Characterization of nitrate-reducing and amino acid-using bacteria prominent in nitrotoxin-enriched equine cecal populations

Authors: ZHANG, YING - Lanzhou University; LONG, RUJUNG - Lanzhou University; Anderson, Robin; Hume, Michael; COVERDALE, JOSIE - Texas A&M University; LATHAM, ELIZABETH - Texas A&M University; Nisbet, David

Submitted to: Journal of Equine Veterinary Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/13/2016
Publication Date: 11/1/2016
Citation: Zhang, Y., Long, R.J., Anderson, R.C., Hume, M.E., Coverdale, J.A., Latham, E.A., Nisbet, D.J. 2016. Characterization of nitrate-reducing and amino acid-using bacteria prominent in nitrotoxin-enriched equine cecal populations. Journal of Equine Veterinary Science. 46:47-53.

Interpretive Summary: Certain plants and molds produce and accumulate toxins known as 3-nitro-1-propionic acid and 3-nitro-1-propanol. Animals and humans can be poisoned if they consume these plants or foods contaminated with the nitro-toxin producing molds. At least one bacterium isolated from the gut of a cow is known to be able to degrade these nitro-toxins thereby contributing to their detoxification. Little is known about the potential for detoxification of the nitro-toxins in other animals. In the present study, we found that populations of gut bacteria collected from a horse were able to degrade the nitro-toxins and this ability was enhanced if the populations were adapted to the nitro-toxins by repeated exposure. We isolated eight different types of bacteria from the adapted populations and these were identified as Streptococcus lutetiensis (5 strains), Escherichia coli (2 strains), and Sporanaerobacter acetigens (1 strain). When grown by themselves, however, none of these were able to degrade the nitro-toxins which suggests that the bacteria may have be able to work together to successfully accomplish this task. Alternatively, it is possible that the bacteria responsible for nitro-toxin degradation were too low in number that their likelihood of being isolated was improbable. Our isolation of the bacterium Sporanaerobacter acetigens (1 strain), however, is a novel finding as this bacterium, which appears to play a major role in anaerobic amino acid-metabolism, has not been found in horses before. Results from this study expand our knowledge of nitro-toxin degradation in different animal populations which ultimately will lead to the development of new strategies to prevent animal and human poisonings by plants and mold that produce these nitro-toxins.

Technical Abstract: In the present study, populations of equine cecal microbes enriched for enhanced rates of 3-nitro-1-propionic acid (NPA) or nitrate metabolism were diluted and cultured for NPA-metabolizing bacteria on a basal enrichment medium (BEM) or tryptose soy agar (TSA) medium supplemented with either 5 mM NPA or nitrate and under H2:CO2 (20:80) as the energy source. After 72 h, separated colonies picked from plates or roll tubes were inoculated into fresh broth medium and cultured again for 72 h. Following culturing, their DNA was extracted and subjected to 16S rRNA sequence analysis. Eight strains isolated from the NPA-enriched populations were identified as Streptococcus lutetiensis (5 strains), Escherichia coli (2 strains) and Sporanaerobacter acetigens (1 strain), respectively. Four strains isolated from nitrate enriched populations were identified as Escherichia coli (1 strain) and Wolinella succinogenes (3 strains), respectively. However, none of the above pure strains degraded NPA. The PCR products of total DNA from NPA- or nitrate-enriched populations of equine cecal microbes, the isolated equine cecal pure strains, as well as the strain of Denitrobacterium detoxificans, a competent NPA-metabolizing microbe from the bovine rumen, were examined using denaturing gradient gel electrophoresis (DGGE). The DGGE analysis indicated that there was no strain in the enriched population of equine cecal microbes which was similar to the known strain of D. detoxificans. However, we report for the first time the isolation of the anaerobic amino acid-utilizing Sporanaerobacter acetigens from the equine cecum.