Bacterial inoculum
Bacterial strains of generic Escherichia coli (E. coli) ATCC 25922, Enterotoxigenic Escherichia coli (ETEC) 3030-2, and Salmonella enterica serotype Typhimurium (S. Typhimurium) ATCC 14028 were grown using Luria Bertani, Campylobacter coli (C. coli) 7A #2016-1 using Mueller-Hinton, and Clostridium perfringens (C. perfringens) 4026 using anaerobic Brain Heart Infusion broth medium at 37°C for 24 h. For E. coli, ETEC, S. Typhimurium, and C. coli, 1 ml of bacterial inoculum was serially diluted using 9 ml of PBS to achieve one concentrations (105 CFU/ml) for each bacterial strain. For Clostridium perfringens, the bacterial concentration was adjusted to 0.5 McFarland Standards using fresh Brain Heart Infusion broth medium per Clinical and Laboratory Standards Institute recommendations.8
Experiment 1 MIC Determination of MCFA
For E. coli, ETEC, S. Typhimurium, and C. coli the compounds tested were C6:0a, C8:0a, C10:0a, and a 1:1:1 blend of C6:0, C8:0, and C10:0. For C. perfringens, the compounds tested were C6:0, C8:0, C10:0, and C12:0a.
The MIC were determined by the micro‐broth dilution method as per Clinical Laboratory Standards Institute CLSI guidelines8 in E. coli, ETEC, S. Typhimurium, and C. coli from 0.1% until an MIC was established, with a maximum tested level of 1.0%. The MIC was also determined using the same method for C. perfringens, with a maximum tested level of 2.0%. There were three replications per product and bacteria combination.
Experiment 2 MCFA Profiles and MIC Determination of Commercially-Based Products
The fatty acid profile of 21 commercially-based products was analyzed, with an emphasis on the MCFA concentration. The 24 products were, 1.) Product Ab, 2.) Product Bc 3.) Product Cb, 4.) Product Dd, 5.) ProductEd, 6.) ProductFd, 7.) Product Gd, 8.) Product He 9.) Product I f, 10.) Product Jf, 11.) Product Kg, 12.) Product L h, 13.) Product Mh, 14.) Product N f, 15.) Product Of, 16.) Product Pf, 17.) Product Qf, 18.) Product Rf, 19.) Coconut Oilg, 20.) Palm Oilg, and 21.) Palm Kernel Oilg. Samples were analyzed according to procedures outlined by Sukhija and Palmquist9. From this analysis, Product A, B, G, H, and a commodity fat source (coconut oil) were selected as having representative MCFA profiles for use in MIC assays. The profiles were selected based on products having the highest concentrations of C6:0 and C8:0 within the fatty acid profile and coconut oil because of its natural source of MCFA and medium chain triglycerides. The MIC were determined as described in Exp. 1 in E. coli, ETEC, S. Typhimurium, and C. coli from 0.1% until an MIC was established, with a maximum tested level of 5.0%. There were three replications per product and bacteria combination.
Experiment 3 Quantification of Enterotoxigenic Escherichia coli-inoculated feed after treatment with two commercially-based MCFA-containing products
Based on their lower MIC compared to other products tested in Exp. 2, Products A and B were selected as treatments to determine their reduction capacity in swine feed inoculated with ETEC. The strain of ETEC was first made resistant to 50 µl/ml nalidixic acid (NalR) antibiotic before being used for inoculation. A complete swine diet was either left un-inoculated and untreated, or mixed with 0.00, 0.25, 0.50, 1.00, or 2.00% Product A or B and inoculated with ETEC. For inoculation, 1 g of each feed sample was mixed with 1 ml of NalRETEC at one of two concentrations (106 or 102 CFU per g of feed) of bacteria. The higher concentration was utilized for quantification of ETEC and the lower for detection. The 10 treatments were: 1.) control feed with no bacteria; 2). Control feed inoculated with bacteria and no addition of an additive; 3.) 0.25% Product A; 4.) 0.5%, Product A; 5.) 1.0%, Product A; 6). 2%. Product A; 7.) 0.5% Product B; 8.) 1.0% Product B; 9.) 2.0% Product B; and 10.) 4.0% Product B. The levels for each product were selected based on the results of Exp. 2. Product A was tested at a lower inclusion level in the feed because of the lower MIC value established in Exp. 2. Product B was then tested at higher inclusion levels because of the higher MIC value that was established in Exp. 2. It was also determined that treatment 1 was confirmed to be negative of ETEC and was not included in the statistical model.
Samples were incubated at 37°C for 24 h. Then, 1 g of the incubated feed containing bacterial inoculum was suspended in 9 ml of PBS, serially diluted, and plated onto MacConkey agar containing nalidixic acid. The plates were incubated at 37°C for 24 h for bacterial enumeration using a standard plate count for viable cells. There were three replications per product and bacteria combination.
Statistical Analysis
Data from each MIC experiment were analyzed as a completely randomized design using PROC GLIMMIX in SASi to evaluate the effect of each treatment within each bacterium. If the MIC value was greater than the detection limit of the analysis, the next logical inclusion level (increase in 0.1% inclusion) was utilized for the statistical analysis. For Exp. 3, the PROC GLIMMIX procedure of SASi was utilized to evaluate linear and quadratic contrasts of increasing product levels. The coefficients for the unequally spaced linear and quadratic contrasts utilized in Exp. 3 were derived using the PROC IML procedure in SASi. In all experiments, results for treatment criteria were considered significant at P ≤ 0.05.