Entero-toxigenic Bacteroides fragilis contributes to intestinal barrier injury and colorectal cancer progression by mediating the BFT/STAT3/ZEB2 pathway.

Our previous findings confirmed the high enrichment of Bacteroides fragilis (BF) in fecal samples from patients with colorectal cancer (CRC). The intestinal mucosal barrier is the first defense of the organism against commensal flora and intestinal pathogens and is closely associated with the occurrence and development of CRC. Therefore, this study aimed to investigate the molecular mechanisms through which BF mediates intestinal barrier injury and CRC progression. SW480 cells and a Caco2 intestinal barrier model were treated with entero-toxigenic BF (ETBF), its enterotoxin (B. fragilis toxin, BFT), and non-toxigenic BF (NTBF). Cell counting kit-8, flow cytometry, wound healing and transwell assays were performed to analyze the proliferation, apoptosis, migration, and invasion of SW480 cells. Transmission electron microscopy, FITC-dextran, and transepithelial electrical resistance (TEER) were used to analyze damage in the Caco2 intestinal barrier model. The Azoxymethane/Dextran Sulfate Sodium (AOM/DSS) animal model was established to evaluate the effect of ETBF on intestinal barrier injury and CRC progression in vivo. ETBF and BFT enhanced the viability, wound healing ratio, invasion, and EMT of SW480 cells. In addition, ETBF and BFT disrupted the tight junctions and villus structure in the intestinal barrier model, resulting in increased permeability and reduced TEER. Similarly, the expression of intestinal barrier-related proteins (MUC2, Occludin and Zo-1) was restricted by ETBF and BFT. Interestingly, the STAT3/ZEB2 axis was activated by ETBF and BFT, and treatment with Brevilin A (a STAT3 inhibitor) or knockdown of ZEB2 limited the promotional effect of ETBF and BFT on the SW480 malignant phenotype. In vivo experiments also confirmed that ETBF colonization accelerated tumor load, carcinogenesis, and intestinal mucosal barrier damage in the colorectum of the AOM/DSS animal model, and that treatment with Brevilin A alleviated these processes. ETBF-secreted BFT accelerated intestinal barrier damage and CRC by activating the STAT3/ZEB2 axis. Our findings provide new insights and perspectives for the application of ETBF in CRC treatment.

A systemic review of the role of enterotoxic Bacteroides fragilis in colorectal cancer.

Enterotoxigenic Bacteroides fragilis (ETBF) has received significant attention for a possible association with, or causal role in, colorectal cancer (CRC). The goal of this review was to assess the status of the published evidence supporting (i) the association between ETBF and CRC and (ii) the causal role of ETBF in CRC. PubMed and Scopus searches were performed in August 2021 to identify human, animal, and cell studies pertaining to the role of ETBF in CRC. Inclusion criteria included the use of cell lines, mice, exposure to BFT or ETBF, and detection of bft. Review studies were excluded, and studies were limited to the English language. Quality of study design and risk of bias analysis was performed on the cell, animal, and human studies using ToxRTools, SYRCLE, and NOS, respectively. Ninety-five eligible studies were identified, this included 22 human studies, 24 animal studies, 43 cell studies, and 6 studies that included both cells and mice studies. We found that a large majority of studies supported an association or causal role of ETBF in CRC, as well as high levels of study bias was detected in the in vitro and in vivo studies. The high-level heterogeneity in study design and reporting made it difficult to synthesize these findings into a unified conclusion, suggesting that the need for future studies that include improved mechanistic models, longitudinal in vitro and in vivo evidence, and appropriate control of confounding factors will be required to confirm whether ETBF has a direct role in CRC etiopathogenesis.

High frequency of enterotoxigenic Bacteroides fragilis and Enterococcus faecalis in the paraffin-embedded tissues of Iranian colorectal cancer patients.

