Bacterial Populations Associated with Different Regions of the Human Colon Wall

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1983, p /83/ $02.00/0 Copyright D 1983, American Society for Microbiology Vol. 45, No. 3 Bacterial Populations Associated with Different Regions of the Human Colon Wall SUSAN C. CROUCHER,t ANNETTE P. HOUSTON, CATHERINE E. BAYLISS,* AND R. J. TURNER Agricultural Research Council, Food Research Institute, Norwich NR4 7UA, United Kingdom Received 17 August 1982/Accepted 10 December 1982 The microorganisms associated with the undiseased human colon wall were examined in material obtained from four sudden-death victims. In traffic accident subjects (aged 45 and 16 years) the anaerobe-aerobe ratio was about 104:1 in all areas of the colon examined, whereas in acute heart failure subjects (aged 74 and 46 years) the ratio was as low as 1.2:1. The flora of each individual was distinct and complex. Although the predominant anaerobes isolated were Bacteroides and Fusobacterium spp., which composed over 50% of the flora in some samples, the species isolated (indicated by morphology and glucose fermentation products) varied between individuals. Other major types observed were gram-positive nonsporing rods, including Bifidobacterium spp., and anaerobic cocci (between 8 and 20% of isolates). Clostridia were only isolated in significant numbers from one individual. Scanning electron microscopy showed that most of the organisms were present below the top surface of the mucin layer overlying the mucosa. The use of several different preparative procedures for microscopy showed a complex microbial structure within the mucus, but major variations in the bacterial populations in different areas of the colon were not found. Spiral-shaped organisms up to 60,um long in the form of double helices were found in two subjects by scanning electron microscopy but were not isolated during the parallel bacteriological investigation. The differences between this and previous studies are discussed in relation to experimental procedures and also in contrast to results with animals that showed a particularly specialized flora associated with the colon wall. Although the fecal flora of humans has been the subject of much research (13, 24), little is known about the composition of the flora associated with the mucous layer between the intestinal epithelium and the lumen. The mucosal flora may be of importance in maintaining the stability of the fecal flora and may have specific functions (10). Results from studies on the flora of the human colon wall conflict (15, 25-27), perhaps because of the difficulties in obtaining suitable material, as biopsies taken during colonoscopic examination of the large bowel are very small and may not be representative (15) and specimens obtained during intestinal surgery from uninvolved tissue (26, 27) may be abnormal due to the clinical condition or as a result of premedication. This report describes a detailed study of the bacterial flora associated with the human colon wall obtained from four sudden-death victims. The objective was to enumerate and characterize the anaerobic and facultative organisms associated with the normal human colonic mucot Present address: 25 Helena Road, Norwich, U.K. sa, to investigate microbial organization in the mucin layer by scanning electron microscopy (SEM), and to discover whether a specialized bacterial flora similar to that found in animals (6, 14, 30) is associated with the intestinal epithelium. MATERIALS AND METHODS Subjects. Specimens of colon wall were obtained from four sudden-death victims, subjects S1 through S4 (Table 1). There was no indication of bowel disease in any of the subjects. Anaerobic methods. The initial isolation and purification of anaerobes was carried out on prereduced media in an anaerobic cabinet (Forma Scientific) containing N2-H2-CO2 (85:10:5, vol/vol). Strict anaerobiosis was maintained by the technique of Hungate (20, 21) Ṗreparation of mucosal samples. A full thickness of colon wall was excised, and the fat was removed. The samples were immediately placed in an anaerobic jar and flushed with H2-C02 (90:10, vol/vol). The jar was transported to the laboratory at ambient temperature and put directly into an anaerobic cabinet, where all subsequent manipulations were carried out. Tissue (0.5 cm2) was also excised and processed for SEM and transmission electron microscopy by methods de- 1025

