Antibiotic Resistance in Enterococci and Enterobacteriaceae from Laboratory-Reared Fresh Mealworm Larvae (Tenebrio molitor L.) and Their Frass.

The occurrence of antibiotic-resistant bacteria in foodstuff involves a human health risk. Edible insects are a precious resource; however, their consumption raises food safety issues. In this study, the occurrence of antibiotic resistant bacteria in laboratory-reared fresh mealworm larvae (Tenebrio molitor L.) and frass was assessed. Antibiotics were not used during the rearing. Enterobacteriaceae and enterococci were isolated from 17 larvae and eight frass samples. In total, 62 and 69 isolates presumed to belong to Enterobacteriaceae and Enterococcus spp., respectively, were obtained and tested for antibiotic susceptibility via disk diffusion. Based on the results, isolates were grouped, and representative resistant isolates were identified at species level through 16S rRNA gene sequencing. For enterococci resistance, percentages higher than 15% were observed for vancomycin and quinupristin-dalfopristin, whereas Enterobacteriaceae resistance higher than 25% was found against cefoxitin, ampicillin, and amoxicillin-clavulanic acid. Based on the species identification, the observed resistances seemed to be intrinsic both for enterococci and Enterobacteriaceae, except for some ß-lactams resistance in Shigella boydii (cefoxitin and aztreonam). These could be due to transferable genetic elements. This study suggests the need for further investigations to clarify the role of edible insects in the spreading of antibiotic resistance determinants through the food chain.

Novel foods, old issues: Metabarcoding revealed mislabeling in insect-based products sold by e-commerce on the EU market.

Insects intended for human consumption are considered Novel Foods according to EU legislation. marketed in form of powders, bars, snacks are increasingly available on the EU market, especially on e-commerce. The commercial form and the way of distribution make IBPs particularly prone to mislabeling. Literature concerning the mislabeling occurrence in IBPs is extremely scarce. In this study, 46 processed IBPs were collected on nine EU e-commerce platforms (e-CO) to be authenticated by metabarcoding. A 200 bp region from 16S rRNA gene was used as molecular target. Sequencing data were processed using DADA2 R package, and sequences were taxonomically assigned through BLAST analysis against GenBank. Procedural blanks and positive controls were included in the analysis, and threshold values were established to filter the final data. The mislabeling rate (i. e. the mismatch between the species declared on the IBP label and the species identified by metabarcoding) was calculated. Overall, a high mislabeling rate (33.3 %) was observed, although this percentage is influenced by the e-CO platform and the insect species, with A. domesticus particularly involved. The use of species not listed in authorized Novel Food (e. g. Gryllus locorojo), and/or the partial replacement of high value species with lower value species was highlighted for the first time in processed IBPs. The presence of insect pests was also detected. Metabarcoding was confirmed as an effective tool for IBPs authentication. Also, outcomes from this study can provide useful data on the main issues involving the EU IBPs' market, that can represent an incentive to reinforce both official controls and FBO's self-controls on these poorly investigated products.

Use of Ozone in Veterinary Dentistry as an Alternative to Conventional Antibiotics and Antiseptics.

This paper aims to assess the disinfecting capacity of a double-distilled ozonated water solution as an alternative to common antibiotic and antiseptic devices. Seventy-five dogs were subjected to the surgical procedures of scaling and dental extraction and included in three study groups: Group 1 subjected to antibiotic pre-treatment (association amoxicillin + clavulanic acid and ampicillin + metronidazole) and disinfection with chlorhexidine, and Group 2 and 3 devoid of preventive anti-microbial treatment in which disinfection was performed, respectively, with ozonated water and chlorhexidine. Sampling by bacteriological buffer was carried out to evaluate the bacterial count in the oral cavity. The analysis of the samples determined the total mesophilic bacterial count by seeding on the culture medium via the inclusion of PCA (Plate Count Agar). The results highlighted the anti-bacterial efficacy of intra-operative ozone as an alternative to pre-surgical antibiotic treatment.

