[CANCER RESEARCH 38, 4458-4462, December 1978]0008-5472/78/0038-0000$02.O0

Fecal Profilesof Anaerobic Microflora of Large Bowel Cancer Patients andPatients with Nonhereditary Large Bowel Polyps1

Anthony J. Mastromarino,2 Bandaru S. Reddy, and Ernst L. Wynder

Division of Nutritional Biochemistry, Naylor Dana Institute, American Health Foundation, Valhalla, New York 10595

ABSTRACT

It has been postulatedthat the intestinal anaerobesplay a role In the etiology of large bowel cancer. Thisstudy was designed to characterize and compare thefecab anaerobes of patients with large bowel cancer,patients wfth nonheredbtarylarge bowel pobyps,andhealthy control subjects. Although some distributionalvariationsof the anaerobicgenerawere observedamongthe studygroups,significantdifferencesin fecal anaerobic microflora and total aerobic counts were not noted.Thissuggeststhat taxonomicgroupingof fecal bacteriaisan inadequate measure of relative risk of developingbargebowel cancer. However,the fecal microbialla-dchydroxylaseand cholesteroldehydrogenaseactivitiesoflarge bowelcancer patientsand patientswith nonhereditary large bowel polyps were significantlyhigher thanthose of healthycontrolsubjects.On the other hand, nosignificantdifference in fecabmicrobial/3-glucuronidaseactivitywas notedamongthe studygroups.It maybe thatassessmentof the total metabolicactivitiesof the intestinalmicroflorawillprovidea betterunderstandingof theirpotentialrole inthe genesisof largebowelcancer.

INTRODUCTION

The worldwide distribution of barge bowel cancer mcidence and mortality shows great variability, being muchmore common in North America and western Europe thanin east Africa, India, and Japan (2, 10, 50). Migrants fromlow- to high-incidence areas soon assume the incidencerates indigenous to the area (21, 44, 48). This implies thatenvironmental factors are more important than geneticfactors. The most commonly suggested environmental factors associated with the development of bargebowel cancerare dietary habits (20, 30, 38, 45). Various nutritional factors, including lack of fiber in the diet (7, 33) and high levelsof refined carbohydrates (12), animal fat (48, 50), and beef(21), have been advanced as significant factors in thevariance of large bowel cancer rates. The incidence of bargebowel cancer among American Mormons and Seventh-DayAdventists is less than in the general population eating amixed Western diet (14, 31, 49).

Diet may influence the fecal microfbora (3, 4, 6, 8, 25, 40,43, 47), fecal bile acids and neutral sterob profiles (23, 25,27, 42), and fecal bacterial enzyme activities in man (32,

I This research was supported by Grant CA 1 6382 awarded by the National

Cancer Institute through the National Large Bowel Cancer Project.2 To whom requests for reprints should be addressed, at National Large

Bowel Cancer Project, The University of Texas System Cancer Center, M. D.Anderson Hospital and Tumor Institute, Texas Medical Center, Houston,Texas 77030.

Received June 2, 1978; accepted August 23, 1978.

39). The intestinal microflora may metabolize bile acids tocarcinogenic and/or cocarcinogenic compounds (1, 27).These secondary bile acids have been reported to act ascolon tumor promoters but not complete carcinogens in ananimal model (41). In one study, Hill et al. (28) reportedsignificant differences in the fecab microbial flora of largebowel cancer patients. Since the putative carcinogenic orcocarcinogenic metabolite(s) of bile acids is formed bybacterial transformation, this study was designed to determine whether differences in fecal bacterial profiles existedamong patients with barge bowel cancer, patients withnonhereditary large bowel polyps, and control subjects.

