Growth Potential of Listeria monocytogenes in Chef-Crafted Ready-to-Eat Fresh Cheese-Filled Pasta Meal Stored in Modified Atmosphere Packaging.

This study evaluated the growth of lactic acid bacteria (LAB) in a fresh, filled-pasta meal, stored in modified atmosphere packaging and the influence of lactic acid (LA) and pH on the growth of Listeria monocytogenes (Lm). Samples were taken from three lots manufactured by a local catering company and stored at both 6 and 14°C. LAB numbers, LA concentration, pH, and the presence of Lm were evaluated at 1, 4, 6, 8, 10, 12, and 14 days of shelf life and the undissociated LA concentration ([LA]) was calculated. The LAB maximum cell density was greater in the products stored at 14°C than those stored at 6°C (10.1 ± 1.1 versus 5.6 ± 1.5 log CFU/g) and [LA] at 14 days was 9 to 21 ppm at 6°C and 509 to 1,887 ppm at 14°C. Challenge tests were made to evaluate the interference of LAB and [LA] on Lm growth. Aliquots of the samples (25 g) were inoculated at 1 to 10 days of shelf life and incubated at 9°C for 7 days, and the difference between Lm numbers at the end and at the beginning of the test (δ) was calculated. Logistic regression was used to model the probability of growth of Lm as a function of LAB and [LA]. The products inoculated at 1 day of shelf life had δ values between 4.2 and 5.6 log CFU/g, but the growth potential was progressively reduced during the shelf life. Lm growth was never observed in the products stored at 14°C. In those stored at 6°C, it grew only in the samples with LAB <5.7 log CFU/g. LAB interaction might thus inhibit the growth of Lm in temperature-abused products and limit its growth in refrigerated products. Logistic regression estimated that the probability of Lm growth was <10% if LAB was >6.6 log CFU/g or log[LA] was >2.2 ppm. The growth or inactivation kinetic of Lm was investigated with a homogenate of three samples with LAB numbers close to the maximum population density. After an initial growth, a subsequent reduction in the number of Lm was observed. This means that the maximum numbers of Lm might not be detected at the end of the product shelf life.

Application of shotgun metagenomics to smoked salmon experimentally spiked: Comparison between sequencing and microbiological data using different bioinformatic approaches.

The aims of this study were i) to evaluate the possibility to detect and possibly quantify microorganisms belonging to different domains experimentally spiked in smoked salmon at known concentrations using shotgun metagenomics; ii) to compare the sequencing results using four bioinformatic tools. The salmon was spiked with six species of bacteria, including potential foodborne pathogens, as well as Cryptosporidium parvum, Saccharomyces cerevisiae and Bovine alphaherpesvirus 1. After spiking, the salmon was kept refrigerated before DNA extraction, library preparation and sequencing at 7 Gbp in paired ends at 150 bp. The bioinformatic tools named MG-RAST, OneCodex, CosmosID and MgMapper were used for the sequence analysis and the data provided were compared using STAMP. All bacteria spiked in the salmon were identified using all bioinformatic tools. Such tools were also able to assign the higher abundances to the species Propionibacterium freudenreichii spiked at the highest concentration in comparison to the other bacteria. Nevertheless, different abundances were quantified for bacteria spiked in the salmon at the same cell concentration. Cryptosporidium parvum was detected by all bioinformatics tools, while Saccharomyces cerevisiae by MG-RAST only. Finally, the DNA virus was detected by CosmosID and OneCodex only. Overall, the results of this study showed that shotgun metagenomics can be applied to detect microorganisms belonging to different domains in the same food sample. Nevertheless, a direct correlation between cell concentration of each spiked microorganism and number of corresponding reads cannot be established yet.

Microbiological profile of chicken carcasses: A comparative analysis using shotgun metagenomic sequencing.

