In Vitro Stimulation of Forage Fiber Degradation by Ruminal Microorganisms with Aspergillus oryzae Fermentation Extract

Aspergillus oryzae fermentation extract (Amaferm) was evaluated for its ability to influence degradation of brome grass and switchgrass fiber fractions by mixed ruminal microorganisms in vitro. Addition of Amaferm at a concentration of 0.067 mg/ml, which is approximately the concentration found in the rumen ecosystem (0.06 mg/ml), increased the degradation of brome grass neutral detergent fiber (NDF) by 28% after fermentation for 12 h (P < 0.01), but had no effect after fermentation for 24 or 48 h. The levels of degradation of both the cellulose and hemicellulose fractions were increased after fermentation for 12 h (P < 0.01). Additions of 0.08 and 8% (vol/vol) Amaferm filtrate (12.5 g/100 ml) stimulated degradation of switchgrass NDF by 12 and 24% (P < 0.01), respectively, after fermentation for 12 h; when 80% filtrate was added, degradation was decreased by 38%. The concentrations of total anaerobes in culture tubes containing 80% filtrate were 5 times greater than the concentrations in the controls; however, the concentrations of cellulolytic organisms were 3.5 times lower than the concentrations in the controls (P < 0.05). These results suggested that the filtrate contained high concentrations of soluble substrate which did not allow the cellulolytic organisms to compete well with other populations. The remaining concentrations of esterified p-coumaric and ferulic acids were lower at 12 h in NDF residues obtained from fermentation mixtures supplemented with Amaferm. Because the total anaerobes were not inhibited in fermentation mixtures containing Amaferm, antibiotics are unlikely to be involved as a mode of action for increasing NDF degradation. The possibility that Amaferm contains enzymes (possibly esterases) that may play a role in stimulating the rate of fiber degradation by mixed ruminal microorganisms by removal of plant cell wall phenolic acid esters is discussed.     

Effects of DL-Malate on In Vitro Forage Fiber Digestion by Mixed Ruminal Microorganisms

The objective of this study was to evaluate the effects of 0, 4, 8, and 12 mM DL-malate on the in vitro mixed ruminal microorganism fermentation of alfalfa hay and Coastal bermudagrass hay. When alfalfa hay was the substrate, 4 and 8 mM DL-malate numerically increased propionate concentration, and 12 mM DL-malate increased (P < 0.10) propionate. All three concentrations of DL-malate decreased (P < 0.05) the acetate:propionate ratio. In Coastal bermudagrass hay fermentations, all three DL-malate concentrations increased (P < 0.05) propionate and decreased (P < 0.05) the acetate:propionate ratio, while 4 and 12 mM DL-malate numerically increased in vitro dry matter disappearance. When mixed ruminal microorganisms were incubated with 6.25 mM DL-lactic acid and alfalfa hay, 8 and 12 mM DL-malate increased (P < 0.05) final pH, and 12 mM DL-malate increased (P < 0.10) propionate and decreased (P < 0.10) the acetate:propionate ratio. DL-Malate treatment had little effect on in vitro dry matter disappearance. Addition of 8 and 12 mM DL-malate to Coastal bermudagrass hay plus DL-lactic acid fermentations increased (P < 0.05) final pH, and 8 mM DL-malate increased (P < 0.10) in vitro dry matter disappearance. Even though DL-malate treatment consistently increased final pH values in fermentations that included DL-lactic acid, there was not a corresponding increase in in vitro dry matter disappearance of either alfalfa hay or Coastal bermudagrass hay in the 48-h batch culture incubations.     