BACKGROUND: The association between specific bacteria and colorectal cancer (CRC) has been proposed. Only a few studies have, however, investigated this relationship directly in colorectal tissue with conflicting results. So, we aimed to quantitate Streptococcus gallolyticus, Fusobacterium spp, Enterococcus faecalis and enterotoxigenic Bacteroides fragilis (ETBF) in formalin-fixed and paraffin-embedded (FFPE) colorectal tissue samples of Iranian CRC patients and healthy controls. METHODS: A total of 80 FFPE colorectal tissue samples of CRC patients (n = 40) and healthy controls (n = 40) were investigated for the presence and copy number of above bacterial species using quantitative PCR. Relative quantification was determined using ΔΔCT method and expressed as relative fold difference compared to reference gene. RESULTS: Relative abundance and copy number of E. faecalis and ETBF were significantly higher in CRC samples compared to control group. E. faecalis was more prevalent than ETBF in tumor samples. Frequency of ETBF and E. faecalis in late stages (III/IV) of cancer was significantly higher than early stages (I/II). We did not detect a significant difference in abundance of S. gallolyticus and Fusobacterium spp between two groups. CONCLUSION: Our study revealed the higher concentration of E. faecalis and ETBF in FFPE samples of CRC patients than controls. However, additional investigations on fecal and fresh colorectal cancer tissue samples are required to substantiate this correlation.

Bacteroides uniformis CECT 7771 alleviates inflammation within the gut-adipose tissue axis involving TLR5 signaling in obese mice.

This study investigated the immune mechanisms whereby administration of Bacteroides uniformis CECT 7771 reduces metabolic dysfunction in obesity. C57BL/6 adult male mice were fed a standard diet or a Western diet high in fat and fructose, supplemented or not with B. uniformis CECT 7771 for 14 weeks. B. uniformis CECT 7771 reduced body weight gain, plasma cholesterol, triglyceride, glucose, and leptin levels; and improved oral glucose tolerance in obese mice. Moreover, B. uniformis CECT 7771 modulated the gut microbiota and immune alterations associated with obesity, increasing Tregs and reducing B cells, total macrophages and the M1/M2 ratio in both the gut and epididymal adipose tissue (EAT) of obese mice. B. uniformis CECT 7771 also increased the concentration of the anti-inflammatory cytokine IL-10 in the gut, EAT and peripheral blood, and protective cytokines TSLP and IL-33, involved in Treg induction and type 2 innate lymphoid cells activation, in the EAT. It also restored the obesity-reduced TLR5 expression in the ileum and EAT. The findings indicate that the administration of a human intestinal bacterium with immunoregulatory properties on the intestinal mucosa helps reverse the immuno-metabolic dysfunction caused by a Western diet acting over the gut-adipose tissue axis.

The uroS and yifB Genes Conserved among Tetrapyrrole Synthesizing-Deficient Bacteroidales Are Involved in Bacteroides fragilis Heme Assimilation and Survival in Experimental Intra-abdominal Infection and Intestinal Colonization.

The human intestinal anaerobic commensal and opportunistic pathogen Bacteroides fragilis does not synthesize the tetrapyrrole protoporphyrin IX in order to form heme that is required for growth stimulation and survival in vivo Consequently, B. fragilis acquires essential heme from host tissues during extraintestinal infection. The absence of several genes necessary for de novo heme biosynthesis is a common characteristic of many anaerobic bacteria; however, the uroS gene, encoding a uroporphyrinogen III synthase for an early step of heme biosynthesis, is conserved among the heme-requiring Bacteroidales that inhabit the mammalian gastrointestinal tract. In this study, we show that the ability of B. fragilis to utilize heme or protoporphyrin IX for growth was greatly reduced in a ΔuroS mutant. This growth defect appears to be linked to the suppression of reverse chelatase and ferrochelatase activities in the absence of uroS In addition, this ΔuroS suppressive effect was enhanced by the deletion of the yifB gene, which encodes an Mg2+-chelatase protein belonging to the ATPases associated with various cellular activities (AAA+) superfamily of proteins. Furthermore, the ΔuroS mutant and the ΔuroS ΔyifB double mutant had a severe survival defect compared to the parent strain in competitive infection assays using animal models of intra-abdominal infection and intestinal colonization. This shows that the presence of the uroS and yifB genes in B. fragilis seems to be linked to pathophysiological and nutritional competitive fitness for survival in host tissues. Genetic complementation studies and enzyme kinetics assays indicate that B. fragilis UroS is functionally different from canonical bacterial UroS proteins. Taken together, these findings show that heme assimilation and metabolism in the anaerobe B. fragilis have diverged from those of aerobic and facultative anaerobic pathogenic bacteria.

