==== Front Intest ResIntest ResIRIntestinal Research1598-91002288-1956Korean Association for the Study of Intestinal Diseases 10.5217/ir.2018.16.3.346Focused Review: Colorectal CancerImpact of microbiota in colorectal carcinogenesis: lessons from experimental models Yu Linda Chia-Hui 1Wei Shu-Chen 2Ni Yen-Hsuan 31 Graduate Institute of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan.2 Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan.3 Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan.Correspondence to Yen-Hsuan Ni, Department of Pediatrics, National Taiwan University Hospital, National Taiwan University College of Medicine, 7 Chung-Shan South Road, Taipei 100, Taiwan. Tel: +886-2-23123456 (ext. 71516), Fax: +886-2-23938871, yhni@ntu.edu.tw7 2018 27 7 2018 16 3 346 357 26 3 2018 28 5 2018 29 5 2018 © Copyright 2018. Korean Association for the Study of Intestinal Diseases.2018Korean Association for the Study of Intestinal DiseasesThis is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.A role of gut microbiota in colorectal cancer (CRC) growth was first suggested in germ-free rats almost 50 years ago, and the existence of disease-associated bacteria (termed pathobionts) had becoming increasingly evident from experimental data of fecal transplantation, and microbial gavage or monoassociation. Altered bacterial compositions in fecal and mucosal specimens were observed in CRC patients compared to healthy subjects. Microbial fluctuations were found at various cancer stages; an increase of bacterial diversity was noted in the adenoma specimens, while a reduction of bacterial richness was documented in CRC samples. The bacterial species enriched in the human cancerous tissues included Escherichia coli, Fusobacterium nucleatum, and enterotoxigenic Bacteroides fragilis. The causal relationship of gut bacteria in tumorigenesis was established by introducing particular bacterial strains in in situ mouse CRC models. Detailed experimental protocols of bacterial gavage and the advantages and caveats of different experimental models are summarized in this review. The microbial genotoxins, enterotoxins, and virulence factors implicated in the mechanisms of bacteria-driven tumorigenesis are described. In conclusion, intestinal microbiota is involved in colon tumorigenesis. Bacteria-targeting intervention would be the next challenge for CRC. Colorectal neoplasmsMicrobiota dysbiosisMucosa-associated bacteriaPathobiontVirulenceNational Health Research Instituteshttp://dx.doi.org/10.13039/501100004737NHRI-EX105-10520BINHRI-EX106-10520BINHRI-EX107-10520BIMinistry of Science and Technology, Taiwanhttp://dx.doi.org/10.13039/501100004663MoST 106-2320-B-002-017 ==== Body INTRODUCTION An ecosystem of microorganisms habituates the human intestinal tract which is defined as the gut microbiota, composed of bacteria, archaea, virus, and fungi.1 The intestinal tract harbors around 1014 bacteria, with the highest amount in the colon.234 Over 1,000 bacterial species belonging mainly to 4 phyla were identified in humans, with each individual possessing more than 160 species.5 The collection of all genomes of the microbiota is termed microbiome, of which the number of intestinal microbial genes has been estimated to be ~100 times more than that of human genes.145 Accumulating evidence demonstrated that gut microbiota is involved in the maintenance of mucosal homeostasis, with an indispensable role in promotion of wound repair and epithelial barrier fortification.6789 While a symbiotic relationship is established between commensal bacteria and the healthy host, the presence of an altered gut microbial population or the emergence of opportunistic bacteria from commensals may contribute to the development of IBD and colorectal cancer (CRC).10111213 Patients with IBD had higher risk of developing cancer later in life, termed colitis-associated CRC.1415 It is generally believed the genetic and dietary factors, as well as chronic inflammation and altered microbial population predispose to tumor development.16171819 DETRIMENTAL AND BENEFICIAL ROLES OF BACTERIA IN EXPERIMENTAL MODELS OF CRC A relationship between commensal bacteria and intestinal carcinogenesis was first suggested in rodent models by Reddy et al. in 1974.20 Germ-free rats showed lower incidence of chemically induced duodenal and colonic tumors compared to those raised in conventional husbandry.20 Reduction of colonic tumor burden was also reported in several chemically induced or genetic deficient mouse models when derived under germ-free conditions.212223 It should be kept in mind that the physiological relevance of germ-free models had been criticized since their mucosal immune systems were undeveloped due to the lack of microfloral establishment.242526 Additional evidence of bacterial involvement in carcinogenesis was supported by studies showing reduction of tumor load after antibiotic manipulation of gut microbiota.2227 The notion of disease-associated microbes originating from commensal-derived opportunistic bacteria (termed pathobionts) was shown by evidence of increased cancer growth in recipient mice after fecal transplantation from high tumor-bearing mouse.2829 These studies suggested the existence of pathobionts with a protumoral characteristic in the fecal bacteria population. On the other hand, presence of beneficial bacteria (termed probionts or probiotics) with tumor-suppressive metabolites was also found in experimental models.1630 Short-chain fatty acids (SCFAs) such as butyrate are bacterial metabolites by fermentation of dietary fibers. Nutritional studies showed that high fiber diets or butyrate in the colon decreased the rate of aberrant crypt foci formation and reduced the tumor burden in animal models.3132 A recent study using a gnotobiotic mouse model also demonstrated that supplementation with high dietary fiber and butyrate-producing bacteria significantly decreased colon tumor growth.30 Taken together, intestinal bacteria are involved in tumorigenesis with both detrimental and beneficial roles. Potential tumorigenic bacteria and their underlying mechanisms to promote cancer growth will be highlighted in this review. For more information on probiotics and cancer, please refer to other review articles.3334 MICROBIOTA DYSBIOSIS AT VARIOUS CANCER STAGES Despite similar fecal bacterial counts between healthy subjects and CRC patients,35 a clear difference in the composition of normal and tumor-associated microbiota was demonstrated. Numerous studies have shown reduction of microbial diversity in the stool samples and mucosal biopsies of patients with IBD363738 (including CD and UC) and in those with CRC.3940 In contrast, higher diversity and greater numbers of bacteria were noted in adenoma biopsies compared to healthy mucosa,4142 suggesting that there existed dynamic changes of microbial population throughout the different tumor stages. It should be noted these cross-sectional studies are based on specimens collected at single time points. Therefore, the changes in gut microbiota only indicated a correlation but did not prove a cause-and-effect relationship for tumor formation in CRC patients. 