Rumen Fungi and Forage Fiber Degradation

The role of anaerobic rumen fungi in in vitro forage fiber degradation was determined in a two forage × two inoculum source × five treatment factorial design. Forages used as substrates for rumen microorganisms were Coastal bermuda grass and alfalfa; inoculum sources were rumen fluid samples from a steer fed Coastal bermuda grass hay or alfalfa hay; treatments were whole rumen fluid (WRF), WRF plus streptomycin (0.2 mg/ml of rumen fluid) and penicillin (1.25 mg/ml of fluid), WRF plus cycloheximide (0.5 mg/ml of fluid), WRF plus streptomycin, penicillin, and cycloheximide, and McDougall buffer. Populations of fungi as shown by sporangial development were greater on bermuda grass leaves than on alfalfa leaflets regardless of inoculum source. However, endogenous fungal populations were greater from the alfalfa hay inoculum. Cycloheximide inhibited the fungi, whereas streptomycin and penicillin, which inhibit bacterial populations, resulted in an increase in numbers of sporangia in the alfalfa inoculum, suggesting an interaction between bacteria and fungi. Bacteria (i.e., WRF plus cycloheximide) were equal to the total population in degrading dry matter, neutral-detergent fiber (NDF), acid-detergent fiber (ADF), and cellulose for both inocula and both forages. Degradation of dry matter, NDF, ADF, and cellulose by anaerobic fungi (i.e., WRF plus streptomycin and penicillin) was less than that due to the total population or bacteria alone. However, NDF, ADF, and cellulose digestion was 1.3, 2.4, and 7.9 percentage units higher, respectively, for bermuda grass substrate with the alfalfa versus bermuda grass inoculum, suggesting a slight benefit by rumen fungi. No substantial loss of lignin (72% H₂SO₄ method) occurred due to fungal degradation. The most active fiber-digesting population in the rumen was the bacteria, even when streptomycin and penicillin treatment resulted in an increase in rumen fungi over untreated WRF. The development of large numbers of sporangia on fiber may not indicate a substantial role as digesters of forage.     

Synergism in Degradation and Utilization of Intact Forage Cellulose, Hemicellulose, and Pectin by Three Pure Cultures of Ruminal Bacteria

Pure cultures of ruminal bacteria characterized as using only a single forage polysaccharide (Fibrobacter succinogenes A3c, cellulolytic; Bacteroides ruminicola H2b, hemicellulolytic; Lachnospira multiparus D15d, pectinolytic) were inoculated separately and in all possible combinations into fermentation tubes containing orchard grass as the sole substrate. Fermentations were run to completion, and then cultures were analyzed for digestion of cellulose plus degradation and utilization of hemicellulose and pectin. Addition of the noncellulolytic organisms, in any combination, to the cellulolytic organism F. succinogenes had little effect on overall cellulose utilization. F. succinogenes degraded but could not utilize hemicellulose; however, when it was combined with B. ruminicola, total utilization of hemicellulose increased markedly over that by B. ruminicola alone. L. multiparus was inactive in hemicellulose digestion, alone or in any combination. Although unable to degrade and utilize purified pectin, B. ruminicola degraded and utilized considerable quantities of the forage pectin. In contrast, L. multiparus was very active against purified pectin, but had extremely limited ability to degrade and utilize pectin from the intact forage. Both degradation and utilization of forage pectin increased when F. succinogenes was combined with B. ruminicola. Sequential addition of two cultures, allowing one to complete its fermentation before adding the second, was used to study synergism between cultures on forage pectin digestion. In general, synergistic effects did not appear to be related to a particular sequence of utilization. The ability of F. succinogenes to degrade and B. ruminicola to degrade and utilize forage pectin contradicts both previous and present data obtained with purified pectin. Thus, isolation and characterization of ruminal bacteria on purified substrates may be misleading with regard to their role in the overall ruminal fermentation.     

