Capacity of the bovine intestinal mucus and its components to support Escherichia coli O157:H7 growth

Escherichia coli O157:H7 contamination of human food products is a major concern for the beef industry. The pathogens responsible for outbreaks often originate from cattle, and E. coli O157:H7 can thrive in healthy cattle. To control contamination in the food chain, it is essential to understand how this pathogen is able to grow and compete with other bacteria in the gastrointestinal tracts of cattle. Previous studies have shown that bovine intestinal mucus supports bacterial colonization and can selectively influence makeup of the bacterial population. Intestinal mucus is made of mucins, which are gel-forming glycoproteins. Mucin molecules contain sialic acid that must be removed by neuraminidase enzyme to allow for complete degradation of mucin. E. coli O157:H7 lacks neuraminidase and should have little ability to degrade the complex mucin molecules. Our objective was to evaluate bovine intestinal mucus and its components in terms of their capacity to support E. coli O157:H7 growth in the presence or absence of feces and to understand the roles various enzymes play in this process.

Capacity of the Bovine Intestinal Mucus and Its Components to Support Escherichia coli O157:H7 Growth1 Introduction Escherichia coli O157:H7 contamination of human food products is a major concern for the beef industry.The pathogens responsible for outbreaks often originate from cattle, and E. coli O157:H7 can thrive in healthy cattle.To control contamination in the food chain, it is essential to understand how this pathogen is able to grow and compete with other bacteria in the gastrointestinal tracts of cattle.
Previous studies have shown that bovine intestinal mucus supports bacterial colonization and can selectively influence makeup of the bacterial population.Intestinal mucus is made of mucins, which are gel-forming glycoproteins.Mucin molecules contain sialic acid that must be removed by neuraminidase enzyme to allow for complete degradation of mucin.E. coli O157:H7 lacks neuraminidase and should have little ability to degrade the complex mucin molecules.Our objective was to evaluate bovine intestinal mucus and its components in terms of their capacity to support E. coli O157:H7 growth in the presence or absence of feces and to understand the roles various enzymes play in this process.

Experimental Procedures
Intestinal tissues from freshly harvested cattle were collected and transported to our laboratory in chilled saline.Sections of the ileum and colon were washed with buffer solution, and mucus was harvested by gently scraping the epithelium.We prepared a mix of five selected strains of Shiga toxin-producing E. coli O157:H7 resistant to nalidixic acid (Nal R ) and added the mix to a buffer or a similar amount of fecal inoculums collected from the rectum of a steer fed a high-grain diet.
Subsequently, we added harvested intestinal mucus or individual mucus components to the culture to assess which components were most capable of supporting Nal R E. coli O157:H7 growth.Intestinal mucus was added at a concentration of 10 mg/mL.Single components of mucus (galactose, D-galacturonic acid, D-gluconic acid, D-glucuronic acid, mannose, L-alpha-phosphatidylserine, sialic acid, and N-acetyl-D-glucosamine) were added at the same concentration, except for L-alpha-phosphatidylserine, which was added at 1 mg/mL.Initial concentrations of E. coli O157:H7 and fecal bacteria in the cultures were 10 3 and 10 4 CFU/mL, respectively.
We also evaluated the impact of adding enzymes and enzyme inhibitors associated with mucus degradation on E. coli O157:H7 growth.Proteases, endoglycosidases, sialidases, or lipases were added to the batches at a concentration of one unit per milliliter.Betagalactosidase inhibitor was added at a final concentration of 200 µM, and protease inhibitor was added at either 0.25 or 2.5 mL/g of E. coli O157.
After 0, 6, 8, and 12 hours of anaerobic incubation at 104°F on a laboratory shaker, we plated 100 µL of culture at different dilutions on agar selective for Nal R E. coli O157:H7.After incubating the plates for 24 hours at 98°F, we counted the Nal R E coli O157:H7 colonies and established the Nal R E coli O157:H7 growth within the different batches (expressed in Log 10 of CFU/mL).

Results and Discussion
There were no significant differences (P>0.10) in E. coli O157:H7 growth between mucus derived from the large intestine (colon) and small intestine (ileum).The bacteria increased from 10 3 CFU/mL of culture at time zero to 10 7 to 10 8 CFU/mL at hour 8.There was an overall time effect on bacteria growth but no significant difference between hour 8 and 12, which drove us to use hour 8 as a point of comparison.Presence or absence of fecal inoculums in the culture affected (P<0.01)E. coli O157:H7 growth.The final concentration of bacteria decreased from 10 7 to 10 5 CFU/mL, which is likely due to competition for nutrients with the fecal bacteria.
Figure 1 depicts E. coli O157:H7 growth after 8 hours of anaerobic incubation without feces but with whole mucus or selected components of mucous as substrates.With the exception of L-alpha-phosphatidylserine, almost all of the mucus components tested increased growth of the bacteria compared with the batch containing only buffer (P<0.05).However, mucus originating from the large and small intestines supported greater growth than the individual mucus fractions (P<0.05).Of the individual mucus components evaluated, only gluconic acid resulted in growth similar to that achieved with whole intestinal mucus.These observations suggest E. coli O157:H7 may need a combination of components to ensure optimal growth or that the bacteria are utilizing a key element present in the mucus that we did not evaluate in this experiment.
In our attempt to analyze the stimulatory effect of mucus-degrading enzymes on growth of Shiga toxin-producing E. coli O157:H7, we found no significant difference in growth of cultures treated with enzymes or protease inhibitors compared with untreated batches (P>0.05).Conversely, as illustrated in Figure 2, addition of betagalactosidase enzyme inhibitor increased the growth of Nal R E. coli O157:H7 cultured with either small or large intestinal mucus (P<0.05).The increase of growth, instead of the expected inhibition, could be due to the bacteria's inability to use the inhibitor as a source of protein.However, the amount of inhibitor added to the culture was very small and, therefore, seems an unlikely explanation.It is equally possible that mucus galactosides that have not been enzymatically degraded are more stimulatory to E. coli O157:H7 growth.Additional controlled experiments are needed to further investigate the increase of growth induced by the beta-galactosidase inhibitors.
Figure 3 illustrates E. coli O157:H7 growth in response to small intestinal mucus or sialic acid substrates in the presence or absence of a fecal inoculum.E. coli O157 growth was significantly lower in sialic acid than in mucus (P<0.01),indicating the pathogen has limited capacity to use sialic acid as a substrate for growth.When fecal inoculum was added to the culture with small intestinal mucus, there was a significant decrease in E. coli growth compared with the same culture without feces.This again suggests the bacteria are competing for nutrients.However, E. coli O157:H7 growth increased when fecal inoculum was added to the culture containing sialic acid.Bacteria present in feces may have the ability to degrade sialic acid, thus allowing E. coli O157:H7 to use the intermediates or end products of the degradation as a substrate for growth.
Cattle fed distillers grains have been shown to have an increase in E. coli O157 shedding, and distillers grains contain a substantial proportion of yeast, which has a high sialic acid content (3% of dry weight).It is possible that sialic acid or other glycoprotein constituents of distillers grains are the active components that stimulate proliferation of E. coli O157:H7 in cattle fed distillers grains.

Implications
This study offers insight regarding the potential of intestinal mucus and its components to promote E. coli O157:H7 growth in cattle.Further investigations are needed to establish whether one of these components could inhibit, or at least regulate, the proliferation of important foodborne pathogens.
Colony forming units/mL, Log