ANTIMICROBIAL SUSCEPTIBILITY OF RUMINAL STRAINS OF BUTYRIVIBRIO FIBRISOL VENS

Marounek M., O. G. Savka: Antimicrobial Susceptibility ojRuminalStrains ojButyrivibrio fibrisolvens. Acta vet. Bmo, 63,1994: 129-132. The susceptibility of five ruminal strains of Butyrivibrio fibrisolvens to 23 antimicrobial compounds was examined to evaluate effects of antimicrobials on this bacterium. All strains were very sensitive to ionophores and inhibitors of protein synthesis, except for aureomycin. On the contrary, butyrivibrios were relatively insensitive to inhibitors of carbohydrate metabolism and uncouplers. The strains studied displayed considerable variation in sensitivity to salinomycin, aureomycin and bacitracin. The substrate used to support the bacterial growth (glucose or xylose) influenced the susceptibility of isolates to antimicrobial agents. In one third of the measurements inhibitory concentrations of antimicrobial compoonds were lower with xylose-grown than with glucose-grown cells. Different response of xylose-grown cultures to antimicrobials may reflect lower energy supply from fermentation of xylose which was metabolized more slowly and with lower biosynthetic efficiency. Rumen, Butyrivibrio fibrisolvens, antimicrobial susceptibility Butyrivibrio fibrisolvens is a butyrate-forming anaerobic bacterium, which stains gram-negatively. but which has a thin gram-positive cell wall (H old e m an et al. 1984). Individual strains that have been isolated from the rumen or various anaerobic habitats can ferment cellulose. xylan, starch, pectin and other plant cell components. In the rumen. B.fibrisolvens belongs to the most common bacteria in animals fed widely different diets (Deh ori t y and Gru b 1977; Van G y Is w Y k and M u rp h y 1993). The purpose of this study was (i) to examine the susceptibility of B.fibrisolvens to antimicrobial compounds, and (ii) to compare glucoseand xylose-grown cultures in this respect. In separate experiments the panuneters of growth of butyrivibrios on glucose and xylose were measured. Most of antimicrobials tested are common growth promoters or drugs effective in prophylaxis and treatment of various animal diseases. Previous studies have shown that B. fibrisolvens is senstitive to ionophores (D e n n i s et al. 1981; Nag ar aj a and T a y lor 1987) and antibiotics that interferre with cell wall synthesis (H e s p e 11 et al. 1993). Antibiotics susceptibility data may be helpful in assessing phenotypic diversity of B. fibrisolvens strains. and also for evaluation of consequences of interactions of antimicrobials with a functionally important rumen bacterium. Materials and Methods B.fibrisolvens. type strain ATCC 19171 was obtained from the American Type Culture Collection. Strain 86 was a gift from the culture collection of the Hannah Research Institute. Ayr. Scotland. Strain Xl was isolated from the rumen fluid of a sheep at this Institute. Strains CBSI and X2D62 were supplied from the National Chemical Research Laboratory. Pretoria. South Africa. Strains were maintained in 20% (v/v) glycerol at -20°C. Bacteria were grown on a vitamin-mineral medium, supplemented with 0.1 % yeast extract, 0.1 % enzymatic hydrolysate of casein and 10% rumen fluid (Marounek et al. 1993). The medium was prepared anaerobically and reduced by 0.025% cysteine. HCl and 0.025% N~S.9~O. Glucose or xylose were added at4g1l (final conc.). The medium was sterilized by autoclaving at 110°C. Parameters of growth on glucose and xylose were followed in batch cultures at pH 6.5 and 39°C in 02-free CO2 atmosphere. Two LF2 fermentors (CSAV Workshop, Prague) were used. The growth was monitored turbidimetrically as optical density at 640 nm. Incubation was completed after reaching the stationary phase. The cell dry weight was determined after centrifugation of the culture. washing with rinsing solutions and drying at 105°C overnight. The following antimicrobialcompound were used: lasalocid (Hoffmann La Roche. Basel, Switzerland). monensin, IJlIfao sin, avilamycin, tylosin (Eli Lilly & Co .• Indianapolis, USA). salinomycin (Hoechst, Frankfurt, Germany). aureomycin, fmazolidone, olaquindox (Farmaceutical Works, Prague, Czech Republic). avoparcin and maduramicin (Cyanamid, Wayne. USA). virginiamycin (Smith Kline Beecham, Genval, Belgium). pentachlorophenol, iodoacete and dicyclohexylcarbodiimide (Flub, Buchs, Switzerland). bacitracin, lincomycin, spiramycin anddimethylamiloride (Sigma. St Louis, USA). nitrovin, picric acid and dinitrophenol (LachemaBmo, Czech Republic). Aureomycin was dissolved in methylcellosolve, avoparcin.lincomycin and iodoacetate in water, fmazolidone, nitrovin, olaquindox, picric acid and dinitrophenol in dimethylsulphoxide. Other antimicrobials were dissolved in ethanol. Solutions were sterilized by filtration through autoclaved asbestos filtering films and added to sterile media containing glucose or xylose to obtain concentrations from 0.01 to 50 !J.g!ml. A control contained an equivalent amount of a solvent Inoculated cultures were incubated in triplicate at 39°C for