1. Microbiota in Stool Samples of CRC Patients The Intestinal microbes are mixed in the outer loose mucus layer and in chyme and feces. Many clinical studies have utilized stool samples for analysis of bacterial composition as a surrogate for intestinal microflora. Although the fecal bacteria may not represent the complete population of gut microbiota, this noninvasive approach has been widely practiced and conveyed crucial information of the bacterial community residing in the intestinal lumen. However, comparison of fecal microbiota composition to that of mucosal specimens and tumor biopsies may provide a more comprehensive view of the changes in the gut ecosystem. Four main phyla were identified in the fecal microbiota of healthy individuals, including Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria, with Bacteroidetes and Firmicutes being the 2 predominant phyla that constitutes more than 90% of the total bacterial population.4344 The percentages of Bacteroidetes and Firmicutes phyla in the fecal microbiota of healthy subjects were ~55% and ~40%, respectively.4344 In CRC patients, these 2 phyla still constitutes the largest proportion in the stool microbiota.4344 Being the 2 predominant phyla with higher total numbers in the individual, it is no doubt that alterations of the bacterial strains in the Bacteroidetes and Firmicutes phyla in the fecal population are easier to spot compared to other bacteria with lower counts. Nevertheless, accumulating evidence indicates that some bacteria belonging to the minor phyla in fecal microbiota may also play protumoral roles. Under the Bacteroidetes phylum, enrichment of genera of Porphyromonas and Prevotella was reported in stool samples of CRC patients compared to healthy volunteers.4445 Conflicting results were shown for the Bacteroides genus; increased percentage were reported in CRC patients in some studies,3543 while others found a significant reduction.45 Among the Bacteroides species, a significant increase of Bacteroides fragilis and a decrease of Bacteroides vulgatus and Bacteroides uniformin were found in fecal samples of CRC patients.4546 Under the Firmicutes phylum, increase of Enterococcus and Streptococcus genus and decrease of genera such as Faecalibacterium, Roseburia and Eubacterium and of Lachnospiraceae family were reported in stool samples of CRC patients compared to healthy volunteers.4547 At the species levels, decreases in Ruminococcus obeum, Ruminococcus albus, Pseudobutyrivibrio ruminus, Lachnospira pectinoschiza, Lachnospira bovis were seen in the fecal microbiota of CRC patients.44 One of the common characteristics of the decreased bacterial genera and species in CRC patients belonging to the Firmicutes phylum is the ability of SCFA production.434445 The altered stool bacterial profiling corresponded to the relatively lower levels of fecal butyrate in CRC patients compared to control subjects.44 In addition, no difference of Lactobacillus was shown in stool samples of cancer patients.35454648 Under the relatively minor phyla in fecal microbiota such as Proteobacteria, Actinobacteria and others, higher percentages of the genera of Escherichia (belonging to Proteobacteria phylum) and Fusobacterium (belonging to Fusobacteria phylum) were found in the stool samples of CRC patients compared to control subjects.434546 The Bifidobacteria genus (belonging to Actinobacteria phylum) in fecal samples were either increased45 or showed no change3546 in CRC patients. 2. Microbiota in Tumor Biopsies of CRC Patients Recent studies have utilized tumor/mucosal biopsies and lavage samples instead of fecal samples to validate and clarify the dysbiosis of gut microbiome. Data obtained from tumor biopsies indicated the alteration in mucosa-associated bacteria. Accumulating evidence showed distinct profiles between mucosal bacteria and fecal microbiota,47495051 whereas microfloral results obtained from mucosal biopsy and similar to those of lavage samples.52 For the evaluation of mucosal microbiota, tumor tissues are usually compared to normal mucosa of healthy individuals or to patient-matched non-cancerous tissues. One report had shown that Proteobacteria was the most predominant phylum (~60%) in the gut mucosa of healthy individuals.49 Another study showed the predominance of Firmicutes, Bacteroidetes, and Proteobacteria, of which the 3 phyla accounts for 80% of the microbiota in the control subjects.50 The findings showed that fecal microbiota as a surrogate only partially reflect the composition of the mucosa-associated bacteria. By using mucosal specimens, the genera of Escherichia and Fusobacterium were found significantly increased in CRC patients compared to healthy controls.495051 It is noteworthy that Escherichia and Fusobacterium constitute a minority of the healthy stool microbiome,4344 and are also of low percentages (0.22% and 0.01%, respectively) of the bacterial population in healthy gut mucosa.49 However, a 15- and 1,000-fold increase of the percentage of Escherichia and Fusobacterium were noted in the microflora of biopsy tissues of CRC patients compared to that of healthy controls.49 When comparing patient-matched cancerous samples and adjacent non-cancerous tissues, abundance of Fusobacterium was also observed in tumors.495051 The enrichment of Escherichia and Fusobacteria in tumor biopsies is in keeping with the findings in stool samples of CRC patients.434546 Higher levels of Bacteroides genus was found in the mucosal microbiota of CRC patients than healthy controls.47 When comparing microbial composition in tumor biopsies to adjacent non-cancerous tissues, the percentage of Bacteroides were decreased in one report50 and increased in another study.49 The inconsistent result of Bacteroides in tumor tissues seems to reflect the fecal microbial composition which also shows conflicting data in the literatures.354345 Decreased mucosal levels of Firmicutes and Actinobacteria phyla were observed in cancerous tissues of CRC patients compared to normal mucosa of healthy subjects or to adjacent non-cancerous tissues.4951 A downregulation of Lachnospiraceae and Ruminococcaceae was observed in cancerous tissues compared to adjacent non-cancerous tissues, which also correlated with the finding of decreased bacterial genus in fecal samples of CRC patients.4550 