Companilactobacillus alimentarius: An extensive characterization of strains isolated from spontaneous fermented sausages

Companilactobacillus alimentarius is a facultatively heterofermentative lactic acid bacterium (LAB) that is a significant constituent within the microbiota of various traditional fermented foods exerting several functions in fermentative or ripening processes. This species has been isolated from Spanish fermented sausages, where its frequency of isolation was comparable to those of Latilactobacillus sakei and Latilactobacillus curvatus. Despite to its presence in several niches, ecological information on this species is still scarce and only few publications report information about its safety features (i.e. antibiotic resistance). Since studies on C. alimentarius concern the analysis of a few individual traits regarding this species, a more extensive work on a larger number of isolates from the same matrix have been performed to allow a clearer interpretation of their phenotypic and technological characteristics. Specifically, 14 strains of C. alimentarius isolated from Mediterranean spontaneously fermented sausages, have been screened for their safety and technological characteristics (such as antibiotic resistance, biogenic amine production, inhibiting potential, growth at different temperatures and NaCl concentrations) and with phenotype microarrays with the aim to elucidate their potential role and contribution to sausage fermentation and ripening. In general, a wide variability was observed in relation to the parameters considered. Several of the tested strains were able to produce histamine, tyramine and putrescine while the antibiotic resistance greatly varied according to the strains, with the exception of vancomycin. In addition, C. alimentarius strains showed a relevant potential to grow in conditions of salt and temperature mimicking those found in fermented foods. In particular, the growth at 10 degrees C and in the presence of salt can explain the presence of C. alimentarius in sausages and its adaptation to fermented meat environment in which low temperature can be applied during ripening. The differentiation of the phenotypic profile reflected the environmental conditions that influenced the isolation source, including those derived by the raw materials. Given the species frequent association with spontaneous fermentations or the ripening microbiota of various products, despite not being intentionally used as starter cultures, the data presented in this study contribute to a deeper comprehension of their role, both advantageous and detrimental, in numerous significant fermented foods

DNA-BASED APPROACHES FOR MICROBIAL FOOD SAFETY AND QUALITY

La contaminazione degli alimenti rappresenta un interesse primario per i consumatori e per produttori coinvolti nella filiera alimentare. Il monitoraggio continuo degli alimenti è quindi fondamentale per prevenire non solo la proliferazione di agenti patogeni, ma anche i microrganismi in grado di produrre un deterioramento influenzando la qualità dei prodotti finali. Da questo punto di vista, l'obiettivo della tesi è di aumentare le conoscenze sulla rilevazione e la caratterizzazione dei batteri di interesse alimentare, utilizzando metodi basati sul DNA, in particolare l'analisi del genoma, e metodi molecolari veloci e facili da usare. In particolare, due capitoli dimostrano come l’analisi del genoma è ormai indispensabile per la determinazione delle specie microbiche, sia per i batteri, sia per i lieviti. I due capitoli seguenti riguardano la valutazione della patogenicità e della resistenza agli antibiotici di Escherichia coli negli alimenti e nella fauna selvatica. I risultati mostrano come WGS fornisca risultati rapidi e approfonditi su argomenti di particolare importanza per la sicurezza alimentare in una prospettiva ‘One Health’. I dati WGS possono essere utilizzati per impostare nuove tecniche molecolari. In questa prospettiva, l'ultimo lavoro riguarda lo sviluppo di un metodo rapido per il rilevamento di un batterio che forma spore e che causa problemi di qualità nei formaggi.Food contamination represents a public concern for the global food chain and a source of awareness for all the consumers. The continuous monitoring of food, from the beginning of the production, is therefore paramount to prevent not only the proliferation of pathogens, but also the spoilage microorganisms that affect the quality of final products. From this perspective, the goal of the PhD thesis is to increase knowledge about the detection and characterization of microbes of food interest, using DNA-based methods, namely genome analysis, and real -time and easy-to -use molecular methods. Two chapters address the evidence that WGS is now indispensable for the microbial species determination, for prokaryotic and eukaryotic microorganisms as well. The following two chapters concern the assessment of pathogenicity and antibiotic resistance of Escherichia coli in food and wildlife, respectively. The results show how WGS provides rapid and in-depth results on topics of particular importance for food safety in a One Health perspective. WGS data can be used to set up new molecular techniques. In this perspective, the latest work concerns the development of a rapid method for the detection of a spore-forming bacterium that causes quality problems in hard cheeses

Development of a droplet digital polymerase chain reaction for rapid and simultaneous identification of common foodborne pathogens in soft cheese

