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

Whole genome sequencing for typing and characterisation of Listeria monocytogenes isolated in a rabbit meat processing plant

Listeria monocytogenes is a food-borne pathogen able to survive and grow in different environments including food processing plants where it can persist for month or years. In the present study the discriminatory power of Whole Genome Sequencing (WGS)-based analysis (cgMLST) was compared to that of molecular typing methods on 34 L. monocytogenes isolates collected over one year in the same rabbit meat processing plant and belonging to three genotypes (ST14, ST121, ST224). Each genotype included isolates indistinguishable by standard molecular typing methods. The virulence potential of all isolates was assessed by Multi Virulence-Locus Sequence Typing (MVLST) and the investigation of a representative database of virulence determinant genes. The whole genome of each isolate was sequenced on a MiSeq platform. The cgMLST, MVLST, and in silico identification of virulence genes were performed using publicly available tools. Draft genomes included a number of contigs ranging from 13 to 28 and N50 ranging from 456298 to 580604. The coverage ranged from 41 to 187X. The cgMLST showed a significantly superior discriminatory power only in comparison to ribotyping, nevertheless it allows the detection of two singletons belonging to ST14 that were not observed by other molecular methods. All ST14 isolates belonged to VT107, which 7-loci concatenated sequence differs for only 4 nucleotides to VT1 (Epidemic clone III). Analysis of virulence genes showed the presence of a fulllength inlA version in all ST14 isolates and of a mutated version including a premature stop codon (PMSC) associated to attenuated virulence in all ST121 isolates

Impact of Cooking Procedures and Storage Practices at Home on Consumer Exposure to Listeria Monocytogenes and Salmonella Due to the Consumption of Pork Meat

[EN] The objective of this research was to analyze the impact of different cooking procedures (i.e., gas hob and traditional static oven) and levels of cooking (i.e., rare, medium, and well-done) on inactivation of Listeria monocytogenes and Salmonella in pork loin chops. Moreover, the consumer's exposure to both microorganisms after simulation of meat leftover storage at home was assessed. The results showed that well-done cooking in a static oven was the only treatment able to inactivate the tested pathogens. The other cooking combinations allowed to reach in the product temperatures always 73.6 degrees C, decreasing both pathogens between 6 log(10) cfu/g and 7 log(10) cfu/g. However, according to simulation results, the few cells surviving cooking treatments can multiply during storage by consumers up to 1 log(10) cfu/g, with probabilities of 0.059 (gas hob) and 0.035 (static oven) for L. monocytogenes and 0.049 (gas hob) and 0.031 (static oven) for Salmonella. The key factors affecting consumer exposure in relation to storage practices were probability of pathogen occurrence after cooking, doneness degree, time of storage, and time of storage at room temperature. The results of this study can be combined with prevalence data and dose-response models in risk assessment models and included in guidelines for consumers on practices to be followed to manage cooking of pork meat at home.The research leading to these results received funding from the E.U. Seventh Framework Programme under grant agreement KBBE 222738BASELINE (Selection and Improving of Fit-for-Purpose Sampling Procedures for Specific Foods and Risks).De Cesare, A.; Doménech Antich, EM.; Comin, D.; Meluzzi, A.; Manfreda, G. (2017). Impact of Cooking Procedures and Storage Practices at Home on Consumer Exposure to Listeria Monocytogenes and Salmonella Due to the Consumption of Pork Meat. Risk Analysis. 38(4):638-652. https://doi.org/10.1111/risa.12882S63865238

Sanitisation of fresh-cut celery and radicchio by gas plasma treatments in water medium

open7noNome progetto: STARTEC: “Decision Support Tools to ensure safe, tasty and nutritious Advanced Ready-to-eat foods for healthy and vulnerable Consumers”.The antimicrobial efficacy of dielectric barrier discharge atmospheric gas plasma (DBD) was tested against Listeria monocytogenes and shigatoxin-producing Escherichia coli serogroups O157 and O26. Challenge tests were carried out with samples of cut celery and radicchio leaves inoculated with a mix of five strains of L. monocytogenes or the two strains of E. coli immersed in deionised water. The treatment efficacy was also assessed considering only the contaminated deionised water. For deionised inoculated water alone, a treatment time-dependent strong effect was observed and a pathogens reduction higher than 6LogCFU/mL was obtained after 40min of treatment. With the vegetables presence in the liquid medium, the efficacy appeared reduced and related to the treatment time, microorganism, substrate and storage duration (reduction up to 2.5 and 3.7LogCFU/cm2 for L. monocytogenes and E. coli, respectively). No significant changes were observed on celery visual attributes, soluble solids content and textural parameters. A significant decrease of the chroma colour parameter during storage was noted in treated radicchio samples respect to control ones.openBerardinelli, Annachiara; Pasquali, Frederique; Cevoli, Chiara; Trevisani, Marcello; Ragni, Luigi; Mancusi, Rocco; Manfreda, GerardoBerardinelli, Annachiara; Pasquali, Frederique; Cevoli, Chiara; Trevisani, Marcello; Ragni, Luigi; Mancusi, Rocco; Manfreda, Gerard

Effect of dietary supplementation with Lactobacillus acidophilus D2/CSL (CECT 4529) on caecum microbioma and productive performance in broiler chickens

