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Background and aims Colonic bacterial biofilms are frequently present in ulcerative colitis (UC) and may increase dysplasia risk through pathogens expressing oncotraits. This prospective cohort study aimed to determine (1) the association of oncotraits and longitudinal biofilm presence with dysplasia risk in UC, and (2) the relation of bacterial composition with biofilms and dysplasia risk. Methods Feces and left- and right-sided colonic biopsies were collected from 80 UC patients and 35 controls. Oncotraits (FadA of Fusobacterium, BFT of Bacteroides fragilis, colibactin (ClbB) and Intimin (Eae) of Escherichia coli) were assessed in fecal DNA with multiplex qPCR. Biopsies were screened for biofilms (n=873) with 16S rRNA fluorescent in situ hybridization. Shotgun metagenomic sequencing (n=265), and ki67-immunohistochemistry were performed. Associations were determined with a mixed-effects regression model. Results Biofilms were highly prevalent in UC patients (90.8%) with a median persistence of 3 years (IQR 2-5 years). Biofilm-positive biopsies showed increased epithelial hypertrophy (p=0.025), a reduced Shannon diversity independent of disease status (p=0.015), however, were not significantly associated with dysplasia in UC (aOR 1.45(95%CI0.63-3.40). In contrast, ClbB independently associated with dysplasia (aOR 7.16 (95%CI1.75-29.28), while FadA and Fusobacteriales were associated with a decreased dysplasia risk in UC (aOR 0.23 (95%CI0.06-0.83), and p Conclusions Biofilms are a hallmark of UC, however, because of their high prevalence a poor biomarker for dysplasia. In contrast, colibactin presence and FadA absence independently associate with dysplasia in UC and might therefore be valuable biomarkers for future risk stratification and intervention strategies.

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Biofilms are polymeric matrices containing bacteria that can express oncotraits and are frequently present in ulcerative colitis (UC). Oncotraits can impact colon epithelial cells directly and may increase dysplasia risk. This study aimed to determine (1) the association of oncotraits and longitudinal biofilm presence with dysplasia risk in UC, and (2) the relation of bacterial composition with biofilms and dysplasia risk.In this prospective cohort study, feces and left- and right-sided colonic biopsies were collected from 80 UC patients and 35 controls. Oncotraits (FadA of Fusobacterium, BFT of Bacteroides fragilis, Colibactin (ClbB) and Intimin (Eae) of Escherichia coli) in fecal DNA were assessed with multiplex qPCR. Biopsies were analyzed for biofilms (n=873) with 16S rRNA fluorescent in situ hybridization and shotgun metagenomic sequencing (n=265), and ki67-immunohistochemistry for cell proliferation. Associations were determined with a regression (mixed) model.ClbB significantly associated with dysplasia in UC (aOR 7.16, (95%CI 1.75-29.28, pFusobacteriales was significantly correlated with a decreased dysplasia risk only in right-sided colonic biopsies (pColibactin and FadA impact dysplasia risk in UC, in contrast to biofilms. These oncotraits are valuable targets for future risk classification and intervention studies.What is already known on this topicBacterial biofilms sometimes contain bacteria with oncogenic traits (oncotraits) and have been associated with colon carcinogenesis in mice and humans. It is yet unknown whether biofilms and oncotraits are involved in early carcinogenesis and could be used as a risk factor for dysplasia in ulcerative colitis patients.What this study addBacterial biofilms associated with lower bacterial diversity and epithelial cell hypertrophy, but did not predict dysplasia. Moreover, in agreement to piling evidence suggesting a role of colibactin in human colorectal cancer, we provide the missing clinical evidence that this oncotrait actually associates with risk for (early) carcinogenesis in human patients. Additionally, dysplasia in UC patients was predicted by absence of Fusobacterium adhesin.How this study might affect research, practice or policyThis prospective cohort study indicates a putative role of bacterial oncotraits in early carcinogenesis, suggesting them as promising targets for future risk classification and intervention studies in ulcerative colitis patients.Lay summaryPatients with ulcerative colitis have an increased risk for colorectal cancer. This study found that bacterial factors in fecal material can predict the development of cancer precursors in these patients.Abstract Figure

G protein-coupled receptor (GPR)35 is highly expressed in the gastro-intestinal tract, predominantly in colon epithelial cells (CEC), and has been associated with inflammatory bowel diseases (IBD), suggesting a role in gastrointestinal inflammation. The enterotoxigenic Bacteroides fragilis (ETBF) toxin (BFT) is an important virulence factor causing gut inflammation in humans and animal models. We identified that BFT signals through GPR35. Blocking GPR35 function in CECs using the GPR35 antagonist ML145, in conjunction with shRNA knock-down and CRISPRcas-mediated knock-out, resulted in reduced CEC-response to BFT as measured by E-cadherin cleavage, beta-arrestin recruitment and IL-8 secretion. Importantly, GPR35 is required for the rapid onset of ETBF-induced colitis in mouse models. GPR35-deficient mice showed reduced death and disease severity compared to wild-type C57Bl6 mice. Our data support a role for GPR35 in the CEC and mucosal response to BFT and underscore the importance of this molecule for sensing ETBF in the colon., Boleij et al. show that G protein-coupled receptor 35 (GPR35) regulates the responses to enterotoxigenic Bacteroides fragilis (ETBF) in colonic epithelial cells. They find that GPR35-deficiency nearly protected mice from ETBF-induced death, suggesting the importance of GPR35 in sensing ETBF in the colon.

