Impact of feed additives and host-related factors on bacterial metabolites, mucosal integrity and immune response in the ileum of broilers.

The present study aimed to investigate the effect of age, breed, and sex of broilers, as well as a probiotic or phytobiotic product on mucosal morphology, bacterial metabolites, and immune traits in the ileum of broilers. A total of 2,880 one-day-old male and female broiler chicks from two breeds (Ross308® and Cobb500®) were randomly assigned to 72 pens. Broilers were offered a wheat-soybean diet without (CO), or with either a probiotic (PO; 2.4 × 109 CFU/kg of Bacillus subtilis DSM32324 and DSM32325 and B. amyloliquefaciens DSM25840) or a phytobiotic (PY; grape extract, 165 ppm procyanidin and 585 ppm polyphenols of the diet) product. The trial was conducted with a 3 × 2 × 2 factorial arrangement of diet, breed, and sex in a completely randomized design (6 replicate-pens per treatment). At day 7, 21, and 35, one chicken per pen was slaughtered for collecting ileal tissue to evaluate of histomorphology and mRNA expression, as well as ileal digesta to measure bacterial metabolites. Data were subjected to ANOVA (the main factors; age, diet, breed, and sex) and Four-Way ANOVA (interactions) using GLM procedure. Overall, the concentration of acetate and total short chain fatty acids reached the peak and lactate decreased to its lowest on day 21, but their concentrations at day 7 and 35 were similar (p > 0.05). Spermine, spermidine, and ammonia decreased after day 7, while putrescine and cadaverine increased after day 21 (p < 0.05). mRNA expression of cytokines, mucin 2 (MUC2) and claudin 5 (CLDN5) was similar; increased from day 7 to 21 and decreased afterward (p < 0.05). Villus height, crypt depth and villus surface area increased with age (p < 0.05). Acidic goblet cells (GC) number and density increased after day 21 (p < 0.05). Ross broilers showed higher D-lactate concentration and IFN-γ expression, while Cobb broilers had greater IL-4, IL-6 and TNF-α expression and higher total GC number (p < 0.05). Female displayed higher villus height and GC number and density (mixed and total GC) than male (p < 0.05). The effect of dietary treatment was not found on any investigated variables (p > 0.05). In conclusion, aging of broilers affected ileal histomorphology, cytokine expression, and barrier integrity, as well as bacterial activity. These observed impacts could be attributed to host-microbiota interaction and the direct effects of bacterial metabolites on intestinal cells and immune system.

Corrigendum: Screening of Host Specific Lactic Acid Bacteria Active Against Escherichia coli From Massive Sample Pools With a Combination of in vitro and ex vivo Methods.

[This corrects the article DOI: 10.3389/fmicb.2019.02705.].

The Impact of Direct-Fed Microbials and Phytogenic Feed Additives on Prevalence and Transfer of Extended-Spectrum Beta-Lactamase Genes in Broiler Chicken.

Poultry frequently account for the highest prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in livestock. To investigate the impact of direct-fed microbials (DFM) and phytobiotic feed additives on prevalence and conjugation of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, an animal trial was conducted. Lactobacillus agilis LA73 and Lactobacillus salivarius LS1 and two commercial phytogenic feed additives (consisting of carvacrol, cinnamaldehyde, and eugenol) were used as feed additives either alone or as a combination of DFM and phytogenic feed additive. An ESBL-producing E. coli donor and a potentially pathogenic Salmonella Typhimurium recipient were inoculated at 5 × 109 cells/mL in cecal contents from 2-week-old broilers. Conjugation frequencies were determined after 4 h aerobic co-incubation at 37 °C and corrected for the impact of the sample matrix on bacterial growth of donor and recipient. Surprisingly, indigenous Enterobacteriaceae acted as recipients instead of the anticipated Salmonella recipient. The observed increase in conjugation frequency was most obvious in the groups fed the combinations of DFM and phytogenic product, but merely up to 0.6 log units. Further, cecal samples were examined for ESBL-producing Enterobacteriaceae on five consecutive days in broilers aged 27-31 days. All samples derived from animals fed the experimental diet showed lower ESBL-prevalence than the control. It is concluded that Lactobacillus spp. and essential oils may help to reduce the prevalence of ESBL-harboring plasmids in broilers, while the effect on horizontal gene transfer is less obvious.

Synergistic Effects of Probiotics and Phytobiotics on the Intestinal Microbiota in Young Broiler Chicken.

Probiotics and phytobiotics have been studied as in-feed antibiotic alternatives for decades, yet there are no studies on their possible symbiotic effects. In the present study, newly hatched chickens were fed with feeds supplemented either with host-specific Lactobacillus strains (L. agilis and L. salivarius), commercial phytobiotics, or combinations of both. After 13 days of life, crops and caecums were analyzed for bacterial composition (16S rDNA sequencing, qPCR) and activity (bacterial metabolites). Crop and caecum samples were also used to study the ex vivo survival of a broiler-derived extended-spectrum beta-lactamase (ESBL) producing Escherichia coli strain. In the crop, combinations of probiotics and phytobiotics, but not their single application, increased the dominance of lactobacilli. The single application of phytobiotics reduced the metabolite concentrations in the crop, but certain combinations synergistically upregulated the metabolites. Changes in the qualitative and quantitative composition of the caecal microbiota were less pronounced than in the crop. Acetate concentrations were significantly lower for phytobiotics or the L. agilis probiotic strain compared to the control group, but the L. salivarius probiotic showed significantly higher acetate concentrations alone or in combination with one phytobiotic. The synergistic effects on the reduction of the ex vivo survival of an ESBL producing E. coli strain in crop or caecum contents were also observed for most combinations. This study shows the beneficial synergistic effects of probiotics and phytobiotics on the intestinal bacterial composition and their metabolic activity in young broilers. The reduced survival of potentially problematic bacteria, such as ESBL-producing E. coli further indicates that combinations of probiotics and phytobiotics may lead to a more enhanced functionality than their individual supplementation.