2 1026 CROUCHER ET AL. TABLE 1. Source of specimens Sub_ Time after death Sub- Age Sex Cause of death that sample was ject Se(Cuerfdet received in no. (yr) ~~~~~~~laboratory (h) S1 45 Male Traffic accident 5 S2 74 Male Acute heart failure 7 S3 16 Female Traffic accident 6 S4 46 Male Acute heart failure 6 scribed previously (6, 8). Samples for bacteriological investigation (0.3 to 0.5 g) were quickly transferred to weighed tubes containing 10 ml of anaerobic dilution solution (ADS) (9), and their weights were recorded before they were washed by being gently inverted 20 times in three changes of 10 ml of ADS. Microorganisms associated with the colon wall were removed from the washed tissue by treating it for 2 min in 20 ml of ADS in a Colworth Stomacher 80 (A. J. Seward, London), which gives a kneading action without macerating the tissue. The resulting solution is referred to as the initial dilution. The possibility that the stomaching procedure did not remove all the aerobic organisms closely associated with the wall tissue was assessed by macerating a sample of stomached tissue in 9 ml of maintenance medium (7) for 2 min with a top-drive miniature homogenizer and enumerating the aerobes. Microscopic examination. The initial dilution from each sample was examined by phase-contrast microscopy as a wet film and by Gram stain, as modified (12). Enumeration of anaerobes. The initial dilution from each sample was serially diluted in ADS and plated in duplicate onto prereduced VLhlf agar (VL medium [3] supplemented with 1,tg of hemin ml ', 5% [vol/vol] liver extract, 10% [vol/vol] chicken fecal extract [4]1 and 12 g of New Zealand agar liter '). The sigmoid Subject TABLE 2. Medium (organism type) colon sample from S2 was also plated onto enriched Trypticase soy agar (BBL Microbiology Systems) (1). The species isolated and the count obtained were unaffected by the medium used. The plates were incubated at 37 C for 5 to 7 days in the anaerobic cabinet, and the number of colonies was recorded. Enumeration of aerobes and lactobacilli. The initial dilution was removed from the cabinet and opened under CO. Further dilutions were carried out in boiled and cooled RCM broths (18) or maintenance medium (for S4) (7). Dilutions were enumerated as follows. Total aerobes: heart infusion agar (HI, Oxoid Ltd.), incubated aerobically at 37'C for 1 day. Enterobacteria: MacConkey 3 agar (Oxoid), incubated aerobically at 37'C for 1 day. Fecal streptococci: thallous acetate-tetrazolium-glucose agar (TLTG) (2), incubated aerobically at 37'C for I day and then at room temperature for several days to confirm that no other types developed. Lactobacilli: rogosa SL agar (Difco Laboratories), incubated under H2-CO2 (90:10, vol/ vol) at 37 C for 2 days. Isolation of anaerobes. All colonies from the highest dilutions showing growth in anaerobic conditions were purified by streaking onto prereduced VLhlf agar. After incubation at 37 C for 2 to 5 days, single colonies were picked into slopes of SM1O agar (4) and incubated at 37 C for 1 to 2 days. Stocks of anaerobes were maintained as freeze-dried cultures. Characterization of anaerobes. Young cultures, grown for I to 2 days in SM10 agar, were examined by Gram stain and as wet films with phase-contrast microscopy. The presence of spores was tested by growing cultures in SM10 broth at 37'C for 7 or 21 days. Portions (3 ml) were heated at 70'C for 10 min, and 1 ml was inoculated into fresh SM1O broth. If growth occurred the culture was reexamined for the position of spores in the cells. Colony counts of colonic mucosal samples from four subjects APPL. ENVIRON. MICROBIOL. No. of organisms (logl>/g [wet wt] of total colon wall) in: Ascending Transverse Descending Sigmoid Cecum colon colon colon colon S1 VLhlf' (total anaerobes) b 8.00 Rogosac (lactobacilli) HI (total aerobes) TLTG (fecal streptococci) 3.15 <2.00 <2.30 <2.23 MacConkey 3 (enterobacteria) S2 VLhlf Rogosa < HI TLTG MacConkey S3 VLhlf Rogosa <2.94 <2.83 <2.78 <2.97 <2.94 HI TLTG MacConkey 3 <2.94 <2.83 < <2.94 S4 VLhlf HI a Prereduced, inoculated, and incubated in an anaerobic cabinet. b, Not done. 'I ncubated under H,-CO (90:10, vol/vol).

