INFECTION AND IMMUNITY, Jan. 1979, p. 128-132 Vol. 23, No. 10019-9567/79/01-0128/05$02.00/0

Adhesion of Commensal Bacteria to the Large Intestine Wallin Humans

C. L. HARTLEY,`* C. S. NEUMANN,2 AND M. H. RICHMOND'Department of Bacteriology, Medical School, University Walk,' and University Department of Medicine,2

Bristol Royal Infirmary, Bristol, England

Received for publication 25 October 1978

Biopsies taken during colonoscopic examination of the human large bowel wereused to examine the relationship of the commensal bacterial to the mucosalepithelial cell surface. Bacteria were seen adhering to the exposed epithelial cellsurface and also to the mucus sheet. Isolation of aerobic organisms showed thatEscherichia coli are closely associated with the gut wall throughout the largeintestine. One strain of E. coli predominated in each biopsy, and this strain waspresent along the whole length of bowel. Adhesion of bacteria to the gut walldoes occur in vivo and may be one of the factors involved in the ability of anorganism to colonize and persist.

Although there have been numerous studieson the bacteriology of the alimentary tracts ofhumans and animals (12, 13) relatively little hasbeen published on the adhesion of bacteria tothe human gut epithelium. Spiral-shaped bac-teria, probably spirochetes, have been reportedin this location by Lee and his colleagues (7),but there seem to be no reports on the adhesionof Escherichia coli to the human gut epitheliumat any point between the stomach and the distalend of the large intestine, even though theseorganisms are common in the lower part of thegut.There has been much interest in the role of

adhesion to mucous membranes as an initialstep in pathogenesis (9). For example, adhesionof Neisseria gonorrhoeae to vaginal cells (8), ofMycoplasma pneumoniae to respiratory tractepithelium (14), and of Vibrio cholerae to brushborder cells of the small intestine (11) have allbeen implicated in the infectious process causedby these organisms, primarily as a meanswhereby the organisms persist in an area abun-dantly washed by secretions. However, there hasbeen little attention paid to the adhesion of theautochthonous flora to epithelial layers in nor-mal healthy individuals. The examination ofhealthy tissue removed during surgery suggeststhat the adhesion of commensal organisms tothe intestine wall of humans occurs in vivo (10,15), but the number of cases examined is limited.Moreover, most of the published studies rely onmicroscopy to detect the microorganism, andthere have been few attempts to grow the ad-hering bacteria and to characterize them.

In this study, we have examined the nature of

the aerobic flora adhering to samples of appar-ently normal gut epithelium taken from humanpatients during colonoscopic examinations ofthelarge bowel.

MATERIALS AND METHODSPatients. Colonoscopy was indicated in 17 patients

to diagnose or determine the extent of bowel disease.In 3, the diagnosis of ulcerative colitis was confirmed,whereas the remaining 14 patients were apparentlyhealthy in that no inflamed or cancerous tissue wasdetected. Fifteen of these patients were not takingantibiotics. The remaining two were taking sulfona-mides, and in those cases the E. coli found attached totheir biopsies were sulfonamide resistant.Biopsy sites. Biopsies of healthy bowel mucosa

were taken at various levels of the large intestine byusing an ACMI long colonoscope and biopsy forceps.Biopsy sites, numbered 1 to 6, were the rectum, sig-moid colon, descending colon, transverse colon, as-cending colon, and cecum, respectively. In 13 patients,biopsies were taken from the upper and lower extrem-ities of the large intestine. In four patients, biopsieswere taken throughout the length of the bowel. Biop-sies were placed in physiological saline and examinedimmediately.

Isolation of aerobic bacteria. The biopsy tissue waswashed with shaking in six changes of physiologicalsaline. Although this procedure is necessarily limitedin its efficiency, organisms which remain in countablenumbers after such extensive washing must be closelyassociated with the tissue. The tissue was blotted dry,weighed, and blended in a 0.1 ml of ground glasshomogenizer in saline. Serial dilutes of the homoge-nate were spread onto McConkey bile lactose agar(Difco Laboratories) plates. The plates were incubatedovermight at 37°C, and total coliform counts weremade. Representative colonies (20 or 100) were pickedinto nutrient broth. Biochemically verified isolates of

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BACTERIA IN THE LARGE BOWEL 129

E. coli were 0-antigen typed (6), and their antibioticresistance pattern was determined.

Light microscopy. In the four patients from whombiopsies were taken along the whole length of the largeintestine, duplicate biopsies were taken at each site.One biopsy was processed as outlined for the isolationof bacteria. The second was fixed in formalin andsubsequently embedded in a paraffin block and sec-

tioned. Sections were stained by Gram stain and were

stained for mucus by Alcian Blue.Scanning electron microscopy. Biopsies were

washed in saline, fixed in 2.5% glutaraldehyde in 0.1 Mcacodylate buffer (pH 7.2), and dehydrated in ethanoland acetone. The specimens were critical-point driedby using liquid carbon dioxide, and they were goldcoated by using a sputter technique. The biopsies wereviewed with a Cambridge S4 Stereoscan electron mi-croscope operating at an acceleration potential of 10kV.

