A Snapshot, Using a Multi-Omic Approach, of the Metabolic Cross-Talk and the Dynamics of the Resident Microbiota in Ripening Cheese Inoculated with Listeria innocua.

Raw milk cheeses harbor complex microbial communities. Some of these microorganisms are technologically essential, but undesirable microorganisms can also be present. While most of the microbial dynamics and cross-talking studies involving interaction between food-derived bacteria have been carried out on agar plates in laboratory-controlled conditions, the present study evaluated the modulation of the resident microbiota and the changes of metabolite production directly in ripening raw milk cheese inoculated with Listeria innocua strains. Using a proxy of the pathogenic Listeria monocytogenes, we aimed to establish the key microbiota players and chemical signals that characterize Latteria raw milk cheese over 60 days of ripening time. The microbiota of both the control and Listeria-inoculated cheeses was analyzed using 16S rRNA targeted amplicon sequencing, while direct analysis in real time mass spectrometry (DART-HRMS) was applied to investigate the differences in the metabolic profiles of the cheeses. The diversity analysis showed the same microbial diversity trend in both the control cheese and the inoculated cheese, while the taxonomic analysis highlighted the most representative genera of bacteria in both the control and inoculated cheese: Lactobacillus and Streptococcus. On the other hand, the metabolic fingerprints revealed that the complex interactions between resident microbiota and L. innocua were governed by continuously changing chemical signals. Changes in the amounts of small organic acids, hydroxyl fatty acids, and antimicrobial compounds, including pyroglutamic acid, hydroxy-isocaproic acid, malic acid, phenyllactic acid, and lactic acid, were observed over time in the L. innocua-inoculated cheese. In cheese that was inoculated with L. innocua, Streptococcus was significantly correlated with the volatile compounds carboxylbenzaldheyde and cyclohexanecarboxylic acid, while Lactobacillus was positively correlated with some volatile and flavor compounds (cyclohexanecarboxylic acid, pyroxidal acid, aminobenzoic acid, and vanillic acid). Therefore, we determined the metabolic markers that characterize a raw milk cheese inoculated with L. innocua, the changes in these markers with the ripening time, and the positive correlation of flavor and volatile compounds with the resident microbiota. This multi-omics approach could suggest innovative food safety strategies based on the enhanced management of undesirable microorganisms by means of strain selection in raw matrices and the addition of specific antimicrobial metabolites to prevent the growth of undesirable microorganisms.

A systematic review on the occurrence of Salmonella in farmed Tenebrio molitor and Acheta domesticus or their derived products.

Insects represent a sustainable and protein-rich food source. This new supply chain requires the study and monitoring of pathogens' presence and impact, as for other farmed animals. Among pathogens, Salmonella is of interest due to the well-established possibility for insects to harbor it. Since Acheta domesticus (cricket) and Tenebrio molitor (mealworm) are the most sold and farmed insect species, the present systematic review aimed to collect, select, and evaluate, in the available scientific literature, studies investigating the occurrence of Salmonella in these species sampled. All available studies published in peer-reviewed journals in English, French, Italian, Portuguese, German, and Spanish were considered. No time limits were imposed. We searched PUBMED, EMBASE, WEB of Science Core Collection, and Food Science and Technology Abstracts. The first date searched was May 10th, 2022; an update of the search was conducted on May 5th, 2023. The data synthesis was presented in tables reporting the number of positives on the number of total analyzed samples with other relevant characteristics of the study. The quality assessment was carried out considering relevant aspects for sampling and the method of analysis for Salmonella detection. At the end of the screening process, 10 and nine studies conducted on crickets and mealworms, respectively, were included for data extraction. The S. serovar Wandsworth and S. serovar Stanley were isolated only in one sample of ready-to-eat crickets. A second study detected OTUs related to S. enterica in cricket and mealworm powders. No studies detected Salmonella in mealworms according to cultural methods. The limitations of the present review are that few studies were retrieved and that included studies had important limitations in terms of study design as sampling was mostly based on convenience and not on a sound statistical basis. The present systematic review underlines the need to obtain reliable data about Salmonella presence in insects considering the growing market and the scaling up of existing farms. This research was funded by the Italian Ministry of Health - Ricerca Corrente IZSVe 03/21. The review protocol was published on the Systematic Reviews for Animals and Food (SYREAF) Web site (https://syreaf.org/protocols/).