Degradation of Bermuda and Orchard Grass by Species of Ruminal Bacteria

Fiber degradation in Bermuda grass and orchard grass was evaluated gravimetrically and by scanning and transmission electron microscopy after incubation with pure cultures of rumen bacteria. Lachnospira multiparus D-32 was unable to degrade plant cell wall components. Butyrivibrio fibrisolvens 49 degraded 6 and 14.9% of the fiber components in Bermuda grass and orchard grass, respectively, and Ruminococcus albus 7 degraded 11.4% orchard grass fiber but none in Bermuda grass. Both B. fibrisolvens and R. albus lacked capsules, did not adhere to fiber, and degraded only portions of the more easily available plant cell walls. R. flavefaciens FD-1 was the most active fiber digester, degrading 8.2 and 55.3% of Bermuda and orchard grass fiber, respectively. The microbe had a distinct capsule and adhered to fiber, especially that which is slowly degraded, but was able to cause erosion and disorganization of the more easily digested cell walls, apparently by extracellular enzymes. Results indicated that more digestible cell walls could be partially degraded by enzymes disassociated from cellulolytic and noncellulolytic bacteria, and data were consistent with the hypothesis that the more slowly degraded plant walls required attachment. Microbial species as well as the cell wall architecture influenced the physical association with and digestion of plant fiber.     

In Vitro Lactate Metabolism by Ruminal Ingesta

Ruminal ingesta (300 ml) obtained from a fistulated cow fed alfalfa hay (H), 3.6 kg of grain mixture with corn silage fed ad libitum (S), 2.5:1 grain-alfalfa hay mixture (G), or a 2.5:1 grain-alfalfa hay mixture providing 545 g of sodium and calcium lactate daily (L) were incubated for 8 hr with nonpolymerized sodium lactate or 17% polymerized lactic acid neutralized to pH 6.7. Polymerization had no effect on the rate of lactate utilization. The initial rates of lactate metabolism for the H, G, S, and L ingesta were 0.72, 0.95, 1.8, and 3.4 meq per 100 ml of rumen fluid per hr, respectively. Lactate-2-(14)C was incubated for 4 hr with each type of ruminal ingesta. Of the label recovered in the volatile fatty acids (VFA), 74.1, 61.2, 49.3, and 38.9% was recovered in acetate, and 9.4, 19.8, 23.3, and 51.9% was recovered in propionate with H, G, S, and L ingesta, respectively. The balance of label was distributed between butyrate and valerate. The titratable VFA did not follow this pattern of production. With the hay ingesta, lactate metabolism resulted in a net loss of acetate and a large increase in butyrate. Little propionate was produced. The G, S, and L ingesta metabolized lactate to yield progressively more propionate and less butyrate. Evidence was gathered to suggest that acetate was the primary end product of lactate metabolism but that oxidation of lactate to pyruvate dictated the synthesis of butyrate from acetate to maintain an oxidation-reduction balance. It was noted that acetate and butyrate production from lactate was pH-dependent, with acetate production maximal at pH 7.4 and butyrate at 6.2. Propionate production was largely unaffected within this pH range.     

In Vitro Activity of Glucosinolates and Their Degradation Products against Brassica-Pathogenic Bacteria and Fungi

Glucosinolates (GSLs) are secondary metabolites found in Brassica vegetables that confer on them resistance against pests and diseases. Both GSLs and glucosinolate hydrolysis products (GHPs) have shown positive effects in reducing soil pathogens. Information about their in vitro biocide effects is scarce, but previous studies have shown sinigrin GSLs and their associated allyl isothiocyanate (AITC) to be soil biocides. The objective of this work was to evaluate the biocide effects of 17 GSLs and GHPs and of leaf methanolic extracts of different GSL-enriched Brassica crops on suppressing in vitro growth of two bacterial (Xanthomonas campestris pv. campestris and Pseudomonas syringae pv. maculicola) and two fungal (Alternariabrassicae and Sclerotiniascletoriorum) Brassica pathogens. GSLs, GHPs, and methanolic leaf extracts inhibited the development of the pathogens tested compared to the control, and the effect was dose dependent. Furthermore, the biocide effects of the different compounds studied were dependent on the species and race of the pathogen. These results indicate that GSLs and their GHPs, as well as extracts of different Brassica species, have potential to inhibit pathogen growth and offer new opportunities to study the use of Brassica crops in biofumigation for the control of multiple diseases.     

