The lack of a full automation and control of environmental parameters might result in potential risk of microbial contamination in small-scale production plants such as artisanal cheese and salami Italian productions. In a previous study, genomes of 33 E. coli isolates were sequenced. In the present study, the pathogenicity potential of E. coli strains was investigated by: (1) phylogenomic comparison with 202 public genomes of human, animal and environmental Italian origin; (2) pathogenicity assessment of strains with virulence patterns predicting specific E. coli pathotypes by using larvae of Galleria mellonella as in vivo infection model. Phylogenetic reconstruction revealed raw material and not the processing environment as source of salami contamination. Moreover, close proximity of some strains isolated from salami production with wild boar and extraintestinal human public strains was observed suggesting pigs and wild boar as potential reservoirs of pathogenic E. coli. The virulome of salami strains revealed the presence of genes already described as gene markers of atypical enteropathogenic E. coli (aEPEC; bfp-, eae+). Interestingly the analysis of virulence genes pointed toward additional genomes which showed genetic markers previously described as strongly associated to and/or extraintestinal pathogenic E. coli (ExPEC). In vivo experiments, confirmed the higher pathogenicity of strain 5STM5 with genetic pattern corresponding to hybrid aEPEC/ExPEC and two strains 3CP1522 and 6MB5 of cheese and salami production, respectively, with virulence genes previously associated to ExPEC pathotype. The combined approach pointed toward two genes espC for aEPEC, as well as malX for ExPEC which were significantly enriched in clinical genomes in comparison to genomes of other origins. These genes are worth of future investigations which could help to assess the risk for consumers after the consumption of contaminated artisanal food.

Chlamydia is the etiological agent of chlamydiosis in wild and domestic birds, mammals, and humans. In this study, Chlamydia reads were detected in the microbiome of the neck skin of 76 broiler carcasses collected in the same slaughterhouse at the end of the chilling tunnel. The carcasses originated from four different flocks of female Ross 308, reared in two broiler houses located in Northern Italy. One flock from each poultry house was sampled in 2019 and one flock in 2023. The carcass neck skin microbiome was investigated by shotgun metagenomic sequencing. Chlamydia reads displayed a mean relative abundance of 7.38%, with significant differences between carcasses obtained from the two poultry houses, sampled at both sampling times. Chlamydia ibidis was the prevalent species among time points and poultry houses. The zoonotic potential of C. ibidis and foodborne transmission have never been demonstrated. However, it is known that the genus Chlamydia has “spore”-like extracellular forms able to survive for months outside the host. Therefore, the presence of C. ibidis reads on broiler carcasses at the end of the chilling tunnel deserves further investigation. The results of this study highlight the feasibility of microbiome investigations to detect unexpected biological hazards in foods.

Citrobacter braakii can colonize the intestinal tract of humans and animals and occasionally act as opportunistic pathogen. Although isolated from food and the environment, its potential as a foodborne pathogen remains uncertain. Twenty C. braakii isolates were previously collected from salami and soft cheese artisanal productions. In the present study, the potentialities of C. braakii as a food safety hazard were explored by a genomic comparison of C. braakii newly sequenced genomes with publicly available genomes, including those of clinical relevance, and a pathogenicity assessment in Galleria mellonella as an in vivo infection model. Phylogenomic reconstruction revealed that one salami clone and two C. braakii genomes of the soft cheese production were closely related (from 11 to 28 core SNP differences) to C. braakii publicly available clinical genomes. All genomes carried the chromosomally located blaCMY and/or qnrB genes and were resistant to cephalosporins and/or had reduced susceptibility to ciprofloxacin. G. mellonella larvae showed 90% mortality after challenge with C. braakii strains carrying the vex and tvi operons coding for the capsular polysaccharide (Vi antigen), in comparison to 40% of strains lacking these two operons. The high mortality rate of vex- and tvi-positive C. braakii isolated from food processing plants suggests C. braakii to be a possible foodborne hazard.