MATERIALSANDMETHODS

The study groups consisted of volunteers from the NewYork metropolitan area who were consuming a normalmixed Western diet. For the bacteriological studies, thecontrol subjects consisted of 7 healthy adults (3 males and4 females with a mean age of 46 years) without evidence ofintestinal problems. In addition, 13 patients (8 males and 5females with a mean age of 58 years) with diagnosedcoborectabcarcinoma and 7 patients (2 males and 5 femaleswith a mean age of 38 years) with nonhereditary large bowelpolyps were studied. None of the subjects had been onantibiotic treatment or other therapy for at beast 4 weeksprior to coblectmonof the fecal samples. No laxatives orother cathartics were used within 1 week of stool collection,and in all cases the specimens were obtained before bowelpreparation was begun. None of the patients had visuallyobserved blood contaminating the feces. Each individualgave informed consent.Fresh fecalspecimens were collectedin sterileplastic

bags at the hospital or at the residence of the subject by anurse-coordinator and immediately placed in a disposableGasPak anaerobic system (Baltimore Biological Laboratory,Cockeysville, Md.). They were then transported to thelaboratory, coded, and processed under strictly anaerobicconditions using a Virginia Polytechnic Institute anaerobicculture system within an hour of defecation. After thoroughmixing of the specimen under a stream of oxygen-free gas(90% N@:10% C02), approximately 1.0-g samples (wetweight) of the fecab homogenate were suspended in each of2 preweighed 9.0-mb d ilution blanks (Scott Laboratories,Fiskevibbe,A. I.) containing a few sterile glass beads. Aftereach tube was reweighed, the contents were homogenizedthoroughly on a vortex. A duplicate set of dilution serieswas prepared in prereduced, anaerobically sterilized dibution blanks to 10@ dilution. One set was heated in a hotwater bath at 80°for 10 mm and then inoculated ontoreinforced cbostridial agar roll tubes and Trypticase soyblood agar plates. Each dilution series in duplicate was

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Fecal constituents of patientswith large bowelTablecancer or1nonhereditarylarge bowelpolyps andhealthycontrols%

recoveryofanaer

Micro Total an obes comscopicaerobicparedtoAerobicDry

mattercountcountmicrocount%dryexcreted(x1011/g(x 10―/gscopic(x108/9matter(g/day)dry

wt)dry wt)countdrywt)Controlsubjects(7)@'26.3±4.6'@28.2±4.63.6±0.72.8±0.773.9±4.529.9±19.6Large

bowel cancer patients17.7 ±3.024.4 ±3.95.6 ±1.53.8 ±1.163.6 ±8.08.4 ±6.6Nonhereditary

polyp pa21 .5 ±@ .725.8 ±5.24.3 ±0.43.0 ±0.371 .5 ±8.53.6 ±1.6

Fecal Flora of Colon Cancer Patients

large bowel cancer, patients with nonhereditary large bowelpobyps, and healthy control subjects were determined aspreviously described (32). The volunteers analyzed for fecalbacteriological profiles are included in these assays.

RESULTS

The general characteristics of the feces obtained for eachgroup of subjects are presented in Table 1. There was nodifference in the percentage of moisture content of thefeces and in the daily fecal dry matter excretion among the3 groups. The mean total microscopic count of the fecalflora was slightly but not significantly higher in barge bowelcancer patients and patients with nonhereditary large bowelpolyps than in controls. Although 2 coborectab cancer patients had relatively bow counts of total anaerobes, themean total anaerobic count was highest in this group ofpatients. The bow counts of fecab anaerobes in these 2coborectal cancer patients may only reflect our inability toculture the isolates rather than the absolute concentrationsof fecal anaerobes. The percentage recovery of fecal anaerobes compared to the microscopic count was a mean valueof 64% or better. The mean concentration of fecab aerobeswas highest in the controls, but individual variation andsmall sample size negate statistically significant differencesamong the groups. The ratio of anaerobes to aerobes was4.7 and 8 times greater among patients with barge bowelcancer and nonhereditary barge bowel polyps compared tocontrols, respectively.

