humanfecal flora: variation in bacterial composition within … · 360 holdeman, good, and moore...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1976, p. 359-375Copyright © 1976 American Society for Microbiology

Vol. 31, No. 3Printed in USA.

Human Fecal Flora: Variation in Bacterial CompositionWithin Individuals and a Possible Effect of Emotional Stress

L. V. HOLDEMAN,* I. J. GOOD, AND W. E. C. MOORE

Anaerobe Laboratory* and Department of Statistics, Virginia Polytechnic Institute and State University,Blacksburg, Virginia 24061

Received for publication 28 August 1975

Data are presented on the distribution of 101 bacterial species and subspeciesamong 1,442 isolates from 25 fecal specimens from three men on: (i) their normaldiet and normal living conditions, (ii) normal living conditions but eating thecontrolled metabolic diet designed for use in the Skylab simulation and mis-sions, and (iii) the Skylab diet in simulated Skylab (isolation) conditions. Thesebacteria represent the most numerous kinds in the fecal flora. Analyses of thekinds of bacteria from each astronaut during the 5-month period showed more

variation in the composition of the flora among the individual astronauts thanamong the eight or nine samples from each person. This observation indicatesthat the variations in fecal flora reported previously, but based on the study ofonly one specimen from each person, more certainly reflect real differences (andnot daily variation) in the types of bacteria maintained by individual people.The proportions of the predominant fecal species in the astronauts were similarto those reported earlier from a Japanese-Hawaiian population and were gener-ally insensitive to changes from the normal North American diet to the Skylabdiet; only two of the most common species were affected by changes in diet.However, one of the predominant species (Bacteroides fragilis subsp. thetaio-taomicron) appeared to be affected during confinement of the men in the Skylabtest chamber. Evidence is presented suggesting that an anger stress situationmay have been responsible for the increase of this species simultaneously in allof the subjects studied. Phenotypic characteristics of some of the less commonisolates are given. The statistical analyses used in interpretation of the resultsare discussed.

This is the second of a series of reports con-cerning human fecal bacterial flora and thefactors that may, or may not, affect the flora. Ina previous work (11), we reported the kinds ofbacteria predominant in the feces of 20 normalindividuals, each sampled once. Work on theNational Aeronautics and Space Administra-tion's Skylab Medical Experiments AltitudeTest (SMEAT) provided an opportunity for us toexamine the bacterial flora of several fecal sam-ples from each of three individuals under nor-mal and test (controlled balanced diet and isola-tion environment) conditions, which should en-able us to determine how well a single speci-men (as examined previously) represents thetypical flora of an individual and to evaluatethe effects of the controlled, balanced, Skylabdiet and/or an isolation environment on thequalitative and quantitative composition of theflora. In addition, we hope to ascertain whetheror not the floras from the normal Japanese-Hawaiian population studied previously could

be considered the same as or similar to thebacterial flora of normal North Americans.The fecal microbiology reported here was one

of many physiological studies on the threeSMEAT crew members. A comprehensive de-scription of the conditions and parameters ofthe SMEAT experimentation is published else-where (12).

MATERIALS AND METHODSSubjects. The Caucasian male astronauts are

called "R" (Commander, age 35), "T" (Scientist-Pilot, age 43), and "B" (Pilot, age 35).

Fecal specimens. To estimate the normal fecalflora of the individuals, fecal specimens were ana-lyzed from each man on days 43, 28, and 1 beforethey started eating the Skylab diet and while theywere working on flight training and living at home.To estimate the effect ofthe Skylab diet per se on theflora, a fourth sample was obtained from each man22 days after the Skylab diet had been initiated butwhile the men were still living at home. Three sam-ples, obtained after 15, 30, and 56 days of confine-

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360 HOLDEMAN, GOOD, AND MOORE

ment in the test chamber while the Skylab diet wasstill being consumed, were studied to evaluate thecombined effect of Skylab diet and environment onthe fecal flora. An eighth specimen was obtainedfrom each man 14 days after egress from the cham-ber and return to normal living conditions, exceptthat the Skylab diet was still being consumed. Aninth specimen was obtained from one of the men(T) 24 days after he returned to his normal diet andnormal living conditions.Chamber conditions. In the chamber, the three

men were under reduced atmospheric pressure (5 lb/in2) and an increased oxygen concentration of 70%oxygen-30% nitrogen. The men had no direct or per-sonal contact with the outside environment. Materi-als were exchanged through air locks and there wasstrict adherence to the dietary regimen.

Skylab diet. The astronaut diet contained a con-siderable variety of foods, which were prepared forlong-term storage. All foods were canned, frozen,irradiated, or dehydrated. The actual menu itemsdiffered for each man at each meal but in generalrepresented the variety and types of food that wouldprobably be consumed normally. Details of the dietscan be found in the complete report of the SMEATstudies (12).

Bacteriological studies. F'ecal specimens werecollected in plastic bags with no precautions to ex-clude air and were transported to the laboratorywith as little delay as possible. However, at times upto several hours elapsed before processing could beinitiated. On receipt in the laboratory, each plasticbag was thoroughly flushed with oxygen-free carbondioxide. The specimen was thoroughly mixed andcultured exactly as described previously (11) by thesame persons who processed the specimens in theearlier study.

After overnight incubation, the rumen fluid-glu-cose-cellobiose agar roll-tube cultures were trans-ported by air (in the passenger cabin) at ambienttemperature from Houston, Tex., to Blacksburg,Va., for reincubation. After 5 days' total incubationtime, 55 colonies were picked in a randomized man-ner from each specimen. Cultural counts, micro-scopic counts, determination ofthe percentage of drymatter of the fecal specimens, and the identificationof all isolates were each performed as described pre-viously (7, 8, 11).

Statistical analyses. (i) Cultural and microscopiccounts. The cultural counts were compared with thedirect microscopic clump counts (DMCC) by using apaired t test. This test was made more accurate bymeans of a square-root transformation, whichmakes the distribution of the counts more closelynormal as can be seen by plotting the data.

(ii) Analyses of variance of predominant species.Because the observed data indicated that the distri-bution of the species was neither normal nor Pois-son, the statistical analyses of the data in Table 4were performed on a transformation of the values togive approximately normal distributions with ap-proximately equal variances. This transformedvalue is equal to the square root of [(observed value/2.1) + (3/8)]. This transformation was used becausethe ratio of the variance to the mean for the normal

APPL. ENVIRON. MICROBIOL.

diet days is 2. 1, and it results in a more conservativetest than would be produced with the observed val-ues. By using [V(x +31/)], one obtains a knownnormalizing and variance-standardizing transfor-mation for Poisson variables (1) for which the vari-ance is, of course, equal to the mean (also see Ap-pendix [b]).

RESULTSCultural and microscopic counts. The cul-

tural counts and DMCC per gram of dry matterof the 25 specimens are given in Table 1. Theaverage values for the 25 specimens were: drymatter, 31%; DMCC, 4.08 x 10"/g of dry matter(standard deviation = 2.73 x 101"); culturalcount, 2.56 x 10"/g of dry matter (standarddeviation = 1.14 x 10"); for an average culturalrecovery of 63.2% of the bacterial cells observedmicroscopically. The paired t test, using asquare-root transformation, gives a single tail-area probability of 0.008 (t = 2.566 with 24 df).(Without the square-root transformation, t =2.717, and hence a single tail-area probabilityof about 0.006. The single tail is justified be-cause it is more likely in advance that thecultural count would be lower than the DMCC.)Therefore, it is probable that the cultural countis low. Neither the DMCC nor the culturalcounts depend significantly on the treatmentstested.Composition of the flora. The 101 different

species and subspecies observed among 1,442isolates from all 25 specimens are listed, indescending order of their frequency (rank or-der), in Table 2. These 101 species account for 95to 98% of the cultivable flora as determined bytwo different analyses: (i) by the sum of thefrequencies calculated by formula Hg of Good(4), and (ii) by the coverage as determined by"1 - number of species observed once/total num-ber of isolates" (also see Appendix [a]).Comparison of astronaut normal flora with

that of the Japanese-Hawaiian population.The frequencies of bacterial species from thethree astronauts (three or four samples each)living under normal conditions are comparedwith those previously reported from the Japa-nese-Hawaiian population (single fecal speci-mens from each of 20 individuals) in Table 3. Ingeneral, the rank order of species bears similar-ity to that for the Japanese-Hawaiian popula-tion, but, because of the large person-to-personvariation noted in both groups, one cannot de-termine with certainty whether the three astro-nauts and the Japanese-Hawaiians belong tothe same population with respect to their fecalflora.Analyses of variance of predominant spe-

cies. The numbers of isolates of predominant

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HUMAN FECAL FLORA 361

TABLE 1. Microscopic and cultural counts per gram of dry matter from 25 fecal specimens

Treatment

Subject Parameter Normal diet Skylab diet Normal dietSubjecParamAmbulatory In chamber 4 Ambulatory

May 100 May 25 June 22 July 20 Aug. 9 Aug. 24 Sept. 19 Oct. 4 Nov. 1

B % DMb 30.8 27.7 27.7 30.2 30.6 33.9 36.0 30.9DMCCC 4.2 3.8 4.2 2.8 10.8 1.7 2.4 1.7Cult.d 2.4 2.2 5.2 3.2 2.2 1.8 1.0 2.0

R % DM 27.6 35.0 15.7 30.9 33.2 32.6 27.0 28.2DMCC 8.9 2.9 3.6 2.1 8.5 1.4 3.0 8.7Cult. 2.4 1.0 3.6 1.6 1.8 3.6 2.7 5.4