RESULTS

Counts of aerobic bacteria. A total of 47biopsies from 17 patients were examined. E. coliwere isolated from 40 biopsies from 15 patients.Among the remainder, no organisms were iso-lated from three biopsies, whereas four biopsies(from two patients) yielded coliforms which werenot E. coli. The counts of E. coli per gram ofmucosal tissue ranged from 103 to 109 (Table 1).The magnitude of the count was not affected bythe site of sampling-counts from the wall of thecaecum were within the same range as thosefrom the rectum.Composition ofthe E. coli wall flora. From

one to three E. coli 0-types were isolated fromeach biopsy, with one 0-type always predomi-nating. In the case of eight biopsies (from fivepatients), 100 E. coli colonies were picked (Table2). From these samples, it emerged that it wassufficient to pick 20 colonies to isolate both thepredominant 0-type(s) and most of the minoritytypes. In the remaining 10 patients, 20 colonieswere therefore picked from each of 16 biopsies.In 75% (12/16) of these biopsies only one or twoE. coli 0-types were isolated (Table 3). In twobiopsies, both from patient 15, there was no

clearly predominating strain. Human fecal spec-imens rarely show more than two distinct 0-

types, with one type predominating (4, 16). Ide-ally, a fecal sample should have heen takenbefore and after colonoscopy to show that theorganisms isolated from the wall were capableof multiplying in the lumen and being excretedand detected in the feces. In practice, such sam-

ples were impossible to obtain.Distribution of 0-types along the large

bowel. In the four patients from whom biopsiesat various points along the length of the largeintestine were obtained, the same E. coli 0-type

was predominant on the wall throughout thebowel.Light microscopy. Sections of colonic tissue

stained for mucin revealed mucus-filled gobletcells and an adherent mucus layer on the epi-

TABLE 1. E. coli counts on biopsy tissueLogE. LogE.

Pa- Sie coli Pa- Sie colitient count/g tient Site count/g

of tissue of tissue

I 1 6.6 6 1 5.92 6.3 6 6.03 6.54 6.5 7 1 6.55 6.6 6 6.36 6.6

2 1 4.3 8 1 3.02 6.8 6 6.63 6.04 6.3 9 1 5.05 6.5 6 7.06 6.3

3 1 4.0 10 1 -

2 3.3 6 4.03 4.0 11 4 7.64 3.0

4 1 4.7 12 1 5.52 5.0 6 5.53 -a 13 1 5.04 - 6 6.35 4.76 4.7

5 1 6.6 14 1 5.56 7.0

4 7.3 15 1 8.76 8.9

a _ No bacteria isolated.

TABLE 2. Sampling of representative E. coli-comparison of 100-colony and 20-colony isolations

100 colonies 20 colonies

No. No.Pa- of Pre- of Pre-tient Si. dif- dominant dif- dominant

ferent 0-type ferent 0-type0- (%) 0- (%)

types types

5 1 2 044 (95) 2 044 (90)4 2 044 (92) 2 044 (100)

6 1 1 038 (100) 1 038 (100)6 3 038 (92) 2 038 (90)

7 1 2 07 (99) 1 07 (100)6 3 07 (90) 3 07 (80)

8 6 3 NT" (70) 2 NT (85)9 6 1 01 (100) 1 01 (100)a NT, Not typable.

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130 HARTLEY, NEUMANN, AND RICHMOND

TABLE 3. Composition of E. coli wall floraaPredominant

Patient Site 0-type Other 0-types(%)

5 1 044 (90) 0754 044 (100)

6 1 038 (100)6 038 (100)

7 1 07 (100)6 07 (80) 09, 023

8 6 NT (85) NT9 6 01 (100)11 4 06 (100)12 1 075 (65) NT, 019

6 075 (95) 08313 6 02 (65) NT14 1 02 (75) 06

6 02 (90) 0615 1 NT (45) 012, 020, 01

6 012 (55) NT, 020, 01, 02,0107

aStrain with a different antibiotic resistance patternfrom that of predominant strain. NT, Not tested.

thelial cell surface and mucus-filled intestinalcrypts. Gram-negative and -positive bacteriawere seen both within and on the surface of theadherent mucus layer and within plugs of mucusat the mouths of the crypts. Gram-negative, rod-shaped bacteria predominated.Scanning electron microscopy. The sur-

face structure of the large intestinal mucosa withbrush border cells and mucus-exuding gobletcells could be clearly seen (Fig. 1). Bacteria couldbe observed in close association with the gutwall (Fig. 2). Organisms were found to be morefrequently associated with mucus, which had notbeen completely removed by the washing pro-cedure, than with the exposed cell surface (Fig.3).