Dairy products can harbor various microorganisms (e.g., Campylobacter spp., Salmonella spp., Listeria monocytogenes, verocytotoxin-producing Escherichia coli) arising from animal reservoirs, and which can become important sources of foodborne illness. Therefore, early detection of food pathogens is crucial to prevent diseases. We wished to develop an accurate quantitative protocol based on a droplet digital polymerase chain reaction (ddPCR) involving eight individual TaqMan™ reactions to detect simultaneously, without selective enrichment, Listeria spp., L. monocytogenes, Salmonella spp., verocytotoxin-producing E. coli and Campylobacter spp. in cheese. ddPCR (a third-generation PCR) provides absolute quantification of target DNAs without requirement of a standard curve, which simplifies experimentation and data comparability. The accuracy, specificity and sensitivity of the developed ddPCR system were assessed using purified DNA from 50 reference pathogenic and non-pathogenic strains from international or Italian collections and analyzing soft cheese samples artificially contaminated with serial dilutions (from 4×106 to 4×101 CFU/g) of pure cultures from the American Type Culture Collection.Finally, the performance of our ddPCR system was compared by parallel testing with quantitative PCR: it gave higher sensitivity (102 CFU/g for the Listeria spp. assay) without the necessity of a standard curve.In conclusion, this is the first ddPCR system developed for simultaneous detection of common foodborne pathogens in cheese using a single set of amplification conditions. As such, it could become a useful strategy for high-throughput screening of microorganisms to evaluate the quality and safety of food products

Wild Boars as an Indicator of Environmental Spread of ESβL-Producing Escherichia coli

Antimicrobial resistance (AMR) represents an increasing issue worldwide, spreading not only in humans and farmed animals but also in wildlife. One of the most relevant problems is represented by Extended-Spectrum Beta-Lactamases (ES beta Ls) producing Escherichia coli because they are the cause of important infections in human. Wild boars (Sus scrofa) as a source of ESK.s attracted attention due to their increasing density and their habits that lead them to be at the human-livestock-wildlife interface. The aim of this study was to increase the knowledge about the ES beta Ls E. coli strains carried by wild boars living in a particularly high-density area of Northern Italy. The analysis of 60 animals allowed to isolate 16 ES beta L-producing E. coli strains (prevalence 23.3%), which were characterised from a phenotypical and molecular point of view. The overall analysis revealed that the 16 isolates were all not only ES beta L producers but also multidrug resistant and carried different types of plasmid replicons. The genome analysis performed on a subset of isolates confirmed the heterogeneity observed with pulsed-field gel electrophoresis (PFGE) and highlighted the presence of two pandemic sequence types, ST131 and ST10, with different collections of virulence factors. The genomic context of ESOL genes further evidenced that all of them were surrounded by transposons and insertion sequences, suggesting the possibility to exchange AMR genes. Overall, this study shows the worrying dissemination of ES beta L-producing E. coli in wild boars in Northern Italy, suggesting the role of these animals as a spreader of AMR and their inclusion in surveillance programmes, to shed light on the "One Health" complex interactions

Development of Coated PLA Films Containing a Commercial Olive Leaf Extract for the Food Packaging Sector

Publisher Copyright: © 2024 by the authors.A commercial olive leaf extract (OL), effective against Salmonella enterica, Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus, was added to three different coating formulations (methylcellulose, MC; chitosan, CT; and alginate, ALG) to produce active polylactic acid (PLA) coated films. Evaluation of these coated PLA films revealed significant inhibition of S. aureus growth, particularly with the MC and CT formulations exhibiting the highest inhibition rates (99.7%). The coated films were then tested for food contact compatibility with three food simulants (A: 10% ethanol; B: 3% acetic acid; D2: olive oil), selected to assess their suitability for pre-cut hams and ready-to-eat vegetables in relation to overall migration. However, coated films with active functions exhibited migration values in simulants A and B above legal limits, while promising results were obtained for simulant D2, highlighting the need to deeply investigate these coatings’ impact on a real food system. Untargeted metabolomics revealed that the type of coating influenced the selective release of certain phenolic classes based on the food simulant tested. The Oxitest analysis of simulant D2 demonstrated that the MC and ALG-coated PLA films slightly slowed down the oxidation of this food simulant, which is an edible vegetable oil.Peer reviewe

Citrus Peel Extracts for Industrial-Scale Production of Bio-Based Active Food Packaging

The thermal stability of four different commercial citrus peel extracts was tested and improved by an encapsulation process with β-cyclodextrins in a spray-dryer. All extracts after the encapsulation process maintained a good antioxidant capacity, with an apparent loss in total phenolic compounds of around 20–25%. In addition, all samples showed good antimicrobial activity (MIC 5–0.625 mg/mL) against Staphylococcus aureus, which was maintained after the encapsulation process (MIC 5–1.25 mg/mL). Based on the antioxidant and antimicrobial activity results, the best-encapsulated citrus extract was selected for incorporation into a polylactic acid/polyhydroxy butyrate (PLA/PHB) film. The latter was then produced on an industrial scale by cast extrusion and was found to be suitable for food contact as it showed overall migration values in different food simulants lower than the legislative limit of 10 mg of non-volatile substances per 1 dm2 of surface area. The UHPLC-HRMS analysis, performed to evaluate the migration of the active compounds, revealed about 13.41% release in food simulant A and 11.02% in food simulant B. Antimicrobial analysis conducted directly on the film showed a growth inhibition activity towards Escherichia coli and Staphylococcus aureus equal to 30 and 60%, respectively