Background
Intestinal microbiota is one of the many factors associated with the cause or complications of the symptoms of inflammatory bowel diseases (IBD). Microbial participation might result from dysbiosis, the action of a particular pathogen, or both. As a dominant facultative aerobe of the colon,
Escherichia coli has long been considered among the candidate IBD pathogens. Results reported thus far[
1‐
3] indicate that IBD patients experience a rise in the population of these bacteria. Yet, due to methodological variations regarding the source of the samples (stools vs. mucosa), quantitation method (culture dependent vs. independent), and histopathological activity of the sampled tissue (inflamed vs. no inflamed), the conclusions of different works are not always coincident. Some reports indicate an increase in mucosa-associated
E. coli population only in Crohn’s disease (CD)[
1]; others sustain that this elevation is also observed in ulcerative colitis (UC)[
3]. The contribution of mucosa-associated bacteria is more relevant than the lumen residing bacteria to the formation of mucosal lesions[
4]. While cultures of mucus depleted colonic biopsies from controls are sterile, cultures under the same treatment of corresponding biopsies taken from CD patients show a high number of
E. coli[
1]. Rise in mucosa-associated
E. coli from CD patients has also been demonstrated in an immunohistological study using polyclonal antibodies directed to
E. coli antigens[
5]. Analyses by temperature gradient gel electrophoresis of 16SrDNA/RNA amplicons from fecal bacteria of UC patients demonstrated a lower bacterial diversity associated with not only the dominance but with the activity of
E. coli[
2]. The proliferation of
E. coli in a given gut mucosal site seems not to correlate with the occurrence of inflammation and the exceeding
E. coli carries few or no traditional virulence markers[
3]. Differences seem to exist regarding the ability of
E. coli isolates from CD and UC cases in triggering the processes responsible for the characteristic lesions of each of these IBD. According to Rhodes[
4] in CD, the bacteria enter the lymphoid tissue, via M cells, persist within regional macrophages leading to granulomatous inflammation, which underlies clinical manifestations, such as ulcerations, obstructions and fistulas. Around 36-40% of
E. coli isolates from CD patients show the ability to invade epithelial cells[
6,
7] and the virulence potential of the adherent and invasive
E. coli (AIEC) is well recognized[
8]. In UC, flagellin mediated bacteria interaction with TLR-5 on mucosa cell surface prompts IL-8 release and ensuing neutrophil recruitment and activation[
4]. The flagellin access to cell basolateral exposed TLR-5 is thought to be facilitated by the abnormally permeable mucosa seen in UC. Recent work reported a high prevalence, in rectal biopsies of UC patients, of enteroaggregative adherent
E. coli (EAEC)[
9], a typically non-invasive pathovar, whose virulence attributes include the induction of mucus secretion by goblet cells, of IL-8 release by mucosal cells, and biofilm formation[
10] – features that could indicate an eventual role for these bacteria in the pathogenesis of UC. Most of studies aiming at determining variation in the concentration of
E. coli in IBD analyses luminal or mucosa associated bacteria. In the present work, by investigating stools and mucosa biopsies samples from distinct gut sites, we show that while no quantitative case–control variation is observed in
E. coli population from stools, the number of these bacteria residing in particular gut mucosal sites of IBD patients, notably those where the lesions often concentrate, is increased.
Discussion
As an indigenous organism of the gut,
E. coli was for long considered unsuspected of causing intestinal diseases[
11] but over last decades, following the description of multiple enteric
E. coli pathovars and their epidemiological association with enteritis, it became one of the most relevant human and animal bacterial enteropathogens, associated with several gastrointestinal diseases ranging from food poisoning to different clinical manifestations of diarrhea[
12]. Accumulated evidences lend support to its involvement with the etiology of IBD as well, in particular with CD, wherein AIEC is thought to be able to invade regional macrophages leading to the formation of the epithelioid granulomas[
8] typically found in histological preparations of gut biopsies from CD patients. Elevation in the number of
E. coli in the gut of IBD patients[
13] and in serum antibodies titers against some of its antigens[
14,
15] are among the first clues of its role in this disease. Rise in
E. coli number was demonstrated in stools[
2] and mucosa of distinct gut sites[
1,
3,
16]. The increase in
E. coli number in IBD patients has also been correlated with disease activity[
13].
To get a broad picture of the quantitative variation in
E. coli population from IBD patients, we have analysed cultures on
E. coli/ coliform agar of samples from at least two distinct clinical materials per patient, consisting of stools and/or biopsies of different gut sites of controls and individuals diagnosed with CD and UC. Statistically significant case-controls differences in the number of bacteria were observed only in
E. coli counting for cultures derived from particular mucosal sites, namely ileum of CD and sigmoid and rectum of both CD and UC patients, which showed a higher bacterial concentration, in comparisons with corresponding sites of controls. In contrast to previous observations[
3], the increase in
E. coli number reported here was not accompanied by a rise in the number of non-
E. coli coliforms.
MacConkey broth cultures from sample suspensions of each clinical material were PCR screened for ten virulence genes, five of which recognized as markers for Diarrheagenic
E. coli (DEC) detection[
12,
17], and five of them corresponding to the SPATE genes
pet, sat, sigA, pic and
sepA. Of the five DEC genes screened, only
eae and
aggR were detected, in 5 of the 67 samples tested (Table
1). The low prevalence of DEC genes in cultures of these IBD clinical samples are not too different from the results of other investigators, who screening bacterial isolates, found no
E. coli positive for any of these markers[
3,
16]. Also, the low prevalence of these classical DEC virulence genes in IBD clinical samples is in agreement with previous observations arguing that IBD
E. coli are more closely related to extra intestinal pathogenic
E. coli (ExPEC)[
3,
4].