3 VOL. 45, 1983 TABLE 3. BACTERIA ASSOCIATED WITH HUMAN COLON WALL 1027 Distribution of major bacterial types in four regions of the human colon wall (subject Si) Glucose No. of isolates from: Major bacterial types (135 isolates) fermentation Ascending Transverse Sigmoid products' Cecum colon colon colon All isolates Clostridia A b Gram-negative nonsporing rods Bacteroides spp. A P iv S Fusobacterium spp. A p B L s Other apbls Gram-positive nonsporing rods Bifidobacterium spp. A L s Other A b L s ab L s A(a)L(l)S Gram-positive cocci/ a L (s) coccobacilli Gram-negative cocci/ A 1 (s) coccobacilli a Capital letters indicate 210,umol/ml; small letters indicate 21 but <10 1Lmol/ml. Products in parentheses were not produced by all isolates. Products (acids): a, acetic; p, propionic; ib, isobutyric; b, butyric; iv, isovaleric; ic, isocaproic; 1, lactic; py, pyruvic; s, succinic. b -, Not done. Glucose fermentation products were determined from cultures grown (37 C, 7 days) in basal SM10, containing 0.5% (wt/vol) glucose but omitting volatile fatty acids. For estimation of volatile acids, 1 ml of ether was added to 4 ml of acidified supernatant (ph <2.0). The procedure of Holdeman and Moore (19) was then followed, except that MgSO4 was not added. For the estimation of nonvolatile acids, 2 ml of boron TABLE 4. Distribution of major bacterial types in four regions of the human colon wall (subject S2) Glucose No. of isolates from: Major bacterial types (54 isolates) fermentation Ascending Transverse Sigmoid productsa Cecum colon colon colon All isolates Clostridia A B l s Apibivicpy Al Gram-negative nonsporing rods Bacteroides spp. A P (p) (iv) S Fusobacterium spp. A p B L S Other b Gram-positive nonsporing rods Bifidobacterium spp. A (p) L s Other A l ALs ab L Gram-positive cocci/ A L coccobacilli A Al bl Aerobes Bacillus Coliforms a See Table 3, footnote a. b, Not done.

4 1028 CROUCHER ET AL. TABLE 5. Distribution of major bacterial types in five regions of the human colon wall (subject S3) Glucose No. of isolates from: Major bacterial types (129 isolates) fermentation Ascending Transverse Descending Sigmoid productsa Cecum colon colon colon colon All isolates Gram-negative nonsporing rods Bacteroides spp. A P (iv) s A P py s ApL ApLS Ap a p (iv) Fusobacterium spp. A p B s BLs pb s Other A p b L apb p b iv S APb b APbivS(s) Gram-positive nonsporing rods Bifidobacterium spp. A p L Other A B ABpL Bs A (a) p Apb Aps Gram-positive cocci/ A p coccobacilli Gram-negative cocci/ A p b coccobacilli A p Gram-variable nonsporing rods A p B 1 s a See Table 3, footnote a. b, Not done. trifluoromethanol complex (BDH Ltd.) was added to 2 ml of acidified supernatant. Samples were mixed gently and allowed to stand for 18 h at 20 C. Chloroform (1 ml) was then added, and the emulsion was mixed by inverting it 20 times. The tubes were spun at 700 x g for 1 min, and the chloroform layer was retained for analysis. Individual acids, both volatile and nonvolatile, were separated and identified by gas-liquid chromatography (5). Formic acid was not detected by this method. RESULTS Bacterial counts. The numbers of anaerobes and aerobes found to be associated with the normal human colon wall are shown in Table 2. The counts for each group of organisms were similar along the length of the colon in subjects Si, S2, and S3, but varied in S4. The number of anaerobes present was between 1.5 x 106 to 2.7 x 108 per g, but the major differences between subjects were in the number of aerobes (3.8 x 103 to 3.9 x 107 per g). Anaerobes were present at higher levels than aerobes in all four subjects. APPL. ENVIRON. MICROBIOL. In S1 and S3 the anaerobe-aerobe ratio was about 104:1, whereas in S2 and S4 the ratio was between 1.2:1 and 10:1. In Si and S3 the anaerobe-aerobe ratio reflected the low levels of aerobes present (3.8 x 103 to 2.0 x 104 per g). Increased numbers of enterobacteria and fecal streptococci accounted for most of the higher number of aerobes in S2. Lactobacilli were present at higher levels in Si than in S2 or S3. The percentage recovery of organisms could not be determined, since the presence of a large amount of tissue debris in the initial dilution meant that an accurate direct microscopic count could not be obtained. The tissue homogenate from S4 contained 7.2 x 105 aerobes per g of macerated tissue, suggesting that a proportion of the flora remained attached after the stomaching procedure. The numbers recovered were lower than those found previously. Bacterial isolates. A total of 318 isolates were obtained from subjects Si, S2, and S3. These isolates were picked from the highest dilutions showing growth on the anaerobic plates. All

