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Klebsiella pneumoniae is a pathogenic bacterium that has been linked to several illnesses acquired in both the community and hospitals. This study aimed to determine the prevalence of the clbA, K1, and K2 genes in K. pneumoniae and their relationship with antibiotic resistance. In this research, 130 clinical samples from patients in hospitals were collected from blood, urine, wounds, burns, and sputum, and only 61 of those samples were positively identified as K. pneumoniae using biochemical tests, selective media, and Vitek II. The method of disc diffusion was utilized to evaluate the 61 isolates' susceptibility to 10 antibiotics, including Imipenem, Meropenem, Cefixime, Ceftazidime, Amoxicillin-clavulanate, Gentamicin, Aztreonam, Azithromycin, Levofloxacin, and Trimethoprim-sulfamethoxazole. The results showed that the highest resistance was towards amoxicillin-clavulanate (98.36%) and the lowest towards meropenem (13.11%), and that 95.08% of the isolates were multidrug resistant (MDR). The clbA, K1, and K2 genes were detected using conventional PCR; the results showed that out of 61 isolates, only 3 were positive for K1, 52 were positive for K2, and none of the isolates were clbA+. (2/3) of the K1 and (51/52) of the K2 isolates were MDR. The finding shows a substantial association between the K1 and K2 genes and resistance to antibiotics, as the capsule is the main virulence factor in this bacteria. [ABSTRACT FROM AUTHOR]

Background The bacterial genotoxin, cytolethal distending toxin (CDT), causes DNA damage in host cells, a risk factor for carcinogenesis. Previous studies have shown that CDT induces phenotypes reminiscent of epithelial to mesenchymal transition (EMT), a process involved in cancer initiation and progression. Methods We investigated different steps of EMT in response to Helicobacter hepaticus CDT and its active CdtB subunit using in vivo and in vitro models. Results Most of the steps of the EMT process were induced by CDT/CdtB and observed throughout the study in murine and epithelial cell culture models. CdtB induced cell-cell junction disassembly, causing individualization of cells and acquisition of a spindle-like morphology. The key transcriptional regulators of EMT (SNAIL and ZEB1) and some EMT markers were upregulated at both RNA and protein levels in response to CDT/CdtB. CdtB increased the expression and proteolytic activity of matrix metalloproteinases, as well as cell migration. A range of these results were confirmed in Helicobacter hepaticus -infected and xenograft murine models. In addition, colibactin, a genotoxic metabolite produced by Escherichia coli , induced EMT-like effects in cell culture. Conclusions Overall, these data show that infection with genotoxin-producing bacteria elicits EMT process activation, supporting their role in tumorigenesis. [ABSTRACT FROM AUTHOR]

Colibactin is a recently characterized pro-carcinogenic genotoxin produced by pks+ Escherichia coli. We hypothesized that cystic fibrosis (CF)-associated dysfunctional mucus structure increases the vulnerability of host mucosa to colibactin-induced DNA damage. In this pilot study, we tested healthy-appearing mucosal biopsy samples obtained during screening and surveillance colonoscopies of adult CF and non-CF patients for the presence of pks+ E. coli, and we investigated the possibility of detecting a novel colibactin-specific DNA adduct that has not been yet been demonstrated in humans. While CF patients had a lower incidence of pks+ E. coli carriage (~8% vs 29%, p = 0.0015), colibactin-induced DNA adduct formation was detected, but only in CF patients and only in those who were not taking CFTR modulator medications. Moreover, the only patient found to have colon cancer during this study had CF, harbored pks+ E. coli, and had colibactin-induced DNA adducts in the mucosal samples. Larger studies with longitudinal follow-up should be done to extend these initial results and further support the development of colibactin-derived DNA adducts to stratify patients and their risk.