The Effect of Nisin and Monensin on Ruminal Fermentations In Vitro

When mixed ruminal bacteria and alfalfa were incubated in vitro, monensin and nisin both inhibited methane production so long as the concentrations were greater than 1 μM. Monensin- and nisin-dependent methane depressions caused a decrease in the acetate to propionate ratio (4.5 to 3.0). Total volatile fatty acid production was decreased by both monensin and nisin addition at concentrations greater than 2 μM. Starch-digesting ruminal bacteria were initially inhibited by monensin and nisin, but this effect disappeared after two to four transfers. Nisin always inhibited cellulolytic bacteria, but the nisin-dependent inhibition of cellulose digestion was no greater than the inhibition caused by monensin. Monensin and nisin also inhibited amino acid degradation, and nisin was more effective than monensin in controlling the growth of Clostridium aminophilum, an obligate amino acid-fermenting ruminal bacterium that can tolerate low concentrations of monensin. Because nisin was as potent as monensin, bacteriocins such as nisin may have potential as feed additives. Received: 2 December 1996 / Accepted: 10 February 1997     

Antibiosis between Ruminal Bacteria and Ruminal Fungi

Cellulose digestion, bacterial numbers, and fungal numbers were monitored over time in vitro by using a purified cellulose medium with and without antibiotics (penicillin and streptomycin). All fermentations were inoculated with a 1:10 dilution of whole rumen contents (WRC). Without antibiotics, cellulose digestion was higher (P < 0.01) at 24, 30, 48, and 72 h; fungi had almost disappeared by 24 h, while bacterial concentrations increased over 100-fold in 24 h and then decreased gradually up to 72 h. In those fermentations with added antibiotics, fungal concentrations increased 4-fold by 30 h and up to 42-fold at 72 h; bacterial concentrations were markedly reduced by 24 h and remained low through 72 h. Similar results were obtained with ground alfalfa as a substrate. In further studies, the in vitro fermentation of purified cellulose without antibiotics was stopped after 18 to 20 h, and the microbial population was killed by autoclaving. Antibiotics were added to half of the tubes, and all tubes were reinoculated with WRC. After 72 h, extensive cellulose digestion had occurred in those tubes without antibiotics, as compared to very low cellulose digestion with added antibiotics. The extent of this inhibition was found to increase in proportion to the length of the initial fermentation period, suggesting the production of a heat-stable inhibitory factor or factors. The inhibitory activity was present in rumen fluid, could be extracted from lyophilized rumen fluid (LRF) with water, and was stable in response to proteolytic enzymes. In addition, the water-extracted residue of LRF was found to contain growth factor activity for rumen fungi in vitro.     

Ruminal Bacterial Degradation of Benzo(b)-thien-4-yl Methylcarbamate (Mobam) and Effect of Mobam on Ruminal Bacteria

Mixtures of ruminal bacteria degraded benzo(b)thien-4-yl methylcarbamate (Mobam) to 4-hydroxybenzothiophene, CO(2), and polar product(s). The metabolite, 4-hydroxybenzothiophene, was identified (after acetylation) by comparative infrared and mass spectrometry with an authentic sample. Carbon dioxide and polar product(s) were produced by degradation of the methylcarbamate moiety. Ten previously characterized strains of ruminal bacteria with diverse physiological capabilities did not degrade Mobam. However, three tributyrin-hydrolyzing strains were isolated that did degrade Mobam. Mobam inhibited growth of two of ten strains isolated on Mobam-free glycerol-tributyrin enrichment medium. One of these strains was also sensitive to 2-carbomethoxy-propene-2yl dimethyl phosphate (Phosdrin). Mobam prevented some ruminal bacteria from producing zones of hydrolysis in tributyrin emulsion media and inhibited some ruminal bacteria from degrading 1-naphthyl acetate and fluorescein-3',6'-diacetate.     