Bsacground: Lynch Syndrome (LS) is an inherited cancer syndrome associated with an increased lifetime risk of colorectal cancer (CRC) and characterised by germline mutations in DNA mismatch repair (MMR) genes. Abnormalities in the function of MMR genes lead to errors during DNA replication, including microsatellite instability. Despite regular colonoscopies, up to 15% of LS patients still develop CRC. Gut microbiome, bowel inflammation, the environment and host genetic and epigenetic factors are involved in CRC development. In this sense, the concomitant analysis of stool host microRNA (miRNA) profiles and gut microbiome composition may arise to the identification of specific fecal markers in LS explaining the molecular basis for the development of cancer in these patients. PURPOSE: In the present study, the fecal miRNome and microbiome were characterized in a time progression in an in vivo model using a mismatch repair deficient mice model that recapitulates human LS. The main aim was to evaluate the changes and crosstalk of miRNAs released in the stool by the host and the microbial population residing in the gut during the carcinogenesis process induced in animals. Findings in mice were compared and correlated to data obtained from a cohort of human subjects affected by LS. Methods: We used an in vivo model for LS based on a conditional knockout mouse with the tissue-specific inactivation of Msh2 (Msh2LoxP) in the intestinal mucosa, combining Msh2LoxP allele with the Villin-Cre transgene (VCMsh2LoxP). Stool from mice were collected at different time points (6, 9 and 12 months of age). Small RNA-sequencing (sRNA-seq) and shotgun metagenomics analyses were performed in stool from all samples. In concomitance, we collected 78 stool samples of clinically diagnosed LS subjects, at different follow up time. At the sampling, 36 resulted negative for colorectal adenomas/lesions, 12 were negative but with a previous history of lesions, 23 presented a neoplasia and 6 developed a lesion in the follow-up. Twenty-two stool samples from healthy subjects were used as controls. Results: In mice, preliminary results from sRNA-seq and metagenomics analyses showed several dysregulated miRNAs and differential microbial relative abundances, either between VCMsh2loxP and controls or at different time points. In humans, we identified 41 and 17 miRNAs respectively up- and downregulated in LS subjects who presented a lesion compared to those not having any lesion at follow-up. In the same comparison the metagenomic data showed significantly different microbial abundances, including those of Eubacterium ramulus. Conclusions: This is the first study characterizing the concomitant alterations in microbial composition and host miRNome overtime in relation to the onset of cancerous/precancerous lesions due to LS. Citation Format: Giulia Francescato, Giulio Ferrero, Marc Beltrà, Sonia Tarallo, Giulia Piaggeschi, Carla Di Battista, Antonio Francavilla, Carlijn Bruggeling, Fabio Penna, Barbara Pardini, Paola Costelli, Annemarie Boleij, Alessio Naccarati. Fecal miRNA profiles and gut metagenome composition in Lynch syndrome: results from a mouse model study and human subjects. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3797.

Introduction: Postoperative complications impact morbidity and mortality of colon cancer patients. Emerging evidence suggests that intestinal microbiota might play a critical role in the development of postoperative complications after gastrointestinal surgery. Here, we studied changes in fecal microbiota composition comparing samples collected before and after colon cancer surgery. Moreover, we examined whether the microbiota composition before surgery was associated with postoperative complications, and we explored potential determinants of the preoperative microbiota in colon cancer patients. Methods: For this study, two fecal samples were provided by 78 patients with colon cancer. The first (preoperative) sample was collected shortly after diagnosis and before (median and interquartile (IQR) range of 4 (2-6) days) colon cancer surgery. The second (postoperative) sample was collected approximately 6 weeks (median and IQR of 42 (37-47) days) after surgery and before start of adjuvant chemotherapy (whenever applicable). Based on 16S ribosomal RNA gene amplicon sequencing, the fecal microbiota diversity and composition were determined. Preoperative microbiota composition of patients who developed postoperative complications in the 30 days following surgery (n=18, 23%) was compared to those who did not (n=60, 77%) using univariate and multivariate analyses. Explorative random forest analyses were conducted to identify predictors of complication status, with relative abundance of the core genera, sex, age, fecal calprotectin levels, dietary fiber intake, body mass index, smoking status, ASA classification, cancer stage and tumor location being considered as potential predictors. Results: After surgery, microbial alpha diversity was reduced compared to the preoperative situation, whereas compositional changes over time only reached statistical significance for Lachnospiraceae NK4A136 and Coprococcus 1, which both decreased in relative abundance after surgery. Already before the start of surgery, relative abundance of the genera Bacteroides (10% vs 6%) and Lachnoclostridium (0.6% vs 0.2%) appeared to be higher in patients with complications versus those with uncomplicated recovery. In a prediction analysis, current smoking and relative abundance of Lachnoclostridium were identified as most profound predictors of complication status. Fecal calprotectin levels and current smoking status explained most (together 6%) of the overall variation in microbiota composition before surgery. Conclusion: In this study, higher relative abundances of the genera Bacteroides and Lachnoclostridium before surgery were associated with postoperative complications in patients with colon cancer. Future studies should expand on the potential causal and pathogenic routes underlying these observations. Citation Format: Dieuwertje E. Kok, Jannigje G. Kers, Hendriek C. Boshuizen, Niels Klaassen, Henk K. van Halteren, Flip M. Kruyt, Marjolein L. Smidt, Johannes H. De Wilt, Annemarie Boleij, Ellen Kampman, Erwin G. Zoetendal. Pre- and postoperative fecal microbiota and its association with complications after surgery in colon cancer patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4380.

ObjectiveThe opportunistic pathogen Streptococcus gallolyticus is one of the few intestinal bacteria that has been consistently linked to colorectal cancer (CRC). This study aimed to identify novel S. gallolyticus-induced pathways in colon epithelial cells that could further explain how S. gallolyticus contributes to CRC development.Design and ResultsTranscription profiling of in vitro cultured CRC cells that were exposed to S. gallolyticus revealed the specific induction of oxidoreductase pathways. Most prominently, CYP1A and ALDH1 genes that encode phase I biotransformation enzymes were responsible for the detoxification or bio-activation of toxic compounds. A common feature is that these enzymes are induced through the Aryl hydrocarbon receptor (AhR). Using the specific inhibitor CH223191, we showed that the induction of CYP1A was dependent on the AhR both in vitro using multiple CRC cell lines as in vivo using wild-type C57bl6 mice colonized with S. gallolyticus. Furthermore, we showed that CYP1 could also be induced by other intestinal bacteria and that a yet unidentified diffusible factor from the S. galloltyicus secretome (SGS) induces CYP1A enzyme activity in an AhR-dependent manner. Importantly, priming CRC cells with SGS increased the DNA damaging effect of the polycyclic aromatic hydrocarbon 3-methylcholanthrene.ConclusionThis study shows that gut bacteria have the potential to modulate the expression of biotransformation pathways in colonic epithelial cells in an AhR-dependent manner. This offers a novel theory on the contribution of intestinal bacteria to the etiology of CRC by modifying the capacity of intestinal epithelial or (pre-)cancerous cells to (de)toxify dietary components, which could alter intestinal susceptibility to DNA damaging events.

