/g in more than 50% of the samples. We isolated 55 strains of presumptive Pseudomonas and carried out their biochemical identification; 67.27% of the isolates were actually Pseudomonas. These data confirm that fresh fish fillets are normally contaminated with Pseudomonas spp. and the FBOs should add it as a "process hygiene criterion" according to EC Regulation n.2073/2005. Furthermore, in food hygiene, it is worth evaluating the prevalence of antimicrobial resistance. A total of 37 Pseudomonas strains were tested against 15 antimicrobials, and they all were identified as being resistant to at least one antimicrobial, mainly penicillin G, ampicillin, amoxicillin, tetracycline, erythromycin, vancomycin, clindamycin and trimethoprim. As many as 76.47% of Pseudomonas fluorescens isolates were multi-drug resistant. Our results confirm that Pseudomonas is becoming increasingly resistant to antimicrobials and thus should be continuously monitored in foods.

Machine Learning Algorithms Highlight tRNA Information Content and Chargaff's Second Parity Rule Score as Important Features in Discriminating Probiotics from Non-Probiotics.

Probiotic bacteria are microorganisms with beneficial effects on human health and are currently used in numerous food supplements. However, no selection process is able to effectively distinguish probiotics from non-probiotic organisms on the basis of their genomic characteristics. In the current study, four Machine Learning algorithms were employed to accurately identify probiotic bacteria based on their DNA characteristics. Although the prediction accuracies of all algorithms were excellent, the Neural Network returned the highest scores in all the evaluation metrics, managing to discriminate probiotics from non-probiotics with an accuracy greater than 90%. Interestingly, our analysis also highlighted the information content of the tRNA sequences as the most important feature in distinguishing the two groups of organisms probably because tRNAs have regulatory functions and might have allowed probiotics to evolve faster in the human gut environment. Through the methodology presented here, it was also possible to identify seven promising new probiotics that have a higher information content in their tRNA sequences compared to non-probiotics. In conclusion, we prove for the first time that Machine Learning methods can discriminate human probiotic from non-probiotic organisms underlining information within tRNA sequences as the most important genomic feature in distinguishing them.

Virulence Characteristics and Distribution of the Pathogen Listeria ivanovii in the Environment and in Food

Listeria ivanovii and L. monocytogenes, are the only pathogenic species of the genus Listeria and share many virulence factors and mechanisms of pathogenicity. L. ivanovii shows host tropism towards small ruminants and rodents and much lower virulence for humans compared to L. monocytogenes. However, severe infections caused by L. ivanovii, resulting in bacteremia, abortion and stillbirth, occasionally occurred in immunocompromised persons and in pregnant women, while in immunocompetent hosts L. ivanovii can cause gastroenteritis. In this review, the updated knowledge on virulence aspects and distribution of L. ivanovii in the environment and in food is summarized. Recent research on its virulence characters at genome level gave indications on how pathogenicity evolved in this bacterial species. As for L. monocytogenes, L. ivanovii infections occurred after the ingestion of contaminated food, so an overview of reports regarding its distribution in food products was carried out to obtain indications on the categories of foods exposed to contamination by L. ivanovii. It was found that a wide variety of food products can be a source of this microorganism and that, like L. monocytogenes, L. ivanovii is able to persist in the food production environment. Studies on its ability to grow in enrichment and isolation media suggested that its occurrence in nature might be underestimated. Moreover, virulence varies among strains for differences in virulence character regulation, presence/absence of genetic regions and the possible instability of a Listeria pathogenicity genomic island, LIPI-2, which is unique to L. ivanovii. We can conclude that L. ivanovii, as a possible pathogen for animals and humans, requires more focused investigations regarding its occurrence in the environment and in food and on intra-species variability of pathogenic potential.

Evaluation of the microbiological and chemical aspects of autochthonous wild snails in Sardinia.

This study was conducted to acquire knowledge on the epidemiology and ecology of some zoonotic agents in snails. Chemical and microbiological analysis was carried out on 46 samples of snails belonging to the species of Helix aspersa and Helix (Eobania) vermiculata. The association between heavy metals and wild snails, a native consumer product in the Region of Sardinia, was determined. The molecular characterisation of Listeria monocytogenes virulence genes has shown a genetic profile that deserves more attention for the improvement of surveillance and risk prevention. Specimens of H. vermiculata showed higher concentrations of cadmium (M=0.80±0.56 mg/kg) than H. aspersa (M=0.61±0.17 mg /kg). A further objective was to determine whether the samples showed significant differences from the point of view of secretion characterisation, in terms of protein content, and to identify species-specific correlations and possible relationships with the environment. The presence of Salmonella enterica sub.sp houtenae (6,14: z4, z23) and Salmonella enterica subsp diarizonae (47: k: e, n, z15) (1 sample), Listeria monocytogenes (2 samples) with Molecular characterization of virulence genes together with the measurement of heavy metals in samples of wild snails has shown a health and hygiene profile that would deserve greater attention for the improvement of the surveillance and prevention of microbiological and chemical risk in such products which currently show a tendency towards increase in consumption.

