In Vitro Evaluation of Brown Seaweed Laminaria spp. as a Source of Antibacterial and Prebiotic Extracts That Could Modulate the Gastrointestinal Microbiota of Weaned Pigs.

Laminaria spp. and their extracts have preventative potential as dietary supplements during weaning in pigs. The first objective of this study was to evaluate increasing concentrations of four whole seaweed biomass samples from two different Laminaria species harvested in two different months in a weaned pig faecal batch fermentation assay. Particularly, February and November whole seaweed biomass samples of L. hyperborea (LHWB-F and LHWB-N) and L. digitata (LDWB-F and LDWB-N) were used. In the next part of the study, the increasing concentrations of four extracts produced from L. hyperborea (LHE1-4) and L. digitata (LDE1-4) were evaluated in individual pure-culture growth assays using a panel of beneficial and pathogenic bacterial strains (second objective). The LHE1-4 and LDE1-4 were obtained using different combinations of temperature, incubation time and volume of solvent within a hydrothermal-assisted extraction methodology (E1-4). In the batch fermentation assay, the L. hyperborea biomass samples, LHWB-F and LHWB-N, lowered Bifidobacterium spp. counts compared to the L. digitata biomass samples, LDWB-F and LDWB-N (p < 0.05). LHWB-F and LDWB-N reduced Enterobacteriaceae counts (p < 0.05). LHWB-F and LDWB-F were selected as the most and least promising sources of antibacterial extracts from which to produce LHE1-4 and LDE1-4. In the pure-culture growth assays, E1- and E4-produced extracts were predominantly associated with antibacterial and bifidogenic activities, respectively. LHE1 reduced both Salmonella Typhimurium and Enterotoxigenic Escherichia coli with LDE1 having a similar effect on both of these pathogenic strains, albeit to a lesser extent (p < 0.05). Both LHE1 and LDE1 reduced B. thermophilum counts (p < 0.05). LDE4 exhibited strong bifidogenic activity (p < 0.05), whereas LHE4 increased Bifidobacterium thermophilum and Lactiplantibacillus plantarum counts (p < 0.05). In conclusion, antibacterial and bifidogenic extracts of Laminaria spp. were identified in vitro with the potential to alleviate gastrointestinal dysbiosis in newly weaned pigs.

Evaluation of the in vitro effects of the increasing inclusion levels of yeast ß-glucan, a casein hydrolysate and its 5 kDa retentate on selected bacterial populations and strains commonly found in the gastrointestinal tract of pigs.

Previously, the 5 kDa retentate (5kDaR) of a casein hydrolysate (CH) and yeast ß-glucan (YBG) were identified as promising anti-inflammatory dietary supplements for supporting intestinal health in pigs post-weaning. However, their direct effects on intestinal bacterial populations are less well-known. The main objectives of this study were to determine if the increasing concentrations of the CH, 5kDaR and YBG individually, can: (1) alter the bacterial and short-chain fatty acid profiles in a weaned pig faecal batch fermentation assay, and (2) directly influence the growth of selected beneficial (Lactobacillus plantarum, L. reuteri, Bifidobacterium thermophilum) and pathogenic (Enterotoxigenic Escherichia coli, Salmonella Typhimurium) bacterial strains in individual pure culture growth assays. The potential of CH as a comparable 5kDaR substitute was also evaluated. The 5kDaR increased lactobacilli counts and butyrate concentration in the batch fermentation assay (P < 0.05) and increased L. plantarum (linear, P < 0.05), L. reuteri (quadratic, P < 0.05) and B. thermophilum (linear, P < 0.05) counts and reduced S. typhimurium (quadratic, P = 0.058) counts in the pure culture growth assays. CH increased butyrate concentration (P < 0.05) in the batch fermentation assay. YBG reduced Prevotella spp. counts (P < 0.05) and butyrate concentration (P < 0.05) in the batch fermentation assay. Both CH and YBG had no major effects in the pure culture growth assays. In conclusion, the 5kDaR had the most beneficial effects associated with increased counts of Lactobacillus and Bifidobacterium genera and butyrate production and reduced S. typhimurium counts in vitro indicating its potential to promote gastrointestinal health.

Evaluation of the Antibacterial and Prebiotic Potential of Ascophyllum nodosum and Its Extracts Using Selected Bacterial Members of the Pig Gastrointestinal Microbiota.