Many studies provided compelling evidence that extracellular vesicles (EVs) are involved in the regulation of the immune response, acting as both enhancers and dampeners of the immune system, depending on the source and type of vesicle. Research, including ours, has shown anti-inflammatory effects of milk-derived EVs, using human breast milk as well as bovine colostrum and store-bought pasteurized cow milk, in in vitro systems as well as therapeutically in animal models. Although it is not completely elucidated which proteins and miRNAs within the milk-derived EVs contribute to these immunosuppressive capacities, one proposed mechanism of action of the EVs is via the modulation of the crosstalk between the (intestinal) microbiome and their host health. There is increasing awareness that the gut plays an important role in many inflammatory diseases. Enhanced intestinal leakiness, dysbiosis of the gut microbiome, and bowel inflammation are not only associated with intestinal diseases like colitis and Crohn’s disease, but also characteristic for systemic inflammatory diseases such as lupus, multiple sclerosis, and rheumatoid arthritis (RA). Strategies to target the gut, and especially its microbiome, are under investigation and hold a promise as a therapeutic intervention for these diseases. The use of milk-derived EVs, either as stand-alone drug or as a drug carrier, is often suggested in recent years. Several research groups have studied the tolerance and safety of using milk-derived EVs in animal models. Due to its composition, milk-derived EVs are highly biocompatible and have limited immunogenicity even cross species. Furthermore, it has been demonstrated that milk-derived EVs, when taken up in the gastro-intestinal tract, stay intact after absorption, indicating excellent stability. These characteristics make milk-derived EVs very suitable as drug carriers, but also by themselves, these EVs already have a substantial immunoregulatory function, and even without loading, these vesicles can act as therapeutics. In this review, we will address the immunomodulating capacity of milk-derived EVs and discuss their potential as therapy for RA patients.Review criteriaThe search terms “extracellular vesicles”, “exosomes”, “microvesicles”, “rheumatoid arthritis”, “gut-joint axis”, “milk”, and “experimental arthritis” were used. English-language full text papers (published between 1980 and 2021) were identified from PubMed and Google Scholar databases. The reference list for each paper was further searched to identify additional relevant articles.

Contains fulltext : 235760.pdf (Publisher’s version ) (Closed access) Breast cancer tissue contains its own unique microbiota. Emerging preclinical data indicates that breast microbiota dysbiosis contributes to breast cancer initiation and progression. Furthermore, the breast microbiota may be a promising biomarker for treatment selection and prognosis. Differences in breast microbiota composition have been found between breast cancer subtypes and disease severities that may contribute to immunosuppression, enabling tumor cells to evade immune destruction. Interactions between breast microbiota, gut microbiota, and immune system are proposed, all forming potential targets to increase therapeutic efficacy. In addition, because the gut microbiota affects the host immune system and systemic availability of estrogen and bile acids known to influence tumor biology, gut microbiota modulation could be used to manipulate breast microbiota composition. Identifying breast and gut microbial compositions that respond positively to certain anticancer therapeutics could significantly reduce cancer burden. Additional research is needed to unravel the complexity of breast microbiota functioning and its interactions with the gut and the immune system. In this review, developments in the understanding of breast microbiota and its interaction with the immune system and the gut microbiota are discussed. Furthermore, the biomarker potential of breast microbiota is evaluated in conjunction with possible strategies to target microbiota in order to improve breast cancer treatment.

Recent advances in microbiome sequencing have rendered new insights into the role of the microbiome in human health with potential clinical implications. Unfortunately, the presence of host DNA in tissue isolates has hampered the analysis of host‐associated bacteria. Here, we present a DNA isolation protocol for tissue, optimized on biopsies from resected human colons (~2–5 mm in size), which includes reduction of human DNA without distortion of relative bacterial abundance at the phylum level. We evaluated which concentrations of Triton and saponin lyse human cells and leave bacterial cells intact, in combination with DNAse treatment to deplete released human DNA. Saponin at a concentration of 0.0125% in PBS lysed host cells, resulting in a 4.5‐fold enrichment of bacterial DNA while preserving the relative abundance of Firmicutes, Bacteroidetes, γ‐Proteobacteria, and Actinobacteria assessed by qPCR. Our optimized protocol was validated in the setting of two large clinical studies on 521 in vivo acquired colon biopsies of 226 patients using shotgun metagenomics. The resulting bacterial profiles exhibited alpha and beta diversities that are similar to the diversities found by 16S rRNA amplicon sequencing. A direct comparison between shotgun metagenomics and 16S rRNA amplicon sequencing of 15 forceps tissue biopsies showed similar bacterial profiles and a similar Shannon diversity index between the sequencing methods. Hereby, we present the first protocol for enriching bacterial DNA from tissue biopsies that allows efficient isolation of all bacteria. Our protocol facilitates analysis of a wide spectrum of bacteria of clinical tissue samples improving their applicability for microbiome research., We present a bacterial DNA isolation method for human colon biopsies which includes reduction of human DNA and does not distort relative bacterial abundance at the phylum level. Shotgun sequencing resulted in 508/521 metagenomes (Shannon index; 2.9, UniFrac distance; 0.56). Fifteen colon biopsies were sequenced by both amplicon and shotgun sequencing resulting in similar bacterial profiles from phylum to genus level. Our protocol facilitates analysis of a wide spectrum of bacteria of clinical tissue samples improving their applicability for microbiome research.