Microplastics in Fish and Fishery Products and Risks for Human Health: A Review.

In recent years, plastic waste has become a universally significant environmental problem. Ingestion of food and water contaminated with microplastics is the main route of human exposure. Fishery products are an important source of microplastics in the human diet. Once ingested, microplastics reach the gastrointestinal tract and can be absorbed causing oxidative stress, cytotoxicity, and translocation to other tissues. Furthermore, microplastics can release chemical substances (organic and inorganic) present in their matrix or previously absorbed from the environment and act as carriers of microorganisms. Additives present in microplastics such as polybrominated diphenyl ethers (PBDE), bisphenol A (BPA), nonylphenol (NP), octylphenol (OP), and potentially toxic elements can be harmful for humans. However, to date, the data we have are not sufficient to perform a reliable assessment of the risks to human health. Further studies on the toxicokinetics and toxicity of microplastics in humans are needed.

Challenge Test as Special Tool to Estimate the Dynamic of Listeria monocytogenes and Other Foodborne Pathogens.

Over 23 million cases of foodborne disease (FBD) occur in Europe each year, with over 4700 deaths. Outbreaks of FBD have a significant impact on our society due to the high economic losses they cause (hospital treatment of affected patients and destruction of contaminated food). Among its health objectives, the European Union has set itself the goal of reducing the incidence of the main FBDs, approving various regulations that codify requirements in order to produce food that is "safe" for human consumption. Among these rules, Regulation 2005/2073 establishes precise food safety criteria for foods that are judged to be most at risk of causing episodes of FBD. The food business operator (FBO) must know their food better and know how to estimate whether a food can support the growth of food pathogens or if they are able to hinder it during the food's shelf life. It is becoming crucial for each FBO to schedule specific laboratory tests (challenge tests) to establish the growth potential of individual pathogens and their maximum growth rate. In 2008 the European Union published the guidelines for programming the challenge tests for Listeria monocytogenes in RTE foods. These guidelines were further implemented in 2014 and again in 2019. In June 2019 the UNI EN ISO 20976-1 was published, which contains indications for setting up and carrying out challenge tests for all foodborne pathogens in all foods. In this article, we compare the three official documents to highlight their common aspects and differences, highlighting the advantages and disadvantages that each of them offers for those who have to set up a challenge test for the various foodborne pathogens. Our conclusion is that the challenge test is today the most effective tool to estimate the dynamics and growth potential of pathogenic microorganisms in food, if it is designed and implemented in a scrupulous way. It is important to develop a rational experimental design for each challenge test, and for each food, and this requires professionals who are experts in this specific field of study and who must be properly trained.

Occurrence, Diversity of Listeria spp. Isolates from Food and Food-Contact Surfaces and the Presence of Virulence Genes.

This study evaluates the hazards posed by foodborne bacteria of the Listeria genus by analyzing the occurrence, diversity and virulence of Listeria spp.in food and food-manufacturing plants. Seventy-five isolates obtained from the routine analysis of 653 samples taken by three diagnostic laboratories in Northern Italy were genotypically differentiated by Repetitive Extragenic Palindrome (rep) PCR, with the GTG5 primer identified by sequencing the 16S rRNA gene and examined by specific PCR tests for the presence of L. monocytogenes virulence determinants occasionally found to occur in other species of the genus. Within this sample, 76% (n = 57) isolates were identified as L. innocua, 16% (n = 12) as L. monocytogenes, 6.6 % (n = 5) as L. welshimeri and 1.3% (n = 1) as L. seeligeri. All L. monocytogenes isolates belonged to the serotype 1/2a and were predicted to be virulent for the presence of the inlJ internalin gene. Potentially virulent strains of L. innocua, L. seeligeri and L. welshimeri, carrying the L. monocytogenesinlA gene and/or hly gene, were identified, and most isolates were found to possess the toxin-antitoxin system mazEF for efficient adaptation to heat shock. Results indicated the need to reinforce food-contamination-prevention measures against all Listeria species by defining efficiently their environmental distribution.

