Journal of Applied Bacteriology 1977,43, 333-344
Vitamin B12 Production by the Gastro-intestinal Microflora of Baboons Fed either a Vitamin B,, Deficient Diet or a Diet Supplemented with Vitamin BI2 PATRICIA F. UPHILL*, FREDERIKA JACOB AND PARVINDER LALL Wellcome Trust Research Laboratories, P.O. Box 43640, Nairobi, Kenya Received 26 March 1 9 7 6 and accepted 28 July 1 9 7 7 The vitamin B ,z-producing capacity of micro-organisms isolated from baboon faeces and gastric contents was measured using Lactobacillus leichmannii. The animals were fed either a diet deficient in or supplemented with vitamin B,, (controls). Samples of gastric and small intestinal contents, obtained at laparotomy from two young vitamin B,,-deprived baboons, contained varying quantities of vitamin BIZ. Many of the organisms isolated from these aspirates produced vitamin BIZin uitro. The highest levels of vitamin B,, were produced by anaerobic organisms. Gastric juice samples from vitamin B 12 deprived and control baboons contained similar types of organisms with like vitamin BIZ-producing capacity. The vitamin B,, content, pH and total bacterial counts of gastric juice samples aspirated after a 6 h fast from vitamin B,, d~eprived baboons were not significantly different from those of the control animals. The pH values of gastric juice samples aspirated 18 h after feeding, however, were significantlylower than those of 6 h fasting samples in both groups. The mean vitamin B,, levels in the total volumes of gastric juice aspirated after each fasting period were similar. The possible involvement of the gastrointestinal flora in the vitamin BIZstatus of the baboon is discussed.
VITAMIN B,,
SYNTHESIS has been shown to occur solely in micro-organisms (Underwood 1956), macro-organisms, in general, requiring an exogenous supply of the vitamin. Bacteria and the blue-green algae are the only groups in which its synthesis has been shown to occur, and not all members of these two groups possess this ability. Many intestinal micro-organisms are known to produce vitamin B,,, often in an amount in excess of the host’s requirements (Mickelsen 1956). However, the site of vitamin B,, production in the intestine is important when considering the potential availability of the vitamin to the host, since vitamin B,, absorption is reported to occur in the upper part of the alimentary tract (Matthews 1967). Ruminants, although feeding on a diet of plant material totally devoid of vitamin BIZ, have been shown to have high levels of the vitamin in their rumen contents (Hungate 1966). Due to the anterior position of the rumen, its contents must pass through the remainder of the alimentary tract, so allowing maximal opportunity for vitamin absorption. In other mammalian species and in birds the densest intestinal microbial populations normally occur in the caecum and colon. Vitamin B,, produced by microbial action in these areas is considered to be excreted, unutilized, in the faeces. Coprophagous species are an exception in that they obtain a considerable part of their vitamin requirements by ingestion of their faeces. The production of vitamin B,, deficiency in animals is known to be difficult (Stokstad 1968) and in this laboratory the feeding of a vitamin B,, deficient diet to baboons over a two year period resulted in the development of a subclinical deficiency of the vitamin (Siddons 1974). It was shown, however, that the vitamin BI2deficiency was more severe
‘Reprint requests should be addressed to: The Zoological Society of London, Nuffield Institute of Comparative Medicine, Regent’s Park, London NW 1 4RY, England.
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P. F. UPHILL, F. JACOB AND P. LALL
in baboons fed a diet containing ampicillin, suggesting that the intestinal flora may play a part in the vitamin B 12 nutrition of the baboon. In addition, a group of young baboons fed a vitamin B,, deficient diet were found to have high serum and liver vitamin B,, levels after 18 months, in comparison with older animals fed the same diet. The intestinal flora of young animals has been shown for many species to be different from that of adult animals (Smith & Crabb 1961). This study was undertaken to determine whether there were any detectable differences in vitamin B,, production by the intestinal flora of the baboons which could explain these findings.
Materials and Methods Experimental animals and diets Twenty-seven male baboons (Papio spp.) were housed and fed individually in cages with wide-mesh wire floors. The cages were hosed down at regular intervals so allowing the animals minimal contact with their excreta. The animals were divided into six experimental and three control groups. Twelve baboons were fed a basal synthetic diet consisting of vitamin-free casein, sucrose, corn oil and a mineral salts mixture, together with a vitamin mixture which was added daily to their drinking water. The composition of this diet has been described (Uphill 1973). Six of these baboons had been fed the diet for three years, and they were large animals weighing 16-20 kg. This group was further subdivided, three animals receiving the diet deficient in vitamin B,, and three animals receiving the diet supplemented with vitamin B,,. The remaining six baboons were young animals weighing only 8-12 kg, and of these, four animals were fed the vitamin B,, deficient diet and two animals were fed the vitamin B,, supplemented diet. The young baboons were fed the diets for 18 months. Fifteen baboons were divided at random into five groups, each of three animals and their basal diet was a modified mixture of that described previously, consisting of vitamin-free casein, starch, sucrose, cellulose, mineral salt mixture and corn oil. These dietary groups have been fully described by Siddons (1974), and were briefly as follows: (1) vitamin B,, deficient basal diet, (2) basal diet supplemented with vitamin B,,, (3) basal diet with the addition of 50 mg ampicillin/kg diet (Penbritin, Beecham Research Laboratories, Brentford, England), (4) basal diet low in fat and containing sodium propionate, ( 5 ) basal diet modified in that the casein was replaced by an equivalent quantity of vitamin-free soya protein. Baboons in groups (l)-(4) were fed their diets for two years. Baboons in group ( 5 ) were fed the soya diet for three months, having previously received the group (1) diet for 21 months. Gastric juice samples Gastric contents were aspirated using a Ryle’s tube from baboons tranquillized with phencyclidine hydrochloride, 1.5 mg/kg (Sernylan Parenteral, Parke Davis and Co., Hounslow, England). Aspirations were carried out after an 18 h fasting period and after a 6 h fasting period. Collection of the 18 h fasting samples required prior histamine stimulation, a dose of 0.025 rng being injected subcutaneously into baboons weighing up to 12 kg, while larger animals received 0.05 mg. A comparison of the vitamin B,, content and pH, and bacteriological examination for the presence of Clostridium perfringens and Lactobacillus spp. was carried out on 18 h and 6 h fasting samples from all baboons. On a separate occasion 6 h fasting samples selected at random from each group of baboons were subjected to a complete aerobic and anaerobic bacterio-
VITAMIN
BIZ
PRODUCTION BY BABOON GUT FLORA
335
logical examination, together with pH estimation and vitamin B,, assay, and the total bacterial count was also calculated. The pH of all gastric samples was measured using a Zeromatic pH meter (Beckman Instruments Ltd., Glenrothes, Fife, Scotland). Total bacterial counts were carried out on gastric juice homogenized by mixing with an aliquot of 4% NaOH. Samples were incubated at 37 "C for 30 min with continuous shaking, then diluted 1 in 5 with 0.85% NaCl and counted in a Helber counting chamber.