Microbial pan-genomes are shaped by a complex combination of stochastic and deterministic forces. Even closely related genomes often exhibit extensive variation in their gene content. Understanding what drives this variation requires exploring the interactions of genes with each other and with their external environments. However, to date, conceptual models of pan-genome dynamics often represent genes as independent units and provide limited information about their mechanistic interactions. Here, we use pan-reactomes as proxies for pan-genomes since they can explicitly represent the interactions between the genes that code for metabolic reactions and simulate complex phenotypes that interact with the metabolic environment. We interpreted pan-reactomes as dynamic pools of metabolic reactions that are potentially gained or lost and simulated the routes along which different lineages lose reactions in alternative environments. We performed these simulations on the pan-reactomes of 46 bacterial and archaeal families covering a broad taxonomic range. These simulations allowed us to disentangle metabolic reactions whose presence does, and does not depend on the metabolite composition of the external environment, allowing us to identify reactions constrained “by nutrition” and “by nature”, respectively. By comparing the frequency of reactions from the first group with their observed frequencies in bacterial and archaeal families, we predicted the metabolic niches that shaped the genomic composition of these lineages in their evolutionary past. Moreover, we found that the lineages that were shaped by a more diverse metabolic niche also occur in more diverse biomes as assessed by global environmental sequencing datasets. Together, we introduce a computational framework for analyzing and interpreting pan-reactomes that provides new insights into the ecological and evolutionary drivers of pan-genome composition.

Recent advances in microbiome sequencing have rendered new insights into the role of the microbiome in human health with potential clinical implications. Unfortunately, developments in the field of tissue microbiomes have been hampered by the presence of host DNA in isolates which interferes with the analysis of the bacterial content. Here, we present a DNA isolation protocol from tissue samples including reduction of host DNA without distortion of microbial abundance profiles. We evaluated which concentrations of Triton and saponin lyse host cells and leave bacterial cells intact, which was combined with DNAse treatment to deplete released host DNA. We applied our protocol to extract microbial DNA from ex vivo and in vivo acquired human colon biopsies (∼2-5 mm in size) and assessed the relative abundance of bacterial and human DNA by qPCR. Saponin at a concentration of 0.0125% in PBS lysed host cells, resulting in a 4.5-fold enrichment of bacterial DNA while preserving the relative abundance of Firmicutes, Bacteroidetes, γ-Proteobacteria and Actinobacteria. Our protocol combined with shotgun metagenomic sequencing revealed a colon tissue microbiome profile with a Shannon diversity index of 3.2 and an UniFrac distance of 0.54, which is comparable to reported numbers based on amplicon sequencing. Hereby, we present the first protocol for enriching bacterial DNA from tissue biopsies that allows efficient isolation of rigid Gram-positive bacteria without depleting the more sensitive Gram-negative bacteria. Our protocol facilitates analysis of a wide spectrum of bacteria of clinical tissue samples improving their applicability for microbiome research.

Bacteroides fragilis has previously been linked to Crohn’s disease (CD) exacerbations, but results are inconsistent and underlying mechanisms unknown. This study investigates the epidemiology of B. fragilis and its virulence factors bft (enterotoxin) and ubiquitin among 181 CD patients and the impact on the intestinal epithelial barrier in vitro. The prevalence of B. fragilis was significantly higher in active (n = 69/88, 78.4%) as compared to remissive (n = 58/93, 62.4%, p = 0.018) CD patients. Moreover, B. fragilis was associated with intestinal strictures. Interestingly, the intestinal barrier function, as examined by transepithelial electrical resistance (TEER) measurements of Caco-2 monolayers, increased when exposed to secretomes of bft-positive (bft-1 and bft-2 isotype; increased TEER ∼160%, p < 0.001) but not when exposed to bft-negative strains. Whole metagenome sequencing and metabolomics, respectively, identified nine coding sequences and two metabolites that discriminated TEER-increasing from non-TEER-increasing strains. This study revealed a higher B. fragilis prevalence during exacerbation. Surprisingly, bft-positive secretomes increased epithelial resistance, but we excluded Bft as the likely causative factor.

Biofilms provide refuge for cancerous bacteria Familial adenomatous polyposis (FAP) causes benign polyps along the colon. If left untreated, FAP leads to a high incidence of colon cancer. To understand how polyps influence tumor formation, Dejea et al. examined the colonic mucosa of FAP patients. They discovered biofilms containing the carcinogenic versions of the bacterial species Escherichia coli and Bacteroides fragilis . Colon tissue from FAP patients exhibited greater expression of two bacterial genes that produce secreted oncotoxins. Studies in mice showed that specific bacteria could work together to induce colon inflammation and tumor formation. Science , this issue p. 592

Streptococcus gallolyticus subspecies gallolyticus (SGG) is potentially associated with colorectal cancer (CRC) and its precursors. A previous case-control study measured antibody responses to SGG pilus proteins Gallo2178 and Gallo2179 and identified significant associations with a small fraction of CRC cases. We aimed at replicating and expanding these findings in an independent study including additional SGG antigens and explored the association with precancerous lesions. We applied multiplex serology to measure antibodies to eleven SGG proteins in serum samples of a screening colonoscopy trial (BliTz study) including participants diagnosed with either non-advanced adenoma (NAA, n = 30), advanced adenoma (AA, n = 100), CRC (n = 50) or controls (n = 228). In addition, we analyzed CRC samples (n = 318) from patients recruited in a clinical setting (DACHSplus study). The association of antibody responses to SGG pilus proteins Gallo2178 and Gallo2179 with CRC was replicated with 4% positive DACHSplus cases compared to 0% positive BliTz controls. Positivity to two or more proteins of a newly defined panel of six SGG markers was significantly associated with CRC in the DACHSplus study (OR: 1.81, 95% CI: 1.07-3.06). Odds for CRC, AA and NAA in the BliTz study were also increased with antibody responses to SGG, and the association was significant for NAA (OR: 2.98, 95% CI: 1.18-7.57). Antibody responses to SGG are associated with CRC and its precursors. The newly identified SGG six-marker panel and associations found with precancerous lesions should be further explored.

