Characterization of bovine ruminal and equine cecal microbial populations enriched for enhanced nitro-toxin metabolizing activity

Authors: ZHANG, YING - Lanzhou University; LONG, RUIJUN - Lanzhou University; WARZECHA, CHRISTINE - Texas A&M University; COVERDALE, JOSIE - Texas A&M University; LATHAM, ELIZABETH - Texas A&M University; Hume, Michael; Callaway, Todd; O'NEIL, MATTHEW - Iowa State University; Beier, Ross; Anderson, Robin; Nisbet, David

Submitted to: Anaerobe
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/17/2013
Publication Date: 1/9/2014
Publication URL: http://handle.nal.usda.gov/10113/58260
Citation: Zhang, Y., Long, R., Warzecha, C.M., Coverdale, J.A., Latham, E.A., Hume, M.E., Callaway, T.R., O'Neil, M.R., Beier, R.C., Anderson, R.C., Nisbet, D.J. 2014. Characterization of bovine ruminal and equine cecal microbial populations enriched for enhanced nitro-toxin metabolizing activity. Anaerobe. 26:7-13.

Interpretive Summary: Plants contain a variety of potential pharmaceutical chemicals. For instance, two chemicals, known as 3-nitro-1-propionic acid (NPA) and 3-nitro-1-propanol (NPOH), are produced by over 150 different species and varieties of grasses potentially grazed by livestock. These compounds are toxic to naive grazing animals but can be safely fed to cattle and sheep if the animals are slowly adapted to the compounds; it is thought that this happens because a unique NPA and NPOH-degrading bacterium which is normally low in numbers becomes more prominent in the gut. However, even at low, residual concentrations, these compounds are potent inhibitors of some other unwanted gut bacteria such as those that produce the greenhouse gas, methane. Evidence suggests, but has not yet proven, that NPA and NPOH inhibit these unwanted bacteria because they inhibit their ability to use hydrogen as a nutrient, but it is not known what effect they may have on other hydrogen-using gut bacteria. Some of these other hydrogen-using bacteria may be beneficial, and others, such as Salmonella, Campylobacter, and Helicobacter, may be dangerous. Moreover, little is known about the metabolism of NPA and NPOH or their effect on hydrogen-using populations in gut habitats of other farm animals. Consequently, the objective of the present study was to test for the presence of NPA and NPOH-metabolizing bacteria in the gut of a horse, chicken, and pig, as well as in a cow. We found that NPA and NPOH were appreciably metabolized by bacteria from the gut of a cow and a horse, but not from a chicken or a pig. Additionally, we observed that the bacterial populations from the cow and horse could be adapted to NPA and NPOH, with rates of metabolism increasing as much as six times and numbers of NPA and NPOH-metabolizing bacteria increasing as much as 100-fold. As expected, NPA was inhibitory to methanogenesis, but nutritional balance estimates indicated that the growth of other hydrogen-using bacteria able to make acetic acid out of carbon dioxide was favored. From an animal nutritional perspective, this is a beneficial result because acetic acid can be used as an energy source by the cow or horse. These results further reveal that NPA and NPOH promoted the growth of nutritionally beneficial hydrogen-using bacteria in the animal gut and set the stage for future studies to examine their effects on hydrogen-using pathogens. Additionally, this research has improved our understanding of the complex interrelationships between bacteria and nutrients in the animal gut which ultimately may lead to the development of methods to more efficiently produce wholesome, microbiologically safe meat and milk at a lower cost for the American consumer.

Technical Abstract: The phytochemicals 3-nitro-1-propionic acid (NPA) and 3-nitro-1-propanol (NPOH) are produced by a wide variety of leguminous plants, including over 150 different species and varieties of Astragalus. These compounds are toxic to naive grazing animals, but can be safely fed to cattle and sheep that have been adapted to the compounds, presumably due to enrichment of NPA- and NPOH-degrading bacteria. However, even at residual concentrations, these compounds are potent inhibitors of rumen methanogenesis, a digestive inefficiency resulting in the loss of up to 15% of an animal’s gross energy intake. Evidence suggests, but has not yet proven, that NPA and NPOH inhibit methanogenic bacteria by inhibiting their ability to use hydrogen as an electron donor, but it is not known what effect these compounds may have on other hydrogen-using gut bacteria, some which are pathogens such as Salmonella, Campylobacter, and Helicobacter. Moreover, little is known about the metabolism of NPA and NPOH or their effect on hydrogen-using populations in gut habitats of other farm animals. In the present study, mixed populations of bovine ruminal and equine cecal microbes, but not populations of avian and porcine cecal microbes, were enriched for NPA-metabolizing bacteria via consecutive 24 to 72 h culture in a basal minimal rumen fluid-based medium supplemented with 4.2 mM NPA and hydrogen as the energy source. Rates of NPA metabolism by the respective populations increased from 58.4 ± 4.8 and 8.6 ± 11.6 nmol NPA/mL per h during initial culture to 88.9 ± 30.6 and 50.2 ± 30.9 nmol NPA/mL per h following enrichment. Results from 3-tube most probable number tests indicated that numbers of NPA-degrading microbes increased 2.1 and 1.8 log10 units during enrichment from numbers measured pre-enrichment (3.9 x 103 and 4.3 x 101 cells/mL for ruminal and equine cecal populations, respectively). Hydrogen, formate, and to a lesser extent, DL-lactic acid served as electron donors to the enriched populations, and CO2 or formate were needed to maintain high rates of NPA-metabolism. The NPA-enriched populations were able to metabolize nitrate which, being a preferred electron acceptor, was antagonistic to NPA metabolism. Supplemental NPA was inhibitory to methanogenesis. Fermentation balance estimates indicated that only 47.6% of carbon available in potential substrates was recovered in headspace CO2, volatile fatty acids, or unmetabolized NPA after 72 h incubation of NPA-enriched populations that had metabolized 98% of 8.4 mM added NPA. Conversely, nitrate-enriched populations grown with NPA in place of nitrate metabolized little of the 8.4 mM added NPA and were highly acetogenic, assimilating > 50 µmol one carbon compounds/mL. Results reveal low level carriage of NPA-metabolizing, CO2, or formate-requiring bacterial populations in the equine cecum.