VenomPred 2.0: A Novel In Silico Platform for an Extended and Human Interpretable Toxicological Profiling of Small Molecules.

The application of artificial intelligence and machine learning (ML) methods is becoming increasingly popular in computational toxicology and drug design; it is considered as a promising solution for assessing the safety profile of compounds, particularly in lead optimization and ADMET studies, and to meet the principles of the 3Rs, which calls for the replacement, reduction, and refinement of animal testing. In this context, we herein present the development of VenomPred 2.0 (http://www.mmvsl.it/wp/venompred2/), the new and improved version of our free of charge web tool for toxicological predictions, which now represents a powerful web-based platform for multifaceted and human-interpretable in silico toxicity profiling of chemicals. VenomPred 2.0 presents an extended set of toxicity endpoints (androgenicity, skin irritation, eye irritation, and acute oral toxicity, in addition to the already available carcinogenicity, mutagenicity, hepatotoxicity, and estrogenicity) that can be evaluated through an exhaustive consensus prediction strategy based on multiple ML models. Moreover, we also implemented a new utility based on the Shapley Additive exPlanations (SHAP) method that allows human interpretable toxicological profiling of small molecules, highlighting the features that strongly contribute to the toxicological predictions in order to derive structural toxicophores.

Behaviour of Staphylococcus aureus in the Rearing Substrate of Tenebrio molitor Larvae.

Tenebrio molitor (mealworm) is one of the most promising insect species to produce sustainable feed and food with high nutritional value. Insects may harbour microorganisms both in the gut and on the exoskeleton originating from the rearing environment. Staphylococcus aureus is a pathogenic microorganism frequently involved in food poisoning due to its enterotoxin production. This study aimed to evaluate the S. aureus growth and enterotoxins production following an experimental inoculation into the T. molitor rearing substrate (about 7 log CFU/g). Analyses on the substrate and larvae were performed over a testing period of seven days. The microbial population dynamics were also evaluated through total viable count and lactic acid bacteria count. The effects of fasting, washing, and cooking on the microbial loads of mealworms were evaluated. The results highlighted that mealworms and substrates can maintain their microbial loads of S. aureus over the tested period. Moreover, fasting and washing were generally not able to significantly reduce (p-value > 0.05) S. aureus count in mealworms. On the other hand, cooking significantly reduced (p-value < 0.001) the microbial load in almost all cases. No production of enterotoxins was revealed during the trial. Therefore, microbiological risks can be reduced by a wise choice of substrate, appropriate control measures, and thermal treatment of larvae.

The future is crawling: Evaluating the potential of insects for food and feed security.

Current estimations showed that the number of people affected by hunger doubled in the last two years, reaching 9.8% of the global population. According to FAO, in order to satisfy the demand for food in the next few years, it will be necessary to double food production. Moreover, the call for a change in dietary patterns has been raised, showing how the food sector is responsible of 1/3 of climate change where meat-based diets or overconsumption of meat play an important role in the negative environmental impact. Consequently, there is a growing concern in how to achieve the goal of increasing food productions without exploiting environmental resources and to explore the production and use of alternative resources, such as insects. Insects are gaining interests both as food and feed not only to reduce the environmental costs in feed production for common livestock, but also to reduce farmers' dependence on traditional protein sources. In this work we aimed to provide an overview of the state-of-the-art upon insect studies, highlighting the most important results obtained from both an industrial and market perspective. The legislative framework concerning edible insects as food and feed is also analyzed, with the final purpose to highlight recent reforms, relevant case-law as well as unsolved regulatory challenges. From a normative perspective, regulatory efforts are still required to fully take advantage of the potentialities of insects-industry. From a consumer point of view, consumers' willingness to pay a premium is going to be a key issue for economic sustainability of the insect farming chain. To meet the food and feed security challenges, insects will have to be considered all-around, including applications in the food, feed, and other sectors. We believe that this review is an important contribution to the field of food science and will be of interest to researchers, food industry professionals, and policymakers in order to prioritize research questions and help communicate scientific knowledge to a broader audience.