The data of mucosa-associated Lactobacillus and Bifidobacterium were inconsistent and a few reports showed no difference of these bacteria between tumor and control samples.3548 Overall, the aforementioned studies all pointed to microbiota dysbiosis in fecal and mucosal samples of CRC patients despite inconsistency of the wax or wane of particular bacterial taxa. The fecal bacterial profiling only partially reflected mucosal microbiota in healthy controls and in CRC. Moreover, a higher ratio of the Proteobacteria and Fusobacteria phyla was observed in CRC patients when the analysis was based on mucosal specimens. POTENTIAL TUMORIGENIC PATHOBIONTS Clinical studies indicated a positive correlation between mucosal bacteria and disease pathogenesis. The gut commensals mostly reside in the intestinal lumen in physiological conditions, separated by the inner mucus layer and are rarely in direct contact with the epithelial cells.53545556 The concept of bacterial adhesion to host epithelial cells was first reported in uropathogenic Escherichia coli in 1908.57 In the past two decades, high numbers of mucosa-associated bacteria were reported in clinical studies of IBD and CRC. Enrichment of mucosa-associated E. coli was found in tissue biopsy of patients with CD and CRC.385859606162 Abundance of mucosa-associated Fusobacterium was noted in CRC patients compared to healthy individuals.4950 Invasive strains of Fusobacterium nucleatum were also isolated from biopsy specimens of CD or acute appendicitis.636465 Moreover, higher levels of enterotoxigenic B. fragilis were found in colonic tumor samples obtained from patients.6667 Evidence of the protumorigenic potentials of E. coli, F. nucleatum, and B. fragilis by using experimental models are discussed. The detailed experimental protocols of bacterial gavage in in situ mouse CRC models are listed in Table 158686970717273747576777879 and the advantages and caveats of different experimental models are summarized as followed. 1. Escherichia coli Adherent-invasive E. coli (AIEC) was first isolated from ileal lesions in CD patients.8081828384 Although AIEC was unable to colonize the intestine of wild type mice, the colitogenic activity of AIEC was shown in transgenic mice expressing human carcinoembryonic antigen-related cell adhesions molecules (CEACAMs; a receptor for type-1 pili or fimbriae [fim]).85 The data suggested that mucosal colonization of AIEC through fimbriae-mediated adhesion was a crucial step for its colitogenic ability as a pathobiont.85 However, there remains no direct evidence for the involvement of AIEC in colon cancer development, except the general link between inflammation and tumorigenesis. It is noteworthy that the fimbrial adhesin is historically known as a common feature for host colonization by various strains of gram-negative bacteria.57 Although fimbriae-mediated adhesion also affected bacterial uptake or promote proinflammatory response in epithelial cells,57 it has not been traditionally regarded as a strain-specific pathogenic virulence.8687 Previous studies demonstrated increased tumor burden in human CEACAM-transgenic mice after azoxymethane (AOM) injections,88 but did not specify the bacterial strains responsible for the fimbriae-dependent tumor growth. It remains undetermined whether the adherent or invasive virulence of E. coli is involved in colorectal tumorigenesis. A clear evidence of E. coli involvement in CRC development was reported with the use of 3 bacterial strains (i.e., NC101, CCR20, and 11G5) which harbors genotoxin-encoding polyketide synthase (pks) pathogenicity islands (Table 1).68697172 The colibactin produced by pks+ E. coli induced DNA damage and cellular senescence in intestinal epithelial cells.68697172 The pks+ NC101 strain was a mouse adherent-invasive E. coli, and the CCR20 and 11G5 strains were clinical isolates from tumor biopsies of CRC patients. Increased colonic tumor load was observed by monoassociation of E. coli NC101 in gnotobiotic interleukin 10 (IL-10)−/− mice given injections of AOM6869 and in adenomatous polyposis coli (APC)Min/+-IL-10−/− mice.70 Moreover, higher tumor susceptibility was found after oral gavage of pks+ E. coli CCR20 to wild type mice administered AOM/dextran sulfate sodium (DSS).7172 Increased tumor numbers and volume were documented in APCMin/+ mice following oral inoculation of pks+ E. coli 11G5 compared to the K12 strain.58 Details of the experimental design are described as followed and also summarized in Table 1. The germ-free IL-10−/− and APCMin/+-IL-10−/− mice were orally gavaged with 108 colony-forming units (CFU) of E. coli NC101 strain in one bolus for bacterial monoassociation.686970 For oral gavage of E. coli CCR20 strain, the wild type mice were raised on specific pathogen free (SPF) conditions and given intraperitoneal injection of AOM, followed by pretreatment of streptomycin (a bactericidal antibiotic to G(−) bacteria to disturb microflora) in drinking water for 2 days prior to gavage of 109 CFU of E. coli CCR20 to facilitate bacterial colonization, and then subjected to 2 cycles of DSS in drinking water to induce colitis.7172 In addition, APCMin/+ raised in SPF conditions were gavaged with 108 CFU of E. coli 11G5 strain 3 days after streptomycin treatment to facilitate bacterial colonization, and tumors were inspected at 7 weeks after infection.58 Due to the nature of these intestinal isolated E. coli which are not pathogens per se, microbiota disturbance by antibiotics prior to bacterial gavage or monoassociation in gnotobiotic mice are necessary to facilitate bacterial colonization to understand their effects on tumor development. Overall, higher tumor loads were found in the mouse groups given pks+ E. coli compared to the pks-deleted bacteria or pks-negative strains, indicating a crucial role of pks operon in promoting carcinogenesis.68697071 Other reports have demonstrated that the human CRC-associated E. coli (11G5 strain) induced colitis and increased crypt cell proliferation in transgenic mice with epithelial overexpression of human CEACAM.73 The E. coli 11G5 strain was gavaged at 2×108 CFU twice a week for 3 weeks after streptomycin and 0.25% DSS treatment in the human CEACAM-transgenic mice.73 Moreover, the human CRC-associated E. coli 11G5 triggered the production of cyclooxygenase (COX)-2 in macrophages after phagocytosis in vitro, in a pks-independent manner.74 Furthermore, human CRC-associated E. coli obtained from tumor biopsies were composed of genotoxin-positive and -negative populations.89 The pks+ E. coli were not adhesive, whereas the highly adherent E. coli were devoid of known genotoxins but caused DNA damage in vitro.89 The findings suggested that aside from causing genotoxicity, the induction of tumor-infiltrating macrophages and other unknown mechanisms may also play indispensable roles in E. coli-driven tumorigenesis. 