Faeces Freshly voided faeces were collected in small wax containers filled to the brim to exclude air, and were immediately placed in the air lock of an anaerobe isolation chamber. Samples were collected from 18 animals selected at random from each dietary group. All samples were cultured aerobically and 12 samples were also cultured anaerobically. Gastro-intestinal contents Gastro-intestinal aspirates were collected at laparotomy from each of two young vitamin B,, deprived animals. Laparotomies were carried out 6 h after feeding. One baboon was anaesthetized with diethyl ether, and the other animal with Halothane, and the laparotomy technique has been described previously (Uphill et al. 1974). The areas sampled were the stomach, duodenum and four areas along the length of the small intestine which were estimated to lie in each quarter of the small intestine. Sampling was started at the quarter immediately proximal to the ileo-caecal valve, and continued working backwards towards the stomach. Each area was sampled in duplicate, two adjacent three inch areas of gut being clamped off with atraumatic clamps, the distal clamp being left in position until the next area had been clamped off. One of each duplicate section was injected with 10 ml 4 strength Ringer's solution, and after mixing by aspiration and re-aspiration the contents were withdrawn for vitamin B,, assay and aerobic cultivation. The adjacent area was injected with 10 ml 4 strength Ringer's solution enriched with 1% vitamin-free casein hydrolysate, 0.5% glucose, 0.05% cysteine HCI, 0.05 yglml menadione and 1 yglml haemin. This solution had been prereduced by storage in an anaerobe isolation chamber for 48 h before usage. After mixing and re-aspiration the contents were immediately placed in the anaerobe isolation chamber for cultivation of the fastidious anaerobes. Bacteriological methods Aerobic organisms Faeces, gastric juice samples and intestinal contents were streaked onto a selection of media for isolation of the aerobes and non-fastidious anaerobes. The media and incubation conditions were as previously described (Uphill 1973). Anaerobic organisms Samples of faeces, gastric juice and intestinal contents were cultured in an anaerobe isolation chamber of the type described by Aranki et al. (1969). Intestinal contents and gastric agpirates were streaked undiluted, faeces were diluted 1 in 10 by homogenizing 1 gm faeces in 9 ml VL broth (Barnes & Impey 197 1). Samples were cultured on a range of enriched and selective media, all of which had been pre-reduced by storage in the
P. F. UPHILL, F. JACOB AND P. LALL
336
chamber for 48 h before use. The media were as follows: Rogosa V agar for Veillonella spp. (Rogosa et al. 1958), Tomato Juice Agar (Oxoid) pH 5.0 for Lactobacillus spp., Tomato Juice Agar adjusted to pH 7.0 for anaerobic sarcinae (Crowther 1971), anaerobic coccus medium + neomycin 100 pg/ml (Thomas & Hare 1954), VL medium + 10% baboon faecal extract and VL medium + menadione 0.5 pg/ml + haemin 1 pg/ml + 1% liver digest (Barnes & Impey 1971), and four selective antibiotic media (Finegold et al. 197 l), namely kanamycin blood agar, kanamycin-vancomycin laked blood agar, neomycin-vancomycin blood agar and rifampicin blood agar. All plates were incubated inside the chamber at 37 OC in a gas mixture consisting of 10% H, and 5% CO, in N, for five days. The organisms were identified broadly on colony and cell shape, reaction to Gram's stain, and redox requirements. Organisms were tested for their ability to grow in the presence of oxygen (aerobes and microaerophiles), and ability to grow anaerobically after previous exposure to oxygen on the laboratory bench for 2 h (non-fastidious anaerobes). Organisms which did not withstand this brief exposure to oxygen were considered to be fastidious anaerobes. The numerous Gram negative anaerobic rods were further grouped by their ability to grow on each of the four antibiotic media. Pure cultures of all organisms were subcultured on to enriched media; VL agar + 10% sheep blood was used for the anaerobes, while the aerobes were cultured on Nutrient Agar (Oxoid) + 7.5% sheep blood. Aerobes, yeasts, lactobacilli and C1. perfringens were inoculated into vitamin B,, assay medium adjusted to pH 7.0, from the enriched agar using a straight wire. The fastidious anaerobes would not grow in this assay medium, and were inoculated into enriched trypticase soy broth (Aranki et al. 1969). The latter medium was found to contain up to 0.04 ng/ml vitamin B,,, and a control medium tube was included in every assay, and its vitamin B,, content subtracted from all assay figures. Both aerobic and anaerobic cultures were incubated at 37 OC for four days prior to assay.
,
Vitamin B assay method One ml of each sample was suspended in 10 mlO.25 M-acetate buffer pH 4-6,then 0.25 ml of 0.1% (w/v) NaCN was added and the solutions stored at -2OOC before assay. Thawed samples were shaken for 5 min, 0.25 ml of 0.5% (w/v) papain was added to each sample and the volume made up to 20 ml with distilled water. The suspensions were then incubated at 60 O C for 1 h followed by autoclaving at 115 "C for 8 min. After centrifuging, the supernatants were assayed microbiologically for vitamin B using Lactobacillus leichmannii (Skeggs et al. 1950; Coates et al. 1953; Spray 1955).
,,
TABLE1 Vitamin B I 2content (ng/gm) of gastro-intestinal aspirates from two young vitamin B,, deprived baboons Small intestinal quarters A
I
Baboon
3
Stomach
Duodenum
First
Second
Third
Fourth
0.32 0.05
0.44
0.93 0.79
0.68 0.06
0.72 2.45
0.69 0
~
A
B
1.09
i
Yeasts Aerobic Gram positive cocci Anaerobic Gram positive cocci Anaerobic Gram negative cocci Micro-aerophilic Gram positive rods CI. perfringens Other anaerobic Gram positive rods
-f -denotes no organisms isolated from this region.
0.03 0.11 0.04 12.70 0.044~152 -
0.10 -
-
W.603 3.22
1.48 -
-
0.07 0.03-0.092 -
CU).154
I
3.20 6.60 0.15-0.18’ 0.10 3.60 0 0.03~~123 0.05-0.142 0 1.08 -
~ 1 . 1 1 4
0.02 03 -
0 03 0.62
0 03 0-1.313 4.33 23.60 0.09 -
0 *OZ
Yeasts Aerobic Gram positive cocci Anaerobic Gram negative cocci Micro-aerophilic Gram positive rods CI.perfringens Other anaerobic Gram positive rods Aerobic Gram negative rods Anaerobic Gram negative rods
--t
Second
First
10.0
Total number of strains isolated*
0 0
68 6
1
0
8 13
1
47
5 0 23 30
32 45 17 296
* Faecal samples collected from 18 animals selected at random from each of the dietary groups. All samples were cultured aerobically and 12 samples were also cultured anaerobically. strains of anaerobic organisms isolated, 48% of cultures contained > 1a 0 ng/ml vitamin B,,, and 23.6% of cultures, mainly Gram negative rods, contained >10 ng/ml. There were no noticeable differences in the types of faecal organisms isolated from controls or baboons fed any of the vitamin B,, deficient diets, with the exception of the group fed ampicillin. The faeces of these animals contained very few aerobic or anaerobic Gram positive organisms, the flora consisting mainly of aerobic and anaerobic Gram negative rods and yeasts. The patterns of vitamin B,, production by the faecal organisms were similar both within the vitamin B,, deficient groups, and between the vitamin deficient and control groups. Table 3 is therefore presented as a composite table showing the ranges of vitamin B,, produced by all the organisms isolated, irrespective of the diets which were fed. Table 4 compares the vitamin B,, produced in cultures of organisms isolated from gastric juice samples aspirated 6 h after feeding, from vitamin B,, deprived and control baboons. The number of isolates obtained from the gastric juice samples from the control group was low in comparison to the vitamin deprived group. There were also fewer isolates producing the higher levels of vitamin B,, obtained from the control baboon gastric samples. No organisms producing >10 ng/ml vitamin B,, were isolated from the gastric juice samples.