T % DM 34.3 26.4 29.6 34.1 32.4 33.le 38.6 35.7 33.7DMCC 5.7 3.7 1.6 2.0 5.7 1.2 1.1 6.7 3.6Cult. 2.5 3.3 3.4 1.5 1.5 2.3 2.6 3.9 1.6

a Sample date ± 1 day. Treatment changes were made as follows: From normal diet to Skylab diet, June27; from ambulatory to chamber, July 25; from chamber to ambulatory, September 20; from Skylab diet tonormal diet, October 8.

bPercent dry matter (DM).c Number given times 101" = direct microscopic clump count (DMCC) per gram of dry matter. Average

DMCC = 4.08 x 10"/g of dry matter; standard deviation (s) = 2.73 x 10".d Number given times 10" = cultural count per gram of dry matter. Average cultural count = 2.56 x 10"/

g of dry matter; s = 1.14 x 10".e Estimated value from average for subject T. There was insufficient sample for direct determination.

species from the 25 individual astronaut fecalspecimens and the significance of the variationare given in Table 4. The values given for thenumber of isolates of a species are the actualnumbers of isolates from each specimen. Forconvenience in the statistical analyses, no cor-rection was made for the variation among thesetotals. Results of analysis of variance obtainedwith the transformed data were similar to thoseobtained using the untransformed data, whichshows that the analysis is robust relative to thestatistical model.Of the 15 predominant species analyzed, 7

showed significant person-person variation, 4showed significant treatment-treatment varia-tion, and 1 showed person-treatment interac-tion. No significant variation was noted with 6of the 15 species analyzed.

DISCUSSIONCultural and microscopic counts. In the

present study, the average cultural recoverywas 63% of the DMCC, whereas in the studyreported previously (11) it was 93% of theDMCC. The only known procedural differencebetween the two studies was a delay in trans-porting the specimens to the laboratory in thepresent study. Delays were unavoidable be-cause the men were in different locations atthe Manned Space Flight Center during thenon-chamber phase of the study and because oflogistics for taking materials through the airlocks during the chamber portion of the study.

The average percentage of dry matter was 10%higher in the present specimens than in thoseexamined previously, but the water contentwas still 61.4 to 84.3% (Table 1). Although wedo not know the effect of the moisture contenton the viability of the bacteria, we postulatethat the prolonged exposure to oxygen was themajor factor affecting the viable bacterialcount.Comparison of astronaut normal flora with

that of the Japanese-Hawaiian population.Gross examination of the data in Table 3 showsthat there is a general similarity of major bac-terial types found in the normal samples fromthe astronauts and in the Japanese-Hawaiianpopulation. The variation of the bacterial floraamong the individual astronauts is also shownin Table 3. To determine whether the astro-nauts' flora differed from that of the Japanese-Hawaiians, we compared the relative fre-quency of species (in rank order, with "1" beingthe most common) in the normal samples fromthe astronauts with the rank order of species ineach of the Japanese-Hawaiians. When therank order of species obtained from the normaldiet specimens from each astronaut was com-pared with the 20 rank orders of species fromthe individual Japanese-Hawaiian specimens,no two specimens were alike. Indeed, even ifone compared only the three most numerousspecies in each sample, or from one individual,no two people or specimens had the same spe-cies in the same rank order. If one compared

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Table 2. Relative frequency of' bacterial species of' the fecal flora of' 3 North A mnerican2 men, each sampled 8 or 9timnes daring a 5-month period, oni niormiial diet, astronazut diet, andLl in land-based Skylab chamiber (1442 isolates)

Rank No. Counta 0% of florab Organism(s)isol.

(x 1010)1 171 3.04(.23)2 144 2.56(.21)3 129 2.29(.20)4 96 1.70(.17)5 87 1.54(.17)6 64 1.13(.14)

(x 109)7 51 8.95(1.3)8 44 7.70(1.2)9 41 7.17(1.1)10 40 6.99(1.1)11 39 6.81(1.1)12-13c 33 5.74(1.0)14 31 5.38(1.0)15 27 4.66(.09)16 25 4.31(.09)17-18 24 4.13(.09)19 22 3.77(.08)20 21 3.59(.08)21 19 3.24(.08)22 18 3.06(.07)23 14 2.35(.06)24 13 2.17(.06)25-26 9 1.46(.05)27-31 8 1.29(.05)

32-35 7 1.11(.04)

(x 108)

11.8(.90)9.9(.83)8.9(.78)6.6(.68)6.0(.64)4.4(.55)

3.5(.49)3.0(.45)2.8(.44)2.7(.43)2.6(.43)2.2(.39)2.1(.38)1.8(.35)1.7(.34)1.6(.33)1.5(.32)1.4(.31)1.3(.29)1.2(.29).91(.25).84(.24).57(.20).50(.18)

.43(.17)

Bactero ides fragilis ss. v ulgaztusEubacteriumn aerofaciensBIactero)ildesfragilis ss. thetainotaoinicron0Peptostreptococcus produictuis 11Ba7cteroides fragilis ss. distasoni.sFiusobLacterium prausnitzii

Coprococcus eutactusEubacteriumn aerofaciens IIIPeptostreptococcus productuis IRumnin(cocclus bro,niiBifidobacteriumii? adolescenitisGem zniger formicilis, Bifido bacteriumiii lo ngug nFubacterlumn siraeuimRuininococcus torquiesF'ubacteriumn rectale III-HEuibacteriumn rectale IV, F'ibacteriumn eligenzsBacteroides eggerthiiClostridiumn leptlum71Bacteroides fr7agilis ss. aFuibacterilum biforiMeBifido bacterilum in/antisEubacteriumiii rectale III-FCoprococclus co nes, Bacteroides caipillosusRumin 0oco ccus albuis, Euabacterium formicigenerans, Eu bacteriumn hallii,Fubacterinini ven triosum I, Fusobacterilum russiiRuminococcus obeunmn, E-ubacteriunm rectale 11, Clostridium ramosum 1,Lactobacilluis leichmann ii

36-37 6 9.39(.40) .36(.16) Ruininococcus callidus, Butyrivibrio crossotus38-41 5 7.67(.36) .30(.14) Acidamniniococclus fermentans, Fuibatcteriuma venitriosumn, Bacteroides

fragilis ss. fragilis, Bacteroides AR42-47 4 5.96(.32) .23(.12) Coprococcus catus, Eubacteriuim hadrian, F. cylindroides, E. ruminan-

tium, Fuibacteriumn CH-1, Staphylococclus epidermidis48-57 3 4.29(.27) .17(.10) Peptostreptococcles BL, F'ubacterium limosumn, Bacteroides praeacutus,

Bacteroiles L, I usobacteriun mortif'erum 1, F. naviforme, Clostridiuminnocuuam, C. ramiiosum, Propiotnibacteriuim acnes, Ruminococcusflavefaciens

58-73 2 2.68(.21) .10(.08) Rurminiococcuis AT, Peptococcuis AU-1, Eubacteriunm AG, -AK, -AL,-AL-1, -AN; Bactero ites fragilis ss. ovatzus, -ss. d, -ss. f; Bacteroides L-1,L-5; Fusobacterium nucleatum, F. mortif'erumn, Escherichia coli,Streptoc occus morbillorium

74-101 1 1.19(.13) .05(.05) Peptococcus mnagnuis, Peptococcuis C, -AU-2; Streptococcus intermned-ius, RiumniniococcuIs lactaris, R uminococcus CO, Gemi iger X, Copro-coccuis BHI, -CC; F:ubacteriuni teniue, Eubacteriumn ramnlus, Eubacter-Iimmi AL, -AG-H, -AG-M, -AJ, -BW-1; Bactero ides clostridiiformis ss.clostridiif'ormis, B. coagulans, B. oralis, B. ruminicola ss. brevis, -ss.runiiuicola, Bacteroides splanchnicus, IDesulfo monas pigra, BacteroidesL-4, -W-1; Ftusobacteriiin H, Lactobacilllus G, Succinivibrio A

a The estimated count per gram ofparentheses).

fecal dry matter (the standard deviation of the estimate is given in

b The percentage of the fecal population (the standard deviation of the estimate is given in parentheses).c Where two rank numbers are listed, each organism cited was detected with equal frequency.


