Adhesion of Commensal Bacteria to the Large Intestine Wall

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1 INFECTION AND IMMUNITY, Jan. 1979, p Vol. 23, No /79/ /05$02.00/0 Adhesion of Commensal Bacteria to the Large Intestine Wall in 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 were used to examine the relationship of the commensal bacterial to the mucosal epithelial cell surface. Bacteria were seen adhering to the exposed epithelial cell surface and also to the mucus sheet. Isolation of aerobic organisms showed that Escherichia coli are closely associated with the gut wall throughout the large intestine. One strain of E. coli predominated in each biopsy, and this strain was present along the whole length of bowel. Adhesion of bacteria to the gut wall does occur in vivo and may be one of the factors involved in the ability of an organism to colonize and persist. Although there have been numerous studies on the bacteriology of the alimentary tracts of humans and animals (12, 13) relatively little has been published on the adhesion of bacteria to the human gut epithelium. Spiral-shaped bacteria, probably spirochetes, have been reported in this location by Lee and his colleagues (7), but there seem to be no reports on the adhesion of Escherichia coli to the human gut epithelium at any point between the stomach and the distal end of the large intestine, even though these organisms are common in the lower part of the gut Ṫhere has been much interest in the role of adhesion to mucous membranes as an initial step in pathogenesis (9). For example, adhesion of Neisseria gonorrhoeae to vaginal cells (8), of Mycoplasma pneumoniae to respiratory tract epithelium (14), and of Vibrio cholerae to brush border cells of the small intestine (11) have all been implicated in the infectious process caused by these organisms, primarily as a means whereby the organisms persist in an area abundantly washed by secretions. However, there has been little attention paid to the adhesion of the autochthonous flora to epithelial layers in normal healthy individuals. The examination of healthy tissue removed during surgery suggests that the adhesion of commensal organisms to the intestine wall of humans occurs in vivo (10, 15), but the number of cases examined is limited. Moreover, most of the published studies rely on microscopy to detect the microorganism, and there have been few attempts to grow the adhering bacteria and to characterize them. In this study, we have examined the nature of 128 the aerobic flora adhering to samples of apparently normal gut epithelium taken from human patients during colonoscopic examinations of the large bowel. MATERIALS AND METHODS Patients. 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 apparently healthy in that no inflamed or cancerous tissue was detected. Fifteen of these patients were not taking antibiotics. The remaining two were taking sulfonamides, and in those cases the E. coli found attached to their biopsies were sulfonamide resistant. Biopsy sites. Biopsies of healthy bowel mucosa were taken at various levels of the large intestine by using an ACMI long colonoscope and biopsy forceps. Biopsy sites, numbered 1 to 6, were the rectum, sigmoid colon, descending colon, transverse colon, ascending colon, and cecum, respectively. In 13 patients, biopsies were taken from the upper and lower extremities of the large intestine. In four patients, biopsies were taken throughout the length of the bowel. Biopsies were placed in physiological saline and examined immediately. Isolation of aerobic bacteria. The biopsy tissue was washed with shaking in six changes of physiological saline. Although this procedure is necessarily limited in its efficiency, organisms which remain in countable numbers after such extensive washing must be closely associated with the tissue. The tissue was blotted dry, weighed, and blended in a 0.1 ml of ground glass homogenizer in saline. Serial dilutes of the homogenate were spread onto McConkey bile lactose agar (Difco Laboratories) plates. The plates were incubated overmight at 37 C, and total coliform counts were made. Representative colonies (20 or 100) were picked into nutrient broth. Biochemically verified isolates of

