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In Vitro Studies of the Effect of Aureomycin and Terramycin on Mixed Suspensions of Bovine Rumen Bacteria1
I N V I T R O S T U D I E S OF T H E
EFFECT
T E R R A M Y C I N ON M I X E D
OF A U R E O M Y C I N "AND
SUSPENSIONS
OF
BOVINE RUMEN BACTERIA 1 lP. J U R T S H U K , JR., R. N. DOETSCH, J. J. M c N E I L L , A~-D J. C. SHAW
Depart~nents of Bacteriology a~td Dairy Httsbaudry, University of Maryland, College JParl:
There are numerous reports in the literature concerning the use of antibiotics for supplementing diets to produce a favorable growth response in ruminants (1~, 22, 25). I n the majority of eases this has been observed when aureomycin was fed to calves (2, 10, 16, 19, 20, 23, 24), and there are a few reports which indicate that terramycin also may act as a growth stimulant (12, 13, 17, 26). In addition, observations made by nmnerous investigators indicate that the stimulating effect of aureomycin in rmninants appears to be greatest during the early part of the growth period (1, 2, 7, 10, 19, 22, 23). This suggests that as the animal matures the effect of aurcolnycill as a growth stimulant decreases; therefore the age of the calf is very significant with regard to its response. I t is suggested that as the rumen develops, some factor or factors are produced which bind the antibiotic in some way and thus inhibit its activity. If present, it was hoped they would be enzyme-like in nature and therefore susceptilfle to detection by conventional techniques. Another study was mldertaken to determine what effect aureomycin and terramycin had on the ability of mixed suspensions of tureen bacteria to utilize various carbohydrates. It was previously reported that such mixed suspensions could utilize xylose, arabinose, glucose, nlaltose, and cellobiose (4). Cultural studies also were conducted to determine the effect of aureomycin and terramycin on actively growing rumen bacteria. Both mixed populations and pure cnltures were exalnined. EXPERIMENTAL PROCEDURE
Effect of tureen bacteria o~l tile pote~wg of a~treomgcS~ atld tcrramyci,. R,umen samples were obtained from a 6-year-old Jersey cow fitted with a rumen fistula. Samples were taken approximately 5 hours after the animal was fed alfalfa hay and a grain mixture containing 16c~ protein. Methodology employed in processing and standardizing the mixed suspension of bovine rmnen bacteria for these experiments has been described previously by Doetseh et al. (4). The bacterial suspension was divided into two equal (12-ml.) portions in 125-ml. Erlemneyer flasks ; the cells in one flask were heated to 80 ° C. for 15 minutes and the cells in the other flask were not heated. One ml. of an aureomycin solution (67.3 nag. aureomycin hydrochloride in 5.0 ml. of M/15 phosphate buffer, p I I 6.9 ~+ 0.1) was then added to 6.0 ml. of both the heated and nonheated cell susReceived for publication July 8, 1954. i Paper No. A-473, contribution No. 2561 of the Maryland Agricultural Experiment Station. 1466
EFFECT
OF A N T I B I O T I C S
ON R U M E N
BACTERIA
1467
pensions in 50-ml. E r l e n m e y e r flasks. H e a t e d a n d n o n h e a t e d cells a n d 1.0 mI. of buffer in place of the antibiotic solution served as controls. The positive control contained 6.0ml. buffer a n d 1.0 ml. of a u r e o m y c i n solution. 3'he flasks were i n c u b a t e d at 39 ° C. u n d e r anaerobic conditions. Anaerobiosis was obtained b y flushing the flasks with n i t r o g e n gas and sealing. A f t e r 14 hours incubation, the cells were c e n t r i f u g e d out (5,400 × G) and the potency of the antibiotic in the s u p e r n a t a n t s in each flask was detcrnfined. This was done by means of sensit i v i t y tests made with sterile filter p a p e r discs and an aureomycin-sensitive s t r a i n of Micrococcus pyogenes vat. a~re~s. These tests were made on n u t r i e n t a g a r plates (B.B.L.) with various dilutions of the s u p e r n a t a n t . The concentration of antibiotic assayed r a n g e d from 960 T p e r m i l l i l i t e r to 1207 p e r m i l l i l i t e r (Table 1). A similar p r o c e d u r e was used to determine tile effect of r u m e n fluid TABLE 1 Zones of iM~ibitio~ prodaced by resid~al a~trcomyci~ after i~wubation with mi~'ed suspects.iotas of bo.vi~te r~men bacteria ~ Aureomycill concentrstion
Nonheated cells + aureomycin
IIeated cells + aureomycin
Buffer + :mreomyein
(7/~,~l) 960 480 320 240 160 120
(ram.) 23 22 21 20 18 16
(ram.) 24 23 20 21 17 J6
(ram.) 33 27 28 26 26 27
" Ineubat,ion period with antibiotic was ] 4 hours at 39 ° (~. p e r se on the a c t i v i t y of aureomycin. A runlen sanlple was s t r a i n e d t h r o u g h cheesecloth a n d 4.0 nil. was i n c u b a t e d with 4.0 nil. of an a u r e o m y c i n solution (3,00'07 per m i l l i l i t e r ) . Necessary controls were included a n d the flasks were i n c u b a t e d a n a e r o b i c a l l y for 3 hours. A f t e r incubation the t u r e e n liquor was c e n t r i f u g e d (5,400 × G ) , and the s u p e r n a t a n t was assayed for antibiotic activity. A n o t h e r e x p e r i m e n t was conducted with t e r r a m y c i n . A slightly modified p r o c e d u r e was used. A solution of terram3\cin was p r e p a r e d (1,000y p e r millil i t e r ) , and 8.0ml. was a d d e d to flasks eontainino~" 8.0ml. of s t a n d a r d i z e d suspension of heated a n d nonbeated r u m e n cells. Coutrols were r u n which contained 8.0 ml. of cell suspension with 8.0 nil. of buffer, and 8.0 ml. of buffer with 8.0 ml. of antibiotic. A f t e r 24 hours i n c u b a t i o n tile cells were c e n t r i f u g e d out (5,400 × G), a n d sensitivity tests were r u n as p r e v i o u s l y described (Table 2). I n each of the above experiments sterile filter p a p e r discs were soaked in the s u p e r n a t a n t s , those c o n t a i n i n g the antibiotic as well as the controls, and then placed on a g a r plates seeded with tile test organism. Effect of aureom.tlcb~, a~d t c r r a m g c i n oJt carbohydrate ~tilizatiou by r~lme~ bacteria. The e x p e r i m e n t a l p r o c e d u r e used for this s t u d y was a modification of the one used by Doetsch et al. (~1). The p r o c e d u r e differs in t h a t a u r e o m y c i n a n d t e r r m u y c i n were a d d e d to flasks containing :3.0 nil. of s t a n d a r d i z e d r u m e n cell
P. JURTSHUK, JR. ET AL
1468
TABLE 2
Zones of inhibition prod~wed by residnal terramycin after incubation with mixed snspensions of bovine tureen bacteria ~ Antibiotic concentration
(~/m~)
Reactants
Buffer + terramycin N o n h e a t e d cells + t e r r a m y c i n H e a t e d cells + t e r r a m y c i n
175
250
(ram.)