Contamination of Zophobas morio Larvae Rearing Substrate with Listeria monocytogenes: A Preliminary Study.

The interest in edible insects is continuously increasing due to their environmental, nutritional, and productive features. The aim of this study was to evaluate the effects and survival of Listeria monocytogenes in Zophobas morio rearing, using two different bacterial loads (2 and 6 log CFU/g). We also considered the effect of washing, fasting, and cooking treatments on the larvae. During the experimental trial, no mortality was observed among the larvae. The L. monocytogenes loads decreased over time, and it was below the detection limit for crates inoculated with 2 log CFU/g, while the substrate inoculated with 6 log CFU/g reached loads of 4.26 (without larvae) and 2.83 log CFU/g (with larvae). Due to the absence of L. monocytogenes in the larvae on day seven, it was not possible to assess the fasting treatment or the washing and the cooking. However, when looking at the total microbial count, significant effects were revealed for all treatments. The unfasted larvae showed no effect of washing, while the total microbial counts decreased after washing in the fasted larvae. This proves that fasting is a good treatment in terms of hygiene assurance for the consumer.

Consumers' acceptance toward whole and processed mealworms: A cross-country study in Belgium, China, Italy, Mexico, and the US.

The interest in edible insects as food is growing, both in traditional and non-traditional insect-eating countries given their advantages in terms of sustainability and nutritional content. However, only a few studies have conducted cross-country investigations on the acceptance of including processed or whole insects in the diet. Thus, this study aimed to examine to which extent consumers were accepting (i) whole and visible mealworms, (ii) processed mealworms in their diet and (iii) to explore the factors affecting the acceptance level of consuming mealworms in countries with and without entomophagy tradition. An online survey was applied to collect responses (3,006) from five countries-i.e., Belgium, China, Italy, Mexico, and the US-using a quota sampling method. Moreover, an information treatment was included with about half of the participants receiving information about the advantages of edible insects as food (ingredient) and the presence of food safety regulations. Across countries, gender was the main factor affecting acceptance level as men accepted mealworms more than women. Entomophagy tradition mainly explained the differences among countries. Countries with entomophagy traditions (Mexico and China) showed higher acceptance of including whole or processed mealworms in the diet compared to countries with no entomophagy traditions (i.e., Belgium, Italy, and the US). While information and age did affect differently the acceptance of including processed mealworms in countries with entomophagy traditions showing that consumer acceptance was affected by information in Mexico and by age in China. Whereas it was found that younger people (below 42 years old) in countries without entomophagy tradition were more open to accepting processed mealworms in their diet. Moreover, across countries, the acceptance of including processed mealworms was higher compared to whole mealworms. These findings provide insights into which consumer segments to target and the potential impact of information when introducing new insect-based foods in countries with and without entomophagy traditions.

A comprehensive suite of earthquake catalogues for the 2016-2017 Central Italy seismic sequence.

The protracted nature of the 2016-2017 central Italy seismic sequence, with multiple damaging earthquakes spaced over months, presented serious challenges for the duty seismologists and emergency managers as they assimilated the growing sequence to advise the local population. Uncertainty concerning where and when it was safe to occupy vulnerable structures highlighted the need for timely delivery of scientifically based understanding of the evolving hazard and risk. Seismic hazard assessment during complex sequences depends critically on up-to-date earthquake catalogues-i.e., data on locations, magnitudes, and activity of earthquakes-to characterize the ongoing seismicity and fuel earthquake forecasting models. Here we document six earthquake catalogues of this sequence that were developed using a variety of methods. The catalogues possess different levels of resolution and completeness resulting from progressive enhancements in the data availability, detection sensitivity, and hypocentral location accuracy. The catalogues range from real-time to advanced machine-learning procedures and highlight both the promises as well as the challenges of implementing advanced workflows in an operational environment.

