The potencies of the ionophores, when added alone or in combination with added Na+, K+ or Ca2+, were compared by determining the concentration at which cell density after

48-h incubation decreased by 50% (IC50; Table 1). Low Na+ Low K+ Low Ca2+ High Na+ Low K+ Low Ca2+ Low Na+ High K+ Low Ca2+ High Na+ High K+ Low Ca2+ Low Na+ Low K+ High Ca2+ High Na+ Low K+ High Ca2+ Low Na+ High K+ High Ca2+ High Na+ High K+ High Ca2+ The clearest and most consistent effect was that increasing the concentration of Na+ increased the potency of both ionophores. The concentration of monensin required to inhibit bacterial growth by 50% was, on average, 35% lower on high compared to low-Na+ medium. With E. ruminantium, S. bovis and P. albensis, the effect of adding Na+ was greatest when K+ was high. K+ alone tended to protect these bacteria from monensin, while the opposite was true with L. casei. Ca2+ had little influence on the potency of monensin, http://www.selleckchem.com/products/BEZ235.html except with L. casei at low [Na+] and GSK1120212 mw [K+] and S. bovis at high [Na+]. Altering

the ionic composition of the medium had little influence on the potency of tetronasin with E. ruminantium (Table 1), but with the other bacteria increasing Ca2+ alone enhanced potency by more than 100%. K+ increased the potency of tetronasin with L. casei but protected P. albensis and S. bovis. When increased cation concentrations were combined, the Ca2+ effect was dominant with L. casei, and Ca2+ remained effective in enhancing potency with these three species in the presence of high [K+]. However, the effects of combining high Na+ and Ca2+ were more complex and species dependent: high [Na+] when [Ca2+] was high did not affect the sensitivity of S. bovis, while the combination was less effective than either cation alone with P. albensis.

The effects of monensin and tetronasin on protonmotive force and ion gradients were determined with late-exponential phase and also with cells that had been in stationary phase for 30 h. The results were similar for both cultures. However, Ca2+ analysis was performed only on stationary-phase cells also (Table 2). The concentrations of ionophores used in this experiment were 0.256 μg monensin and 0.064 μg tetronasin mL−1, concentrations sufficient to cause severe inhibition of growth (Table 1). When these concentrations of monensin and tetronasin were added to exponentially growing E. ruminantium, growth ceased within 30 min (results not shown). Eubacterium ruminantium had an internal volume of 3.4 ± 0.47 μL mg protein−1, as calculated from the inulin exclusion volume. This was not affected by the presence of either ionophore. Both monensin and tetronasin caused increases in the internal pH of E. ruminantium, coupled with a slight decrease in the electrical potential (Δψ) (Table 2). The total Δp therefore fell by about 20 mV with both ionophores over the 2-h incubation period. Both ionophores resulted in the movement of Na+ and K+.