In Vitro Antimicrobial Activities of Organic Acids and Their Derivatives on Several Species of Gram-Negative and Gram-Positive Bacteria.

The objective of this study was to determine the in vitro antimicrobial activity of several organic acids and their derivatives against Gram-positive (G+) and Gram-negative (G-) bacteria. Butyric acid, valeric acid, monopropionin, monobutyrin, monovalerin, monolaurin, sodium formate, and ProPhorce-a mixture of sodium formate and formic acid (40:60 w/v)-were tested at 8 to 16 concentrations from 10 to 50,000 mg/L. The tested bacteria included G- bacteria (Escherichia coli, Salmonella enterica Typhimurium, and Campylobacter jejuni) and G+ bacteria (Enterococcus faecalis, Clostridium perfringens, Streptococcus pneumoniae, and Streptococcus suis). Antimicrobial activity was expressed as minimum inhibitory concentration (MIC) of tested compounds that prevented growth of tested bacteria in treated culture broth. The MICs of butyric acid, valeric acid, and ProPhorce varied among bacterial strains with the lowest MIC of 500-1000 mg/L on two strains of Campylobacter. Sodium formate at highest tested concentrations (20,000 mg/L) did not inhibit the growth of Escherichia coli, Salmonella Typhimurium, and Enterococcus faecalis, but sodium formate inhibited the growth of other tested bacteria with MIC values from 2000 to 18,800 mg/L. The MIC values of monovalerin, monolaurin, and monobutyrin ranged from 2500 to 15,000 mg/L in the majority of bacterial strains. Monopropionin did not inhibit the growth of all tested bacteria, with the exception that the MIC of monopropionin was 11,300 mg/L on Clostridia perfringens. Monolaurin strongly inhibited G+ bacteria, with the MIC value of 10 mg/L against Streptococcus pneumoniae. The MIC tests indicated that organic acids and their derivatives exhibit promising antimicrobial effects in vitro against G- and G+ bacteria that are resistant to antimicrobial drugs. The acid forms had stronger in vitro antimicrobial activities than ester forms, except that the medium chain fatty acid ester monolaurin exhibited strong inhibitory effects on G+ bacteria

Antimicrobial Effects of Organic Acids and Their Derivatives

The benefits of dietary organic acids have been utilized for decades to increase the shelf life of animal feed and promote animal growth and health. As products of fiber fermentation, or components in biological sources such as milk, organic acids are natural antimicrobial compounds. Since the 1930s, humans have administered antibiotics to prevent and treat bacterial infections in humans and animals. Although antibiotics were revolutionary and contributed to our success, they lose efficacy over time due to antibiotic resistance in bacteria. The development of antibiotic resistance has occurred naturally over millennia and is exacerbated by the misuse and/or overuse of antibiotics in humans and animals. The use of in-feed antibiotics for animal growth promotion in the United States has been banned since 2017. Livestock animals had received various protections and health benefits by consuming in-feed antibiotics or antimicrobial growth promoters (AGPs) prior to their banning. Therefore, research on the beneficial effects of non-antibiotic feed additives is increasing in order to potentially replace the growth-promoting effects and health benefits observed when fed AGPs is needed. To address this need, the aim of this experiment was to evaluate the potential of using organic acids and their derivatives as alternative antibiotic feed additives to promote animal growth. The selected organic acids, including butyric acid, valeric acid, propionate glycerides, butyrate glycerides, valerate glycerides, monolaurin, sodium formate, and ProPhorce, a mixture of sodium formate and formic acid (40:60 w/v), were tested at 8 to 16 concentrations from 10 to 50,000 mg/L. Their minimum inhibitory concentrations (MICs) for Gram-negative bacteria (Escherichia coli, Salmonella Typhimurium, and Campylobacter jejuni) and Gram-positive bacteria (Enterococcus faecalis, Clostridium perfringens, Streptococcus pneumoniae, and Streptococcus suis) were evaluated using the micro-broth dilution method (in vitro; Stoddard et al., 2008; Li et al., 2014; Li et al., 2015). The MICs of butyric acid, valeric acid, and ProPhorce varied among bacterial strains with the lowest MIC of 500 mg/L for two strains of Campylobacter. Sodium formate at highest tested concentrations (20,000 mg/L) did not inhibit the growth of Escherichia coli, Salmonella Typhimurium, and Enterococcus faecalis, but sodium formate inhibited the growth of other tested bacteria with MIC values from 2,000 to 18,800 mg/L. The MIC values of valerate glycerides, monolaurin, and butyrate glycerides ranged from 2,500 to 15,000 mg/L against the majority of bacterial strains. Propionate glycerides did not inhibit the growth of all investigated bacteria with the exception that the MIC of propionate glycerides was 11,300 mg/L on Clostridia perfringens. Monolaurin strongly inhibited Gram-positive bacteria with the MIC value of 10 mg/L against Streptococcus pneumoniae, and 300 mg/L against Clostridium perfringens. The MIC results indicate that organic acids and their derivatives exhibit promising antimicrobial effects in vitro against Gram-negative and Gram-positive bacteria that are resistant to antibiotics. Monoglyceride derivatives are inhibitory, although they may be less effective compared with their free fatty acid forms. In particular, medium chain fatty acid ester, monolaurin, exhibited very strong inhibitory effect on Gram-positive bacteria. In summary, this study suggests that certain organic acids and their derivatives have promising antimicrobial properties and are candidates for partially replacing antibiotics in feed. Future in vivo research will be needed in order to determine their benefits on promoting the overall health and performance in pig and poultry

