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.

Antibacterial activity in egg samples from small-spotted catshark Scyliorhinus canicula and nursehound Scyliorhinus stellaris: A preliminary investigation.

The study aims to identify antibacterial properties in unfertilized eggs of Scyliorhinus canicula and Scyliorhinus stellaris. Despite challenging marine conditions, these eggs retain their integrity for extended periods and remain unaffected by pathogens. The antibacterial activity was measured using minimum inhibitory and minimum bactericidal concentration analysis. The eggs of S. stellaris exhibited a slight inhibitory effect against Staphylococcus aureus and Listeria monocytogenes, whereas both species' eggs showed no activity against gram-negative microorganisms.

Exploitation of Marginal Hilly Land in Tuscany through the Cultivation of Lavandula angustifolia Mill.: Characterization of Its Essential Oil and Antibacterial Activity.

Lavandula angustifolia Mill., known as one of the best essential oil-bearing plants, is an aromatic plant that is well cultivated in many Mediterranean regions due to its adaptability to variations in climatic and edaphic conditions. Therefore, its essential oil (EO) composition and its antimicrobial activity change as a consequence of abiotic and biotic factors. The chemical composition of L. angustifolia EO collected during four consecutive years of growth was one of the aims of this work. The volatile profile evidenced the prevalence of linalool and linalool acetate even though they switched their positions according to age. Plants in their first year were characterized by a high amount of sesquiterpene compounds (22.1% of the identified fraction). This percentage decreased during plant growth, not representing more than 5.3% in the fourth year. It is interesting to note that both the third- and fourth-year plants showed a content of monoterpenes that exceeded 90% of the total identified constituents. The EO extracted from the oldest plants evidenced higher activity on the studied strains, with more sensitivity on the Gram-positive ones. Tuscan lavender EO, especially that obtained from the four-year-old plants, is of great interest for its potential industrial applications and constitutes an example for the valorization of marginal Tuscan land and good-quality production.

New Insights into the Biological and Pharmaceutical Properties of Royal Jelly.

Royal jelly (RJ) is a yellowish-white and acidic secretion of hypopharyngeal and mandibular glands of nurse bees used to feed young worker larvae during the first three days and the entire life of queen bees. RJ is one of the most appreciated and valued natural product which has been mainly used in traditional medicines, health foods, and cosmetics for a long time in different parts of the world. It is also the most studied bee product, aimed at unravelling its bioactivities, such as antimicrobial, antioxidant, anti-aging, immunomodulatory, and general tonic action against laboratory animals, microbial organisms, farm animals, and clinical trials. It is commonly used to supplement various diseases, including cancer, diabetes, cardiovascular, and Alzheimer's disease. Here, we highlight the recent research advances on the main bioactive compounds of RJ, such as proteins, peptides, fatty acids, and phenolics, for a comprehensive understanding of the biochemistry, biological, and pharmaceutical responses to human health promotion and life benefits. This is potentially important to gain novel insight into the biological and pharmaceutical properties of RJ.

Listeria monocytogenes contamination of Tenebrio molitor larvae rearing substrate: Preliminary evaluations.

Today, edible insects represent a hot topic as an emerging and eco-friendly source of protein. The mealworm (Tenebrio molitor L.) is among the most employed insects for human consumption and feed purposes. So far Listeria monocytogenes, have never been detected either in products sold on the market or during the rearing process. In this study, the substrate employed for mealworm rearing was deliberately contaminated with L. monocytogenes and the bacterium was enumerated during the rearing period and after technological treatments of the larvae. L. monocytogenes persisted during the rearing period. Washing the larvae did not produce any significant effect, while fasting the larvae for 24 or 48 h reduced the L. monocytogenes load (P < 0.001). Oven cooking eliminated L. monocytogenes cells from the product, reducing the risk associated to this foodborne pathogen to zero.

Genome scan for the possibility of identifying candidate resistance genes for goat lentiviral infections in the Italian Garfagnina goat breed.

Small ruminant lentiviruses (SRLVs) are a heterogeneous group of viruses of sheep, goat, and wild ruminants responsible of lifelong persistent infection leading to a multisystem chronic disease. Increased evidences indicate that host genetic factors could influence the individual SRLV resistance. The present study was conducted on the Garfagnina goat breed, an Italian goat population registered on the Tuscan regional repertory of genetic resources at risk of extinction. Forty-eight adult goats belonging to a single flock were studied. SRLV diagnosis was achieved by serological tests and 21 serologically positive animals were identified. All animals were genotyped with the Illumina GoatSNP60 BeadChip and a genome-wide scan was then performed on the individual marker genotypes, in an attempt to identify genomic regions associated with the infection. One SNP was found significant (P < 5 × 10-5) on CHR 18 at 62,360,918 bp. The SNP was an intron of the zinc finger protein 331 (ZNF331) protein. In the region 1 Mb upstream the significant SNP, the NLRP12 (NLR family pyrin domain containing 12), the PRKCG (protein kinase C gamma), and the CACNG7 (calcium voltage-gated channel auxiliary subunit gamma 7) were found.