Enterotoxigenic Bacteroides fragilis infection exacerbates tumorigenesis in AOM/DSS mouse model.

The azoxymethane (AOM)/dextran sulfate sodium (DSS) murine model is commonly used to study colitis-associated cancer. The human commensal bacterium, enterotoxigenic Bacteroides fragilis (ETBF) secretes the Bacteroides fragilis toxin (BFT) which is necessary and sufficient to cause colitis. We report that BALB/c mice infected with WT-ETBF and administered three cycles of AOM/DSS developed numerous, large-sized polyps predominantly in the colorectal region. In addition, AOM/DSS-treated BALB/c mice orally inoculated with wild-type nontoxigenic Bacteroides fragilis (WT-NTBF) overexpressing bft (rETBF) developed numerous polyps whereas mice infected with WT-NTBF overexpressing a biologically inactive bft (rNTBF) did not promote polyp formation. Unexpectedly, the combination of AOM+ETBF did not induce polyp formation whereas ETBF+DSS did induce polyp development in a subset of BALB/c mice. In conclusion, WT-ETBF promoted polyp development in AOM/DSS murine model with increased colitis in BALB/c mice. The model described herein provides an experimental platform for understanding ETBF-induced colonic tumorigenesis and studying colorectal cancer in wild-type mice.

The impact of the gut microbiota on prognosis after surgery for colorectal cancer - a systematic review and meta-analysis.

The aim of this study was to conduct a systematic review of the association between gut microbiota and prognosis after colorectal cancer surgery. The review was conducted according to the PRISMA guidelines. A systematic literature search was conducted in PubMed, Embase, and Scopus. Studies examining the association between gut microbiota and survival after colorectal cancer surgery were identified. Secondary outcomes were association with cancer stage and immune infiltration of tumor. A total of 27 studies were included in the review. Fusobacterium nucleatum was the most frequently examined bacterium, and the meta-analysis showed that high level of F. nucleatum was significantly associated with decreased overall survival, hazard ratio of 1.63 (95% confidence interval 1.23-2.16) for unadjusted data, and hazard ratio of 1.47 (95% confidence interval 1.08-1.98) for adjusted data. Association between higher tumor stage and F. nucleatum was reported in ten studies, and two studies found an association with unfavorable tumor infiltration of immune cells. Three out of five studies examining Bacteroides fragilis found an association with decreased survival, advanced tumor stage, or unfavorable immune infiltration of tumor. High levels of F. nucleatum and possibly B. fragilis were associated with worse prognosis after surgery for colorectal cancer.

Promoter orientation of the immunomodulatory Bacteroides fragilis capsular polysaccharide A (PSA) is off in individuals with inflammatory bowel disease (IBD).

Bacteroides fragilis is a member of the normal microbiota of the lower gastrointestinal tract, but some strains produce the putative tumourigenic B. fragilis toxin (BFT). In addition, B. fragilis can produce multiple capsular polysaccharides that comprise a microcapsule layer, including an immunomodulatory, zwitterionic, polysaccharide A (PSA) capable of stimulating anti-inflammatory interleukin-10 (IL-10) production. It is known that the PSA promoter can undergo inversion, thereby regulating the expression of PSA. A PCR digestion technique was used to investigate B. fragilis capsular PSA promoter orientation using human samples for the first time. It was found that approximately half of the B. fragilis population in a healthy patient population had PSA orientated in the 'ON' position. However, individuals with inflammatory bowel disease (IBD) had a significantly lower percentage of the B. fragilis population with PSA orientated 'ON' in comparison with the other patient cohorts studied. Similarly, the putative tumourigenic bft-positive B. fragilis populations were significantly associated with a lower proportion of the PSA promoter orientated 'ON'. These results suggest that the proportion of the B. fragilis population with the PSA promoter 'ON' may be an indicator of gastrointestinal health.

Orthopedic infections caused by obligatory anaerobic Gram-negative rods: report of two cases.

