Effects of potassium ion concentrations on the antimicrobial activities of ionophores against ruminal anaerobes.

The antimicrobial activities of monensin and lasalocid against representative strains of ruminal bacteria were evaluated in medium containing three different concentrations of potassium (1.3, 7.9, or 23.3 mM). The growth of Eubacterium ruminantium was inhibited by low concentrations of ionophores (less than or equal to 0.16 mg/liter), while the strain of Streptococcus bovis tested was resistant to high concentrations of ionophores (40 mg/liter) at all potassium concentrations tested. The MICs of the ionophores for strains of Bacteroides succinogenes, Butyrivibrio fibrisolvens, Ruminococcus albus, and Ruminococcus flavefaciens and for one strain of Bacteroides ruminicola increased with increasing potassium concentrations in the medium. High concentrations of ionophores (40 mg/liter) decreased the maximum cell yields or increased the lag times or both in cultures of one strain of Bacteroides ruminicola and two strains of Selenomonas ruminantium but did not completely inhibit the growth of these organisms. Increased potassium concentrations in the medium (from 7.9 to 23.3 mM) decreased the lag times or increased the cell yields or both when these three strains were grown in ionophore-containing medium, while the activities of lasalocid and monensin against these organisms were enhanced in the medium containing low potassium concentrations (1.3 mM). The data from this study suggest that extracellular potassium concentrations may influence the antimicrobial activities of ionophores in the rumen.     

Use of chitinase to assess ruminal fungi associated with plant residues in vitro.

Treatment of fibrous residue from in vitro digestion trials with chitinase was evaluated gravimetrically and microscopically to determine the associated fungal biomass. The percent dry weight removed by chitinase with time paralleled changes in the number of sporangia associated with leaves. The fungal biomass contributed about 12% dry matter to the residue of leaves incubated with ruminal fluid plus streptomycin and penicillin.     

Effects of potassium ion concentrations on the antimicrobial activities of ionophores against ruminal anaerobes

The antimicrobial activities of monensin and lasalocid against representative strains of ruminal bacteria were evaluated in medium containing three different concentrations of potassium (1.3, 7.9, or 23.3 mM). The growth of Eubacterium ruminantium was inhibited by low concentrations of ionophores (less than or equal to 0.16 mg/liter), while the strain of Streptococcus bovis tested was resistant to high concentrations of ionophores (40 mg/liter) at all potassium concentrations tested. The MICs of the ionophores for strains of Bacteroides succinogenes, Butyrivibrio fibrisolvens, Ruminococcus albus, and Ruminococcus flavefaciens and for one strain of Bacteroides ruminicola increased with increasing potassium concentrations in the medium. High concentrations of ionophores (40 mg/liter) decreased the maximum cell yields or increased the lag times or both in cultures of one strain of Bacteroides ruminicola and two strains of Selenomonas ruminantium but did not completely inhibit the growth of these organisms. Increased potassium concentrations in the medium (from 7.9 to 23.3 mM) decreased the lag times or increased the cell yields or both when these three strains were grown in ionophore-containing medium, while the activities of lasalocid and monensin against these organisms were enhanced in the medium containing low potassium concentrations (1.3 mM). The data from this study suggest that extracellular potassium concentrations may influence the antimicrobial activities of ionophores in the rumen.     

Use of chitinase to assess ruminal fungi associated with plant residues in vitro

Treatment of fibrous residue from in vitro digestion trials with chitinase was evaluated gravimetrically and microscopically to determine the associated fungal biomass. The percent dry weight removed by chitinase with time paralleled changes in the number of sporangia associated with leaves. The fungal biomass contributed about 12% dry matter to the residue of leaves incubated with ruminal fluid plus streptomycin and penicillin.     

