Campylobacter jejuni response when inoculated in bovine in vitro fecal microbial consortia incubations in the presence of metabolic inhibitors

Authors: DITTOE, DANA - University Of Wyoming; Anderson, Robin; KRUEGER, NATHAN - Blinn College; Harvey, Roger; Poole, Toni; Crippen, Tawni - Tc; CALLAWAY, TODD - University Of Georgia; RICKE, STEVEN - University Of Wisconsin

Submitted to: Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/23/2023
Publication Date: 11/26/2023
Citation: Dittoe, D.K., Anderson, R.C., Krueger, N.A., Harvey, R.B., Poole, T.L., Crippen, T.L., Callaway, T.R., Ricke, S.C. 2023. Campylobacter jejuni response when inoculated in bovine in vitro fecal microbial consortia incubations in the presence of metabolic inhibitors. Pathogens. 12. Article 1391. https://doi.org/10.3390/pathogens12121391.
DOI: https://doi.org/10.3390/pathogens12121391

Interpretive Summary: Campylobacter jejuni is the number one cause of bacterial foodborne disease in the United States. This bacterial pathogen can reside in the gut of food producing animals, including cattle, but factors affecting its ability to persist in the intestinal tract are not clearly understood. For instance, Campylobacter jejuni are known to be inadequate in their ability to use carbohydrates as their energy source for growth but rather are thought to use hydrogen gas or amino acids produced during digestion by other gut microbes. We hypothesized that this may be why Campylobacter jejuni are rarely preset within the pregastric rumen compartment of cattle but are frequently recovered at high numbers from the large intestine. In support of this hypothesis, we found that when cultured under conditions simulating an intestinal environment deficient in amino acids, the number of Campylobacter jejuni cells at the end of the 48-h incubation period were 100-times lower in cultures inoculated with hydrogen-using microbes from the rumen than in cultures inoculated with feces which contain far fewer hydrogen-using microbes. In follow up experiments intended to further examine the potential effects of hydrogen and hydrogen-consuming microbes on Campylobacter jejuni in simulated amino acid rich intestinal environments, freshly collected feces were incubated under either a 100% carbon dioxide or 50:50 carbon dioxide:hydrogen gas phase. We found numbers of Campylobacter jejuni do not differ whether grown under low or high hydrogen conditions likely because of the absence of potential hydrogen-using competitors and the ability of Campylobacter jejuni to grow by using amino acids as an energy source which were readily available under the conditions of these follow up experiments. These results indicate amino acids are a more important energy source than hydrogen for Campylobacter jejuni in the intestinal tract of cattle. Ultimately, this research will enable scientists to develop strategies to eliminate Campylobacter jejuni from cattle thereby helping producers provide safe and wholesome meat and milk for the American consumer.

Technical Abstract: Foodborne pathogen Campylobacter infection is the leading bacterial cause of human foodborne illness in the United States. The objectives of this experiment were to test the hypothesis that mixed microbial populations from the bovine rumen may be better at excluding Campylobacter than populations from freshly voided feces and to explore potential reasons as to why the rumen may be a less favorable environment for Campylobacter than feces. In an initial experiment, C. jejuni cultures inoculated without or with freshly collected bovine rumen fluid, bovin feces, or their combination were cultured micro-aerobically for 48 h. Results revealed that C. jejuni grew at similar growth rates during the first 6 h of incubation regardless of inoculation with the rumen or fecal contents, with rates ranging from 0.178 to 0.222/h. However, C. jejuni counts (Log10 colony forming units/mL) at the end of the 48-h incubation were lowest in cultures inoculated with rumen fluid (5.73 Log10 CFU/mL), intermediate in cultures inoculated with feces or both feces and rumen fluid (7.16 and 6.36 Log10 CFU/mL) and highest in pure culture controls that had not been inoculated with the rumen or fecal contents (8.32 Log10 CFU/mL). In follow up experiments intended to examine the potential effects of hydrogen and hydrogen-consuming methanogens on C. jejuni, freshly collected bovine feces, suspended in anaerobic buffer, were incubated anaerobically under either a 100% carbon dioxide or 50:50 carbon dioxide:hydrogen gas mix. While C. jejuni viability decreased < 1 Log10 CFU/mL during incubation of the fecal suspensions, this did not differ whether under low or high hydrogen accumulations or whether the suspensions were treated without or with the mechanistically distinct methanogen inhibitors, 5 mM nitrate, 0.05 mM 2-bromosulfonate or 0.001 mM monensin. These results suggest that little if any competition between C. jejuni and hydrogen-consuming methanogens exists in the bovine intestine.