Colonization of the Human Gut by E. coli and Colorectal ...Human Cancer Biology Colonization of the...

Human Cancer Biology

Colonization of the Human Gut by E. coli and ColorectalCancer Risk

Mathilde Bonnet1,2, Emmanuel Buc1,2,3, Pierre Sauvanet1,2,3, Claude Darcha3, Damien Dubois1,2,3,Bruno Pereira3, Pierre D�echelotte3, Richard Bonnet1,2,3, Denis Pezet1,2,3, and Arlette Darfeuille-Michaud1,2,3

AbstractPurpose: The intestinal microbiota is potentially involved in the development of colorectal carcinoma

via various mechanisms. Escherichia coli are commensal bacteria of the human gut microbiota, but some

pathogenic strains have acquired the ability to induce chronic inflammation and/or produce toxins, such as

cyclomodulin, which could participate in the carcinogenesis process. Here, we analyzed the E. coli

population associated with mucosa of patients with colon cancer in relation to clinicopathologic char-

acteristics. We assessed carcinogenic properties of a colon cancer–associated E. coli strain in multiple

intestinal neoplasia (Min) mice.

Experimental design:Mucosa-associated or internalized E. coli were quantified and characterized from

tumors and mucosa of patients with colon cancer and the healthy mucosa of diverticulosis controls. Min

micewere inoculatedwith a colon cancer–associatedE. coli strain (11G5). Thenumber of colonic polypswas

evaluated at 7 weeks after infection.

Results: An increased level of mucosa-associated and internalized E. coli was observed in the tumors

compared with normal tissue. A relationship between poor prognostic factors for colon cancer (tumor–

node–metastasis stage) and colonization of mucosa by E. coli was observed. Pathogenic cyclomodulin-

positive E. coli strains were more prevalent on mucosa of patients with stages III/IV than those with stage I

colon cancer. Proliferative index and E. coli colonization level of the mucosa distant from the tumor

significantly correlated. Min mice infected with the E. coli strain 11G5 displayed a marked increase in the

number of visible colonic polyps compared with controls.

Conclusion: These findings support that pathogenic E. coli could be a cofactor in pathogenesis of

colorectal cancer. Clin Cancer Res; 20(4); 859–67. �2013 AACR.

IntroductionColorectal cancer is the third most commonly diag-

nosed cancer worldwide and is responsible for more than600,000 deaths every year. Because of its high incidenceand mortality rate, colorectal cancer is a major publichealth problem. Survival and risk of recurrence are con-ditioned by the extent of the tumor, stratified according tothe prognostic classification of the American Joint Com-

mittee on Cancer (AJCC; ref. 1). When the tumor isconfined to the colon (stages I and II), surgery is curativewith a 5-year survival rate up to 80%; however, in the caseof dissemination (nodal involvement, metastatic disease)the prognosis is dramatically reduced (2). Thus, thedevelopment of new diagnostic tools for the early detec-tion of lesions is attractive.

Colorectal carcinogenesis was first defined as a classicadenoma–carcinoma sequence by Fearon and Vogelsteinin 1990 and several risk factors were identified to favor thistumoral development (3). Among them, pathogens seemto play a major role. Indeed, approximately 20% of theglobal cancer burden can be linked to infectious agents(4, 5). Bacteria and their related products could participatein initiation or progression of sporadic colon cancer bydriving a variety of mechanisms, including the induction ofproinflammatory and procarcinogenic pathways in epithe-lial cells, the production of genotoxins and reactive oxygenspecies, and the conversion of procarcinogenic dietaryfactors into carcinogens (6–8). In colorectal cancer, variousbacteria have been associated with carcinogenesis, includ-ing Streptococcus bovis, Enterococcus spp., Helicobacter pylori,the enterotoxigenic Bacteroides fragilis, and various

Authors' Affiliations: 1Clermont Universit�e, UMR 1071Inserm/Universit�ed'Auvergne; 2 Institut National de la Recherche Agronomique (INRA), USC-2018; and 3Centre Hospitalier Universitaire, Clermont-Ferrand, France

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

M. Bonnet and E. Buc contributed equally to this work.

Corresponding Author: Mathilde Bonnet, Clermont Universit�e, UMR1071Inserm/Universit�e d'Auvergne, Centre Biomedical de Recherche etValorisation, 28 Place Henri Dunant, INRA, USC-2018, 63005 Clermont-Ferrand, France. Phone: 33-473178376; Fax: 33-473178371; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-13-1343

�2013 American Association for Cancer Research.