Escherichia coli can harbor a broad repertoire of virulence and antimicrobial resistance (AMR) genes, which can be exchanged across the human gastrointestinal microflora, thus posing a public health risk. In this study, six batches of artisanal soft cheese and a 6-month ripened fermented dried sausage were investigated to assess the occurrence, phylogeny, and genomic traits (AMR, virulence, and mobilome) of E. coli. Thirty and three strains isolated from salami and cheese food chains, respectively, were confirmed as E. coli by whole genome sequencing. The accumulation of single nucleotide polymorphism differences within small clusters of strains encompassing batches or processing stages, combined with high serotype and phylogroup diversity, suggested the occurrence of different contamination phenomena among the facilities. A total of eight isolates harbored plasmid-mediated resistance genes, including one cheese strain that carried an IncQ1 plasmid carrying AMR determinants to macrolides [mph(B)], sulfonamides (sul1, sul2), trimethoprim (dfrA1), and aminoglycosides [aph(3'')-Ib and aph(6)-Id]. A pool of virulence-associated genes in the class of adhesion, colonization, iron uptake, and toxins, putative ColV-positive iron uptake systems sit, iro, or iuc (eight salami and two cheese), plasmid-encoded hemolysin operon hlyABCD (one salami), and potential atypical enteropathogenic E. coli (three salami environment) were reported. Overall, our findings underscore the importance of routine surveillance of E. coli in the artisanal food chain to prevent the dissemination of AMR and virulence.

In this pilot study, we compared the metagenomic profiles of different types of artisanal fermented meat products collected in Italy, Greece, Portugal, and Morocco to investigate their taxonomic profile, also in relation to the presence of foodborne pathogens and antimicrobial resistance genes. In addition, technical replicates of the same biological sample were tested to estimate the reproducibility of shotgun metagenomics. The taxonomic analysis showed a high level of variability between different fermented meat products at both the phylum and genus levels. Staphylococcus aureus was identified with the highest abundance in Italian fermented meat; Escherichia coli in fermented meat from Morocco; Salmonella enterica in fermented meat from Greece; Klebsiella pneumoniae and Yersinia enterocolitica in fermented meat from Portugal. The fungi Aspergillus, Neosartoria, Emericella, Penicillum and Debaryomyces showed a negative correlation with Lactococcus, Enterococcus, Streptococcus, Leuconostoc and Lactobacillus. The resistome analysis indicated that genes conferring resistance to aminoglycoside, macrolide, and tetracycline were widely spread in all the samples. Our results showed that the reproducibility between technical replicates tested by shotgun metagenomic was very high under the same conditions of analysis (either DNA extraction, library preparation, sequencing analysis, and bioinformatic analysis), considering both the degree of overlapping and the pairwise correlation.

In the PRIMA project ArtiSaneFood, the microbiological parameters of several artisanal cheeses produced in the Mediterranean area have been quantified. In this pilot study, we selected four of these artisanal cheese products from Italy, Portugal, Spain, and Morocco to investigate and compare their microbiomes in terms of taxonomic composition, presence of reads of foodborne pathogens, as well as virulence and antimicrobial resistance genes. Lactococcus, Streptococcus and Lactobacillus were the most represented genera in the Portuguese and Spanish cheeses, Streptococcus in the Italian cheese, and Enterococcus, Klebsiella, Escherichia, and Citrobacter in the Moroccan products. The correlation analysis indicated a negative association between the abundance of some lactic acid bacteria ( i.e. , Lactococcus, Lactobacillus, Streptococcus, and Leuconostoc) and foodborne pathogenic genera, like Escherichia and Salmonella. The analysis of pathogen abundance, virulence factors, and antimicrobial resistance genes showed a strong clusterization based on the cheese type, confirming that the presence of potential human health risk determinants was higher in the artisanal products derived from unpasteurized milk that underwent spontaneous fermentation.

