Characterization of the effects of nitrate and tungstate, alone or combined, and Salmonella Newport inoculation on rumen microbiota, fermentation, and Salmonella survivability in vitro.

Nitrate consumption by ruminants may enrich nitrate-respiring Salmonella in the gut. To test whether tungstate, an inhibitor of nitrate reductase, may prevent nitrate-promoted enrichment of Salmonella, ruminal microbes inoculated with or without 104 colony-forming units (CFU)/mL of Salmonella Newport were incubated for 26 h under simulated rumen conditions with or without 10 mM nitrate, 100 mM tungstate, or their combination. Results indicated more nitrate was metabolized (p < 0.05) by ruminal populations supplemented with nitrate alone than with nitrate and tungstate combined, with means (± standard deviations) being 8.55 ± 0.74 and 3.97 ± 0.67 μmol nitrate/mL, respectively. Nitrite accumulations were affected (p < 0.05) by tungstate treatment, achieving 4.96 ± 0.26 and 0.18 ± 0.07 μmol/mL in populations supplemented with nitrate alone or combined with tungstate, respectively, when not inoculated with S. Newport and achieving 1.95 ± 0.18 and 0.06 ± 0.01 μmol/mL, respectively, when inoculated with S. Newport. Salmonella increased (p < 0.05) in populations treated with tungstate, alone or combined with nitrate (5.82 ± 0.10 and 5.57 ± 0.15 log10 CFU/mL, respectively), compared to controls or nitrate-only supplemented populations (1.63 ± 0.58 and 2.84 ± 0.21 log10 CFU/mL, respectively). In populations not inoculated with S. Newport, the addition of nitrate, tungstate, or their combination increased (p < 0.05) wild-type coliforms by 1.4 to 3.9 log10 units compared to untreated controls (2.67 ± 0.18 log10 CFU/mL). In S. Newport-inoculated populations, tungstate treatment, alone or combined with nitrate, increased (p < 0.05) coliforms by 2.8 to 3.2 log10 units compared to control and nitrate-only supplemented populations (3.06 ± 0.48 and 3.49 ± 0.06 log10 CFU/mL, respectively). Wildtype lactic acid bacteria were enriched by tungstate treatment and nitrate supplementation, alone or combined to 0.2 to 1.1 log10 units, compared to untreated controls (6.84 ± 0.03 and 6.68 ± 0.16 log10 CFU/mL, respectively). Methane production decreased by 70% (p < 0.05) in populations supplemented with nitrate, whether alone or combined with tungstate, compared to untreated or tungstate-only treated populations (29.85 ± 5.05 and 23.08 ± 5.49 μmol methane/mL of incubation fluid, respectively). These results indicate that tungstate treatment marginally decreased nitrate metabolism in the ruminal populations but surprisingly promoted Salmonella enrichment.