VITAMIN Biz PRODUCTION BY BABOON GUT FLORA
339
TABLE 4
Vitamin B,, production by organisms isolated from baboon gastric juice collected 6 h afterfeeding either a diet dejicient in vitamin B , , or a vitamin B , , supplemented diet
--Number of strains in each range of vitamin B production (ng/ml culture) A
r
Vitamin B,, deprived diet*
0-0.1
0.11-1.0
Aerobic Gram positive cocci Anaerobic Gram positive cocci Anaerobic Gram negative cocci Yeasts Micro-aerophilic Gram positive rods Anaerobic Gram positive rods Aerobic Gram negative rods Anaerobic Gram negative rods
33
5
0
7
1
0
0 14
2 0
0
12
6 9 3
4
4
4
0
2
Total number of strains
78
30
13
22
10
Organisms
3 5
1.01-10.0
Vitamin B ,* supplemented diet?' 04.1
0.11-1.0
\
1.01-10.0
1 1 5 0
4 2 1
1 6 0
* Ten animals sampled. t Five animals sampled.
The Gram negative anaerobic species producing the highest levels of vitamin B,, were not identified. However, four type cultures (National Collection of Type Cultures, Central Public Health Laboratory, Colindale Avenue, Colindale, London NW9 5HT) were assayed with the following results: Bacferoidesfragilis (NCTC 8560), 0.13 ng/ml; Fusobacterium polymorphum (NCTC 10562), 72 8 nglml; Sphaeropherus varians (NCTC lO560), 69.6 ng/ml; and anaerobic Gram negative coccus (NCTC 9805), 0.14 ng/ml. Cultures obtained from Gram negative rods originally isolated on rifampicin blood agar and on neomycin-vancomycin blood agar, always produced TABLE5
Vitamin B,, content (nglgm),p H values and total bacterial counts (x109/mlgastric juice) of baboon gastric juice samples taken 6 h after feeding either a vitamin B,, deficient diet or a vitamin B,, supplemented diet
--
Vitamin B,, deprived animals*
~~
PH Vitamin B,, content Total bacterial count
* There
~
Control animals
Meant
S.E.
Mean
S.E.
3.53 1.14 1.78
0.54
3.13 1.62 1.83
0.29 0.40 1.20
0.27 0.33
were no significant differences between mean values from vitamin B,, deficient or vitamin B,, supplemented animals, P > 0.10. t Mean values with their standard errors for ten animals fed a vitamin B,, deficient diet and five control animals.
P. F. UPHILL, F. JACOB AND P. LALL
340
TABLE6
Comparison of p H values and vitamin B , , content (nglgm aspirate) in gastric juice samples taken after an 18 h fast or a 6 h fast from baboons fed either a diet deficient in vitamin B , , or a diet supplemented with vitamin B , , ~~~
~
~~~
~
18 h fast Diet
6 h fast
&
Factors measured
&
Mean*
S.E.
Mean
S.E.
B,, deficient
[ PH
I B,, content
1.64 3.90
0.14 0.58
3.07 0.98
0.23 0.13
B,, supplemented
{ gf content
1.50 2.54
0.09 0.55
3.28 1.so
0.30 0.10
>o. lot >o. 10
B,, deficient B,, supplemented
0.01
(0.001
B,, deficient
B,, supplemented
>o. 10 o. lot
0.01
>o. 10 >o. 10
B,, deficient B,, supplemented
* Mean values with their standard errors for 18 animals fed a vitamin B,, deficient diet and 8 animals fed a vitamin B,, supplemented diet.
t Probability values.
many of the 6 h fasting samples in both dietary groups, but from few of the 18 h samples.
Discussion Since it is well established that microbial synthesis provides the main source of vitamin B,, for the ruminant (Hungate 1966), it seems logical to assume that the organisms most likely to produce the vitamin in quantity would be anaerobic. The vitamin B,, producing capacity of micro-organisms has not been extensively documented, and comparison of the reported results is difficult due to the use of differing assay methods. The variable composition of culture media in which isolates were grown for assay must also affect the quantities of vitamin produced. Kon et al. (1969) reviewed vitamin B,, production by rumen micro-organisms and studied 615 strains of 15 species of rumen bacteria, of which 150 strains showed activity when assayed with Escherichia coli. In two studies on non-ruminants, however, aerobic organisms have been reported to synthesize quantities of vitamin B,, when assayed with L . leichmannii. Halbrook et al. (1950) isolated strains of Aerobacter aerogenes, yeasts, Bacillus megatherium and some moulds from poultry house litter and droppings, which produced >Om4 ng/ml vitamin B,2. In measurements of vitamin B,, production in horse gut contents, Davies (1971) found that the highest concentration of the vitamin was produced by a strain of E. coli (1 2 ng/ml). In a study of anaerobic organisms isolated from human faeces, Gall (1970) showed that 16 type cultures produced OvO1-0-32 ng/ml vitamin B,,. The aerobic isolates obtained from baboon faeces and gastric contents in this study produced little or no vitamin BIZ,although all these organisms grew well in the vitamin B,,-free assay medium in which the cultures were prepared. The highest levels of vitamin B,, were produced by the anaerobic isolates, in particular by C1. perfringens and some of the anaerobic Gram negative rods. It has been shown that vitamin B,, was present in the stomach and upper intestine of two of the young vitamin B,, deprived baboons. In addition, some of the micro-
342
P. F. UPHILL, F. JACOB AND P. LALL
organisms isolated from the gastro-intestinal contents of these baboons were capable of producing in vitro large quantities of vitamin BIZ.However, gastric juice samples from all the vitamin deprived and control baboons contained organisms capable of producing vitamin B 12 in vitro. No differences were detected due to different diets or age of animal, with the exception of the baboons receiving ampicillin. Therefore, the unusually high serum and liver vitamin B,, levels found in the young animals, but not in older baboons, remains unexplainable. The cultivation of such a variety of aerobic, microaerophilic and anaerobic organisms from the gastric contents of baboons fasted for 6 h may be due to the type of synthetic diet fed, and to the possession by baboons of facial cheek pouches in which food can be stored (Uphill et al. 1974). A fasting period would date from the moment that the pouch was empty, rather than from the time of withholding food. The lack of difference in vitamin B,, content between gastric samples taken from vitamin B,, deprived and control baboons 6 h after feeding would suggest that digestion was complete, since the control baboons received 2 pg vitamin B ,,/day. Comparative examination of gastric contents taken after withholding food for 6 h or 18 h, however, suggested that the baboon stomach had not completely returned to the resting state 6 h after feeding. The mean gastric pH of both vitamin deprived and control baboons being significantly lower after an 18 h fast. Evidence that the stomach and intestines, after a 6 h fast, contained more viable organisms than at 18 h is provided by the increased percentage isolation of CI.perfringens and Lactobacillus spp. at 6 h. The numbers of viable bacteria present in the 6 h fasting samples is unknown, but the total counts were high, being only 100 times lower than the total bacterial counts found in baboon faeces (unpublished data). This indicates that considerable bacterial multiplication had taken place in the stomach at some time before sampling. Vitamin B found in the gastro-intestinal contents of vitamin B,, deprived baboons could be derived from ingested food, desquamated epithelial cells, digestive secretions or from the bodies and/or the secretions of the gastro-intestinal microflora. Siddons (1974) reported that the vitamin-free casein used in these studies contained 0.004 pg/gm L. leichmannii growth-promoting ability, which, if it was due to vitamin B,,, could result in each baboon receiving 0.01 pg vitamin B,,/day. The three baboons fed the soya protein diet did