The total mean concentration of the fecab anaerobicgenera recovered and the number of subjects in each groupfrom whom these genera were isolated are presented inTable 2. Although some variation is evident, in general, theconcentrations of fecab anaerobes among the groups werecomparable.

The percentage compositions and distributions of thefecab anaerobic flora in the 3 study groups are presented inTable 3. Bacteroides, Bifidobacterium, Clostridium, Eubacterium and Lactobacillus species were most commonlyisolated. Anaerobic cocci, Propionibacterium, and Fusobacterium species were found less commonly, and othergenera were encountered infrequently. Despite great diligence, a number of specimens contained isolates that wewere unable to characterize. These organisms are groupedtogether in Table 2 as “unidentified.―Species of Bifidobacterium, although generally constituting a predominant

inoculated anaerobically into brain-heart infusion agar andrumen fluid-glucose cebbobiose agar roll tubes, Trypticasesoy blood agar plates, chopped meat glucose, choppedmeat, and thiogbycolbate fluid. The anaerobic brain-heartinfusion agar, rumen fluid-glucose celbobiose agar, andreinforced cbostridial agar roll tubes were used to quantitatetotal anaerobic and cbostridiab counts, respectively. Corrected colony counts based on dry weight were obtained bythe methods described in the Virginia Polytechnic InstituteAnaerobe Manual (29). Duplicate direct microscopic clumpcounts were prepared from the 10@ dFlution and comparedto the observed colony counts to determine the percentageof recovery of anaerobes. Trypticase soy blood agar plateswere also inoculated and incubated aerobically at 37°for 24to 48 hr and used to quantitate the fecal aerobes based oncolony counts.

The dilution series were kept on ice during the procedures and were then passed into a Coy anaerobic chamber(85% N@:10%CO2:5% H2)containing plates of general andselected media anaerobically reduced for a minimum of 24to 48 hr. The dilution series were inoculated in duplicateonto the following media: Trypticase soy blood agar, Forget-Fredette agar, Cbostrisel agar, phenybethyl alcoholblood agar, neomycin blood agar, kanamycin-vancomycinblood agar, neomycin-vancomycin blood agar, Columbiacolistin nalidixic acid agar, Veillonella-vancomycin agar,sulfite polymyxin sulfadiazine agar, and brain-heart chloramphenicol cycboheximide agar. The plates were allowedto incubate at 37°in vented GasPak jars for 5 to 7 days,after which primary isolation of anaerobes was achieved bypicking about 50 representative colonies in the anaerobicchamber to chopped meat and chopped meat glucosemedia. After good growth, generally 48 hr, the isolates werestained and, if pure, transferred to peptone yeast glucoseand chopped meat agar slants. Definitive identification ofeach isolate was based on Gram reaction (Kopeboff modification), morphology, spore test, gas-liquid chromatographyprofiles of ether, and methylated extracts from peptoneyeast glucose cultures, with the use of a Hewlett-Packardgas chromatograph fitted with a Resoflex column, and thenecessary biochemical determinations (29), with both theAPI 20 anaerobic system (Analytab Products, Inc., Plainview, N. Y.) and commercially prepared anaerobic media(Scott Laboratories, Fiskeville, R. I.).

Fecal bacterial 7a-dehydroxybase, cholesterol dehydrogenase, and f3-glucuronidase activities in patients with

(C Numbers in parentheses, number of subjects.

b Mean ± S.E.