2 VOL. 23, 1979 E. coli were 0-antigen typed (6), and their antibiotic resistance pattern was determined. Light microscopy. In the four patients from whom biopsies were taken along the whole length of the large intestine, duplicate biopsies were taken at each site. One biopsy was processed as outlined for the isolation of bacteria. The second was fixed in formalin and subsequently embedded in a paraffin block and sectioned. 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 M cacodylate buffer (ph 7.2), and dehydrated in ethanol and acetone. The specimens were critical-point dried by using liquid carbon dioxide, and they were gold coated by using a sputter technique. The biopsies were viewed with a Cambridge S4 Stereoscan electron microscope operating at an acceleration potential of 10 kv. RESULTS Counts of aerobic bacteria. A total of 47 biopsies from 17 patients were examined. E. coli were isolated from 40 biopsies from 15 patients. Among the remainder, no organisms were isolated from three biopsies, whereas four biopsies (from two patients) yielded coliforms which were not E. coli. The counts of E. coli per gram of mucosal tissue ranged from 103 to 109 (Table 1). The magnitude of the count was not affected by the site of sampling-counts from the wall of the caecum were within the same range as those from the rectum. Composition of the E. coli wall flora. From one to three E. coli 0-types were isolated from each biopsy, with one 0-type always predominating. In the case of eight biopsies (from five patients), 100 E. coli colonies were picked (Table 2). From these samples, it emerged that it was sufficient to pick 20 colonies to isolate both the predominant 0-type(s) and most of the minority types. In the remaining 10 patients, 20 colonies were therefore picked from each of 16 biopsies. In 75% (12/16) of these biopsies only one or two E. coli 0-types were isolated (Table 3). In two biopsies, both from patient 15, there was no clearly predominating strain. Human fecal specimens rarely show more than two distinct 0- types, with one type predominating (4, 16). Ideally, a fecal sample should have heen taken before and after colonoscopy to show that the organisms isolated from the wall were capable of multiplying in the lumen and being excreted and detected in the feces. In practice, such samples were impossible to obtain. Distribution of 0-types along the large bowel. In the four patients from whom biopsies at various points along the length of the large intestine were obtained, the same E. coli 0-type BACTERIA IN THE LARGE BOWEL 129 was predominant on the wall throughout the bowel. Light microscopy. Sections of colonic tissue stained for mucin revealed mucus-filled goblet cells and an adherent mucus layer on the epi- TABLE 1. E. coli counts on biopsy tissue LogE. LogE. Pa- Sie coli Pa- coli Sie tient count/g tient Site count/g of tissue of tissue I a a _ No bacteria isolated. TABLE 2. Sampling of representative E. colicomparison of 100-colony and 20-colony isolations 100 colonies 20 colonies No. No. Pa- of Pre- of Pretient Si. dif- dominant dif- dominant ferent 0-type ferent 0-type 0- (%) 0- (%) types types (95) (90) (92) (100) (100) (100) (92) (90) (99) 1 07 (100) (90) 3 07 (80) NT" (70) 2 NT (85) (100) 1 01 (100) a NT, Not typable.

3 130 HARTLEY, NEUMANN, AND RICHMOND TABLE 3. Composition of E. coli wall floraa Predominant Patient Site 0-type Other 0-types (%) (90) (100) (100) (100) (100) 6 07 (80) 09, NT (85) NT (100) (100) (65) NT, (95) (65) NT (75) (90) NT (45) 012, 020, (55) NT, 020, 01, 02, 0107 astrain with a different antibiotic resistance pattern from that of predominant strain. NT, Not tested. thelial cell surface and mucus-filled intestinal crypts. Gram-negative and -positive bacteria were seen both within and on the surface of the adherent mucus layer and within plugs of mucus at the mouths of the crypts. Gram-negative, rodshaped bacteria predominated. Scanning electron microscopy. The surface structure of the large intestinal mucosa with brush border cells and mucus-exuding goblet cells could be clearly seen (Fig. 1). Bacteria could be observed in close association with the gut wall (Fig. 2). Organisms were found to be more frequently associated with mucus, which had not been completely removed by the washing procedure, than with the exposed cell surface (Fig. 3). DISCUSSION For obvious reasons, it is difficult to study the adhesion of bacteria to normal human gut epithelium, and all the experimental approaches that have been attempted so far have their inherent disadvantages (12). Surgical removal of material usually only occurs in the presence of diagnosed disease and after premedication, and material obtained after fatal accidents is usually not available soon enough for one to be confident that postmortem changes have not taken place. The main objections to the use of biopsy material of the type studied here are, first, that the samples represent only a very small proportion of the total area under consideration (12, 13) and, second, the risk of contamination of the samples that occurs during colonoscopy. In the studies reported here, the same E. coli 0-antigen types were usually found adhering very tightly to the excised epithelium from a number of sites in the gut of the patient. This uniformity of bacterial adhesion at a number of sites indicates that the small biopsy samples are likely to be representative of wide areas of gut epithelium. As far as the second objection is concerned, if the adhesion occurs by contamination, it does seem to require some rather special pleading to suggest that the epithelium had no strongly attached E. coli when in situ, despite the large number of these organisms present in the gut lumen, but that on all occasions it became contaminated by a limited number of strongly adhering 0-antigen types in the course of retrieval 4 / ~~~~~~~~~~~~ 1~ ~~~,i INFECT. IMMUN. 4" -i FIG. 1. Surface structure of the large intestine. (a) Polygonal unit with central crypt, X650; (b) individual brush border cells and goblet cells exuding mucus, X2,700.

4 VOL. 23, 1979 BACTERIA IN THE LARGE BOWEL 131 thelium is the presence of mucus, and it is interesting that the bacteria which resist washing are often associated with the mucus sheet. It could be that an ability to metabolize mucin is a key feature in the ability of bacteria to live in this situation. Even while degrading the mucus, such -*A^ ;;,> zi bacteria may provide a benign protection for the ~ _,gepithelium proper against external influences. ab K-ses f- 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 } * w 0 V*t -$> flora, could be isolated from the extensively washed biopsy tissue. Perhaps the most striking 5"*4w,- > BB feature of this work is the uniformity of the fft ~~~~~~~~~~~~~t U ii,*wn a e save Downloaded from #'/.+ ;r->.;r * FIG 2 Bateiain close association with the,. 'S '~' brush border cells. (a) x6,000; (b) x12,ooo0 L!9 4{ f e of the biopsy forceps. j The present study has shown that gram-neg- it e 3w ative bacilli can be closely associated with the _5K,rp mucosal 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.r lumen-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 ;O face. Microscopy shows bacteria both adheringqr7 to the exposed epithelial surface and closely FIG. 3. (a) Mucus sheet which has not been reassociated with the mucous sheath. An impor- moved by the washing process, x650; (b) Many orgatant feature of the surface of the large gut epi- nisms associated with the mucus sheet x2,400. on July 22, 2018 by guest