not receive even this minimal supplementation however, but the mean 18 h and 6 h fasting gastric samples also contained high levels of the vitamin. The vitamin B,, content of desquamated epithelial cells or digestive secretions is unlikely to account for all of the vitamin B 12 found in the stomach contents of baboons deprived of the vitamin. These animals were showing evidence of vitamin B,, deprivation in their low serum and liver levels (Siddons 1974), but the mean vitamin B,, levels in their stomach contents 18 h after feeding were not significantly lower than those of control baboons. It would be impossible to state conclusively that the intestinal flora were largely responsible for the vitamin BIZfound in the baboon stomach and intestine, since production of the vitamin by an organism in vitro does not necessarily mean that comparable levels would be produced in viva However, this study does provide presumptive evidence that the vitamin B found in the baboon stomach and upper intestine could have been produced by microbial action. An increased vitamin B deficiency found in baboons fed 2 vitamin B deficient diet containing ampicillin (Siddons 1974) could be explained by the observed inhibition by the antibiotic of all the Gram positive flora. All the isolated strains of C1. perfringens produced 1-10 ng/ml vitamin B,, and many of the lactobacilli produced varying
,*
VITAMIN
BIZ
PRODUCTION BY BABOON GUT FLORA
343
amounts of the vitamin. Assuming that the vitamin B,, produced by these organisms in vitro was also being produced in vivo in the baboon stomach and upper intestine, it is possible that the vitamin was being absorbed and utilized to meet part of the animal’s nutritional requirements. Vitamin B,, produced by the Gram positive flora was unavailable to the baboons fed ampicillin and their vitamin BI2deficiency was increased. The faecal vitamin B,, content was not reduced in the ampicillin-fed baboons (Siddons 1974) and although the numbers of C1.perfringens and lactobacilli in their faeces were greatly reduced, there was a large increase in the number of anaerobic Gram negative rods, many of which produced > 10 ng/ml vitamin B,, in vitro (unpublished data). This study suggests that the chances of producing a vitamin B I Zdeficiency in the baboon might be improved by a change in its feeding habits. Smith (1965), showed that in fowls with ablated crops, and in ducks which have no crops, the bacterial content of the alimentary tract was lower than in ordinary fowls. Closure of the baboon facial pouches might also lead to a reduced intestinal bacterial population. In addition, the baboons used in this study were fed their daily ration in two meals with an interval of 5 h between. The results show that vitamin BIZ-producingorganisms would therefore be present in the upper intestine for at least 12 out of every 24 h. The feeding of only one large meal/day and a change of diet to a type less likely to cause an increase in the putrefactive and potentially vitamin B,,-producing flora could speed the development of vitamin B,, deficiency in the baboon. Alternatively the Gram positive vitamin B12producing flora can be eliminated by the daily feeding of small quantities of ampicillin. We should like to thank Dr P. Sayer for doing the laparotomies, Mrs A. Aplin for technical assistance, Mr R. A. Whittingham for arranging the construction of the anaerobic chamber and for animal maintenance and Dr R. Siddons for assistance in the preparation of this manuscript.
References ARANKI, A., SYED,S. A., KENNEY,E. B. & FRETER, R. 1969 Isolation of anaerobic bacteria from human gingiva and mouse caecum by means of a simplified glove box procedure. Applied Microbiology 17,568-576. BARNES,E. M. & IMPEY,C. S. 1971 The isolation of the anaerobic bacteria from chicken caeca with particular reference to members of the family Bacteroidaceae. In Isolation of’ Anaerobes The Society for Applied Bacteriology Technical Series 5, ed. Shapton, D. A. & Board, R. G. pp. 115-123, London & New York: Academic Press. COATES,M. E., FORD,J. E., HARRISON, G. F., KON,S. K. & PORTER, J. W. G. 1953 Vitamin B,,-like compounds. I. Vitamin B,, activity for chicks and for different micro-organisms of gut contents and faeces. British Journal of Nutrition 7,3 19-326. CRowTnER, J. s. 1971 Sarcina uentriculi in human faeces. Journal of Medical Microbiology 4, 343-3 50. DAVIES,M. E. 197 1 Production of vitamin B,, in the horse. British Veterinary Journal 127,3436. FINEGOLD, S. M., SUGIHARA, P. T. & SUTTER, V. L. 197 1 Use of selective media for isolation of anaerobes from humans. In Isolation of Anaerobes The Society for Applied Bacteriology Technical Series 5, ed. Shapton, D. A. & Board, R. G. pp. 99-108, London & New York: Academic Press. GALL,L. S. 1970 Normal faecal flora of man. American Journal of Clinical Nutrition 23, 14571465.
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HALBROOK, E. R., CORDS,F., WINTER,A. R. & SUTTON, T. S. 1950 Vitamin B,, production by micro-organisms isolated from poultry house litter and droppings. Journal of Nutrition 41, 555-563. HUNGATE, R. E. 1966 The Rumen and its Microbes London & New York: Academic Press. KON,S. K., PORTER,J. W. G., THORLEY, C. M. & WILLIAMS, A. P. 1969 The possible origin of vitamin B,, activity in the rumen. Vpamet na Akademik Ignat Emanuilov: trudore burkhy izbrani problemi nu mikrobiologiyata, enzimologiyata i biokhimiyata. Pashev, I. et al. pp. 203-207, Sofiya: Bulgarska Akademiya na Naukite. MATTHEWS, D. M. 1967 Absorption of water-soluble vitamins. British Medical Bulletin 23, 258-262. MICKELSON,0. 1956 Intestinal synthesis of vitamins in the non-ruminant. Vitamins and Hormones 14,l-95. ROGOSA,M., FITZGERALD, R. J., MACKINTOSH, M. E. & BEAMAN,A. J. 1958 Improved medium for selective isolation of Veillonella. Journal of Bacteriology 76,455-456. SIDDONS,R. C. 1974 The experimental production of vitamin B,, deficiency in the baboon (Papio cynocephalus). A two-year study. British Journal of Nutrition 32,2 19-228. SKEGGS,H., NEPPLE,H. M., VALENTIK,K. A., HUFF, J. W. & WRIGHT,L. D. 1950 Observations on the use of Lactobacillus leichmannii 4797 in the microbiological assay of vitamin B,y Journal of Biological Chemistry 184,211-22 1. SMITH,H. W. & CRABB,W. E. 1961 The faecal bacterial flora of animals and man: its development in the young. Journal of Pathology and Bacteriology 82,53-66. SMITH,H. W. 1965 Observations on the flora of the alimentary tract of animals and factors affecting its composition. Journal of Pathology and Bacteriology 89,95-122. SPRAY,G. H. 1955 An improved method for the rapid estimation of vitamin B,, in serum. Clinical Science 14,661-667. STOKSTAD, E. L. R. 1968 Experimental anaemias in animals resulting from folic acid and vitamin B,, deficiencies. Vitamins and Hormones 26,443-463. THOMAS, C. G. A. & HARE, R. 1954 The classification of anaerobic streptococci and their isolation in normal human beings and pathological processes. Journal of Clinical Pathology 7,300-304. UNDERWOOD, E. J. 1956 Trace Elements in Human andAnima1 Nutrition (2nd edn. 1962) New York: Academic Press. UPHILL,P. F. 1973 A quantitative comparison of the faecal microflora of baboons fed a natural diet or a synthetic diet complete or deficient in pyridoxine or riboflavine. Journal of Applied Bacteriology 36,501-5 1 1 . UPHILL,P. F., WILDE, J. K. €3. & BERGER,J. 1974 Repeated examinations, using the laparotomy sampling technique, of the gastro-intestinal microflora of baboons fed a natural or a synthetic diet. Journal of Applied Bacteriology 37,309-3 17.