Anaerobic bone and joint infections are uncommon, although the number of anaerobic infections is presumably underestimated because of difficulties with isolation and identification of obligate anaerobes. This study describes two cases of complicated Bacteroides fragilis peri-implant infection of the lumbar spine, infection of the hip and osteomyelitis. Bacteria were identified with the use of a mass spectrometer, VITEK MS system. Drug susceptibility was performed with the use of E-test. The EUCAST breakpoints were used for interpretation with B. fragilis ATCC 25285 as a control. In the two described cases clinical samples were collected for microbiological examination intraoperatively and simultaneously empirical treatment was applied. B. fragilis was isolated in monoculture or in a combination with other bacteria. The treatment was continued according to the susceptibility tests. In a case one clindamycin failure was observed and clindamycin resistance of the isolate was likely due to inadequate time of therapy. Difficulties in collecting an adequate samples and culturing anaerobic bacteria cause that not all infections are properly recognized. In a successful therapy, identification and determination of the susceptibility of the pathogen are essential as well as an appropriate surgical debridement.

Fatal sepsis caused by multidrug-resistant Bacteroides fragilis, harboring a cfiA gene and an upstream insertion sequence element, in Japan.

Here, we report a case of fatal sepsis resulting from an intra-abdominal infection caused by a Bacteroides fragilis strain containing a CfiA4 metallo-ß-lactamase and an upstream insertion sequence (IS) element. Meropenem was used as empiric therapy for septic shock as a result of the intra-abdominal infection, although two rounds of carbapenem treatment had been administered previously. B. fragilis was isolated from two anaerobic blood culture bottles 4 days after the onset of septic shock. Susceptibility testing revealed that the isolate was non-susceptible to all tested agents except metronidazole and tigecycline. The isolate gave a positive result in ethylenediaminetetraacetic acid and carbapenem inactivation tests, but a negative result in a double-disk synergy test using sodium mercaptoacetate. Next-generation whole-genome sequencing indicated the presence of the cfiA4, emrG and emrF genes. PCR indicated the presence of an IS element upstream of the cifA4 gene. Although carbapenem-resistant B. fragilis isolates have previously been reported, clinical sepsis by this organism is considered rare. In Japan, as in most countries worldwide, routine susceptibility testing and the detection of metallo-ß-lactamases is not carried out in anaerobic organisms, including B. fragilis. The emergence of carbapenem resistance during therapy should be monitored, as B. fragilis strains containing the cfiA gene show decreased sensitivity during carbapenem therapy. Therefore, susceptibility testing and appropriate antibiotic stewardship are required in cases of anaerobic bacterial infections.

First case report of a human sepsis involving a recently identified anaerobic agent: Bacteroides faecis.

Up until now, Bacteroides faecis, a Gram-negative, anaerobic, non-motile, nonsporeforming rod has been principally described as a commensal microbe isolated from the feces of healthy adults. We report the first case of human Bacteroides faecis sepsis after removal of suspected post-colonic ischemia colonized epicardic electrodes. Electrodes and blood cultures both grew Gram-negative anaerobic rods but usual phenotypic methods and 16S rARN gene sequencing failed to ensure its species identification. B. faecis was finally identified using hsp60 gene sequencing. Because this species is not well-known and is difficult to identify, it may have been overlooked or misidentified in previous studies.

A case of Bacteroides pyogenes bacteremia secondary to liver abscess.

Bacteroides pyogenes, a non-spore-forming, anaerobic, gram-negative rod, is a component of the oral flora of animals and has, on occasion, been reported to cause human infection through dog or cat bites. We report the first case of B. pyogenes bacteremia secondary to liver abscess with no history of an animal bite. The microorganism was identified by 16S rRNA sequencing.

Bacteroides pyogenes causing serious human wound infection from animal bites.

Bacteroides pyogenes is part of the normal oral flora of domestic animals. There is one previous report of human infection, with B. pyogenes bacteremia following a cat bite (Madsen 2011). We report seven severe human infections where B. pyogenes was identified by Bruker matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDTI-TOF MS), but not by VITEK MS and was misidentified by VITEK ANC card.

The Dysregulation of Polyamine Metabolism in Colorectal Cancer Is Associated with Overexpression of c-Myc and C/EBPß rather than Enterotoxigenic Bacteroides fragilis Infection.

