Determining the Minimum Inhibitory Concentration of Medium Chain Fatty Acids for Generic Escherichia coli, Enterotoxigenic Escherichia coli, Salmonella Typhimurium, and Campylobacter coli

Research has demonstrated that medium chain fatty acids (MCFA) can serve as reduction strategies for bacterial and viral pathogens in animal feed and ingredients. However, it is unknown how the type or level of MCFA impact bacteria growth. This can be tested through a minimum inhibitory concentration (MIC) benchtop assay, which identifies the lowest concentration of a chemical that prevents visible growth of a bacterium. The objective of this study was to 1) determine the MCFA MIC of C6:0, C8:0, C10:0, and C12:0 for generic Escherichia coli, Enterotoxigenic Escherichia coli, Salmonella Typhimurium, Campylobacter coli, and Clostridium perfringens; 2) determine the MIC of commercial based MCFA products against the same bacteria; and 3) determine the effect of 2 commercial based MCFA products on the quantification of Enterotoxigenic Escherichia coli. For Exp. 1 and 2, MIC were determined by modified microbroth dilution method using a 96 well microtiter plate with a concentration of 105 CFU/mL for each bacterial strain. For Exp. 3, the two products selected for quantification were mixed with a complete swine diet and inoculated with two concentrations (106 or 102 CFU/g of feed) of a NalR strain of Enterotoxigenic Escherichia coli (ETEC) for bacterial enumeration. From Exp. 1, the MIC of MCFA varied among bacteria species. The lowest MIC of the MCFA was 0.43% of a 1:1:1 blend of C6:0, C8:0, and C10:0 for Campylobacter coli, 0.25% C12:0 for Clostridium perfringens, 0.60% 1:1:1 blend for generic Escherichia coli, 0.53% C6:0 for ETEC, and 0.40% C6:0 for Salmonella Typhimurium. In Exp. 2, products containing high concentrations of C6:0 or C8:0 had lower MIC in gram negative bacteria. In Exp. 3, feed containing either of the commercial based MCFA products reduced (linear, P < 0.05) quantifiable ETEC. Overall, the inhibitory efficacy of MCFA varies among bacteria species. This suggests that MCFA mixtures may provide a wider spectrum of bacterial control. As commercial products containing MCFA become available for livestock, it is important to consider the interaction between MCFA chain length and concentration on the potential to effectively mitigate various feed-based bacteria.


Summary
Research has demonstrated that medium chain fatty acids (MCFA) can serve as reduction strategies for bacterial and viral pathogens in animal feed and ingredients. However, it is unknown how the type or level of MCFA impact bacteria growth. This can be tested through a minimum inhibitory concentration (MIC) benchtop assay, which identifies the lowest concentration of a chemical that prevents visible growth of a bacterium. The objective of this study was to 1) determine the MCFA MIC of C6:0, C8:0, C10:0, and C12:0 for generic Escherichia coli, Enterotoxigenic Escherichia coli, Salmonella Typhimurium, Campylobacter coli, and Clostridium perfringens; 2) determine the MIC of commercial based MCFA products against the same bacteria; and 3) determine the effect of 2 commercial based MCFA products on the quantification of Enterotoxigenic Escherichia coli. For Exp. 1 and 2, MIC were determined by modified microbroth dilution method using a 96 well microtiter plate with a concentration of 10 5 CFU/mL for each bacterial strain. For Exp. 3, the two products selected for quantification were mixed with a complete swine diet and inoculated with two concentrations (10 6 or 10 2 CFU/g of feed) of a Nal R strain of Enterotoxigenic products containing MCFA become available for livestock, it is important to consider the interaction between MCFA chain length and concentration on the potential to effectively mitigate various feed-based bacteria.