Vitamin B12 Production by the Gastro-intestinal Microflora of Baboons Fed either a Vitamin B,, Deficient Diet or a Diet Supplemented with Vitamin BI2 PATRICIA F. UPHILL*, FREDERIKA JACOB AND PARVINDER LALL Wellcome Trust Research Laboratories, P.O. Box 43640, Nairobi, Kenya Received 26 March 1 9 7 6 and accepted 28 July 1 9 7 7 The vitamin B ,z-producing capacity of micro-organisms isolated from baboon faeces and gastric contents was measured using Lactobacillus leichmannii. The animals were fed either a diet deficient in or supplemented with vitamin B,, (controls). Samples of gastric and small intestinal contents, obtained at laparotomy from two young vitamin B,,-deprived baboons, contained varying quantities of vitamin BIZ. Many of the organisms isolated from these aspirates produced vitamin BIZin uitro. The highest levels of vitamin B,, were produced by anaerobic organisms. Gastric juice samples from vitamin B 12 deprived and control baboons contained similar types of organisms with like vitamin BIZ-producing capacity. The vitamin B,, content, pH and total bacterial counts of gastric juice samples aspirated after a 6 h fast from vitamin B,, d~eprived baboons were not significantly different from those of the control animals. The pH values of gastric juice samples aspirated 18 h after feeding, however, were significantlylower than those of 6 h fasting samples in both groups. The mean vitamin B,, levels in the total volumes of gastric juice aspirated after each fasting period were similar. The possible involvement of the gastrointestinal flora in the vitamin BIZstatus of the baboon is discussed.
VITAMIN B,,
SYNTHESIS has been shown to occur solely in micro-organisms (Underwood 1956), macro-organisms, in general, requiring an exogenous supply of the vitamin. Bacteria and the blue-green algae are the only groups in which its synthesis has been shown to occur, and not all members of these two groups possess this ability. Many intestinal micro-organisms are known to produce vitamin B,,, often in an amount in excess of the host’s requirements (Mickelsen 1956). However, the site of vitamin B,, production in the intestine is important when considering the potential availability of the vitamin to the host, since vitamin B,, absorption is reported to occur in the upper part of the alimentary tract (Matthews 1967). Ruminants, although feeding on a diet of plant material totally devoid of vitamin BIZ, have been shown to have high levels of the vitamin in their rumen contents (Hungate 1966). Due to the anterior position of the rumen, its contents must pass through the remainder of the alimentary tract, so allowing maximal opportunity for vitamin absorption. In other mammalian species and in birds the densest intestinal microbial populations normally occur in the caecum and colon. Vitamin B,, produced by microbial action in these areas is considered to be excreted, unutilized, in the faeces. Coprophagous species are an exception in that they obtain a considerable part of their vitamin requirements by ingestion of their faeces. The production of vitamin B,, deficiency in animals is known to be difficult (Stokstad 1968) and in this laboratory the feeding of a vitamin B,, deficient diet to baboons over a two year period resulted in the development of a subclinical deficiency of the vitamin (Siddons 1974). It was shown, however, that the vitamin BI2deficiency was more severe
‘Reprint requests should be addressed to: The Zoological Society of London, Nuffield Institute of Comparative Medicine, Regent’s Park, London NW 1 4RY, England.
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P. F. UPHILL, F. JACOB AND P. LALL
in baboons fed a diet containing ampicillin, suggesting that the intestinal flora may play a part in the vitamin B 12 nutrition of the baboon. In addition, a group of young baboons fed a vitamin B,, deficient diet were found to have high serum and liver vitamin B,, levels after 18 months, in comparison with older animals fed the same diet. The intestinal flora of young animals has been shown for many species to be different from that of adult animals (Smith & Crabb 1961). This study was undertaken to determine whether there were any detectable differences in vitamin B,, production by the intestinal flora of the baboons which could explain these findings.
Materials and Methods Experimental animals and diets Twenty-seven male baboons (Papio spp.) were housed and fed individually in cages with wide-mesh wire floors. The cages were hosed down at regular intervals so allowing the animals minimal contact with their excreta. The animals were divided into six experimental and three control groups. Twelve baboons were fed a basal synthetic diet consisting of vitamin-free casein, sucrose, corn oil and a mineral salts mixture, together with a vitamin mixture which was added daily to their drinking water. The composition of this diet has been described (Uphill 1973). Six of these baboons had been fed the diet for three years, and they were large animals weighing 16-20 kg. This group was further subdivided, three animals receiving the diet deficient in vitamin B,, and three animals receiving the diet supplemented with vitamin B,,. The remaining six baboons were young animals weighing only 8-12 kg, and of these, four animals were fed the vitamin B,, deficient diet and two animals were fed the vitamin B,, supplemented diet. The young baboons were fed the diets for 18 months. Fifteen baboons were divided at random into five groups, each of three animals and their basal diet was a modified mixture of that described previously, consisting of vitamin-free casein, starch, sucrose, cellulose, mineral salt mixture and corn oil. These dietary groups have been fully described by Siddons (1974), and were briefly as follows: (1) vitamin B,, deficient basal diet, (2) basal diet supplemented with vitamin B,,, (3) basal diet with the addition of 50 mg ampicillin/kg diet (Penbritin, Beecham Research Laboratories, Brentford, England), (4) basal diet low in fat and containing sodium propionate, ( 5 ) basal diet modified in that the casein was replaced by an equivalent quantity of vitamin-free soya protein. Baboons in groups (l)-(4) were fed their diets for two years. Baboons in group ( 5 ) were fed the soya diet for three months, having previously received the group (1) diet for 21 months. Gastric juice samples Gastric contents were aspirated using a Ryle’s tube from baboons tranquillized with phencyclidine hydrochloride, 1.5 mg/kg (Sernylan Parenteral, Parke Davis and Co., Hounslow, England). Aspirations were carried out after an 18 h fasting period and after a 6 h fasting period. Collection of the 18 h fasting samples required prior histamine stimulation, a dose of 0.025 rng being injected subcutaneously into baboons weighing up to 12 kg, while larger animals received 0.05 mg. A comparison of the vitamin B,, content and pH, and bacteriological examination for the presence of Clostridium perfringens and Lactobacillus spp. was carried out on 18 h and 6 h fasting samples from all baboons. On a separate occasion 6 h fasting samples selected at random from each group of baboons were subjected to a complete aerobic and anaerobic bacterio-
VITAMIN
BIZ
PRODUCTION BY BABOON GUT FLORA
335
logical examination, together with pH estimation and vitamin B,, assay, and the total bacterial count was also calculated. The pH of all gastric samples was measured using a Zeromatic pH meter (Beckman Instruments Ltd., Glenrothes, Fife, Scotland). Total bacterial counts were carried out on gastric juice homogenized by mixing with an aliquot of 4% NaOH. Samples were incubated at 37 "C for 30 min with continuous shaking, then diluted 1 in 5 with 0.85% NaCl and counted in a Helber counting chamber.
