As an indigenous organism of the gut, E. coli was for long considered unsuspected of causing intestinal diseases 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. 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 typically found in histological preparations of gut biopsies from CD patients. Elevation in the number of E. coli in the gut of IBD patients 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 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.
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, 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 (Table1). 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 but also the variety of biological activities of these proteases, which could eventually play some role in the pathogenesis of IBD. Furthermore, EAEC, a pathovar showing high prevalence of SPATE genes were recently found to be dominant among IBD E. coli isolates. 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., 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. On the other hand, AIEC has being detected in proportion as high in early as in chronic ileal lesions of CD patients. It is believed that in CD, the bacterial adhesion is favoured by abnormal receptors exposition in cell surface of genetically susceptible hosts. Soluble plant fibers, to which a therapeutic potential in treatment of CD has been attributed, are able to inhibit this bacterial adhesion.
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, 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. 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, 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. 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.