Among food-borne pathogens, Listeria monocytogenes continues to pose concerns to food business operators due to its capacity to form biofilm in processing environments. Ozone may be an eco-friendly technology to control microbial contaminations, but data concerning its effect on Listeria monocytogenes biofilm are still limited. In this study, the effect of gaseous ozone at 50 ppm on planktonic cells and biofilm of reference and food-related Listeria monocytogenes strains was evaluated. Ozone caused a reduction in microbial loads of 3.7 ± 0.4 and 3.9 ± 0.4 Log10 CFU/mL after 10 and 30 min, respectively. A complete inactivation of planktonic cells after 6 h of treatment was observed. Biofilm inhibition and eradication treatments (50 ppm, 6 h) resulted in a significant decrease of the biofilm biomass for 59% of the strains tested, whilst a slight dampening of live cell loads in the biofilm state was observed. In conclusion, gaseous ozone is not sufficient to completely counteract Listeria monocytogenes biofilm, but it may be useful as an additional tool to contrast Listeria monocytogenes free-living cells and to improve the existing sanitization procedures in food processing environments.

Background: Six Sarcocystis species are known to use cattle (Bos taurus) as the intermediate host, two of which, S. hominis and S. heydorni, are zoonotic. There is a need for a method that will enable rapid identification of the Sarcocystis species in cattle. Methods: The diaphragm muscles of 102 cattle from Lithuania were examined for the presence of Sarcocystis spp., using two different methods for species identification. Individual sarcocysts were isolated from squash preparations of the diaphragm muscle under the light microscope, followed by genetic characterisation of excised cysts using sequence analysis of the 18S rRNA (18S rRNA) and cytochrome c oxidase subunit I (cox1) genes. The same cattle muscle samples were digested and species-specific PCR analyses targeting cox1 were developed to identify the Sarcocystis isolates to the species level. Results: Under the light microscope, sarcocysts were detected in 87.3% of animals, and Sarcocystis infection was verified in all digested samples. Three species, namely S. cruzi (n = 20), S. bovifelis (n = 23) and S. hirsuta (n = 6), were identified by DNA sequence analysis of isolated sarcocysts. Based on sequence analysis of cox1, the level of genetic variability depended on Sarcocystis species and geographical location. Four Sarcocystis species, S. cruzi (96.1%), S. bovifelis (71.6%), S. hirsuta (30.4%) and S. hominis (13.7%), were confirmed in the digested samples. In individual samples, the most common finding was two species of Sarcocystis (44.1%), followed by three species (26.5%), a single species (24.5%) and four species (4.9%). Conclusions: Although examination of tissue preparations under the light microscrope did not detect any sarcocysts belonging to S. hominis, this species was identified in the digested samples subjected to a cox1-specific PCR analysis. These results demonstrate the need for effective molecular diagnosis techniques to detect Sarcocystis spp., which may be present at a lower prevalence and not detectable among the limited number of sarcocysts identified individually under the light microscope. Graphical

Over the past decades, antimicrobial resistance (AMR) has been recognized as one of the most serious threats to public health. Although originally considered a problem to human health, the emerging crisis of AMR requires a “One Health” approach, considering human, animal, and environmental reservoirs. In this regard, the extensive use of antibiotics in the livestock production systems to treat mastitis and other bacterial diseases can lead to the presence of AMR genes in bacteria that contaminate or naturally occur in milk and dairy products, thereby introducing them into the food chain. The recent development of high-throughput next-generation sequencing (NGS) technologies is improving the fast characterization of microbial communities and their functional capabilities. In this context, whole metagenome sequencing (WMS), also called shotgun metagenomic sequencing, allows the generation of a vast amount of data which can be interrogated to generate the desired evidence, including the resistome. However, the amount of host DNA poses a major challenge to metagenome analysis. Given the current absence of literature concerning the application of WMS on milk to detect the presence of AMR genes, in the present study, we evaluated the effect of different sequencing depths, host DNA depletion methods and matrices to characterize the resistome of a milk production environment. WMS was conducted on three aliquots of bulk tank milk and three aliquots of the in-line milk filter collected from a single dairy farm; a fourth aliquot of milk and milk filter was bioinformatically subsampled. Two commercially available host DNA depletion methods were applied, and metagenomic DNA was sequenced to two different sequencing depth. Milk filters proved to be the most suitable matrices to evaluate the presence of AMR genes; besides, the pre-extraction host DNA depletion method was the most efficient approach to remove host reads. To our knowledge, this is the first study to evaluate the limitations posed by the host DNA in investigating the milk resistome with a WMS approach, confirming the circulation of AMR genes in the milk production environment.