Antibodies to Streptococcus gallolyticus subspecies gallolyticus (SGG) have been associated with colorectal cancer (CRC). Because SGG may correlate with impaired gut epithelia, we assessed the association of antibodies to bacterial flagellin C (FliC), a measure potentially related to this impairment, with CRC and the CRC-specific interaction with antibodies to SGG proteins. Antibodies to FliC and SGG pilus proteins Gallo2178 and Gallo2179 were measured in two independent studies, a combined study from Nijmegen and Detroit (93 CRC cases, 74 controls) and a replication data set including 576 cases and 576 controls from the Spanish multicenter multicase-control study (MCC-Spain). Logistic regression was applied to assess whether antibodies to FliC were associated with CRC and modified the association of antibodies to SGG proteins with CRC. Antibodies to FliC were associated with those to SGG Gallo2178 among CRC cases, resulting in an interaction in the association of antibodies to Gallo2178 with CRC (p = 0.007). This association was only present among individuals with high antibody responses to FliC (OR: 2.42, 95% CI: 1.45–4.06). In conclusion, our findings suggest that colorectal tumorigenesis could be accompanied by an impaired integrity of the epithelium that could result in associated increased antibody responses to bacterial proteins., We thank Shaynoor Dramsi and Camille Danne (Department of Microbiology, Institut Pasteur, Paris, France) for providing the purified proteins Gallo2178 and Gallo2179. This work was supported by the National Institutes of Health (grant number R01-CA93817, Ikuko Kato); The Netherlands Organization for Scientific Research (NWO 016.166.089 to A. B.); the "Accion Transversal del Cancer", approved on the Spanish Ministry Council on the 11th October 2007; the Instituto de Salud Carlos III-FEDER funds -a way to build Europe-(grants numbers PI08/1770, PI08/0533, PI08/1359, PS09/00773, PS09/01662, PS09/01286, PS09/01903, PS09/02078, PI11/00226, PI11/01403, PI11/01810, PI11/01889, PI11/02213, PI12/00150, PI12/00265, PI12/00488, PI12/00715, PI12/01270, PI14/00613, PI17/00092); the Fundacion Marques de Valdecilla (grant number API 10/09); Obra Social CAJASTUR (grant number SV-CAJASTUR-1); Recercaixa (grant number 2010ACUP 00310); Spanish Association Against Cancer (AECC) Scientific Foundation; Agencia de Gestio d'Ajuts Universitaris i de Recerca (AGAUR) -Generalitat de Catalunya (Catalonian Government) (grant numbers 2009SGR1026, 2009SGR1465 and 2017SGR723); and Junta de Castilla y Leon (grant number LE022A10-2). Sample collection and storage was partially supported by the Instituto de Salud Carlos III-FEDER (grant number RD09/0076/00036), Xarxa de Bancs de Tumors de Catalunya sponsored by Pla Director d'Oncologia de Catalunya (XBTC).

Several bacteria in the human gut microbiome have been associated with colorectal cancer (CRC) by high-throughput screens. In some cases, molecular mechanisms have been elucidated that drive tumorigenesis, including bacterial membrane proteins or secreted molecules that interact with the human cancer cells. For most gut bacteria, however, it remains unknown if they enhance or inhibit cancer cell growth. Here, we screened bacteria-free supernatants (secretomes) and inactivated cells of over 150 cultured bacterial strains for their effect on CRC cell growth. Five CRC cell lines and one noncancerous kidney cell line were selected based on their differences in mutational landscapes. We observed family-level and strain-level effects that often differed between bacterial cells and secretomes, suggesting that different molecular mechanisms are at play. Secretomes of Bacteroidaceae, Enterobacteriaceae, and Erysipelotrichaceae enhanced CRC cell growth, while most Fusobacteriaceae cells and secretomes inhibited growth, contrasting prior findings. For some bacterial families, strong cell line-dependent effects were obsevered, such as for Streptococcaceae and HT29 cells. In some bacteria, the presence of specific functional genes was associated with CRC cell growth rates, including the virulence genes TcdA in Clostridiales and FadA in Fusobacteriaceae, which both inhibited growth. Bacteroidaceae cells that enhanced growth were enriched for genes of the cobalamin synthesis pathway, while Fusobacteriaceae cells that inhibit growth were enriched for genes in ethanolamine utilization pathway. Together, our results reveal how different gut bacteria have wide-ranging effects on cancer cells, contribute a better understanding of the effects of the gut microbiome on the human host, and provide a valuable resource for identifying candidate target genes for potential microbiome-based diagnostics and treatment strategies. Citation Format: Rahwa Taddese, Daniel R. Garza, Lilian Ruiter, Clara Belzer, Steven Aalvink, Iris D. Nagtegaal, Bas E. Dutilh, Annemarie Boleij. Growth rate alterations of human colorectal cancer cells by 157 gut bacteria [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A05.

Colonic bacterial biofilms are an emerging manifestation in colorectal cancer (CRC); they exhibit carcinogenic properties and are frequently present on right-sided cancerous lesions. Whether bacterial biofilms propose a risk factor for early carcinogenesis in humans is yet unresolved. Therefore, we studied bacterial biofilms in tandem with adenoma formation in patients with Lynch syndrome (LS). LS patients carry a pathogenic germline variant in one of the DNA mismatch repair (MMR) genes, resulting in a variable predisposition to develop colonic cancerous lesions. A total of 100 LS patients were included in our study, consisting of 23 MLH1, 24 MSH2, 36 MSH6, and 17 PMS2 MMR variants. During regular screening colonoscopies, normal-appearing forceps biopsies were taken from colon ascendens (right colon) and descendens (left colon). Biopsies were screened for bacterial biofilms using fluorescent in situ hybridization by targeting bacterial 16s rRNA. The frequency of colorectal adenomas (tubular adenomas and [tubulo]villous adenomas) before and during colonoscopy was registered. Overall, 60% of patients presented with a biofilm, of which most were right-sided (right-sided: 25%, both sides: 21%, left-sided: 14%). Interestingly, adenomas were more frequently present in patients with a right-sided biofilm (right-sided: 64%, both sides: 58%) than in patients with a left-sided biofilm (29%) or no biofilm (38%). The occurrence of bacterial biofilms was not correlated with age, BMI, or MMR-variant. Statistical analysis revealed that right-sided bacterial biofilms correlated with right-sided adenomas (Pearson: 0.272, p=0.007) and left-sided adenomas (Pearson: 0.227, p=0.026), while left-sided biofilms were not correlated with left- or right-sided adenomas (Pearson: 0.037, p=0.718 and -.127, p=.213). To model the probability of right-sided adenoma formation, we performed a binary logistic regression analysis and found that age (odds ratio: 1.065 [CI: 1.024; 1.108, p=0.002]) and right-sided biofilms (odds ratio: 3.020 [CI: 1.151; 7.926, p=0.025]) significantly contributed. Our data suggest that right-sided bacterial biofilms are a hallmark for high-risk LS patients and may play a role in early carcinogenesis. This abstract is also being presented as Poster A06. Citation Format: Carlijn Bruggeling, Vera Witjes, Daniel Garza, Milou Fransen, Joyce Krekels, Tanya Bisseling, Mariëtte van Kouwen, Nicoline Hoogerbrugge, Sebastian Lücker, Bas Dutilh, Iris Nagtegaal, Annemarie Boleij. Right-sided colonic biofilms are associated with adenoma formation in patients with Lynch syndrome [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr PR01.