Background:The results of omic methodologies are often reported as separate datasets. In this study we applied for the first time multi-omic features clustering and pathway enrichment to clarify the biological impact of vitamin B2 supplementation on broiler caeca microbiome. Methods:The caeca contents of broilers fed +50 and +100 mg/kg vitamin B2 were analyzed by shotgun metagenomic and metabolomic. Latent variables extracted from NMR spectra, as well as taxonomic and functional features profiled from metagenomes, were integrated to characterize the effect of vitamin B2 in modulating caeca microbiome. A pathway-based network was obtained by mapping the observed input genes and compounds, highlighting connected strands of metabolic ways through pathway-enrichment analysis. Results:At day 14, the taxonomic, functional and metabolomic features in the caeca of tested broilers showed some degree of separation between control and treated groups, becoming fully clear at 28 days and persisting up to 42 days. In the caeca of birds belonging to the control group Alistipes spp. was the signature species, while the signature species in the caeca of broilers fed +50 and +100 mg/kg vitamin B2 were Bacteroides fragilis and Lactobacillus crispatus, Lactobacillus reuteri, Ruminococcus torques, Subdoligranum spp., respectively. The pathway enrichment analysis highlighted that the specific biochemical pathways enhanced by the supplementations of vitamin B2 were N-Formyl-L-aspartate amidohydrolase, producing Aspartate and Formate; L-Alanine:2-oxoglutarate amino transferase, supporting the conversion of L-Alanine and 2-Oxoglutarate in Pyruvate and L-Glutamate; 1D-myo-inositol 1/4 phosphate phosphohydrolase, converting Inositol 1/4-phosphate and water in myo-Inositol and Orthophosphate. The results of this study demonstrated that the caeca of birds fed +50 and + 100 mg/kg were those characterized by taxonomic groups more beneficial to the host and with a higher concentration of myo-inositol, formic acid, amino acids and pyruvate involved in glycolysis and amino acid biosynthesis. Conclusion:In this study we demonstrated how to perform multi-omic features integration to describe the biochemical mechanisms enhanced by the supplementation of different concentrations of vitamin B2 in the poultry diet. The relationship between vitamin B2 supplementation and myo-inositol production was highlighted in our study for the first time.

The aim of the present study was to investigate the genetic diversity and antimicrobial resistance (AMR) of E. coli during enrofloxacin therapy in broilers affected by colisepticemia. Three unrelated farms with ongoing colibacillosis outbreaks were sampled at day 1 before treatment and at days 5, 10 and 24 post-treatment. A total of 179 E. coli isolates were collected from extraintestinal organs and submitted to serotyping, PFGE and the minimum inhibitory concentration (MIC) against enrofloxacin. PFGE clusters shifted from 3-6 at D1 to 10-16 at D5, D10 and D24, suggesting an increased population diversity after the treatment. The majority of strains belonged to NT or O78 and to ST117 or ST23. PFGE results were confirmed with SNP calling: no persistent isolates were identified. An increase in resistance to fluoroquinolones in E. coli isolates was observed along the treatment. Resistome analyses revealed qnrB19 and qnrS1 genes along with mutations in the gyrA, parC and parE genes. Interestingly, despite a fluoroquinolone selective pressure, qnr-carrying plasmids did not persist. On the contrary, two conjugative AMR plasmid clusters (AB233 and AA474) harboring AMR genes other than qnr were persistent since they were identified in both D1 and D10 genomes in two farms. Further studies should be performed in order to confirm plasmid persistence not associated (in vivo) to antimicrobial selective pressure.

The present study aimed at assessing the occurrence of microbiological hazards (Listeria monocytogenes, Staphylococcus aureus, Salmonella spp. and Escherichia coli O157) in an artisanal soft cheese produced in northern Italy. In the same product total bacterial count, lactic acid bacteria and Enterobacteriaceae were enumerated, and pH and water activity measured in two batches sampled in summer and winter. Samples of raw materials, environmental swabs from the production processes and cheese during 15 days of storage at 2 and 8°C as well as dynamic temperature of 2°C for 5 days and 8°C for 10 days were collected and tested. The load of total bacterial count was significantly higher in the winter batch in comparison to the summer one, with a significant increase at the end of the storage period also noticed for lactic acid bacteria. Statistical higher values of pH were registered in raw materials and end of storage in winter batch. S. aureus was confirmed only in the winter batch within samples (n=4) of stored cheese. On plates used for E. coli O157 detection, colonies of Klebsiella pneumoniae and Klebsiella oxytoca were isolated. The results suggest that the highest bacterial population in the winter batch was associated to a higher pH in stored cheese and a higher number of biological hazards identified. Their isolation started in the maturation room suggesting this step as relevant for possible cheese contamination.

