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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2012  |  Volume : 55  |  Issue : 4  |  Page : 485-489
Adherent Escherichia coli in colorectal mucosal biopsies: A histological and ultrastructural evaluation


1 Global Hospitals, Chennai, Tamil Nadu, India
2 MIOT, Chennai, Tamil Nadu, India
3 CMC, Vellore, Tamil Nadu, India

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Date of Web Publication4-Mar-2013
 

   Abstract 

Background: Colorectal mucosal biopsies occasionally demonstrate the presence of bacteria adherent to the epithelium. This study evaluated the histological and ultrastructural correlates of such bacterial adherence. Materials and Methods: Rectal mucosal biopsies from eight patients in whom histopathological examination of biopsies had earlier demonstrated adherent bacteria were examined by electron microscopy and by bacterial culture. Colorectal biopsies of 69 patients with adherent bacteria detected histologically were retrospectively evaluated for histological changes at sites proximal and distant to adherent bacteria. Results: Escherichia coli of different serogroups were isolated from 7 of 8 rectal biopsies demonstrating bacterial adherence. All isolates showed diffuse or focal adherence to HEp-2 cell monolayers. Ultrastructural changes noted included microvillus damage, pedestal formation, actin web condensation, and protrusions of the apical cytoplasm of epithelial cells into the lumen towards the bacteria. Histological changes noted at light microscopy included reduction in epithelial cell height, focal epithelial cell degeneration, cryptitis and neutrophil infiltration at sites of bacterial adherence whereas these were usually absent at sites distant to adherent bacteria. Bacterial adherence was noted more often in biopsies from Crohn's disease patients than in patients without this diagnosis (P < 0.001). Conclusion: Adherent Escherichia coli in colorectal biopsies were associated with focal epithelial damage and showed an association with Crohn's disease.

Keywords: Colon, Crohns disease, enteroadherent Escherichia coli, mucosal biopsy, rectum

How to cite this article:
Patra S, Samal SC, Kang G, Pulimood A, Mathan M, Ramakrishna BS. Adherent Escherichia coli in colorectal mucosal biopsies: A histological and ultrastructural evaluation. Indian J Pathol Microbiol 2012;55:485-9

How to cite this URL:
Patra S, Samal SC, Kang G, Pulimood A, Mathan M, Ramakrishna BS. Adherent Escherichia coli in colorectal mucosal biopsies: A histological and ultrastructural evaluation. Indian J Pathol Microbiol [serial online] 2012 [cited 2019 Sep 18];55:485-9. Available from: http://www.ijpmonline.org/text.asp?2012/55/4/485/107786



   Introduction Top


Adhesion of bacteria to mucosal surfaces is an important mechanism in the pathogenesis of infections in the gastrointestinal tract. Gram negative bacteria adhering to the colonic epithelium are occasionally noted during routine examination of colorectal mucosal biopsies. However, the literature does not attach any special significance to their presence in mucosal biopsies. Certain pathogenic strains of  Escherichia More Details coli have the ability to adhere to enterocyte and colonocyte apical membranes and produce characteristic morphologic alterations. [1] Enteroadherent E. coli (EAEC) have been implicated in the causation of persistent diarrhoea in children and adults. [2],[3] Adherent E. coli have also been implicated in the pathogenesis of ulcerative colitis and Crohn's disease. [4],[5] The present study identified adherent Escherichia coli by culture of rectal mucosal biopsies in patients with a histological finding of bacterial adherence to the surface epithelium. A retrospective analysis of histological and ultrastructural changes in biopsies showing bacterial adherence is also reported.