(n,~n.)
15 16 16
18 19 19
500
(~.) 20 22 21
I n c u b a t i o n p e r i o d w i t h a n t i b i o t i c w a s 24 hours a t 39 ° C.
suspension and 4.0 ml. of carbohydrate substrate. The carbohydrates (200~M per flask) used were xylose, arabinose, glucose, maltose, and eellobiose. The concentrations of aureomycin and t e r r a m y c i n used were 100 and 2007 per milliliter per flask, and only 1.0 nil. was added to the flasks receiving the antibiotic. I u these macro-fermentation tests, controls consisted of cells plus substrate without antibiotic and cells with antibiotic but without substrate. Buffer was added to each flask to make the final volume 11.0 nil. At various intervals the contents of the flasks were removed and tests were conducted for the presence of carbohydrates with Benedict's reagent for reducing sugars.
Cultural studies. Aureomycin and t e r r a m y c i n were added to H u n g a t e ' s medium (9) prior to inoculation with dilutions of a r m n e n sample so t h a t the final concentration of antibiotic in the medium was 25,/ per milliliter. P r e p a r a tion of medium (No. 1) and dilution blanks, in addition to the processing procedure, has been described previously (6). A f t e r inoculation of both media containing antibiotic and controls without the antibiotic, the tubes were incubated at 39 ° C. for 48 hours and colony counts were made (Table 3). I n addition, penicillin G (crystalline potassimn salt), aureomycin (hydroehloride), terramycin (hydroehloride), d i h y d r o s t r e p t o m y c i n sulfate, and chloromyeetin (Chloramphenicol) were incorporated into modified E u g o n medium (6), omitting a g a r and sulfide, in concentrations (micrograms per milliliter), respectively, of 1, 1, 2, 20, and 1. Qualitative estimations for growth were made a f t e r 24, 48, and 96 hours incubation at 39 ° C. on 16 p u r e cultures isolated from the bovine tureen. RESULTS
Results of experiments on the effect of mixed suspensions of bovine r u m e n bacteria on aureomycin are presented in Table 1. Upon comparing zones of inhibition, no significant difference is observed between the heated and nonheated bacterial cells. However, a difference in zone diameter is observed between the aureomycin incubated with buffer and the aureomycin incubated with both the heated and nonheated bacterial suspensions. This indicates some nonspecific type of inactivation, most likely due to tb~ protein nature of the cells. Inhibition of the test organism was not observed in the control flasks in the absence of antibiotic.
EFFECT
OF ANTIBIOTICS
ON RUMEN
BACTERIA
1469
S i m i l a r r e s u l t s were o b t a i n e d w h e n t u r e e n fluid was i n c u b a t e d w i t h aure,)m y c i n . The c o n c e n t r a t i o n of a u r e o m y c i n r a n g e d f r o m 200~ p e r m i l l i l i t e r to 1,5007 p e r m i l l i l i t e r , a n d a nonspecific t y p e of i n a c t i v a t i o n also was observed. A u r e o m y c i u w h e n i n c u b a t e d w i t h p h o s p h a t e b u f f e r r e t a i n e d m o r e of its a c t i v i t y , a f t e r i n c u b a t i o n f o r 3 hours, t h a n d i d the a u r e o m y c i n i n c u b a t e d w i t h t h e h e a t e d a n d n o n h e a t e d r u m e n fluid. The r e s u l t s of the s e n s i t i v i t y tests in w h i c h t e r r a n i y c i n was u s e d a r e p r e s e n t e d i n ] ' a b l e 2. The r e s u l t s i n d i c a t e t h a t t h e r e was no s i g n i f i c a n t or c o n s i s t e n t difference b e t w e e n h e a t e d a n d n o n h e a t e d cells w i t h r e g a r d to i n a c t i v a t i o n of terramycin. M i x e d s u s p e n s i o n s of r u m e n b a c t e r i a in t h e " r e s t i n g s t a t e , " w h e n i n c u b a t e d w i t h a u r e o m y c i n or t e r r a m y c i n (100 or 2007 p e r n l i l l i l i t e r p e r flask) d i d n o t u t i l i z e xylose, a r a b i n o s e , glucose, maltose, a n d eellobiose as r e a d i l y as d i d the controls. TABLE 3 The effect of a~trco.myeb~ a~d terramycin on activel9 growi~g r ~ e n
Hungate's medium cont.~ining Terramycin (257/m1") Aureomycin (25y/ml) No antibiotic (control)
bacteria
× 10~
Colony count × 1O;
× 10s
0,0,0,0" 0,0,0,0,0 130, 175,b 95, 75
0,0,0,0 0,0,0,0 40, 24, 35, 120
0,0,0,0,0 0,0,0,0,0 3, 13, 6, 2, 2, 2, 0
No growth. b Nmnbers represent actual number of colonies counted. W i t h r e g a r d to t h e c u l t u r a l studies, t h e effects of a u r e o m y c i n a n d t e r r a m y c i n oil a c t i v e l y g r o w i n g r u n i e n b a c t e r i a a r e s u m m a r i z e d i n T a b l e 3. I t was o b s e r v e d t h a t w h e n a n t i b i o t i c s w e r e a d d e d to I I u n g a t e ' s m e d i u m (final c o n c e n t r a t i o n of a n t i b i o t i c 257 p e r n I i l l i l i t e r ) the g r o w t h of t u r e e n b a c t e r i a was c o m p l e t e l y i n h i b i t e d . F r o m t h i s one can c o n c l u d e t h a t a u r e o m y c i n a n d t e r r a m y c i n a r e d e f i n i t e l y d e t r i m e n t a l to in v i t r o g r o w t h of r m n e n b a c t e r i a . S i x t e e n p u r e c u l t u r e s were selected as r e p r e s e n t a t i v e f r o m a m u c h l a r g e r collection on the basis of m o r p h o l o g y a n d g r o w t h r a t e s . The r e s u l t s of a n t i b i o t i c s on the g r o w t h of these c u l t u r e s a r e s h o w n i n T a b l e 4. P e n i c i l l i n a n d t e r r a m y c i n w e r e v e r y active, w h e r e a s the t h r e e o t h e r a n t i b i o t i c s showed s l i g h t a c t i v i t y . I a no case was s t i n i u l a t i o n of g r o w t h o b s e r v e d w i t h a n y of t h e a n t i b i o t i c s . TABLE 4 Effect of vario~ls a~*tibiotics oJt growth of pt~re mdttlres of tureen bacteria