In the last few years metagenomic and 16S rRNA sequencing have completly changed the microbiological investigations of food products. In this preliminary study, the microbiological profile of chicken carcasses collected from animals fed with different diets were tested by using shotgun metagenomic sequencing. A total of 15 carcasses have been collected at the slaughetrhouse at the end of the refrigeration tunnel from chickens reared for 35 days and fed with a control diet (n=5), a diet supplemented with 1500 FTU/kg of commercial phytase (n=5) and a diet supplemented with 1500 FTU/kg of commercial phytase and 3g/kg of inositol (n=5). Ten grams of neck and breast skin were obtained from each carcass and submited to total DNA extraction by using the DNeasy Blood & Tissue Kit (Qiagen). Sequencing libraries have been prepared by using the Nextera XT DNA Library Preparation Kit (Illumina) and sequenced in a HiScanSQ (Illumina) at 100 bp in paired ends. A number of sequences ranging between 5 and 9 million was obtained for each sample. Sequence analysis showed that Proteobacteria and Firmicutes represented more than 98% of whole bacterial populations associated to carcass skin in all groups but their abundances were different between groups. Moraxellaceae and other degradative bacteria showed a significantly higher abundance in the control compared to the treated groups. Furthermore, Clostridium perfringens showed a relative frequency of abundance significantly higher in the group fed with phytase and Salmonella enterica in the group fed with phytase plus inositol. The results of this preliminary study showed that metagenome sequencing is suitable to investigate and monitor carcass microbiota in order to detect specific pathogenic and/or degradative populations.

Whole genome sequencing based typing and characterisation of Shiga-toxin producing Escherichia coli strains belonging to O157 and O26 serotypes and isolated in dairy farms.

In the present study, the genetic relationships as well as the virulome and resistome of newly sequenced O26 and O157 Shiga-toxin producing E. coli (STEC) isolates, collected from dairy farms in Italy, were investigated in comparison to publicly available genomes collected worldwide. The whole genome of Italian isolates was sequenced on Illumina MiSeq Platform. Reads quality control, de novo draft genome assembly, species confirmation and the 7- loci Multi-Locus Sequence Type assignment were performed using INNUca pipeline. Reference-based SNPs calling was performed on O157 and O26 genomes, separately, mapping contigs to high-quality finished genomes. Virulence and antimicrobial resistance determinants were detected in silico using the tool ABRicate. Phylogenetic reconstructions revealed that genomes clustered mainly based on their 7-loci MLST type. The virulome of tested genomes included 190 determinants. O157 genomes carried chu genes associated to heme mediated iron uptake, whereas O26 genomes harboured genes ybt associated to siderophore mediated iron uptake. Resistome analysis showed the presence of tet(34) on all but one O157 genomes and on only one O26 genomes. Only 4 genomes carried genes associated to multiresistance. In the present study, the genes chu and ybt were identified as potential biomarker for the differentiation of O157 and O26 serotypes.

Microbiota analysis and microbiological hazard assessment in poultry carcasses from conventional and antibiotic free farms.

The aim of this study was to assess microbiota and microbiological hazards in poultry carcasses from animals reared in conventional (n=15) and antibiotic free (n=15) farms. An aliquot of neck and breast skin was obtained from each individual carcass at the end of the refrigeration tunnel and submitted to DNA extraction. Total DNA was sequenced in the 16S rRNA and reads analysed with MG-RAST to classify the colonising bacteria up to the genus level and compare each taxonomic group in terms of mean relative frequency of abundance in conventional and antibiotic free carcasses. Firmicutes displayed abundances always higher than 38% but did not show statistically significative differences between conventional and antibiotic free carcasses. On the contrary, Bacteroidetes and Actinobacteria were significantly higher in antibiotic free then conventional carcasses (21.57 vs 10.95%; 19.29 vs 12.05%), whereas Proteobacteria were higher in the latter (33.19 vs 19.52%). The genera significantly higher in antibiotic free than conventional carcasses were Chryseobacterium (10.07 vs 1.94%), Rothia (3.08 vs 0.77%) and Micrococcus (1.12 vs 0.16%), while Shewanella was significantly higher in conventional carcasses (1.38 vs 0.26%). Among Firmicutes, the genera significantly higher in conventional carcasses were Ureibacillus (1.45 vs 0.11%) and Bacillus (3.28 vs 0.56%). The higher abundance of Proteobacteria in conventional carcasses might suggest that hygienic conditions in conventional farms are worse than antibiotic free farms. However, from a food safety point of view, Salmonella was not detected in both kinds of carcasses and the Campylobacter mean relative frequency of abundance was always lower than 0.4%.