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Fecalanaerobicmicroflora ofnoncontrol

subjectsand patients with large bowel cancerorhereditary largebowelpolypsConcentration

of organisms/g drywtNonhereditaryControl

sub- Largebowel can- largebowelGenusjectscer patients polyppatientsBacteroides

(x 10―)1 .5 ±0.4@(7)b i .4 ±0.3 (12) 1.2 ±0.2(7)Fusobacterium(x 1010)1 .7 ±1.1 (5) 0.1 ±0.1 (5) 1 .0 ±0.4(3)Bifidobacterium(x 1010)5.4 ±1.9 (6) 2.9 ±1.1 (8) 7.1 ±2.3(6)Eubacterium

(x 10'°)2.1 ±1.3 (6) 10.9±6.8 (13) 4.1 ±1.0(6)Clostridium(x 1010)1 .2 ±0.7 (7) 4.9 ±2.9 (13) 0.8 ±0.4(7)Lactobacillus

(x 10'°)0.5 ±0.2 (4) 1 .5 ±0.4 (12) 2.4 ±0.7(7)Propionibacterium(x 10'°)1 .2 ±0.6 (6) 2.1 ±1.3 (9) 0.5 ±0.2(3)Anaerobic

cocci (x 1010)0.4 ±0.2 (4) 1.5 ±0.9 (10) 1.3 ±0.5(5)Lachnospira(x 10)0 (0) 1.0 ±0.9 (1) 0.6 ±0.5(1)Actinomyces(x 10)0.6 ±0.5 (1) 0.1 ±0.06 (2) 0(0)Butyrivibrio

(x 10w)0 (0) 0 (0) 8.2 ±7.6(1)Unidentified(x 10'°)1 .4 ±0.7 (6) 0.1 ±0.04 (4) 0(0)Total

meanconcentration2.8 ±0.7 3.8 ±1.1 3.0 ±0.3(x10―)

Table3Percentagecomposition and distribution of fecalanaerobicgenera

compared to total fecal microscopic anaerobic countincontrolsubjects and patients with large bowel cancerornonhereditary

large bowelpolypsLarge

Nonheredibowel can- tarybargeControl

cer pa- bowelpolypGenussubjects tientspatientsBacteroides

52.0 46.540.2Fusobacterium6.3 1.63.8Bifidobacterium

18.2 11.021.7Eubacterium8.8 19.714.9Clostridium3.2 11.83.4Lactobacillus1.5 6.18.9Propionibacterium3.5 2.52.1Anaerobic

cocci 1.9 2.34.7Lachnospira0 0.30.3Actinomyces0.2 0.020Butyrivibrio0 00.04Unidentified5.1 0.3 0

A. J. Mastromarino et al.

Table 2

a Mean ±SE.b Numbers in parentheses, number of subjects from whom genus was isolated.

mum, and Clostridium tertium , were greater in large bowelcancer patients. One or more of the lecithinase-negativespecies of Clostridium tertium was recovered from thestools of 5 of 13 large bowel cancer patients (38.5%),whereas only 1 of 7 control specimens (14.3%) and 1 of 7stools of patients with nonhereditary large bowel polyps(14.3%) yielded similar organisms. Moreover, the meanconcentration of this group of organisms was 2 logs greaterin large bowel cancer patients than in the other groups.These differences, however, were not statistically significant.

Despite a thorough quantitative and qualitative study ofthe fecab anaerobic flora of control subjects and patientswith large bowel cancer or nonhereditary large bowelpobyps, no clearly discernible statistically significant relationships between the fecab profile of the bacterial flora andcolon cancer were evident. However, the metabolic activities of the fecal bacterial flora among these groups ofsubjects differed significantly. Although the mean fecabbacterial 7a-dehydroxylase and cholesterol dehydrogenaseactivities were not significantly different between patientswith large bowel cancer and nonhereditary barge bowelpolyps, their mean activities were significantly increased inboth groups compared to healthy control subjects (Table4). fJ-Glucuronidase activities, however, were similaramong the study groups (Table 4). The data presented inthis series represent an elaboration of results previouslypublished (32) and include additional large bowel cancerpatients.