   Materials and Methods Top


Eight patients whose rectal mucosal biopsies showed histological evidence of bacterial adhesion underwent repeat biopsy of the rectal mucosa within a week of the first biopsy. The biopsies were preceded by oral colonic lavage involving the intake of four litres of polyethylene glycol electrolyte solution (PEGLEC, Tablets India, Chennai) along with two tablets of Bisacodyl the previous night. Colonoscopic examinations were not performed unless the feces was clear and watery without flecks of fecal matter. Biopsies were taken using sterilized endoscopic biopsy forceps and two bits were fixed for electron microscopy in buffered glutaraldehyde and processed in Araldite. One-micron sections were stained with toluidine blue to select areas suitable for electron microscopic examination, and ultrathin sections stained with aqueous uranyl and lead salts were examined in a Philips EM 201 electron microscope. Two biopsy bits for these patients were also taken for bacterial culture. These biopsy bits were washed vigorously with sterile saline, ground and plated onto MacConkey agar and blood agar plates and incubated overnight at 37°C. Appropriate biochemical reactions were used for bacterial identification. At least 5 colonies of non-mucoid lactose fermenters on MacConkey agar plates were serogrouped to identify enteropathogenic serotypes of Escherichia coli by slide agglutination using Biotec polygrouping antisera and Wellcome individual EPEC antisera. All Escherichia coli that could not be grouped thus were serogrouped at the Central Public Health Laboratory, Colindale, London, U.K. All Escherichia coli isolated were tested for adherence using HEp-2 cell monolayers. [6]

The retrospective component of the study was the study of 69 colorectal biopsies that had been reported to show histological evidence of bacterial adhesion over a three and a half year period. All the biopsies had been processed routinely and stained with haematoxylin and eosin, and examined by one of three histopathologists who only evaluated gastrointestinal mucosal biopsies. Routinely these three specialist histopathologists reported upon the presence of bacteria adherent to the surface epithelial cells, and all 69 biopsies reported to have adherent bacteria were chosen for the study. Biopsies from these 69 patients were re-evaluated for histological changes related to sites of bacterial adherence. We specifically looked for the following features - epithelial cell degeneration, neutrophil infiltration of surface and crypt epithelium, mucus depletion, regenerative activity, lamina propria cellularity and neutrophil margination in the microvasculature. All changes were noted in relation to areas with adherent bacteria, and were noted also in the same biopsy at sites distant to bacterial adhesion, and in biopsies from the same individual where no bacteria were identified. Epithelial cell height was measured for an average of 10 cells per high power field using a calibrated eye piece graticule. Lamina propria cellularity and neutrophil infiltration were subjectively graded on a scale of 0 to 2. The final clinical diagnosis was recorded from review of the hospital records. The studies were approved by the Research and Ethics Committee of the Christian Medical College, Vellore and informed consent was obtained from the patients.


   Results Top


In all 8 patients (6 with a diagnosis of irritable bowel syndrome and 2 with a diagnosis of Crohn's disease) in whom biopsies were obtained for examination, adherent bacteria were reconfirmed in the second biopsy by light microscopy. Culture of these biopsies grew Escherichia coli in 7 of the 8 biopsies. All the isolates showed adherence to HEp-2 cell monolayers. They were all of different serotypes. Adherence pattern of the different serotypes was as follows: Diffuse adherence (O10), localized adherence (O2:K60, O158, O160) or stacked brick pattern of aggregative adherence (O19ab, O86, O200).

Examination of ultrathin sections by light microscopy showed bacteria covering the epithelial surface either diffusely [Figure 1] or focally [Figure 2]. On electron microscopy, degenerative changes were noted in epithelial cells [Figure 3]. Bacteria were noted close to the microvilli [Figure 4] sometimes showing pedestal formation resembling the attaching-effacing lesions of enteropathogenic Escherichia coli infection [Figure 5]. Cells at sites of adherence showed excess production of microvillus glycocalyces [Figure 6] reaching towards the bacteria. Microvilli appeared short, irregularly grouped and deformed, or were lost with protrusion of the apical cytoplasm [Figure 6]. Actin polymerization near the terminal web region and swelling of mitochondria and dilatation of rough endoplasmic reticulum were also noted.
Figure 1: Colonic mucosa with microcolonies of adherent bacteria diffusely covering the surface epithelium, which shows degenerative changes. (EM, × 1218)

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Figure 2: Colonic surface epithelium showing bacteria close to the microvillus border. Note the degenerative change and tufting (arrow) of the cells (EM, × 1137)

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Figure 3: Apical region of surface colonocytes with bacteria adjacent to microvillus border. Microvilli are short, irregular and grouped. Cytoplasm shows vacuolization and damaged mitochondria (EM, × 4063)