Colorectal cancer is one of the most common cancers in the world. It is well known that the chronic inflammation can promote the progression of colorectal cancer (CRC). Recently, a number of studies revealed a potential association between colorectal inflammation, cancer progression, and infection caused by enterotoxigenic Bacteroides fragilis (ETBF). Bacterial enterotoxin activates spermine oxidase (SMO), which produces spermidine and H2O2 as byproducts of polyamine catabolism, which, in turn, enhances inflammation and tissue injury. Using qPCR analysis, we estimated the expression of SMOX gene and ETBF colonization in CRC patients. We found no statistically significant associations between them. Then we selected genes involved in polyamine metabolism, metabolic reprogramming, and inflammation regulation and estimated their expression in CRC. We observed overexpression of SMOX, ODC1, SRM, SMS, MTAP, c-Myc, C/EBPß (CREBP), and other genes. We found that two mediators of metabolic reprogramming, inflammation, and cell proliferation c-Myc and C/EBPß may serve as regulators of polyamine metabolism genes (SMOX, AZIN1, MTAP, SRM, ODC1, AMD1, and AGMAT) as they are overexpressed in tumors, have binding site according to ENCODE ChIP-Seq data, and demonstrate strong coexpression with their targets. Thus, increased polyamine metabolism in CRC could be driven by c-Myc and C/EBPß rather than ETBF infection.

[DYNAMICS OF INDICES OF A LOCAL IMMUNITY IN AN ACUTE APPENDICITIS].

Abstract The results of investigation on dynamics of a local immunity indices in an acute appendicitis, depending on the pathological process stage as well as on bacteriological investigation of parietal microflora of processus vermicularis, were adduced. The sIgA and lisocymal dynamics have witnessed that while a destructive process progressing their concentration was enhanced, and in a gangrenous acute appendicitis they practically disappeared. Due to affection of a barrier function of the processus vermicularis wall a favorable conditions were created for the microorganisms intramural translocation as well as to abdominal cavity.

Screening for enterotoxigenic Bacteroides fragilis in stool samples.

Bacteroides fragilis is a commensal bacterium found in the gut of most humans, however enterotoxigenic B. fragilis strains (ETBF) have been associated with diarrhoea and colorectal cancer (CRC). The purpose of this study was to establish a method of screening for the Bacteroides fragilis toxin (bft) gene in stool samples, as a means of determining if carriage of ETBF is detected more often in CRC patients than in age-matched healthy controls. Stool samples from 71 patients recently diagnosed with CRC, and 71 age-matched controls, were screened by standard and quantitative PCR using primers specific for the detection of the bft gene. Bacterial template DNA from stool samples was prepared by two methods: a sweep, where all colonies growing on Bacteroides Bile Esculin agar following stool culture for 48 h at 37 °C in an anaerobic environment were swept into sterile water and heat treated; and a direct DNA extraction from each stool sample. The bft gene was detected more frequently from DNA isolated from bacterial sweeps than from matched direct DNA extractions. qPCR was found to be more sensitive than standard PCR in detecting bft. The cumulative total of positive qPCR assays from both sample types revealed that 19 of the CRC patients had evidence of the toxin gene in their stool sample (27%), compared to seven of the age-matched controls (10%). This difference was significant (P = 0.016). Overall, ETBF carriage was detected more often in CRC patient stool samples compared to controls, but disparate findings from the different DNA preparations and testing methods suggests that poor sensitivity may limit molecular detection of ETBF in stool samples.

[VERTEBRAL OSTEOMYELITIS CAUSED BY RARE PATHOGENS--THE NEED FOR HIGH INDEX OF SUSPICION].

Vertebral OsteomyeLitis (V.O.) is a rare event that usually presents insidiously and follows an indolent clinical course, making early diagnosis difficult. The most important infecting organism in V.O. is Staphylococcus aureus, followed by gram-negative bacilli. We describe herein two cases of V.O. hospitalized in our department during the same week, caused by rare pathogens--Streptococcus sanguis (viridans) and Bacteroides fragilis. V.O. must be recognized rapidly because delay in diagnosis and treatment can result in neurologic compromise and high mortality. Its prompt and accurate diagnosis depends on detailed knowledge of the disease along with a high index of suspicion, even in face of rare pathogens on bacteriology results.

Antimicrobial susceptibility of clinical isolates of Bacteroides fragilis group organisms recovered from 2009 to 2012 in a Korean hospital.

