Routes of dispersion of antibiotic resistance genes from the poultry farm system.

Poultry farms are hotspots for the development and spread of antibiotic resistance genes (ARGs), due to high stocking densities and extensive use of antibiotics, posing a threat of spread and contagion to workers and the external environment. Here, we applied shotgun metagenome sequencing to characterize the gut microbiome and resistome of poultry, workers and their households - also including microbiomes from the internal and external farm environment - in three different farms in Italy during a complete rearing cycle. Our results highlighted a relevant overlap among the microbiomes of poultry, workers, and their families (gut and skin), with clinically relevant ARGs and associated mobile elements shared in both poultry and human samples. On a finer scale, the reconstruction of species-level genome bins (SGBs) allowed us to delineate the dynamics of microorganism and ARGs dispersion from farm systems. We found the associations with worker microbiomes representing the main route of ARGs dispersion from poultry to human populations. Collectively, our findings clearly demonstrate the urgent need to implement more effective procedures to counteract ARGs dispersion from poultry food systems and the relevance of metagenomics-based metacommunity approaches to monitor the ARGs dispersion process for the safety of the working environment on farms.

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.

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.

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.

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.

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.

Survey on Carbapenem-Resistant Bacteria in Pigs at Slaughter and Comparison with Human Clinical Isolates in Italy.

This study is focused on resistance to carbapenems and third-generation cephalosporins in Gram-negative microorganisms isolated from swine, whose transmission to humans via pork consumption cannot be excluded. In addition, the common carriage of carbapenem-resistant (CR) bacteria between humans and pigs was evaluated. Sampling involved 300 faecal samples collected from slaughtered pigs and 300 urine samples collected from 187 hospitalised patients in Parma Province (Italy). In swine, MIC testing confirmed resistance to meropenem for isolates of Pseudomonas aeruginosa and Pseudomonas oryzihabitans and resistance to cefotaxime and ceftazidime for Escherichia coli, Ewingella americana, Enterobacter agglomerans, and Citrobacter freundii. For Acinetobacter lwoffii, Aeromonas hydrofila, Burkolderia cepacia, Corynebacterium indologenes, Flavobacterium odoratum, and Stenotrophomonas maltophilia, no EUCAST MIC breakpoints were available. However, ESBL genes (blaCTXM-1, blaCTX-M-2, blaTEM-1, and blaSHV) and AmpC genes (blaCIT, blaACC, and blaEBC) were found in 38 and 16 isolates, respectively. P. aeruginosa was the only CR species shared by pigs (4/300 pigs; 1.3%) and patients (2/187; 1.1%). P. aeruginosa ST938 carrying blaPAO and blaOXA396 was detected in one pig as well as an 83-year-old patient. Although no direct epidemiological link was demonstrable, SNP calling and cgMLST showed a genetic relationship of the isolates (86 SNPs and 661 allele difference), thus suggesting possible circulation of CR bacteria between swine and humans.

A multi-omics approach to elucidate the mechanisms of action of a dietary muramidase administered to broiler chickens.

A novel dietary muramidase has been shown to have positive effects on broiler chickens. However, very little is known about its mechanisms of action. The present multi-omics investigation sought to address this knowledge gap. A total of 2,340 day-old male broilers were assigned to 3 groups (12 replicates each) fed, from 0 to 42 d, a basal diet (control group-CON) or the basal diet supplemented with muramidase at 25,000 (low-dose group-MUL) or 45,000 LSU(F)/kg feed (high-dose group-MUH). MUH significantly outperformed CON in terms of cumulative feed intake (4,798 vs 4,705 g), body weight (2,906 vs 2,775 g), and feed conversion ratio (1.686 vs 1.729), while MUL exhibited intermediate performance. At caecal level, MUH showed the lowest alpha diversity, a significantly different beta diversity, a reduction in Firmicutes, and a rise in Bacteroidetes, especially compared with MUL. MUH also exhibited a considerable decrease in Clostridiaceae and an overrepresentation of Bacteroidaceae and Lactobacillaceae. At blood level, MUH had lower hypoxanthine-probably due to its drop at caecal level-histidine, and uracil, while greater pyruvate, 2-oxoglutarate, and glucose. This study sheds light on the mode of action of this muramidase and lays the groundwork for future investigations on its effects on the intestinal ecosystem and systemic metabolism of broiler chickens.

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.

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.

Comparison between 16S rRNA and shotgun sequencing data for the taxonomic characterization of the gut microbiota.

In this paper we compared taxonomic results obtained by metataxonomics (16S rRNA gene sequencing) and metagenomics (whole shotgun metagenomic sequencing) to investigate their reliability for bacteria profiling, studying the chicken gut as a model system. The experimental conditions included two compartments of gastrointestinal tracts and two sampling times. We compared the relative abundance distributions obtained with the two sequencing strategies and then tested their capability to distinguish the experimental conditions. The results showed that 16S rRNA gene sequencing detects only part of the gut microbiota community revealed by shotgun sequencing. Specifically, when a sufficient number of reads is available, Shotgun sequencing has more power to identify less abundant taxa than 16S sequencing. Finally, we showed that the less abundant genera detected only by shotgun sequencing are biologically meaningful, being able to discriminate between the experimental conditions as much as the more abundant genera detected by both sequencing strategies.

Metagenomics-Based Proficiency Test of Smoked Salmon Spiked with a Mock Community.