5 132 HARTLEY, NEUMANN, AND RICHMOND aerobic bacterial colonization of the gut epithelium. Even in those cases where biopsies were taken at widely separated points in the large bowel, each site yielded the same E. coli 0- types. This observation suggests that each individual has something approaching a pure culture of coliforms coating his gut epithelium at any one time. Unfortunately, we are unable to comment on the attachment of anaerobes as yet. It was also impossible in this survey, because of lack of availability of pre- and post-colonoscopy fecal specimens, to determine whether the restricted number of 0-types found in close association with the surface of the large bowel were the same as those which constituted the majority flora in the lumen of the gut. However, experiments with chickens (unpublished data) have shown that the bacteria adhering to the wall are indeed the same as those forming the majority strains in the lumen. It is impossible to determine from this study the precise role of adhesion in colonization and persistence. Previous work has shown that strains of E. coli vary greatly in their ability to survive as majority components in the human gastrointestinal tract (4, 6). The persistence of organisms in the gut may involve both an ability to survive and multiply under the conditions prevailing in the large bowel on the one hand, and adhesion to the gut wall on the other. Nothing is known about the relative colonizing abilities of commensal anaerobic enteric bacteria, and very little is known about aerobes except that certain 0-types of E. coli are more prevalent in the gut than others (1). Whether "good colonizers" first adhere to the gut wall and are thereby shed into the lumen or whether the organisms possess an enhanced ability to multiply in the conditions provided in the gut and subsequently adhere to the wall, is as yet not clear. The resistance of adhering bacteria to removal by mechanical means shown in the study would suggest adhesion to the gut wall was more than a transitory, consequential phenomenon. Since only a minority of bacterial strains adhered strongly in this way, and since they were able to resist the "wash-out" effects of catharsis and other procedures before colonoscopy, this suggests that adhesion, either to the epithelial cells or to the mucus layer, does play a role in colonization. ACKNOWLEDGMENTS We thank Adrian Manning and Robin Teague and their patients for cooperation in obtaining biopsy material. We are indebted to R. Porter for his excellent technical assistance in the use of the scanning electron microscope. This work was supported by a grant from the Medical Research Council. LITERATURE CITED INFECT. IMMUN. 1. Ewing, W. H., and B. R. Davis The 0 antigen groups of Escherichia coli cultures from various sources, p Communicable Disease Centre Publication, Atlanta. 2. Fuller, R Nature of the determinant responsible for the adhesion of lactobacilli to chicken crop epithelial cells. J. Gen. Microbiol. 87: Gibbons, R. J., and J. Van Houte Selective bacterial adherence to oral epithelial surfaces and its role as an ecological determinant. Infect. Immun. 3: Hartley, C. L., H. M. Clements, and K. B. Linton Escherichia coli in the faecal flora of man. J. Appl. Bacteriol. 43: Hartley, C. L., K. Howe, A. H. Linton, K. B. Linton, and M. H. Richmond Distribution of R plasmids among the 0-antigen types of Escherichia coli isolated from human and animal sources. Antimicrob. Agents Chemother. 8: Hartley, C. L., and M. H. Richmond Antibiotic resistance and survival of E. coli in the alimentary tract. Br. Med. J. 4: Lee, F. D., A. Kraszewski, J. Gordon, J. G. R. Howie, D. McSeveny, and W. A. Harland Intestinal spirochaetosis. Gut 12: Mardh, P.-A., and L. Westrom Adherence of bacteria to vaginal epithelial cells. Infect. Immun. 13: Mims, C. A The pathogenesis of infectious disease, p Academic Press Inc., London. 10. Nelson, D. P., and L. J. Mata Bacterial flora associated with the human gastrointestinal mucosa. Gastroenterology 58: Nelson, E. T., J. D. Clements, and R. A. Finkelstein Vibrio cholerae adherence and colonization in experimental studies: electron microscope studies. Infect. Immun. 14: Savage, D. C Microbial ecology of the gastrointestinal tract. Annu. Rev. Microbiol. 31: Savage, D. C Survival on mucosal epithelia, epithelial penetration and growth in tissues of pathogenic bacteria. Symp. Soc. Gen. Microbiol. 22: Sobeslavsky, O., B. Prescott, and R. M. Chanock Adsorption of Mycoplasma pneumoniae to neuraminic acid receptors of various cells and possible role in virulence. J. Bacteriol. 96: Tabaqchali, S., A. Howard, C. H. Teoh-chan, K. A. Bettelheim, and S. L. Gorbach Escherichia coli serotypes throughout the gastrointestinal tract in patients with intestinal disorders. Gut 18: Weidemann, B., and H. Knothe Untersuchungen uber die stabilitat der koliflora des gesunden menschen. Arch. Hyg. 153: