Contribution of natural milk culture to microbiota, safety and hygiene of raw milk cheese produced in alpine malga

Processing of alpine milk in malga farms is carried out under conditions that can favor contamination by coliforms, coagulase-positive staphylococci, or pathogens such as Listeria monocytogenes. With the aim to improve the hygienic characteristics and safety of cheese produced in four malga farms the use of lyophilized Natural Milk Culture prepared with selected strains was tested.. Two cheesemaking tests were carried out in the same day always starting from the same milk: in the first case following the malga recipe that uses either Natural Whey Culture or without the addition of a starter, in the second one using a Natural Milk Culture. Cheesemaking were carried out in four malga farms located in the west area of Trentino region within the same week. For hygienic and safety evaluation, aerobic colony count, coagulase-positive staphylococci, Escherichia coli, staphylococcal toxins, Listeria monocytogenes, and Salmonella spp, pH and aw were determined in raw milk from evening and morning milking, curd in vat, curd after extraction and two months-ripened cheese. Pathogens or toxins, high values of coagulase- positive staphylococci and E. coli were not found in cheese samples. However, in the curd coagulase-positive staphylococci reached values almost of 5 Log CFU/g in the two malga without starter cultures. The use of Natural Milk Culture reduced E. coli counts. In addition, DNA was extracted from cheese samples and from Natural Milk Culture and the composition of the microbial community determined by Next Generation Sequencing method. The determination of cheese microbial communities demonstrated that the use of Natural Milk Culture exerted different effects in the different malga, in any case preserving bacterial biodiversity

Characterisation of the thermostable protease AprX in strains of Pseudomonas fluorescens and impact on the shelf-life of dairy products: preliminary results

Bacterial proteases are involved in food spoilage and shelf-life reduction. Among the bacterial proteases, a predominant role in spoilage of dairy products seems to be played by the thermostable metallo-protease AprX, which is produced by various strains of Pseudomonas fluorescens. Differences in AprX enzyme activity among different strains were highlighted, but the most proteolytic strains were not identified. In this study, the presence of the aprX gene was evaluated in 69 strains isolated from food matrices and 18 reference strains belonging to the P. fluorescens group ,which had been previously typed by the multi locus sequence typing method. Subsequently, a subset of reference strains was inoculated in ultra-high temperature milk, and the expression of the aprX gene was evaluated at 22 and 6°C. On the same milk samples, the proteolytic activity was then evaluated through Azocasein and trinitrobenzenesulfonic acid solution assays. Finally, to assess the applicability of the former assay directly on dairy products the proteolityc activity was tested on industrial ricotta samples using the Azocasein assay. These results demonstrate the spread of aprX gene in most strains tested and the applicability of Azocasein assay to monitor the proteolytic activity in dairy products

The use of Unmanned Aerial Vehicles (UAVs) to sample the blow microbiome of small cetaceans

Recent studies describe the use of UAVs in collecting blow samples from large whales to analyze the microbial and viral community in exhaled air. Unfortunately, attempts to collect blow from small cetaceans have not been successful due to their swimming and diving behavior. In order to overcome these limitations, in this study we investigated the application of a specific sampling tool attached to a UAV to analyze the blow from small cetaceans and their respiratory microbiome. Preliminary trials to set up the sampling tool were conducted on a group of 6 bottlenose dolphins (Tursiops truncatus) under human care, housed at Acquario di Genova, with approximately 1 meter distance between the blowing animal and the tool to obtain suitable samples. The same sampling kit, suspended via a 2 meter rope assembled on a waterproof UAV, flying 3 meters above the animals, was used to sample the blows of 5 wild bottlenose dolphins in the Gulf of Ambracia (Greece) and a sperm whale (Physeter macrocephalus) in the southern Tyrrhenian Sea (Italy), to investigate whether this experimental assembly also works for large whale sampling. In order to distinguish between blow-associated microbes and seawater microbes, we pooled 5 seawater samples from the same area where blow samples\u2019 collection were carried out. The the respiratory microbiota was assessed by using the V3-V4 region of the 16S rRNA gene via Illumina Amplicon Sequencing. The pooled water samples contained more bacterial taxa than the blow samples of both wild animals and the sequenced dolphin maintained under human care. The composition of the bacterial community differed between the water samples and between the blow samples of wild cetaceans and that under human care, but these differences may have been mediated by different microbial communities between seawater and aquarium water. The sperm whale\u2019s respiratory microbiome was more similar to the results obtained from wild bottlenose dolphins. Although the number of samples used in this study was limited and sampling and analyses were impaired by several limitations, the results are rather encouraging, as shown by the evident microbial differences between seawater and blow samples, confirmed also by the meta-analysis carried out comparing our results with those obtained in previous studies. Collecting exhaled air from small cetaceans using drones is a challenging process, both logistically and technically. The success in obtaining samples from small cetacean blow in this study in comparison to previous studies is likely due to the distance the sampling kit is suspended from the drone, which reduced the likelihood that the turbulence of the drone propeller interfered with successfully sampling blow, suggested as a factor leading to poor success in previous studies