Dissimilation of Tryptophan and Related Indolic Compounds by Ruminal Microorganisms In Vitro

Intraruminal doses of L-tryptophan cause acute pulmonary edema and emphysema in cattle. The D and L isomers of tryptophan and 22 related indolic compounds were incubated with ruminal microorganisms in vitro. Incubation of L-[U-benzene ring-(14)C]tryptophan with ruminal microorganisms for 24 h resulted in 39% of the added radioactivity being incorporated into skatole, 7% into indole, and 4% into indoleacetate (IAA). D-Tryptophan was not degraded to any of these metabolites. The major pathway of skatole formation from L-tryptophan appeared to be by the decarboxylation of IAA. Incubation of [2-(14)C]IAA with ruminal microorganisms for 24 h resulted in 38% incorporation into skatole. L-[5-Hydroxy]tryptophan was degraded to 5-hydroxyskatole and 5-hydroxyindole, whereas 5-hydroxyindoleacetate was degraded to only 5-hydroxyskatole. Incubation of indolepyruvate, indolelactate, and indolealdehyde with ruminal microorganisms resulted in the formation of both skatole and indole. Under similar conditions, indoleacetaldehyde was converted to IAA and tryptophol. The addition of increasing concentrations of glucose (0 to 110 mM) reduced the formation of both skatole and indole from L-tryptophan and resulted in the accumulation of IAA. Antibiotics reduced the degradation of L-tryptophan to skatole and indole, with kanamycin and neomycin particularly effective in reducing the decarboxylation of IAA to skatole.     

In Vitro Susceptibility of Selected Bacteria to Cefaclor

Cefaclor is an orally absorbed cephalosporin antibiotic chemically and pharmacologically similar to cephalexin. It appears to be more active than cephalexin against susceptible strains. The in vitro sensitivity of 230 clinical bacterial isolates to cefaclor was studied. Most isolates of S. aureus, K. pneumoniae, E. coli, and indole negative Proteus species were inhibited at clinically attainable serum and urine concentrations. Like cephalexin, cefaclor was less active against isolates of Enterobacter species, indole positive Proteus species and enterococci although many of these isolates were inhibited at concentrations achievable in urine.     

Tryptophan Biosynthesis and Production of Other Related Compounds from Indole and L-Serine by Mixed Ruminal Bacteria, Protozoa, and Their Mixture In Vitro

Tryptophan (Trp) biosynthesis and production of other related compounds from 1 mM each of indole (IND), L-serine (Ser), and IND plus Ser by mixed ruminal bacteria (B), protozoa (P), and their mixture (BP) in an in vitro system were quantitatively investigated. Ruminal microorganisms were anaerobically incubated at 39°C for 12 h. Trp and other related compounds produced in both the supernatants and microbial hydrolyzates of the incubation were analyzed by HPLC. B, P, and BP suspensions were not able to produce Trp when incubated with only IND or Ser. Appreciable amounts of Trp (9.8, 3.1, and 6.6% of substrate) were produced from IND plus Ser after 12 h by B, P, and BP suspensions, respectively. Trp produced from IND + Ser in B was found only in the hydrolyzate, whereas it was found in the medium as a free form in P after a 12-h incubation period. Rumen bacteria and protozoa were separately demonstrated for the first time to produce Trp from IND plus Ser, and the ability of P to produce Trp from IND plus Ser was about one-third that of B in 12 h. Trp produced from IND plus Ser by B, P, and BP suspensions was simultaneously degraded into its related compounds, and, among them, indoleacetic acid (IAA) was a major product found in B. Production of IAA was 4.3, 0.3, and 3.2% of IND in 12 h by B, P, and BP suspensions, respectively. A small amount of skatole (SKT) (1.1 and 2.5% in B and BP, respectively) and p-cresol (CRL) (2.4 and 3.4% in B and BP, respectively) were also produced from IND plus Ser during 12-h incubation. P suspension produced no SKT or CRL from IND plus Ser in 12-h incubation. These results suggested for the first time that both rumen bacteria and protozoa have an ability to produce Trp from IND plus Ser, and the ability was higher in B than in P. The ratios of Trp produced from IND plus Ser to that from indolepyruvic acid by B, P, and BP were 1:3.4, 1:14.2, and 1:6.6 during 12-h incubation period. From these results, the degree of importance of producing Trp from IND plus Ser in the rumen was indicated. Received: 18 February 1999 / Accepted: 18 May 1999     

Quantitative Antibiotic Sensitivities of Ruminal Bacteria

Fifteen species of ruminal bacteria were tested against 10 antibiotics in concentrations ranging from 0.1 to 200 mug/ml in an anaerobic tube dilution system.     