5 VOL. 45, 1983 BACTERIA ASSOCIATED WITH HUMAN COLON WALL 1029 _wlf _-N" FIG. 1. SEM of bacteria associated with mucin layers in the colon. Tissue samples from S2 were excised from (a) cecum, (b) transverse, (c) descending, and (d) sigmoid colon, fixed with osmium vapor and freeze-dried (a) or dehydrated with acetone (b, c, d) and critical-point dried. Bar = 10 (a, b, c) or 1,um (d). U _ isolates were examined microscopically and checked for aerobic growth. Organisms representing all morphological types isolated from each specimen were tested for the presence of spores and glucose fermentation products. Some differences were observed in the bacteria isolated from the three subjects. In S1 and S3 all isolates were strict anaerobes, but three isolates from S2 were facultative anaerobes. All four samples from S1 yielded similar organisms (Table 3). These were predominantly gram-negative anaerobic rods, including Bacteroides and Fusobacterium spp. (49% of isolates). Other major types observed were gram-

6 1030 CROUCHER ET AL. APPL. ENVIRON. MICROBIOL. FIG. 2. SEM of spiral-shaped organisms associated with mucin layers. Tissue sample from S2 was excised from descending colon, fixed with osmium vapor, dehydrated with alcohol, and critical-point dried. Bar = 10,m. positive nonsporing rods (27%), including Bifidobacterium spp., and anaerobic cocci (20%). A few clostridia were isolated (4%). A total of 54 isolates were examined from four specimens from S2 (Table 4). These included one Bacillus sp. and two aerobic coliforms obtained from the sigmoid colon, where the highest aerobic count was recorded. Clostridia were present at higher levels (16% of isolates) than in specimens from Si, and gram-negative anaerobic rods were isolated from only two of the specimens, comprising 38% of the isolates from the ascending colon and 73% of those from the sigmoid colon. Gram-positive nonsporing rods were obtained only from the cecum and ascending colon (25% of total isolates). Anaerobic cocci were found in the same proportions as in S1 (20%). Compared with isolates from S1 and S2, those from S3 showed a much greater range of morphology and glucose fermentation products and included gram-negative nonsporing rods (54% of isolates), some of which formed a palisade arrangement, and gram-positive nonsporing rods in each region of the colon (38%) (Table 5). In addition, anaerobic cocci were found at lower levels than for Si or S2 (8%), and no clostridia were isolated. SEM. SEM examination of samples from Si (6) had suggested that most of the organisms associated with the wall were under the surface of the mucin. Surface examination of tissue from S2 prepared by freeze-drying without prior chemical fixation showed an almost featureless mucous surface in which the crypts were not visible. Exposure of the samples to osmium vapor before freeze-drying revealed bacteria in localized areas, embedded in mucin or protruding through the surface (Fig. la). However, acetone dehydration and critical-point drying led to shrinkage and revealed rods and cocci within the mucin (Fig. lb, c, and d). Solvent-dehydrated specimens from which some mucin had been removed showed spiral-shaped organisms in association with long rods (Fig. 2) rather than with the communities shown in Fig. 1. Spiral organisms up to 60,um long (Fig. 3) were present in all samples from S2 and S3 and appeared as double helices with bifurcated ends (Fig. 4) and a pitch of approximately 1.0,um. These organisms were deep within the mucin layer but apparently above the surface of the epithelium. DISCUSSION The results from this study of colonic mucosal specimens from four sudden-death victims indicate that the predominant flora associated with the normal human colon wall is anaerobic. The anaerobe-aerobe ratio varied but depended