ClinicalCancer

Research

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Published OnlineFirst December 13, 2013; DOI: 10.1158/1078-0432.CCR-13-1343

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pathogenic Escherichia coli (7–11). Despite the fact that E.coli is a commensal bacteria of the human microbiota andrepresents the most common cultivable, gram-negative,aero–anaerobic bacteria, various studies have demon-strated a clear link between mucosa-adherent E. coli andcolorectal cancer (8, 10, 11). E. coli are divided into fourphylogenic groups (A, B1, B2, and D) according to theacquisition of factors of virulence. E. coli from phy-logroups A and B1 are generally not pathogenic, whereasphylogroups B2 and D are involved in intestinal andextra-intestinal diseases. Interestingly, some strains ofE. coli from phylogroup B2 are associated with Crohndisease, a chronic inflammatory bowel disease known tobe a risk factor for colorectal cancer (12, 13). Moreover,two studies showed that mucosa-associated and mucosa-internalized E. coli were observed more often in patientswith colorectal cancer than in controls, supporting thecentral role of these bacteria in the development ofcolorectal cancer (10, 11).

Pathogenic E. coli strains synthesize various virulencefactors, including several toxins called cyclomodulins suchas cytolethal distending toxins (CDT), cytotoxic necrotizingfactor (CNF), cycle inhibiting factor, and colibactin (6).Cyclomodulins are genotoxic and/or modulate cell-cycleprogression, proliferation, cell differentiation, and apopto-sis (14–19). In particular, colibactin, a hybrid polyketide-nonribosomal peptide encoded by the pks genomic island,has genotoxic properties causingDNAdouble-strandbreaksand chromosomal instability inhumaneukaryotic cells (18,19). With regard to the cyclomodulin-producing E. coli, arecent study revealed an increased prevalence of cyclomo-dulin-producing B2 E. coli in colon tumor biopsies, suggest-ing a possible role of such pathogenic E. coli in coloncarcinogenesis (20).

The aim of the present study is to compare the bacterialcolonization of the colonic mucosa in patients screened forcolorectal cancer with noncolon cancer controls. Given thatE. coli represent themajority of the cultivable bacteria in thecolon and are positively selected for during chronic inflam-mation (21), we restricted our analysis to these bacteria. Weevaluated two parameters of mucosa-associated and inter-nalized E. coli in the normal mucosa and tumors of patientswith colon cancer compared with the normal mucosaof patients receiving an operation due to diverticulosis,a nontumoral intestinal pathology. We analyzed the rela-tionship between the levels of mucosa-associated E. coli,including genotypic characterization of the isolated strains(phylogroup and search for cyclomodulin-encoding genes),and clinicopathologic characteristics. Finally, we assessedthe carcinogenic properties of an E. coli strain colonizing thetumor and themucosa in a patient with colon cancer, usingthemultiple intestinal neoplasia (Min)mice animalmodel.

Materials and MethodsPatients and resection specimen

Patients with resectable colon cancer (n ¼ 50) anduncomplicated diverticulosis (noncancer controls; n ¼33) were prospectively included in the Surgical DigestiveUnit of Clermont-Ferrand (France) between March 2007and July 2010. All patients were adult volunteers andsigned informed consent. Patients with diverticulosiswere operated on to prevent any inflammatory recurrenceat least 10 weeks after an episode of diverticulitis. Exclu-sion criteria included neo-adjuvant chemotherapy, histo-ry of previous colonic resection, emergency surgery, anduse of antibiotics within 4 weeks before surgery. Theclinical data were collected from medical files and aresummarized in Tables 1 and 2. The sex ratio (M/F) was1.22 and 0.74 for patients with colon cancer and diver-ticulosis, respectively. The age range was 35 to 95 years forcancer patients (median age, 70 years) and 34 to 81 yearsfor controls (median age, 58 years). Of note, the diver-ticulosis group comprised younger subjects than thecolorectal cancer group (Table 1).