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Table 3. Frequency of'bacterial species in feces of'difj'erenit individuials unider- normal conditions

Person B 20 Person R 20 Person T 20Three normal specimens I-H" Three normal specimens J-H Four normal specimens J-H

No. Species Isol. / No. Species Isol. / No. Species Isol. /isol. persons Isol. persons isol. persons

30 f'. aerofaciens20 B. f'ragilis ss. znlgatus18 P. productus 1111 B. f'ragilis ss.

thetalotaomicron8 E. rectale 111-H7 F. prausnitzii7 C. eutactus6 R. torques5 P. productus I5 B. f'ragilis ss. aS B. f'ragilis ss. distasoni.s3 G. f'ormicilis3 Rumninococcu.s obelin3 B. eggerthii3 B. adolescentis3 L. leichmannii2 R. albus2 R. callidlus2 R. flavefaciens2 E. formicigenerans2 Eubacteriumn badrum2 Bacteroides L-12 B. longum2 B. capillosus1 C. comnes1 P. magnus1 Peptostrep. BL1 F. aerofacien?s III1 F. ballii1 E. rectale III Fubacterilum AG1 fulibacterium AG-M1 B. coagulans1 B. fragilis ss. f'ragilis1 Bacteroides AR1 F. navif'orme1 F. russii

70/13140/1951/17

52/165/2

80/145/39/5

39/1526/9

28/1121/814/91/1

75/146/5

13/65/30/04/40/01/1

22/72/26/40/00/0

29/91/1

27/112/20/00/08/40/00/04/2

23 F'. aerofjacienis 70/1320 R. bromnii 17/917 B. fragilis ss. viulgatuis 140/1911 B. adolescentis 75/1411 B. longum 22/710 B. fragilis ss. distasoni.s 28/117 P. productus 11 51/176 B. fragilis ss.

tbetaiotao nicroni 52/165 F. prausnlitzii 80/144 El3bacteriuzia CH-1 0/04 R umiOCOCcUS obeumI 14/9b3 F. rectale 1ll-Il 5/23 P. productus I 39/153 R. albuis 13/63 B. praeacutius 0/02 R. torques 9/52 F. aerofacienis lll 70/132 F. f'orinicigetnerants 4/42 Fubacteriumiz AL-1 0/02 B. capillosus 2/22 Bacteroides L 4/32 F. russii 4/22 L. leicb,nannlii 6/51 C. catuis 6/31 CG. f'ormiiicilis 21/81 R uminococcus lactaris 0/01 Rumniinococcus AT 0/01 F. limosum 1/11 F-'. rectale Ill-F 10/61 Fubacterium siraeum 0/01 B. fragilis ss. f 3/31 B. oralis 0/01 Bacteroides L-4 0/01 Bacteroides L-5 0/01 F. mtiortiferumn I 0/01 Clostridininii leptiuni 0/01 B. infantis 18/41 Lactobacillus G 0/01 S. epidermidis 2/2

35 B. f'ragilis ss. vulgatus29 P. productus II16 F. pralusnitZii14 F. biforme14 E. rectale IV13 B. f'ragilis ss.

tbetaiotaomicron12 C. eutactus11 F. eligens10 B. f'ragilis ss. distasonis8 E. rectale Ill-H7 B. eggerthii6 Buityrivibrio crossotlus5 C. comnes5 G;. formicilis5 P. productus I4 R. brom??1ii4 E. aerof'acien.s lI3 1.. aer-ofacienis2 R. callidlus2 K. torques2 Pep tococcus AU-12 F. f'ormnicigenieranis2 Eubacteriiumii badrumii2 F. russiiz2 C. ramosum2 P. acnes1 C. catuis1 R. flavefacienis1 Rumnin7ococcus CO1 F. balliu1 F. rectalc 111 F. ruminantinm1 F. tenie1 E. ventriosimi I1 Eubacterium siraeumn1 F'ubacteriumti AE1 Fubacteriuim AJ1 Fubacteriumn AK1 Fubacteriuim AL1 B. fragilis ss. fragilis1 B. f'ragilis ss. d1 B. ruminicola ss. brevis1 B. " ss. rilninicola1 Bacteroides AR1 Succinivibrio A1 F. naviforme1 F, unlxcleatuon1 Fusobacterilum H1 B. lotngumn1 L. leichmnanniii1 S. epidermidis

140/1951/17b80/1438/1023/6

52/165/3

43/1228/11

5/21/10/06/4

21/839/1517/929/9

70/135/39/50/04/40/04/23/13/26/30/00/01/1

27/112/20/00/00/01/10/04/30/08/45/42/10/00/00/00/00/00/0

22/76/52/2

isolated in the Japanese-Hawaiian

b Ruminococcus obeumn was not differentiated from Peptostreptococcuis productus 11 in our report of the

Japanese-Hawaiian population (11).

a Number of isolates/number of persons from which this species was

population of 20 people (II).

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TABLE 4. Numbers of isolates ofpredominant species from individual specimens

Treatment

Species Normal diet Astronaut diet Astronaut diet in chamber pa

Bb R T B R T B R T

B. fragilis subsp. 5 10 5 10 4 3 11 6 8 10 7 3 1 3 12 7 5 4 6 8 6 3 14 17 3 *pvulgatus

E. aerofaciens 14 8 8 10 13 - - 3 -- 15 16 2 4 - 1 15 13 12 1 3 1 2 2 1 ***PB. fragilis subsp. 4 5 2 4 1 1 3 4 4 2 1 4 2 3 7 5 5 19 1 7 12 4 6 13 10 *T

thetaiotaomicronP. productus II 3 2 13 5 1 1 3 13 8 5 7 - 5 5 6 5 1 1 - 2 2 1 7 *TB. fragilis subsp. 1 2 2 2 2 6 2 3 1 4 - 5 4 4 5 5 1 5 2 5 5 4 6 8 3 NS

distasonisF.prausnitzii -2 5 3 1 1 1 3 4 8 3 3 2 7 - 5 - 2 3 1 2 5 - 2 1 NSC. eutactus 3 - 4 --- 2 1 4 5 6 1 -- 3 7 11-- - - 2 2 *pE aerofaciens III - 1 - 1 1 - 1 2 1 - 1 - 6 4 5 - 6 5 2 3 1 4 **P*TP.productus I 2 2 1 1 2 - 1 - 3 1 2 2 6 2 - 1 4 1 1 1 4 1 3-- NSR. bromii --- 1 1 18 --4 - 1 - 1 3-4 1 6 *pB. adolescentis 2 - 1 3 8 - - - 1 2 - 1 - 2 3 2 3 7 - - 4 - NSG. formicilis --3 1 2 1 2 - 2 2 1 2 4 1 2 2 1 3 - 2 2 NSB.Iongum 1 1 - 3 7 1--- -- - 7 3 2 4 2 --- *pE siraeum 1 - 1 -- 6 7 - 1 7 7 1 ***P**TIR. torques 1 3 2 - - 2 ---2 2 - 1 1 - 1 212 2 1 - 2 - 2 NS

Total no. of isolates 50 55 61 54 61 47 49 58 56 79 66 54 59 56 53 57 1 57 72 50 55 66 51 55 66 55_a *, Significant at 0.05 level; **, significant at 0.01 level;

treatment; I, interaction.b Person tested.

only the most numerous two species found inany one individual, only astronaut B and Japa-nese-Hawaiian specimens 10 and 22 were thesame, with Eubacterium aerofaciens and Bac-teroides fragilis subsp. vulgatus having thesame rank order (Table 3; 11). The several nextmost frequent species were dissimilar. There-fore, because of the large person-to-person vari-ation in the flora among both populations, wecannot say that the astronauts and the Japa-nese-Hawaiians do, or do not, belong to thesame population with respect to their fecalflora. In these comparisons, the greatest simi-larity was seen in isolates from the multiplesamples from individual astronauts.

Person-to-person variation. To determinewhether the apparent person-to-person varia-tion in composition of the flora was real or onlyreflected small sample sizes or simply largedaily variation within individuals, an analysisof variance was performed on the 15 most nu-merous species in the astronaut population (Ta-ble 4). For a number of species, the frequencywas reasonably constant between differentspecimens and also between different men. Fora number of other predominant species, varia-tion between men was significantly greaterthan variation between different sampleswithin men. Indeed, Coprococcus eutactus didnot appear at all in subject R, and this is clearlyvery strong evidence by itself that the floradiffers from one individual to another. There-

***, significant at 0.001 level; NS, not significant; P, person; T,

fore, these analyses indicate that there are sta-tistical differences in the composition of theflora of the three men. Additionally, it appearsvirtually certain that the differences observedpreviously in the composition of the flora ofdifferent individuals, each sampled only once,do reflect a real difference in the floras thatdifferent persons tend to maintain and do notresult from sampling problems or wide dailyvariation within each individual.The analyses of variance were performed in-

dependently on the 15 most frequent species.Therefore, one or more of the differences de-tected could have occurred by chance alone.However, the statistical differences in the con-centrations of E. aerofaciens, C. eutactus, E.aerofaciens II, Ruminococcus bromii, and Bifi-dobacterium longum are also similar to thedifferences among the specimens from differentindividuals reported previously (11). It also ap-pears that the species responded independentlyof each other to person, treatment, or person-treatment interaction.

Effect of diet change on the flora. The anal-yses of data shown in Table 4 also indicate thatsome species were sensitive to a change fromthe astronauts' normal diet to the Skylab diet,whereas other intestinal species were appar-ently indifferent to the dietary change.The response of E. aerofaciens III to treat-

ment appeared to be related to the diet ratherthan to conditions in the chamber. The propor-


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tions of this species increased noticeably in twoof the men and reached 10.2 and 10.9% of theisolates from single specimens in one of themen (B) while on the Skylab diet. However,this bacterial species constituted 14.5 and 11.1%of the isolates from two individuals ofthe Japa-nese-Hawaiian population on their normaldiets; therefore, it is unlikely that its increasewas caused by unique properties of the Skylabdiet per se.

Eubacterium siraeum also responded to thechange of diet in one (R) of the two astronautsin which it was present when on normal diet.This species was not detected in the Japanese-Hawaiian population. Therefore, the signifi-cance of its response to the Skylab diet in one ofthe men is not known.

Effect of conditions in the test chamber onthe flora. One species (B. fragilis subsp. the-taiotaomicron) was apparently affected by en-vironmental changes in the test chamber.Changes in the proportion of this species in allthree men are illustrated in Fig. 1. For a moreanalytical approach to the significance of thissimultaneous increase in all three astronauts,see Appendix (c) and (f). Figure 2 shows thecorresponding graphs for Peptostreptococcusproductus II, but the effect illustrated in Fig. 2may be mere coincidence, as is shown in Ap-pendix (c), which takes into account the specificselection of the species.When the increase in the proportion of B.