DISCUSSION

The present study does not suggest any obvious shift inthe profile of the microbial flora in individuals at increasedrisk for barge bowel cancer. Fmnegoldet a!. (16) observedsignificantly different concentrations of some fecab organisms in cobonic polyp patients and matched controls, butthe organisms differed from those found to be different in aprevious study of Japanese Americans or Japanese on

group of organisms in control subjects and in patients withnonhereditary large bowel polyps, were less frequentlyisolated from large bowel cancer patients; species of Fusobacterium were more frequently isolated from healthy controls. Species of Bacteroides were found in highest concentrations in all but one specimen, and constituted the bulk ofthe anaerobic fecab flora in each group of subjects (40 to52%). Bifidobacterium and Eubacterium were recovered inthe next highest concentrations. Species of Eubacteriumrepresented a greater percentage of the fecal anaerobicflora of large bowel cancer patients (20%) and patients withnonhereditary large bowel polyps (15%) than controls (9%).Species of Clostridium constituted a relatively greater percentage of the fecab flora in patients with large bowelcancer (12%) than in patients with nonhereditary largebowel polyps (3%) or control subjects (3%).

The results also revealed that the frequencies and converted mean concentrations of lecithmnase-negative speciesof Clostridium, i.e., Clostriduim butyricum, Clostridiumindolis, Clostridium paraputrificum, Clostridium sartago for

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Fecalmicrobial 7a-dehydroxylase,cholesterol dehydrogenase,andfi-glucuronidaseactivitiesin patients with large bowel cancer, patients with nonhereditary largebowelpolyps,

and healthy control subjectsCholesterol

de- f3-GlucuroniGroup 7a-Dehydroxybas&' hydrogenasedaseLarge

bowel cancer pa- 74.0 ±6@2bC.1 (@/J)(I 59.8 ±6.0' (15) 255 ±40'(20)tientsNonhereditary

large 53.4 ±6.2' (9) 32.3 ±4.9' (6) 192 ±48'(10)bowelpolyppatientsHealthy

controls 38.3 ±2.92 (26) 20.8 ±2.72(15) 257 ±35' (24)

Fecal Flora of Colon Cancer Patients

Table 4

a 7a-Dehydroxylase activity is expressed as percentage of cholic acid microbially

converted to deoxycholic acid per 100 mg dry feces per 2-hr incubation; cholesteroldehydrogenaseactivity is expressedas percentageof cholesterol microbially convertedto coprostanol and coprostanoneper 100mg dry feces per 24-hr incubation; f3-gbucuronidase activity is expressed as @tgphenobphthabein glucuronide hydrolyzed per mg proteinper 4-hr incubation.

b Mean ± S.E.

C Mean with a differing numbered superscript, 1 and 2, between groups in a vertical

row is significant, p < 0.05 (Student'st test).(I Numbers in parentheses, number of subjects analyzed.

Western diets (15). A recent study by Finegold et al. (17)demonstrated that Seventh-Day Adventists had fewer C/ostridium septicum and C. tertium isolates than did nonAdventists. Although the amount of fecal secondary bileacids and f3-glucuronidase activity is decreased in vegetarians (42), no significant difference was found between thetypes or numbers of bacteria in their stools and those of thecontrol group. Several studies (13, 15, 16, 18, 22, 34, 46),therefore, support our finding that gross differences in thecomposition of the fecab flora are not apparent betweenhigh- and low-risk populations. These data are not incomplete agreement with those of Hill et al. (28), although,bike Finegold et al. (15, 16), we report a slightly higherfrequency of the becithinase-negative, nuclear-dehydrogenating clostridia among large bowel cancer patients thancontrol subjects. Hill et al. (28) have found a correlationbetween bile acid metabolites and increased concentrations of the nuclear-dehydrogenating cbostridia in patientswith barge bowel cancer.