Number of cultures Antibiotic
Concentration
Complete inhibition
Partial inhibition
Unaffected
15 0 13 0 0
0 4 2 3 4
1 12 1 13 12
(y/toO Penicillin Aureomycin Terramycin Streptomycin Chloromyeetin
] 1 2 20 1
1470
P. J U R T S H U K , Jl~. ET AL
DISC U S S I O N
The purpose of the foregoing experiments was to determine whether some explanation eould be found for the failure of antibiotic feeding to remain effective after the development of the r u m e n and its eoncomitant bacterial flora. F r o m tile results obtained il~ these relatively simple experiments little evidence call be offered that implicates enzymatic inactivation of aureomycin and t e r r a m y c i n either by mixed suspensions of bovine r u m e n bacteria or by r m n e n liquor. Rather high levels of antibiotic were employed in these experiments to facilitate assay and because r a p i d decrease in potency occurs in the presence of proteinaeeous material at 39 ° C. (21, 27). This fact m a y p a r t l y explain the nonspecifie inactivation observed with both rumen bacterial cells and r u m e n liquor. I n addition, aureomycin inactivation m a y occur in the presence of riboflavin (8), and this also m a y be a contributory factor since this vitamin is •present in rumen liquor (18). I n vitro studies have shown that aureomycin is detrimental to certain r u m e n bacteria when initially administered to normal calves (J5). Constant supplementation of the ration probably involves some adaptation of r u m e n bacteria to antibiotics as the previously cited s t u d y revealed. I f data obtained by these in vitro experiments can be t r a n s f e r r e d to the intact animal it would seem that aureomycin a n d terramycin, at the levels employed in this work, interfere with the r a p i d utilization of carbohydrates most likely to be found in the plant materials of the feed. I t is interesting to note that 257 per milliliter of aureomycin or t e r r a m y c i n added to a r m n e u sample has little effect on the total count obtained (tl). This is iI~terpretert to mean t h a t the action of these antibiotics is bacteriostatie and not bactericidal. On the other hand, 257 per milliliter of aureomycin or t e r r a m y c i n added directly to the plating medium has a p r o f o u n d effect on the total count, and if it is assumed t h a t a wide v a r i e t y of authentic r u m e n bacteria can be cultured on this medium, it is obvious t h a t the effect of these antibiotics on growth is general. The results with p u r e cultures bear out this contention with regard to terramycin. The effect of antibiotic feeding on certain "marker" organisms in the r m n e n and intestinal tract of animals has been touched upon in a few investigations (3, 24). There is still need for more detailed studies in this direction, especially with organisms known to be involved in the physiological reactions of the r u m e n (Veillondla, Propionibacterium, Lactobacilhts, Bacteriodes. etc.) and more particularly on the effect of antibiotics on the over-all reactions brought about by the mixed bacterial flora. Such data could be obtained b y using methods previously described b y this group (4), and studies might well include the effect of antibiotics on aspartate, suecinate, and formate dissimilations, as well as polysaccharide synthesis and lactic acid formation from carbohydrates (5). As this group has repeatedly stressed, it seems that in this direction the most f r u i t f u l results will be obtained for the efforts expended.
E F F E C T OF A N T I B I O T I C S ON RUSIEN B.kCTEI%IX
1471
SUMMARY
No s i g n i f i c a n t or c o n s i s t e n t d i f f e r e n c e was o b s e r v e d b e t w e e n h e a t e d a n d n o n h e a t e d s u s p e n s i o n of b o v i n e r m n e n b a c t e r i a in t h e i r a b i l i t y to i n a c t i v a t e a u r e o m y c i n a n d t e r r a m y c i n . T h e p r e s e n c e of a specific h e a t - l a b i l e i n h i b i t o r such as an " a u r e o m y e i n a s e " could n o t be d e m o n s t r a t e d in e i t h e r the m i x e d suspensions of r u m e n cells or in t h e t u r e e n fluid itself. A nonspecific t y p e of i n a c t i v a t i o n was o b s e r v e d in some i n s t a n c e s w i t h a u r e o m y c i n , a n d this was m o s t p r o b a b l y due to t h e p r o t e i n n a t u r e of t h e m i x e d s u s p e n s i o n s of r u m e n cells. Th e p r e s e n c e of both a u r e o m y c i n a n d t e r r a m y c i n (100 v p e r m i l l i l i t e r ) defin i t e l y d e c r e a s e d the a b i l i t y of r u m e n b a c t e r i a in the " r e s t i n g s t a t e " to u t i l i ze t h e c a r b o h y d r a t e s xylose, arabinose, glucose, maltose, a n d eellobiose i n v i t r o . A u r e o n 4 y e i n a n d t e r r a m y c i n w h e n a d d e d to a p l a t i n g m e d i m n (final concent r a t i o n 257 p e r m i l l i l i t e r ) c o m p l e t e l y i n h i b i t e d the g r o w t h of b o v i n e r u m e n b a c t e r i a , w h e r e a s w h e n this c o n c e n t r a t i o n was a d d e d to r u m e n fluid, t h e n p l a t e d , it d i d not. S u g g e s t i o n s f o r the d i r e c t i o n in w h i c h f u r t h e r