2. Fusobacterium nucleatum Enrichment of F. nucleatum was demonstrated in the stool and tissue samples of CRC patients.43495066909192 F. nucleatum is known as a commensal G(−) species residing in the oral cavity, and has been reported as an invasive strain associated with inflammatory disease in the mouth such as gingivitis.9394 Invasive strains of F. nucleatum were also isolated from inflamed biopsy tissues from patients with CD or acute appendicitis.636465 Recent data showed that orogavage of F. nucleatum increased tumor burden in APCMin/+ mice or in wild type mice given AOM/DSS (Table 1).757695 A number of mechanisms were proposed by in situ CRC mouse models, including β-catenin and nuclear factor-κB signaling via activation of Toll-like receptor 4 (TLR4) (a cell surface innate receptor for lipopolysaccharide [LPS]),7595 and recruitment of tumor-infiltrating myeloid cells.76 Other studies by using human CRC cell lines and a xenograft model showed that FadA adhesin expressed on F. nucleatum, via extracellular binding to E-cadherin, induced nuclear translocation of β-catenin for oncogenic transcription and epithelial hyperproliferation.9697 The invasiveness of F. nucleatum was not a requirement for its effect on oncogenic transcription but was associated with proinflammatory signals which could be indirectly involved in cancer development.96 So far, whether F. nucleatum infection and its pro-tumorigenic ability are associated with inflammation is still controversial. Although some reported that the histopathological features of colitis and enteritis were not induced,76 others showed upregulation of proinflammatory cytokines and chemokines following inoculation of F. nucleatum.75 It is noteworthy that the in situ CRC model was pre-fed with streptomycin (a bactericidal antibiotic to G(−) bacteria) to disturb the normal flora, followed by inoculation with a very high number of F. nucleatum for a long period of time (109 CFU by gavage per day for 20 weeks or 108 CFU by gavage per day for 8 weeks).7576 With such a high bacterial load given continuously, the roles of F. nucleatum as a driver or a passenger for tumorigenesis remain in doubt. A recent study demonstrated that gavage of a single strain or multiple strains of F. nucleatum obtained from clinical specimens of CRC patients to gnotobiotic APCMin/+ mice or APCMin/+-IL-10−/− mice did not increase colitis severity nor enhance colon tumor burden.70 The germ-free mice were gavaged weekly with a mixture of 6 F. nucleatum clinical isolates at 108 CFU per strain per mice, or were transferred to SPF microbiota prior to gavage of F. nucleatum mixtures and sacrificed 16 weeks later to examine tumor growth. However, no increase in tumors was observed in either protocols.70 The inconsistent data of F. nucleatum suggested that other unidentified mechanisms, such as interaction with other bacteria, may partly contribute to its protumoral characteristics. 3. Bacteroides fragilis A subclass of the human commensal Bacteroides species, enterotoxigenic Bacteroides fragilis (ETBF), was associated with acute inflammatory diarrheal disease and CRC in patients.669899 Increased percentage of B. fragilis was reported in fecal microbiota.4546 The levels of ETBF and enterotoxin gene (bft) in tumor and stool samples were significantly higher in late stages (III/IV) of CRC compared to control tissues.666799 Despite reported as an obligate anaerobe, abundant literature showed invasive traits of the G(−) bacteria B. fragilis. The most frequent anaerobe isolated in clinical cases of peritonitis, intra-abdominal abscess or bacteremia is B. fragilis, suggesting its aerotolerance and invasiveness.100101102 Previous studies have demonstrated that ETBF triggered colitis and accelerated tumor growth in APCMin/+ mice (Table 1).7778 The colonization of ETBF (108 CFU by oral gavage in one dose) was facilitated by pretreatment of clindamycin (an antibiotic against G(+) bacteria) and streptomycin (an antibiotic against G(−) bacteria) or gentamicin (a broad-spectrum antibiotic) to disturb the gut microbiota.777879 Following antibiotic manipulation of microbiota, colonization with ETBF but not its nontoxigenic counterparts increased tumor burden in APCMin/+ mice.7778 The B. fragilis enterotoxin (also known as fragilysin) acts as a metalloprotease that causes oxidative DNA damage, E-cadherin cleavage, epithelial barrier damage, and activation of STAT3/Th17 immune responses, and generation of protumoral monocytic myeloid suppressor cells.777879103104 Studies in vitro showed that fragilysin stimulated the production of spermine oxidase in intestinal epithelial cell lines, suggesting a direct role of the enterotoxin on epithelial free radical production and DNA damage.78 Taken together, the findings indicated that ETBF was a strong colitogenic infectious agent that promoted tumorigenesis through both direct and indirect mechanisms. UNANSWERED QUESTIONS AND FUTURE DIRECTIONS A number of hints and questions arose through the review of protumorigenic pathobionts. The protumorigenic bacteria (e.g., E. coli, F. nucleatum, and B. fragilis) identified so far in in situ CRC models were all G(−) bacteria with mucosal colonization characteristics. First, the tumor-promoting bacteria are enriched in the mucosa and belong to the category of G(−) microbes, indicating that the juxtaposition of G(−) bacteria and its wall component (i.e., LPS) recognized by mucosal innate receptors are likely to contribute partly to the tumor susceptibility.33105106107 The findings re-emphasized the role of abnormal expression or upregulation of epithelial immune receptors in tumor predisposition.107108109 Second, microbial colonization and active invasion was shown to facilitate other commensal bacteria to be internalized into epithelium,110111112 and may cause secondary bystanders to pass the epithelial barrier through paracellular spaces.113114115116 The active or passive entry of bacteria could further fuel inflammation-associated tumor growth or may induce DNA damage and hyperproliferative signals in host cells. In addition, the common features of mucosal colonization among protumorigenic bacteria suggest that there may be shared adherence/invasion-associated virulence factors to induce epithelial damages, which may partly underlie bacterial mechanisms of epithelial malignant transformation. Third, physiological mucosal defense against pathogen colonization and invasion included free radical production117 and autophagy of infected organelles in the epithelial cells.118119 The mechanisms underlying the incompentent clearance