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Figure 4: Bacteria close to the microvilli with glycocalyx attached to them (arrow). Vacuolization of apical cytoplasm and mitochondria is seen (EM, × 9856)

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Figure 5: Apical border of colonocyte with bacteria adherent to cell border showing characteristic pedestal formation (arrow). Dilated endoplasmic reticulum and altered mitochondria are seen (EM, × 19710)

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Figure 6: Apical border of colonocyte with protrusion of cell borders (arrow). There is actin polymerization at the rootlets of microvilli (arrow head). Prominent glycocalyx can be noticed (open arrow) (EM, × 11519)

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Biopsies from the cecum, ascending colon, transverse colon, descending colon, sigmoid colon or rectum did not show significant differences in prevalence of adherent bacteria [Table 1]. Epithelial cell height was significantly reduced at sites of bacterial attachment (9.74 ± 3.15 microns, mean ± SD), compared to sites remote from bacterial attachment (11.87 ± 5.28) or to control biopsies without adherent bacteria (15 ± 3.2) (P < 0.001) [Figure 7]. Areas with bacterial attachment showed focal epithelial cell degeneration and neutrophil infiltration of the epithelium [Table 2], [Figure 8]. Cryptitis and neutrophilic infiltration of the lamina propria were also more frequent in biopsies demonstrating bacterial adherence compared to biopsies without bacterial adherence [Table 2], [Figure 8].
Figure 7: Epithelial cell height in microns (mean and SD are shown) of cells adjacent to adherent bacteria, cells in areas distant to adherent bacteria in the same biopsy, and cells in biopsies from the same patient without adherent bacteria. Significant P values are shown (ANOVA with post-hoc Tukey's test)

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Figure 8: Mucosal changes in the biopsies in sites adjoining adherent bacteria and at sites distant from adherent bacteria. Changes are shown as percentages of sites examined. Significant P values (Fisher's exact test) are shown

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Table 1: Percentage of biopsies showing bacterial adhesion to the epithelium, in relation to the segment biopsied

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Table 2: Prevalence of histological abnormalities associated with bacterial attachment. Comparison of areas with and without bacterial attachment

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The final clinical diagnoses in these 69 patients included irritable bowel syndrome (22), Crohn's disease (14), ulcerative colitis (4), tuberculosis of the small bowel (2), infective colitis (7), and miscellaneous conditions (20). None of them was HIV seropositive. During this same time period, colorectal biopsies were obtained from a total of 2223 patients. These included biopsies from 40 patients who were diagnosed with Crohn's disease. The distribution of biopsies with bacterial adherence in relation to the patient's diagnosis is shown in [Table 3]. The proportion of Crohn's disease patients (14/40, 35%) who had biopsies demonstrating bacterial adherence was significantly higher than the proportion of patients with other diagnoses in whom biopsies showed bacterial adherence (Chi squared test, P < 0.001).
Table 3: Frequency of detection of adherent bacteria in relation to the clinical diagnosis

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   Discussion Top


In this study adherent Escherichia coli (EAEC) in colorectal mucosal biopsies were associated with histological evidence of mucosal inflammation and ultrastructural evidence of epithelial cell damage. The ultrastructural changes resemble those reported in E. coli (RDEC) infection in rabbits. [7] Pedestal formation, reported in EPEC infection, [1] was noted in 2 of 8 biopsies. EAEC were first implicated in the causation of persistent diarrhoea in children, [1],[2] and later in chronic enteropathy in patients with HIV/AIDS. [8],[9] Recent studies have implicated adherent-invasive E. coli in the causation of ileal Crohn's disease. [10],[11] These bacteria exhibited phenomena of both adherence and invasiveness and were largely found in ileal biopsies although similar bacteria could occasionally be found in the colon. Our studies did not demonstrate evidence of invasiveness of the bacteria.