r e s e a r c h on t h i s p r o b l e m
s h o u l d p r o c e e d are p r e s e n t e d . REFERENCES (1) BAI%TLEY,E. E., EOUNTAINE,F. C., AND ATKINSON, W. F. Effect on Growth of Feeding Aureomycin to Dairy Calves from Birth to Thirteen Months of Age. (Abs.) J. Dairy Sci., 36: 604. 1953. (2) BLOO~, S., AND KNOi)T, C. B. The Value of Aureomycin and Vitamin B~2 in Milk Replacement Formulas for Dairy Calves. J. Dairy Sei., 35: 910. 1952. (3) CHANCE,C. M., S~ITH, C. K., HUrF~AN, C. F., AN1) DUNCAN,C. W. Antibiotics in Rumen Digestion and Synthesis. III. The Effect of Aureomycin on Rumen Microorganisms, with Special Reference to the Streptococci and Coliform Groups. J. Dairy Sci., 36: 743. 1953. (4) DOETSCH, !R. N., ROBINSON, R. Q., BRO~VN,R. E., AND SHA~V, J. C. Catabolic Reactions of Mixed Suspensions of Bovine Rumen Bacteria. J. Dairy Sci., 36: 825. 1953. (5) DOETSCH, R. N., ROBINSON, R. Q., McNEILL, ,I. ,]-., JURTSHUK, P., BRO~N, R. E., AND SHAW, J. C. Physiological Studies on Mixed Suspensions of Rumen Bacteria. i~ld. Agr. Expt. Sta., Misc. P~tbl. 205. 1954. (6) DOBTSCH, R. N., ROBINSON, R. Q., AND SHA~V, J. C. Techniques Employed in Cultural Investigations of the Bacteriology of the Bovine Rumen. J. Animal Sci., 11: 536. 1952. (7) FINCHAM, R. C., ~iND VOELKER, H. H. The Long-Time Effects of Aureomycin Feeding to Dairy Heifers. (Abs.) J. Dairy Sci., 35: 594. 1953. (8) FOSTEI%,J. W., AND PITTILL0, R. F. Metabolite Reversal of Antibiotic Inhibition, Especially Reversal of Aureomycin Ilflfibition by Riboflavin. J. Baeteriol., 66: 478. 1953. (9) HUNGATE, R. E. The Anaerobic Mesophilie Cellulolytie Bacteria. Bacteriol. Boys., 14: 1. 1950. (10) JACOBSON,N. L., KAlVFENTZAKIS,J. G., AND MURLEY, W. R. Response of " R u m i n a t i n g " Dairy Calves to Aureomycin Feeding. J. An~mai Sci., 10: 1950. 1951. (11) JUKTSHUK,P., KELLEY, N., AND DOETSCH, R. N. Unpublished Results. 1954. (12) KESLE~, E. M. Effect of Terramycin Fed to Holstein Calves on Growth, I n Vitro Cellulose Digestion and B-Vitamin Synthesis. J. Ani~t~al Sci., 13: 10. 1954. (13) KESLEI{, E. M., AND KNODT, C. B. Effect of Terramycin on Growth and Rumen Function in Holstein Calves. (Abs.) J. Animal Sci., 11: 768. 1952.
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(14) KNODT, C. B. Antibiotics in the Growth of Ruminant Animals. ~ntibiotics and Chettwtherapy, 3: 442. 1953. (15) LODGE, J. R., MILES, J. T., AND JACOBSON, N. L. The Effect of Aureomycin Feeding to Dairy Cattle on the I n Vitro Digestion of Cellulose by Rumen Microorganisms. (Abs.) J. Dairy Sci., 37: 663. 1954. (16) MAcKaY, A. M., RIDDELL, W. H., AND FITZSI~I}Z0NS, R. Feed Supplement Containing Aureomycin and Vitamin B~_ofor Dairy Calves. J. Animal Sci., 11: 341. 1952. (17) MACKAY, A. M., RIDDELL, W. I~., AND FITZSII~II~IONS, R. Terramycin Supplement for Dairy Calves. J. A~tb~al Sci., 11: 19. 1953. (18) MCELROY, L. W., AND GROSS, H. Quantitative Study of Vitamins in Rumen Contents of Sheep and Cows Fed Vitamin Low Diets. 1. Riboflavin and Vitamin K . J. N~ltri~ tion, 20: 527. 1940. (19) MUELEY, W. R., JACOBSON, N. L., A~'D ALLEN, l~. S. Effect of Aureomycin Supplementation on Growth and Feed Utilization of Young Dairy Calves. J. Dairy Sci., 35: 846. 1952. (20) MURLEY, W. R., JACOBSON, N. L., WING, J. M., AND STODDAI~D, G. E. The Response of Aureomycin Supplementation of Young Dairy Calves Fed Various ~ ' P r a c t i c a l ' ' and Restricted Diets. (Abs.) J. Dairy Sci., 34: 500. 1951. (21) PAINE, T. F., JR., COLLINS, H. S., AND FINLAND, M. Bacteriologic Studies on Aureomycin. J. BaeterioL, 55: 489. 1948. (22) }~En), J. T., WARNER, R. G., AND LOOSLI, J. K. Antibiotics in the Nutrition of Ruminants. Agr. Food Chem., 2: 186. 1954. (23) RUSO~F, L. L. A.P.F. Supplements for Calves. (Abs.) J. At~i~aal Sci., 9: 666. 1950. (24) RUS0FF, L. L., ALFOI~D, J. H., AND HYDE, C. E. Effect of Type of Protein on the Response of Young Dairy Calves to Aureomycin with Data on the Intestinal Microflora. J. Dairy Sci., 36: 45. 1953. (25) S~OKS~AI), E. L. R. Antibiotics in Animal Nutrition. PhysioT. Revs., 34: 25. 1954. (26) VOELKEa, It. It., AND C.XSON, J. L. Antibiotic Studies with Dairy Calves. (Abs.) J. Anin~al Sc~., 10: 1056. 1951. (27) WAISBREN,B. A., CARa, C., AND DUNNETTE, 5. The Tube Dilution Method of Determining Bacterial Sensitivity to Antibiotics. ~4~a. J. Clin. PathoL, 21" 884. 1951.