Antibiotic Susceptibility of Anaerobic Ruminal Bacteria

This study demonstrated that 15 species of ruminal bacteria with no previous history of contact with antibiotics are susceptible to bacitracin, chloramphenicol, chlortetracycline, erythromycin, novobiocin, oleandomycin, oxytetracycline, penicillin, tetracycline, tylosin, and vancomycin. A number of the species were not inhibited by kanamycin, neomycin, polymyxin, and streptomycin. The data suggest that antibiotic-resistant cells occur within susceptible cultures of these species. Streptococcus bovis FD-10 and a nonruminal anaerobe, Bacteroides melaninogenicus BE-1, showed similar antibiotic susceptibilities.     

Edman Degradation on In Vitro Biosynthesized Peptidoglycans from Staphylococcus epidermidis

Edman degradations were performed on the pentapeptide bridges of peptidoglycans which were biosynthesized in vitro by using six different species of seryl-transfer ribonucleic acid (tRNA). The ratio of glycine to serine in each of the five bridge positions varied with the concentration of crude glycyl-tRNA contained in the reaction mixtures, and serine appeared to be incorporated in a random manner. No single species of seryl-tRNA could be identified as the one active in in vivo pentapeptide bridge synthesis; however, it can be speculated that seryl-tRNA(s) from category I could be involved in the in vivo nonrandom incorporation of serine in the bridge.     

Susceptibility of Phytopathogenic Bacteria to Wheat Purothionins In Vitro

Purothionins are basic polypeptides with antimicrobial properties that are present in the endosperm of wheat and other cereal species. Susceptibility to wheat purothionins among phytopathogenic bacteria of the genera Pseudomonas, Xanthomonas, Agrobacterium, Erwinia, and Corynebacterium has been investigated. Sensitive strains have been found in all of these genera except Agrobacterium (the only strain of A. tumefaciens available proved to be resistant). Minimal inhibitory concentrations (MIC) with partially purified crude purothionins ranged from 1 μg/ml for C. sepedonicum (C.5) to 540 μg/ml for E. amylovora (E.3). Minimal bactericidal concentrations (MBC) were not higher than twice the MIC value, except for C. poinsettiae (C.4) (MBC/MIC = 8). Purothionins α and β, obtained by carboxymethyl-cellulose column chromatography, were tested against P. solanacearum (P.2) and X. phaseoli (X.2); α purothionin was more active than β against X.2, and β more active than α against P.2. This suggests a relationship between polypeptide sequence and specificity of action.     

Relative Contributions of Bacteria, Protozoa, and Fungi to In Vitro Degradation of Orchard Grass Cell Walls and Their Interactions

To assess the relative contributions of microbial groups (bacteria, protozoa, and fungi) in rumen fluids to the overall process of plant cell wall digestion in the rumen, representatives of these groups were selected by physical and chemical treatments of whole rumen fluid and used to construct an artificial rumen ecosystem. Physical treatments involved homogenization, centrifugation, filtration, and heat sterilization. Chemical treatments involved the addition of antibiotics and various chemicals to rumen fluid. To evaluate the potential activity and relative contribution to degradation of cell walls by specific microbial groups, the following fractions were prepared: a positive system (whole ruminal fluid), a bacterial (B) system, a protozoal (P) system, a fungal (F) system, and a negative system (cell-free rumen fluid). To assess the interactions between specific microbial fractions, mixed cultures (B+P, B+F, and P+F systems) were also assigned. Patterns of degradation due to the various treatments resulted in three distinct groups of data based on the degradation rate of cell wall material and on cell wall-degrading enzyme activities. The order of degradation was as follows: positive and F systems > B system > negative and P systems. Therefore, fungal activity was responsible for most of the cell wall degradation. Cell wall degradation by the anaerobic bacterial fraction was significantly less than by the fungal fraction, and the protozoal fraction failed to grow under the conditions used. In general, in the mixed culture systems the coculture systems demonstrated a decrease in cellulolysis compared with that of the monoculture systems. When one microbial fraction was associated with another microbial fraction, two types of results were obtained. The protozoal fraction inhibited cellulolysis of cell wall material by both the bacterial and the fungal fractions, while in the coculture between the bacterial fraction and the fungal fraction a synergistic interaction was detected.     