Ascophyllum nodosum and its extracts are promising antibacterial and prebiotic dietary supplements for pigs. The objectives of this study were to evaluate the effects of the increasing concentrations of: (1) two whole biomass samples of A. nodosum with different harvest seasons, February (ANWB-F) and November (ANWB-N), in a weaned pig faecal batch fermentation assay, and (2) A. nodosum extracts produced using four different extraction conditions of a hydrothermal-assisted extraction methodology (ANE1-4) and conventional extraction methods with water (ANWE) and ethanol (ANEE) as solvent in individual pure culture growth assays using a panel of beneficial and pathogenic bacterial strains. In the batch fermentation assay, ANWB-F reduced Bifidobacterium spp. counts (p < 0.05) while ANWB-N increased total bacterial counts and reduced Bifidobacterium spp. and Enterobacteriaceae counts (p < 0.05). Of the ANE1-4, produced from ANWB-F, ANWE and ANEE that were evaluated in the pure culture growth assays, the most interesting extracts were the ANE1 that reduced Salmonella Typhimurium, enterotoxigenic Escherichia coli and B. thermophilum counts and the ANE4 that stimulated B. thermophilum growth (p < 0.05). In conclusion, the extraction method and conditions influenced the bioactivities of the A. nodosum extracts with ANE1 and ANE4 exhibiting distinct antibacterial and prebiotic properties in vitro, respectively, that merit further exploration.

Effect of a Laminarin Rich Macroalgal Extract on the Caecal and Colonic Microbiota in the Post-Weaned Pig.

Dietary supplementation with 300 ppm of a laminarin rich macroalgal extract reduces post-weaning intestinal dysfunction in pigs. A comprehensive analysis of the impact of laminarin on the intestinal microbiome during this period is essential to inform on the mode of action of this bioactivity. The objective of this study was to evaluate the effects of supplementing the diet of newly weaned pigs with 300 ppm of a laminarin rich extract, on animal performance, volatile fatty acids, and the intestinal microbiota using 16S rRNA gene sequencing. Pigs fed the laminarin-supplemented diet had higher average daily feed intake, growth rate, and body weight compared to pigs fed the control diet (p < 0.05). Pigs fed the laminarin-supplemented diet had reduced abundance of OTUs assigned to Enterobacteriaceae and increased abundance of OTUs assigned to the genus Prevotella (p < 0.05) compared to pigs fed the control diet. Enterobacteriaceae had negative relationships (p < 0.05) with average daily feed intake (ADFI), average daily gain (ADG), and butyric acid concentrations. In contrast, Prevotellaceae were positively correlated (p < 0.05) with ADFI, ADG, total VFA, acetic, propionic, butyric acids, and negatively correlated with isovaleric acid. Hence supplementation with a laminarin enriched extract potentially improves performance during the post-weaning period by promoting the proliferation of bacterial taxa such as Prevotella that favourably enhance nutrient digestion while reducing the load of potentially pathogenic bacterial taxa including Enterobacteriaceae.

Evaluation of carvacrol for the control of Escherichia coli O157 on cattle hide and carcass cuts.

The antimicrobial activity of carvacrol against Escherichia coli O157 was assessed on cattle hide and beef carcass cuts. Carvacrol (0, 10, 20, and 30 mg/mL) was applied using a spray bottle to cattle hide and beef carcass cuts, inoculated with a cocktail of E. coli O157 (5-6 log(10) CFU/cm(2)) and left in contact for 10 min. Following treatment, hide and carcass cuts were sampled using a swab method, and E. coli O157 were enumerated by plate counts on selective media. Carvacrol (30 mg/mL) significantly (p < 0.05) reduced inoculated E. coli O157 (1.4 and 1.58 log(10) CFU/cm(2)) compared to the no wash and water wash (0 mg/mL) controls on both carcass cuts and hide respectively. This preliminary study shows that carvacrol has the potential to control E. coli O157 on bovine hide and carcass cuts, but further research with larger scale trials is needed.

Inhibition of verocytotoxigenic Escherichia coli by antimicrobial peptides caseicin A and B and the factors affecting their antimicrobial activities.