Increasing reports on K. pneumoniae strains with antimicrobial resistance and virulence traits from food and farm animals are raising concerns about the potential role of Klebsiella spp. as a foodborne pathogen. This study aimed to report and characterize Klebsiella spp. isolates from two artisanal ready-to-eat food (soft cheese and salami) producing facilities, and to track similar genotypes in different ecological niches. Over 1170 samples were collected during the whole production chain of different food batches. The overall Klebsiella prevalence was 6%. Strains were classified into the three Klebsiella species complexes: K. pneumoniae (KpSC, n = 17), K. oxytoca (KoSC, n = 38) and K. planticola (KplaSC, n = 18). Despite high genetic diversity we found in terms of known and new sequence types (STs), core genome phylogeny revealed clonal strains persisting in the same processing setting for over 14 months, isolated from the environment, raw materials and end-products. Strains showed a natural antimicrobial resistance phenotype-genotype. K. pneumoniae strains showed the highest virulence potential, with sequence types ST4242 and ST107 strains carrying yersiniabactin ybt16 and aerobactin iuc3 . The latter was detected in all K. pneumoniae from salami and was located on a large conjugative plasmid highly similar (97% identity) to iuc 3 + plasmids from human and pig strains circulating in nearby regions of Italy. While identical genotypes may persist along the whole food production process, different genotypes from distinct sources in the same facility shared an iuc 3-plasmid. Surveillance in the food chain will be crucial to obtain a more comprehensive picture of the circulation of Klebsiella strains with pathogenic potential.

Artisanal salami is produced in small-scale production plants, where the lack of full automation might result in higher variability in food intrinsic properties. The aim of the present study was to evaluate the inter- and intra-batch variability in physicochemical parameters and its impact on microbial quality and occurrence of foodborne pathogens on 480 samples collected from six batches of an artisanal Italian production of organic salami. Relatively high total bacterial counts (TBC) were found on the surface of the table in the stuffing room (4.29 ± 0.40 log cfu/cm2). High loads of Enterobacteriaceae in the meat mixture of batch 2 and TBC in batch 5 were associated with a higher occurrence of bacterial pathogens. During ripening, water activity (aw) and pH failed to reach values lower than 0.86 and 5.3, respectively. Six Staphylococcus aureus and four Listeria monocytogenes isolates were collected from the salami meat mixture during ripening and the processing environment. A total of 126 isolates of Enterobacteriaceae were characterized at a species level, with Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Citrobacter freundii isolated from the final products. Results suggest the relevance of first steps of production in terms of the hygiene of raw materials and handling during stuffing procedures, especially when the physicochemical parameters of the final products do not reach values that represent hurdles for foodborne pathogens.

Artisanal cheeses are produced in small-scale production plants, where the lack of full automation and control of environmental and processing parameters suggests a potential risk of microbial contamination. The aim of this study was to perform a longitudinal survey in an Italian artisanal factory producing a spreadable soft cheese with no rind to evaluate the inter- and intra-batch variability of physicochemical and microbial parameters on a total of 720 environmental and cheese samples. Specifically on cheese samples, the evaluation was additionally performed on physicochemical parameters. Cheese samples were additionally collected during 15 days of storage at constant temperatures of 2 and 8°C, as well as a dynamic profile of 2°C for 5 days and 8°C for 10 days. Furthermore, Enterobacteriaceae isolates were identified at species level to have a better knowledge of the environmental and cheese microbiota potentially harboring human pathogens. High inter-batch variability was observed for lactic acid bacteria (LAB) and total bacteria count (TBC) in cheese at the end of production but not for pH and water activity. A temperature of 8°C was associated with a significantly higher load of Enterobacteriaceae in cheeses belonging to batch 6 at the end of storage, and this temperature also corresponded with the highest increase in LAB and TBC loads over cheese shelf life. Results from generalized linear mixed models (GLMMs) indicated that drains in the warm room and the packaging area were associated with higher levels of TBC and Enterobacteriaceae in cheese. Regarding foodborne pathogens, no sample was positive for verotoxigenic Escherichia coli (VTEC) or Listeria monocytogenes, whereas six Staphylococcus aureus and one Salmonella pullorum isolates were collected in cheese samples during storage and processing, respectively. Regarding Enterobacteriaceae, 166 isolates were identified at species level from all batches, with most isolates belonging to Klebsiella oxytoca and pneumoniae, Enterobacter cloacae, Hafnia alvei, and Citrobacter freundii evidencing the need to focus on standardizing the microbial quality of cow milk and on hygienic procedures for cleaning and disinfection especially in warm and maturation rooms. Further studies should be performed to investigate the potential pathogenicity and antimicrobial resistance of the identified Enterobacteriaceae species in artisanal cheeses.