Phenotypic and Phylogenetic Characterization of Ruminal Tannin-Tolerant Bacteria

The 16S rRNA sequences and selected phenotypic characteristics were determined for six recently isolated bacteria that can tolerate high levels of hydrolyzable and condensed tannins. Bacteria were isolated from the ruminal contents of animals in different geographic locations, including Sardinian sheep (Ovis aries), Honduran and Colombian goats (Capra hircus), white-tail deer (Odocoileus virginianus) from upstate New York, and Rocky Mountain elk (Cervus elaphus nelsoni) from Oregon. Nearly complete sequences of the small-subunit rRNA genes, which were obtained by PCR amplification, cloning, and sequencing, were used for phylogenetic characterization. Comparisons of the 16S rRNA of the six isolates showed that four of the isolates were members of the genus Streptococcus and were most closely related to ruminal strains of Streptococcus bovis and the recently described organism Streptococcus gallolyticus. One of the other isolates, a gram-positive rod, clustered with the clostridia in the low-G+C-content group of gram-positive bacteria. The sixth isolate, a gram-negative rod, was a member of the family Enterobacteriaceae in the gamma subdivision of the class Proteobacteria. None of the 16S rRNA sequences of the tannin-tolerant bacteria examined was identical to the sequence of any previously described microorganism or to the sequence of any of the other organisms examined in this study. Three phylogenetically distinct groups of ruminal bacteria were isolated from four species of ruminants in Europe, North America, and South America. The presence of tannin-tolerant bacteria is not restricted by climate, geography, or host animal, although attempts to isolate tannin-tolerant bacteria from cows on low-tannin diets failed.The toxicity of phenolic compounds in the environment has fostered studies of bacteria that are able to tolerate and/or metabolize high levels of these compounds, particularly under anaerobic conditions (1, 4, 14, 21, 30, 36). Tannins are secondary polyphenolic compounds known primarily for their ability to bind to and precipitate proteins and other macromolecules. Tannins have been found in many habitats, including sewage sludge, forest litter, and the rumen (9, 14, 15, 28). Bacteria capable of degrading or tolerating tannins have been isolated from sewage sludge (14) and from the alimentary tracts of koalas (Phascolarctos cinereus) (33), goats (Capra hircus) (4, 30), and horses (Equus caballus) (31). Most of the isolates have been characterized phenotypically, and phylogenetic characterization has been limited to studies conducted in Australia (4, 34, 35) and Japan (31). Little is known about the geographic diversity and host species diversity of tannin-tolerant and tannin-degrading bacteria.The objective of this study was to characterize six recently isolated tannin-tolerant bacteria by examining their phenotypic characteristics and molecular phylogeny. These bacteria were isolated from the ruminal contents of goats (C. hircus), sheep (Ovis aries), white-tail deer (Odocoileus virginianus), and Rocky Mountain elk (Cervus elaphus nelsoni), all of which had consumed forage containing tannins. Our goal was to genetically and biochemically characterize tannin-tolerant bacteria isolated from different host animals in various geographic locations.     

In Vitro Activity of Doxycycline Against Bacteria from Clinical Material

Doxycycline (alpha-6-deoxy-oxytetracycline) was tested against various bacteria of recent clinical origin with 30-mug discs. The antibiotic susceptibility of these bacteria to commonly used antimicrobial agents was also established. Those bacteria which responded with equivocal zones of inhibition about the tetracycline compounds were tested by the tube dilution technique. Staphylococci and enterococci consistently displayed greater in vitro susceptibility to doxycycline than to tetracycline or demethylchlortetracycline.