of intracellular microbes remain poorly understood. A recent paper suggested that increased levels of microRNA 106B caused a reduction of autophagy-related gene transcription in intestinal epithelia of CD may account for the failure of intracellular bacterial clearance.120 It remains unclear whether epithelial recognition of pathobionts derived from commensals may activate suppressive/tolerant or defensive mechanisms. The involvement of epithelial barrier impairment in tumorigenesis warrants further studies. In summary, it is widely recognized nowadays that intestinal microbiota is involved in colorectal tumorigenesis. Protumorigenic bacteria (e.g., E. coli and F. nucleatum) which are not predominant species in fecal microflora are enriched in the cancerous tissues, and may promote tumorigenesis by expression of genotoxins and virulence factors. Further understanding of the host-microbe interaction and how the bacterial factors fit into the genetic and molecular paradigm of tumor development will shed light to the development of novel microbe-targeting therapy. Specific elimination of pathobionts with sparing of beneficial symbionts would be the next challenge. FINANCIAL SUPPORT: This study was supported by the National Health Research Institute, Taiwan (NHRI-EX105-10520BI, NHRI-EX106-10520BI, NHRI-EX107-10520BI) and Ministry of Science and Technology (MoST 106-2320-B-002-017). CONFLICT OF INTEREST: No potential conflict of interest relevant to this article was reported. AUTHOR CONTRIBUTION: Writing - original draft: LCY. Writing - review and editing: LCY, SCW, YHN. Approval of final manuscript: LCY, SCW, YHN. Table 1 Bacteria Promoted Tumorigenesis in in situ Mouse CRC Models Bacterial strain Animal models Housing and pretreatment Dose and time for bacterial gavage Outcome Proposed mechanisms Reference Escherichia coli NC101 (a mouse adherentinvasive E. coli) IL-10−/− mice administered AOM GF Monoassociation by gavage at 108 CFU, followed by 6 wk AOM injections starting at 1–4 wk postmonoassociation Mice showed more severe colitis and invasive mucinous adenocarcinoma Colibactin encoded by pks operon caused DNA damage 686970 APCMin/+-IL-10−/− mice GF Monoassociation by gavage at 108CFU, and tumor inspection after 16 wk Mice showed more severe colitis and higher colon tumor loads Colibactin encoded by pks operon caused DNA damage 70 CCR20 (a clinical isolate of E. coli from CRC samples) Wild type mice administered AOM/DSS SPF; streptomycin for 2 day starting on the first day of AOM injection Single gavage at 109 CFU 1 day after the end of antibiotic pretreatment Mice developed higher tumor numbers Colibactin encoded by pks operon induced cellular senescence 7172 11G5 (a clinical isolate of E. coli from CRC samples) APCMin/+ mice SPF; streptomycin Single gavage at 108 CFU after antibiotic pretreatment, and tumor inspection at 7 wk after infection Mice showed increase tumor numbers and volume Colibactin encoded by pks operon caused DNA damage 58 Transgenic mice expressing epithelial-specific human CEACAM SPF; streptomycin and 0.25% DSS Gavage twice wk at 2×108 CFU for 3 wk, after antibiotic and DSS pretreatment Mice displayed more severe colitis, epithelial damage and proliferation E. coli induced epithelial proliferation, partly via induction of macrophage COX-2 synthesis in a pks-independent manner 7374 Fusobacterium nucleatum A TCC25586 (a clinical isolate of cervical-facial lesion) APCMin/+ mice; wild type mice administered AOM/ DSS AOM/DSS SPF; streptomycin for 3 day Daily gavage at 109 CFU for 20 wk Mice displayed higher tumor burden Activation of TLR4/NF-κB signaling and upregulation of miR-21 75 EAVG_002; 7/1 (a clinical isolate from sigmoid colon of a CD patient) APCMin/+ mice SPF Daily gavage at 108 CFU for 8 wk Mice showed accelerated onset and higher numbers of colonic tumors, but with no sign of colitis Recruitment of tumor-infiltrating myeloid cells 76 IL-10−/− mice; T-bet−/−Rags−/− mice SPF Daily gavage at 108 CFU for 8 wk No increase in tumor load or colitis severity 76 EAVG_002; 7/1 APCMin/+ mice SPF Transfer from GF to SPF conditions, gavage with SPF microbiota followed by weekly gavage at 108 CFU for 20 wk No increase in tumor load compared to SPF controls gavaged with broth 70 C53, CC7/3JVN3C1, CC7/5JVN1A4, CC2/3Fmu1, CC2/3FmuA, CC7/4Fmu3 (all clinical isolates from CRC patients) APCMin/+-IL-10−/− mice GF Transfer from GF to SPF conditions, gavage with SPF microbiota followed by weekly gavage of 6 bacterial mixtures (108 CFU per strain per mouse) for 16 wk No increase in tumor load compared to SPF controls gavaged with broth 70 APCMin/+ mice GF Polyassociation with 6 bacterial mixtures by gavage of 101 CFU per strain per mouse, and sacrificed 16 wk later No increase in tumor load compared to GF controls gavaged with broth 70 Bacteroides fragilis Enterotoxigenic B. fragilis strain 86-5443-2-2 (a piglet isolate) APCMin/+ mice SPF; clindamycin/streptomycin for 3–5 day Single gavage at 108 CFU Mice displayed rapid colitis and increased tumor numbers Enterotoxin fragilysin caused DNA damage, activated STAT3/Th17 cell response, and increased protumoral monocytic myeloid-derived suppressor cells to promote carcinogenesis 777879 CRC, colorectal cancer; IL, interleukin; AOM, azoxymethane; GF, germ free; CFU, colony-forming units; pks, polyketide synthase; APC, adenomatous polyposis coli; DSS, dextran sulfate sodium; SPF, specific pathogen-free; CEACAM, carcinoembryonic antigen-related cell adhesion molecule; COX, clyclooxygenase; TLR, Toll-like receptor; NF-κB, nuclear factor κB. ==== Refs 1 Lynch SV Pedersen O The human intestinal microbiome in health and disease N Engl J Med 2016 375 2369 2379 27974040 2 Yu LC Wang JT Wei SC Ni YH Host-microbial interactions and regulation of intestinal epithelial barrier function: from physiology to pathology World J Gastrointest Pathophysiol 2012 3 27 43 22368784 3 Ley RE Peterson DA Gordon JI Ecological and evolutionary forces shaping microbial diversity in the human intestine Cell 2006 124 837 848 16497592 4 Sender R Fuchs S Milo R Revised estimates for the number of human and bacteria cells in the body PLoS Biol 2016 14 e1002533 10.1371/journal.pbio.1002533 27541692 5 Qin J Li R Raes J A human gut microbial gene catalogue established by metagenomic sequencing Nature 2010 464 59 65 20203603 6 Sommer F Anderson JM Bharti R Raes J Rosenstiel P The resilience of the intestinal microbiota influences health and disease Nat Rev Microbiol 2017 15 630 638 28626231 7 Owyang C Wu GD The gut microbiome in health and disease Gastroenterology 2014 146 1433 1436 24675436 8 Roy S Trinchieri G Microbiota: a key orchestrator of cancer therapy Nat Rev Cancer 2017 17 271 285 28303904 9 Nagao-Kitamoto H Kitamoto