The isolation of rumen enterococci strains along with high potential utilizing cyanide

Abstract

Cyanogenic glycosides in forage species and the possibility of cyanide (CN) poisoning can have undesirable effects on ruminants. The literature estimates that unknown rumen bacteria with rhodanese activity are key factors in the animal detoxification of cyanogenic glycosides, as they are capable of transforming CN into the less toxic thiocyanate. Therefore, identifying these bacteria will enhance our understanding of how to improve animal health with this natural CN detoxification process. In this study, a rhodanese activity screening assay revealed 6 of 44 candidate rumen bacterial strains isolated from domestic buffalo, dairy cattle, and beef cattle, each with a different colony morphology. These strains were identified as belonging to the species Enterococcus faecium and E. gallinarum by 16S ribosomal DNA sequence analysis. A CN-thiocyanate transformation assay showed that the thiocyanate formation capacity of the strains after a 12 h incubation ranged from 4.42 to 25.49 mg hydrogen CN equivalent/L. In addition, thiocyanate degradation resulted in the production of ammonia nitrogen and acetic acid in different strains. This study showed that certain strains of enterococci substantially contribute to CN metabolism in ruminants. Our results may serve as a starting point for research aimed at improving ruminant production systems in relation to CN metabolism.

Figure 1

Rhodanese activity screening in rumen enterococci strains. SCN⁻, thiocyanate anion; error bar, standard deviation. In this rhodanese assay, the crude enzyme samples (n = 3) from each strain were extracted and incubated with the reaction mixture containing KCN. The negative control group (n = 3) consisted of rhodanese samples that were boiled to inactivate their activity. The rhodanese activity of the isolated strains ranged from 4.35 to 6.60 µmol of SCN^– production/min/mg protein.

Figure 2

Phylogenetic trees of E. faecium (a) and E. gallinarum (b) type strains based on 16S rRNA gene sequences. The strains studied in our experiment are marked (red circle) and referred to the NCBI accession number. The nucleotide sequences were used to construct the highest log likelihood trees (− 14,901.92 for E. faecium and − 11,697.18 for E. gallinarum) with Lactobacillus plantarum WCFS1 (KC429782) as an outgroup. The trees were constructed using MEGA X, employing the Maximum Likelihood method and Tamura-Nei model, with 10,000 bootstrap iterations. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Bars indicated sequence divergences.

Figure 3

Scanning electron microscopy (SEM) at 150,000× magnifications of rumen enterococci strains. The image displays the cell morphology of pure strains. These bacteria have an ovoid shape with a cell diameter of approximately 1.0 µm.

Figure 4

Growth at different pH, temperatures, and CN substrate concentrations in MRS broth after incubation for 24 h. Bar, standard error of the mean; ***, p < 0.001; ns, p > 0.05. The statistical difference among the six strains at each pH, temperature, or CN substrate concentration was analyzed using one-way ANOVA with three replicates. No strain grew at a pH of 4.0, and the optimal pH range for most strains was 6‒9. At a pH of 10.0, abundant growth of KKU-BF7 and KKU-BC8 strains was observed compared with the other four strains (p < 0.001). The bacteria did not grow at 4 °C and 60 °C. The optimal temperature range for most strains was 30‒40 °C, and KKU-DC6 and KKU-BC2 strains had an OD > 1.5, which was greater than any other strain (p < 0.001). The results showed that the susceptibility of the KKU-BC15 strain to CN was significantly lower than that of all other strains at high levels of CN (400 mg/L) (p < 0.001).

Figure 5

CN-thiocyanate transformation assay of rumen enterococci strains using an in vitro rumen fermentation technique. (a) Total CN concentration during the 48 h fermentation. Error bar, standard error of the mean; **, p < 0.01; ***, p < 0.001. (b) Pearson’s correlation heatmap of thiocyanate concentration and fermentation end-products for 12–48 h of incubation. The color scheme indicates the strength of correlation of thiocyanate degradation and fermentation end-products in each strain (n = 9). *, p < 0.05; OD₆₀₀, optical density at 600 nm; VFAs, volatile fatty acids. In this assay, a cell-free rumen fluid medium containing KCN substrate was utilized to evaluate the ability of each strain to produce thiocyanate from the CN source. The fermentation process involved analyzing the CN compounds (KCN and thiocyanate) in the medium using a distillation method, and the results are reported in mg of HCN equivalent/L. The assimilation of thiocyanate within the cytoplasm of bacterial cells is thought to occur through their rhodanese activity. To differentiate thiocyanate from the remaining KCN substrate, a baseline reference (negative control without bacteria or a rhodanese source) was used during the incubation period. Compared with bacterial fermentation, the baseline KCN substrate releases free CN (HCN and CN⁻) instead of thiocyanate, as it possesses hydrolytic abilities in the medium. In (a), the total CN concentration in the medium was measured at 3, 6, 12, 24, and 48 h of incubation for each strain (n = 3) and baseline (n = 3). After 12 h of incubation, the baseline (plotted in the red line) showed a remarkably low concentration of CN from an initial concentration of 80 mg HCN equivalent/L. This suggests that there was minimal interference from the remaining KCN substrate on thiocyanate production after the strains’ rhodanese activity. The statistical difference among the six strains at each time of incubation was analyzed using one-way ANOVA with three replicates. The results indicated that the KKU-BF7 strain exhibited the highest capacity for CN-thiocyanate transformation, with a minimum of 25.49 mg HCN equivalent/L during the 12 h incubation period. This value was significantly greater than that of all other strains (p < 0.001). In (b) the fermentation end-products from each strain at 12, 24, and 48 h of incubation were observed to analyze their correlation with the thiocyanate values using Pearson’s correlation coefficient. The results indicate a significant association between the degradation of thiocyanate and the production of ammonia nitrogen (p < 0.05) and/or acetic acid (p < 0.05) in certain strains.