A systematic review of experimental studies on Salmonella persistence in insects.

The consumption of insects as food and feed has been recently suggested as a possible alternative to the rising global food need, thus it is crucial to monitor any potential food safety hazards in the insect supply chain. The aims of this systematic review were to collect, select, and evaluate studies investigating the persistence of Salmonella in insects. We searched PUBMED, EMBASE, WEB of Science Core Collection, and Food Science and Technology Abstracts. In total, 36 papers investigating the persistence of Salmonella in insects (both holometabolous and heterometabolous) were included after screening. Regarding complete metamorphosis insects, the longest Salmonella persistence was reported in Phormia regina, in which the pathogen persisted for 29 days at 5 °C. Similarly, Salmonella persisted in the feces of Alphitobius diaperinus for 28 days. The incomplete metamorphosis insect showing the longest Salmonella persistence (>10 months) was Blatella germanica. Periplaneta americana excreted Salmonella via feces for 44 days until all the insects were dead. The retrieved data on the persistence of Salmonella can be useful for further analysis by risk assessors and decision-makers involved in the safety of insect-based food, contributing to defining the sanitary requirements and risk mitigation measures along the supply chain. The review protocol is registered in PROSPERO database (CRD42022329213).

Tetrodotoxin in bivalve mollusks: An integrated study towards the comprehension of the influencing factors of a newly native phenomenon.

Tetrodotoxins (TTXs) are potent neurotoxins named after the Tetraodontidae fish family. The ingestion of TTX-contaminated flesh can cause neurotoxic symptoms and can lead to death. In 2017 symptoms the European Food Safety Authority (EFSA) recognized the threat to food safety resulting from TTX exposure via food consumption and, thus, proposed a safety limit of 44 µg/kg of TTX in marine gastropods and bivalves. To date, however, TTXs have not yet been included in the list of biotoxins to be monitored within the European Union, even though, in a few cases, levels of TTX found were higher than the EFSA limit. The origin of TTX production is debated and the roles of both biotic and abiotic factors on TTX-mediated toxic events remain unclear. In order to meet these knowledge requests the present study was aimed to investigate the role of seawater temperature, pH, water conductivity, and oxygen saturation, along with the marine phytoplankton community and the bacterial community of mussels and oysters on the accumulation of TTX and analogues in the bivalves. Abiotic parameters were measured by means of a multi-parametric probe, phytoplankton community was analyzed by optic microscopy while microbial community was described by amplicon metataxonomic sequencing, TTXs concentration in the collected matrices were measured by HILIC-MS/MS. A possible role of seawater pH and temperature, among the investigated abiotic factors, in regulating the occurrence of TTXs was found. Regarding biotic variables, a possible influence of Vibrio, Shewanella and Flavobacteriaceae in the occurrence of TTXs was found. Concurrently, Prorocentrum cordatum cell numbers were correlated to the incidence of TTX in mussels. The results herein collected suggest that environmental variables play a consistent part in the occurrence of TTX in the edible bivalve habitats, and there are also indications of a potential role played by specific bacteria taxa in association with phytoplankton.

Bombyx mori from a food safety perspective: A systematic review.

Bombyx mori (BM) is an economically important insect for silk production, and it is also farmed and used as food in different countries around the world. The present systematic review aims to assess the suitability of BM as an edible insect, retrieving data from scientific papers reporting microbiological, chemical, and allergenic hazards of silkworm consumed as either whole insects or insect derivatives. We considered all studies published in peer-reviewed journals in English, French, and Spanish languages. No time limits were imposed. We searched PUBMED, WEB of Science Core Collection, and EMBASE databases. The last literature search was carried out on May 5th, 2021. Data were collected in pre-defined tabular forms for the aforementioned hazards. In total, 65 records investigating the safety aspects were included after screening: 32 on microbiological hazards; 27 on chemical hazards; 16 on allergenic hazards. Concerning microbiological aspects, a high presence of Enterococcus in raw insects (5.00 % to 70.10 %) was reported through metagenomic analysis. Through non-metagenomic methods (classical and biomolecular microbiology techniques), Bacillus cereus and Pseudomonas fluorescens were the most commonly investigated and detected bacteria in the unprocessed insects, while B. cereus and Enterobacteriaceae were studied and reported in insect-based food. The foodborne pathogens Listeria monocytogenes and Salmonella spp. were never detected. Concerning toxicological aspects, three studies assessed the toxicity of BM powder in laboratory animals, but no negative effects were observed. Regarding heavy metal bioaccumulation in BM, evidence was reported for As, Cu, and, Zn. Allergic reactions following the ingestion of BM or derivative products are due to proteins that are widespread in arthropods. Furthermore, BM proteins can undergo possible cross-reactions with proteins of other insect species or crustaceans. However, heat treatments do not seem to reduce the allergenic potential of the silkworm proteins. The major limitation of the present review is that we could include only scientific literature published in Western languages, while the majority of relevant studies were conducted in Asian countries and part of them are published in Asian languages. In conclusion, scientific evidence regarding microbiological and chemical hazards of BM relevant for food safety is very limited. In the present work microbiological and chemical hazards relevant for food safety were identified in BM, however their presence do not impair the use as food but suggest the need for a risk assessment under specific conditions of production and use. Allergic reactions are possible in sensitive individuals following the ingestion of edible BM.