26

24

22-

20

18

16

0 14

120

at 10

8.

6.

4.

2.

* = B* = R* = T

May May June July Aug Aug Sept Oct Nov10 25 22 20 9 24 19 4 1

|H Skylab diet~- chamber

FIG. 1. Percentage of isolates, from different fecalspecimens, that were B. fragilis subsp. thetaiotaomi-cron.

24

22

20-

18-

16-

5 14-

- 12-0-

ae 10-

8-

6-

4-

* = B= R

* = T

I \ I

'N

/ I

\\- I",May May June July Aug Aug Sept Oct Nov10 25 22 20 9 24 19 4 1

1 - Skylab diet -*- chamber -

FIG. 2. Percentage of isolates, from different fecalspecimens, that were P. productus II.

fragilis subsp. thetaiotaomicron in the chamberwas first noted, we wondered whether it repre-sented an increase in the strains normallymaintained by each man or whether there was"cross contamination" due to the confined andisolated conditions in the chamber. In prelimi-nary testing we had noted that the strains ofB.fragilis subsp. thetaiotaomicron isolated fromone astronaut were resistant to tetracycline attest levels (17) and those from the other twoastronauts were susceptible. Therefore, repre-sentative strains of this species from each manwere tested by broth disk analyses (17) for theirresistance to tetracycline. Throughout thetrial, the tested strains from one man wereresistant and those from the other two menwere susceptible, indicating that there was aproliferation of each individual's own strainsrather than cross contamination between themen in close confinement.The rise in the proportion ofB. fragilis subsp.

thetaiotaomicron did not correlate with cham-ber conditions per se, since there was a simulta-neous decrease in this species while the menwere still in the chamber. The only factor thatappeared to correspond with both the simulta-neous increases and decreases in the proportionof this species is that the proportion increasedin response to an anger stress situation thatdeveloped during the test and decreased as thatsituation was reconciled. This would indeed bea very hypothetical and tenuous surmise if asimilar change in flora had not been independ-ently noted by another investigator and also by

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us in two other studies. Because of the potentialsignificance of this observation, and of the var-ious inherent problems in defining "stress," wewish to pursue this point in some detail and citethree other apparently similar cases.

Effect of stress on the flora. Soon after weobserved the high level of B. fragilis subsp.thetaiotaomicron in the three men simultane-ously, we observed a similar high value fromone of five other persons who were participatingin a different diet study (Abstr. Annu. Meet.Am. Soc. Microbiol. 1973, G109, p. 44). At thetime (March, 1973) that this person had a highlevel ofB. fragilis subsp. thetaiotaomicron (Ta-ble 5), she was experiencing a strong personal-ity conflict with her co-workers and was underconsideration for dismissal. Subsequently theproblems resolved and the proportion of thisspecies was lower in later specimens. The sta-tistical significance of 11 strains of B. fragilissubsp. thetaiotaomicron of 57 isolates in thesecond specimen (3/5/73) can be described ashaving a tail-area probability of 1:16, as pre-sented in more detail in the Appendix (d).

Coincident observations of the high propor-tion of B. fragilis subsp. thetaiotaomicron insome specimens from the astronauts and in onespecimen from the person in the diet studysuggested that stress might influence the pro-portion of this species in fecal specimens. Totest this hypothesis, specimens were obtainedfrom graduate students on the day of their pre-liminary oral examinations (a stress time) andagain 2 weeks later. There was no significantdifference in the proportion ofB. fragilis subsp.thetaiotaomicron isolated from the specimensobtained the day of examination and those ob-tained later (Table 6), and the relative numbersof the isolates of this species from the graduatestudents were within the range observed formost people. Clearly, stress per se did not in-crease the levels of B. fragilis subsp. thetaio-taomicron in these individuals. We realized, inretrospect, that the stress experienced by thestudents was of a different nature from that of

TABLE 5. Proportion of B. fragilis subsp.thetaiotaomicron in fecal specimens from one

individuala

No. of B. fragilisDate subsp. thetaio- % of isolatesDate ~taomicron/total %ofilae

no. of isolates

1/10/73 0/60 0.03/05/73 11/57 19.45/22/73 6/59 10.29/17/73 6/57 10.5

a Only normal diet is considered in this table.

TABLE 6. Proportion of B. fragilis subsp.thetaiotaomicron in fecal samples from three

graduate students

No. of isolates ofB. fragilis subsp. the-

Subject taiotaomicron/total no. of isolates

Day of exam Ca. 2 weeks later

S-09 2/53 2/52S-10 1/61 0/58S-11 3/63 Not tested

the astronauts and of the person on the dietstudy. It appeared that an anger stress situa-tion might be the correlating factor. The casescited below are in support of this hypothesis.

Recently we found high levels of B. fragilissubsp. thetaiotaomicron in the intestinal tractof a 19-year-old-woman who was shot by herhusband who, in turn, committed suicide. Inthis person the proportions of this species were:ascending colon, 17/51 (33%); transverse colon,8/5o (16%); descending colon, 4/57 (7%); rectum,5/55 (9%). Since the proportions of observed spe-cies in these different areas of the tract wereremarkably uniform in other individuals(Abstr. Annu. Meet. Am. Soc. Microbiol. 1975,D57, p. 61), the high proportion of B. fragilissubsp. thetaiotaomicron in the ascending colonof this woman suggests a rather rapid responseto probable anger or fear stress for a periodprevious to death. The statistical significance ofthe high level in the ascending colon can bedescribed as having a single-tailed probabilityof 1:37 (details of the analysis are presented inAppendix [e]).More recently, Bruce Maier, University of

Missouri, in an independent study, observed acontinual increase in the proportion of B. fra-gilis subsp. thetaiotaomicron in feces from 12individuals who were fed a monotonous diet for60 days (personal communication). These indi-viduals became extremely angry and argumen-tative with the test supervisors concerning theconditions of the test and the requirement toconsume the exact same menu day after day.The rise in B. fragilis subsp. thetaiotaomicronwas accentuated as the experiment continuedthrough a change in diet from low meat to highmeat. Dr. Maier is preparing a detailed reportof the microbiology of this study for publication.Because anger stress appeared to be a com-

mon factor in any specimens in which we noteda high relative frequency of B. fragilis subsp.thetaiotaomicron, we investigated in more de-tail the conditions in the NASA SMEAT trial.An understandable anger stress situation ap-parently did exist during the study, especiallyfor one astronaut (T) who felt his diet had been


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miscalculated (which it proved to be; he lost 19lbs. during the study), and because of equip-ment failure and misunderstandings betweenthe astronauts and the chamber support per-sonnel. Stress aspects of this study are de-scribed in the SPT report (16): ". . . At no timewas an angry or irritable word exchanged be-tween crew members.... The most obviouslystressful aspects of the test were those timeswhen it appeared that data were being lostafter considerable effort on the part of the crewto gather it [sic], especially if it had involveddifficulty on their part. Examples of this were:continued runs with faulty gear which wasapparently not being repaired; obviously erro-neous lab data such as the polyethylene glycol[one capsule was taken at each meal during thestudy and used as a non-absorbable fecal recov-ery standard for nutrient balance] when manyhours were expended counting pills to checkthat the material was correctly consumed ateach meal (a nuisance in itself); or seeing incor-rect data repeating that had been previouslycorrected by the subjects. Also stressful was theimpression that arose from some situationsthat the crew were being used as experimentalanimals rather than participating investiga-tors in an experiment. Some [outside] investi-gators were never able to accept or tolerate anyview of the situation other than investigator-subject. There was unquestionably some polari-zation of 'we' (the crew) against 'them' (theoutside world)...."The flight surgeon verified that an anger

stress situation did exist and indicated (per-sonal communication) that crew discussionswith responsible "outside" personnel, in whichproblems were recognized and assurances weregiven of problem solution, "if not by the end ofthe test, at least before Skylab actually flew,"served to reassure the crew during the laterphase of the test that their personal dedicationwas not a wasted effort. The decrease in theproportion of B. fragilis subsp. thetaiotaomi-cron in the feces of the astronauts appeared tocoincide with the time that these assuranceswere given, except for subject T, whose diet wasinsufficient and who believed the balance trialsthat had established his dietary allowance hadbeen incorrectly interpreted.The effect of emotional stress on the intes-

tinal health of astronauts in the NASA MannedSpace Flight program was recognized as a seri-ous consideration (13). Anger or fright, anddepression, reportedly produce distinct effectsupon the colon and upper gastrointestinal tract.Emotional stress affects epinephrine and gas-trin secretion which, in turn, affect intestinal

motility, intestinal blood flow, gastric and co-lonic mucin secretion, nutrient and bile deriva-tive absorption, and possibly bile secretion.These changes are known to be related to co-lonic irritability and colitis, as well as to gastricand duodenal ulcers (2, 3, 9, 15). To the manyamong us who have experienced ulcers or coli-tis, it would be surprising if some importantmembers of the colonic flora were not affectedby physiological changes that result from stressdue to anger or emotional conflict.A significance test for the hypothesis that an

anger stress situation tends to produce a highcount for the species B. fragilis subsp. thetaio-taomicron shows a composite tail-area proba-bility of 1:3,000 (also see Appendix [f]).When the proportion ofB. fragilis subsp. the-

taiotamicron increased in the astronauts (Fig.1), the proportion of P. productus II decreased(Fig. 2). There was no obvious in vitro interre-lationship between the strains of these two spe-cies when cross streaked on agar plates. Thus,they appear to have responded independentlyto changes in the host intestinal physiologythat may have occurred during anger stress.Concurrent low proportions of P. productus IIwere not observed in the two other anger stresscases we have examined. This observation andthe simultaneous occurrence of relatively highproportions of both species in astronaut T onJuly 20 and September 19 (Fig. 1 and 2) furtherindicate that the responses of B. fragilis subsp.thetaiotaomicron and P. productus II are inde-pendent.