Mint Oils: In Vitro Ability to Perform Anti-Inflammatory, Antioxidant, and Antimicrobial Activities and to Enhance Intestinal Barrier Integrity

The objectives of the study were to test the biological activities of peppermint and spearmint oils via (i) measuring in vitro anti-inflammatory effects with porcine alveolar macrophages (PAMs), (ii) determining the barrier integrity of IPEC-J2 by analyzing transepithelial electrical resistance (TEER), (iii) testing their antioxidant activities, and (iv) investigating the antimicrobial activity against enterotoxigenic Escherichia coli (ETEC) F18+. Briefly, (i) macrophages were seeded at 106 cells/mL and treated (24 h) with mint oils and lipopolysaccharide (LPS). The treatments were 2 (0 or 1 μg/mL of LPS) × 5 (0, 25, 50, 100, 200 µg/mL of mint oils). The supernatants were collected for TNF-α and IL-1β measurement by ELISA; (ii) IPEC-J2 cells were seeded at 5 × 105 cells/mL and treated with mint oils (0, 25, 50, 100, and 200 μg/mL). TEER (Ωcm2) was measured at 0, 24, 48, and 72 h; (iii) the antioxidant activity was assessed (0, 1, 50, 100, 200, 500, and 600 mg/mL) using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and reducing power assays; (iv) overnight-grown ETEC F18+ were quantified (CFU/mL) after supplementing with peppermint and spearmint oils (0, 1.44, 2.87, 5.75, 11.50, and 23.00 mg/mL). All data were analyzed using the MIXED procedure. Both mint oils significantly inhibited (p < 0.05) IL-1β and TNF-α secretion from LPS-stimulated PAMs. Mint oil treatments did not affect TEER in IPEC-J2. Spearmint and peppermint oils exhibited (p < 0.05) strong antioxidant activities in DPPH and reducing power assays. Both mint oils also dose-dependently inhibited (p < 0.05) the growth of ETEC F18+ in vitro. The results of the study indicated that both mint oils are great candidate feed additives due to their in vitro anti-inflammatory, antioxidant, and antimicrobial effects. Further research is needed to evaluate their efficacy in vivo

Trace amounts of antibiotic exacerbated diarrhea and systemic inflammation of weaned pigs infected with a pathogenic E. coli