BACKGROUND: Periodic monitoring of antimicrobial resistance trends of clinically important anaerobic bacteria such as Bacteroides fragilis group organisms is required. We determined the antimicrobial susceptibilities of clinical isolates of B. fragilis group organisms recovered from 2009 to 2012 in a tertiary-care hospital in Korea. METHODS: A total of 180 nonduplicate clinical isolates of B. fragilis group organisms were collected in a tertiary care hospital. The species were identified by conventional methods: the ATB 32A rapid identification system (bioMérieux, France) and the Vitek MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (bioMérieux). Antimicrobial susceptibility was determined by the CLSI agar dilution method. RESULTS: Imipenem and meropenem resistance rates were 0-6% for B. fragilis group isolates. The rate of resistance to piperacillin-tazobactam was 2% for B. fragilis and 0% for other Bacteroides species, but 17% for B. thetaiotaomicron isolates. High resistance rates to piperacillin (72% and 69%), cefotetan (89% and 58%), and clindamycin (83% and 69%) were observed for B. thetaiotaomicron and other Bacteroides spp. The moxifloxacin resistance rate was 27% for other Bacteroides spp. The MIC50 and MIC90 of tigecycline were 2-4 µg/mL and 8-16 µg/mL, respectively. No isolates were resistant to chloramphenicol or metronidazole. CONCLUSIONS: Imipenem, meropenem, chloramphenicol, and metronidazole remain active against B. fragilis group isolates. Moxifloxacin and tigecycline resistance rates are 2-27% and 8-15% for B. fragilis group isolates, respectively.

Stat3 activation in murine colitis induced by enterotoxigenic Bacteroides fragilis.

BACKGROUND: Enterotoxigenic Bacteroides fragilis (ETBF), a molecular subclass of the common human commensal, B. fragilis, has been associated with inflammatory bowel disease. ETBF colitis is characterized by the activation of Stat3 and a Th17 immune response in the colonic mucosa. This study was designed to investigate the time course and cellular distribution of Stat3 activation in ETBF-colonized mice. METHODS: C57BL/6 wild-type, C57BL/6, or Rag-1 mice were inoculated with saline, nontoxigenic B. fragilis or ETBF. Histologic diagnosis and mucosal Stat activation (immunohistochemistry, Western blot, and/or electrophorectic mobility shift assay) were evaluated over time (6-24 h, 1-7 d, and 1-18 mo after inoculation). Mucosal permeability was evaluated at 16 hours, 1 day, and 3 days. Mucosal immune responses were evaluated at 1 week, and 12 and 18 months. RESULTS: ETBF induced rapid-onset colitis that persisted for up to 1 year. Stat3 activation (pStat3) was noted in the mucosal immune cells within 16 hours, with colonic epithelial cell activation evident at 24 hours after inoculation. ETBF-induced increased mucosal permeability was first observed at 24 hours after inoculation, after which the initial immune cell pStat3 activation was noted. Immune cell pStat3 was present in the absence of epithelial pStat3 (C57BL/6). Epithelial pStat3 was present in the absence of T and B cells (Rag-1 mice). pStat3 persisted in the epithelial and immune cells for 1 year, characterized by isolated pStat3-positive cell clusters, with varying intensity distributed through the proximal and distal colon. Similarly, mucosal Th17 immune responses persisted for up to 1 year. Loss of fecal ETBF colonization was associated with the loss of mucosal pStat3 and Th17 immune responses. CONCLUSIONS: ETBF rapidly induces immune cell pStat3, which is independent of epithelial pStat3. This occurs before ETBF-induced mucosal permeability, suggesting that ETBF, likely through B. fragilis toxin and its action on the colonic epithelial cell, triggers mucosal immune cell Stat3 activation. Peak mucosal Stat3 activation (immune and epithelial cells) occurs subsequently when other colonic bacteria may contribute to the ETBF-initiated immune response due to barrier dysfunction. ETBF induces long-lived, focal colonic Stat3 activation and Th17 immune responses dependent on the ongoing ETBF colonization. Further study is needed to evaluate the early mucosal signaling events, resulting in epithelial Stat3 activation and the sequelae of long-term colonic Stat3 activation.