Nutritional Composition of Bombyx mori Pupae: A Systematic Review.

As insects have started to enter the eating habits of Western countries, an increasing amount of literature regarding the mulberry silkworm (Bombyx mori) prospective application as food has been published. Despite this growing interest, there is currently no systematic review of silkworm nutritional composition available. In this paper, we performed a systematic review of the recent available literature on the nutrient composition of mulberry silkworm pupae. After screening the titles and abstracts of 14,008 studies retrieved from three scientific databases, data about nutrients was extracted from 29 selected papers, together with their related variables. This systematic review provides an overview of the variety of data reported in the literature and highlights that many elements contribute to hindering a sound comparison of the different nutritional values reported for silkworm pupae. The observed variability of the composition data reported could be due to differences in diet, strains, pretreatments, and origin of the silkworm analyzed. However, all these variables were not always available and should be reported in future studies to simplify the data comparison.

Tetrodotoxin in live bivalve mollusks from Europe: Is it to be considered an emerging concern for food safety?

Tetrodotoxins (TTXs) are a group of potent neurotoxins named after the Tetraodontidae fish family (pufferfish). TTXs have been reported in several animal taxa, both terrestrial and marine. The ingestion of TTX-contaminated flesh can cause serious neurotoxic symptomatology and can eventually lead to death. Traditionally, TTXs have been associated with Asian countries, in particular with pufferfish consumption. However, they have also been reported in bivalve mollusks farmed in the Pacific area and, recently, in European seas. In Europe, different countries have reported TTXs, especially those bordering the Mediterranean Sea. As a consequence, in 2017 the European Food Safety Authority (EFSA) released an opinion with reference to TTX present in marine gastropods and bivalves, proposing a safety limit of 44 µg/kg TTXs in shellfish meat, below which no adverse effects should be observed in humans. Nevertheless, this limit has been exceeded on many occasions in European shellfish and, while for bivalves there have been no registered human intoxications, that is not the case for marine gastropods. However, TTXs have not yet been included in the list of marine biotoxins officially monitored in live bivalve mollusks within the European Union (EU). Thus, the aims of this manuscript are to discuss the increasing occurrence of TTXs in live bivalve mollusks from European sea waters, to acknowledge the still ongoing knowledge gaps that should be covered and to stimulate constructive debate on the eventuality of adopting a shared regulatory context, at least in the EU, for monitoring and managing this potential threat to food safety.

Female sperm storage mediates post-copulatory costs and benefits of ejaculate anticipatory plasticity in the guppy.