EFFECT
T E R R A M Y C I N ON M I X E D
OF A U R E O M Y C I N "AND
SUSPENSIONS
OF
BOVINE RUMEN BACTERIA 1 lP. J U R T S H U K , JR., R. N. DOETSCH, J. J. M c N E I L L , A~-D J. C. SHAW
Depart~nents of Bacteriology a~td Dairy Httsbaudry, University of Maryland, College JParl:
There are numerous reports in the literature concerning the use of antibiotics for supplementing diets to produce a favorable growth response in ruminants (1~, 22, 25). I n the majority of eases this has been observed when aureomycin was fed to calves (2, 10, 16, 19, 20, 23, 24), and there are a few reports which indicate that terramycin also may act as a growth stimulant (12, 13, 17, 26). In addition, observations made by nmnerous investigators indicate that the stimulating effect of aureomycin in rmninants appears to be greatest during the early part of the growth period (1, 2, 7, 10, 19, 22, 23). This suggests that as the animal matures the effect of aurcolnycill as a growth stimulant decreases; therefore the age of the calf is very significant with regard to its response. I t is suggested that as the rumen develops, some factor or factors are produced which bind the antibiotic in some way and thus inhibit its activity. If present, it was hoped they would be enzyme-like in nature and therefore susceptilfle to detection by conventional techniques. Another study was mldertaken to determine what effect aureomycin and terramycin had on the ability of mixed suspensions of tureen bacteria to utilize various carbohydrates. It was previously reported that such mixed suspensions could utilize xylose, arabinose, glucose, nlaltose, and cellobiose (4). Cultural studies also were conducted to determine the effect of aureomycin and terramycin on actively growing rumen bacteria. Both mixed populations and pure cnltures were exalnined. EXPERIMENTAL PROCEDURE
Effect of tureen bacteria o~l tile pote~wg of a~treomgcS~ atld tcrramyci,. R,umen samples were obtained from a 6-year-old Jersey cow fitted with a rumen fistula. Samples were taken approximately 5 hours after the animal was fed alfalfa hay and a grain mixture containing 16c~ protein. Methodology employed in processing and standardizing the mixed suspension of bovine rmnen bacteria for these experiments has been described previously by Doetseh et al. (4). The bacterial suspension was divided into two equal (12-ml.) portions in 125-ml. Erlemneyer flasks ; the cells in one flask were heated to 80 ° C. for 15 minutes and the cells in the other flask were not heated. One ml. of an aureomycin solution (67.3 nag. aureomycin hydrochloride in 5.0 ml. of M/15 phosphate buffer, p I I 6.9 ~+ 0.1) was then added to 6.0 ml. of both the heated and nonheated cell susReceived for publication July 8, 1954. i Paper No. A-473, contribution No. 2561 of the Maryland Agricultural Experiment Station. 1466
EFFECT
OF A N T I B I O T I C S
ON R U M E N
BACTERIA
1467
pensions in 50-ml. E r l e n m e y e r flasks. H e a t e d a n d n o n h e a t e d cells a n d 1.0 mI. of buffer in place of the antibiotic solution served as controls. The positive control contained 6.0ml. buffer a n d 1.0 ml. of a u r e o m y c i n solution. 3'he flasks were i n c u b a t e d at 39 ° C. u n d e r anaerobic conditions. Anaerobiosis was obtained b y flushing the flasks with n i t r o g e n gas and sealing. A f t e r 14 hours incubation, the cells were c e n t r i f u g e d out (5,400 × G) and the potency of the antibiotic in the s u p e r n a t a n t s in each flask was detcrnfined. This was done by means of sensit i v i t y tests made with sterile filter p a p e r discs and an aureomycin-sensitive s t r a i n of Micrococcus pyogenes vat. a~re~s. These tests were made on n u t r i e n t a g a r plates (B.B.L.) with various dilutions of the s u p e r n a t a n t . The concentration of antibiotic assayed r a n g e d from 960 T p e r m i l l i l i t e r to 1207 p e r m i l l i l i t e r (Table 1). A similar p r o c e d u r e was used to determine tile effect of r u m e n fluid TABLE 1 Zones of iM~ibitio~ prodaced by resid~al a~trcomyci~ after i~wubation with mi~'ed suspects.iotas of bo.vi~te r~men bacteria ~ Aureomycill concentrstion
Nonheated cells + aureomycin
IIeated cells + aureomycin
Buffer + :mreomyein
(7/~,~l) 960 480 320 240 160 120
(ram.) 23 22 21 20 18 16
(ram.) 24 23 20 21 17 J6
(ram.) 33 27 28 26 26 27
" Ineubat,ion period with antibiotic was ] 4 hours at 39 ° (~. p e r se on the a c t i v i t y of aureomycin. A runlen sanlple was s t r a i n e d t h r o u g h cheesecloth a n d 4.0 nil. was i n c u b a t e d with 4.0 nil. of an a u r e o m y c i n solution (3,00'07 per m i l l i l i t e r ) . Necessary controls were included a n d the flasks were i n c u b a t e d a n a e r o b i c a l l y for 3 hours. A f t e r incubation the t u r e e n liquor was c e n t r i f u g e d (5,400 × G ) , and the s u p e r n a t a n t was assayed for antibiotic activity. A n o t h e r e x p e r i m e n t was conducted with t e r r a m y c i n . A slightly modified p r o c e d u r e was used. A solution of terram3\cin was p r e p a r e d (1,000y p e r millil i t e r ) , and 8.0ml. was a d d e d to flasks eontainino~" 8.0ml. of s t a n d a r d i z e d suspension of heated a n d nonbeated r u m e n cells. Coutrols were r u n which contained 8.0 ml. of cell suspension with 8.0 nil. of buffer, and 8.0 ml. of buffer with 8.0 ml. of antibiotic. A f t e r 24 hours i n c u b a t i o n tile cells were c e n t r i f u g e d out (5,400 × G), a n d sensitivity tests were r u n as p r e v i o u s l y described (Table 2). I n each of the above experiments sterile filter p a p e r discs were soaked in the s u p e r n a t a n t s , those c o n t a i n i n g the antibiotic as well as the controls, and then placed on a g a r plates seeded with tile test organism. Effect of aureom.tlcb~, a~d t c r r a m g c i n oJt carbohydrate ~tilizatiou by r~lme~ bacteria. The e x p e r i m e n t a l p r o c e d u r e used for this s t u d y was a modification of the one used by Doetsch et al. (~1). The p r o c e d u r e differs in t h a t a u r e o m y c i n a n d t e r r m u y c i n were a d d e d to flasks containing :3.0 nil. of s t a n d a r d i z e d r u m e n cell
P. JURTSHUK, JR. ET AL
1468
TABLE 2
Zones of inhibition prod~wed by residnal terramycin after incubation with mixed snspensions of bovine tureen bacteria ~ Antibiotic concentration
(~/m~)
Reactants
Buffer + terramycin N o n h e a t e d cells + t e r r a m y c i n H e a t e d cells + t e r r a m y c i n
175
250
(ram.)