Surgery was performed either by laparotomy or laparos-copy. None of the patients received mechanical bowelpreparation before the surgery. All patients received cefox-itin (2 g i.v.) at the time of incision. A specimen of mac-roscopically normal mucosa (absence of diverticules) wascollected from the patients with diverticulosis and a non-necrotic fragment from the peripheral areas of the tumorwas collected from the patients with colon cancer. In par-allel, fresh normal mucosa samples adjacent to the tumor(10 cm from the tumor) were collected from the patientswith colon cancer.

Pathologic analysisAfter colonic resection, fresh specimens were transported

to the pathology laboratory, fixed in buffered 4% formalin,embedded in paraffin, cut into 5-mmslices, and stainedwithhematoxylin/eosin/safranin. For the patients with coloncancer, tumors were staged according to the seventh

Translational RelevanceThe gut microbiota and dysbiosis have recently been

associated with colorectal cancer. We observed here asignificant relationship between the colonization ofmucosa by pathogenic Escherichia coli and poor prognos-tic factors for colorectal cancer, such as tumor–node–metastasis stage, suggesting that this colonization by E.coli could be an additional prognostic factor of pooroutcome in patients. In addition, a strong associationwasobservedbetween theproliferative indexof epithelialcells and the colonization of the mucosa by E. coli,suggesting a role for these bacteria in regenerativeepithelial cell growth and tumor progression. We dem-onstrated that infection with a pathogenic colon cancer–associated E. coli strain accelerates tumor development inmultiple intestinal neoplasia mice, thereby providingevidence of the strain’s procarcinogenic properties. Ourfindings show thatmucosa colonizationbypathogenicE.coli could be a crucial factor in colorectal carcinogenesisand serve as a new prognostic marker.

Bonnet et al.

Clin Cancer Res; 20(4) February 15, 2014 Clinical Cancer Research860




AJCC/Union for International Cancer Control (UICC)tumor–node–metastasis classification (TNM stage; ref. 1).The acronym is indicative of the following criteria: (T)tumor size and nearby tissue invasion, (N) regional lymphnode involvement, and (M) distant metastasis. The degreeof tumor differentiation (poor, moderate, or high), peri-neural invasion, vascular embols, the mucinous colloidcomponent, and the presence of necrosiswere also assessed.In patients with diverticulosis, the extent ofmucosal inflam-mation (congestion of the mucosa or the submucosa,ulcerations, polynuclear infiltration, or abscesses) and pres-ence of polyps or tumors were assessed.Immunohistochemical staining of the human Ki-67 anti-

gen was performed on the colonic mucosa sections frompatients with colon cancer. Five-micrometer sections ofparaffin-embedded, healthy mucosa were labeled with theanti–Ki-67 MIB-1 antibody (Dako) and Ultra View Detec-tion Kit (Ventana) on Benchmark XT stainer (Ventana).Sections were counterstained with Harris’s hematoxylin.

For each patient, the proliferation index was calculated asthe percentage of Ki-67–positive epithelial cells in propor-tion to the total number of cells in a minimum of 10 entirecrypt columns as previously reported (22). Ki-67 expressionwas then classified as a low (Ki-67–positive cells �10%) orhigh (Ki-67–positive cells >10%) proliferative index.

Microbiologic analysesSpecimens (tumor, normal mucosa) were immediately

processed for the analysis of mucosa-associated and inter-nalized E. coli colonization as previously described (20).The genotypic characteristics (phylogroup typing and pres-ence of cyclomodulin-encoded genes) of these E. coli strainswere assessed using PCR, and cyclomodulin expression wasdetermined by assessing a specific cytopathic effect oncultured epithelial HeLa cells (20).