7 VOL. 45, 1983 BACTERIA ASSOCIATED WITH HUMAN COLON WALL 1031 OT1 C) -3I CA ot Cn C- C' 30. CA R3A 0 P) 11 0 is

8 1032 CROUCHER ET AL. APPL. ENVIRON. MICROBIOL. FIG. 4. Tissue sample from S3, excised from sigmoid colon. Bar = 1,um. mainly on the level of aerobes present, since the number of anaerobes isolated from the four subjects only varied between 1.5 x 106 and 2.7 x 108 per g, whereas the aerobes varied by 104- fold. The apparent association between a low anaerobe-aerobe ratio and death from acute heart failure needs further investigation. Peach et al. (27), using samples of uninvolved tissue from patients undergoing surgery for Crohn's disease or for carcinoma of the colon, found that anaerobes were associated with the colonic mucosa at levels of 103 to 108 per g and that the anaerobe-aerobe ratio did not exceed 1.5:1. The predominant anaerobes present in uninvolved colon tissue specimens were Bacteroides spp. Other anaerobes isolated were gram-positive rods, gram-positive cocci, and bifidobacteria. The predominant aerobic isolates were gramnegative rods and gram-positive cocci. The lower counts of anaerobes generally obtained in their study may have been due to the use of different isolation techniques. For example, isolation plates were incubated for only 2 days and, as the authors indicated, slow-growing anaerobes may not have been detected. It is also possible that transport and storage of specimens frozen in glycerol broth affected the recovery of anaerobes (17). In another study (26), undiseased colon wall specimens were obtained from 27 Guatemalan patients undergoing surgery. All tissue homogenates contained at least 107 anaerobes and 106 aerobes per g, and the most frequently isolated organisms were anaerobic and microaerophilic streptococci and enterobacteria. It is unlikely that the higher counts of aerobes obtained in these two previous studies were due to the use of different techniques; it is more likely that the mucosal flora was altered as a result of preoperative treatment, and Edmiston et al. (15) recently suggested that, in colonoscopy specimens, the decreased numbers of anaerobes associated with the mucosa of the distal colon may result from the routine enemas given before investigation. In the present study a wider range of organisms was found, and the flora of each subject was quite distinct when individual groups of organisms were compared. Microscopic examination of specimens obtained from colonoscopic examination of the human large bowel showed that bacteria adhere to the exposed epithelial surface and to the mucous sheet (16). Gram-negative and gram-positive bacteria were observed within and on the surface of the mucous layer and within plugs of mucus at the mouths of the crypts. Gram-negative rods were the predominant types. Other workers have observed spirochetes associated with and apparently altering the architecture of the intestinal epithelium of the human colon (22, 23). In preliminary reports of our observations on specimens from SI, it was noted that organisms were more frequently seen within the mucin (6, 11) than on the surface. Examination of specimens from S2 by SEM showed a complex structure within the mucin layer; however, it was not possible to differentiate between areas of colon by SEM examination of the bacterial population. SEM revealed some distinct groups of organisms that were not isolated in the parallel bacteriological investigation. These included a group of characteristic spiral-shaped organisms that have not previously been described. The differences between this study and previous work may reflect the sample sources, for

9 VOL. 45, 1983 BACTERIA ASSOCIATED WITH HUMAN COLON WALL 1033 example, sudden-death victims, patients undergoing surgery, or colonoscopy specimens. It is possible that the flora present on the human colonic mucosa may change within several hours after death, although Moore et al. (25) established that the gastrointestinal contents of hogs were bacteriologically stable for at least 4 h although considerable desquamation of the gut epithelium had taken place. Transmission electron microscopy of specimens from S2 showed similar changes, and it is possible that desquamated epithelial cells and the attached microorganisms were removed from the wall tissue during washing. Bacteriological studies of populations associated with the human colon wall are subject to many limitations, including differences in sampling, washing and culture media, and methods. The results presented here show that anaerobic bacteria are abundant on the normal human colonic mucosa, and the flora appears to be as complex as in feces and colon contents (24, 25). This is contrary to the findings obtained by a number of workers with laboratory animals, in which a specialized flora colonizes the gastrointestinal wall (6, 28, 29). Further investigations will be necessary to determine whether the trends observed in the four subjects reported here are typical of sudden-death victims. ACKNOWLEDGMENTS We thank J. H. Cummings (Medical Research Council Dunn Clinical Nutrition Unit, Cambridge. U.K.) for cooperation in providing samples and for useful discussions and N. Horn and S. G. Wharf for excellent technical assistance. LITERATURE CITED 1. Aranki, A., S. A. Syed, E. B. Kenney, and R. Freter Isolation of anaerobic bacteria from human gingiva and mouse cecum by means of a simplified glove box procedure. AppI. Microbiol. 17: Barnes, E. 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