(n,~n.)
15 16 16
18 19 19
500
(~.) 20 22 21
I n c u b a t i o n p e r i o d w i t h a n t i b i o t i c w a s 24 hours a t 39 ° C.
suspension and 4.0 ml. of carbohydrate substrate. The carbohydrates (200~M per flask) used were xylose, arabinose, glucose, maltose, and eellobiose. The concentrations of aureomycin and t e r r a m y c i n used were 100 and 2007 per milliliter per flask, and only 1.0 nil. was added to the flasks receiving the antibiotic. I u these macro-fermentation tests, controls consisted of cells plus substrate without antibiotic and cells with antibiotic but without substrate. Buffer was added to each flask to make the final volume 11.0 nil. At various intervals the contents of the flasks were removed and tests were conducted for the presence of carbohydrates with Benedict's reagent for reducing sugars.
Cultural studies. Aureomycin and t e r r a m y c i n were added to H u n g a t e ' s medium (9) prior to inoculation with dilutions of a r m n e n sample so t h a t the final concentration of antibiotic in the medium was 25,/ per milliliter. P r e p a r a tion of medium (No. 1) and dilution blanks, in addition to the processing procedure, has been described previously (6). A f t e r inoculation of both media containing antibiotic and controls without the antibiotic, the tubes were incubated at 39 ° C. for 48 hours and colony counts were made (Table 3). I n addition, penicillin G (crystalline potassimn salt), aureomycin (hydroehloride), terramycin (hydroehloride), d i h y d r o s t r e p t o m y c i n sulfate, and chloromyeetin (Chloramphenicol) were incorporated into modified E u g o n medium (6), omitting a g a r and sulfide, in concentrations (micrograms per milliliter), respectively, of 1, 1, 2, 20, and 1. Qualitative estimations for growth were made a f t e r 24, 48, and 96 hours incubation at 39 ° C. on 16 p u r e cultures isolated from the bovine tureen. RESULTS
Results of experiments on the effect of mixed suspensions of bovine r u m e n bacteria on aureomycin are presented in Table 1. Upon comparing zones of inhibition, no significant difference is observed between the heated and nonheated bacterial cells. However, a difference in zone diameter is observed between the aureomycin incubated with buffer and the aureomycin incubated with both the heated and nonheated bacterial suspensions. This indicates some nonspecific type of inactivation, most likely due to tb~ protein nature of the cells. Inhibition of the test organism was not observed in the control flasks in the absence of antibiotic.
EFFECT
OF ANTIBIOTICS
ON RUMEN
BACTERIA
1469
S i m i l a r r e s u l t s were o b t a i n e d w h e n t u r e e n fluid was i n c u b a t e d w i t h aure,)m y c i n . The c o n c e n t r a t i o n of a u r e o m y c i n r a n g e d f r o m 200~ p e r m i l l i l i t e r to 1,5007 p e r m i l l i l i t e r , a n d a nonspecific t y p e of i n a c t i v a t i o n also was observed. A u r e o m y c i u w h e n i n c u b a t e d w i t h p h o s p h a t e b u f f e r r e t a i n e d m o r e of its a c t i v i t y , a f t e r i n c u b a t i o n f o r 3 hours, t h a n d i d the a u r e o m y c i n i n c u b a t e d w i t h t h e h e a t e d a n d n o n h e a t e d r u m e n fluid. The r e s u l t s of the s e n s i t i v i t y tests in w h i c h t e r r a n i y c i n was u s e d a r e p r e s e n t e d i n ] ' a b l e 2. The r e s u l t s i n d i c a t e t h a t t h e r e was no s i g n i f i c a n t or c o n s i s t e n t difference b e t w e e n h e a t e d a n d n o n h e a t e d cells w i t h r e g a r d to i n a c t i v a t i o n of terramycin. M i x e d s u s p e n s i o n s of r u m e n b a c t e r i a in t h e " r e s t i n g s t a t e , " w h e n i n c u b a t e d w i t h a u r e o m y c i n or t e r r a m y c i n (100 or 2007 p e r n l i l l i l i t e r p e r flask) d i d n o t u t i l i z e xylose, a r a b i n o s e , glucose, maltose, a n d eellobiose as r e a d i l y as d i d the controls. TABLE 3 The effect of a~trco.myeb~ a~d terramycin on activel9 growi~g r ~ e n
Hungate's medium cont.~ining Terramycin (257/m1") Aureomycin (25y/ml) No antibiotic (control)
bacteria
× 10~
Colony count × 1O;
× 10s
0,0,0,0" 0,0,0,0,0 130, 175,b 95, 75
0,0,0,0 0,0,0,0 40, 24, 35, 120
0,0,0,0,0 0,0,0,0,0 3, 13, 6, 2, 2, 2, 0
No growth. b Nmnbers represent actual number of colonies counted. W i t h r e g a r d to t h e c u l t u r a l studies, t h e effects of a u r e o m y c i n a n d t e r r a m y c i n oil a c t i v e l y g r o w i n g r u n i e n b a c t e r i a a r e s u m m a r i z e d i n T a b l e 3. I t was o b s e r v e d t h a t w h e n a n t i b i o t i c s w e r e a d d e d to I I u n g a t e ' s m e d i u m (final c o n c e n t r a t i o n of a n t i b i o t i c 257 p e r n I i l l i l i t e r ) the g r o w t h of t u r e e n b a c t e r i a was c o m p l e t e l y i n h i b i t e d . F r o m t h i s one can c o n c l u d e t h a t a u r e o m y c i n a n d t e r r a m y c i n a r e d e f i n i t e l y d e t r i m e n t a l to in v i t r o g r o w t h of r m n e n b a c t e r i a . S i x t e e n p u r e c u l t u r e s were selected as r e p r e s e n t a t i v e f r o m a m u c h l a r g e r collection on the basis of m o r p h o l o g y a n d g r o w t h r a t e s . The r e s u l t s of a n t i b i o t i c s on the g r o w t h of these c u l t u r e s a r e s h o w n i n T a b l e 4. P e n i c i l l i n a n d t e r r a m y c i n w e r e v e r y active, w h e r e a s the t h r e e o t h e r a n t i b i o t i c s showed s l i g h t a c t i v i t y . I a no case was s t i n i u l a t i o n of g r o w t h o b s e r v e d w i t h a n y of t h e a n t i b i o t i c s . TABLE 4 Effect of vario~ls a~*tibiotics oJt growth of pt~re mdttlres of tureen bacteria