Electron microscopyHuman epithelial cell (T84) monolayers were infected

with the colon cancer–associated E. coli strain 11G5 in vitrofor 3hours at amultiplicity of infection (MOI)of 50bacteriaper cell. The cells were then fixed in 1.6% glutaraldehydefor 1 hour, washed in cacodylate buffer (0.2mol/L, pH7.2),and postfixed in 1% OsO4 for 1 hour. After washing,the samples were dehydrated in graded alcohol and embed-ded in epoxy resin. Thin sections (60 nm) cut with adiamond knife in an ultramicrotome were stained withuranyl acetate and lead citrate and observed using a HitachiH7650 TEM (Hitachi High Technologies Corporation).Sample preparations and observations were performed inthe Centre Imagerie Cellulaire Sant�e (CICS) platform.

Animal modelStudies were performed using 5- to 6-week-old C57BL/

6J-ApcMin/þ (Min mice; The Jackson Laboratory) or wild-type (WT) C57BL/6J female mice in accordance with theFrench and European Economic Community guidelines(86-60, EEC) for care of laboratory animals. Studies wereapproved by the French Regional Ethical Animal UseCommittee (No. CE-2912). All mice were housed inspecific pathogen-free conditions at the animal care facil-ity of the Universit�e d’Auvergne (Clermont-Ferrand,France). To enhance E. coli strain colonization, we admin-istered streptomycin (2.5 g/L) for 3 days before inocula-tions of peroral bacteria (�1 � 108 bacteria in PBS) orPBS alone (controls).

Two E. coli strains were tested: the ampicillin- and kana-mycin-resistant E. coli strain 11G5 isolated from a patientwith colon cancer as the colon cancer–associated strain andthe commensal rifampicin-resistant E. coli strain K-12MG1655 (laboratory stock) as the nonpathogenic control.We periodically quantified fecal bacterial colonization ascolony-forming units (CFU) per stool as previouslydescribed (23). Fifty days after bacterial inoculation, themice were sacrificed. The colons were then removed fromthe caecum to the rectum, flushed in PBS, and splayedlongitudinally. Then, the tumors were counted. The colonswere swiss-rolled from the distal to proximal end and fixed

Table 1. Clinical data from patients with coloncancer and diverticulosis in the search formucosa- or tumor-associated and internalizedE. coli

Patients withcolon cancer

Patients withdiverticulosis

Patients, n 50 33Age 70.5 � 11.4 58.0 � 11.9b

Sex, male/female 28/22 13/20

Presence of colon-associated E. coli, n (%)Mucosa Yes 45 (90) 29 (88)

No 5 (10) 4 (12)Tumora Yes 41 (93)

No 3 (7)

Presence of colon-internalized E. coli, n (%)Mucosa Yes 27 (54) 16 (48)

No 23 (46) 17 (52)Tumora Yes 38 (86)c

No 6 (14)

Presence of B2 phylotype E. coli strainsYes 31 (65)d 13 (39)No 17 (35) 20 (61)

Presence of cyclomodulin-positive E. coli strainsYes 26 (60)e 6 (26)No 17 (40) 17 (74)

aSix are undetermined.bP ¼ 0.03.cP < 0.0002; tumor versus mucosa of patients with coloncancer or diverticulosis.dP ¼ 0.04; in both the mucosa and tumors of patients withcolon cancer versus diverticulosis.eP ¼ 0.01; in both the mucosa and tumors of patients withcolon cancer versus diverticulosis.

Colonization of Human Gut by E. coli and Colorectal Cancer

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overnight in 10% formalin. Paraffin-embedded sectionswere cut into 5 mm sections, and the tissue sections wereprepared for hematoxylin–phloxin staining and routinepathologic analysis.

Statistical analysisThe bacterial colony counts among multiple disease

populations were compared using the Kruskal–Wallis non-parametric analysis followed by the Dunn posttest. TheFisher exact test or x2 analysis was used for comparisonsof the proportion of patient samples positive for E. coliacross multiple disease populations. Unpaired Student ttests andMann–Whitney tests were used for comparisons of2 groups where appropriate (normality verified by theShapiro–Wilk test and homoscedasticity by the Fisher–Snedecor test). All tests were performed using Graph PadPrism 5 STATA (StataCorp) or R software (http://cran.r-project.org/). We considered P values of <0.05 as statisti-cally significant.