DISCUSSIONFor obvious reasons, it is difficult to study the

adhesion of bacteria to normal human gut epi-thelium, and all the experimental approachesthat have been attempted so far have their in-herent disadvantages (12). Surgical removal ofmaterial usually only occurs in the presence ofdiagnosed disease and after premedication, andmaterial obtained after fatal accidents is usuallynot available soon enough for one to be confidentthat postmortem changes have not taken place.The main objections to the use of biopsy

material of the type studied here are, first, thatthe samples represent only a very small propor-tion of the total area under consideration (12,13) and, second, the risk of contamination of thesamples that occurs during colonoscopy. In thestudies reported here, the same E. coli 0-antigen

types were usually found adhering very tightlyto the excised epithelium from a number of sitesin the gut of the patient. This uniformity ofbacterial adhesion at a number of sites indicatesthat the small biopsy samples are likely to berepresentative of wide areas of gut epithelium.As far as the second objection is concerned, ifthe adhesion occurs by contamination, it doesseem to require some rather special pleading tosuggest that the epithelium had no strongly at-tached E. coli when in situ, despite the largenumber of these organisms present in the gutlumen, but that on all occasions it became con-taminated by a limited number of strongly ad-hering 0-antigen types in the course of retrieval

4" -i4 / ~~~~~~~~~~~~ 1~~~~,i

FIG. 1. Surface structure of the large intestine. (a)Polygonal unit with central crypt, X650; (b) individ-ual brush border cells and goblet cells exuding mu-cus, X2,700.

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BACTERIA IN THE LARGE BOWEL 131

thelium is the presence of mucus, and it is inter-esting that the bacteria which resist washing areoften associated with the mucus sheet. It couldbe that an ability to metabolize mucin is a keyfeature in the ability of bacteria to live in thissituation. Even while degrading the mucus, such

-*A ;;,> zi bacteria may provide a benign protection for the~ _,gepithelium proper against external influences.

ab__K-sesf- Although the vast majority of bacteria that->*w.,y,,.- v were visualized with light electron microscopy

were probably anaerobes, aerobic organisms,w_8sJr-e * which form only 1% or less of the large gut lumen

} * Vw 0 *t -$>§ flora, could be isolated from the extensivelywashed biopsy tissue. Perhaps the most striking

5"*4w,- > BB feature of this work is the uniformity of the

fft ~~~~~~~~~~~~~tU iI

,*WN a e saVe

;r->#'/.+ .;r *

FIG2 Bateiain close association with the ,. '~''Sbrush border cells. (a) x6,000; (b) x12,OOO0 L!9 4{ f e

of the biopsy forceps. jThe present study has shown that gram-neg- it e 3w

ative bacilli can be closely associated with the _5K,rpmucosal surface of the large intestine in humans. b iJJ.;4;All epithelial surfaces in contact with the "out- .vA0 ;side" of an animnal are vulnerable to external -influences and, despite its "internal" situation, w ; l-. >the epithelium of the large gut can be no excep- _tion. In this case the immediate exterior-the _w at"'.-.(s.rlumen-is very rich in bacteria, and it is perhaps v>e^-' _not surprising that the epithelium provides itself _=L5]with a coating of bacteria to constitute an inter- r _ - k ;Oface. Microscopy shows bacteria both adheringQR7to the exposed epithelial surface and closely FIG. 3. (a) Mucus sheet which has not been re-associated with the mucous sheath. An impor- moved by the washing process, x650; (b) Many orga-tant feature of the surface of the large gut epi- nisms associated with the mucus sheet x2,400.

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132 HARTLEY, NEUMANN, AND RICHMOND

aerobic bacterial colonization of the gut epithe-lium. Even in those cases where biopsies weretaken at widely separated points in the largebowel, each site yielded the same E. coli 0-types. This observation suggests that each indi-vidual has something approaching a pure cultureof coliforms coating his gut epithelium at anyone time. Unfortunately, we are unable to com-ment on the attachment of anaerobes as yet. Itwas also impossible in this survey, because oflack of availability of pre- and post-colonoscopyfecal specimens, to determine whether the re-stricted number of 0-types found in close asso-ciation with the surface of the large bowel werethe same as those which constituted the major-ity flora in the lumen of the gut. However,experiments with chickens (unpublished data)have shown that the bacteria adhering to thewall are indeed the same as those forming themajority strains in the lumen.

