Modulating Gut Microbiota Prevents Anastomotic Leak to Reduce Local Implantation and Dissemination of Colorectal Cancer Cells after Surgery.

PURPOSE: Anastomotic leak (AL) is a major complication in colorectal cancer surgery and consists of the leakage of intestinal content through a poorly healed colonic wound. Colorectal cancer recurrence after surgery is a major determinant of survival. We hypothesize that AL may allow cancer cells to escape the gut and lead to cancer recurrence and that improving anastomotic healing may prevent local implantation and metastatic dissemination of cancer cells. EXPERIMENTAL DESIGN: We investigated the association between AL and postoperative outcomes in patients with colorectal cancer. Using mouse models of poor anastomotic healing, we assessed the processes of local implantation and dissemination of cancer cells. The effect of dietary supplementation with inulin and 5-aminosalicylate (5-ASA), which activate PPAR-γ in the gut, on local anastomotic tumors was assessed in mice undergoing colonic surgery. Inulin and 5-ASA were also assessed in a mouse model of liver metastasis. RESULTS: Patients experiencing AL displayed lower overall and oncologic survival than non-AL patients. Poor anastomotic healing in mice led to larger anastomotic and peritoneal tumors. The microbiota of patients with AL displays a lower capacity to activate the antineoplastic PPAR-γ in the gut. Modulation of gut microbiota using dietary inulin and 5-ASA reinforced the gut barrier and prevented anastomotic tumors and metastatic spread in mice. CONCLUSIONS: Our findings reinforce the hypothesis that preventing AL is paramount to improving oncologic outcomes after colorectal cancer surgery. Furthermore, they pave the way toward dietary targeting of PPAR-γ as a novel way to enhance healing and diminish cancer recurrence.

Inulin impacts tumorigenesis promotion by colibactin-producing Escherichia coli in ApcMin/+ mice.

Introduction: The prebiotic inulin has previously shown both protective and tumor-promoting effects in colorectal cancer (CRC). These inconsistencies may be due to the gut microbial composition as several bacteria have been associated with CRC. Specifically, polyketide synthase-positive (pks+) Escherichia coli promotes carcinogenesis and facilitates CRC progression through the production of colibactin, a genotoxin that induces double-strand DNA breaks (DSBs). We investigated whether colibactin-producing Escherichia coli changed the protection conferred by inulin against tumor growth and progression using the ApcMin/+ mouse model of CRC. Methods: Mice received a 2% dextran sodium sulfate (DSS) solution followed by oral gavage with the murine pks + E. coli strain NC101 (EcNC101) and were fed a diet supplemented with 10% cellulose as control or 10% inulin for 4 weeks. Results: Inulin supplementation led to increase EcNC101 colonization compared to mice receiving the control diet. The increased colonization of EcNC101 resulted in more DSBs, tumor burden, and tumor progression in ApcMin/+ mice. The tumorigenic effect of EcN101 in ApcMin/+ mice mediated by inulin was dependent on colibactin production. Pasteurized E. coli Nissle 1917 (EcN), a probiotic, suppressed the inulin-driven EcNC101 expansion and impacted tumor progression. Discussion: Our results suggest that the presence of pks + E. coli influences the outcome of inulin supplementation in CRC and that microbiota-targeted interventions may mitigate this effect. Given the prevalence of pks + E. coli in both healthy and CRC populations and the importance of a fiber-rich diet, inulin supplementation in individuals colonized with pks + bacteria should be considered with caution.

Gut microbiota influence anastomotic healing in colorectal cancer surgery through modulation of mucosal proinflammatory cytokines.

OBJECTIVE: Colorectal cancer (CRC) is the third most diagnosed cancer, and requires surgical resection and reconnection, or anastomosis, of the remaining bowel to re-establish intestinal continuity. Anastomotic leak (AL) is a major complication that increases mortality and cancer recurrence. Our objective is to assess the causal role of gut microbiota in anastomotic healing. DESIGN: The causal role of gut microbiota was assessed in a murine AL model receiving faecal microbiota transplantation (FMT) from patients with CRC collected before surgery and who later developed or not, AL. Anastomotic healing and gut barrier integrity were assessed after surgery. Bacterial candidates implicated in anastomotic healing were identified using 16S rRNA gene sequencing and were isolated from faecal samples to be tested both in vitro and in vivo. RESULTS: Mice receiving FMT from patients that developed AL displayed poor anastomotic healing. Profiling of gut microbiota of patients and mice after FMT revealed correlations between healing parameters and the relative abundance of Alistipes onderdonkii and Parabacteroides goldsteinii. Oral supplementation with A. onderdonkii resulted in a higher rate of leaks in mice, while gavage with P. goldsteinii improved healing by exerting an anti-inflammatory effect. Patients with AL and mice receiving FMT from AL patients presented upregulation of mucosal MIP-1α, MIP-2, MCP-1 and IL-17A/F before surgery. Retrospective analysis revealed that patients with AL present higher circulating neutrophil and monocyte counts before surgery. CONCLUSION: Gut microbiota plays an important role in surgical colonic healing in patients with CRC. The impact of these findings may extend to a vast array of invasive gastrointestinal procedures.

Prevalence of pks + bacteria and enterotoxigenic Bacteroides fragilis in patients with colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the third most diagnosed cancer and the second most common cause of cancer deaths worldwide. CRC patients present with an increase in pathogens in their gut microbiota, such as polyketide synthase-positive bacteria (pks +) and enterotoxigenic Bacteroides fragilis (ETBF). The pks + Escherichia coli promotes carcinogenesis and facilitates CRC progression through the production of colibactin, a genotoxin that induces double-strand DNA breaks (DSBs). ETBF is a procarcinogenic bacterium producing the B. fragilis toxin (bft) that promotes colorectal carcinogenesis by modulating the mucosal immune response and inducing epithelial cell changes. METHODS: Fecal samples were collected from healthy controls (N = 62) and CRC patients (N = 94) from the province of Québec (Canada), and a bacterial DNA extraction was performed. Fecal DNA samples were then examined for the presence of the pks island gene and bft using conventional qualitative PCR. RESULTS: We found that a high proportion of healthy controls are colonized by pks + bacteria (42%) and that these levels were similar in CRC patients (46%). bft was detected in 21% of healthy controls and 32% of CRC patients, while double colonization by both pks + bacteria and ETBF occurred in 8% of the healthy controls and 13% of the CRC patients. Most importantly, we found that early-onset CRC (< 50 years) patients were significantly less colonized with pks + bacteria (20%) compared to late-onset CRC patients (52%). CONCLUSIONS: Healthy controls had similar levels of pks + bacteria and ETBF colonization as CRC patients, and their elevated levels may place both groups at greater risk of developing CRC. Colonization with pks + bacteria was less prevalent in early-compared to late-onset CRC.

Oral iron supplementation after antibiotic exposure induces a deleterious recovery of the gut microbiota.

BACKGROUND: Oral iron supplementation is commonly prescribed for anemia and may play an important role in the gut microbiota recovery of anemic individuals who received antibiotic treatment. This study aims to investigate the effects of iron supplementation on gut microbiota recovery after antibiotics exposure. RESULTS: Mice were subjected to oral antibiotic treatment with neomycin and metronidazole and were fed diets with different concentrations of iron. The composition of the gut microbiota was followed throughout treatment by 16S rRNA sequencing of DNA extracted from fecal samples. Gut microbiota functions were inferred using PICRUSt2, and short-chain fatty acid concentration in fecal samples was assessed by liquid-chromatography mass spectrometry. Iron supplementation after antibiotic exposure shifted the gut microbiota composition towards a Bacteroidetes phylum-dominant composition. At the genus level, the iron-supplemented diet induced an increase in the abundance of Parasutterella and Bacteroides, and a decrease of Bilophila and Akkermansia. Parasutterella excrementihominis, Bacteroides vulgatus, and Alistipes finegoldii, were more abundant with the iron excess diet. Iron-induced shifts in microbiota composition were accompanied by functional modifications, including an enhancement of the biosynthesis of primary bile acids, nitrogen metabolism, cyanoamino acid metabolism and pentose phosphate pathways. Recovery after antibiotic treatment increased propionate levels independent of luminal iron levels, whereas butyrate levels were diminished by excess iron. CONCLUSIONS: Oral iron supplementation after antibiotic therapy in mice may lead to deleterious changes in the recovery of the gut microbiota. Our results have implications on the use of oral iron supplementation after antibiotic exposure and justify further studies on alternative treatments for anemia in these settings.