ResultsPatients and pathologic data

Fifty patients with adenocarcinoma (22 proximal and 28distal samples) and 33 with diverticulosis were prospec-tively included.Most patients with colon cancer had tumorsinvading the peritoneal serosa (T3 stage, 60%) and negativelymph nodes (N0, 62%; Supplementary Table SA). Tumorsweremost often classified asmoderate orwell-differentiated(42% and 54%, respectively) adenocarcinoma as well aslacking perinervous encasement (91%), vascular embols

(82%), mucinous component (84%), or intratumoralnecrosis (64%; Supplementary Table SA). Acute (n ¼ 5) orchronic (n ¼ 6) inflammation was observed in 11 patientswith diverticulosis. None of the patients with diverticulosisdisplayed signs of neoplastic transformation or adenoma-tous polyps.

Mucosa-associated and internalized E. coli in coloncancer and noncancerous tissues

Mucosa-associatedE. coliwere detected in 88%, 90%, and93% of diverticulosis mucosa, colon cancer mucosa, andcolon cancer tumor specimens, respectively (Table 1). Asignificant increase in mucosa-associated E. coli wasobserved in colon cancer tumors compared with coloncancer and diverticulosis mucosa (P ¼ 0.01 and P <0.001, respectively; Fig. 1A). Mucosa-internalized E. coliwere detected in 48%, 54%, and 86% of diverticulosis,colon cancer mucosa, and colon cancer tumor specimens,respectively (P < 0.0002). In addition, the number ofinternalized E. coli was significantly increased in coloncancer tumors compared with colon cancer and diverticu-losis mucosa (P < 0.001; Fig. 1B).

Colonization and clinical dataColonization by associated and internalized E. coli was

assessed according to the patients’ clinical data (Supple-mentary Table SA). The sex and the age of the patients withdiverticulosis and colon cancer did not influence the col-onization by associated and internalized E. coli (data notshown).

Table 2. Characteristics of E. coli strains isolated from the colon cancer samples according to TNM status

TNM status

I II III/IV

Number of patientsa, n 14 15 19

Presence of E. coli, n (% of patients) No 3 (21%)� 1 (7%) 1 (5%)Yes 11 (79%) 14 (93%) 18 (95%)

Phylogroup, n (% of patients) A 6 (43%) 2 (13%) 6 (32%)B1 5 (36%) 4 (27%) 4 (21%)B2 6 (43%) 12 (80%)c 13 (68%)c

D 5 (36%) 4 (27%) 5 (26%)

Cyclomodulin-positive strainb, n (% of patients) þ 5 (45%) 9 (64%)c 12 (67%)c

� 6 (55%) 5 (36%) 6 (33%)

pks-positive strain, n (% of patients) þ 4 (36%) 8 (57%)d 9 (50%)d

� 7 (64%) 6 (43%) 9 (50%)

aUndetermined for 2 patients (one patient with stage I tumor and other with stage III tumor).bThe presence of genes coding for the cyclomodulins CNF, CDT, CIF, and colibactin (pks island) was analyzed.cP < 0.05.dP < 0.05.�P < 0.05.

Bonnet et al.





Colonizationwas associatedwith the TNMstage and eachparameter separately (T, N). Given the low number ofpatients with metastasis (n¼ 3), statistical analysis was notperformed on this category. Colonization by associated E.

coliwas significantly increased in thenormalmucosa distantfrom the tumor at advanced stages (stages III/IV) comparedwith stage I (P < 0.01; Fig. 2A). No significant variation ininternalized bacteriawas observed in relation to TNMstages

Figure 1. Mucosa-associated andinternalized E. coli are significantlyincreased in colon cancer tumors.Quantification of colon-associated(A) and internalized (B) E. coli inhuman colonic mucosa of patientswith diverticulosis and coloncancer as well as the tumorsof patients with colon cancer.�, P < 0.05.

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Figure 2. Significant relationshipbetween mucosal colonization byE. coli and TNMstage classificationor its parameters T and N. A,mucosa-associated E. coliaccording to the TNM stage (fromthe TNM classification of AJCC/UICC). B and C, mucosa-associatedE. coli according to T (B)or N status (C). D, mucosa-internalized E. coli according to Tstatus. �, P < 0.05.