S Kuffa P Kamada N Pathogenic role of the gut microbiota in gastrointestinal diseases Intest Res 2016 14 127 138 27175113 10 Schwabe RF Jobin C The microbiome and cancer Nat Rev Cancer 2013 13 800 812 24132111 11 Guinane CM Cotter PD Role of the gut microbiota in health and chronic gastrointestinal disease: understanding a hidden metabolic organ Therap Adv Gastroenterol 2013 6 295 308 12 Drewes JL Housseau F Sears CL Sporadic colorectal cancer: microbial contributors to disease prevention, development and therapy Br J Cancer 2016 115 273 280 27380134 13 Abreu MT Peek RM Jr Gastrointestinal malignancy and the microbiome Gastroenterology 2014 146 1534 1546.e3 24406471 14 Munkholm P Review article: the incidence and prevalence of colorectal cancer in inflammatory bowel disease Aliment Pharmacol Ther 2003 18 Suppl 2 1 5 15 Brackmann S Andersen SN Aamodt G Relationship between clinical parameters and the colitis-colorectal cancer interval in a cohort of patients with colorectal cancer in inflammatory bowel disease Scand J Gastroenterol 2009 44 46 55 18609187 16 Bultman SJ Interplay between diet, gut microbiota, epigenetic events, and colorectal cancer Mol Nutr Food Res 2017 61 1500902 10.1002/mnfr.201500902 17 Tözün N Vardareli E Gut microbiome and gastrointestinal cancer: les liaisons dangereuses J Clin Gastroenterol 2016 50 Suppl S191 S196 27741173 18 Hullar MA Burnett-Hartman AN Lampe JW Gut microbes, diet, and cancer Cancer Treat Res 2014 159 377 399 24114492 19 Hiley CT Swanton C Pruning cancer's evolutionary tree with lesion-directed therapy Cancer Discov 2016 6 122 124 26851181 20 Reddy BS Weisburger JH Narisawa T Wynder EL Colon carcinogenesis in germ-free rats with 1,2-dimethylhydrazine and N-methyl-n'-nitro-N-nitrosoguanidine Cancer Res 1974 34 2368 2372 4843537 21 Uronis JM Mühlbauer M Herfarth HH Rubinas TC Jones GS Jobin C Modulation of the intestinal microbiota alters colitisassociated colorectal cancer susceptibility PLoS One 2009 4 e6026 10.1371/journal.pone.0006026 19551144 22 Zackular JP Baxter NT Iverson KD The gut microbiome modulates colon tumorigenesis MBio 2013 4 e00692 e00613 10.1128/mBio.00692-13 24194538 23 Li Y Kundu P Seow SW Gut microbiota accelerate tumor growth via c-jun and STAT3 phosphorylation in APCMin/+ mice Carcinogenesis 2012 33 1231 1238 22461519 24 Chung H Pamp SJ Hill JA Gut immune maturation depends on colonization with a host-specific microbiota Cell 2012 149 1578 1593 22726443 25 Vannucci L Stepankova R Kozakova H Fiserova A Rossmann P Tlaskalova-Hogenova H Colorectal carcinogenesis in germfree and conventionally reared rats: different intestinal environments affect the systemic immunity Int J Oncol 2008 32 609 617 18292938 26 Rhee KJ Sethupathi P Driks A Lanning DK Knight KL Role of commensal bacteria in development of gut-associated lymphoid tissues and preimmune antibody repertoire J Immunol 2004 172 1118 1124 14707086 27 Zackular JP Baxter NT Chen GY Schloss PD Manipulation of the gut microbiota reveals role in colon tumorigenesis mSphere 2015 1 e00001 e00015 10.1128/mSphere.00001-15 27303681 28 Couturier-Maillard A Secher T Rehman A NOD2-mediated dysbiosis predisposes mice to transmissible colitis and colorectal cancer J Clin Invest 2013 123 700 711 23281400 29 Hu B Elinav E Huber S Microbiota-induced activation of epithelial IL-6 signaling links inflammasome-driven inflammation with transmissible cancer Proc Natl Acad Sci U S A 2013 110 9862 9867 23696660 30 Donohoe DR Holley D Collins LB A gnotobiotic mouse model demonstrates that dietary fiber protects against colorectal tumorigenesis in a microbiota- and butyrate-dependent manner Cancer Discov 2014 4 1387 1397 25266735 31 Perrin P Pierre F Patry Y Only fibres promoting a stable butyrate producing colonic ecosystem decrease the rate of aberrant crypt foci in rats Gut 2001 48 53 61 11115823 32 Singh N Gurav A Sivaprakasam S Activation of Gpr109a, receptor for niacin and the commensal metabolite butyrate, suppresses colonic inflammation and carcinogenesis Immunity 2014 40 128 139 24412617 33 So SS Wan ML El-Nezami H Probiotics-mediated suppression of cancer Curr Opin Oncol 2017 29 62 72 27792053 34 Zhu Y Michelle Luo T Jobin C Young HA Gut microbiota and probiotics in colon tumorigenesis Cancer Lett 2011 309 119 127 21741763 35 Sobhani I Tap J Roudot-Thoraval F Microbial dysbiosis in colorectal cancer (CRC) patients PLoS One 2011 6 e16393 10.1371/journal.pone.0016393 21297998 36 Walker AW Sanderson JD Churcher C High-throughput clone library analysis of the mucosa-associated microbiota reveals dysbiosis and differences between inflamed and non-inflamed regions of the intestine in inflammatory bowel disease BMC Microbiol 2011 11 7 21219646 37 Hansen R Russell RK Reiff C Microbiota of de-novo pediatric IBD: increased Faecalibacterium prausnitzii and reduced bacterial diversity in Crohn's but not in ulcerative colitis Am J Gastroenterol 2012 107 1913 1922 23044767 38 Lepage P Häsler R Spehlmann ME Twin study indicates loss of interaction between microbiota and mucosa of patients with ulcerative colitis Gastroenterology 2011 141 227 236 21621540 39 Ahn J Sinha R Pei Z Human gut microbiome and risk for colorectal cancer J Natl Cancer Inst 2013 105 1907 1911 24316595 40 Huipeng W Lifeng G Chuang G Jiaying Z Yuankun C The differences in colonic mucosal microbiota between normal individual and colon cancer patients by polymerase chain reaction-denaturing gradient gel electrophoresis J Clin Gastroenterol 2014 48 138 144 24162169 41 Shen XJ Rawls JF Randall T Molecular characterization of mucosal adherent bacteria and associations with colorectal adenomas Gut Microbes 2010 1 138 147 20740058 42 Sanapareddy N Legge RM Jovov B Increased rectal microbial richness is associated with the presence of colorectal adenomas in humans ISME J 2012 6 1858 1868 22622349 43 Wu N Yang X Zhang R Dysbiosis signature of fecal microbiota in colorectal cancer patients Microb Ecol 2013 66 462 470 23733170 44 Weir TL Manter DK Sheflin AM Barnett BA Heuberger AL Ryan EP Stool microbiome and metabolome differences between colorectal cancer patients and healthy adults PLoS One 2013 8 e70803 10.1371/journal.pone.0070803 23940645 45 Wang T Cai G Qiu Y Structural segregation of gut microbiota between colorectal cancer patients and healthy volunteers ISME J 2012 6 320 329 21850056 46 Kasai C Sugimoto K Moritani I Comparison of human gut microbiota in control subjects and patients with colorectal carcinoma in adenoma: terminal restriction fragment length polymorphism and next-generation sequencing analyses Oncol Rep 2016 35 325 333 26549775 47 Flemer B Lynch DB Brown JM Tumour-associated and non-tumour-associated microbiota in colorectal cancer Gut 2017 66 633 643 26992426 48 Gueimonde