The aim of the present study was to investigate the resistome and virulome diversity of 43 isolates of Listeria monocytogenes, Salmonella enterica and S. aureus collected from artisanal fermented meat and dairy products and their production environments in Portugal, Spain, Italy and Morocco. After DNA extraction, genomes were sequenced, and de novo assembled. Genetic relationships among genomes were investigated by SNP calling and in silico 7- loci MLST. Genomes of the same species belonged to different ST-types demonstrating the circulation of different clones in in the same artisanal production plant. One specific clone included genomes of S. Paratyphi B belonging to ST43 and repeatedly isolated for more than a year in an artisanal sausage production plant. No genomes but three (belonging to Salmonella enterica), were predicted as multiresistant to different antimicrobials classes. Regarding virulence, genomes of L. monocytogenes belonging to ST1, ST3 and ST489, as well as genomes of S.enterica enterica (ST43, ST33, ST314, ST3667, ST1818, ST198) and ST121 S. aureus were predicted as virulent and hypervirulent. The occurrence of virulent and hypervirulent L. monocytogenes, Salmonella enterica and S. aureus strains in artisanal fermented meat and dairy productions as well as in their finished products suggests the need for a specific focus on prevention and control measures able to reduce the risk of these biological hazards in artisanal food productions.

Antibiotic free farms are increasing in the poultry sector in order to address new EU regulations and consumer concerns. In this pilot study, we investigated whether the efforts of raising chickens without the use antibiotics make any difference in the microbiome of poultry meat eaten by consumers. To this aim we compared the microbiomes characterizing caeca and the corresponding carcasses of two groups of chickens reared, one reared on a conventional farm and one on an antibiotic-free intensive farm. The results showed a clear separation between the taxonomic, functional and antibiotic resistant genes in the caeca of the birds reared on the conventional and antibiotic free farm. However, that separation was completely lost on carcasses belonging to the two groups. The antibiotic-free production resulted in statistically significant lower antimicrobial resistance load in the caeca in comparison to the conventional production. Moreover, the antimicrobial resistance load on carcasses was much higher than in the caeca, without any significant difference between carcasses coming from the two types of farms. All in all, the results of this research highlighted the need to reduce sources of microbial contamination and antimicrobial resistance not only at the farm level but also at the post-harvest one.

Several strategies have been in place in food animal production to reduce the unnecessary use of antimicrobial agents. Beyond the monitoring of their use, the evaluation of the effect of these strategies on the occurrence and types of antimicrobial resistance (AMR) associated genes is crucial to untangle the potential emergence and spread of AMR to humans through the food chain. In the present study, the occurrence of these genes was evaluated in commensal Escherichia coli isolated from broiler carcasses "produced without the use of antibiotics" in 3 antibiotic-free (AB-free) farms in Italy in 2019. Sequenced data were analyzed along with publicly available genomes of E. coli collected in Italy from the broiler food chain from previous years (2017-2018). The genetic relationships among all 93 genomes were assessed on de novo assemblies by in silico MLST and SNP calling. Moreover, the resistomes of all genomes were investigated. According to SNP calling, genomes were gathered in three clades. Clade A encompassed, among others, ST117, ST8070, and ST1011 genomes. ST10 belonged to clade B, whereas Clade C included ST58, ST297, ST1101, and ST23 among others. Regarding the occurrence of AMR genes, a statistically significant lower occurrence of these genes in the genomes of this study in comparison to the public genomes was observed considering the whole group of genes as well as genes specifically conferring resistance to aminoglycosides, β-lactams, phenicols, trimethoprim, and lincosamides. Moreover, significant reductions were observed by comparing the whole group of AMR associated mutations, as well as those specifically for fluoroquinolones and fosfomycin resistance. Although the identification of 3° generation cephalosporin resistance associated genes in AB-free E. coli is a concern, this study provides a first indication of the impact of a more prudent use of antimicrobial agents on the occurrence of AMR genes in Italian broiler production chain. More studies are needed in next years on a higher number of genomes to confirm this preliminary observation.