(data not shown). When each parameter of the TNM clas-sification was considered, colonization by associated E. coliwas significantly increased in nontumormucosa of patientswith locally advanced tumors (T3 and T4; P ¼ 0.004) andlymphatic spreading (Nþ; P ¼ 0.03; Fig. 2B and C). Withregard to associated E. coli, the level of internalized bacteriawas increased in nontumor mucosa of patients withadvanced tumors (T3 and T4) compared with noninvasivetumor (T1 and T2; P ¼ 0.045; Fig. 2D). In the tumor, nosignificant variation in the colonization by either associatedor internalized E. coli was observed in relation to TNMclassification or parameters. For other clinical data (differ-entiation status, perinervous encasement, vascular inva-sion, mucinous component, and intratumoral necrosis),no significant association was observed with associated orinternalized bacteria colonization in either tumor or nor-malmucosa. In addition,wemeasured the percentage of theKi-67 positive cells in the normal mucosa of patients withcolon cancer (Fig. 3). The proliferative index of the mucosadid not correlate with the TNM characteristics of the corre-sponding tumors (Supplementary Fig. SA). However, the

proliferative index significantly correlated with the levels ofboth mucosa-associated and internalized E. coli (P < 0.02and P < 0.04, respectively; Fig. 3).

We then extended the study by searching for a correlationbetween the presence and characteristics of the E. coli strainswith regards to TNM classification. Of note, the frozensubcultures ofmucosa-associatedbacteria from twopatients,one with TNM stage I and the other with stage III, were notviable.NoE. coliwas isolated in21%,7%, and5%ofpatientswith stage I (3 of 14), II (1 of 15), and III/IV (1 of 18) tumors,respectively (Table 2). In most cases, similar clones of E. colistrainswere found in the normalmucosa and tumor tissue ofpatients with colon cancer andmost strainswere from the B2phylogroup. Interestingly, the proportion of mucosa andtumor colonizationbyB2phylogroup E.coliwas significantlyincreased in patients with stage II (80%) and III/IV disease(68%) compared with stage I (43%; P < 0.05; cf. Table 2).TNM stage was not associated with other phylogroups. Aspreviously reported for tumor tissue (20), E. coli strainsharboring cyclomodulin-encoding genes (cdt, cif, and/orcnf) and/or the pks island were significantly more fre-quently observed in normal mucosa of patients withcolon cancer (26/43) than diverticulosis (6 of 23; 60%vs. 26%, respectively; P ¼ 0.01). Of great interest, theproportion of mucosa and tumor colonization by cyclo-modulin-positive E. coli strains was significantly higher inTNM stage II (64%) and III/IV (67%) patients than stage Ipatients (45%; P < 0.05; cf. Table 2). The presence of B2phylogroup or cyclomodulin-positive E. coli did notinfluence the overall level of E. coli colonization in themucosa or tumors (Supplementary Figs. SB and SC).

Colon cancer–associated E. coli 11G5 stimulates colontumor development in Min mice

The E. coli strain 11G5, which exhibited high level col-onization in the healthy mucosa and tumor tissue of apatient with an advanced stage tumor (T4) and liver metas-tasis, was selected as a representative colon cancer–associ-ated E. coli strain. The strain belongs to phylogroup B2 andpossesses the pks island encoding colibactin. Electronmicroscopy analysis of infected T84 intestinal cells indicat-ed that E. coli 11G5 induces the elongation of eukaryotic cellmembranes at bacterial adhesion sites. In addition, bacteriawere internalizedwithin endocytic vacuoles (Fig. 4A). Infec-tion of Min mice with E. coli 11G5 resulted in high-levelcolonization of the gut as indicated by the number ofbacteria in the feces (4.95 � 109 CFUs per gram feces) 2days after infection (Fig. 4B). Similar levels of colonizationweremeasured in the stools ofWT andMinmice (P¼ 0.79).In contrast, significantly reducedbacterial levels (1.69�107