Diversity within Italian Cheesemaking Brine-Associated Bacterial Communities Evidenced by Massive Parallel 16S rRNA Gene Tag Sequencing

This study explored the bacterial diversity of brines used for cheesemaking in Italy, as well as their physicochemical characteristics. In this context, 19 brines used to salt soft, semi-hard, and hard Italian cheeses were collected in 14 commercial cheese plants and analyzed using a culture-independent amplicon sequencing approach in order to describe their bacterial microbiota. Large NaCl concentration variations were observed among the selected brines, with hard cheese brines exhibiting the highest values. Acidity values showed a great variability too, probably in relation to the brine use prior to sampling. Despite their high salt content, brine microbial loads ranged from 2.11 to 6.51 log CFU/mL for the total mesophilic count. Microbial community profiling assessed by 16S rRNA gene sequencing showed that these ecosystems were dominated by Firmicutes and Proteobacteria, followed by Actinobacteria and Bacteroidetes. Cheese type and brine salinity seem to be the main parameters accountable for brine microbial diversity. On the contrary, brine pH, acidity and protein concentration, correlated to cheese brine age, did not have any selective effect on the microbiota composition. Nine major genera were present in all analyzed brines, indicating that they might compose the core microbiome of cheese brines. Staphylococcus aureus was occasionally detected in brines using selective culture media. Interestingly, bacterial genera associated with a functional and technological use were frequently detected. Indeed Bifidobacteriaceae, which might be valuable probiotic candidates, and specific microbial genera such as Tetragenococcus, Corynebacterium and non-pathogenic Staphylococcus, which can contribute to sensorial properties of ripened cheeses, were widespread within brines. \ua9 2017 Marino, Innocente, Maifreni, Mounier, Cobo-D\uedaz, Coton, Carraro and Cardazzo

Agricultural by-products with bioactive effects: A multivariate approach to evaluate microbial and physicochemical changes in a fresh pork sausage enriched with phenolic compounds from olive vegetation water

The use of phenolic compounds derived from agricultural by-products could be considered as an eco-friendly strategy for food preservation. In this study a purified phenol extract from olive vegetation water (PEOVW) was explored as a potential bioactive ingredient for meat products using Italian fresh sausage as food model. The research was developed in two steps: first, an in vitro delineation of the extract antimicrobial activities was performed, then, the PEOVW was tested in the food model to investigate the possible application in food manufacturing. The in vitro tests showed that PEOVW clearly inhibits the growth of food-borne pathogens such as Listeria monocytogenes and Staphylococcus aureus. The major part of Gram-positive strains was inhibited at the low concentrations (0.375–3 mg/mL). In the production of raw sausages, two concentrates of PEOVW (L1:0.075% and L2: 0.15%) were used taking into account both organoleptic traits and the bactericidal effects. A multivariate statistical approach allowed the definition of the microbial and physicochemical changes of sausages during the shelf life (14 days). In general, the inclusion of the L2 concentration reduced the growth of several microbial targets, especially Staphylococcus spp. and LABs (2 log10 CFU/g reduction),while the increasing the growth of yeasts was observed. The reduction of microbial growth could be involved in the reduced lipolysis of raw sausages supplemented with PEOVWas highlighted by the lower amount of diacylglycerols. Moisture and aw had a significant effect on the variability of microbiological features,while food matrix (the sausages' environment) can mask the effects of PEOVW on other targets (e.g. Pseudomonas). Moreover, the molecular identification of the main representative taxa collected during the experimentation allowed the evaluation of the effects of phenols on the selection of bacteria. Genetic data suggested a possible strain selection based on storage time and the addition of phenol compounds especially on LABs and Staphylococcus spp. The modulation effects on lipolysis and the reduction of several microbial targets in a naturally contaminated product indicates that PEOVW may be useful as an ingredient in fresh sausages for improving food safety and quality