Multi-Analyte MS Based Investigation in Relation to the Illicit Treatment of Fish Products with Hydrogen Peroxide.

Fishery products are perishable due to the action of many enzymes, both endogenous and exogenous. The latter are produced by bacteria that may contaminate the products. When fishes age, there is a massive bacteria growth that causes the appearance of off-flavor. In order to obtain "false" freshness of fishery products, an illicit treatment with hydrogen peroxide is reported to be used. Residues of hydrogen peroxide in food may be of toxicology concern. We developed two mass spectrometry based methodologies to identify and quantify molecules related to the treatment of fishes with hydrogen peroxide. With ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) we evaluated the concentration of trimethylamine-N-oxide (TMAO), trimethylamine (TMA), dimethylamine (DMA), and cadaverine (CAD) in fish products. After evaluating LOQ, we measured and validated the lower limits of quantification (LLOQs as first levels of calibration curves) values of 50 (TMAO), 70 (TMA), 45 (DMA), and 40 (CAD) ng/mL. A high ratio between TMAO and TMA species indicated the freshness of the food. With a GC-MS method we confirmed the illicit treatment measuring the levels of H2O2 after an analytical reaction with anisole to give 2-hydroxyanisole as a marker. This latter product was detected in the headspace of the homogenized sample with simplification of the work-up. A LLOQ of 50 ng/mL was checked and validated. When fish products were whitened and refreshed with hydrogen peroxide, the detected amount of the product 2-hydroxyanisole could be very important, (larger than 100 mg/kg). The developed analytical methods were suitable to detect the illicit management of fishery products with hydrogen peroxide; they resulted as sensitive, selective, and robust.

Distribution of Listeria spp. on Carcasses of Regularly Slaughtered Swine for Italian Dry Cured Ham.

HIGHLIGHTS: Swine carcasses are often contaminated with Listeria spp. Heads are more contaminated than shoulders and thighs. Lairage time higher than 10 h is a risk factor for Listeria spp. contamination. Closed-cycle farms presented greater carcass contamination.

Undeclared animal species in dry and wet novel and hydrolyzed protein diets for dogs and cats detected by microarray analysis.

BACKGROUND: Although the European Pet Food Industry Federation (FEDIAF) stated that labels must be accurate and provide detailed information on the ingredients, mislabeling of pet food has been documented by several authors. This phenomenon is of particular concern when related to products used as elimination diets for the diagnosis of adverse food reaction (AFR) in dogs and cats because the presence of undeclared ingredients may negatively interfere with the trial and prevent the veterinarian from making an appropriate diagnosis. The aim of this study was to shed light upon the problem of contamination and mislabeling in both dry and wet novel protein diets (NPDs) and hydrolyzed protein diets (HPDs) using a microarray-based commercial kit which tests for the presence of 19 animal species. RESULTS: Of the 40 analyzed products (9 dry NPDs, 22 wet NPDs, 6 dry HPDs and 3 wet HPDs), ten presented a content that correctly matched the label, while five did not contain the declared animal species, twenty-three revealed the presence of undeclared animal species, and two had a vague label that did not allow the evaluation of its accuracy. The most frequently contaminants identified in both dry and wet pet foods were pork, chicken and turkey. The presence of undeclared animal species was higher in dry than wet pet foods; furthermore, a lower number of contaminating animal species was identified in HPDs than NPDs (4 vs 10), and a lower number of contaminated HPDs (6 out of 9, 67%) than contaminated NPDs was detected (24 out of 31, 77%). Thirteen out of 14 brands tested presented at least one mislabeled product. CONCLUSIONS: Mislabeling seems to be a widespread issue in pet foods used as elimination diets. Contamination can occur in all types of products used for the purpose, although dry NPDs are the main issue. Due to the high risk of contamination, particular attention should be given to both the selection of raw material suppliers and the production process.

Effect of diet and packaging system on the microbial status, pH, color and sensory traits of rabbit meat evaluated during chilled storage.

The combined effect of two dietary lipid sources (sunflower vs linseed oil) and three packaging methods (PVC film - BAG, modified atmosphere CO2 20% + 80% O2 - MAP, and vacuum - VAC) on the shelf-life (pH, color, microbial count, sensory traits) of rabbit loins was tested at days 1, 4 and 10 of refrigerated storage. Linseed oil did not adversely affect neither the physical or sensory traits, nor the microbiological quality of rabbit meat throughout the shelf-life trial. The three packaging methods were all similarly effective in maintaining the physical and sensory quality of the meat, irrespective of the different dietary lipid sources and thus different susceptibility to oxidation. MAP and VAC were more effective than BAG in preserving rabbit meat hygienic quality, as highlighted by the microbial count analyzed at day 4 of shelf-life (P < 0.001). At day 10, the microbiological quality didn't differ between MAP and VAC rabbit meat samples.