In Vitro Anthelmintic Activity of Sea Buckthorn (Hippophae rhamnoides) Berry Juice against Gastrointestinal Nematodes of Small Ruminants

Gastrointestinal nematodes are one of the major threats in small ruminant breeding. Their control is difficult due to the development of anthelmintic resistance, and the search for new molecules endowed with anthelmintic activity (AH) is considered a priority. In this context, we evaluated the in vitro AH activity of two commercial sea buckthorn (Hippophae rhamnoides) berry juices, namely SBT and SBF. The in vitro evaluation was based on the egg-hatch test and larval exsheathment assay at different concentrations. Data were statistically analysed, and the EC50 was calculated. Chemical analyses were performed to evaluate the total polyphenol content of the juices and chemical profile of the most represented compounds. The role of the polyphenolic fraction in the anthelmintic activity of the juices was also assessed. At the highest concentrations, the activity of SBT was high in both tests and comparable to that observed in the thiabendazole-treated positive controls, while SBF showed a lower efficacy. Glycosylated isorhamnetin and quercetin were the most represented polyphenolic compounds in both juices. In conclusion, both H. rhamnoides berry juices tested in this study showed interesting anthelmintic properties in vitro.

Effects of Dietary Supplementation with Honeybee Pollen and Its Supercritical Fluid Extract on Immune Response and Fillet's Quality of Farmed Gilthead Seabream (Sparus aurata).

The awareness of the correlation between administered diet, fish health and products' quality has led to the increase in the research for innovative and functional feed ingredients. Herein, a plant-derived product rich in bioactive compounds, such as honeybee pollen (HBP), was included as raw (HBP) and as Supercritical Fluid Extracted (SFE) pollen (HBP_SFE) in the diet for gilthead seabream (Sparus aurata). The experiment was carried out on 90 fish with an average body weight of 294.7 ± 12.8 g, divided into five groups, according to the administration of five diets for 30 days: control diet (CTR); two diets containing HBP at 5% (P5) and at 10% (P10) level of inclusion; two diets containing HBP_SFE, at 0.5% (E0.5) and at 1% (E1) level of inclusion. Their effects were evaluated on 60 specimens (336.2 ± 11.4 g average final body weight) considering the fish growth, the expression of some hepatic genes involved in the inflammatory response (il-1ß, il-6 and il-8) through quantitative real-time PCR, and physico-chemical characterization (namely color, texture, water holding capacity, fatty acid profile and lipid peroxidation) of the fish fillets monitored at the beginning (day 0) and after 110 days of storage at −20 °C. The results obtained showed that the treatment with diet E1 determined the up-regulation of il-1ß, il-6, and il-8 (p < 0.05); however, this supplementation did not significantly contribute to limiting the oxidative stress. Nevertheless, no detrimental effect on color and the other physical characteristics was observed. These results suggest that a low level of HBP_SFE could be potentially utilized in aquaculture as an immunostimulant more than an antioxidant, but further investigation is necessary.

Exploring the Future of Edible Insects in Europe.

The effects of population increase and food production on the environment have prompted various international organizations to focus on the future potential for more environmentally friendly and alternative protein products. One of those alternatives might be edible insects. Entomophagy, the practice of eating insects by humans, is common in some places but has traditionally been shunned in others, such as European countries. The last decade has seen a growing interest from the public and private sectors to the research in the sphere of edible insects, as well as significant steps forward from the legislative perspective. In the EU, edible insects are considered novel foods, therefore a specific request and procedure must be followed to place them in the market; in fact, until now, four requests regarding insects as a novel food have been approved. Insects could also be used as feed for livestock, helping to increase food production without burdening the environment (indirect entomophagy). Market perspectives for the middle of this decade indicate that most of the demand will be from the feed sector (as pet food or livestock feed production). Undoubtedly, this sector is gaining momentum and its potential relies not only in food, but also in feed in the context of a circular economy.

Probiotics in Rabbit Farming: Growth Performance, Health Status, and Meat Quality.