ABSTRACTHuman colorectal cancers (CRCs) are readily colonized by colibactin-producing E. coli (CoPEC). CoPEC induces DNA double-strand breaks, DNA mutations, genomic instability, and cellular senescence. Infected cells produce a senescence-associated secretory phenotype (SASP), which is involved in the increase in tumorigenesis observed in CRC mouse models infected with CoPEC. This study investigated whether CoPEC, and the SASP derived from CoPEC-infected cells, impacted chemotherapeutic resistance. Human intestinal epithelial cells were infected with the CoPEC clinical 11G5 strain or with its isogenic mutant, which is unable to produce colibactin. Chemotherapeutic resistance was assessed in vitro and in a xenograft mouse model. Expressions of cancer stem cell (CSC) markers in infected cells were investigated. Data were validated using a CRC mouse model and human clinical samples. Both 11G5-infected cells, and uninfected cells incubated with the SASP produced by 11G5-infected cells exhibited an increased resistance to chemotherapeutic drugs in vitro and in vivo. This finding correlated with the induction of the epithelial to mesenchymal transition (EMT), which led to the emergence of cells exhibiting CSC features. They grew on ultra-low attachment plates, formed colonies in soft agar, and overexpressed several CSC markers (e.g. CD133, OCT-3/4, and NANOG). In agreement with these results, murine and human CRC biopsies colonized with CoPEC exhibited higher expression levels of OCT-3/4 and NANOG than biopsies devoid of CoPEC. Conclusion: CoPEC might aggravate CRCs by inducing the emergence of cancer stem cells that are highly resistant to chemotherapy.

Fecal carriage of the colibactin (clb) gene in Escherichia coli is described as a source that could promote carcinogenesis, progressing to colorectal cancer. The present study investigated the demographic, dietary, and antibiotic consumption variables as correlates for fecal carriage of clb +/ E coli among the student populace. In a randomized cross-sectional survey, E coli (N = 136) from the fecal samples of eligible students were characterized and evaluated for antibiotic resistance, β-lactamase (blm), biofilm, virulence factor production, and strain tryptophan reverse mutagenic activity. The encoded clb +/ E coli were analyzed for correlates with principal component analysis. Of all the E coli strains, a low rate of 2 clb+/E coli (1.5%) and higher rates of biofilm (13.2%) and blm producers (11.8%) were recorded among the mutant strains as compared with the nonmutant types. All the clb+ / E coli showed complete resistance to amoxicillin, Augmentin (amoxicillin and clavulanate), gentamicin, and trimethoprim/sulfamethoxazole. The fecal clb -encoded E coli (1.5%) were not associated with demographic status, fiber-based food (odds ratio [OR], 1.03; 95% CI, 56.74–138.7; P =.213), alcohol (OR, 1.27; 95% CI, 61.74–147.1; P =.221), antibiotic consumptions (OR, 1.11; 95% CI, 61.29–145.3; P =.222), and handwashing (OR, 1.17; 95% CI, 60.19–145.5; P =.216). The hierarchical cluster of blm+/ E coli revealed high-level resistance with a multiantibiotic resistance index ≥0.2 (P <.05). Only 12% of all strains were tryptophan mutant/ blm +, and 1.5% of clb +/ECblm+ were observed in fecal samples with a 452–base pair size. Trimethoprim/sulfamethoxazole and biofilm production positively regressed with clb expression (P >.05). Principal component analysis score plot indicated an association of clb +/ECblm+ with dietary pattern, alcohol, blm, and hemolysin production. The combined activity of blm and biofilm production in the gut microbiota could promote clb +/ E coli colonization, facilitating genotoxin production and possible colorectal cancer induction. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is the second-highest cause of cancer-associated mortality among both men and women worldwide. One of the risk factors for CRC is obesity, which is correlated with a high-fat diet prevalent in Western dietary habits. The association between an obesogenic high-fat diet and CRC has been established for several decades; however, the mechanisms by which a high-fat diet increases the risk of CRC remain unclear. Recent studies indicate that gut microbiota strongly influence the pathogenesis of both high-fat diet-induced obesity and CRC. The gut microbiota is composed of hundreds of bacterial species, some of which are implicated in CRC. In particular, the expansion of facultative anaerobic Enterobacteriaceae, which is considered a microbial signature of intestinal microbiota functional imbalance (dysbiosis), is associated with both high-fat diet-induced obesity and CRC. Here, we review the interaction between the gut microbiome and its metabolic byproducts in the context of colorectal cancer (CRC) during high-fat diet-induced obesity. In addition, we will cover how a high-fat diet can drive the expansion of genotoxin-producing Escherichia coli by altering intestinal epithelial cell metabolism during gut inflammation conditions. [ABSTRACT FROM AUTHOR]