There are many approaches available to inactivate bacteria, each with a different efficacy, impact on cell integrity, and potential for application in high-throughput. The aim of this study was to compare these approaches and develop a standardized protocol for generation of intact Gram-positive as well as on Gram-negative “bacterial zombies”, i.e. cells that are metabolically dead with retained cellular integrity. Here, we introduce the term “bacterial zombies” in addition to “bacterial ghosts” to differentiate inactivated bacteria with preserved cellular integrity from those with perforated membranes, where DNA and cytoplasmic contents have been released. This differentiation of inactivated bacteria is important if the cell content is the subject of study, or if cell contents in the media may cause unwanted effects in downstream applications. We inactivated eight different bacterial species by treatment with beta-propiolactone, ethanol, formalin, sodium hydroxide, and pasteurization. Inactivation efficacy was determined by culturing, and cell wall integrity assessed by quantifying released DNA and visualization by scanning electron microscopy. Based on these results, we discuss the choice of bacterial inactivation methods, and conclude that beta-propiolactone and ethanol are the most promising approaches for standardized generation of bacterial zombies.IMPORTANCEFor applications such as vaccination or analyses that are sensitive to bacterial growth, inactivated bacteria are preferred because they simplify the analyses and the interpretation of results. This study compared various bacterial inactivation treatments that maintain cell integrity and may be used in high-throughput. Our results demonstrated that beta-propiolactone and 70% ethanol were the best techniques to achieve these goals.

The colonic opportunist Streptococcus gallolyticus subspecies gallolyticus (SGG) is potentially associated with colorectal cancer (CRC). Large-scale seroepidemiological data for SGG antibodies and their possible association with CRC is currently missing. Associations between CRC and antibody responses to SGG were examined in 576 CRC cases and 576 controls matched by sex, age and province from a population-based multicase-control project (MCC-Spain). MCC-Spain was conducted between 2008 and 2013 in 12 Spanish provinces. Antibody responses to recombinant affinity-purified SGG pilus proteins Gallo1569, 2039, 2178 and 2179 were analysed by multiplex serology. Polyomavirus (PyV) JC VP1 and PyV 6 VP1 proteins served as disease-specificity controls. In the control population, antibody responses to pilus proteins were mostly weak. Antibody responses to individual pilus proteins Gallo2039 (OR: 1.58, 95% CI: 1.09-2.28), Gallo2178 (OR: 1.58, 95% CI: 1.09-2.30) and Gallo2179 (OR: 1.45, 95% CI: 1.00-2.11) were significantly associated with CRC risk. The association was stronger for positivity to two or more pilus proteins of Gallo1569, Gallo2178 and Gallo2179 (OR:1.93, 95% CI: 1.04-3.56) and for double-positivity to Gallo2178 and Gallo2179 (OR: 3.54, 95% CI: 1.49-8.44). The association between SGG infection and CRC risk was stronger among individuals younger than 65 years. For the first time we demonstrated a statistically significant association of exposure to SGG antigens and CRC in a large seroepidemiological study. These results should stimulate further studies on the role of SGG in CRC pathogenesis.

Item does not contain fulltext BACKGROUND: Bacteremia with Clostridium septicum (CS) and Streptococcus gallolyticus subsp. gallolyticus (SGG) have both been associated with colorectal neoplasms (CRN) and colonoscopic examination is advised, however the differences and similarities in colorectal findings are not well known. METHODS: This is a multicenter, comparative study of patients with CS bacteremia [44 of 664 cases (6.6%) of Clostridium spp.] and SGG bacteremia [257 of 596 cases (44.2%) of S. bovis group], carried out in three hospitals from Spain. Clinical findings related to bacteremia and associated CRN were collected. RESULTS: The main sources of infection were abdominal (77.7%) for CS bacteremia and endovascular (75%) for SGG bacteremia. CS bacteremia was more often associated with malignancies, (72.6% vs. 19.4%) and neutropenia (29.5% vs. 3.1%), and was more acute, with shock at presentation (63.6% vs. 3.9%) and higher 30-day mortality (47.7% vs. 9.7%) compared to SGG (P

Item does not contain fulltext Bacteria constitute about 90% of all cells in the human body. The densest and most complex bacterial community is in the large intestine. This population is quite stable in healthy intestines, but intestinal disease distorts the ecological balance and induces dysbiosis. Results of studies have indicated that the epithelial and metabolic changes that occur with colorectal cancer provide a competitive advantage to a subset of intestinal bacteria. Strikingly, however, Streptococcus gallolyticus gallolyticus (previously known as Streptococcus bovis biotype I) is one of the very few opportunistic pathogens that has been clinically linked to colonic malignant diseases. In this Personal View we describe how S. gallolyticus gallolyticus exploits its unique range of virulence features to cause infections in patients with colorectal cancer. We postulate that distinct virulence factors on one hand enable this bacterium to establish a symptomatic infection in susceptible individuals, and on the other hand make its ability to do this dependent on pre-existing colonic abnormalities. We believe that our current reconstruction of this route of infection aids understanding of how S. gallolyticus gallolyticus infections can be best exploited for early detection of colorectal cancer.