Effect of potassium concentration and ouabain on the renal adaptation to potassium depletion in isolated perfused rat kidney

Renal adaptation for potassium (K) conservation has been demonstrated in isolated perfused kidneys from rats within 3 days of K depletion and appears to be independent of aldosterone and sodium excretion. This study was designed to investigate whether the renal adaptation for K conservation is independent of ambient [K] and renal tissue levels of K and whether ouabain may have effects on K excretion, which are in contrast to the effects on K excretion in normal animals. In the first study, rats K depleted for 3 days received 2500 mu equiv. KCI intraperitoneally, while other K-depleted rats and a group of control diet animals received intraperitoneal H2O alone to determine whether simple restoration of K deficits would reverse the renal adaptation for K conservation. Intraperitoneal KCI increased plasma [K] and kidney tissue K significantly within 3 h in the K-repleted group compared with the K-depleted rats. Isolated Kidneys were perfused from the three groups of rats 3 h after intraperitoneal injection. Despite K repletion in vivo, perfused kidneys from the K-repleted group still had significantly decreased K excretion (1.28 +/- 0.085 mu equiv./min) compared with controls (2.05 +/- 0.291 mu equiv./min), and K excretion was still not different from the K-depleted group (0.57 +/- 0.134 mu equiv./min). However, fractional K excretion by the kidneys from K-repleted rats was increased above K-depleted kidneys (0.48 +/- 0.051 vs. 0.18 +/- 0.034, p less than 0.01). Despite the increased renal tissue K in K-repleted kidneys at the start of perfusion (285 +/- 5.1 vs. 257 +/- 5.4 mu equiv./g), by the end of the perfusion tissue K in perfused kidneys was identical in all three groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inducible bacteriophages from ruminal bacteria.

The incidence of temperate bacteriophage in a wide range of ruminal bacteria was investigated by means of induction with mitomycin C. Supernatant liquid from treated cultures was examined for phagelike particles by using transmission electron microscopy. Of 38 ruminal bacteria studied, nine organisms (23.7%) representing five genera (Eubacteria, Bacteroides, Butyrivibrio, Ruminococcus, and Streptococcus) produced phagelike particles. Filamentous particles from Butyrivibrio fibrisolvens are the first of this morphological type reported from ruminal bacteria. All of the other particles obtained possessed polyhedral heads and long, noncontractile tails (group B-type phage). The limited range of morphological types produced by mitomycin C induction cannot yet account for the much wider range of types found in ruminal contents by direct examination. The presence of viral genetic material in a significant percentage of the bacteria tested, as well as in a range of different genera, indicates that viral genetic material may be a normal constituent of the genome of appreciable numbers of ruminal bacteria.     

Superoxide dismutase in ruminal bacteria.

Of 13 species of anaerobic ruminal bacteria examined, 11 were found to contain measurable levels of superoxide dismutase activity. Four of five other strict anaerobic species studied for comparison were found to contain superoxide dismutase activity.     

Intracellular pH of acid-tolerant ruminal bacteria.

Acid-tolerant ruminal bacteria (Bacteroides ruminicola B1(4), Selenomonas ruminantium HD4, Streptococcus bovis JB1, Megasphaera elsdenii B159, and strain F) allowed their intracellular pH to decline as a function of extracellular pH and did not generate a large pH gradient across the cell membrane until the extracellular pH was low (less than 5.2). This decline in intracellular pH prevented an accumulation of volatile fatty acid anions inside the cells.     

Intracellular pH of acid-tolerant ruminal bacteria.

Acid-tolerant ruminal bacteria (Bacteroides ruminicola B1(4), Selenomonas ruminantium HD4, Streptococcus bovis JB1, Megasphaera elsdenii B159, and strain F) allowed their intracellular pH to decline as a function of extracellular pH and did not generate a large pH gradient across the cell membrane until the extracellular pH was low (less than 5.2). This decline in intracellular pH prevented an accumulation of volatile fatty acid anions inside the cells.     

Bacteriocins of ruminal bacteria

Similar sequences of distribution of structural genes encoding enterocin A (isolated from the ruminal strainE. faecium BC25) and enterolysin A (isolated from the ruminal amylolytic strainS. bovis II/I) were demonstrated by PCR using oligonucleotide primers specific for these bacteriocins within the ruminal enterococcal and streptococcal strains. Variable occurrence of these bacteriocins was found within the populations of Gram-positive ruminal cocci. An erratum to this article is available at .     

Susceptibility and resistance of ruminal bacteria to antimicrobial feed additives.