Males of many species evolved the capability of adjusting their ejaculate phenotype in response to social cues to match the expected mating conditions. When females store sperm for a prolonged time, the expected fitness return of plastic adjustments of ejaculate phenotype may depend on the interval between mating and fertilization. Although prolonged female sperm storage (FSS) increases the opportunity for sperm competition, as a consequence of the longer temporal overlap of ejaculates from several males, it may also create variable selective forces on ejaculate phenotype, for example by exposing trade-offs between sperm velocity and sperm survival. We evaluated the relationship between the plasticity of ejaculate quality and FSS in the guppy, Poecilia reticulata, a polyandrous live-bearing fish in which females store sperm for several months and where stored sperm contribute significantly to a male's lifelong reproductive success. In this species, males respond to the perception of future mating opportunities by increasing the quantity (number) and quality (swimming velocity) of ready-to-use sperm (an anticipatory response called 'sperm priming'). Here we investigated (a) the effect of sperm priming on in vitro sperm viability at stripping and its temporal decline (as an estimate of sperm survival), and (b) the in vivo competitive fertilization success in relation to female sperm storage using artificial insemination. As expected, sperm-primed males produced more numerous and faster sperm, but with a reduced in vitro sperm viability at stripping and after 4 hr, compared with their counterparts. Artificial insemination revealed that the small (nonsignificant) advantage of primed sperm when fertilization immediately follows insemination is reversed when eggs are fertilized by female-stored sperm, weeks after insemination. By suggesting a plastic trade-off between sperm velocity and viability, these results demonstrate that prolonged female sperm storage generates divergent selection pressures on ejaculate phenotype.

Genes conferring resistance to critically important antimicrobials in Salmonella enterica isolated from animals and food: A systematic review of the literature, 2013-2017.

Antimicrobial resistance is a major public health concern, and food systems are a crucial point in the epidemiology of these resistances. Among antimicrobials, critically important ones are therapeutic drugs that should be primarily safeguarded to allow successful outcomes against important bacterial infections in humans. The most important source of antimicrobial resistance has been recognized in the inappropriate use of antimicrobials in human and animal medicine, with farming being a critical stage. Products of animal origin are the link between animal and humans and can contribute to the spread of antimicrobial resistance, in particular through bacteria such as Enterobacteriaceae, commonly present in both animals' gut and food. Salmonella is an important member of this bacterial family due to its pathogenicity, its noteworthy prevalence and the frequent detection of resistance genes in different isolates. In the present systematic review, the distribution of antimicrobial resistance determinants among Salmonella enterica serovars in pigs, cattle and poultry production was investigated in the European context. A comprehensive literature search was carried out in three different databases, and 7955 papers were identified as relevant. After the different steps of the review process, 31 papers were considered eligible for data extraction to gain insight about sources and reservoirs for such genes. Results suggest that despite the increasing attention directed toward antimicrobial resistance in animal production, a wide plethora of genes still exist and further actions should be undertaken to face this challenge.

Different Resolution Power of Multilocus Variable-Number Tandem Repeat Analysis and Whole-Genome Sequencing in the Characterization of S. 1,4,[5],12:i:- Isolates.

Salmonella enterica serovar 1,4,[5],12:i:- has emerged over the last two decades as one of the most common serovars causing human salmonellosis in Europe. It is supposed to originate from Salmonella enterica serovar Typhimurium due to antigenic and genotypic similarities between the two serovars. Due to the high level of similarity, the multilocus variable-number tandem repeat analysis (MLVA) protocol designed for Salmonella Typhimurium routine typing is commonly used also for the characterization of S. 1,4,[5],12:i. Nevertheless, the Salmonella Typhimurium-based MLVA protocol often shows poor discriminatory power for S. 1,4,[5],12:i. Indeed, only a limited number of MLVA profiles have been described for S. 1,4,[5],12:i:-. Moreover, based on the MLVA clustering, S. 1,4,[5],12:i:- is supposed to display high clonality. The aim of the present work was to assess whether the five loci of Salmonella Typhimurium investigated by MLVA are sufficiently accurate to correctly assign S. 1,4,[5],12:i:- isolates. For this purpose, 38 epidemiologically unrelated S. 1,4,[5],12:i:- were subjected to whole-genome sequencing. Isolates were selected among a collection of monophasic strains isolated in Italy from different sources over the period 2014-2016 and belonging to the five most commonly detected MLVA profiles. Results confirmed the possible clonality for S. 1,4,[5],12:i:- serovar in the light of the scarce difference observed in terms of single-nucleotide polymorphisms (SNPs) among investigated isolates. Nevertheless, unrelated isolates on the basis of the difference of SNP number were characterized as indistinguishable by MLVA profile, thus suggesting an insufficient resolution of MLVA. Hence, we can conclude that MLVA-based approach does not seem a valuable proxy to deepen into the epidemiological relationship among S. 1,4,[5],12:i:- isolates. These evidences can be useful to avoid incorrect assignment especially when surveillance data are used for outbreak investigations.