Currently there is no evidence concerningany possible medical significance of thechanges in fecal flora observed during theSMEAT trials.

Identification of fecal isolates. Manystrains isolated in the studies of human fecalflora do not have characteristics that corre-spond to those of any described species. Al-though it is legal to describe a new species onthe basis of a single strain, we believe that thispractice has caused much taxonomic confusion.Therefore we prefer not to assign full taxonomicrecognition to those less frequent species ofwhich we have only a few strains. However, weare proposing full taxonomic recognition forthose species for which we have at least nineisolates from at least six different persons. De-scriptions of eight new species (Desulfomonaspigra, Clostridium leptum, Eubacterium had-rum, Eubacterium ramulus, Eubacterium sir-aeum, Ruminococcus lactaris, Ruminococcusobeum, and Butyrivibrio crossotus) that arecited in the work will be published elsewhere(W. E. C. Moore, J. L. Johnson, and L. V.

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Holdeman, Int. J. Syst. Bacteriol., in press).Characteristics of other recently described spe-cies cited in this publication can be found inreferences 5, 7, and 10.

Also cited in this study are (i) phenotypicgroups within recognized species and (ii) spe-cies for which we have too few strains to war-rant full taxononiic recognition. Criteria usedto differentiate among these strains are pre-sented below.

(i) New phenotypic groups within recog-nized species. Among some species, the recog-nition of subspecies or biotypes on the basis ofdistinct clusters of strains within the specieshas proved to be of value. In B. fragilis, subspe-cies designated on this basis have since beenshown to be genetically distinct entities (6) thatshould merit species rank. In this work we haverecognized distinct clusters within species.Some were described previously (11). The clus-ters of strains within species that were deter-mined in the present study, but not describedpreviously, are given below.Fusobacterium mortiferum I: The biochemi-

cal activities of this organism are similar tothose described for F. mortiferum. However, nogrowth is produced on the surface of blood agar(BAP) or supplemented brain heart infusionagar plates incubated in anaerobe jars, cellularmorphology is generally uniform, and little orno gas and no hydrogen is produced. Knownstrains of F. mortiferum usually exhibit ex-treme pleomorphism, especially from growthon BAP, and produce copious gas with morethan 2% hydrogen in the head space.Eubacterium ventriosum I: The species E.

ventriosum and E. ruminantium have similaracid products. We have differentiated E. rumi-nantium from E. ventriosum on the basis ofhydrogen production in at least small amountsby strains of E. ventriosum. E. ventriosum ap-pears to contain three groups of strains whichwe currently refer to as E. ventriosum, E. ven-triosum I, and E. ventriosum II. These groupsare differentiated according to the characteris-tics listed in Table 7.

(ii) Characteristics of strains that do notbelong to described species. We have assignedletter designations within the appropriate ge-nus to those strains that do not have character-istics of any described species and of which wehave only a few representatives. Some of thesewere described previously (11). Characteristicsof other infrequently isolated species are givenin Tables 8 and 9 and in the descriptions below.None of these species grew aerobically on

BAP, grew in a candle jar, produced detectablecatalase, or reduced nitrate. Unless specifically


TABLE 7. Differential reactions of Eubacteriumventriosum subgroups

SubgroupaSubstrate E. ven- E. ven- E. ven-

triosum triosum I triosum II

Amygdalin - -A AArabinose - -ACellobiose - A wMannose A- -w AMelibiose - Aw

a A, Acid reaction, pH below 5.5; -, negativereaction; w, weak reaction, pH between 5.5 and 6.0.

TABLE 8. Differential reactions of somefermentative, sucrose-negative, indole-positive

species of the genus BacteroidesBacteroides sp.a

Test B. eg- Bacte- B. Bacte- Bacte-gerthgi roides splanch- roides roidesgrhi AR nicus W-1 W-2

Arabinose Aw - A A ACellobiose Aw -w - - AGlycogen A- -A -A AMaltose A wA - A AMannose A wA A ARham- Aw -w - - A

noseStarchpH A v - Aw AStarch + v - + +

hydrol-ysis

Gelatin v + v +Hydrogen 1 1 2,3 2,4Bile 3,1 4,1 4 4 -1growtha A, Acid reaction, pH below 5.5; w, weak reac-

tion, pH between 5.5 and 6.0; -, negative reaction;+, positive reaction. Numbers, hydrogen productionon "- to 4+" scale; growth in bile on "- to 4+" scale.

stated below, they did not grow in 6.5% NaCl,produce propionate from threonine, or utilizelactate. Unless otherwise noted, subsurface col-ony descriptions are from rumen fluid-glucose-cellobiose agar cultures and surface colony de-scriptions are from cultures on BAP incubatedin an anaerobe jar and from cultures on brainheart infusion agar roll streak tubes (8).Coprococcus. Coprococcus BH: Subsurface

colonies are 1 mm and translucent with a rhi-zoid center. Surface colonies are 0.5 to 2 mm,buff, flat with opaque or spotted centers orraised with entire to slightly irregular edges.Glucose broth (peptone-yeast extract-glucose[PYG]) cultures are turbid with white floccu-lent sediment and a pH of 4.4 to 4.9 in 5 days.Acetylmethylcarbinol is not produced. Lactate

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is converted to acetate and butyrate. Threonineis converted to propionate. Growth is enhancedby Tween 80 and by heme. Moderate growth issometimes produced in the absence of ferment-able carbohydrate. Production of formic acid,fermentation of melezitose, and hydrolysis ofstarch differentiate this species from C. comes.Fermentation of arabinose, mannitol, and sor-bitol differentiate this species from C. eutactus.Coprococcus CC: Subsurface colonies are 0.2

to 0.5 mm, lenticular to raspberry shaped,translucent to transparent. Surface colonies are0.5 to 1 mm, circular, entire, convex, translu-cent to opaque, glistening, and white to yellow.PYG cultures have smooth white sedimentwithout turbidity and a pH of 5.8 to 5.9 in 1 to 5days. The pH in fructose or inositol broth is 4.6to 5.4 in 5 days. Production of indole and fer-mentation of fructose, glucose, and inositol dif-ferentiate this species from others in the genus.Gemmiger. Gemmiger X: Subsurface colo-

nies are 0.5 mm, tan, lenticular, and translu-cent to opaque. No growth was produced on thesurface of BAP or brain heart infusion agarplates incubated in anaerobe jars. In sweet E(8) agar streak tubes, surface colonies were 2.0mm, fried-egg shaped, opaque, white, andsmooth. Maltose broth cultures have ropy sedi-ment without turbidity in 4 days. Growth isusually best in maltose and occasionally is mod-erate in cellobiose, arabinose, or another carbo-hydrate broth. However, most carbohydratemedia have little or no growth. Little or nogrowth was produced in 6.5% NaCl. This spe-cies appears to differ from Gemmiger formicilisin producing poorer growth. Growth is notstimulated by rumen fluid or Tween 80, and nodetectable formic acid is produced. The cellularmorphology of the two species is similar.

Peptococcus. Peptococcus G: Subsurface col-onies are 0.5 mm, white, translucent and lentic-ular. Surface colonies are pinpoint to 1 mm,circular, entire, convex, glistening, and white.PYG cultures are turbid with a smooth sedi-ment and a pH of 5.0 to 5.4 in 4 to 5 days. Thecarbohydrates fermented and production of iso-butyric and isovaleric acids differentiate thisspecies from others in Peptococcus or Pepto-streptococcus.Peptococcus AU-1: Subsurface colonies are

0.5 mm, lenticular, and translucent. Surfacecolonies are 0.5 to 1 mm, circular, entire, lowconvex, opaque, and grayish white. PYG cul-tures have a granular sediment with no turbid-ity in 4 days; the pH is 5.8 to 6.2. This speciesdiffers from P. magnus in that longer chains ofcells are produced, milk is digested, and growthis not stimulated by Tween 80.

Peptococcus AU-2: Subsurface colonies are0.2 mm, white, lenticular, and translucent.Surface colonies are punctiform to 0.5 mm, cir-cular, entire, low convex, semi-opaque, white,glistening, and smooth. PYG cultures havemoderate growth with little turbidity, floccu-lent sediment, and a pH of 5.9 to 6.0 in 2 days.This species is differentiated from Peptostrepto-coccus micros, which it closely resembles, by itsproduction of hydrogen and trace amounts ofisobutyric, n-butyric, and isovaleric acids.