Faeces Freshly voided faeces were collected in small wax containers filled to the brim to exclude air, and were immediately placed in the air lock of an anaerobe isolation chamber. Samples were collected from 18 animals selected at random from each dietary group. All samples were cultured aerobically and 12 samples were also cultured anaerobically. Gastro-intestinal contents Gastro-intestinal aspirates were collected at laparotomy from each of two young vitamin B,, deprived animals. Laparotomies were carried out 6 h after feeding. One baboon was anaesthetized with diethyl ether, and the other animal with Halothane, and the laparotomy technique has been described previously (Uphill et al. 1974). The areas sampled were the stomach, duodenum and four areas along the length of the small intestine which were estimated to lie in each quarter of the small intestine. Sampling was started at the quarter immediately proximal to the ileo-caecal valve, and continued working backwards towards the stomach. Each area was sampled in duplicate, two adjacent three inch areas of gut being clamped off with atraumatic clamps, the distal clamp being left in position until the next area had been clamped off. One of each duplicate section was injected with 10 ml 4 strength Ringer's solution, and after mixing by aspiration and re-aspiration the contents were withdrawn for vitamin B,, assay and aerobic cultivation. The adjacent area was injected with 10 ml 4 strength Ringer's solution enriched with 1% vitamin-free casein hydrolysate, 0.5% glucose, 0.05% cysteine HCI, 0.05 yglml menadione and 1 yglml haemin. This solution had been prereduced by storage in an anaerobe isolation chamber for 48 h before usage. After mixing and re-aspiration the contents were immediately placed in the anaerobe isolation chamber for cultivation of the fastidious anaerobes. Bacteriological methods Aerobic organisms Faeces, gastric juice samples and intestinal contents were streaked onto a selection of media for isolation of the aerobes and non-fastidious anaerobes. The media and incubation conditions were as previously described (Uphill 1973). Anaerobic organisms Samples of faeces, gastric juice and intestinal contents were cultured in an anaerobe isolation chamber of the type described by Aranki et al. (1969). Intestinal contents and gastric agpirates were streaked undiluted, faeces were diluted 1 in 10 by homogenizing 1 gm faeces in 9 ml VL broth (Barnes & Impey 197 1). Samples were cultured on a range of enriched and selective media, all of which had been pre-reduced by storage in the
P. F. UPHILL, F. JACOB AND P. LALL
336
chamber for 48 h before use. The media were as follows: Rogosa V agar for Veillonella spp. (Rogosa et al. 1958), Tomato Juice Agar (Oxoid) pH 5.0 for Lactobacillus spp., Tomato Juice Agar adjusted to pH 7.0 for anaerobic sarcinae (Crowther 1971), anaerobic coccus medium + neomycin 100 pg/ml (Thomas & Hare 1954), VL medium + 10% baboon faecal extract and VL medium + menadione 0.5 pg/ml + haemin 1 pg/ml + 1% liver digest (Barnes & Impey 1971), and four selective antibiotic media (Finegold et al. 197 l), namely kanamycin blood agar, kanamycin-vancomycin laked blood agar, neomycin-vancomycin blood agar and rifampicin blood agar. All plates were incubated inside the chamber at 37 OC in a gas mixture consisting of 10% H, and 5% CO, in N, for five days. The organisms were identified broadly on colony and cell shape, reaction to Gram's stain, and redox requirements. Organisms were tested for their ability to grow in the presence of oxygen (aerobes and microaerophiles), and ability to grow anaerobically after previous exposure to oxygen on the laboratory bench for 2 h (non-fastidious anaerobes). Organisms which did not withstand this brief exposure to oxygen were considered to be fastidious anaerobes. The numerous Gram negative anaerobic rods were further grouped by their ability to grow on each of the four antibiotic media. Pure cultures of all organisms were subcultured on to enriched media; VL agar + 10% sheep blood was used for the anaerobes, while the aerobes were cultured on Nutrient Agar (Oxoid) + 7.5% sheep blood. Aerobes, yeasts, lactobacilli and C1. perfringens were inoculated into vitamin B,, assay medium adjusted to pH 7.0, from the enriched agar using a straight wire. The fastidious anaerobes would not grow in this assay medium, and were inoculated into enriched trypticase soy broth (Aranki et al. 1969). The latter medium was found to contain up to 0.04 ng/ml vitamin B,,, and a control medium tube was included in every assay, and its vitamin B,, content subtracted from all assay figures. Both aerobic and anaerobic cultures were incubated at 37 OC for four days prior to assay.
,
Vitamin B assay method One ml of each sample was suspended in 10 mlO.25 M-acetate buffer pH 4-6,then 0.25 ml of 0.1% (w/v) NaCN was added and the solutions stored at -2OOC before assay. Thawed samples were shaken for 5 min, 0.25 ml of 0.5% (w/v) papain was added to each sample and the volume made up to 20 ml with distilled water. The suspensions were then incubated at 60 O C for 1 h followed by autoclaving at 115 "C for 8 min. After centrifuging, the supernatants were assayed microbiologically for vitamin B using Lactobacillus leichmannii (Skeggs et al. 1950; Coates et al. 1953; Spray 1955).
,,
TABLE1 Vitamin B I 2content (ng/gm) of gastro-intestinal aspirates from two young vitamin B,, deprived baboons Small intestinal quarters A
I
Baboon
3
Stomach
Duodenum
First
Second
Third
Fourth
0.32 0.05
0.44
0.93 0.79
0.68 0.06
0.72 2.45
0.69 0
~
A
B
1.09
i
Yeasts Aerobic Gram positive cocci Anaerobic Gram positive cocci Anaerobic Gram negative cocci Micro-aerophilic Gram positive rods CI. perfringens Other anaerobic Gram positive rods
-f -denotes no organisms isolated from this region.
0.03 0.11 0.04 12.70 0.044~152 -
0.10 -
-
W.603 3.22
1.48 -
-
0.07 0.03-0.092 -
CU).154
I
3.20 6.60 0.15-0.18’ 0.10 3.60 0 0.03~~123 0.05-0.142 0 1.08 -
~ 1 . 1 1 4
0.02 03 -
0 03 0.62
0 03 0-1.313 4.33 23.60 0.09 -
0 *OZ
Yeasts Aerobic Gram positive cocci Anaerobic Gram negative cocci Micro-aerophilic Gram positive rods CI.perfringens Other anaerobic Gram positive rods Aerobic Gram negative rods Anaerobic Gram negative rods
--t
Second
First
10.0
Total number of strains isolated*
0 0
68 6
1
0
8 13
1
47
5 0 23 30
32 45 17 296
* Faecal samples collected from 18 animals selected at random from each of the dietary groups. All samples were cultured aerobically and 12 samples were also cultured anaerobically. strains of anaerobic organisms isolated, 48% of cultures contained > 1a 0 ng/ml vitamin B,,, and 23.6% of cultures, mainly Gram negative rods, contained >10 ng/ml. There were no noticeable differences in the types of faecal organisms isolated from controls or baboons fed any of the vitamin B,, deficient diets, with the exception of the group fed ampicillin. The faeces of these animals contained very few aerobic or anaerobic Gram positive organisms, the flora consisting mainly of aerobic and anaerobic Gram negative rods and yeasts. The patterns of vitamin B,, production by the faecal organisms were similar both within the vitamin B,, deficient groups, and between the vitamin deficient and control groups. Table 3 is therefore presented as a composite table showing the ranges of vitamin B,, produced by all the organisms isolated, irrespective of the diets which were fed. Table 4 compares the vitamin B,, produced in cultures of organisms isolated from gastric juice samples aspirated 6 h after feeding, from vitamin B,, deprived and control baboons. The number of isolates obtained from the gastric juice samples from the control group was low in comparison to the vitamin deprived group. There were also fewer isolates producing the higher levels of vitamin B,, obtained from the control baboon gastric samples. No organisms producing >10 ng/ml vitamin B,, were isolated from the gastric juice samples.