M Ouwehand A Huhtinen H Salminen E Salminen S Qualitative and quantitative analyses of the Bifidobacterial microbiota in the colonic mucosa of patients with colorectal cancer, diverticulitis and inflammatory bowel disease World J Gastroenterol 2007 13 3985 3989 17663515 49 Gao Z Guo B Gao R Zhu Q Qin H Microbiota disbiosis is associated with colorectal cancer Front Microbiol 2015 6 20 25699023 50 Burns MB Lynch J Starr TK Knights D Blekhman R Virulence genes are a signature of the microbiome in the colorectal tumor microenvironment Genome Med 2015 7 55 26170900 51 Gao R Kong C Huang L Mucosa-associated microbiota signature in colorectal cancer Eur J Clin Microbiol Infect Dis 2017 36 2073 2083 28600626 52 Watt E Gemmell MR Berry S Extending colonic mucosal microbiome analysis-assessment of colonic lavage as a proxy for endoscopic colonic biopsies Microbiome 2016 4 61 27884202 53 Yu LC Commensal bacterial internalization by epithelial cells: an alternative portal for gut leakiness Tissue Barriers 2015 3 e1008895 10.1080/21688370.2015.1008895 26451337 54 Wu LL Peng WH Kuo WT Commensal bacterial endocytosis in epithelial cells is dependent on myosin light chain kinase-activated brush border fanning by interferon-gamma Am J Pathol 2014 184 2260 2274 24911373 55 Johansson ME Sjövall H Hansson GC The gastrointestinal mucus system in health and disease Nat Rev Gastroenterol Hepatol 2013 10 352 361 23478383 56 Johansson ME Gustafsson JK Holmén-Larsson J Bacteria penetrate the normally impenetrable inner colon mucus layer in both murine colitis models and patients with ulcerative colitis Gut 2014 63 281 291 23426893 57 Kline KA Fälker S Dahlberg S Normark S Henriques-Normark B Bacterial adhesins in host-microbe interactions Cell Host Microbe 2009 5 580 592 19527885 58 Bonnet M Buc E Sauvanet P Colonization of the human gut by E. coli and colorectal cancer risk Clin Cancer Res 2014 20 859 867 24334760 59 Prorok-Hamon M Friswell MK Alswied A Colonic mucosa-associated diffusely adherent afaC+ Escherichia coli expressing lpfA and pks are increased in inflammatory bowel disease and colon cancer Gut 2014 63 761 770 23846483 60 Chassaing B Rolhion N de Vallée A Crohn disease: associated adherent-invasive E. coli bacteria target mouse and human Peyer's patches via long polar fimbriae J Clin Invest 2011 121 966 975 21339647 61 Martin HM Campbell BJ Hart CA Enhanced Escherichia coli adherence and invasion in Crohn's disease and colon cancer Gastroenterology 2004 127 80 93 15236175 62 Kleessen B Kroesen AJ Buhr HJ Blaut M Mucosal and invading bacteria in patients with inflammatory bowel disease compared with controls Scand J Gastroenterol 2002 37 1034 1041 12374228 63 Strauss J Kaplan GG Beck PL Invasive potential of gut mucosa-derived Fusobacterium nucleatum positively correlates with IBD status of the host Inflamm Bowel Dis 2011 17 1971 1978 21830275 64 Swidsinski A Dörffel Y Loening-Baucke V Mucosal invasion by fusobacteria is a common feature of acute appendicitis in Germany, Russia, and China Saudi J Gastroenterol 2012 18 55 58 22249094 65 Dharmani P Strauss J Ambrose C Allen-Vercoe E Chadee K Fusobacterium nucleatum infection of colonic cells stimulates MUC2 mucin and tumor necrosis factor alpha Infect Immun 2011 79 2597 2607 21536792 66 Viljoen KS Dakshinamurthy A Goldberg P Blackburn JM Quantitative profiling of colorectal cancer-associated bacteria reveals associations between fusobacterium spp., enterotoxigenic Bacteroides fragilis (ETBF) and clinicopathological features of colorectal cancer PLoS One 2015 10 e0119462 10.1371/journal.pone.0119462 25751261 67 Boleij A Hechenbleikner EM Goodwin AC The Bacteroides fragilis toxin gene is prevalent in the colon mucosa of colorectal cancer patients Clin Infect Dis 2015 60 208 215 25305284 68 Arthur JC Perez-Chanona E Mühlbauer M Intestinal inflammation targets cancer-inducing activity of the microbiota Science 2012 338 120 123 22903521 69 Arthur JC Gharaibeh RZ Mühlbauer M Microbial genomic analysis reveals the essential role of inflammation in bacteria-induced colorectal cancer Nat Commun 2014 5 4724 25182170 70 Tomkovich S Yang Y Winglee K Locoregional effects of microbiota in a preclinical model of colon carcinogenesis Cancer Res 2017 77 2620 2632 28416491 71 Cougnoux A Dalmasso G Martinez R Bacterial genotoxin colibactin promotes colon tumour growth by inducing a senescence-associated secretory phenotype Gut 2014 63 1932 1942 24658599 72 Dalmasso G Cougnoux A Delmas J Darfeuille-Michaud A Bonnet R The bacterial genotoxin colibactin promotes colon tumor growth by modifying the tumor microenvironment Gut Microbes 2014 5 675 680 25483338 73 Raisch J Buc E Bonnet M Colon cancer-associated B2 Escherichia coli colonize gut mucosa and promote cell proliferation World J Gastroenterol 2014 20 6560 6572 24914378 74 Raisch J Rolhion N Dubois A Darfeuille-Michaud A Bringer MA Intracellular colon cancer-associated Escherichia coli promote protumoral activities of human macrophages by inducing sustained COX-2 expression Lab Invest 2015 95 296 307 25545478 75 Yang Y Weng W Peng J Fusobacterium nucleatum increases proliferation of colorectal cancer cells and tumor development in mice by activating toll-like receptor 4 signaling to nuclear factor-kappaB, and up-regulating expression of microRNA-21 Gastroenterology 2017 152 851 866.e24 27876571 76 Kostic AD Chun E Robertson L Fusobacterium nucleatum potentiates intestinal tumorigenesis and modulates the tumor-immune microenvironment Cell Host Microbe 2013 14 207 215 23954159 77 Wu S Rhee KJ Albesiano E A human colonic commensal promotes colon tumorigenesis via activation of T helper type 17 T cell responses Nat Med 2009 15 1016 1022 19701202 78 Goodwin AC Destefano Shields CE Wu S Polyamine catabolism contributes to enterotoxigenic Bacteroides fragilis-induced colon tumorigenesis Proc Natl Acad Sci U S A 2011 108 15354 15359 21876161 79 Thiele Orberg E Fan H Tam AJ The myeloid immune signature of enterotoxigenic Bacteroides fragilis-induced murine colon tumorigenesis Mucosal Immunol 2017 10 421 433 27301879 80 Darfeuille-Michaud A Neut C Barnich N Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn's disease Gastroenterology 1998 115 1405 1413 9834268 81 Darfeuille-Michaud A Boudeau J Bulois P High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn's disease Gastroenterology 2004 127 412 421 15300573 82 Mimouna S Gonçalvès D Barnich N Darfeuille-Michaud A Hofman P Vouret-Craviari V Crohn disease-associated Escherichia coli promote gastrointestinal inflammatory disorders by activation of HIF-dependent responses Gut Microbes 2011 2 335 346 22157238 83 Martinez-Medina M Aldeguer X Lopez-Siles M Molecular diversity of Escherichia coli in the human gut: new ecological evidence