Peptostreptococcus. Peptostreptococcus BL:Subsurface colonies are 0.5 to 1 mm, lenticular,and off-white. No growth is produced on thesurface of BAP or brain heart infusion agarplates incubated in anaerobe jars. Surface colo-nies on sweet E streak tubes are punctiform to 1mm in diameter, circular, entire, raised, andtranslucent to opaque. PYG cultures have faintto moderate turbidity and no change in pHafter 5 days. This indole-positive species differsfrom Peptococcus asaccharolyticus in morphol-ogy and arrangement of cells and by digestinggelatin and sometimes milk. Growth is notstimulated by Tween 80.Ruminococcus. Ruminococcus AT: Subsur-

face colonies are 1 mm, lenticular, translucent,and white. Surface colonies are 1 to 2 mm,circular, entire, convex, opaque, glistening,and smooth. PYG cultures have slight turbiditywith ropy sediment and a pH of 4.9 to 5.2 in 5days. This species differs from RuminococcusAQ described earlier (11) by fermenting salicinand trehalose.Ruminococcus CO: Subsurface colonies are

0.1 to 2 mm, tan to brown, lenticular, translu-cent, and sometimes have a diffuse edge. Sur-face colonies are 1 to 2 mm, low convex to fried-egg shaped, circular, entire, smooth, and glis-tening. PYG cultures have slight turbidity witha mucoid or crumby sediment and a pH of 4.6 to5.4 in 5 days. Growth is sometimes enhancedwith Tween 80 or rumen fluid. This speciesdiffers from R. lactaris by the production of acidfrom sucrose and trehalose.

Lactobacillus. Lactobacillus G: Subsurfacecolonies are 0.5 mm, lenticular, and translu-cent to opaque. Surface colonies are punctiformto 1 mm, circular, entire, convex, translucent toopaque, smooth, and glistening. PYG cultureshave moderate turbidity with smooth whitesediment and a pH of 5.2 to 5.9 in 4 to 5 days.Hydrolysis of esculin and occasional fermenta-tion of maltose, salicin, rhamnose, or xylosedifferentiate this species from L. rogosae.

Fusobacterium. Fusobacterium H: Subsur-face colonies are 0.5 mm, spherical, fuzzy, andwhite. Surface colonies are punctiform, circu-

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370 HOLDEMAN, GOOD, AND MOORE APPL. ENVIRON. MICROBIOL.

TABLE 9. Characteristics of less common bacteria of the fecal floral

3: U

Characteristic.<

i.,-

No. strains 1 4/4

No. peopleAdonitol wAmygdalin wArabinose ACellobiose ADextrin A

Esculin pH -w

Esculin hyd. +

Fructose A AwGalactose AGlucose A wA

Glycerol wGlycogen - -

Inositol - AInulin ALactose A

Maltose AMannitol AMannose AMelezitose AMelibiose A

Raffinose ARhamnoseRibose -w

Salicin ASorbitol Aw

Sorbose wStarch pH AStarch hyd. +Sucrose ATrehalose - -

Xylose A

Gelatin dig.Milk c -

Indole - +EYA react.Hemolysis - -a

Gas 4 2,4Hydrogen 4 4PY-growth 2 1PY-CHO gr. 4 4PYG-bile gr. 4 1-

Resaz. red. + vPyruvate Ab BAGluconateMotility - -

Morphology

*.10 *,e

,microns * 5

S%

cU <

2/2 2/2 2/1

-A _ _w- Aw -

-w- -

- Aw -

- A w--A w -

w- A w-

-w

w- Aw -

-w A -

A -

-w A -

w

-w

- As -

-A A- -

- A -

v w- -

- - cd

-1 3 -

1 -

1 2 12,1 3,4 2,41- 2,3 1

B(A) A -

\ :* ~~SI ._

1/1

-W

-W

2

*0

*:

,*R'*5

O

< <

U~~~- AtL 4w _

;~~~&; t< x< <

4/3 2/1 3/2 3/3 1/1

w ~ ~ A

- - -W AW -

_ _ A- -- + +- + _

- A A A- -- w A - -- A A Aw -

- - - - Aw

- w Aw w- -- A A -w --- A- - -

- - A - -

- w - _

- Aw - _w~~~

_A w

- wA -w A- -

- A A - -- Aw A- - -

_- w-

+

-d c- c -c -

- 2,4 3 - -- 4 4,1 - -1 1 1 -1 21,2 4 4 4 3-1 4 4,3 -1 -_ + + _+ _

- A - AF Ba+

\ Im (g*, e' t I 1

-

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VOL. 31, 1976 HUMAN FECAL FLORA 371

Table 9. cont'd.

< < < < <3

Characteristic *;x c E .i

<<W. < < L;; < m x x xt~~~~~<_~~~~~~~~~~~~<

No. strains / 4/4 1/1 1/1 6/3 2/1 2/2 7/5 2/2 2/2 6/4 1/1 1/1

No.people~ ~ ~ ~ ~ . -.

Adonitol - w- w

Amygdalin - - - A- Aw - -w -W--wArabinose - - / / 6 Aw 7 2 2 6 ACellobiose -w - - -s - - -w -

Dextrin - - - - - - w- - - Aw A -

Esculin pH - - - w- w w - - - w- -w -

Esculin hyd. - - - + + v - - - + + -

Fructose w- - A As wA Aw A - Aw w w -

Galactose - - A - wA Aw A - A w- w -

Glucose - - A As A Aw A - A wA Aw -

Glycerol - - - - - -wGlycogen - - - - - - - - - -A AwInositolInulinLactose - - - w- - -w A - - Aw w

Maltose - - - -w - -w A - - Aw AMannitol -

Mannose - - w- - - w- -w - Sw Aw AwMelezitose -

Melibiose - - - - - - - - - -w w

Raffinose -

Rhamnose - - A - - -ARibose - - Aw - - w- -w - - -w wSalicin - - w A- A w-Sorbitol - - - - - -w A-

SorboseStarch pH - - - - - - w- - - Aw AwStarch hyd. - - - - - - - _ +_ +Sucrose - - - Aw A - vTrehalose - - w -A - A- -

Xylose A- - - w- v Aw

Gelatin dig. - - - - - - - - - + +Milk - - - - - c- c- - - c c -Indole - - - - - - _ + - + +

EYA react.Hemolysis - - - - - b- -

Gas -1 -1 3 -2 2- v 4,2 - v -

Hydrogen 4 4 4 -1 - 4 3,4 - 2,1 - 3 2PY-growth 1- 1- 1 .1- 1 1 1 1- 1 1,2 2,3 1PY-CHO gr. 2,1 2 4 4,3 4 4,3 4 1- 4 4,3 3,4 2PYG-bile gr. - - 4 v 1 3,1 3 -1 2- 4 4,2 -1

Resaz. red. - - + + + + + - +- - + -

Pyruvate - - A a Ab(F) AF AF - A A ABibiv ASGluconate - - - - - - _ +

Motility - + - - - - - - - - - +

Morphology j /( , ,.

10 F~1/~

micronsJ,~\i~D '

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lar, slightly erose, low convex, glistening, andoff-white. Inositol broth cultures have denseflocculent and ropy sediment with no turbidityand a pH of 5.3 to 5.4 in 4 days. This species isunusual in that it grows well in basal mediumwith added heme and ferments only inositol.Eubacterium. Eubacterium AG-H: Subsur-

face colonies are 0.2 to 0.5 mm, off-white to tan,transparent to translucent, and may havedarkened centers. Surface colonies are puncti-form, circular, and transparent. Carbohydratebroth cultures have slight smooth sediment, noturbidity, and no decrease in pH in 2 to 5 days.This species differs from Eubacterium AG (11)by producing copious hydrogen.Eubacterium AG-M: Subsurface colonies are

0.5-mm rhizoid spheres. Maltose broth cultureshave very faint sediment in 10 days. Poor tomoderate growth is produced in 6.5% NaCl.This species differs from Eubacterium AG-H inmorphology, and it is motile and amphitri-chous. It differs from Eubacterium AG (11) bymotility and hydrogen production.Eubacterium AJ: Subsurface colonies are 0.5

mm, lenticular, and white. Surface colonies are0.5 to 1 mm, circular, low convex, and semi-opaque to opaque, with a granular surface.PYG cultures have heavy turbidity with smoothsediment and a pH of 5.2 to 5.3 in 6 days. Thisspecies differs from E. eligens by producingcopious hydrogen. It differs from E. formici-generans in morphology and failure to ferment(or grow well in) maltose broth.Eubacterium AL: Subsurface colonies are

punctiform to 0.5 mm, off-white, lenticular, andtranslucent. Surface colonies are 1 to 2 mm,circular, entire, convex, and opaque to translu-cent. PYG cultures have mucoid sedimentwithout turbidity and a pH of 4.7 to 5.7 in 4 to 7days. In some PYG cultures pH below 6.0 is notobtained, but growth is superior to that ob-tained with many other carbohydrates or thebasal medium. Growth may also be stimulatedby Tween 80. This species differs from E. cylin-droides in morphology and by lack of fermenta-tion of mannose. It differs from E. tortuosum inmorphology and by the lack of hydrogen pro-duction.