An inter-laboratory proficiency test was organized to assess the ability of participants to perform shotgun metagenomic sequencing of cold smoked salmon, experimentally spiked with a mock community composed of six bacteria, one parasite, one yeast, one DNA, and two RNA viruses. Each participant applied its in-house wet-lab workflow(s) to obtain the metagenomic dataset(s), which were then collected and analyzed using MG-RAST. A total of 27 datasets were analyzed. Sample pre-processing, DNA extraction protocol, library preparation kit, and sequencing platform, influenced the abundance of specific microorganisms of the mock community. Our results highlight that despite differences in wet-lab protocols, the reads corresponding to the mock community members spiked in the cold smoked salmon, were both detected and quantified in terms of relative abundance, in the metagenomic datasets, proving the suitability of shotgun metagenomic sequencing as a genomic tool to detect microorganisms belonging to different domains in the same food matrix. The implementation of standardized wet-lab protocols would highly facilitate the comparability of shotgun metagenomic sequencing dataset across laboratories and sectors. Moreover, there is a need for clearly defining a sequencing reads threshold, to consider pathogens as detected or undetected in a food sample.

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.

Effects of Alternative Administration Programs of a Synbiotic Supplement on Broiler Performance, Foot Pad Dermatitis, Caecal Microbiota, and Blood Metabolites.

This research investigated the effects of different synbiotic administration programs on broiler productive performance and foot pad dermatitis (FPD). Molecular insights on caecal microbiota and plasma metabolomics were also performed. - A total of 1000 one-day-old male chicks were grouped by the synbiotic treatment. The synbiotic was either sprayed as gel droplets onto newly hatched chicks at the hatchery (100 g/10,000 birds) or supplemented in-feed during the entire rearing period (1000, 500, and 250 g/ton according to feeding phase), or both. Only the treatments' combination produced significant results in comparison with the control group (untreated), improving feed conversion ratio from 14 to 29 d and in the overall period of the trial (1.570 vs. 1.509 and 1.643 vs. 1.596, respectively; p < 0.05) while lowering FPD occurrence at slaughter (17% vs. 5%; p < 0.05). These findings can be related to significant variations of caecal microbiota, like higher Firmicutes to Bacteroidetes ratio (with favorable implications for host's energy-harvesting potential from the diet) and more beneficial microbial consortium presumably sustaining eubiosis. Overall, these results indicate that administering synbiotics through gel droplets at the hatchery combined to in-feed supplementation for the whole growing cycle positively affects broiler feed efficiency and welfare.

Effect of Lactobacillus acidophilus D2/CSL (CECT 4529) supplementation in drinking water on chicken crop and caeca microbiome.

In this study we gained insights into the effects of the supplementation with Lactobacillus acidophilus D2/CSL (CECT 4529) in the chicken drinking water on crop and caeca microbiomes. The probiotic was supplemented at the concentrations of 0.2 g Lactobacillus acidophilus/day/bird and 0.02 g Lactobacillus acidophilus/day/bird and its effect on the crop and caeca microbiomes was assessed at 14 and 35 days of rearing. The results showed that mean relative abundance of Lactobacillus acidophilus in the caeca did not show significative differences in the treated and control birds, although Lactobacillus acidophilus as well as Faecalibacterium prausnitzii, Lactobacillus crispatus and Lactobacillus reuteri significantly increased over time. Moreover, the treatment with the high dose of probiotic significantly increased the abundance of Clostridium asparagiforme, Clostridium hathewayi and Clostridium saccharolyticum producing butyrate and other organic acids supporting the chicken health. Finally, at 35 days, the Cell division protein FtsH (EC 3.4.24.-) and the Site-specific recombinase genes were significantly increased in the caeca of birds treated with the high dose of probiotic in comparison to the control group. The results of this study showed that Lactobacillus acidophilus D2/CSL (CECT 4529) supplementation in the drinking water at the concentrations of 0.2 and 0.02 g Lactobacillus acidophilus/day/bird improved beneficial microbes and functional genes in broiler crops and caeca. Nevertheless, the main site of action of the probiotic is the crop, at least in the early stage of the chicken life. Indeed, at 14 days Lactobacillus acidophilus was significantly higher in the crops of chickens treated with the high dose of LA in comparison to the control (14.094 vs 1.741%, p = 0.036).

Network Approach to Source Attribution of Salmonella enterica Serovar Typhimurium and Its Monophasic Variant.

Salmonella enterica subspecies enterica serovar Typhimurium and its monophasic variant are among the most common Salmonella serovars associated with human salmonellosis each year. Related infections are often due to consumption of contaminated meat of pig, cattle, and poultry origin. In order to evaluate novel microbial subtyping methods for source attribution, an approach based on weighted networks was applied on 141 human and 210 food and animal isolates of pigs, broilers, layers, ducks, and cattle collected in Denmark from 2013 to 2014. A whole-genome SNP calling was performed along with cgMLST and wgMLST. Based on these genomic input data, pairwise distance matrices were built and used as input for construction of a weighted network where nodes represent genomes and links to distances. Analyzing food and animal Typhimurium genomes, the coherence of source clustering ranged from 89 to 90% for animal source, from 84 to 85% for country, and from 63 to 65% for year of isolation and was equal to 82% for serotype, suggesting animal source as the first driver of clustering formation. Adding human isolate genomes to the network, a percentage between 93.6 and 97.2% clustered with the existing component and only a percentage between 2.8 and 6.4% appeared as not attributed to any animal sources. The majority of human genomes were attributed to pigs with probabilities ranging from 83.9 to 84.5%, followed by broilers, ducks, cattle, and layers in descending order. In conclusion, a weighted network approach based on pairwise SNPs, cgMLST, and wgMLST matrices showed promising results for source attribution studies.