The study of the microbiome in broiler chickens holds great promise for the development of strategies for health maintenance and performance improvement. Nutritional strategies aimed at modulating the microbiota—host relationship can improve chickens’ immunological status and metabolic fitness. Here, we present the results of a pilot trial aimed at analyzing the effects of a nutritional strategy involving vitamin B2 supplementation on the ileum, caeca and litter microbiota of Ross 308 broilers, as well as on the metabolic profile of the caecal content. Three groups of chickens were administered control diets and diets supplemented with two different dosages of vitamin B2. Ileum, caeca, and litter samples were obtained from subgroups of birds at three time points along the productive cycle. Sequencing of the 16S rRNA V3–V4 region and NMR metabolomics were used to explore microbiota composition and the concentration of metabolites of interest, including short-chain fatty acids. Vitamin B2 supplementation significantly modulated caeca microbiota, with the highest dosage being more effective in increasing the abundance of health-promoting bacterial groups, including Bifidobacterium, resulting in boosted production of butyrate, a well-known health-promoting metabolite, in the caeca environment.

In the present study, the antimicrobial effect of Cannabis sativa Futura 75 was evaluated both in vitro against foodborne bacterial pathogens, and on food against naturally occurring microbial groups of minced meat stored for 8 days at 4°C. Ethanol extraction was performed on the grind of the inflorescence. After extraction, ethanol was completely evaporated and substituted by water. Serial dilutions of the extract, the grind and cannabidiol 99% were added to Nutrient Agar and spotted with Listeria monocytogenes, Salmonella Typhimurium, Escherichia coli and Staphylococcus spp. Regarding the evaluation on food, 50 mL of extract, characterised by CBD at concentration of 322,70 μg/mL, were added to 2.5 kg of minced beef meat. Meat was divided into aliquots and stored for 8 days at 4°C. At 0, 1, 2, 3, 4, 7, and 8 days, aerobic bacteria, enterobacteria, coliforms and E. coli were enumerated. All tested products were efficient against Gram +. In particular, extract corresponding to CBD concentration of 0.017 and 0.3 mg/mL were effective against L. monocytogenes and Staphylococcus spp. respectively. After 8 days of storage at 4°C, treated minced meat showed a bright red colour in comparison to a brownish control meat. Moreover, Enterobacteriaceae and coliforms were significantly reduced of 2.3 log CFU/g and 1.6 log CFU/g respectively in treated meat in comparison to the control. Although preliminary, the present study suggests the antimicrobial properties of the extract of Cannabis sativa both in vitro and in minced meat.

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.

This study investigated for the first time the decontamination efficacy of a probiotic-based cleaning product containing Bacillus subtilis, Bacillus pumilus, and Bacillus megaterium spores on fresh and reused broiler litters during 3 rearing cycles of 6 wk each. Moreover, the impact of reused litters treated with the cleaning product on the chicken caeca microbiota was assessed at the end of the rearing cycles in comparison to untreated litter. The Bacillus spores provided with the cleaning treatment were able to successfully colonize the reused poultry litters, decreasing the mean counts of total aerobic bacteria, Enterobacteriaceae, and coagulase positive Staphylococci. The decrease of Enterobacteriaceae, mainly represented by the genus Escherichia, was also observed in the caeca of broilers reared on reused litters treated with the cleaning product. Moreover, the treatment retained the caeca content of Ruminococcaceae and Faecalibacterium as well as the level of biodiversity among the bacteria genera colonizing the caeca of animals reared on reused litter. Overall, the results of this study highlight a positive effect of the probiotic-based cleaning strategy on the microbial decontamination of reused litters and on broiler caeca stability, thereby enhancing animal health and prevention of poultry diseases.

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.