Susceptibility and resistance of ruminal bacterial species to avoparcin, narasin, salinomycin, thiopeptin, tylosin, virginiamycin, and two new ionophore antibiotics, RO22-6924/004 and RO21-6447/009, were determined. Generally, antimicrobial compounds were inhibitory to gram-positive bacteria and those bacteria that have gram-positive-like cell wall structure. MICs ranged from 0.09 to 24.0 micrograms/ml. Gram-negative bacteria were resistant at the highest concentration tested (48.0 micrograms/ml). On the basis of their fermentation products, ruminal bacteria that produce lactic acid, butyric acid, formic acid, or hydrogen were susceptible and bacteria that produce succinic acid or ferment lactic acid were resistant to the antimicrobial compounds. Selenomonas ruminantium was the only major lactic acid-producing bacteria resistant to all the antimicrobial compounds tested. Avoparcin and tylosin appeared to be less inhibitory (MIC greater than 6.0 micrograms/ml) than the other compounds to the two major lactic acid-producing bacteria, Streptococcus bovis and Lactobacillus sp. Ionophore compounds seemed to be more inhibitory (MIC, 0.09 to 1.50 micrograms/ml) than nonionophore compounds (MIC, 0.75 to 12.0 micrograms/ml) to the major butyric acid-producing bacteria. Treponema bryantii, an anaerobic rumen spirochete, was less sensitive to virginiamycin than to the other antimicrobial compounds. Ionophore compounds were generally bacteriostatic, and nonionophore compounds were bactericidal. The specific growth rate of Bacteroides ruminicola was reduced by all the antimicrobial compounds except avoparcin. The antibacterial spectra of the feed additives were remarkably similar, and it appears that MICs may not be good indicators of the potency of the compounds in altering ruminal fermentation characteristics.     

Improved assay for quantitating adherence of ruminal bacteria to cellulose.

A quantitative technique suitable for the determination of adherence of ruminal bacteria to cellulose was developed. This technique employs adherence of cells to cellulose disks and alleviates the problem of nonspecific cell entrapment within cellulose particles. By using this technique, it was demonstrated that the adherence of Ruminococcus flavefaciens FD1 to cellulose was inhibited by formaldehyde, methylcellulose, and carboxymethyl cellulose. Adherence was unaffected by acid hydrolysates of methylcellulose, glucose, and cellobiose.     

Use of fluorescent probes to assess physiological functions of bacteria at single-cell level

A wide diversity of fluorescent probes is currently available to assess the physiological state of microorganisms. The recent development of techniques such as solid-phase cytometry, the increasing sensitivity of fluorescence tools and multiparametric approaches combining taxonomic and physiological probes have improved the effectiveness of direct methods in environmental and industrial microbiology.     

Effect of phenolic monomers on ruminal bacteria.

Ruminal bacteria were subjected to a series of phenolic compounds in various concentrations to acquire fundamental information on the influence on growth and the potential limits to forage utilization by phenolic monomers. Ruminococcus albus 7, Ruminococcus flavefaciens FD-1, Butyrivibrio fibrisolvens 49, and Lachnospira multiparus D-32 were tested against 1, 5, and 10 mM concentrations of sinapic acid, syringaldehyde, syringic acid, ferulic acid, vanillin, vanillic acid, p-coumaric acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, and hydrocinnamic acid. Responses were variable and dependent on the phenolic compound and microbial species. Compounds especially toxic (i.e., resulting in poor growth, effect on several species, dose-related response) were p-coumaric acid and p-hydroxybenzaldehyde, and adaptation to the toxins did not occur after three 24-h periods. Syringic, p-hydroxybenzoic, and hydrocinnamic acids stimulated growth of all four species and also stimulated filter paper degradation by R. flavefaciens. None of the stimulatory compounds supported microbial growth in the absence of carbohydrates. In vitro dry matter digestibility of cellulose (Solka-Floc) was not stimulated by any of the phenolic compounds (10 mM), but the cinnamic acids and benzoic aldehydes (10 mM) reduced (P less than 0.05) digestion by the mixed population in ruminal fluid. Growth of R. flavefaciens in the presence of p-hydroxybenzoic acid (10 mM) or p-coumaric acid (5 mM) resulted in recognizable alterations in cell ultrastructure. Both phenolics caused a reduction in cell size (P less than 0.05), and p-coumaric acid caused a reduction in capsular size (P less than 0.05) and produced occasional pleomorphic cells.     