Eubacterium AL-1: Subsurface colonies are 1to 2 mm, tan, lenticular, and translucent. Sur-face colonies are 1 mm, circular, entire to erose,convex, and opaque. PYG cultures have heavyturbidity with smooth white sediment and a pHof 4.4 to 4.8 in 3 to 5 days. Poor growth issometimes produced in 6.5% NaCl. This speciesdiffers from Eubacterium AL and E. cylin-droides in morphology and from E. tortuosumin morphology and by the absence of hydrogenproduction.Eubacterium BW-1: Subsurface colonies are

0.5 mm, lenticular, white, and translucent.Surface colonies are punctiform to 1 mm, cir-cular, entire, convex, translucent, off-white,sometimes with transparent edges, smooth,and glistening. PYG cultures have a dense floc-culent sediment with little or no turbidity and apH of 5.1 to 5.5 in 5 days. Absence of fermenta-tion of mannitol, sorbitol, and xylose differen-tiate this species from Eubacterium AY de-scribed previously (11).Eubacterium CH-1: Subsurface colonies are

0.5 to 1 mm, lenticular, multifoliate or rhizoid,white to tan, and semi-opaque. Surface coloniesare 1 to 2 mm, circular, entire to erose, convex,and opaque or translucent with dense centers,and off-white in color. PYG cultures have denseflocculent or mucoid sediment and dense tur-bidity with a pH of 5.0 to 5.3 in 5 to 7 days.Scant growth is produced in noncarbohydratemedia, and cells in these cultures are coccoidand arranged in long chains. Elongated cells aswell as cocci are produced in the same chains ofcells in those carbohydrate media that are at-tacked. This species resembles Ruminococcusspecies in its requirement for fermentable car-bohydrate, acid products,' and production of coc-coid forms. However, the presence of definiterods within the chains indicates it should beassociated with Eubacterium species.

Bacteroides. Bacteroides L-4: Subsurface col-onies are 1 mm, tan, and lenticular. No growthis produced on the surface of agar media incu-bated in anaerobe jars. Scant growth withslight turbidity and no sediment is produced inbroth media. Terminal pH in carbohydratebroth is the same as in the basal medium. This

a Acids and alcohols produced in PY-carbohydrate (usually glucose or fructose) broth cultures are given below the nameof the organism. Those produced from pyruvate are indicated in the table. Capital letters indicate 1 or more meq/100 ml;small letters indicate less than 1 meq/100 ml. Products in parentheses are not uniformly detected. Abbreviations forproducts: a, acetic acid; b, butyric acid; f, formic acid; ib, isobutyric acid; iv, isovaleric acid; 1, lactic acid; p, propionic acid;py, pyruvic acid; s, succinic acid; 2, ethanol; i4, isobutyl alcohol; 4, butyl alcohol; i5, isoamyl alcohol. Abbreviations andsymbols for other reactions: A (carbohydrate cultures), acid (below pH 5.5); a (hemolysis), alpha; b (hemolysis), beta; c(milk), curd; +, positive reaction; -, negative reaction; -, no visible growth or only slight growth and negative reaction; w,weak reaction or pH between 5.5 and 6.0; a, growth is stimulated; v, variable reaction; numbers (growth and gas), amountestimated on "- to 4+" scale. Where two reactions are given (e.g., Aw), the first is the more usual and the second isobserved less frequently. EYA, Egg yolk agar (modified McClung-Toabe).


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species differs from other unreactive Bacte-roides species in that it produces indole but nohydrogen and fails to coagulate milk.Bacteroides L-5: Subsurface colonies are 0.5

mm, lenticular, or rhizoid. Surface colonies are0.5 to 2 mm, circular, entire, convex to slightlypeaked, translucent, buff, and smooth. PYGcultures are turbid with a flocculent sedimentand a pH of 5.2 to 5.5 in 4 to 6 days. This speciesdiffers from Bacteroides species described pre-viously (11) by the production of acid (pH below5.5) from glucose and fructose and the produc-tion of hydrogen. Growth is stimulated in thepresence of fructose, glucose, galactose, andmannose. Hydrogen sulfide is not produced inSIM (BBL) medium.Bacteroides AR: Subsurface colonies are 0.5

to 2 mm, lenticular, translucent, and white.Surface colonies are punctiform to 1 mm indiameter, circular, entire, convex, translucentto opaque, glistening, smooth, and off-white tobuff. PYG cultures have moderate to slight tur-bidity with smooth sediment and a pH of 5.3 to5.6 in 5 to 8 days. Threonine is converted topropionate. This species is similar to B. egger-thii except that arabinose is not fermented andthe cells are thinner.Bacteroides W-1: Subsurface colonies are 1-

mm, white wooly balls. Surface colonies are 0.5to 2 mm, circular, entire, low convex, andtranslucent. PYG cultures have heavy turbid-ity with smooth white sediment and a pH of 5.2to 5.9 in 4 to 5 days. Threonine is converted topropionate and lactate is utilized. The fermen-tative, sucrose-negative, indole-positive spe-cies, including B. splanchnicus, B. eggerthii,Bacteroides AR, Bacteroids W-1, and Bacte-roides W-2 (11), are differentiated according tothe characteristics shown in Table 8.

Succinivibrio. Succinivibrio A: Subsurfacecolonies are 0.5 mm, white, and rhizoid. Sur-face colonies are 0.5 mm, circular, entire, trans-parent, low convex, smooth, and glistening.PYG cultures have a slight turbidity and slightsediment with no decrease in pH in 4 days.Monotrichous curved cells and production ofsuccinic acid from pyruvate indicate that thisspecies is a member of the genus Succinivibrio.Production of large amounts of acetic acid andsmaller amounts of propionic acid and butyricacid without a measurable decrease in pH ofcarbohydrate media differentiate this speciesfrom others of the genus.

Variable reactions among all the species de-scribed above are listed in Table 9.

APPENDIX(a) Composition of the flora. Let the number of

species represented by r isolates in the sample be

denoted by nr, the "frequency of the frequency r." Ithas been shown by Good (4) that the "coverage" of asample is approximately 1 - n,/N, where N is thetotal number lrn, of isolates, and the coverage isthe probability that the next isolate sampled willbelong to a species that has already orcurred. In ourproblem, as mentioned in Results, we had N =1,442. Further, we had n, = 28, so that the estimate1 - n,IN of the coverage is 98%. But we consider thata species represented by only two or three isolatesmight, in some cases, be found to correspond to morethan one species if the sample were much increased,so that n, is probably an underestimate of its trueexpected value. Accordingly, we consider that theformula Hg in Good (4), which is nr oc xr/r(r + 1)(xclose to 1) and which fits well for r 2 2, may wellgive a more realistic estimate of the coverage. Itgives the lower figure of 95% mentioned in Results.A good fit to the observed values of nr, including

ni, can be obtained by Fisher's formula (H3 of refer-ence 4), nr = f3x /r, with /8 = 24.744 andx = 0.98313,and this hypothesis would again lead to the estimateof 98.3% for the coverage. An even more exact fit tothe observed nr values could of course be obtained bymeans of the more general hypothesis H4, whichcontains three parameters. For the determination ofthe parameters see Sichel (16), but for the purpose ofestimating the coverage the use of this hypothesis isunnecessary.On the available evidence it is impossible to de-

cide whether 98 or 95% is the better estimate of thecoverage. This becomes especially clear when it isheld in mind that it is unimportant for a smoothingto be good for large values of r for the purpose ofestimating the coverage.

(b) Note on the transformation. V/(xI2.1 + 3/8).The choice of this transformation was based on thefrequencies x of isolates (Table 4) corresponding tothe eight common species (B. fragilis subsp. vulga-tus, E. aerofaciens, B. fragilis subsp. thetaiotaomi-cron, P. productus II, B. fragilis subsp. distasonis, F.prausnitzii, C. eutactus, and P. productus I). Aftermaking the transformation, the following summarystatistics were computed for the data as brokendown into (i) normal diet, (ii) Skylab diet, not inchamber, and (iii) Skylab diet, in chamber (with theentries 19, 12, and 13 for the period August 24 re-moved to help with the analysis discussed in sectionc, below): (i) 79 df, x = 1.3785, standard deviation (s)= 0.55267; (ii) 47 df, x = 1.4489, s = 0.53818; (iii) 69df, x = 1.3188, s = 0.59860. The three means for thethree diets certainly do not differ significantly [allthree t values, for comparison of [i] with [ii], [i] with[iii], and [ii] with [iii], are less than 0.25). Bartlett'stest for homogeneity of variances gives X2[2 df] =0.758, so there is certainly no reason to suspect thatthe variances depend on the diet. The pooled meanis 1.3604 and the pooled variance is 0.32015 (194 df),corresponding to an estimated standard deviation of0.5658. The pooled variance is used in section c(below).

(c) An analytical discussion corresponding toFig. 1 and 2. The graphical approach of Fig. 1 and 2could be misleading, so we here discuss the dataconcerning B. fragilis subsp. thetaiotaomicron and

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P. productus II analytically. The frequency counts ofisolates for the former, classified both by periods andby person, were:

B 4 5 2 1 5 19 1 4R 4 1 1 2 7 12 4 3T 3 4 4 7 6 13 10 5 2

where the nine columns correspond to the nine pe-riods specified in Fig. 1. Now the totals (shown inthe last row of Table 4) were high corresponding tocolumn 6 (August 24), namely 72, 66, and 66; so webegin by replacing the column [19, 12, 13] by [19 x56/72, 12 x 56/66, and 13 x 56/66]. If we then apply thenormalizing transformation J(x/2. 1 + 3/8), the databecome

B 1.5 1.7 1.2 0.9 1.7 2.7 0.9 1.5R 1.5 0.9 0.9 1.2 1.9 2.29* 1.5 1.3T 1.3 1.5 1.5 1.9 1.8 2.4 2.27* 1.7 1.2

Totals 4.3 4.1 3.6 4.0 5.4 7.4 4.7 4.5(* The reason for giving two of the entries to twodecimal places is to help in the nonparametric testdescribed below. These entries are both regarded asequal to 2.3 for all other purposes.)By chance, 1/1i/3times the sample standard devia-

tion ofthe totals 4.3, 4.1, ..., 4.5 (excluding the total7.4 for August 24) is smaller than that for individualentries 1.5, 1.7, . . . This in its turn is smaller thanthe estimated standard deviation, 0.5658, based on194 df, as found in section b (above). To avoid over-stating our thesis we have used the latter estimate.We then regard the seven numbers 4.3, 4.1, 3.6, 4.0,5.4, 4.7, and 4.5 as normal variables of unknownmean, and standard deviation estimated by 0.5658 x3= 0.980. If the total on August 24 had the same

expectation as that in the other periods, the proba-bility that a difference as large as 7.4 - (4.3 + . . . +4.5)/7 = 3.029 would be attained would be approxi-mately that of a Student's t equal to 3.029/[0.980

(1 + 1/7)J = 2.891, where the denominator has 194df. The corresponding single tail-area probability is1/468. It might be thought that a double tail-areashould be used and that a penalty should be paid forthe special selection of the species and of the periodAugust 24. The reason for not paying these penaltiesis explained in section f.

