Listeria monocytogenes Circulating in Rabbit Meat Products and Slaughterhouses in Italy: Prevalence Data and Comparison Among Typing Results.

Rabbit meat has outstanding dietetic and nutritional properties. However, few data on microbiological hazards associated with rabbit productions are available. In this study, the presence of Listeria monocytogenes was determined in 430 rabbit carcasses, 256 rabbit meat cuts and products, and 599 environmental sponges collected from four Italian rabbit slaughterhouses over a period of 1 year. Prevalence of L. monocytogenes among the 1285 rabbit meat and environmental samples was 11%, with statistically significant differences between slaughterhouses. The highest prevalence (33.6%) was observed in rabbit meat cuts and products; the majority of positive environmental samples were collected from conveyor belts. Overall, 27.9% and 14.3% of rabbit cuts and carcasses, respectively, had L. monocytogenes counts higher than 1 colony-forming unit (CFU)/10 g. A selection of 123 isolates from positive samples was genotyped and serotyped to determine genetic profiles and diversity among L. monocytogenes isolates contaminating different slaughterhouses and classes of products investigated. Discriminatory power and concordance among the results obtained using multilocus variable-number tandem-repeat analysis (MLVA), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), automated EcoRI ribotyping, and serotyping were assessed. The isolates selected for typing were classified into serotypes 1/2a (52.8%), 1/2c (32.5%), and 1/2b (14.6%). The majority of the isolates were classified as ST14 (34.1%), ST9 (35.5%), ST121 (17.9%), and ST224 (14.6%). The greatest discriminatory power was observed with the MLVA typing, followed by MLST, PFGE, and ribotyping. The best bidirectional concordance was achieved between PFGE and MLST. There was 100% correlation between both MLST and MLVA with serotype. Moreover, a high unidirectional correspondence was observed between MLVA and both MLST and PFGE, as well as between PFGE and both MLST and serotyping. The results of this study show for the first time in Italy prevalence and genetic profiles of L. monocytogenes isolated in rabbit products and slaughterhouses.

Comparison between Salmonella enterica Serotype Enteritidis Genotyping Methods and Phage Type.

A quantitative comparison between discriminatory indexes and concordance among multilocus variable-number tandem-repeat analysis (MLVA), pulsed-field gel electrophoresis (PFGE), automated ribotyping, and phage typing has been performed, testing 238 Salmonella enterica serotype Enteritidis isolates not epidemiologically correlated. The results show that MLVA is the best choice, but each typing method provides a piece of information for establishing clonal relationships between the isolates.

Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii Circulation in a Dairy Farm and Sources of Milk Contamination.

Even though dairy cows are known carriers of Arcobacter species and raw or minimally processed foods are recognized as the main sources of human Arcobacter infections in industrialized countries, data on Arcobacter excretion patterns in cows and in milk are scant. This study aimed to identify potentially pathogenic Arcobacter species in a dairy herd and to investigate the routes of Arcobacter transmission among animals and the potential sources of cattle infection and milk contamination. A strategy of sampling the same 50 dairy animals, feed, water, and milk every month for a 10-month period, as well as the sampling of quarter milk, animal teats, the milking environment, and animals living on the farm (pigeons and cats), was used to evaluate, by pulsed-field gel electrophoresis (PFGE), the characteristic patterns in animals, their living environment, and the raw milk they produced. Of the 463 samples collected, 105 (22.6%) were positive for Arcobacter spp. by culture examination. All the matrices except quarter milk and pigeon gut samples were positive, with prevalences ranging from 15 to 83% depending on the sample. Only three Arcobacter species, Arcobacter cryaerophilus (54.2%), A. butzleri (34.2%), and A. skirrowii (32.3%), were detected. PFGE analysis of 370 isolates from positive samples provided strong evidence of Arcobacter circulation in the herd: cattle likely acquire the microorganisms by orofecal transmission, either by direct contact or from the environment, or both. Water appears to be a major source of animal infection. Raw milk produced by the farm and collected from a bulk tank was frequently contaminated (80%) by A. butzleri; our PFGE findings excluded primary contamination of milk, whereas teats and milking machine surfaces could be sources of Arcobacter milk contamination.