Functional Electrical Stimulation of Intrinsic Laryngeal Muscles under Varying Loads in Exercising Horses

Bilateral vocal fold paralysis (BVCP) is a life threatening condition and appears to be a good candidate for therapy using functional electrical stimulation (FES). Developing a working FES system has been technically difficult due to the inaccessible location and small size of the sole arytenoid abductor, the posterior cricoarytenoid (PCA) muscle. A naturally-occurring disease in horses shares many functional and etiological features with BVCP. In this study, the feasibility of FES for equine vocal fold paralysis was explored by testing arytenoid abduction evoked by electrical stimulation of the PCA muscle. Rheobase and chronaxie were determined for innervated PCA muscle. We then tested the hypothesis that direct muscle stimulation can maintain airway patency during strenuous exercise in horses with induced transient conduction block of the laryngeal motor nerve. Six adult horses were instrumented with a single bipolar intra-muscular electrode in the left PCA muscle. Rheobase and chronaxie were within the normal range for innervated muscle at 0.55±0.38 v and 0.38±0.19 ms respectively. Intramuscular stimulation of the PCA muscle significantly improved arytenoid abduction at all levels of exercise intensity and there was no significant difference between the level of abduction achieved with stimulation and control values under moderate loads. The equine larynx may provide a useful model for the study of bilateral fold paralysis

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5-11 December, to 17.5% (25/143 samples) in the week 12-18, to 65.9% (89/135 samples) in the week 19-25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool

Caratterizzazione molecolare delle comunità batteriche coinvolte nella maturazione del formaggio Montasio D.O.P.

This study provides a complete view of the composition of the microbial community in Montasio cheese obtained using a culture-independent approach. By molecular direct methods, microbial DNA and RNA were extracted from cheese matrixes without any culturing step. To identify the bacteria populations of raw milk and cheese samples, clone libraries were constructed from the 16S rRNA PCR amplified from DNA (and cDNA). T-RFLP (terminal restriction fragment length polymorphism) was used to identify clones and to investigate the community structure. real-time quantitative PCR (qRT-PCR) assays were also developed to detect and quantify three species and one genus of lactic-acid bacteria (Streptococcus thermophilus, Lactobacillus casei, Pediococcus pentosaceus, Enterococcus spp.). One qRT-PCR assay was developed to detect Pseudomonas spp., a food spoilage related bacteria. Pseudomonas spp., Acinetobacter spp. and Enterobacter spp., the most frequently reported psychrotrophs in raw milk, were found also in our milk samples. Streptococcus thermophilus, added as starter, was the predominant LAB species throughout the whole ripening period of Montasio cheese. Enterococci were also found and they resulted probably from milk. Instead, Pediococcus pentosaceus and Lactobacillus casei were detected only in the mature cheeses. Expression analysis of TCS (two component regulatory system) genes were also performed by qRT-PCR. This work was a prelimanary study to evaluate the quality and quantity of RNA (extracted from cheese matrix) in gene expression studies.In questo studio è stata analizzata la biodiversità microbica durante le fasi di produzione del formaggio Montasio D.O.P utilizzando un approccio coltura-indipendente. Con le metodiche molecolari è possibile estrarre il DNA e l'RNA microbico direttamente dalla matrice formaggio senza utilizzare alcuna metodica colturale di microbiologia classica. Per identificare le popolazioni batteriche del latte crudo e dei campioni di formaggio sono state costruite delle librerie 16S rRNA a partire da DNA e RNA. Il sequenziamento e il T-RFLP (Terminal Restriction Fragment Length Polymorphism) sono stati impiegati per l'analisi dei cloni. Il T-RFLP è stato applicato anche per l'analisi diretta delle comunità microbiche presenti nei campioni di Montasio. Saggi in real-timePCR quantitativa (qRT-PCR) sono stati messi a punto per rilevare e quantificare tre specie e un genere di batteri lattici (Streptococcus thermophilus, Lactobacillus casei, Pediococcus pentosaceus, Enterococcus spp.). Un saggio qRT-PCR è stato sviluppato per rilevare Pseudomonas spp. Acinetobacter spp., Pseudomonas spp. e Enterobacter spp., i batteri psicotrofi più comunemente riportati nel latte crudo, sono stati trovati anche nei campioni di latte analizzati in questo studio. Streptococcus thermophilus, aggiunto come starter, è stato la specie LAB (Lactic Acid Bacteria) predominante attraverso tutto il periodo di maturazione del formaggio Montasio. Anche enterococchi sono stati ritrovati durante le fasi di stagionatura e questi, probabilmente, derivavano dal latte crudo. Pediococcus pentosaceus e Lactobacillus casei sono stati rilevati solo nei campioni di formaggio stagionato. Sono stati messi inoltre a punto dei saggi in real-timePCR per studiare l’espressione di otto geni implicati nel sistema quorum-sensing in Streptococcus thermophilus. Questo è stato fatto per verificare l’applicabilità di studi di espressione genica su RNA batterico estratto direttamente dalla matrice formaggio con l'intenzione, in futuro, di utilizzare questa tipologia di studi per la caratterizzazione di funzioni geniche interessanti per la produzione di prodotti lattiero-caseari