The antimic robial activities of caseicin A and B antimicrobial peptides (AMPs) were assessed against a selection of verocytotoxigenic Escherichia coli (VTEC) strains (n=11), other bacterial pathogenic and spoilage bacteria (n=7), using a model broth system. The ability of the AMPs to retain their antimicrobial activities against a strain of E. coli O157:H7 380-94 under various test conditions (pH, temperature, water activity, sodium chloride concentrations, inoculum size and the presence of competitive microflora) was assessed and the minimum inhibitory concentrations (MIC) and number of surviving E. coli O157:H7 calculated. The mean number of VTEC surviving after exposure to 2 mg/ml caseicin A and B was reduced by 4.96 and 4.19 log(10) cfu/ml compared to the respective controls. The susceptibility of E. coli O157:H7 to the caseicin AMPs decreased as temperature, pH, water activity and inoculum size were reduced. The presence of sodium chloride (0.5-2.5%) did not affect the activity of caseicin A (p>0.05), however it did inhibit the activity of caseicin B. The presence of a competitive microflora cocktail did not significantly (p>0.05) affect the activities of the AMPs for the majority of the concentrations tested. Using a quantitative PCR assay, the levels of verotoxins (vt1 and vt2) expressed by E. coli O157:H7 following exposure to a sub-inhibitory concentration (0.5 mg/ml) of caseicin A showed that the verotoxin levels did not differ from the levels produced by the control cultures. The antimicrobial activity of caseicin A against E. coli O157:H7 was also tested in a model rumen system, however concentrations of ≥2 mg/ml did not significantly (p>0.05) reduce E. coli O157:H7 numbers in the model system over a 24 h period. The application of caseicin AMPs in food and/or animal production may be valuable in combination with other antimicrobials although further research is required.

In vivo and ex vivo evaluations of bacteriophages e11/2 and e4/1c for use in the control of Escherichia coli O157:H7.

This study investigated the effect of bacteriophages (phages) e11/2 and e4/1c against Escherichia coli O157:H7 in an ex vivo rumen model and in cattle in vivo. In the ex vivo rumen model, samples were inoculated with either 10³ or 106 CFU/ml inoculum of E. coli O157:H7 and challenged separately with each bacteriophage. In the presence of phage e11/2, the numbers of E. coli O157:H7 bacteria were significantly (P < 0.05) reduced to below the limit of detection within 1 h. Phage e4/1c significantly (P < 0.05) reduced E. coli O157:H7 numbers within 2 h of incubation, but the number of surviving E. coli O157:H7 bacteria then remained unchanged over a further 22-h incubation period. The ability of a phage cocktail of e11/2 and e4/1c to reduce the fecal shedding of E. coli O157:H7 in experimentally inoculated cattle was then investigated in two cattle trials. Cattle (yearlings, n = 20 for trial one; adult fistulated cattle, n = 2 for trial two) were orally inoculated with 10(10) CFU of E. coli O157:H7. Animals (n = 10 for trial one; n = 1 for trial two) were dosed daily with a bacteriophage cocktail of 10(11) PFU for 3 days postinoculation. E. coli O157:H7 and phage numbers in fecal and/or rumen samples were determined over 7 days postinoculation. E. coli O157:H7 numbers rapidly declined in all animals within 24 to 48 h; however, there was no significant difference (P > 0.05) between the numbers of E. coli O157:H7 bacteria shed by the phage-treated or control animals. Phages were recovered from the rumen but not from the feces of the adult fistulated animal in trial two but were recovered from the feces of the yearling animals in trial one. While the results from the rumen model suggest that phages are effective in the rumen, further research is required to improve the antimicrobial effectiveness of phages for the elimination of E. coli O157:H7 in vivo.

Inhibition of verocytotoxigenic Escherichia coli in model broth and rumen systems by carvacrol and thymol.

The antimicrobial activities of thymol and carvacrol were assessed against a selection of verocytotoxigenic Escherichia coli (VTEC) strains (n=11) and other bacterial species and spoilage bacteria (n=7) using a model broth system. The effects of pH, temperature, water activity, sodium chloride concentrations, inoculum size and the presence of competing microflora on the activities of thymol and carvacrol against E. coli O157:H7 strain 380-94 were also determined. The minimum inhibitory and bactericidal concentrations (MIC and MBC, respectively) and numbers of surviving E. coli O157:H7 were determined following incubation. The mean numbers of VTEC surviving exposure to thymol or carvacrol at concentrations of >/=500mug/ml were between 2.0 and 7.8log cfu/ml less than the numbers in the corresponding controls. The susceptibility of E. coli O157:H7 to carvacrol or thymol was found to increase with decreasing storage temperature, water activity, pH and E. coli O157:H7 inoculum size. Sodium chloride (0.5-2.5%) and the presence of a microflora cocktail did not significantly (p>0.05) affect the antimicrobial activities of thymol or carvacrol against E. coli O157:H7. The antimicrobial activity of carvacrol against E. coli O157:H7 was also tested in a model rumen system. A MIC of 500mug/ml carvacrol reduced E. coli O157:H7 inoculated at levels of 10(3) and 10(6)cfu/ml to undetectable levels in the system after 24h incubation. This concentration of carvacrol significantly (p<0.05) decreased the total gas production and volatile fatty acid concentrations in the model rumen assay.