Butyrivibrio fibrisolvens is a butyrate-forming anaerobic bacterium, which stains gram-negatively.but which has a thin gram-positive cell wall (H old e m an et al. 1984).Individual strains that have been isolated from the rumen or various anaerobic habitats can ferment cellulose.xylan, starch, pectin and other plant cell components.In the rumen.B.fibrisolvens belongs to the most common bacteria in animals fed widely different diets (Deh ori t y and Gru b 1977; Van G y Is w Y k and M u rp h y 1993).The purpose of this study was (i) to examine the susceptibility of B.fibrisolvens to antimicrobial compounds, and (ii) to compare glucose-and xylose-grown cultures in this respect.In separate experiments the panuneters of growth of butyrivibrios on glucose and xylose were measured.Most of antimicrobials tested are common growth promoters or drugs effective in prophylaxis and treatment of various animal diseases.
Previous studies have shown that B. fibrisolvens is senstitive to ionophores (D e n n i s et al. 1981; Nag ar aj a and T a y lor 1987) and antibiotics that interferre with cell wall synthesis (H e s p e 11 et al. 1993).Antibiotics susceptibility data may be helpful in assessing phenotypic diversity of B. fibrisolvens strains.and also for evaluation of consequences of interactions of antimicrobials with a functionally important rumen bacterium.
Materials and Methods B.fibrisolvens. type strain ATCC 19171 was obtained from the American Type Culture Collection.Strain 86 was a gift from the culture collection of the Hannah Research Institute.Ayr.Scotland.Strain Xl was isolated from the rumen fluid of a sheep at this Institute.Strains CBSI and X2D62 were supplied from the National Chemical Research Laboratory.Pretoria.South Africa.Strains were maintained in 20% (v/v) glycerol at -20°C.
Bacteria were grown on a vitamin-mineral medium, supplemented with 0.1 % yeast extract, 0.1 % enzymatic hydrolysate of casein and 10% rumen fluid (Marounek et al. 1993).The medium was prepared anaerobically and reduced by 0.025% cysteine.HCl and 0.025% N~S.9~O.Glucose or xylose were added at4g1l (final conc.).The medium was sterilized by autoclaving at 110°C.Parameters of growth on glucose and xylose were followed in batch cultures at pH 6.5 and 39°C in 02-free CO 2 atmosphere.Two LF2 fermentors (CSAV Workshop, Prague) were used.The growth was monitored turbidimetrically as optical density at 640 nm.Incubation was completed after reaching the stationary phase.The cell dry weight was determined after centrifugation of the culture.washing with rinsing solutions and drying at 105°C overnight.

Results
The average time of growth, i. e. the time interval between the inoculation and substrate depletion varied between 5.2-14.5 h and 8.5-15.7 h in glucose-and xylose-grown cultures of B.fibrisolvens strains, resp.(Table 1).The average production of cell dry matter in these strains ranged from 920 to 1140 mgll and from 724 to 854 mgll in glucose-and xylose--grown cultures, resp.Table 1 presents averages of three incubations.The susceptibility of  B. fibrisolvens to antimicrobial compounds is shown in Table 2.The strains studied were very sensitive to ionophores, spiramycin and tylosin.On the other hand, they were more or less resistant to furazolidone, chlorpromazin, dicyclohexylcarbodiimide, dinitrophenol, pentachlorophenol, picric acid and dimetylamiloride.Table 2 presents both MICs found in glucose-grown cultures and MICs found in xylose-grown cultures.

Discussion
Several authors examined the susceptibility of B. fibrisolvens to ionophores (0 e n n i s et al. 1981;Nagaraja and Taylor 1987), non-ionophore antimicrobial feed additives (EI Akkad and Hobson 1966;Wang et al. 1969;Stewart et al. 1983; Nagaraj a and T a y lor 1987) and some uncouplers and inhibitors (0 a w son et al. 1979; Yo k 0 ya m a et al. 1988).In this study the minimum inhibitory concentrations of ionophores (lasalocid, maduramicin, monensin, narasin, salinomycin), inhibitors of protein synthesis (aureomycin, lincomycin, spiramycin, tylosin, virginiamycin), cell wall synthesis (avoparcin, bacitracin), carbohydrate metabolism (furazolidone, iodoacetate, nitrovin) and uncouplers (dicyclohexylcarbodiimide, dinitrophenol, picric acid) were determined.Chlorpromazin is an electron transport inhibitor.Pentachlorophenol, which is a widely used biocide, functions as uncoupler and protonophore.Dimethylamiloride is a selective inhibitor of Na+/H+ antiport.All strains tested were sensitive to ionophores and inhibitors of protein synthesis, except aureomycin.On the contrary, they were relatively insensitive to inhibitors of carbohydrate metabolism and uncouplers.Failure of uncouplers to inhibit growth ofbutyrivibrios indicates the unimportance of electron transport phosphorylation or the impermeability of the cell wall to these compounds.There was a considerable strain-to-strain variation, esp.as far as the susceptibility to salinomycin, aureomycin and bacitracin is concerned.
It is worth noting that MICs found in cultures grown on xylose were often lower than in cultures grown on glucose (in about one third of measurements).These differences probably reflect lower energy gain (per unit of time) in growth on xylose, which was metabolized more slowly than glucose in all strains examined.In the author's opinion, bacterial cells damaged by antimicrobial compounds are able to equilibrate 'energy-consuming and energy-producing processes to some extent, provided that the energy supply is sufficient.The differences between MICs on glucose and xylose were most frequent in the ionophore group of antimicrobials, which comprises of compounds influencing directly the energy balance of cells.