Contains fulltext : 110363.pdf (Publisher’s version ) (Closed access) Cancer has long been considered a genetic disease. However, accumulating evidence supports the involvement of infectious agents in the development of cancer, especially in those organs that are continuously exposed to microorganisms, such as the large intestine. Recent next-generation sequencing studies of the intestinal microbiota now offer an unprecedented view of the aetiology of sporadic colorectal cancer and have revealed that the microbiota associated with colorectal cancer contains bacterial species that differ in their temporal associations with developing tumours. Here, we propose a bacterial driver-passenger model for microbial involvement in the development of colorectal cancer and suggest that this model be incorporated into the genetic paradigm of cancer progression.

Contains fulltext : 96731.pdf (Publisher’s version ) (Closed access) BACKGROUND: Streptococcus bovis has long been associated with colorectal cancer (CRC). However, not all genospecies are as closely related to CRC. With this systematic review, we aim to increase the awareness of the association between S. bovis biotype I (Streptococcus gallolyticus) and CRC and urge for uniform molecular microbiological classification. METHODS: In January 2011, the PubMed database was searched for all studies that investigated the association between S. bovis, infective endocarditis (IE), and CRC. A total of 191 studies were screened for eligibility and yielded 52 case reports and 31 case series, of which 11 were used for meta-analysis on the association between S. bovis biotype, IE, and adenomas/carcinomas (CRC). RESULTS: Among the S. bovis-infected patients who underwent colonic evaluation, the median percentage of patients who had concomitant adenomas/carcinomas was 60% (interquartile range, 22%), which largely exceeds the disease rate reported in the general asymptomatic population. Meta-analysis showed that patients with S. bovis biotype I infection had a strongly increased risk of having CRC (pooled odds ratio [OR], 7.26; 95% confidence interval [CI], 3.94-13.36) and IE (pooled OR, 16.61; 95% CI, 8.85-31.16), compared with S. bovis biotype II-infected patients. Notably, CRC occurred more often among patients with S. bovis IE than among patients with S. bovis infection at other sites (pooled OR, 3.72; 95% CI, 2.03-6.81). CONCLUSIONS: Our meta-analysis clearly indicates that S. bovis should no longer be regarded as a single species in clinical practice, because S. gallolyticus (S. bovis biotype I) infection, in particular, has an unambiguous association with CRC.

Systemic chemotherapy has limited success in treating liver metastasis of colorectal cancer. Alternative approaches such as hepatic arterial infusion or trans arterial chemoembolisation aim to deliver the chemotherapy locally to address the predominant liver disease. Chemoembolisation with drug eluting beads (DEB) designed to deliver drug at the target over a protracted period of time is a new strategy to reduce the tumor burden of liver metastases. To test this hypothesis, DEB possessing anionic groups capable of ionically complexing with cationic drugs were synthesised by a suspension polymerisation method and were fractionated to produce an average size of 75 microm. The DEB were loaded with the desired concentration of either doxorubicin hydrochloride or irinotecan hydrochloride prior to administration by immersion in the drug solution, yielding essentially 100% loading efficiency. To determine their effect in vivo, a transplantable orthotopic and isogenic rat liver metastasis model was used which is based on intraportal injection of 4 x 10(6) beta-galactosidase transfected CC531 rat colorectal cancer cells into male WAG/Rij rats. By MTT assay, the cells were shown to be sensitive to both drugs in vitro with the IC(50) being by two orders of magnitude lower for doxorubicin (110 nM after 72 h) compared to irinotecan (25 microM after 72 h). For the in vivo phase, a differential expression of the ERK MAP kinase between tumor cells cultured in vitro and those inoculated in vivo was noted using Western blotting techniques. This was considered to be indicative of passage-induced cell senescence that reduced the sensitivity of the tumor cells to DEB chemoembolisation. This notwithstanding, administration of DEB loaded with irinotecan or doxorubicin by single injection into the hepatic artery showed significant anticancer activity, as measured by a reduction in the tumor burden of the liver and a corresponding reduction in liver weight. Comparing the two agents, irinotecan appears more advantageous because of its significant activity and excellent tolerability following administration at two dosages of either 20 or 30 mg/kg. Doxorubicin showed a narrower window of activity, being effective at 4 mg/kg but ineffective at the lower dose of 2 mg/kg. We conclude that chemoembolisation with DEB with either agent may have potential for treating patients with colorectal liver metastasis, although irinotecan DEB appeared to have a more favourable safety profile.

Background. Enterotoxigenic Bacteroides fragilis (ETBF) produces the Bacteroides fragilis toxin, which has been associated with acute diarrheal disease, inflammatory bowel disease, and colorectal cancer (CRC). ETBF induces colon carcinogenesis in experimental models. Previous human studies have demonstrated frequent asymptomatic fecal colonization with ETBF, but no study has investigated mucosal colonization that is expected to impact colon carcinogenesis. Methods. We compared the presence of the bft gene in mucosal samples from colorectal neoplasia patients (cases, n = 49) to a control group undergoing outpatient colonoscopy for CRC screening or diagnostic workup (controls, n = 49). Single bacterial colonies isolated anaerobically from mucosal colon tissue were tested for the bft gene with touch-down polymerase chain reaction. Results. The mucosa of cases was significantly more often bft-positive on left (85.7%) and right (91.7%) tumor and/or paired normal tissues compared with left and right control biopsies (53.1%; P = .033 and 55.5%; P = .04, respectively). Detection of bft was concordant in most paired mucosal samples from individual cases or controls (75% cases; 67% controls). There was a trend toward increased bft positivity in mucosa from late- vs early-stage CRC patients (100% vs 72.7%, respectively; P = .093). In contrast to ETBF diarrheal disease where bft-1 detection dominates, bft-2 was the most frequent toxin isotype identified in both cases and controls, whereas multiple bft isotypes were detected more frequently in cases (P ≤ .02). Conclusions. The bft gene is associated with colorectal neoplasia, especially in late-stage CRC. Our results suggest that mucosal bft exposure is common and may be a risk factor for developing CRC.