A Limited Survey of Heavy Metal Concentrations in Fresh and Frozen Cuttlefish Ink and Mantle Used As Food.

Cuttlefish ink is consumed as a delicacy worldwide. The current study is the first assessment of heavy metal concentrations in cuttlefish ink versus mantle under different storage methods. A total of 212 samples (64 of fresh mantle, 42 of frozen mantle, 64 of fresh ink, and 42 of frozen ink) were analyzed for the detection of the following heavy metals: arsenic (As), chromium (Cr), iron (Fe), lead (Pb), mercury (Hg), and cadmium (Cd). The median As concentrations were 12.9 mg/kg for fresh mantle, 8.63 mg/kg for frozen mantle, 10.8 mg/kg for frozen ink, and 0.41 mg/kg for fresh ink. The median Cr concentrations were 0.06 mg/kg for fresh mantle and frozen ink, 0.03 mg/kg for frozen mantle, and below the limit of quantification (LOQ) for fresh ink. The median Fe concentrations were 4.08 mg/kg for frozen ink, 1.51 mg/kg for fresh mantle, 0.73 mg/kg for frozen mantle, and below the LOQ for fresh ink. The median Pb concentrations of almost all samples were below the LOQ; only two frozen ink, one fresh ink, one frozen mantle, and one fresh mantle sample exceeded the limit stipulated by the European Union. The Hg concentrations were statistically similar among the four categories of samples; the median Hg concentrations were below the LOQ, and the maximum concentrations were found in frozen ink, at 1.62 mg/kg. The median Cd concentrations were 0.69 mg/kg for frozen ink and 0.11 mg/kg for frozen mantle, fresh mantle and fresh ink concentrations were below the LOQ, and in 11.3% of the tested samples, Cd concentrations were higher than the European Union limit. The probability of samples having a Cd concentration above the legal limit was 35.75 times higher in frozen than in fresh products. Fresh ink had significantly lower concentrations of As, Cr, Fe, and Cd, but the concentrations of Hg and Pb were not significantly different from those of other products. Frozen ink had significantly higher concentrations of Cd, Cr, and Fe, but concentrations of As were lower than those in fresh mantle, pointing out a possible role for the freezing process and for different fishing zones as risk factors for heavy metal contamination.

Antimicrobial resistance: A global emerging threat to public health systems.

Antimicrobial resistance (AMR) became in the last two decades a global threat to public health systems in the world. Since the antibiotic era, with the discovery of the first antibiotics that provided consistent health benefits to human medicine, the misuse and abuse of antimicrobials in veterinary and human medicine have accelerated the growing worldwide phenomenon of AMR. This article presents an extensive overview of the epidemiology of AMR, with a focus on the link between food producing-animals and humans and on the legal framework and policies currently implemented at the EU level and globally. The ways of responding to the AMR challenges foresee an array of measures that include: designing more effective preventive measures at farm level to reduce the use of antimicrobials; development of novel antimicrobials; strengthening of AMR surveillance system in animal and human populations; better knowledge of the ecology of resistant bacteria and resistant genes; increased awareness of stakeholders on the prudent use of antibiotics in animal productions and clinical arena; and the public health and environmental consequences of AMR. Based on the global nature of AMR and considering that bacterial resistance does not recognize barriers and can spread to people and the environment, the article ends with specific recommendations structured around a holistic approach and targeted to different stakeholders.

Prevalence and quantification of thermophilic Campylobacter spp. in Italian retail poultry meat: Analysis of influencing factors.

Retail poultry meat is a crucial vehicle for consumers' exposure to Campylobacters, but no official controls are currently applied in Italy. The aim of this study was the evaluation of Campylobacter contamination of a wide range of poultry meats marketed in Italy. N. 472 chicken and turkey meat samples (sectioned meats, offal, meat preparations and products) were taken from slaughterhouses, deboning plants and different retailers and submitted to detection/enumeration of Campylobacter spp. The isolates were identified by phenotypic and biomolecular techniques. Campylobacter spp. was detected in 34.1% of the samples, with general low counts. Higher values were observed in offal (especially liver) and sectioned meats, with significantly higher rates in skin-on samples (86.8% vs 32.7%). Minced meat preparations showed lower prevalence (22.4% vs 58.3%) and counts than whole pieces. Decreasing rates were observed among slaughterhouses (80%), deboning plants (49%), butcher's shops (37%) and large scale retailers (25%). Sectioned chicken meats were significantly more contaminated than turkey meats. Almost all the isolates were identified as C. jejuni or C. coli, with similar prevalences (18.4% and 20.5%, respectively); C. jejuni was predominant only in samples from slaughterhouses/deboning plants. For setting future control programs, meat typology should be considered the main critical factor.