It is urgent to expand the market of biodegradable alternatives to oil-derived plastics owing to (i) increasingly limited oil availability/accessibility, and (ii) the dramatic impact of traditional plastics on aquatic life, the food chain, all Earth ecosystems, and ultimately, human health. Polyhydroxyalkanoates (PHAs) are promising biodegradable polymers that can be obtained through microbial fermentation of agro-industrial byproducts, e.g., milk and cheese whey. Here, the PHA-accumulating efficiency of a mixed microbial culture (MMC, derived from activated sludges) grown on dairy byproducts (cheese and scotta whey) was measured. Bioreactor tests featuring temperature and pH control showed that both scotta and pre-treated Toma cheese whey could be used for PHA production by MMC, although scotta cheese whey supported higher PHA yield and productivity. The advantages of open MMCs include their plasticity and versatility to fast changing conditions; furthermore, no growth-medium sterilization is needed prior to fermentation. However, the use of pure cultures of efficient PHA producers may support better metabolic performances. Therefore, PHA-producing strains were isolated from a MMC, leading to the satisfactory identification of two bacterial strains, Citrobacter freundii and Leuconostoc spp., whose ability to accumulate PHAs in synthetic media was confirmed. A more detailed investigation by mass spectrometry revealed that the strain was L. mesenteroides. Although the validation of L. mesenteroides potential to produce PHA through fermentation of agro-industrial byproducts requires further investigations, this is the first study reporting PHA production with the Leuconostoc genus.

Since the number of studies of the microbial communities related to food and food-associated matrices almost completely reliant on next-generation sequencing techniques is rising, evaluations of these high-throughput methods are critical. Currently, the two most used sequencing methods to profile the microbiota of complex samples, including food and food-related matrices, are the 16S ribosomal RNA (rRNA) metabarcoding and the whole metagenome sequencing (WMS), both of which are powerful tools for the monitoring of foodborne pathogens and the investigation of the microbiome. Herein, the microbial profiles of 20 bulk tank milk filters from different dairy farms were investigated using both the full-length 16S (FL-16S) rRNA metabarcoding, a third-generation sequencing method whose application in food and food-related matrices is yet in its infancy, and the WMS, to evaluate the correlation and the reliability of these two methods to explore the microbiome of food-related matrices. Metabarcoding and metagenomic data were generated on a MinION platform (Oxford Nanopore Technologies) and on a Illumina NovaSeq 6000 platform, respectively. Our findings support the greater resolution of WMS in terms of both increased detection of bacterial taxa and enhanced detection of diversity; in contrast, FL-16S rRNA metabarcoding has proven to be a promising, less expensive, and more practical tool to profile most abundant taxa. The significant correlation of the two technologies both in terms of taxa diversity and richness, together with the similar profiles defined for both highly abundant taxa and core microbiomes, including Acinetobacter , Bacillus, and Escherichia genera, highlights the possible application of both methods for different purposes. This study allowed the first comparison of FL-16S rRNA sequencing and WMS to investigate the microbial composition of a food-related matrix, pointing out the advantageous use of FL-16S rRNA to identify dominant microorganisms and the superior power of WMS for the taxonomic detection of low abundant microorganisms and to perform functional analysis of the microbial communities.