Arcobacter butzleri in sheep ricotta cheese at retail and related sources of contamination in an industrial dairy plant.

This study aimed to evaluate Arcobacter species contamination of industrial sheep ricotta cheese purchased at retail and to establish if the dairy plant environment may represent a source of contamination. A total of 32 sheep ricotta cheeses (1.5 kg/pack) packed in a modified atmosphere were purchased at retail, and 30 samples were collected in two sampling sessions performed in the cheese factory from surfaces in contact with food and from surfaces not in contact with food. Seven out of 32 samples (21.9%) of ricotta cheese collected at retail tested positive for Arcobacter butzleri at cultural examination; all positive samples were collected during the same sampling and belonged to the same batch. Ten surface samples (33.3%) collected in the dairy plant were positive for A. butzleri. Cluster analysis identified 32 pulsed-field gel electrophoresis (PFGE) patterns. The same PFGE pattern was isolated from more than one ricotta cheese sample, indicating a common source of contamination, while more PFGE patterns could be isolated in single samples, indicating different sources of contamination. The results of the environmental sampling showed that A. butzleri may be commonly isolated from the dairy processing plant investigated and may survive over time, as confirmed by the isolation of the same PFGE pattern in different industrial plant surface samples. Floor contamination may represent a source of A. butzleri spread to different areas of the dairy plant, as demonstrated by isolation of the same PFGE pattern in different production areas. Isolation of the same PFGE pattern from surface samples in the dairy plant and from ricotta cheese purchased at retail showed that plant surfaces may represent a source of A. butzleri postprocessing contamination in cheeses produced in industrial dairy plants.

Occurrence and genomic characterization of antimicrobial-resistant and potential pathogenic Escherichia coli from Italian artisanal food productions of animal origin.

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, 6 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. 30 and 3 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 8 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 (8 salami and 2 cheese), plasmid-encoded hemolysin operon hlyABCD (one salami), and potential atypical enteropathogenic E. coli (3 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.

Shotgun metagenomic investigation of foodborne pathogens and antimicrobial resistance genes in artisanal fermented meat products from the Mediterranean area.

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 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.

Occurrence and genetic diversity of Arcobacter butzleri in an artisanal dairy plant in Italy.

The present study aimed to investigate the presence, distribution, and persistence of Arcobacter spp. in an artisanal dairy plant and to test the isolates to determine their different genotypes in the processing plant and in foods. Samples were collected in an artisanal cheese factory on four occasions between October and December 2012. Food samples (raw milk, ricotta cheese, mozzarella cheese, and conditioning liquid), water samples, and environmental samples were analyzed by the culture method; isolates were identified by multiplex PCR and genotyped by pulsed-field gel electrophoresis (PFGE) analysis. Arcobacter butzleri was isolated from 29 out of 59 samples (46.6%), 22 of which were from environmental samples and 7 of which were from food samples. Cluster analysis divided the strains into 47 PFGE patterns: 14 PFGE clusters and 33 unique types. Our findings indicate that the plant harbored numerous A. butzleri pulsotypes and that the manual cleaning and sanitation in the studied dairy plant do not effectively remove Arcobacter. The recurrent isolation of A. butzleri suggests that the environmental conditions in the dairy plant constitute a good ecological niche for the colonization of this microorganism. In some cases, the presence of indistinguishable strains isolated from the same facilities on different sampling days showed that these strains were persistent in the processing environment.

Investigation on the microbiological hazards in an artisanal salami produced in Northern Italy and its production environment in different seasonal periods.

