How low should the pH go?

Acidifying the drinking water to decrease/prevent the growth of E. coli.

By Carly BatesColibacillosis or Escherichia coli (E. coli) infection is a leading cause of morbidity and mortality in weaned pigs. The disease interrupts the intestinal barrier which causes diarrhea, enterotoxemia and/or sudden death. Antimicrobial resistant E. coli is a growing concern as many challenging strains are multidrug resistant. Alternative treatments are being explored to combat the disease that do not involve use of antimicrobials, one of which is acidifying the drinking water to decrease/prevent the growth of E. coli. This experiment’s goal is to understand what pH should be targeted or at what acidic pH does growth inhibition of the organism begin.The study was completed in two phases. Phase one was to determine the target pH levels of media without contamination. Phase two was to determine growth outcomes in the various pH levels of media.Phase one
Eighty-eight percent lactic acid and sterile water were mixed at a 1 to 10 dilution as a stock solution. The stock was then added to 100 mL of tryptic soy broth (TSB) in .05-2mL increments until 3, 4, 5, 6 and 7 pH levels were met and confirmed using a pH meter. The final amounts are listed in table 1. This was utilized to accurately assess the pH levels of the TSB without contaminating the test media. This phase was repeated twice for accuracy.

Phase two
After proper pH levels had been produced in each of the TSB containers (3, 4, 5, 6 and 7 pH) one colony of a F18 strain E. coli from a clinical case was obtained on an inoculating loop and place in the TSB and swirled vigorously. TSB without lactic acid added was inoculated and plated to act as a positive control. The TSB test containers were incubated for 24 hours at 37 degrees F. After incubation, the samples were removed from the incubator and diluted 1:10, 1:100, 1:1,000, 1:10,000 and 1:100,000 dilutions. These dilutions and the original inoculum were plated using .5 mL of the solution spread evenly over 5% bovine blood agar plates. Plates were incubated and evaluated after 12 hours. Colony counts and pictures were taken for a quantitative and visual representation of growth.Results and discussion
All plates at pH of 5, 6 and 7 had noticeable surface growth whereas plates at a pH of 3 and 4 had no visual surface growth. Providing evidence that the E. coli isolate’s growth inhibiting point is at a pH of 4. At the 1:100,000 dilution, 5 pH had 398 colonies of growth while 6 and 7 had TNTC colonies indicating that a mild inhibition of growth begins near a pH of 5. Next steps are to understand E. coli’s growth inhibition at pH’s incrementally from 4.0 to 5.0 and gaining an understanding of in vivo enteric luminal pH when pigs are administered water of various pH’s.

This picture represents how to TSB looked 24 hours after the broth had been inoculated with one colony of E. coli. At a pH of 3 and 4 the TSB was still clear whereas 5, 6, 7 and the negative control had a very cloudy after 24 hours of incubation which could be indicating growth.

All plates at the 1:1000 dilution. There was surface growth on 4 of the 6 plates with a clear cut off between a pH of 4 and 5.

Closer comparison at a 1:1000 dilution comparing a pH of 4 with no surface growth and a pH of 5 which had significant amounts of surface growth.

Bates is a third-year student at Iowa State University College of Veterinary Medicine and received the 2023 Morrison Swine Innovator Prize for her presentation, “How low should the pH go? How pH levels affect E. coli growth.”