Number of cultures Antibiotic
Concentration
Complete inhibition
Partial inhibition
Unaffected
15 0 13 0 0
0 4 2 3 4
1 12 1 13 12
(y/toO Penicillin Aureomycin Terramycin Streptomycin Chloromyeetin
] 1 2 20 1
1470
P. J U R T S H U K , Jl~. ET AL
DISC U S S I O N
The purpose of the foregoing experiments was to determine whether some explanation eould be found for the failure of antibiotic feeding to remain effective after the development of the r u m e n and its eoncomitant bacterial flora. F r o m tile results obtained il~ these relatively simple experiments little evidence call be offered that implicates enzymatic inactivation of aureomycin and t e r r a m y c i n either by mixed suspensions of bovine r u m e n bacteria or by r m n e n liquor. Rather high levels of antibiotic were employed in these experiments to facilitate assay and because r a p i d decrease in potency occurs in the presence of proteinaeeous material at 39 ° C. (21, 27). This fact m a y p a r t l y explain the nonspecifie inactivation observed with both rumen bacterial cells and r u m e n liquor. I n addition, aureomycin inactivation m a y occur in the presence of riboflavin (8), and this also m a y be a contributory factor since this vitamin is •present in rumen liquor (18). I n vitro studies have shown that aureomycin is detrimental to certain r u m e n bacteria when initially administered to normal calves (J5). Constant supplementation of the ration probably involves some adaptation of r u m e n bacteria to antibiotics as the previously cited s t u d y revealed. I f data obtained by these in vitro experiments can be t r a n s f e r r e d to the intact animal it would seem that aureomycin a n d terramycin, at the levels employed in this work, interfere with the r a p i d utilization of carbohydrates most likely to be found in the plant materials of the feed. I t is interesting to note that 257 per milliliter of aureomycin or t e r r a m y c i n added to a r m n e u sample has little effect on the total count obtained (tl). This is iI~terpretert to mean t h a t the action of these antibiotics is bacteriostatie and not bactericidal. On the other hand, 257 per milliliter of aureomycin or t e r r a m y c i n added directly to the plating medium has a p r o f o u n d effect on the total count, and if it is assumed t h a t a wide v a r i e t y of authentic r u m e n bacteria can be cultured on this medium, it is obvious t h a t the effect of these antibiotics on growth is general. The results with p u r e cultures bear out this contention with regard to terramycin. The effect of antibiotic feeding on certain "marker" organisms in the r m n e n and intestinal tract of animals has been touched upon in a few investigations (3, 24). There is still need for more detailed studies in this direction, especially with organisms known to be involved in the physiological reactions of the r u m e n (Veillondla, Propionibacterium, Lactobacilhts, Bacteriodes. etc.) and more particularly on the effect of antibiotics on the over-all reactions brought about by the mixed bacterial flora. Such data could be obtained b y using methods previously described b y this group (4), and studies might well include the effect of antibiotics on aspartate, suecinate, and formate dissimilations, as well as polysaccharide synthesis and lactic acid formation from carbohydrates (5). As this group has repeatedly stressed, it seems that in this direction the most f r u i t f u l results will be obtained for the efforts expended.
E F F E C T OF A N T I B I O T I C S ON RUSIEN B.kCTEI%IX
1471
SUMMARY
No s i g n i f i c a n t or c o n s i s t e n t d i f f e r e n c e was o b s e r v e d b e t w e e n h e a t e d a n d n o n h e a t e d s u s p e n s i o n of b o v i n e r m n e n b a c t e r i a in t h e i r a b i l i t y to i n a c t i v a t e a u r e o m y c i n a n d t e r r a m y c i n . T h e p r e s e n c e of a specific h e a t - l a b i l e i n h i b i t o r such as an " a u r e o m y e i n a s e " could n o t be d e m o n s t r a t e d in e i t h e r the m i x e d suspensions of r u m e n cells or in t h e t u r e e n fluid itself. A nonspecific t y p e of i n a c t i v a t i o n was o b s e r v e d in some i n s t a n c e s w i t h a u r e o m y c i n , a n d this was m o s t p r o b a b l y due to t h e p r o t e i n n a t u r e of t h e m i x e d s u s p e n s i o n s of r u m e n cells. Th e p r e s e n c e of both a u r e o m y c i n a n d t e r r a m y c i n (100 v p e r m i l l i l i t e r ) defin i t e l y d e c r e a s e d the a b i l i t y of r u m e n b a c t e r i a in the " r e s t i n g s t a t e " to u t i l i ze t h e c a r b o h y d r a t e s xylose, arabinose, glucose, maltose, a n d eellobiose i n v i t r o . A u r e o n 4 y e i n a n d t e r r a m y c i n w h e n a d d e d to a p l a t i n g m e d i m n (final concent r a t i o n 257 p e r m i l l i l i t e r ) c o m p l e t e l y i n h i b i t e d the g r o w t h of b o v i n e r u m e n b a c t e r i a , w h e r e a s w h e n this c o n c e n t r a t i o n was a d d e d to r u m e n fluid, t h e n p l a t e d , it d i d not. S u g g e s t i o n s f o r the d i r e c t i o n in w h i c h f u r t h e r