In the present study, the occurrence of Listeria monocytogenes, Staphylococcus aureus, Salmonella spp. and Escherichia coli VTEC was investigated in two batches of artisanal Italian salami tested in winter and summer. Moreover, enumerations of total bacterial count, lactic acid bacteria and Enterobacteriaceae were performed as well as monitoring of water activity and pH. Samples were taken from raw materials, production process environment, semi-finished product and finished products. The results revealed an overall increase of total bacterial count and lactic acid bacteria during the ripening period, along with a decrease of Enterobacteriaceae, pH and water activity. No significant difference was observed between the two batches. The enterobacterial load appeared to decrease during the maturation period mainly due to a decrease in pH and water activity below the limits that allow the growth of these bacteria. E. coli VTEC, Salmonella spp. or L. monocytogenes were not detected in both winter and summer batches. However, Klebsiella pneumoniae was detected in both summer and winter products. Except for one isolate, no biological hazards were detected in the finished salami, proving the efficacy of the ripening period in controlling the occurrence of microbiological hazard in ripened salami. Further studies are required to assess the virulence potential of the Klebsiella pneumoniae isolates.

Genomic features of Klebsiella isolates from artisanal ready-to-eat food production facilities.

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 iuc3+ 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 iuc3-plasmid. Surveillance in the food chain will be crucial to obtain a more comprehensive picture of the circulation of Klebsiella strains with pathogenic potential.

Genetic Diversity and Antimicrobial Resistance of Extraintestinal E. coli Populations Pre- and Post-Antimicrobial Therapy on Broilers Affected by Colisepticemia.

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.

Variability in Physicochemical Parameters and Its Impact on Microbiological Quality and Occurrence of Foodborne Pathogens in Artisanal Italian Organic Salami.

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.

Application of multi-omic features clustering and pathway enrichment to clarify the impact of vitamin B2 supplementation on broiler caeca microbiome.

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.

Investigation on the microbiological hazards in an artisanal soft cheese produced in northern Italy and its production environment in different seasonal periods.

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.

Research note: The resistome of commensal Escherichia coli isolated from broiler carcasses "produced without the use of antibiotics"a.

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, ß-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.

Pilot Study on Poultry Meat from Antibiotic Free and Conventional Farms: Can Metagenomics Detect Any Difference?

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.

Occurrence of foodborne pathogens in Italian soft artisanal cheeses displaying different intra- and inter-batch variability of physicochemical and microbiological parameters.

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.

Resistome and virulome diversity of foodborne pathogens isolated from artisanal food production chain of animal origin in the Mediterranean region.

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.

Prevalence of Helicobacter pullorum in conventional, organic, and free-range broilers and typing of isolates.

Helicobacter pullorum represents a potential food-borne pathogen, and avian species appear to be a relevant reservoir of this organism. In this study, the prevalence of H. pullorum was investigated at 30 conventional farms where 169 ceca from 34 flocks were tested, at eight organic farms where 39 ceca from eight flocks were tested, and at seven free-range farms where 40 ceca from eight flocks were tested. All of the ceca were obtained from healthy broiler chickens. Moreover, amplified fragment length polymorphism, pulsed-field gel electrophoresis, and automated ribotyping were employed to estimate the levels of genetic variability of H. pullorum broiler isolates within and between flocks. Overall, Gram-negative, slender, curved rods, identified as H. pullorum by PCR, were isolated at 93.3% of the farms tested. The percentage of positive free-range farms (54.2%) was significantly lower than that of conventional (100%) or organic (100%) farms (P < 0.001). The level of within-flock genetic variability, calculated as the number of flocks colonized by isolates genetically different by all of the typing methods, was 34.9%. Isolates showing identical profiles by each typing method were observed in 11.6% of the flocks, but they were never detected between flocks. However, groups of isolates clustered together with an overall similarity level of ≥85%. Our results suggest that even though a high level of genetic variability is attributable to H. pullorum broiler isolates, their hierarchical genotyping produces data useful for epidemiological investigations.

Preliminary data on the antimicrobial effect of Cannabis sativa L. variety Futura 75 against food-borne pathogens in vitro as well as against naturally occurring microbial populations on minced meat during storage.

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.

Effects of Vitamin B2 Supplementation in Broilers Microbiota and Metabolome.

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.