The rabbit's complex microbiota of the gastrointestinal tract (GIT) plays a critical role in feed digestion, in vitamin production, in fermentative activity with production of volatile fatty acids, and stimulation of immune response, as well as in the infection defence against pathogens and countering environmental stresses. To prevent digestive disorders of this fragile ecosystem, rabbit breeders adopt suitable diets supplemented with additives such as probiotics. Probiotics can interact with the host and with the other microflora leading to an increased health status. A review on the effects of probiotics on rabbit growth performance, health status, and meat quality was conducted to reduce the incidence of digestive diseases and enhance productive performance. Some authors observed that the supplementation of probiotics to the diet improved feed conversion ratio and growth and digestion coefficients, while other authors reported a lack of effect on the live performance. Benefits derived from the use of probiotics were observed on the mortality and the morbidity. The studies performed, to evaluate the effects of probiotic supplementation in diets on the immune response, showed variations in the results. Some authors reported no significant effect on haematological parameters, such as total protein, immunoglobulins, and IgG, while others observed a significant increase or decrease of the same parameters. Most of the research reported significant modifications of intestinal morphology and positive effects on the GIT microbiota, supporting the host's natural defences. Regarding the carcass and meat quality, the studies reported only partial and opposing results.

Mealworm (Tenebrio molitor): Potential and Challenges to Promote Circular Economy.

Over the last few years, the concept of Circular Economy (CE) has received a lot of attention due to its potential contribution to the Sustainable Development Goals (SDGs), especially by reconciling economic growth with the protection of the environment through its grow-make-use-restore approach. The use of insects in circular production systems has been a good example of this concept as insects can transform a wide range of organic waste and by-products into nutritious feedstuffs, which then go back into the production cycle. This paper explores the potential of mealworms (Tenebrio molitor) in circular production systems by reviewing their use and applicability in several industries such as pharmaceuticals, agriculture, food, etc. Despite the high versatility of this insect and its potential as a substitute source of nutrients and other valuable components, there are still many legislative and behavioural challenges that hinder its adoption and acceptance.

Edible Insects and Sustainable Development Goals.

The insect sector can become an important component of sustainable circular agriculture by closing nutrient and energy cycles, fostering food security, and minimising climate change and biodiversity loss, thereby contributing to SDGs. The high levels of the interaction of the insect sector with the SDGs is clearly illustrated inside the review, analysing all of the SDGs that can have direct and indirect effects on insects. Mapping the interactions between the SDGs goals and insect sector offers a starting point, from which it could be possible to define practical next steps for better insect policy.

Effect of Cooking Techniques on the in vitro Protein Digestibility, Fatty Acid Profile, and Oxidative Status of Mealworms (Tenebrio molitor).

Tenebrio molitor (T. molitor) (mealworm) larvae are one of the most promising insects for feed-food purposes. Mealworms are rich in several macro and micro nutritional elements and can be practically reared on side stream substrates. In this study, the effects of seven different cooking techniques were tested on the nutritional value of mealworms focusing the attention on protein digestibility, fatty acid (FA) profile, and oxidative status. Uncooked larvae (UC) were used as control and compared to two combinations of temperature/time in oven cooking (70°C for 30 min, OC70-30, 150°C for 10 min, OC150-10), two methods of frying (mealworms fried in sunflower oil as deep fry, DF, or pan fry, PF), microwaving (MW), boiling (in plastic bag under vacuum, BO), and steaming (ST). Proximate composition, in vitro digestibility (gastric and duodenal), FA profile, and oxidative status (tocopherol and tocotrienol, carbonyl, and lipid oxidation) were then tested. Cooking technique affected all the tested parameters. As expected, cooking affected proximate composition in relation to the method applied (dry matter increased after oven cooking and frying; lipids increased by frying). In vitro digestion revealed the highest value for the OC70-30 method, followed by UC and ST. Deep frying revealed the worst digestibility percentage. FA profile was deeply affected by the cooking technique, with general decrease in SFA and MUFA. The highest modifications in FA profile were revealed in ST larvae with an increased percentage of linoleic acid linked to the lowering of SFA and MUFA contents. Furthermore, deep frying larvae in sunflower oil increased the relative abundance of PUFAs. Tocols values were higher in DF and MW groups than PF (about 6-fold more) and all other groups (7-fold more). Carbonyls increased with oven cooking (OC150-10 and OC70-30), whereas the values were lower with frying and similar to ST and UC. Lipid oxidation was highest as well in OC150-10 but similar to frying methods (DF and PF). Based on the obtained results, it can be concluded that mealworm larvae surely meet human nutritional requirements, but the cooking method must be carefully chosen to maintain a high nutritional value.