The experiment was conducted to investigate the effects of trace amounts of antibiotic on growth performance, diarrhea, systemic immunity, and intestinal health of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli. Weaned pigs (n = 34, 6.88 ± 1.03 kg body weight [BW]) were individually housed in disease containment rooms and randomly allotted to one of the three dietary treatments: nursery basal diet (CON) and two additional diets supplemented with 0.5 or 50 mg/kg carbadox to the nursery basal diet (TRA or REC), respectively. The experiment lasted 18 d with 7 d before and 11 d after the first E. coli inoculation. The E. coli F18 inoculum was orally provided to all pigs with a dose of 1010 colony-forming unit (CFU)/3 mL for three consecutive days. Fecal and blood samples were collected on day 0 before inoculation and days 2, 5, 8, and 11 postinoculation (PI) to test the percentage of β-hemolytic coliforms in total coliforms and complete blood cell count, respectively. Sixteen pigs were euthanized on day 5 PI, whereas the remaining pigs were euthanized at the end of the experiment to collect the jejunal and ileal mucosa and mesenteric lymph node for gene expression and bacterial translocation, respectively. Pigs in REC had greater (P < 0.05) final BW and lower (P < 0.05) overall frequency of diarrhea compared with pigs in the CON and TRA groups. Pigs in TRA had the lowest (P < 0.05) average daily gain and feed efficiency from day 0 to 5 PI, highest (P < 0.05) percentage of β-hemolytic coliforms in fecal samples on days 2 and 5 PI, and greatest (P < 0.05) bacterial colonies in mesenteric lymph nodes on day 11 PI compared with pigs in the CON and REC groups. Pigs in TRA had the greatest (P < 0.05) neutrophils on day 5 PI and higher (P < 0.05) white blood cell counts and lymphocytes than other groups on day 11 PI. Pigs in TRA had the greatest (P < 0.05) serum C-reactive protein on days 2 and 5 PI and serum tumor necrosis factor-α on day 5 PI, compared with pigs in the CON and REC groups. Pigs fed REC had increased (P < 0.05) mRNA expression of zona occludens-1 (ZO-1) and occludin (OCDN) and reduced (P < 0.05) interleukin-1 beta (IL1B), interleukin-6 (IL6), and tumor necrosis factor-alpha (TNFA) in ileal mucosa on day 5 PI, compared with the CON, whereas TRA upregulated (P < 0.05) mRNA expression of IL1B, IL6, and cyclooxygenase-2 (COX2) in the ileal mucosa on day 11 PI, compared with the REC. In conclusion, trace amounts of antibiotic may exacerbate the detrimental effects of E. coli infection on pig performance by increasing diarrhea and systemic inflammation of weanling pigs

Supplementation of oligosaccharide-based polymer enhanced growth and disease resistance of weaned pigs by modulating intestinal integrity and systemic immunity.

BACKGROUND: There is a great demand for antibiotic alternatives to maintain animal health and productivity. The objective of this experiment was to determine the efficacy of dietary supplementation of a blood group A6 type 1 antigen oligosaccharides-based polymer (Coligo) on growth performance, diarrhea severity, intestinal health, and systemic immunity of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli (ETEC), when compared with antibiotics. RESULTS: Pigs in antibiotic carbadox or Coligo treatment groups had greater (P < 0.05) body weight on d 5 or d 11 post-inoculation (PI) than pigs in the control group, respectively. Supplementation of antibiotics or Coligo enhanced (P < 0.05) feed efficiency from d 0 to 5 PI and reduced (P < 0.05) frequency of diarrhea throughout the experiment, compared with pigs in the control group. Supplementation of antibiotics reduced (P < 0.05) fecal β-hemolytic coliforms on d 2, 5, and 8 PI. Pigs in antibiotics or Coligo groups had reduced (P < 0.05) neutrophil counts and serum haptoglobin concentration compared to pigs in the control group on d 2 and 5 PI. Pigs in Coligo had reduced (P < 0.05) total coliforms in mesenteric lymph nodes on d 5 and 11 PI, whereas pigs in antibiotics or Coligo groups had reduced (P < 0.05) total coliforms in spleen on d 11 PI compared with pigs in the control group. On d 5 PI, pigs in the Coligo group had greater (P < 0.05) gene expression of ZO1 in jejunal mucosa, but less (P < 0.05) mRNA expression of IL1B, IL6, and TNF in ileal mucosa, in comparison with pigs in the control group. Supplementation of antibiotics enhanced (P < 0.05) the gene expression of OCLN in jejunal mucosa but decreased (P < 0.05) IL1B and IL6 gene expression in ileal mucosa, compared with the control. On d 11 PI, supplementation of antibiotics or Coligo up-regulated (P < 0.05) gene expression of CLDN1 in jejunal mucosa, but Coligo reduced (P < 0.05) IL6 gene expression in ileal mucosa compared to pigs in the control group. CONCLUSIONS: Supplementation of Coligo improved growth performance, alleviated diarrhea severity, and enhanced gut health in weaned pigs infected with ETEC F18 in a manner similar to in-feed antibiotics

Supplementation of oligosaccharide-based polymer enhanced growth and disease resistance of weaned pigs by modulating intestinal integrity and systemic immunity

