Emerging of Shiga toxin-producing Escherichia coli O177:H11 and O177:H25 from cattle at slaughter in Italy.

Shiga toxin-producing Escherichia coli (STEC) are zoonotic pathogens frequently carried by cattle, responsible in humans of mild to bloody diarrhoea, haemolytic uraemic syndrome (HUS) and even death. In 2023-2024, a study on STEC contamination of hide and carcasses of dairy cattle at slaughter was planned in Emilia-Romagna region (northern Italy). When the study was still in progress and 60 animals were sampled, the detection of STEC O177 isolates reached high rates and gained our attention. A total of five O177 STEC strains were detected, namely four from three carcasses (5.0 %) and one from a hide sample (1.7 %). The isolates were typed by WGS as following: 1) STEC O177:H11 sequence type (ST) 765 (stx2a+, eae+), detected from one carcass; 2) STEC O177:H25 ST659 (stx2c+, eae+) detected from three carcasses and one hide sample. One carcass was contaminated by both STEC serotypes. The isolates carried other virulence determinants often found in STEC strains associated with HUS, namely the exha, astA and espP genes, together with genes for adhesion to the epithelial cells of the gut (lpfA, fdeC, fimH) and non-Locus for Enterocyte Effacement (LEE) effector protein genes (nleA, nleB). The STEC O177:H11 isolate harboured antimicrobial resistance (AMR) genes to ß-lactams (blaTEM-1A), aminoglycosides (aadA1, aph(3″)-Ib, aph(6)-Id), trimethoprim (dfrA1), sulphonamides (sul1, sul2), tetracyclines (tetA), (tetB), streptothricin (sat2), and quaternary ammonium compounds (qacEdelta1). On the contrary, the STEC O177:H25 isolates carried no AMR genes. Persistent carriage of STEC O177:H25 ST659 (stx2c+, eae+) at farm level was assessed by testing animals of the same herd sent to slaughter. Interestingly, the colonies of STEC O177:H11 and STEC O177:H25 had different morphology on CHROMagar™ STEC plates, being mauve and colourless, respectively. Since mauve is the colour STEC colonies commonly have on the CHROMagar™ STEC medium, our findings can help microbiologists in the selection of uncommon serotypes. To the best of our knowledge, this is the first detection of STEC O177 from carcasses and hides of dairy cattle at slaughter. Noteworthy, the STEC-positive hide was classified as "very dirty" thus stressing the need of clean animals entering the slaughter chain, as required by Regulation (EC) No 853/2004. Since STEC O177 has been responsible of HUS in Europe, our data could add information on the source of uncommon serogroups in human infections.

Isolation and Molecular Evidence of Tunisian Sheep-like Pestivirus (Pestivirus N) in Persistently Infected Sheep in Northern Italy, 2023.

Over the last few decades, several pestiviruses have been discovered in ruminants, pigs, and, more recently, in non-ungulate hosts. Consequently, the nomenclature and taxonomy of pestiviruses have been updated. The Tunisian sheep-like pestivirus (TSV, Pestivirus N) is an additional ovine pestivirus genetically closely related to classical swine fever virus (CSFV). In this study, during a survey of pestivirus infections in ovine farms in the Lombardy region of Northern Italy, we identified and isolated a pestivirus strain from a sheep that was found to belong to Pestivirus N species based on its genomic nucleotide identity. The sheep itself and its lamb were found to be persistently infected. We performed molecular characterization and phylogenetic analysis of three viral genomic regions (a fragment of 5'-UTR, partial Npro, and the whole E2 region). In conclusion, these results confirmed circulating TSV in Northern Italy after notification in Sicily, Italy, and France. Correlation with Italian, Tunisian, and French strains showed that detection might have resulted from the trading of live animals between countries, which supports the need for health control measures.

Is the metabarcoding ripe enough to be applied to the authentication of foodstuff of animal origin? A systematic review.

Food authentication using molecular techniques is of great importance to fight food fraud. Metabarcoding, based on the next-generation sequencing (NGS) technologies, allowing large-scale taxonomic identification of complex samples via massive parallel sequencing of fragments (called DNA barcodes) simultaneously, has become increasingly popular in many scientific fields. A systematic review to answer the question "Is the metabarcoding ripe enough to be applied to the authentication of foodstuff of animal origin?" is presented. The inclusion criteria were focused on the selection of scientific papers (SPs) only applying metabarcoding to foodstuff of animal origin collected on the market. The 23 included SPs were first analyzed with respect to the metabarcoding phases: library preparation (target genes, primer pairs, and fragment length), sequencing (NGS platforms), and final data analysis (bioinformatic pipelines). Given the importance of primer selection, the taxonomic coverage of the used primers was also evaluated. In addition, the SPs were scored based on the use of quality control measures (procedural blanks, positive controls, replicates, curated databases, and thresholds to filter the data). A lack of standardized protocols, especially with respect to the target barcode/s and the universal primer/s, and the infrequent application of the quality control measures, leads to answer that metabarcoding is not ripe enough for authenticating foodstuff of animal origin. However, the observed trend of the SP quality improvement over the years is encouraging. Concluding, a proper protocol standardization would allow a wider use of metabarcoding by both official and private laboratories, enabling this method to become the primary for the authentication of foodstuffs of animal origin.

Evaluation of the Virulence Potential of Listeria monocytogenes through the Characterization of the Truncated Forms of Internalin A.

Listeria monocytogenes is a widespread Gram-positive pathogenic bacterium that causes listeriosis, a rather rare but severe foodborne disease. Pregnant women, infants, the elderly, and immunocompromised individuals are considered particularly at risk. L. monocytogenes can contaminate food and food-processing environments. In particular, ready-to-eat (RTE) products are the most common source associated with listeriosis. L. monocytogenes virulence factors include internalin A (InlA), a surface protein known to facilitate bacterial uptake by human intestinal epithelial cells that express the E-cadherin receptor. Previous studies have demonstrated that the presence of premature stop codon (PMSC) mutations naturally occurring in inlA lead to the production of a truncated protein correlated with attenuate virulence. In this study, 849 L. monocytogenes isolates, collected from food, food-processing plants, and clinical cases in Italy, were typed and analyzed for the presence of PMSCs in the inlA gene using Sanger sequencing or whole-genome sequencing (WGS). PMSC mutations were found in 27% of the isolates, predominantly in those belonging to hypovirulent clones (ST9 and ST121). The presence of inlA PMSC mutations in food and environmental isolates was higher than that in clinical isolates. The results reveal the distribution of the virulence potential of L. monocytogenes circulating in Italy and could help to improve risk assessment approaches.

Newly Designed Primers for the Sequencing of the inlA Gene of Lineage I and II Listeria monocytogenes Isolates.

Listeria monocytogenes is a major human foodborne pathogen responsible for listeriosis. The virulence factor Internalin A (inlA) has a key role in the invasion of L. monocytogenes into the human intestinal epithelium, and the presence of premature stop-codons (PMSC) mutations in the inlA gene sequence is correlated with attenuated virulence. The inlA sequencing process is carried out by dividing the gene into three sections which are then reassembled to obtain the full gene. The primers available however were only able to entirely amplify the lineage II isolates. In this study, we present a set of new primers which allow inlA sequencing of isolates belonging to both lineages, since lineage I isolates are the ones most frequently associated to clinical cases. Using newly designed primers, we assessed the presence of inlA PMSCs in food, food processing environments and clinical isolates.