Exploring the Acceptance of Entomophagy: A Survey of Italian Consumers.

Insect-based food is not common in Europe, because most people do not consider insects to be edible, but rather a threat and a health risk. Fear and refusal to eat a new food product introduced into a culture is called food neophobia, which results in a hesitation to trying and experimenting with new foods. Although there is significant interest in this novel sector, there is a lack of research on the link between rejection, the level of food neophobia, and consumer behavior related to the introduction of insects into the diet. In this study, through 420 questionnaires, a specific experimental scale of insects was introduced which, together with a neophobia scale, analyzed the probability and the intention of respondents to consume insects. Another issue tested has been their intention to eat food containing insects. We observed that the analyses of the two scales produced different results, confirming the need for a specific scale to measure "insect phobia". This is important, since knowledge about consumer preferences for and barriers to using insects as human food sources is limited but necessary in order to set up commercialization strategies. The development of insect-based food offers physical health benefits and also improves the sustainability of the food industry.

Intestinal Strongyle Genera in Different Typology of Donkey Farms in Tuscany, Central Italy.

Intestinal strongyles are common helminths of donkeys, in which they may be responsible for disease and poor performance. This study aimed to identify intestinal strongyle genera in 55 naturally infected donkeys from three different farm typologies in Tuscany, central Italy, using morphological and metrical analysis of third stage larvae (L3) obtained from faecal cultures. Larvae were identified using two previous reported morphological identification keys. Moreover, eggs per gram (EPG) data were also evaluated to assess differences, if any, according to the farm typology, sex, and age of the examined donkeys. The results showed that small strongyles were prevalent in all donkey farms. In all examined farms, most (92-100%) of L3 were identified as cyathostomin species of the genera Cylicocyclus spp. and Cylicostephanus spp. Large strongyles of the genera Strongylus spp. and Triodontophorus spp., were identified at low percentage (8%), only in the single organic farm included in the study. A high agreement was observed between the two different morphometric keys used. No significant differences were found for EPG according to farm typology, and sex and age from the examined donkeys. This is the first report about genera identification of intestinal strongyles infecting donkeys in Tuscany, Italy.

In Vitro Antibacterial Activity of Manuka (Leptospermum scoparium J.R. et G. Forst) and winter Savory (Satureja montana L.) Essential Oils and Their Blends against Pathogenic E. coli Isolates from Pigs.

Neonatal diarrhoea (ND), post-weaning diarrhoea (PWD) and oedema disease (OD) are among the most important diseases affecting pig farming due to economic losses. Among the main aetiological agents, strains of Escherichia coli are identified as the major responsible pathogens involved. Several strategies have been put in place to prevent these infections and, today, research is increasingly studying alternative methods to antibiotics to reduce the antibiotic resistance phenomenon. Essential oils (EOs) are among the alternative tools that are being investigated. In this study, the in vitro effectiveness of winter savory and manuka essential oils and their mixtures in different proportions against strains of E. coli isolated from episodes of disease in pigs was evaluated. The EOs alone demonstrated slight antibacterial effectiveness, whereas the blends, by virtue of their synergistic action, showed remarkable activity, especially the 70%-30% winter savory-manuka blend, showing itself as a potential tool for prevention and therapy.