It is impossible to determine from this studythe precise role of adhesion in colonization andpersistence. Previous work has shown thatstrains of E. coli vary greatly in their ability tosurvive as majority components in the humangastrointestinal tract (4, 6). The persistence oforganisms in the gut may involve both an abilityto survive and multiply under the conditionsprevailing in the large bowel on the one hand,and adhesion to the gut wall on the other. Noth-ing is known about the relative colonizing abili-ties of commensal anaerobic enteric bacteria,and very little is known about aerobes exceptthat certain 0-types of E. coli are more preva-lent in the gut than others (1). Whether "goodcolonizers" first adhere to the gut wall and arethereby shed into the lumen or whether theorganisms possess an enhanced ability to multi-ply in the conditions provided in the gut andsubsequently adhere to the wall, is as yet notclear. The resistance of adhering bacteria toremoval by mechanical means shown in thestudy would suggest adhesion to the gut wallwas more than a transitory, consequential phe-nomenon. Since only a minority of bacterialstrains adhered strongly in this way, and sincethey were able to resist the "wash-out" effectsof catharsis and other procedures before colon-oscopy, this suggests that adhesion, either to theepithelial cells or to the mucus layer, does playa role in colonization.

ACKNOWLEDGMENTS

We thank Adrian Manning and Robin Teague and theirpatients for cooperation in obtaining biopsy material. We areindebted to R. Porter for his excellent technical assistance inthe use of the scanning electron microscope.

This work was supported by a grant from the MedicalResearch Council.

LITERATURE CITED

1. Ewing, W. H., and B. R. Davis. 1961. The 0 antigengroups of Escherichia coli cultures from varioussources, p. 1-21. Communicable Disease Centre Publi-cation, Atlanta.

2. Fuller, R. 1975. Nature of the determinant responsiblefor the adhesion of lactobacilli to chicken crop epithelialcells. J. Gen. Microbiol. 87:245-250.

3. Gibbons, R. J., and J. Van Houte. 1971. Selectivebacterial adherence to oral epithelial surfaces and itsrole as an ecological determinant. Infect. Immun. 3:567-573.

4. Hartley, C. L., H. M. Clements, and K. B. Linton.1977. Escherichia coli in the faecal flora of man. J.Appl. Bacteriol. 43:261-269.

5. Hartley, C. L., K. Howe, A. H. Linton, K. B. Linton,and M. H. Richmond. 1975. Distribution of R plasmidsamong the 0-antigen types of Escherichia coli isolatedfrom human and animal sources. Antimicrob. AgentsChemother. 8:122-131.

6. Hartley, C. L., and M. H. Richmond. 1975. Antibioticresistance and survival of E. coli in the alimentary tract.Br. Med. J. 4:71-74.

7. Lee, F. D., A. Kraszewski, J. Gordon, J. G. R. Howie,D. McSeveny, and W. A. Harland. 1971. Intestinalspirochaetosis. Gut 12:126-133.

8. Mardh, P.-A., and L. Westrom. 1976. Adherence ofbacteria to vaginal epithelial cells. Infect. Immun. 13:661-666.

9. Mims, C. A. 1977. The pathogenesis of infectious disease,p. 14-17. Academic Press Inc., London.

10. Nelson, D. P., and L. J. Mata. 1970. Bacterial floraassociated with the human gastrointestinal mucosa.Gastroenterology 58:56-61.

11. Nelson, E. T., J. D. Clements, and R. A. Finkelstein.1976. Vibrio cholerae adherence and colonization inexperimental studies: electron microscope studies. In-fect. Immun. 14:527-547.

12. Savage, D. C. 1977. Microbial ecology of the gastrointes-tinal tract. Annu. Rev. Microbiol. 31:107-133.

13. Savage, D. C. 1972. Survival on mucosal epithelia, epi-thelial penetration and growth in tissues of pathogenicbacteria. Symp. Soc. Gen. Microbiol. 22:25-57.

14. Sobeslavsky, O., B. Prescott, and R. M. Chanock.1968. Adsorption of Mycoplasma pneumoniae to neu-raminic acid receptors of various cells and possible rolein virulence. J. Bacteriol. 96:695-705.

15. Tabaqchali, S., A. Howard, C. H. Teoh-chan, K. A.Bettelheim, and S. L. Gorbach. 1977. Escherichiacoli serotypes throughout the gastrointestinal tract inpatients with intestinal disorders. Gut 18:351-355.

16. Weidemann, B., and H. Knothe. 1969. Untersuchungenuber die stabilitat der koliflora des gesunden menschen.Arch. Hyg. 153:342-348.

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