r e s e a r c h on t h i s p r o b l e m
s h o u l d p r o c e e d are p r e s e n t e d . REFERENCES (1) BAI%TLEY,E. E., EOUNTAINE,F. C., AND ATKINSON, W. F. Effect on Growth of Feeding Aureomycin to Dairy Calves from Birth to Thirteen Months of Age. (Abs.) J. Dairy Sci., 36: 604. 1953. (2) BLOO~, S., AND KNOi)T, C. B. The Value of Aureomycin and Vitamin B~2 in Milk Replacement Formulas for Dairy Calves. J. Dairy Sei., 35: 910. 1952. (3) CHANCE,C. M., S~ITH, C. K., HUrF~AN, C. F., AN1) DUNCAN,C. W. Antibiotics in Rumen Digestion and Synthesis. III. The Effect of Aureomycin on Rumen Microorganisms, with Special Reference to the Streptococci and Coliform Groups. J. Dairy Sci., 36: 743. 1953. (4) DOETSCH, !R. N., ROBINSON, R. Q., BRO~VN,R. E., AND SHA~V, J. C. Catabolic Reactions of Mixed Suspensions of Bovine Rumen Bacteria. J. Dairy Sci., 36: 825. 1953. (5) DOETSCH, R. N., ROBINSON, R. Q., McNEILL, ,I. ,]-., JURTSHUK, P., BRO~N, R. E., AND SHAW, J. C. Physiological Studies on Mixed Suspensions of Rumen Bacteria. i~ld. Agr. Expt. Sta., Misc. P~tbl. 205. 1954. (6) DOBTSCH, R. N., ROBINSON, R. Q., AND SHA~V, J. C. Techniques Employed in Cultural Investigations of the Bacteriology of the Bovine Rumen. J. Animal Sci., 11: 536. 1952. (7) FINCHAM, R. C., ~iND VOELKER, H. H. The Long-Time Effects of Aureomycin Feeding to Dairy Heifers. (Abs.) J. Dairy Sci., 35: 594. 1953. (8) FOSTEI%,J. W., AND PITTILL0, R. F. Metabolite Reversal of Antibiotic Inhibition, Especially Reversal of Aureomycin Ilflfibition by Riboflavin. J. Baeteriol., 66: 478. 1953. (9) HUNGATE, R. E. The Anaerobic Mesophilie Cellulolytie Bacteria. Bacteriol. Boys., 14: 1. 1950. (10) JACOBSON,N. L., KAlVFENTZAKIS,J. G., AND MURLEY, W. R. Response of " R u m i n a t i n g " Dairy Calves to Aureomycin Feeding. J. An~mai Sci., 10: 1950. 1951. (11) JUKTSHUK,P., KELLEY, N., AND DOETSCH, R. N. Unpublished Results. 1954. (12) KESLE~, E. M. Effect of Terramycin Fed to Holstein Calves on Growth, I n Vitro Cellulose Digestion and B-Vitamin Synthesis. J. Ani~t~al Sci., 13: 10. 1954. (13) KESLEI{, E. M., AND KNODT, C. B. Effect of Terramycin on Growth and Rumen Function in Holstein Calves. (Abs.) J. Animal Sci., 11: 768. 1952.
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(14) KNODT, C. B. Antibiotics in the Growth of Ruminant Animals. ~ntibiotics and Chettwtherapy, 3: 442. 1953. (15) LODGE, J. R., MILES, J. T., AND JACOBSON, N. L. The Effect of Aureomycin Feeding to Dairy Cattle on the I n Vitro Digestion of Cellulose by Rumen Microorganisms. (Abs.) J. Dairy Sci., 37: 663. 1954. (16) MAcKaY, A. M., RIDDELL, W. H., AND FITZSI~I}Z0NS, R. Feed Supplement Containing Aureomycin and Vitamin B~_ofor Dairy Calves. J. Animal Sci., 11: 341. 1952. (17) MACKAY, A. M., RIDDELL, W. I~., AND FITZSII~II~IONS, R. Terramycin Supplement for Dairy Calves. J. A~tb~al Sci., 11: 19. 1953. (18) MCELROY, L. W., AND GROSS, H. Quantitative Study of Vitamins in Rumen Contents of Sheep and Cows Fed Vitamin Low Diets. 1. Riboflavin and Vitamin K . J. N~ltri~ tion, 20: 527. 1940. (19) MUELEY, W. R., JACOBSON, N. L., A~'D ALLEN, l~. S. Effect of Aureomycin Supplementation on Growth and Feed Utilization of Young Dairy Calves. J. Dairy Sci., 35: 846. 1952. (20) MURLEY, W. R., JACOBSON, N. L., WING, J. M., AND STODDAI~D, G. E. The Response of Aureomycin Supplementation of Young Dairy Calves Fed Various ~ ' P r a c t i c a l ' ' and Restricted Diets. (Abs.) J. Dairy Sci., 34: 500. 1951. (21) PAINE, T. F., JR., COLLINS, H. S., AND FINLAND, M. Bacteriologic Studies on Aureomycin. J. BaeterioL, 55: 489. 1948. (22) }~En), J. T., WARNER, R. G., AND LOOSLI, J. K. Antibiotics in the Nutrition of Ruminants. Agr. Food Chem., 2: 186. 1954. (23) RUSO~F, L. L. A.P.F. Supplements for Calves. (Abs.) J. At~i~aal Sci., 9: 666. 1950. (24) RUS0FF, L. L., ALFOI~D, J. H., AND HYDE, C. E. Effect of Type of Protein on the Response of Young Dairy Calves to Aureomycin with Data on the Intestinal Microflora. J. Dairy Sci., 36: 45. 1953. (25) S~OKS~AI), E. L. R. Antibiotics in Animal Nutrition. PhysioT. Revs., 34: 25. 1954. (26) VOELKEa, It. It., AND C.XSON, J. L. Antibiotic Studies with Dairy Calves. (Abs.) J. Anin~al Sc~., 10: 1056. 1951. (27) WAISBREN,B. A., CARa, C., AND DUNNETTE, 5. The Tube Dilution Method of Determining Bacterial Sensitivity to Antibiotics. ~4~a. J. Clin. PathoL, 21" 884. 1951.