Proteinase pattern of honeybee prepupae from healthy and American Foulbrood infected bees investigated by zymography.

American foulbrood disease (AFB) is the main devastating disease that affects honeybees' brood, caused by Paenibacillus larvae. The trend of the research on AFB has addressed the mechanisms by which P. larvae bacteria kill honeybee larvae. Since prepupae could react to the infection of AFB by increasing protease synthesis, the aim of this work was to compare protease activity in worker prepupae belonging to healthy colonies and to colonies affected by AFB. This investigation was performed by zymography. In gel, proteolytic activity was observed in prepupae extracts belonging only to the healthy colonies. In the prepupae extracts, 2D zimography followed by protein identification by MS allowed to detect Trypsin-1 and Chymotrypsin-1, which were not observed in diseased specimens. Further investigations are needed to clarify the involvement of these proteinases in the immune response of honeybee larvae and the mechanisms by which P. larvae inhibits protease production in its host.

Identification of candidate genes for paratuberculosis resistance in the native Italian Garfagnina goat breed.

Paratuberculosis disease is a chronic bacterial disease infection of ruminants of global relevance, caused by MAP (Mycobacterium avium subsp. paratuberculosis). The present study was conducted on the Garfagnina goat breed that is an Italian native goat population registered on the Tuscan regional repertory of genetic resources at risk of extinction. Forty-eight adult goats (27 serologically positive to MAP-positive and 21 serologically negative to MAP-negative) belonging to a single flock that had experienced annual mortalities due to MAP infection were identified and genotyped with the Illumina GoatSNP60 BeadChip. Diagnosis was achieved by serological tests, as well as post-mortem examination of affected animals. A genome-wide scan was then performed on the individual marker genotypes, in an attempt to identify genomic regions associated with MAP infection disease. Nine significant markers were highlighted and they were located within, or nearby, annotated genes. Two genes found in this study encode are linked to protein kinases that are among the most important enzymes involved in the immune response to Johne's disease, and four genes are involved in the functions of the Golgi complex.

Detection of Mycobacterium avium subsp. paratuberculosis in a sheep flock in Tuscany.

Paratuberculosis is an infectious disease which affects ruminants. In this work, a sheep flock in Garfagnana district (Tuscany, Italy) was examined by agar gel immune-diffusion (AGID) tests, culture, and PCR from feces, milk, tissue samples, and cheeses. At the first AGID test, 7/280 (2.5 %) animals were positive. From these animals, feces and milk samples were collected: 4/7 feces (57.14 %) and 2/7 milk samples (28.57 %) were positive to culture and 7/7 (100 %) feces samples and 1/7 milk samples (14.28 %) were positive to PCR; 2/4 (50 %) cheeses ripened for 14 days and 1/3 (33.33 %) cheese ripened for 20 days were positive to PCR, from which no viable microorganisms were isolated. Then, the AGID-positive animals were slaughtered and tissue samples were taken from one sheep with PCR-positive feces and milk: the liver, intestine, mesenteric lymph nodes, but not the spleen and mammary lymph nodes were positive to culture; all these samples were instead PCR-positive. After 1 year, a second AGID survey was performed on the remaining animals: 6/244 (2.45 %) subjects were positive to this test. Data obtained revealed the presence of paratuberculosis in a sheep population in Garfagnana.

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.

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.

Exploring the Volatile Composition and Antibacterial Activity of Edible Flower Hydrosols with Insights into Their Spontaneous Emissions and Essential Oil Chemistry.

In the circular economy framework, hydrosols, by-products of the essential oil industry, are gaining attention for their potential in waste reduction and resource reuse. This study analyzed hydrosols from six edible flowers, investigating their chemical composition (VOC-Hyd) and antibacterial properties alongside volatile organic compounds of fresh flowers (VOC-Fs) and essential oils (EOs). Antirrhinum majus exhibited ketones as major VOC-Fs (62.6%) and VOC-Hyd (41.4%), while apocarotenoids dominated its EOs (68.0%). Begonia cucullata showed alkanes (33.7%) and aldehydes (25.7%) as primary VOC-Fs, while alkanes were prevalent in both extracts (65.6% and 91.7% in VOC-Hyd and in EOs, respectively). Calandula officinalis had monoterpenoids in VOC-Fs and VOC-Hyd (89.3% and 49.7%, respectively), while its EOs were rich in sesquiterpenoids (59.7%). Dahlia hortensis displayed monoterpenoid richness in both VOC-Fs and extracts. Monocots species' VOC-Fs (Polianthes tuberosa, Tulbaghia cominsii) were esters-rich, replaced by monoterpenoids in VOC-Hyd. P. tuberosa EO maintained ester richness, while T. cominsii EOs contained a significant percentage of sulfur compounds (38.1%). Antibacterial assays indicated comparable minimum inhibitory concentration profiles across VOC-Hyd: B. calcullata and P. tuberosa against Staphylococcus aureus and Salmonella enterica ser. typhimurium, T. cominsii against Escherichia coli and S. enterica, A. majus and C. officinalis against S. aureus, and D. hortensis against S. enterica.