supporting the role of adherent-invasive E. coli (AIEC) in Crohn's disease Inflamm Bowel Dis 2009 15 872 882 19235912 84 Baumgart M Dogan B Rishniw M Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn's disease involving the ileum ISME J 2007 1 403 418 18043660 85 Carvalho FA Barnich N Sivignon A Crohn's disease adherent-invasive Escherichia coli colonize and induce strong gut inflammation in transgenic mice expressing human CEACAM J Exp Med 2009 206 2179 2189 19737864 86 Thanassi DG Bliska JB Christie PJ Surface organelles assembled by secretion systems of Gram-negative bacteria: diversity in structure and function FEMS Microbiol Rev 2012 36 1046 1082 22545799 87 Lillington J Geibel S Waksman G Biogenesis and adhesion of type 1 and P pili Biochim Biophys Acta 2014 1840 2783 2793 24797039 88 Chan CH Cook D Stanners CP Increased colon tumor susceptibility in azoxymethane treated CEABAC transgenic mice Carcinogenesis 2006 27 1909 1916 16632476 89 Buc E Dubois D Sauvanet P High prevalence of mucosaassociated E. coli producing cyclomodulin and genotoxin in colon cancer PLoS One 2013 8 e56964 10.1371/journal.pone.0056964 23457644 90 Kostic AD Gevers D Pedamallu CS Genomic analysis identifies association of Fusobacterium with colorectal carcinoma Genome Res 2012 22 292 298 22009990 91 Yu J Feng Q Wong SH Metagenomic analysis of faecal microbiome as a tool towards targeted non-invasive biomarkers for colorectal cancer Gut 2017 66 70 78 26408641 92 Purcell RV Pearson J Aitchison A Dixon L Frizelle FA Keenan JI Colonization with enterotoxigenic Bacteroides fragilis is associated with early-stage colorectal neoplasia PLoS One 2017 12 e0171602 10.1371/journal.pone.0171602 28151975 93 Fredricks DN Schubert MM Myerson D Molecular identification of an invasive gingival bacterial community Clin Infect Dis 2005 41 e1 e4 10.1086/430824 15937752 94 Matsuo T Shirakami T Ozaki K Nakanishi T Yumoto H Ebisu S An immunohistological study of the localization of bacteria invading root pulpal walls of teeth with periapical lesions J Endod 2003 29 194 200 12669880 95 Chen Y Peng Y Yu J Invasive Fusobacterium nucleatum activates beta-catenin signaling in colorectal cancer via a TLR4/P-PAK1 cascade Oncotarget 2017 8 31802 31814 28423670 96 Rubinstein MR Wang X Liu W Hao Y Cai G Han YW Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating E-cadherin/beta-catenin signaling via its FadA adhesin Cell Host Microbe 2013 14 195 206 23954158 97 Fardini Y Wang X Témoin S Fusobacterium nucleatum adhesin FadA binds vascular endothelial cadherin and alters endothelial integrity Mol Microbiol 2011 82 1468 1480 22040113 98 Sears CL Islam S Saha A Association of enterotoxigenic Bacteroides fragilis infection with inflammatory diarrhea Clin Infect Dis 2008 47 797 803 18680416 99 Toprak NU Yagci A Gulluoglu BM A possible role of Bacteroides fragilis enterotoxin in the aetiology of colorectal cancer Clin Microbiol Infect 2006 12 782 786 16842574 100 Rocha ER Smith CJ Ferritin-like family proteins in the anaerobe Bacteroides fragilis: when an oxygen storm is coming, take your iron to the shelter Biometals 2013 26 577 591 23842847 101 Betteken MI Rocha ER Smith CJ Dps and DpsL mediate survival in vitro and in vivo during the prolonged oxidative stress response in Bacteroides fragilis J Bacteriol 2015 197 3329 3338 26260459 102 Park Y Choi JY Yong D Lee K Kim JM Clinical features and prognostic factors of anaerobic infections: a 7-year retrospective study Korean J Intern Med 2009 24 13 18 19270476 103 Rhee KJ Wu S Wu X Induction of persistent colitis by a human commensal, enterotoxigenic Bacteroides fragilis, in wild-type C57BL/6 mice Infect Immun 2009 77 1708 1718 19188353 104 Wick EC Rabizadeh S Albesiano E Stat3 activation in murine colitis induced by enterotoxigenic Bacteroides fragilis Inflamm Bowel Dis 2014 20 821 834 24704822 105 Kuo WT Lee TC Yang HY LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis Cell Death Differ 2015 22 1590 1604 25633197 106 Kuo WT Lee TC Yu LC Eritoran suppresses colon cancer by altering a functional balance in Toll-like receptors that bind lipopolysaccharide Cancer Res 2016 76 4684 4695 27328732 107 Kuo WT Lee TC Yu LC Janus-faced bacterial regulation of epithelial cell death and survival: association with colon carcinogenesis Mol Cell Oncol 2015 3 e1029064 10.1080/23723556.2015.1029064 27308544 108 Fukata M Abreu MT TLR4 signalling in the intestine in health and disease Biochem Soc Trans 2007 35 1473 1478 18031248 109 Yu LC Wei SC Ni YH Interplay between the gut microbiota and epithelial innate signaling in colitis-associated colon carcinogenesis Cancer Res Front 2017 3 1 28 110 Chen TL Chen S Wu HW Persistent gut barrier damage and commensal bacterial influx following eradication of Giardia infection in mice Gut Pathog 2013 5 26 23991642 111 Kalischuk LD Leggett F Inglis GD Campylobacter jejuni induces transcytosis of commensal bacteria across the intestinal epithelium through M-like cells Gut Pathog 2010 2 14 21040540 112 Kalischuk LD Inglis GD Buret AG Campylobacter jejuni induces transcellular translocation of commensal bacteria via lipid rafts Gut Pathog 2009 1 2 19338680 113 Denizot J Sivignon A Barreau F Adherent-invasive Escherichia coli induce claudin-2 expression and barrier defect in CEABAC10 mice and Crohn's disease patients Inflamm Bowel Dis 2012 18 294 304 21688348 114 Lapointe TK O'Connor PM Jones NL Menard D Buret AG Interleukin-1 receptor phosphorylation activates Rho kinase to disrupt human gastric tight junctional claudin-4 during Helicobacter pylori infection Cell Microbiol 2010 12 692 703 20070312 115 Fedwick JP Lapointe TK Meddings JB Sherman PM Buret AG Helicobacter pylori activates myosin light-chain kinase to disrupt claudin-4 and claudin-5 and increase epithelial permeability Infect Immun 2005 73 7844 7852 16299274 116 Wei SC Yang-Yen HF Tsao PN SHANK3 regulates intestinal barrier function through modulating ZO-1 expression through the PKCε-dependent pathway Inflamm Bowel Dis 2017 23 1730 1740 28906292 117 Salzman AL Eaves-Pyles T Linn SC Denenberg AG Szabó C Bacterial induction of inducible nitric oxide synthase in cultured human intestinal epithelial cells Gastroenterology 1998 114 93 102 9428223 118 Negroni A Colantoni E Vitali R NOD2 induces autophagy to control AIEC bacteria infectiveness in intestinal epithelial cells Inflamm Res 2016 65 803 813 27335178 119 Huang FC De Novo sphingolipid synthesis is essential for Salmonella-induced autophagy and human beta-defensin 2 expression in intestinal epithelial cells Gut Pathog 2016 8 5 26893616 120 Lu C Chen J Xu HG MIR106B and MIR93 prevent removal of bacteria from epithelial cells by disrupting ATG16L1-mediated autophagy Gastroenterology 2014 146 188 199 24036151