Adhesion of cellulolytic ruminal bacteria to barley straw

Adhesion of the cellulolytic ruminal bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes to barley straw was measured by incubating bacterial suspensions with hammer-milled straw for 30 min, filtering the mixtures through sintered glass filters, and measuring the optical densities of the filtrates. Maximum adhesion of both species occurred at pH 6.0 and during mid- to late-exponential phase. Adhesion was saturable at 33 and 23 mg (dry weight) g of straw for R. flavefaciens and F. succinogenes, respectively. Methyl cellulose and carboxymethyl cellulose inhibited adhesion by 24 to 33%. Competition between species was determined by measuring characteristic cell-associated enzyme activities in filtrates of mixtures incubated with straw; p-nitrophenyl-beta-d-lactopyranoside hydrolysis was used as a marker for F. succinogenes, while either beta-xylosidase or carboxymethyl cellulase was used for R. flavefaciens, depending on the other species present. R. flavefaciens had no influence on F. succinogenes adhesion, and F. succinogenes had only a minor (<20%) effect on R. flavefaciens adhesion. The noncellulolytic ruminal bacteria Bacteroides ruminicola and Selenomonas ruminantium had no influence on adhesion of either cellulolytic species, although these organisms also adhered to the straw. We concluded that R. flavefaciens and F. succinogenes have separate, specific adhesion sites on barley straw that are not obscured by competition with non-cellulolytic species.     

Utilization of xylooligosaccharides by selected ruminal bacteria.

The ability of ruminal bacteria to utilize xylooligosaccharides was examined. Xylooligosaccharides were prepared by partially hydrolyzing oat spelt xylan in phosphoric acid. This substrate solution was added (0.2%, wt/vol) to a complex medium containing yeast extract and Trypticase that was inoculated with individual species of ruminal bacteria, and growth and utilization were monitored over time. All of the xylanolytic bacteria examined were able to utilize this oligosaccharide mixture as a growth substrate. Butyrivibrio fibrisolvens, Eubacterium ruminantium, and Ruminococcus albus used xylooligosaccharides and whole, unhydrolyzed xylan to similar extents, while Prevotella ruminicola used twice as much xylooligosaccharides as xylan (76 versus 34%). Strains of Selenomonas ruminantium were the only nonxylanolytic species that were able to grow on xylooligosaccharides. The ability of individual S. ruminantium strains to utilize xylooligosaccharides was correlated with the presence of xylosidase and arabinosidases activities.     

Amylolytic activity of selected species of ruminal bacteria.

A variety of species of ruminal bacteria were screened for the ability to grow in starch-containing medium and produce amylase. Of those tested, the highest levels of amylase were produced by Streptococcus bovis JB1 and Ruminobacter amylophilus H18. Other strains that grew well on starch and produced amylase included Butyrivibrio fibrisolvens A38 and 49 and Bacteroides ruminicola 23 and B14. Varying the carbohydrate source provided for growth resulted in changes in the growth rate and level of amylase produced by these strains. All strains grew rapidly in starch-containing medium, and the rates of growth were generally more rapid than those observed for maltose-grown cultures. For S. bovis JB1, B. ruminicola 23 and B14, and B. fibrisolvens 49 and A38, amylase was produced when growth was on maltose or starch, but this activity was greatly reduced in glucose-grown cultures. The distribution of amylolytic activity between cellular and extracellular fractions was sometimes affected by the carbohydrate provided for growth. If S. bovis JB1 and B. fibrisolvens 49 were grown on starch, amylase was largely associated with cell pellets; however, if grown on maltose these strains produced activities that were almost entirely present in the extracellular fluid fractions. Although not as dramatic, a similar shift in the location of amylase activities was noted for the two B. ruminicola strains when grown on the same substrates. Growth on maltose or starch had little influence on either the predominantly cell-associated activity of B. fibrisolvens A38 or the activity of R. amylophilus H18, which was equally divided between cell pellet and extracellular fluid fractions.(ABSTRACT TRUNCATED AT 250 WORDS)