Item does not contain fulltext Smoking and high red meat intake have been associated with colorectal cancer (CRC) risk. Increased iron exposure may be a common factor, favoring the colonization of certain bacterial pathogens that preferentially grow in an iron-rich luminal environment. We analyzed the data from a population-based case-control study of CRC and measured antibody levels against flagelin of Salmonella (FliC), one of the irontrophic bacteria, in 2 independent blood collections. The risk of CRC synergistically increased by combined exposures to heme iron intake and pack-yr (PY) of cigarette smoking (P value for the interaction = 0.039 on the continuous scale). There was a marginally significant interaction between heme iron intake and PY in increasing FliC antibody in the U.S. control subjects (P = 0.055), although no iron or smoking data were available for Dutch samples. Furthermore, FliC antibody levels were significantly higher in patients with colorectal polyps and cancer than in controls in both Dutch (3.93 vs. 2.23) (P = 0.014) and U.S. samples (6.65 vs. 4.37) (P < 0.001). Potential roles of iron from cigarette smoking and dietary heme in CRC through altering irontrophic luminal bacterial population may warrant further investigation.

A human body contains at least tenfold more bacteria cells than human cells and the most abundant and diverse microbial community (also known as microbiota or microbiome) resides in the large intestine (colon). It is estimated that this colonic microbiome is composed of ∼1014 bacterial cells, comprising >103 species (Dethlefsen et al., 2006; Qin et al., 2010). Intestinal microbiomes differ from individual to individual but remain relatively stable during adult life (Green et al., 2006; Arumugam et al., 2011). The resident microbiome provides the host with core functions that are essential for digestion of food and control of intestinal epithelial homeostasis. Conversely, an increasing body of evidence supports a relationship between infective agents and human colorectal cancer (CRC) by production of DNA damaging metabolites or toxins, and the induction of cell proliferation and procarcinogenesis pathways by a subpopulation of the intestinal microbiota. It could be speculated that the intrinsic intestinal microbiome of a certain individual may contain an unfavorable number of disease-inducing bacteria. On the long term, their activities may override the health-promoting activities of the commensal bacterial population. On the other hand, the dramatic physiological alterations that result from colon carcinogenesis itself (Hirayama et al., 2009) disturbs the local intestinal microenvironment and causes (local) shifts in the microbiota composition and provides a portal of infection for certain opportunistic pathogens. The latter phenomenon could explain why some uncommon bacterial infections are often associated with CRC. In this chapter we will discuss the mechanisms by which intestinal bacteria may drive the initiation and progression of sporadic CRC, but also the driving forces of intestinal carcinogenesis on local microbial dysbiosis and the consequences thereof will be reviewed.

Item does not contain fulltext A healthy human body contains at least tenfold more bacterial cells than human cells and the most abundant and diverse microbial community resides in the intestinal tract. Intestinal health is not only maintained by the human intestine itself and by dietary factors, but is also largely supported by this resident microbial community. Conversely, however, a large body of evidence supports a relationship between bacteria, bacterial activities and human colorectal cancer. Symbiosis in this multifaceted organ is thus crucial to maintain a healthy balance within the host-diet-microbiota triangle and accordingly, changes in any of these three factors may drive a healthy situation into a state of disease. In this review, the factors that sustain health or drive this complex intestinal system into dysbiosis are discussed. Emphasis is on the role of the intestinal microbiota and related mechanisms that can drive the initiation and progression of sporadic colorectal cancer (CRC). These mechanisms comprise the induction of pro-inflammatory and pro-carcinogenic pathways in epithelial cells as well as the production of (geno)toxins and the conversion of pro-carcinogenic dietary factors into carcinogens. A thorough understanding of these processes will provide leads for future research and may ultimately aid in development of new strategies for CRC diagnosis and prevention.

Streptococcus gallolyticus subsp. gallolyticus (previously called Streptococcus bovis biotype I) infections have long been associated with colorectal cancer (CRC). This work aimed to investigate the CRC-associated humoral immune response to four pilus proteins of this bacterium by newly developed ELISAs. Pilus proteins are interesting diagnostic targets as they are the building blocks of pilin-like structures that mediate bacterial virulence and are readily exposed to the host immune system upon infection. The presence of serum antibodies against these pilus proteins was evaluated in Dutch and American populations. These analyses showed that an immune response to these antigens was specific for clinical S. gallolyticus subsp. gallolyticus infections, but that increased serum antibody titers to multiple pilus proteins in single individuals were rarely observed. However, a multiplex approach based on antibody titers against any of these four antigens resulted in assay sensitivities between 16% and 43% for the detection of early-stage CRC. Together these findings underscore the potential of a multi-antigen approach to complement diagnosis of S. gallolyticus subsp. gallolyticus–associated CRC. Cancer Prev Res; 5(2); 260–5. ©2011 AACR.

Item does not contain fulltext One of the few bacteria that have been consistently linked to colorectal cancer (CRC) is the opportunistic pathogen Streptococcus gallolyticus. Infections with this bacterium are generally regarded as an indicator for colonic malignancy, while the carriage rate of this bacterium in the healthy large intestine is relatively low. We speculated that the physiological changes accompanying the development of CRC might favor the colonization of this bacterium. To investigate whether colon tumor cells can support the survival of S. gallolyticus, this bacterium was grown in spent medium of malignant colonocytes to simulate the altered metabolic conditions in the CRC microenvironment. These in vitro simulations indicated that S. gallolyticus had a significant growth advantage in these spent media, which was not observed for other intestinal bacteria. Under these conditions, bacterial responses were profiled by proteome analysis and metabolic shifts were analyzed by (1)H-NMR-spectroscopy. In silico pathway analysis of the differentially expressed proteins and metabolite analysis indicated that this advantage resulted from the increased utilization of glucose, glucose derivates, and alanine. Together, these data suggest that tumor cell metabolites facilitate the survival of S. gallolyticus, favoring its local outgrowth and providing a possible explanation for the specific association of S. gallolyticus with colonic malignancy.