Cannabis sativa L. essential oil: chemical characterisation and antimicrobial activity against methicillin-resistant Staphylococcus pseudintermedius.

Cannabis sativa L. essential oil has attracted the interest of the scientific community thanks to its numerous biological activities. Several studies have evaluated EOs as alternative therapeutic approaches to limit the use of antibiotics; the present study aimed to evaluate the in vitro inhibitory and bactericidal activity of the essential oils obtained from the leaves and inflorescences of two hemp genotypes against twenty-one multidrug-resistant, methicillin-resistant Staphylococcus pseudintermedius strains isolated from canine clinical samples. Both EOs were mainly represented by sesquiterpene hydrocarbons, with a prevalence of ß-caryophyllene and α-humulene. However, different relative amounts of phytocannabinoids were also detected. Microbiological results evidenced better outcomes for the EO characterised by the highest content of phytocannabinoids, which in turn showed no differences among the tested strains. Nevertheless, both the EOs showed better inhibitory and bactericidal activities than their main constituent, ß-caryophyllene, tested individually, highlighting the presence of synergistic effects among the EO compounds.

The role of diallyl sulfides and dipropyl sulfides in the in vitro antimicrobial activity of the essential oil of garlic, Allium sativum L., and leek, Allium porrum L.

The in vitro antibacterial activity of essential oils (EOs) obtained from fresh bulbs of garlic, Allium sativum L., and leek, Allium porrum L. ( Alliaceae), was studied. A. sativum (garlic) EO showed a good antimicrobial activity against Staphylococcus aureus (inhibition zone 14.8 mm), Pseudomonas aeruginosa (inhibition zone 21.1 mm), and Escherichia coli (inhibition zone 11.0 mm), whereas the EO of A. porrum (leek) had no antimicrobial activity. The main constituents of the garlic EO were diallyl monosulfide, diallyl disulfide (DADS), diallyl trisulfide, and diallyl tetrasulfide. The EO of A. porrum was characterized by the presence of dipropyl disulfide (DPDS), dipropyl trisulfide, and dipropyl tetrasulfide. The antimicrobial activities of the DADS and DPDS were also studied. The results obtained suggest that the presence of the allyl group is fundamental for the antimicrobial activity of these sulfide derivatives when they are present in Allium or in other species (DADS inhibition zone on S. aureus 15.9 mm, P. aeruginosa 21.9 mm, E. coli 11.4 mm).

Preliminary evaluation of probiotic potential of Lactobacillus plantarum strains isolated from Italian food products.

The aim of this study was to investigate some probiotic properties of 42 wild Lactobacillus plantarum strains isolated from different Italian foods of animal origin. The strains were first screened for their antibiotic resistance profile (chloramphenicol, erythromycin, gentamicin, and tetracycline), subsequently they were tested for their in vitro resistance to lysozyme (100 mg L⁻¹), low pH (3.0, 2.5 and 2.0) and bile salts (0.3, 0.5 and 1.0 %). Moreover, agglutination property was studied (adhesion to Saccharomyces cerevisiae cells), as well as the presence of bsh and msa genes. The strains with the best characteristics were subjected to a further trial in order to evaluate their ability to survive to multiple stresses over time (lysozyme, low pH and bile salts) and the effect of these treatments on adhesion to yeast cells. All the strains were susceptible to chloramphenicol, erythromycin and gentamicin, while 6 strains were excluded from further evaluation because of their resistant phenotype against tetracycline. All the strains were able to grow in presence of lysozyme, as well as in MRS broth at pH 3.0. Only 4 strains showed a growth rate lower than 80 % when grown in MRS broth at pH 2.5, while a relevant growth rate decrease was observed after exposure to pH 2.0. Bile salts didn't affect the viability of the L. plantarum cells. Twenty-one strains out of 33 tested strains were able to adhere to S. cerevisiae cells. Presence of both bsh and msa genes was detected in 6 strains. The strains resistant to all the stresses, positive to agglutination with S. cerevisiae and showing bsh and msa genes were selected for further evaluation and subjected to different stress treatments over time. The assessment of growth rates showed that exposure to lysozyme significantly increased low pH resistance in L. plantarum. This increase ranged from 2.35 to 15.57 %. The consequential lysozyme and low pH exposures didn't affect the growth rate values after bile salts treatment, as well as the ability of the strains to adhere to yeast cells wasn't modified by previous treatments (lysozyme, low pH and bile salts). The present work allows to increase knowledge about non starter lactic acid bacteria from Italian food products. The studied L. plantarum strains showed a good potential for their use as probiotic cultures. However, more in vivo tests are necessary to confirm this potentiality.