Optimization of five qPCR protocols toward the detection and the quantification of antimicrobial resistance genes in environmental samples

none4Here, we describe the optimization and validation of five quantitati ve PCR (qPCR) assays by employing the SYBRGreen chemistry paired with melting curve analysis to detect and quantify clinically relevant antimicrobial resistance genes (ARGs) (i.e. ermB, bla CTXM1-like , bla CMY-2 , qnrA and qnrS ) from environmental samples (i.e. soil and manure) . These five protocols accurately detected and quantified the aforementioned ARGs in complex environmental matrices and represent useful tools for both diagnostic and monitoring activities of resistant bacteria and ARGs into the environment.restrictedRoberta Tolosi, Lisa Carraro, Andrea Laconi, Alessandra PiccirilloTolosi, Roberta; Carraro, Lisa; Laconi, Andrea; Piccirillo, Alessandr

Active Rumen Bacterial and Protozoal Communities Revealed by RNA-Based Amplicon Sequencing on Dairy Cows Fed Different Diets at Three Physiological Stages

Seven Italian Simmental cows were monitored during three different physiological stages, namely late lactation (LL), dry period (DP), and postpartum (PP), to evaluate modifications in their metabolically-active rumen bacterial and protozoal communities using the RNA-based amplicon sequencing method. The bacterial community was dominated by seven phyla: Proteobacteria, Bacteroidetes, Firmicutes, Spirochaetes, Fibrobacteres, Verrucomicrobia, and Tenericutes. The relative abundance of the phylum Proteobacteria decreased from 47.60 to 28.15% from LL to DP and then increased to 33.24% in PP. An opposite pattern in LL, DP, and PP stages was observed for phyla Verrucomicrobia (from 0.96 to 4.30 to 1.69%), Elusimicrobia (from 0.32 to 2.84 to 0.25%), and SR1 (from 0.50 to 2.08 to 0.79%). The relative abundance of families Succinivibrionaceae and Prevotellaceae decreased in the DP, while Ruminococcaceae increased. Bacterial genera Prevotella and Treponema were least abundant in the DP as compared to LL and PP, while Ruminobacter and Succinimonas were most abundant in the DP. The rumen eukaryotic community was dominated by protozoal phylum Ciliophora, which showed a significant decrease in relative abundance from 97.6 to 93.9 to 92.6 in LL, DP, and PP, respectively. In conclusion, the physiological stage-dependent dietary changes resulted in a clear shift in metabolically-active rumen microbial communities