This study examines the effects of the dietary supplementation with Lactobacillus acidophilus D2/CSL (CECT 4529) (LA) on productive performances, incidence of foot pad dermatitis and caecum microbioma in broiler chickens. A total of 1,100 one-day old male Ross 308 chicks were divided into 2 groups of 16 replicates with 25 birds each and reared from 1-41 d. One group was fed a basal diet (CON) and the other group the same diet supplemented with LA. Caecum contents were collected from 4 selected birds at day one and 5 selected birds at the end of the rearing period. Then, they were submitted to DNA extraction and whole DNA shotgun metagenomic sequencing. Overall, the LA supplementation produced a significant beneficial effect on body weight gain between 15-28 d and improved feed conversion rate in the overall period. On the contrary, litter moisture, pH and incidence of the foot pad lesions were not affected by LA. Birds treated with LA showed a lower occurrence of pasty vent at both 14 and 28 d. At the end of the rearing period, Lachanospiraceae were significantly higher in LA birds in comparison to CON (17.07 vs 14.39%; P = 0.036). Moreover, Ruminococcus obeum, Clostridium clostridioforme, Roseburia intestinalis, Lachnos-piraceae bacterium 14-2T and Coprococcus eutactuswere significantly higher in LA birds in comparison to CON. The relative abundance of Lactobacillus acidophilus was comparable between LA and CON groups. However, a positive effect was observed in relation to the metabolic functions in the treated group, with particular reference to the higher abundance of \u3b2-glucosidase. In conclusion, the LA supplementation improved broiler productive performances and metabolic functions promoting animal health

Impact of a probiotic-based cleaning product on the microbiological profile of broiler litters and chicken caeca microbiota

ABSTRACT 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

Effect of a low protein diet on chicken ceca microbiome and productive performances

ABSTRACT The aim of this study was to investigate the impact of supplementation of a low protein diet on ceca microbiome and productive performances of broiler chickens. A total of 1,170 one-day-old male chicks (Ross 308) were divided in 2 diet groups and reared in the same conditions up to 42 D. Birds belonging to the control group were fed a basal diet. Birds belonging to the low protein group the basal diet with a reduced level of crude protein (–7%). Cecum contents from randomly selected birds were collected at 14 and 42 D within each diet group, submitted to DNA extraction and then tested by shotgun metagenomic sequencing. Abundances of species belonging to Actinobacteria and Proteobacteria were mainly affected by the diet as well as interaction between diet and time, while species belonging to Firmicutes and Cyanobacteria changed mainly according to the age of the birds. At family level, Lactobacillaceae significantly decreased in the low protein group up to 14 D. However, at the end of the rearing period the same family was significantly higher in the low protein group. The most abundant functional genes, represented by cystine desulfurase, alpha-galactosidase, and serine hydroxymethyltransferase, displayed comparable abundances in both diet groups, although significative differences were identified for less abundant functional genes at both sampling times. Birds fed control and low protein diets showed similar productive performances. However, in the finisher phase, feed conversion rate was significantly better in chickens fed the low protein diet. Overall, this study showed that a reduced intake of crude protein in broilers increases the abundance of Lactobacillaceae in the ceca over time and this seems to be linked to a better feed conversion rate between 36 and 42 D. A reduced intake of crude protein in chicken production can help to improve exploitation of edible resources, while reducing the emission of nitrogen pollutants in the environment

Depression and Microbiome—Study on the Relation and Contiguity between Dogs and Humans

Behavioral studies demonstrate that not only humans, but all other animals including dogs, can suffer from depression. A quantitative molecular evaluation of fatty acids in human and animal platelets has already evidenced similarities between people suffering from depression and German Shepherds, suggesting that domestication has led dogs to be similar to humans. In order to verify whether humans and dogs suffering from similar pathologies also share similar microorganisms at the intestinal level, in this study the gut-microbiota composition of 12 German Shepherds was compared to that of 15 dogs belonging to mixed breeds which do not suffer from depression. Moreover, the relation between the microbiota of the German Shepherd\u2019s group and that of patients with depression has been investigated. The results indicate that the German Shepherd\u2019s gut-microbiota has a different composition compared to other dog breeds and is characterized by microbial groups identified in humans with depression, highlighting the existence of a \u201ccore\u201d microbiota associated with depression

Evaluation of Real-Time PCR to complement ISO 6579:2004 method for the detection of Salmonella in pork cuts

According to Commission Regulation (EC) No 2073/2005 of 15 November 2005 on microbiological crite-ria for foodstuff , the analytical reference method for the detection of Salmonella in food is ISO 6579:2004. However this long and labor-intensive method is not in line with the short production times of the food industry. In the last years, Real-Time PCR is used more and more by scientists for the relia-ble, fast and specific detection of bacterial pathogens in food. The aim of the present study was to eval-uate the Salmonella detection capability of a validated Real-Time PCR assay on naturally contaminated pork cuts in comparison with the reference method ISO 6579:2004. Three sampling were performed and included 16 pork cut packaging. From each packaging, three aliquots of 10 g each were tested separate-ly by ISO 6579:2004 method and by Real-Time PCR. In particular this molecular method was applied on DNA samples extracted from pre-enrichment broth after 1 and 18 hours of incubation. Within the three sampling periods, Real-Time PCR detected Salmonella in 81%, 100% e 62,5% of pork cut samples respectively, whereas the corresponding percentages of detection of the reference method were 56%, 81% e 62,5% respectively. In conclusion the Real-Time PCR assay used in the present study might be a reliable tool for a fast detection of Salmonella on pork cuts, especially when large number of samples needs to be tested. The reference method might be applied only on positive samples for isolation purpos-es mandatory in epidemiological investigations

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

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