CFUs per gram feces) were detected in stools of miceinfected with E. coli K-12 (P < 0.001; Fig. 4C). At the timeof sacrifice (50 days after infection), colonic polyps weredetected in 92%ofMinmice infectedwithE. coli11G5, 57%of Min mice with K-12 E. coli, and 50% of uninfected Minmice (P < 0.05). The histologic analysis indicated that allpolyps were adenocarcinomas (Fig. 4D) and no sign ofobvious inflammation in the 11G5-colonized mice was

Figure 3. Significant relationship between colonization of the mucosa byE. coli and the mucosal proliferative index. A, representative Ki-67immunostainings from the normal colonic mucosa of patients with coloncancer with a low (left) or high (right) proliferative index. B andC,mucosa-associated (B) and mucosa-internalized (C) E. coli according to theproliferative index.

Bonnet et al.





detected. Infection experiments with strain 11G5 in WTmice did not lead to any neoplastic changes (data notshown). Markedly significant increases in the number andin the size of tumors were observed in the 11G5-colonizedMin mice (Fig. 4E and F).

DiscussionBacterial and viral infections have long been established

as important factors in the etiologyof severalhumancancers(4, 5). More recently, the gutmicrobiota and dysbiosis havebeen associated with colon cancers. This article presentsevidence for the presence of pathogenic E. coli strains in themucosa and tumors of patients with colon cancer that couldplay a role in colorectal carcinogenesis. We observed that

mucosa-adherent E. coliwere not restricted to the tumor siteof carcinoma as previously reported (8, 10, 24). We dem-onstrated that colon cancer tumors have increased numbersof associated E. coli than colon cancer and diverticulosismucosa. It could be argued that local modifications of themucosal properties inducedby the tumor, such as loss of themucosal barrier and epithelial cell polarity as well as expres-sion of abnormal surface antigens in patients with cancer,promote increased adhesion and internalization of bacteriain the tumor tissue. Although colon cancer–specific surfaceantigens remain uncharacterized, we can hypothesize thatthe carcino-embryonic antigen cell adhesion-related mole-cule 6 (CEACAM6), which is upregulated in patients withcolorectal cancer (25), could be involved. Indeed, we have

Figure 4. The colon cancer–associated E. coli strain 11G5persists in intestinal epithelial cellsand stimulates colon tumordevelopment in Min mice. A,ultrastructural analysis of T84intestinal epithelial cells infectedwith the E. coli strain 11G5 (MOI ¼50 per cell) for 3 hours. Analysis bytransmission electron microscopyshows internalization of 11G5bacteria located within endocyticvacuoles (a and b) and control,noninfected T84 cells (c). B, level ofcolonizationmeasured in the stoolsof mice infected with the 11G5colon cancer–strain or K12commensal bacteria 2 dayspostinfection (p.i.). C, the level ofcolonization by the 11G5 strainmeasured in the stools of WT andMin mice. D, histologic analysis ofcolonic polyps observed in K12- or11G5-infectedmice. E, the numberof visible colonic tumors in miceinfected with the colon cancer E.coli strain 11G5, the K12-commensal E. coli strain, or controlPBS. F, the size of tumors ininfected mice. �, P < 0.05.






previously reported, in Crohn disease that type I pili areamong the bacterial virulence factors potentially implicatedin increased E. coli adhesion based on binding to CEA-CAM6, which is abnormally expressed in mucosa of thesepatients (26). Despite noting no significant variation in thetotal number of E. coli between the normal mucosa ofpatients with colon cancer and diverticulosis, we observedchanges in the E. coli populations. We previously showedthat pathogenic cyclomodulin-positive E. coli strainsbelonging to B2 phylogroup were significantly more prev-alent in patients with colon cancer tumors than in themucosal samples of diverticulosis (20). We demonstratedhere that pathogenic E. coli strains were also detected onnormal mucosa of patients with colon cancer. Our findingsare consistent with results by Arthur and colleagues in asmaller cohort of patients with colon cancer (8). Thisfinding prompted us to explore the effect of colon can-cer–associated E. coli infection in the Min mice modelfor which the microbiota has been reported to play arole in carcinogenesis (27). The representative pathogenicpks-positive E. coli strain (11G5) significantly inducedtumorigenicity with an increase in the size and number ofadenocarcinoma tumor. The genotoxic effects of colibactinrequires bacteria–host cell contact (18, 19), and it has beenreported that mRNA expression of the pks island occurs inthe bacteria in closest contact with the epithelial cells (28).We observed similar levels of colonization with E. coli 11G5in Min and WT mice, indicating that tumor developmentinduced by infection with pks-positive bacteria requires afirst hit, such as an apc mutation which is consistent withresults previously observed in the interleukin (IL)-10�/�

murine model (8, 29, 30). In parallel to these resultsobserved in mice, a strong association between the levelsof E. coli colonization in human normal mucosa and theproliferative index (Ki-67 immunostaining) was noted,suggesting that these bacteria play a role in regenerativeepithelial cell growth and tumor progression which ispreviously demonstrated in murine models (24, 27). Wealso observed a relationship between the levels of mucosa-associated E. coli colonization and the tumor cell dissem-ination process (axillary lymph node invasion and invasivetumor status) and a higher prevalence of the pathogenicstrains in aggressive form of cancer. Given that abnormalbacterial colonization was previously reported on themucosa of patients with adenoma (10, 24), all these dataon human mucosa suggest that pathogenic E. coli couldpromote the development of an aggressive form of carci-noma at the early steps of enterocyte transformation bycyclomodulin genotoxic agent effect, such as colibactin,and/or the induction of chronic inflammatory reactions.

We report, for the first time, a statistically significantrelationship between high levels of mucosa-associated E.

coli and poor colorectal carcinoma prognostic factors, suchas TNM stages. The TNM staging system is still the mostwidely used in patient selection for adjuvant chemotherapyafter curative resection. At present, it is not possible toaccurately differentiate between patients with a favorableor poor prognosis in stages II and III colon cancer (31).Various clinical factors have been associated with a higherrisk of recurrence in stage II and III colon cancer, includingtumor obstruction and/or perforation, the number oflymph nodes identified in the surgical specimen, T4 stage,lymphovascular invasion, undifferentiated histology, andlymph node micrometastasis (32–36). None of these mar-kers are able to clearly separate patients into distinct prog-nostic groups. Although othermicrobes likely participate inthe progression of colon cancer, our findings demonstratethat mucosal colonization by pathogenic E. coli could serveas a new and crucial prognostic factor of poor outcome.Large multicenter prospective clinical studies are needed toevaluate whether pathogenic E. coli colonization in themucosa is a prognostic marker.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Bonnet, E. Buc, R. Bonnet, A. Darfeuille-MichaudDevelopment of methodology: M. Bonnet, P. Sauvanet, P. D�echelotte, R.BonnetAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): M. Bonnet, E. Buc, P. Sauvanet, D. Dubois, P.D�echelotte, D. PezetAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis):M.Bonnet, E. Buc, P. Sauvanet, B. Pereira,A. Darfeuille-MichaudWriting, review, and/or revision of themanuscript:M. Bonnet, E. Buc, P.Sauvanet, B. Pereira, R. Bonnet, A. Darfeuille-MichaudAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M. Bonnet, E. Buc, P. Sauvanet, D.PezetStudy supervision: D. Pezet, A. Darfeuille-MichaudHistologic analysis: C. Darcha

AcknowledgmentsThe authors thank the CICS platforms from Universit�e d’Auvergne for

assistance with the transmission electron microscopy, as well as AlexandraRezard and Abdelkrim Alloui (Animal Facilities) for animal care.

Grant SupportThis study was supported by the Minist�ere de la Recherche et de la

Technologie, Inserm and Universit�e d’Auvergne (UMR1071), INRA (USC-2018), and grants from the Conseil R�egional d’Auvergne.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received May 15, 2013; revised November 22, 2013; accepted November26, 2013; published OnlineFirst December 13, 2013.

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2014;20:859-867. Published OnlineFirst December 13, 2013.Clin Cancer Res Mathilde Bonnet, Emmanuel Buc, Pierre Sauvanet, et al. Risk

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