Improvement of colonic healing and surgical recovery with perioperative supplementation of inulin and galacto-oligosaccharides.

BACKGROUND AND AIMS: Anastomotic leak (AL) is a major complication in colorectal surgery. Recent evidence suggests that the gut microbiota may affect healing and may cause or prevent AL. Butyrate is a beneficial short-chain fatty acid (SCFA) that is produced as a result of bacterial fermentation of dietary oligosaccharides and has been described as beneficial in the maintenance of colonic health. To assess the impact of oligosaccharides on colonic anastomotic healing in mice, we propose to modulate the microbiota with oligosaccharides to increase butyrate production via enhancement of butyrate-producing bacteria and, consequently, improve anastomotic healing in mice. METHODS: Animal experiments were conducted in mice that were subjected to diets supplemented with inulin, galacto-oligosaccharides (GOS) or cellulose, as a control, for two weeks before undergoing a surgical colonic anastomosis. Macroscopic and histological assessment of the anastomosis was performed. Extent of epithelial proliferation was assessed by Ki-67 immunohistochemistry. Gelatin zymography was used to evaluate the extent of matrix metalloproteinase (MMP) hydrolytic activity. RESULTS: Inulin and GOS diets were associated with increased butyrate production and better anastomotic healing. Histological analysis revealed an enhanced mucosal continuity, and this was associated with an increased re-epithelialization of the wound as determined by increased epithelial proliferation. Collagen concentration in peri-anastomotic tissue was higher with inulin and GOS diets and MMP activity, a marker of collagen degradation, was lower with both oligosaccharides. Inulin and GOS diets were further associated with lower bacterial translocation. CONCLUSIONS: Dietary supplementation with inulin and GOS may improve anastomotic healing and reinforce the gut barrier in mice.

Oligosaccharides increase the genotoxic effect of colibactin produced by pks+ Escherichia coli strains.

BACKGROUND: Colibactin is a genotoxin that induces DNA double-strand breaks that may lead to carcinogenesis and is produced by Escherichia coli strains harboring the pks island. Human and animal studies have shown that colibactin-producing gut bacteria promote carcinogenesis and enhance the progression of colorectal cancer through cellular senescence and chromosomal abnormalities. In this study, we investigated the impact of prebiotics on the genotoxicity of colibactin-producing E. coli strains Nissle 1917 and NC101. METHODS: Bacteria were grown in medium supplemented with 20, 30 and 40 mg/mL of prebiotics inulin or galacto-oligosaccharide, and with or without 5 µM, 25 µM and 125 µM of ferrous sulfate. Colibactin expression was assessed by luciferase reporter assay for the clbA gene, essential for colibactin production, in E. coli Nissle 1917 and by RT-PCR in E. coli NC101. The human epithelial colorectal adenocarcinoma cell line, Caco-2, was used to assess colibactin-induced megalocytosis by methylene blue binding assay and genotoxicity by γ-H2AX immunofluorescence analysis. RESULTS: Inulin and galacto-oligosaccharide enhanced the expression of clbA in pks+ E. coli. However, the addition of 125 µM of ferrous sulfate inhibited the expression of clbA triggered by oligosaccharides. In the presence of either oligosaccharide, E. coli NC101 increased dysplasia and DNA double-strand breaks in Caco-2 cells compared to untreated cells. CONCLUSION: Our results suggest that, in vitro, prebiotic oligosaccharides exacerbate DNA damage induced by colibactin-producing bacteria. Further studies are necessary to establish whether oligosaccharide supplementation may lead to increased colorectal tumorigenesis in animal models colonized with pks+ E. coli.