VITAMIN Biz PRODUCTION BY BABOON GUT FLORA
339
TABLE 4
Vitamin B,, production by organisms isolated from baboon gastric juice collected 6 h afterfeeding either a diet dejicient in vitamin B , , or a vitamin B , , supplemented diet
--Number of strains in each range of vitamin B production (ng/ml culture) A
r
Vitamin B,, deprived diet*
0-0.1
0.11-1.0
Aerobic Gram positive cocci Anaerobic Gram positive cocci Anaerobic Gram negative cocci Yeasts Micro-aerophilic Gram positive rods Anaerobic Gram positive rods Aerobic Gram negative rods Anaerobic Gram negative rods
33
5
0
7
1
0
0 14
2 0
0
12
6 9 3
4
4
4
0
2
Total number of strains
78
30
13
22
10
Organisms
3 5
1.01-10.0
Vitamin B ,* supplemented diet?' 04.1
0.11-1.0
\
1.01-10.0
1 1 5 0
4 2 1
1 6 0
* Ten animals sampled. t Five animals sampled.
The Gram negative anaerobic species producing the highest levels of vitamin B,, were not identified. However, four type cultures (National Collection of Type Cultures, Central Public Health Laboratory, Colindale Avenue, Colindale, London NW9 5HT) were assayed with the following results: Bacferoidesfragilis (NCTC 8560), 0.13 ng/ml; Fusobacterium polymorphum (NCTC 10562), 72 8 nglml; Sphaeropherus varians (NCTC lO560), 69.6 ng/ml; and anaerobic Gram negative coccus (NCTC 9805), 0.14 ng/ml. Cultures obtained from Gram negative rods originally isolated on rifampicin blood agar and on neomycin-vancomycin blood agar, always produced TABLE5
Vitamin B,, content (nglgm),p H values and total bacterial counts (x109/mlgastric juice) of baboon gastric juice samples taken 6 h after feeding either a vitamin B,, deficient diet or a vitamin B,, supplemented diet
--
Vitamin B,, deprived animals*
~~
PH Vitamin B,, content Total bacterial count
* There
~
Control animals
Meant
S.E.
Mean
S.E.
3.53 1.14 1.78
0.54
3.13 1.62 1.83
0.29 0.40 1.20
0.27 0.33
were no significant differences between mean values from vitamin B,, deficient or vitamin B,, supplemented animals, P > 0.10. t Mean values with their standard errors for ten animals fed a vitamin B,, deficient diet and five control animals.
P. F. UPHILL, F. JACOB AND P. LALL
340
TABLE6
Comparison of p H values and vitamin B , , content (nglgm aspirate) in gastric juice samples taken after an 18 h fast or a 6 h fast from baboons fed either a diet deficient in vitamin B , , or a diet supplemented with vitamin B , , ~~~
~
~~~
~
18 h fast Diet
6 h fast
&
Factors measured
&
Mean*
S.E.
Mean
S.E.
B,, deficient
[ PH
I B,, content
1.64 3.90
0.14 0.58
3.07 0.98
0.23 0.13
B,, supplemented
{ gf content
1.50 2.54
0.09 0.55
3.28 1.so
0.30 0.10
>o. lot >o. 10
B,, deficient B,, supplemented
0.01
(0.001
B,, deficient
B,, supplemented
>o. 10 o. lot
0.01
>o. 10 >o. 10
B,, deficient B,, supplemented
* Mean values with their standard errors for 18 animals fed a vitamin B,, deficient diet and 8 animals fed a vitamin B,, supplemented diet.
t Probability values.
many of the 6 h fasting samples in both dietary groups, but from few of the 18 h samples.
Discussion Since it is well established that microbial synthesis provides the main source of vitamin B,, for the ruminant (Hungate 1966), it seems logical to assume that the organisms most likely to produce the vitamin in quantity would be anaerobic. The vitamin B,, producing capacity of micro-organisms has not been extensively documented, and comparison of the reported results is difficult due to the use of differing assay methods. The variable composition of culture media in which isolates were grown for assay must also affect the quantities of vitamin produced. Kon et al. (1969) reviewed vitamin B,, production by rumen micro-organisms and studied 615 strains of 15 species of rumen bacteria, of which 150 strains showed activity when assayed with Escherichia coli. In two studies on non-ruminants, however, aerobic organisms have been reported to synthesize quantities of vitamin B,, when assayed with L . leichmannii. Halbrook et al. (1950) isolated strains of Aerobacter aerogenes, yeasts, Bacillus megatherium and some moulds from poultry house litter and droppings, which produced >Om4 ng/ml vitamin B,2. In measurements of vitamin B,, production in horse gut contents, Davies (1971) found that the highest concentration of the vitamin was produced by a strain of E. coli (1 2 ng/ml). In a study of anaerobic organisms isolated from human faeces, Gall (1970) showed that 16 type cultures produced OvO1-0-32 ng/ml vitamin B,,. The aerobic isolates obtained from baboon faeces and gastric contents in this study produced little or no vitamin BIZ,although all these organisms grew well in the vitamin B,,-free assay medium in which the cultures were prepared. The highest levels of vitamin B,, were produced by the anaerobic isolates, in particular by C1. perfringens and some of the anaerobic Gram negative rods. It has been shown that vitamin B,, was present in the stomach and upper intestine of two of the young vitamin B,, deprived baboons. In addition, some of the micro-
342
P. F. UPHILL, F. JACOB AND P. LALL
organisms isolated from the gastro-intestinal contents of these baboons were capable of producing in vitro large quantities of vitamin BIZ.However, gastric juice samples from all the vitamin deprived and control baboons contained organisms capable of producing vitamin B 12 in vitro. No differences were detected due to different diets or age of animal, with the exception of the baboons receiving ampicillin. Therefore, the unusually high serum and liver vitamin B,, levels found in the young animals, but not in older baboons, remains unexplainable. The cultivation of such a variety of aerobic, microaerophilic and anaerobic organisms from the gastric contents of baboons fasted for 6 h may be due to the type of synthetic diet fed, and to the possession by baboons of facial cheek pouches in which food can be stored (Uphill et al. 1974). A fasting period would date from the moment that the pouch was empty, rather than from the time of withholding food. The lack of difference in vitamin B,, content between gastric samples taken from vitamin B,, deprived and control baboons 6 h after feeding would suggest that digestion was complete, since the control baboons received 2 pg vitamin B ,,/day. Comparative examination of gastric contents taken after withholding food for 6 h or 18 h, however, suggested that the baboon stomach had not completely returned to the resting state 6 h after feeding. The mean gastric pH of both vitamin deprived and control baboons being significantly lower after an 18 h fast. Evidence that the stomach and intestines, after a 6 h fast, contained more viable organisms than at 18 h is provided by the increased percentage isolation of CI.perfringens and Lactobacillus spp. at 6 h. The numbers of viable bacteria present in the 6 h fasting samples is unknown, but the total counts were high, being only 100 times lower than the total bacterial counts found in baboon faeces (unpublished data). This indicates that considerable bacterial multiplication had taken place in the stomach at some time before sampling. Vitamin B found in the gastro-intestinal contents of vitamin B,, deprived baboons could be derived from ingested food, desquamated epithelial cells, digestive secretions or from the bodies and/or the secretions of the gastro-intestinal microflora. Siddons (1974) reported that the vitamin-free casein used in these studies contained 0.004 pg/gm L. leichmannii growth-promoting ability, which, if it was due to vitamin B,,, could result in each baboon receiving 0.01 pg vitamin B,,/day. The three baboons fed the soya protein diet did