The reasoning for the search of SPATE genes among IBD clinical samples took in account not only the wide distribution of SPATE among DEC and ExPEC isolates[
18] but also the variety of biological activities of these proteases[
19], which could eventually play some role in the pathogenesis of IBD. Furthermore, EAEC
, a pathovar showing high prevalence of SPATE genes[
20] were recently found to be dominant among IBD
E. coli isolates[
9]. With the exception of
pic, all the remaining of the five SPATE genes searched for were found among the samples screened, with
sat and
sigA identified in nearly all of them. Nevertheless, no statistically significant differences could be observed in the prevalence of any of the SPATE genes identified (
pet, sat, sepA and
sigA) among the different clinical samples or groups of patients investigated. In the work by Kotlowski et al.[
3], where a PCR screening for 8 SPATE genes was carried out, a higher prevalence of SPATE positive
E. coli were found among isolates from IBD patients as compared to controls. Since in their analyses they used pure
E. coli cultures instead of total Gram negative cultures from the samples, the nature of the PCR target as well as the number of genes searched for might explain the observed divergence in relation to our results.
The proliferation of
E. coli raises the question whether the exceeding bacteria plays an active or secondary role in IBD etiopathogenesis. The prevalence of haemolytic and necrotoxic
E. coli strains has been shown to be higher in patients in relapsing UC, but follow-up analyses demonstrated that these bacteria tended to follow rather than precede the onset of relapse attack[
13]. On the other hand, AIEC has being detected in proportion as high in early as in chronic ileal lesions of CD patients[
6]. It is believed that in CD, the bacterial adhesion is favoured by abnormal receptors exposition in cell surface of genetically susceptible hosts[
8]. Soluble plant fibers, to which a therapeutic potential in treatment of CD has been attributed, are able to inhibit this bacterial adhesion[
1].
Although previous report suggests an active rather than secondary role for
E. coli in IBD, on the basis of a high prevalence of strains possessing virulence markers in IBD patients[
3], this indication is not supported by the data of the present work. By using as PCR target gross (total) Gram negative bacteria cultures which are more representative than a few colonies of the bacterial population in the samples, we were unable to detect difference in the prevalence of the
E. coli virulence genes searched for among distinct groups of patients or clinical material investigated here. The genes consisted of five markers traditionally used for identification of DEC and five SPATEs genes, categories of proteases which up to date were not found in non-pathogenic organisms[
18]. Moreover, the bacteria proliferation in IBD patients was noticed in sites where the lesions are prominent and usual, namely the ileum, sigmoid and rectum in CD and the sigmoid and rectum in UC. The apparently non-random
E. coli abundance in these sites and its unaltered number in stools do not preclude the possibility that they represent secondary local colonizers. Also, in view of their localization, luminal bacteria could be a more direct target for drugs interfering with bacterial species composition of the gut, and thus the history of drugs intake could explain why the increment in the number of mucosa colonizing
E. coli was not also observed for luminal population of these bacteria, among IBD patients. Despite of the low number of genes searched for and the restricted quantity of some samples, such as left side colon, the uniform distribution of virulence genes among the various clinical materials in controls and IBD patients might indicate an irrelevance of these factors and/or the
E. coli pathovars marked by them for the pathogenesis of IBD.
There are numerous evidences to support the involvement of
E. coli with CD and hence AIEC is recognized as new pathovar within the species[
21], but the link of
E. coli with UC is not as clear. We speculate that in UC, and eventually in CD, aggregative adherent
E. coli might play some role, at least in aggravating already established inflammatory processes. This group of bacteria, which is extremely heterogeneous in regard to expression of virulence factors and pathogenic potential, is classified in typical and atypical EAEC, based upon the presence of the
aggR transcriptional activator of virulence genes in the first and absence in the last[
21]. Atypical EAEC includes commensal, intestinal and extra-intestinal pathogenic
E. coli. Testing some
E. coli isolates derived from the Gram negative cultures studied here for the ability to adhere to Hep-2 indicated the presence of atypical EAEC in samples from IBD cases (data not shown). With the characterization of these bacteria presently under way in our laboratory we hope to get relevant information which will be of help in understanding their proliferation in the gut mucosa of IBD patients.
Competing interests
No competing interests declared.
Authors’ contributions
HLdS and VRC worked in collection, preservation and processing of the samples for bacterial quantitation and E. coli detection. RK assisted HLdS in PCR technical issues. LYS got and managed the patients’ clinical information. FGR conducted the patients’ colonoscopic examinations. JR supervised laboratory work, compiled the data and wrote the manuscript. All authors read and approved the final manuscript.