In case the assumptions ofapproximate normalityare not accurate enough, we can also evaluate thesignificance of the "sixth column" by a nonparamet-ric test. Even after multiplying the three counts inthat column by 56/72, 56/66f, and 56/66, we find that theygive the top three among 25 numbers. The probabil-ity of this, if the effect were merely random, wouldbe 1/2300, which is even smaller than 1/468. But 2.29 isonly just larger than 2.27 so this nonparametric testoverevaluates the statistical significance.

For P. productus II, the table of transformed dataisB 1.3 1.2 2.6 1.9 0.9 0.6 0.6 0.6R 1.7 0.9 0.9 1.7 0.9 0.6 1.2 1.7T 1.3 2.6 2.0 1.8 1.2 0.9 1.9 1.7

Totals 4.3 4.7 4.5 5.4 3.0 2.1 3.7 4.0

The difference between the (low) total for August 24and the mean of the other seven totals is now 4.229- 2.1 = 2.129 and the corresponding Student t is2.032, corresponding to a double tail-area probabil-ity of about 1/23. Here we do not need to allow for thespecial selection of the period, but some allowancemust be made for the selection of the species, so thatthe result for P. productus II is not striking. (Theresults are slightly better than this argument sug-gests because the samples were large for the periodAugust 24.)

(d) Statistical interpretation of Table 5. The highvalue of 11 out of 57 in Table 5 can be evaluated bythe same method as in section c (above). After apply-ing the transformation /(x/2. 1 + 3/8) we can inter-pret the deviation 2.4 - (0.6 + 1.8 + 1.8)/3 = 1.0 byregarding 1.0/[0.5658v(1 + 1/3)] = 1.53 as being a tdistribution in which the denominator has 194 df.This corresponds to a single tail-area probability of1/16.

(e) Statistical significance of the ascending colonobservation. Of the observations 17/51, 8/50, 4/57, and5/55, the first referred to the ascending colon. For thesake of accuracy we can first replace the frequencies17, 8, 4, and 5 by 17 x 56/51, 8 x 56/50, 4 x 56/57, and5 x 56/55 and then apply the transformation0(x/2.1 + 3/8) to them. This gives the numbers 3.04,2.154, 1.50, and 1.67, and the significance of thedifference 3.04 - (2.154 + 1.50 + 1.67)/3 = 1.267 canbe measured, as in section d, as that of a t value of1.267/[0.56587(1 T+ 13= 1.939, where the denomi-nator has 194 df. The corresponding single-tailedprobability is 1/37.

(f) Significance of the anger stress hypothesis.Here we attempt to evaluate the significance of thehypothesis that an anger-stress situation tends tolead to a high count for the species B. fragilis subsp.thetaiotaomicron, based on the combination of allthe observations.The statistical evaluation of the data cannot logi-

cally depend on the chronological order of the obser-vations. Accordingly, it is legitimate to start withthe independent observations by Bruce Maier, men-tioned in the text. We can regard his observations asimplying a prima facie case for the hypothesis underconsideration. Had this information been availableto us before we analyzed the astronaut data, and hadwe known of the stressful situation of the astronautsin a period including August 24, we might haveexpected high counts of B. fragilis subsp. thetaio-taomicron in that period. This period probably cov-ered most of the time in the chamber. It should benoted that the totals 4.3, 4.1.... in section c (above)are largest for the three sample days in this period.For simplicity in the analysis we decided to test thesignificance of the hypothesis by using just the high-est of these three totals. Although this involves alittle special selection, we feel that this is compen-sated by the high totals in the two adjacent sampledates. Accordingly we regard it as justifiable to com-bine the pieces of evidence of section c, d, and e as ifthe hypothesis under consideration had been formu-lated in advance.The individual single-tailed P values, given in

sections c, d, and e, were 1/468, 1/16, and 1/37,


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375

respectively. We combine these by Fisher's methodto obtain a composite tail-area probability of 1/3,000.

Thus, the hypothesis appears probable in thelight of all the available evidence. It may well bethat there is also an effect on some of the otherspecies, but for most ofthem the samples may be toosmall for the effect to be noticed.

ACKNOWLEDGMENTSWe are indebted to Ella W. Beaver, Sarah L. Chalgren,

Dale D. Charles, Ruth L. Chiles, Gail K. Harrington, Caro-lyn L. Hubbard, Julia J. Hylton, Susan M. Sieg, Dianne M.Wall, and Catherine A. Waters for technological assistancein analysis of the bacterial isolates, and to Elizabeth P.Cato and others of our laboratory who performed work on orfor this project. We are also indebted to Bruce R. Maier forpermission to cite his independent observations, and toNASA for financial support of this work under contractNASA 9-12601. Statistical aspects of the work were sup-ported in part by Public Health Service grant RO1-GM18870from the National Institute of General Medical Sciences.We wish to thank James L. McQueen (then technical moni-tor for NASA) for his assistance with logistics, C. E. Ross(crew surgeon) for his observations on stress conditions, andthe three astronauts to whom our work contributed prob-lems.

LITERATURE CITED

1. Anscombe, F. J. 1948. The transformation of Poisson,binomial and negative-binomial data. Biometrika35:246-254.

2. Christensen, J. 1972. Motility, p. 389-406. In E. D.Frohlich (ed.), Pathophysiology. Altered regulatorymechanisms in disease. J. B. Lippincott Co., Phila-delphia.

3. Davenport, H. W. 1972. Mechanisms of gastric andpancreatic secretion, p. 407-422. In E. D. Frohlich(ed.), Pathophysiology. Altered regulatory mecha-nisms in disease. J. B. Lippincott Co., Philadelphia.

4. Good, I. J. 1953. The population frequencies of speciesand the estimation of population parameters. Biome-trika 40:237-264.

5. Gossling, J., and W. E. C. Moore. 1975. Gemmiger

formicilis, n.gen., n.sp., an anaerobic budding bacte-rium from intestines. Int. J. Syst. Bacteriol. 25:202-207.

6. Johnson, J. L. 1973. Use of nucleic acid homologies inthe taxonomy of anaerobic bacteria. Int. J. Syst. Bac-teriol. 23:308-315.

7. Holdeman, L. V., and W. E. C. Moore. 1974. Newgenus, Coprococcus, twelve new species, andemended descriptions of bacteria from human feces.Int. J. Syst. Bacteriol. 24:260-277.

8. Holdeman, L. V., and W. E. C. Moore (ed.). 1975.Anaerobe laboratory manual, 3rd ed. Virginia Poly-technic Institute and State University, Blacksburg.

9. Lundgren, 0. 1972. Autonomic control of gastrointes-tinal function, p. 615-630. In E. D. Frohlich (ed.),Pathophysiology. Altered regulatory mechanisms indisease. J. B. Lippincott Co., Philadelphia.

10. Moore, W. E. C., E. P. Cato, and L. V. Holdeman.1972. Ruminococcus bromii sp.n. and emendation ofthe description of Ruminococcus Sijpesteijn. Int. J.Syst. Bacteriol. 22:78-80.

11. Moore, W. E. C., and L. V. Holdeman. 1974. Humanfecal flora: the normal flora of 20 Japanese-Hawaii-ans. Appl. Microbiol. 27:961-979.

12. National Aeronautics and Space Administration. 1973.Skylab medical experiments altitude test (SMEAT).NASA Publ. TM X-58115. Lyndon B. Johnson SpaceCenter, Houston, Tex.

13. Palmer, E. D. 1970. Upper gastrointestinal hemor-rhage. Charles C Thomas, Springfield, Ill.

14. Sichel, H. S. 1975. On a distribution law for word fre-quencies. J. Am. Stat. Assoc. 70:542-547.

15. Sodeman, W. A., Jr., and D. W. Watson. 1974. Thelarge intestine, p. 767-788. In W. A. Sodeman, Jr.and W. A. Sodeman (ed.), Pathologic physiologymechanisms of disease. W. B. Saunders Co., Phila-delphia.

16. Thornton, W. E. 1973. Some medical aspects ofSMEATas observed by the crew-member physician, p. 19-6.In Skylab medical experiments altitude test(SMEAT). NASA Publ. TM X-58115. Lyndon B. John-son Space Center, Houston, Tex.

17. Wilkins, T. D., and T. Thiel. 1973. A modified broth-disc method for testing the antibiotic susceptibility ofanaerobic bacteria. Antimicrob. Agents Chemother.3:350-356.

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