Study of the Population Dynamics of Listeria Monocytogenes and Pseudomonas Fluorescens in Buffalo Mozzarella by Means of Challenge Testing.

Campania's buffalo mozzarella is a greatly appreciated cheese in Italy and worldwide. From a microbiological standpoint, it is a highly perishable food and potentially at risk of contamination by pathogens such as Listeria monocytogenes (L. monocytogenes). The present paper reports the results of a challenge test carried out to assess the population dynamics of L. monocytogenes, alone and in presence of Pseudomonas fluorescens (P. fluorescens), in buffalo mozzarella. For this purpose buffalo mozzarella samples were contaminated with L. monocytogenes alone or combined with P. fluorescens. In samples where L. monocytogenes was inoculated alone, the bacterial load remained unchanged. On the contrary, in samples contaminated with L. monocytogenes and P. fluorescens, the growth of L. monocytogenes increased.

Resistance to Biocides in Listeria monocytogenes Collected in Meat-Processing Environments.

The emergence of microorganisms exerting resistance to biocides is a challenge to meat-processing environments. Bacteria can be intrinsically resistant to biocides but resistance can also be acquired by adaptation to their sub-lethal concentrations. Moreover, the presence of biocide resistance determinants, which is closely linked to antibiotic resistance determinants, could lead to co-selection during disinfection practices along the food chain, and select cross-resistant foodborne pathogens. The purpose of this work was to test the resistance of wild strains of Listeria monocytogenes, isolated from pork meat processing plants, toward benzalkonium chloride (BC), used as proxy of quaternary ammonium compounds. Furthermore, the expression of two non-specific efflux pumps genes (lde and mdrL) under biocide exposure was evaluated. L. monocytogenes were isolated from five processing plants located in the Veneto region (northeast of Italy) before and after cleaning and disinfection (C&D) procedures. A total of 45 strains were collected: 36 strains before and nine after the C&D procedures. Collected strains were typed according to MLST and ERIC profiles. Strains sampled in the same site, isolated before, and after the C&D procedures and displaying the same MLST and ERIC profiles were tested for their sensitivity to different concentrations of BC, in a time course assay. The expression of non-specific efflux pumps was evaluated at each time point by qPCR using tufA gene as housekeeping. A differential expression of the two investigated genes was observed: lde was found to be more expressed by the strains isolated before C&D procedures while its expression was dose-dependent in the case of the post C&D procedures strain. On the contrary, the expression of mdrL was inhibited under low biocidal stress (10 ppm BC) and enhanced in the presence of high stress (100 ppm BC). These findings suggests a possible role for C&D procedures to select L. monocytogenes persisters, pointing out the importance of dealing with the identification of risk factors in food plants sanification procedures that might select more tolerant strains.

Students' Consumption of Beverages and Snacks at School and Away from School: A Case Study in the North East of Italy.

In North-East Italy (the Veneto region), several public school nutrition policies have been developed to reduce the consumption of high-caloric snacks and beverages. However, little is known about whether the policies actually influence students' dietary behaviors. In order to address this point, a multi-center cross-sectional survey of 691 Italian students was conducted. Students completed the Beverage and Snack Questionnaire, which assesses the consumption of beverages and snacks at school and out of school. Three-level Poisson Models with random intercept with students (level 1 units) nested into classroom (level 2 units), and nested into schools (level 3 units), were used to examine the influence of the school setting vs. the out of school environment (independent variable) on students' consumption of sweet beverages, snacks, milk-based beverages, low-carbohydrate drinks, fruit, and vegetables (dependent variable) (p ≤ 0.05). The results showed a significantly higher consumption of sweet beverages, snacks, milk-based beverages, low-carbohydrate drinks, fruit, and vegetables out-of-the school, suggesting a school-protective association Thus, the policies aimed to limit or deny access to unhealthy foods in the school environment may play an important role in promoting more healthful dietary patterns for school children. Additional studies should be conducted to compare students' dietary behaviors between schools with nutrition policies to those without nutrition policies.