Introduction
Medium chain fatty acids (MCFA) have been demonstrated to significantly reduce problematic bacterial and viral contamination in animals, animal feed, and feed ingredients. 3,4,5 Compared to other feed additives, MCFA are unique in their potential mode of action. It is thought that the MCFA carry bacteriostatic and bactericidal properties by causing a destabilization of the bacterial double phospholipid bilayer membrane and causing leakage of intracellular content. 3 It is also thought that the MCFA can acidify the cell by liberating H + ions, leading to cell death. 3 More recently, a 2% inclusion of a 1:1:1 ratio of C6:0, C8:0, and C10:0 reduced Salmonella enterica subsp. enterica serovar Typhimurium from 2.35 to 0.66 log CFU/g within 1 day. 4 The MCFA were also very effective on the initial inoculation day compared to the inoculated feed matrices containing no mitigation additives (2.35 vs. 5.45 log CFU/g, respectively). 4 However, there is limited information regarding which specific MCFA is the most effective, whether combinations of different MCFA exhibit additive effects, and what the optimal level of MCFA is that will impact various bacteria associated with animal production. This can be determined utilizing a minimum inhibitory concentration (MIC) benchtop assay, which identifies the lowest concentration of a treatment that prevents visible growth of a bacterium. Therefore, the objective of this study was to determine the minimum inhibitory concentration of specific MCFA and commercial products for Campylobacter coli, Clostridium perfringens, generic Escherichia coli, Enterotoxigenic Escherichia coli, and Salmonella Typhimurium as well as their potential application in feed as a reduction strategy.
The MIC were determined by the micro-broth dilution method as per CLSI guidelines 8 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  ; 20) Palm oil g ; and 21) Palm kernel oil g . Samples were analyzed according to procedures outlined by Sukhija and Palmquist. 7 From this analysis, products 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 coliinoculated 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 (Nal R ) 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 Nal R ETEC at one of two concentrations (10 6 or 10 2 CFU/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 concentration in the feed because of the lower MIC value established in Exp. 2. Product B was then tested at higher concentrations 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 SAS version 9.4 (SAS Inst. Inc., Cary, NC, USA) 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 concentration (increase in 0.1% addition) was utilized for the statistical analysis. For Exp. 3, the PROC GLIMMIX procedure of SAS 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 SAS. In all experiments, results for treatment criteria were considered significant at P ≤ 0.05.

Experiment 2
The fatty acid profile varied widely in the 21 commercially-based products (Table 2). Based on these analyses, Product A, B, F, G, and coconut oil were selected as candidate products for MIC determination in gram negative bacteria due to their high concentrations of C6:0 and C8:0. In C. coli, the MIC for Product B was lower (P < 0.05) than either Product F or G, with Product A intermediate (Table 3). Product A and B had lower (P < 0.05) MIC in generic E. coli, ETEC, and Salmonella Typhimurium than other tested products. The MIC for coconut oil was not detected in any bacteria as it was greater than the maximum tested level of 5.0%.

Experiment 3
Due to their efficacy in the MIC determination, products A and B were selected as treatments to determine their effect on detectable or quantifiable ETEC in feed. In the higher concentration of bacteria, Product A resulted in a linear decrease (linear, P < 0.05) in the number of quantifiable bacteria (Table 4). For Product B, as the level increased, the number of quantifiable bacteria decreased (quadratic, P < 0.05).
In the lower concentration of bacteria, Product A again resulted in a decrease (linear, P < 0.05) in the number of quantifiable bacteria (Table 5). However, in Product B, no linear or quadratic response was observed (P > 0.10).
In summary, MCFA mixtures may provide a wider spectrum of bacterial control. As commercial products containing MCFA become available for livestock, it is important to consider the interaction between MCFA chain length and concentration on the potential to effectively mitigate various feed-based bacteria. Minimum inhibitory concentration for C6:0, C8:0, C10:0, and a 1:1:1 blend of C6:0, C8:0, and C10:0 were tested in E. coli, ETEC, S. Typhimurium, and C. coli using a 96 well microtiter plate with a concentration of 10 5 CFU/mL for each bacterial strain. For C. perfringens, the compounds tested were C6:0, C8:0, C10:0, and C12:0 utilizing a 96 well microtiter plate with a concentration of 0.5 McFarland Standards for each well. Each value is represented by an N=3.
2 Minimum inhibitory concentration was above the tested detection limit and therefore the next logical inclusion level (increase in 0.1% inclusion) was utilized for the statistical analysis. abcd Means within a bacterial species lacking a common superscript differ (P < 0.05).  Minimum inhibitory concentration was above the tested detection limit and therefore the next logical inclusion level (increase in 0.1% inclusion) was utilized for the statistical analysis. abcd Means within a bacteria species lacking a common superscript differ (P < 0.05). Products A and B were tested in a concentration of 10 6 CFU/g of feed ETEC in a complete swine diet in order to determine the growth of that bacteria using MacConkey agar containing nalidixic acid for bacterial enumeration.  Products A and B were tested in a concentration of 10 2 CFU/g of feed ETEC in a complete swine diet in order to determine the growth of that bacteria using MacConkey agar containing nalidixic acid for bacterial enumeration.