not receive even this minimal supplementation however, but the mean 18 h and 6 h fasting gastric samples also contained high levels of the vitamin. The vitamin B,, content of desquamated epithelial cells or digestive secretions is unlikely to account for all of the vitamin B 12 found in the stomach contents of baboons deprived of the vitamin. These animals were showing evidence of vitamin B,, deprivation in their low serum and liver levels (Siddons 1974), but the mean vitamin B,, levels in their stomach contents 18 h after feeding were not significantly lower than those of control baboons. It would be impossible to state conclusively that the intestinal flora were largely responsible for the vitamin BIZfound in the baboon stomach and intestine, since production of the vitamin by an organism in vitro does not necessarily mean that comparable levels would be produced in viva However, this study does provide presumptive evidence that the vitamin B found in the baboon stomach and upper intestine could have been produced by microbial action. An increased vitamin B deficiency found in baboons fed 2 vitamin B deficient diet containing ampicillin (Siddons 1974) could be explained by the observed inhibition by the antibiotic of all the Gram positive flora. All the isolated strains of C1. perfringens produced 1-10 ng/ml vitamin B,, and many of the lactobacilli produced varying
,*
VITAMIN
BIZ
PRODUCTION BY BABOON GUT FLORA
343
amounts of the vitamin. Assuming that the vitamin B,, produced by these organisms in vitro was also being produced in vivo in the baboon stomach and upper intestine, it is possible that the vitamin was being absorbed and utilized to meet part of the animal’s nutritional requirements. Vitamin B,, produced by the Gram positive flora was unavailable to the baboons fed ampicillin and their vitamin BI2deficiency was increased. The faecal vitamin B,, content was not reduced in the ampicillin-fed baboons (Siddons 1974) and although the numbers of C1.perfringens and lactobacilli in their faeces were greatly reduced, there was a large increase in the number of anaerobic Gram negative rods, many of which produced > 10 ng/ml vitamin B,, in vitro (unpublished data). This study suggests that the chances of producing a vitamin B I Zdeficiency in the baboon might be improved by a change in its feeding habits. Smith (1965), showed that in fowls with ablated crops, and in ducks which have no crops, the bacterial content of the alimentary tract was lower than in ordinary fowls. Closure of the baboon facial pouches might also lead to a reduced intestinal bacterial population. In addition, the baboons used in this study were fed their daily ration in two meals with an interval of 5 h between. The results show that vitamin BIZ-producingorganisms would therefore be present in the upper intestine for at least 12 out of every 24 h. The feeding of only one large meal/day and a change of diet to a type less likely to cause an increase in the putrefactive and potentially vitamin B,,-producing flora could speed the development of vitamin B,, deficiency in the baboon. Alternatively the Gram positive vitamin B12producing flora can be eliminated by the daily feeding of small quantities of ampicillin. We should like to thank Dr P. Sayer for doing the laparotomies, Mrs A. Aplin for technical assistance, Mr R. A. Whittingham for arranging the construction of the anaerobic chamber and for animal maintenance and Dr R. Siddons for assistance in the preparation of this manuscript.
References ARANKI, A., SYED,S. A., KENNEY,E. B. & FRETER, R. 1969 Isolation of anaerobic bacteria from human gingiva and mouse caecum by means of a simplified glove box procedure. Applied Microbiology 17,568-576. BARNES,E. M. & IMPEY,C. S. 1971 The isolation of the anaerobic bacteria from chicken caeca with particular reference to members of the family Bacteroidaceae. In Isolation of’ Anaerobes The Society for Applied Bacteriology Technical Series 5, ed. Shapton, D. A. & Board, R. G. pp. 115-123, London & New York: Academic Press. COATES,M. E., FORD,J. E., HARRISON, G. F., KON,S. K. & PORTER, J. W. G. 1953 Vitamin B,,-like compounds. I. Vitamin B,, activity for chicks and for different micro-organisms of gut contents and faeces. British Journal of Nutrition 7,3 19-326. CRowTnER, J. s. 1971 Sarcina uentriculi in human faeces. Journal of Medical Microbiology 4, 343-3 50. DAVIES,M. E. 197 1 Production of vitamin B,, in the horse. British Veterinary Journal 127,3436. FINEGOLD, S. M., SUGIHARA, P. T. & SUTTER, V. L. 197 1 Use of selective media for isolation of anaerobes from humans. In Isolation of Anaerobes The Society for Applied Bacteriology Technical Series 5, ed. Shapton, D. A. & Board, R. G. pp. 99-108, London & New York: Academic Press. GALL,L. S. 1970 Normal faecal flora of man. American Journal of Clinical Nutrition 23, 14571465.
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HALBROOK, E. R., CORDS,F., WINTER,A. R. & SUTTON, T. S. 1950 Vitamin B,, production by micro-organisms isolated from poultry house litter and droppings. Journal of Nutrition 41, 555-563. HUNGATE, R. E. 1966 The Rumen and its Microbes London & New York: Academic Press. KON,S. K., PORTER,J. W. G., THORLEY, C. M. & WILLIAMS, A. P. 1969 The possible origin of vitamin B,, activity in the rumen. Vpamet na Akademik Ignat Emanuilov: trudore burkhy izbrani problemi nu mikrobiologiyata, enzimologiyata i biokhimiyata. Pashev, I. et al. pp. 203-207, Sofiya: Bulgarska Akademiya na Naukite. MATTHEWS, D. M. 1967 Absorption of water-soluble vitamins. British Medical Bulletin 23, 258-262. MICKELSON,0. 1956 Intestinal synthesis of vitamins in the non-ruminant. Vitamins and Hormones 14,l-95. ROGOSA,M., FITZGERALD, R. J., MACKINTOSH, M. E. & BEAMAN,A. J. 1958 Improved medium for selective isolation of Veillonella. Journal of Bacteriology 76,455-456. SIDDONS,R. C. 1974 The experimental production of vitamin B,, deficiency in the baboon (Papio cynocephalus). A two-year study. British Journal of Nutrition 32,2 19-228. SKEGGS,H., NEPPLE,H. M., VALENTIK,K. A., HUFF, J. W. & WRIGHT,L. D. 1950 Observations on the use of Lactobacillus leichmannii 4797 in the microbiological assay of vitamin B,y Journal of Biological Chemistry 184,211-22 1. SMITH,H. W. & CRABB,W. E. 1961 The faecal bacterial flora of animals and man: its development in the young. Journal of Pathology and Bacteriology 82,53-66. SMITH,H. W. 1965 Observations on the flora of the alimentary tract of animals and factors affecting its composition. Journal of Pathology and Bacteriology 89,95-122. SPRAY,G. H. 1955 An improved method for the rapid estimation of vitamin B,, in serum. Clinical Science 14,661-667. STOKSTAD, E. L. R. 1968 Experimental anaemias in animals resulting from folic acid and vitamin B,, deficiencies. Vitamins and Hormones 26,443-463. THOMAS, C. G. A. & HARE, R. 1954 The classification of anaerobic streptococci and their isolation in normal human beings and pathological processes. Journal of Clinical Pathology 7,300-304. UNDERWOOD, E. J. 1956 Trace Elements in Human andAnima1 Nutrition (2nd edn. 1962) New York: Academic Press. UPHILL,P. F. 1973 A quantitative comparison of the faecal microflora of baboons fed a natural diet or a synthetic diet complete or deficient in pyridoxine or riboflavine. Journal of Applied Bacteriology 36,501-5 1 1 . UPHILL,P. F., WILDE, J. K. €3. & BERGER,J. 1974 Repeated examinations, using the laparotomy sampling technique, of the gastro-intestinal microflora of baboons fed a natural or a synthetic diet. Journal of Applied Bacteriology 37,309-3 17.