Background There is a great demand for antibiotic alternatives to maintain animal health and productivity. The objective of this experiment was to determine the efficacy of dietary supplementation of a blood group A6 type 1 antigen oligosaccharides-based polymer (Coligo) on growth performance, diarrhea severity, intestinal health, and systemic immunity of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli (ETEC), when compared with antibiotics. Results Pigs in antibiotic carbadox or Coligo treatment groups had greater (P < 0.05) body weight on d 5 or d 11 post-inoculation (PI) than pigs in the control group, respectively. Supplementation of antibiotics or Coligo enhanced (P < 0.05) feed efficiency from d 0 to 5 PI and reduced (P < 0.05) frequency of diarrhea throughout the experiment, compared with pigs in the control group. Supplementation of antibiotics reduced (P < 0.05) fecal beta-hemolytic coliforms on d 2, 5, and 8 PI. Pigs in antibiotics or Coligo groups had reduced (P < 0.05) neutrophil counts and serum haptoglobin concentration compared to pigs in the control group on d 2 and 5 PI. Pigs in Coligo had reduced (P < 0.05) total coliforms in mesenteric lymph nodes on d 5 and 11 PI, whereas pigs in antibiotics or Coligo groups had reduced (P < 0.05) total coliforms in spleen on d 11 PI compared with pigs in the control group. On d 5 PI, pigs in the Coligo group had greater (P < 0.05) gene expression of ZO1 in jejunal mucosa, but less (P < 0.05) mRNA expression of IL1B, IL6, and TNF in ileal mucosa, in comparison with pigs in the control group. Supplementation of antibiotics enhanced (P < 0.05) the gene expression of OCLN in jejunal mucosa but decreased (P < 0.05) IL1B and IL6 gene expression in ileal mucosa, compared with the control. On d 11 PI, supplementation of antibiotics or Coligo up-regulated (P < 0.05) gene expression of CLDN1 in jejunal mucosa, but Coligo reduced (P < 0.05) IL6 gene expression in ileal mucosa compared to pigs in the control group. Conclusions Supplementation of Coligo improved growth performance, alleviated diarrhea severity, and enhanced gut health in weaned pigs infected with ETEC F18 in a manner similar to in-feed antibiotics

Bacillus subtilis: a potential growth promoter in weaned pigs in comparison to carbadox.

The study was conducted to investigate the efficacy of a probiotic Bacillus subtilis strain on growth performance, diarrhea, systemic immunity, and intestinal health of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli and to compare the efficacy of B. subtilis with that of carbadox. Weaned pigs (n = 48, 6.17 ± 0.36 kg body weight [BW]) were individually housed in disease containment rooms and randomly allotted to one of four dietary treatments: negative control (NC, control diet without E. coli challenge), positive control (PC, control diet with E. coli challenge), and supplementation of 50 mg/kg of carbadox (antibiotic growth promotor [AGP]) or 2.56 × 109 CFU/kg of B. subtilis probiotics (PRO). The experiment lasted for 28 d with 7 d before and 21 d after the first E. coli inoculation. Fecal and blood samples were collected on days 0, 3, 7, 14, and 21 post inoculation (PI) to analyze β-hemolytic coliforms and complete blood cell count, respectively. Diarrhea score was recorded daily for each pig to calculate the frequency of diarrhea. All pigs were euthanized at day 21 PI to collect jejunal and ileal mucosa for gene expression analysis. Pigs in AGP had greater (P &lt; 0.05) BW on days 7, 14, and 21 PI than pigs in PC and PRO groups. Supplementation of PRO enhanced pigs' BW on day 21 PI compared with the PC. Escherichia coli F18 challenge reduced (P &lt; 0.05) average daily gain (ADG) and feed efficiency from day 0 to 21 PI, while supplementation of carbadox or PRO enhanced ADG and feed efficiency in E. coli F18-challenged pigs from day 0 to 21 PI. Pigs in AGP and PRO groups had reduced (P &lt; 0.05) frequency of diarrhea throughout the experiment and fecal β-hemolytic coliforms on day 7 PI than pigs in the PC. Pigs in PRO had greater (P &lt; 0.05) gene expression of CLDN1 in jejunal mucosa than pigs in the PC. Supplementation of carbadox or PRO reduced (P &lt; 0.05) the gene expression of IL6 and PTGS2 in ileal mucosa of E. coli-infected pigs compared with pigs in the PC. Pigs in the PRO group had lower (P &lt; 0.05) white blood cell number and neutrophil count, and serum haptoglobin concentration on day 7 PI, and less (P &lt; 0.05) monocyte count on day 14 PI, compared with PC. In conclusion, supplementation of probiotic B. subtilis could enhance disease resistance and promote the growth performance of weaned pigs under disease challenge conditions. The potential mechanisms include but not limited to enhanced gut barrier integrity and local and systemic immune responses of weaned pigs