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.

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.

Occurrence of Campylobacter in Faeces, Livers and Carcasses of Wild Boars Hunted in Tuscany (Italy) and Evaluation of MALDI-TOF MS for the Identification of Campylobacter Species.

A total of 193 wild boars hunted in Tuscany, an Italian region with a high presence of wild ungulates, were examined to assess the occurrence of Campylobacter species in faeces, bile, liver and carcasses, with the aim of clarifying their contribution to human infection through the food chain. Campylobacter spp. were found in 44.56% of the animals, 42.62% of the faecal samples, 18.18% of the carcass samples, 4.81% of the liver tissues and 1.97% of the bile samples. The Campylobacter species genotypically identified were C. coli, C. lanienae, C. jejuni and C. hyointestinalis. The prevalent species transpired to be C. coli and C. lanienae, which were isolated from all the matrices; C. jejuni was present in faeces and liver, while C. hyointestinalis only in faeces. Identification was carried out by matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) on 66 out of 100 isolates identified genotypically, and the technique yielded unsatisfactory results in the case of C. lanienae, which is responsible for sporadic human disease cases. The level of Campylobacter spp. contamination of meat and liver underlines the need to provide appropriate food safety information to hunters and consumers.

Chemical Composition and Antimicrobial Activity against the Listeria monocytogenes of Essential Oils from Seven Salvia Species.

In recent years, essential oils (EOs) have received interest due to their antibacterial properties. Accordingly, the present study aimed to investigate the effectiveness of the EOs obtained from seven species of Salvia on three strains of Listeria monocytogenes (two serotyped wild strains and one ATCC strain), a bacterium able to contaminate food products and cause foodborne disease in humans. The Salvia species analysed in the present study were cultivated at the Botanic Garden and Museum of the University of Pisa, and their air-dried aerial parts were subjected to hydrodistillation using a Clevenger apparatus. The obtained EOs were analysed via gas chromatography coupled with mass spectrometry for the evaluation of their chemical composition, and they were tested for their inhibitory and bactericidal activities by means of MIC and MBC. The tested Eos showed promising results, and the best outcomes were reached by S. chamaedryoides EO, showing an MIC of 1:256 and an MBC of 1:64. The predominant compounds of this EO were the sesquiterpenes caryophyllene oxide and ß-caryophyllene, together with the monoterpenes bornyl acetate and borneol. These results suggest that these EOs may possibly be used in the food industry as preservatives of natural origins.

Antimicrobial and anti-biofilm activity of manuka essential oil against Listeria monocytogenes and Staphylococcus aureus of food origin.

The activity of manuka (Leptospermum scoparium) essential oil (EO) on biofilms of foodborne Listeria monocytogenes and Staphylococcus aureus has been studied. Seven strains of L. monocytogenes and 7 of S. aureus (5 methicillin-resistant) were tested. EO minimal inhibitory concentration (MIC), EO minimal bactericidal concentration (MBC) and biofilm production quantification were determined for each strain by microtiter methods. Moreover, EO Minimum Biofilm Inhibitory Concentration (MBIC) and Minimum Biofilm Eradicating Concentration (MBEC) were determined on 2 L. monocytogenes and 3 S. aureus that showed the best biofilm production. Finally, on 4 strains out of 5 (2 L. monocytogenes and 2 S. aureus) EO Biofilm Reduction Percentage (BRP) vs. untreated controls was assessed after a treatment with EO subinhibitory concentrations. The chemical composition of manuka essential oil was determined by Gas Chromatography- Electron Impact Mass Spectrometry (GCEIMS). The manuka EO demonstrated good antimicrobial activity: L. monocytogenes MIC and MBC were 0.466 mg/ml and 0.933 mg/ml, respectively, whereas S. aureus MIC and MBC were 0.233 mg/ml and 0.466 mg/ml, respectively. Furthermore, L. monocytogenes showed a MBIC of 0.933 mg/ml and a MBEC in the range of 0.933-1.865 mg/ml, whereas S. aureus had a MBIC in the range of 7.461-14.922 mg/ml and a MBEC of 14.922 mg/ml. L. monocytogenes revealed no significant BRP after the treatment with manuka EO, whereas S. aureus showed a BRP higher than 50% with MIC/2 and MIC/4 EO concentrations. These results provide information for feasible manuka EO applications in food production systems.