However, work reported from England (1, 9, 11, 24, 27,36) suggests differences in the fecal microflora of high- andlow-risk populations. When high-risk subjects (British andAmerican) and bow-risk subjects (Ugandans, Indians, andJapanese) were compared, the former group was found tohave more fecal Bacteroides and Bifidobacterium , whereasthe latter group had more aerobic streptococci and enterobacteria (1, 27). Further studies by this group reveal moreGram-negative anaerobes (Bacteroides and Fusobacterium)in subjects consuming a Western diet, and a greater proportion of Gram-positive organisms, especially Eubacterium, among subjects from low-risk areas (36). Steroidnuclear-dehyd rogenating clostrid ia have been characterized in the stools of high-risk populations more frequentlythan in low-risk groups (24, 28).

Although some minor quantitative variations in intestinalflora were observed between high-risk populations (polyppatients and North Americans, and Japanese-Americans onWestern diets) and bow-risk populations [rural Japanese,Africans of the Tswana tribe, and Japanese-Americanseating a traditional Japanese diet (15, 16, 35)], no singleorganism or group of organisms was characteristic of high

or low-risk groups. Notwithstanding the unequivocal epidemiological clues of the importance of diet in large bowelcancer (2, 10, 21, 48, 50), attempts to alter the compositionof the fecal flora by alterations in dietary intake havegenerally proved unsuccessful (13, 22, 35). Yet markeddifferences can be demonstrated among the flora of individuals (34-36).

The observed elevation in enzyme activity of the fecalflora to metabolize bile acids (7a-dehydroxylase) and chobesterol (cholesterol dehydrogenase) in patients with largebowel cancer or nonhereditary large bowel polyps compared to that in healthy control subjects may be due toaltered physiological conditions in the colon affecting thebacteria that act on bile acids and cholesterol, or to quantitative and/or qualitative alterations in the cholesterol andbile acid-degrading anaerobic bacteria. Although the bacteriobogicab profiles generated in this study do not appearto substantiate significant differences in the flora per se,this study and others (5, 9, 26, 28, 37, 46) have indirectlyconfirmed differences in fecal bacterial enzyme activities asindicated by the fecal excretion of bacterial degradationproducts of bile acids and cholesterol. In contrast, nodifferences in the fecal bacterial f3-gbucuronidase activitywere observed among the study groups. Previous studies(19, 39) have demonstrated a relationship between diet andfecal microbial fJ-glucuronidase activity. In the presentstudy, all volunteers, including the patients, were consuming a mixed Western diet, suggesting that the compositionof the diet is a major factor in determining the bacterial f3-glucuronidase activity.

An important point that arises from these studies is thattaxonomic grouping of bacteria may be inadequate whenmeasuring the effect of diet on the intestinal microfbora.Since the intestinal bacteria contain many adaptive andconstitutive enzymes, the metabolic activity of the gutbacteria can be modified appreciably by diet or otherenvironmental factors. It appears that the overall metabolicactivities of intestinal bacteria, regardless of species, needto be examined under different dietary conditions, as wellas in groups consuming different diets, to understand therobeof gut bacteria in the etiology of colon cancer. Such an

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A. J. Mastromarino et al.

approach has been proposed by several investigators (13,19, 32, 35). Elucidation of the metabolic activities of theorganisms should provide a better understanding of thepotential role of the intestinal microflora in the genesis oflarge bowel cancer. Correlations between the compositionof the fecal microbial flora, fecal metabolities, and bacterialenzyme activities are being investigated.

ACKNOWLEDGMENTS

The authors wish to thank C. Sharma, B. Schimpf, and A. Molless for theirexpert laboratory technical assistance; C. Gustafson, R. N., nurse-coordinator for specimen collection; D. Conroy for manuscript preparation; and thevolunteers as well as Dr. M. Stearns and Dr. P. Sherlock and staff ofMemorial Hospital, New York, who collaborated and cooperated in thisstudy.

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4462 CANCER RESEARCH VOL. 38

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1978;38:4458-4462. Cancer Res   Anthony J. Mastromarino, Bandaru S. Reddy and Ernst L. Wynder  Patients and Patients with Nonhereditary Large Bowel PolypsFecal Profiles of Anaerobic Microflora of Large Bowel Cancer

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