Ultrastructural examination showed evidence of pedestal formation in areas and evidence of epithelial cell damage including mirovillus damage, mitochondrial damage, actin condensation in the terminal web region, and interestingly showed cytoplasmic processes extending towards the adherent bacteria. Whether these processes are responsible for sampling bacterial molecules such as the pathogen associated molecular patterns is speculative and was not examined. Histological examination showed that there was epithelial damage localized to sites of bacterial adherence, without evidence of damage at sites distant from bacterial adherence or in biopsies without adherent bacteria. This suggests that these bacteria are not merely incidental findings in colonic biopsies but are likely to be associated with pathology in the bowel wall. Adherent bacteria were detected on light microscopy by experienced pathologists who dealt only with gastrointestinal mucosal biopsies. We did not use special stains to detect the bacteria on light microscopy in the first instance. While the detection rate is very high when specialist pathologists examine mucosal biopsies, it is possible that the use of special stains may have increased the yield of adherent bacteria in the study. The preliminary finding of an association with Crohn's disease in this study is very interesting.

In summary, adherent bacteria (EAEC) were detected in 3.1% of colorectal mucosal biopsies and were associated with epithelial cell degeneration and shortening, cryptitis and inflammatory infiltration of the lamina propria as well as the epithelium. These bacteria showed a positive association with Crohn's disease. Characterisation of the strains by O antigen serotyping showed that they belonged to different strains, but they all exhibited the characteristic adherence patterns of enteroadherent Escherichia coli in cell line adherence assays. Further, studies are necessary to confirm these associations and to elucidate their role in disease pathogenesis.

 
   References Top

1.Phillips AD, Frankel G. Mechanisms of gut damage by Escherichia coli. Baillieres Clin Gastroenterol 1997;11:465-83.  Back to cited text no. 1
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2.Bhan MK, Raj P, Levine MM, Kaper JB, Bhandari N, Srivastava R, et al. Enteroaggregative Escherichia coli associated with persistent diarrhea in a cohort of rural children in India. J Infect Dis 1989;159:1061-4.  Back to cited text no. 2
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3.Cravioto A, Tello A, Navarro A, Ruiz J, Villafán H, Uribe F, et al. Association of Escherichia coli HEp-2 adherence patterns with type and duration of diarrhoea. Lancet 1991;337:262-4.  Back to cited text no. 3
    
4.Burke DA, Axon AT. Adhesive Escherichia coli in inflammatory bowel disease and infective diarrhoea. BMJ 1988;297:102-4.  Back to cited text no. 4
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5.Darfeuille-Michaud A, Boudeau J, Bulois P, Neut C, Glasser AL, Barnich N, et al. High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn's disease. Gastroenterology 2004;127:412-21.  Back to cited text no. 5
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6.Kang G, Mathan MM, Mathan VI. Evaluation of a simplified HEp-2 cell adherence assay for Escherichia coli isolated from south Indian children with acute diarrhea and controls. J Clin Microbiol 1995;33:2204-5.  Back to cited text no. 6
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7.Takeuchi A, Inman LR, O'Hanley PD, Cantey JR, Lushbaugh WB. Scanning and transmission electron microscopic study of Escherichia coli 015 (RDEC-1) enteric infection in rabbits. Infect Immun 1978;19:686-94.  Back to cited text no. 7
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8.Kotler DP, Giang TT, Thiim M, Nataro JP, Sordillo EM, Orenstein JM. Chronic bacterial enteropathy in patients with AIDS. J Infect Dis 1995;171:552-8.  Back to cited text no. 8
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9.Mathewson JJ, Jiang ZD, Zumla A, Chintu C, Luo N, Calamari SR, et al. HEp-2 cell-adherent Escherichia coli in patients with human immunodeficiency virus-associated diarrhea. J. Infect Dis 1995;171:1636-9.  Back to cited text no. 9
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10.Rolhion N, Darfeuille-Michaud A. Adherent-invasive Escherichia coli in inflammatory bowel disease. Inflamm Bowel Dis 2007;13:1277-83.  Back to cited text no. 10
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11.Chassaing B, Rolhion N, de Vallée A, Salim SY, Prorok-Hamon M, Neut C, et al. Crohn disease-associated adherent-invasive E. coli bacteria target mouse and human Peyer's patches via long polar fimbriae. J. Clin Invest 2011;121:966-75.  Back to cited text no. 11
    

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Correspondence Address:
Balakrishnan S Ramakrishna
Department of Gastrointestinal Sciences, Christian Medical College, Vellore 632004
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.107786

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]

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