ABSTRACTColibactin, a bacterial genotoxin produced by E. coli strains harboring the pks genomic island, induces cytopathic effects, such as DNA breaks, cell cycle arrest, and apoptosis. Patients with inflammatory bowel diseases, such as ulcerative colitis, display changes in their microbiota with the expansion of E. coli. Whether and how colibactin affects the integrity of the colonic mucosa and whether pks+ E. coli contributes to the pathogenesis of colitis is not clear. Using a gnotobiotic mouse model, we show that under homeostatic conditions, pks+ E. coli do not directly interact with the epithelium or affect colonic integrity. However, upon short-term chemical disruption of mucosal integrity, pks+ E. coli gain direct access to the epithelium, causing epithelial injury and chronic colitis, while mice colonized with an isogenic ΔclbR mutant incapable of producing colibactin show a rapid recovery. pks+ E. coli colonized mice are unable to reestablish a functional barrier. In turn, pks+ E. coli remains in direct contact with the epithelium, perpetuating the process and triggering chronic mucosal inflammation that morphologically and transcriptionally resembles human ulcerative colitis. This state is characterized by impaired epithelial differentiation and high proliferative activity, which is associated with high levels of stromal R-spondin 3. Genetic overexpression of R-spondin 3 in colon myofibroblasts is sufficient to mimic barrier disruption and expansion of E. coli. Together, our data reveal that pks+ E. coli are pathobionts that promote severe injury and initiate a proinflammatory trajectory upon contact with the colonic epithelium, resulting in a chronic impairment of tissue integrity.

ABSTRACTColorectal cancer (CRC) patients are frequently colonized by colibactin-producing Escherichia coli (CoPEC) (>40%), which enhances tumorigenesis in mouse models of CRC. We observed that 50% of CoPEC also contains the cnf1 gene, which encodes cytotoxic necrotizing factor-1 (CNF1), an enhancer of the eukaryotic cell cycle. The impact of its co-occurrence with colibactin (Clb) has not yet been investigated. We evaluated the impact of CNF1 on colorectal tumorigenesis using human colonic epithelial HT-29 cells and CRC-susceptible ApcMin/+ mice inoculated with the CoPEC 21F8 clinical strain (Clb+Cnf+) or 21F8 isogenic mutants (Clb+Cnf-, Clb-Cnf+ and Clb-Cnf-). Infection with the Clb+Cnf- strain induced higher levels of inflammatory cytokines and senescence markers both in vitro and in vivo compared to those induced by infection with the Clb+Cnf+ strain. In contrast, the Clb+Cnf- and Clb+Cnf+ strains generated similar levels of DNA damage in HT-29 cells and in colonic murine tissues. Furthermore, the ApcMin/+ mice inoculated with the Clb+Cnf- strain developed significantly more tumors than the mice inoculated with the Clb+Cnf+ strain or the isogenic mutants, and the composition of their microbiota was changed. Finally, rectal administration of the CNF1 protein in ApcMin/+ mice inoculated with the Clb+Cnf- strain significantly decreased tumorigenesis and inflammation. Overall, this study provides evidence that CNF1 decreases the carcinogenic effects of CoPEC in ApcMin/+ mice by decreasing CoPEC-induced cellular senescence and inflammation.

ABSTRACTUp to 25% of the E. coli strains isolated from the feces of healthy humans harbor the pks genomic island encoding the synthesis of colibactin, a genotoxic metabolite. Evidence is accumulating for an etiologic role of colibactin in colorectal cancer. Little is known about the conditions of expression of colibactin in the gut. The intestine is characterized by a unique oxygenation profile, with a steep gradient between the physiological hypoxic epithelial surface and the anaerobic lumen, which favors the dominance of obligate anaerobes. Here, we report that colibactin production is maximal under anoxic conditions and decreases with increased oxygen concentration. We show that the aerobic respiration control (ArcA) positively regulates colibactin production and genotoxicity of pks+ E. coli in response to oxygen availability. Thus, colibactin synthesis is inhibited by oxygen, indicating that the pks biosynthetic pathway is adapted to the anoxic intestinal lumen and to the hypoxic infected or tumor tissue.

Herein, we report the synthesis of a series of colibactin warhead model compounds using two newly developed metal‐free photocatalytic cyclopropanation reactions. These mild cyclopropanations expand the known applications of eosin within synthesis. A halogen atom transfer reaction mode has been harnessed so that dihalides can be used as the cyclopropanating agents. The colibactin warhead models were then used to provide new insight into two key mechanisms in colibactin chemistry. An explanation is provided for why the colibactin warhead sometimes undergoes a ring expansion‐addition reaction to give fused cyclobutyl products while at other times nucleophiles add directly to the cyclopropyl unit (as when DNA adds to colibactin). Finally, we provide some evidence that Cu(II) chelated to colibactin may catalyze an important oxidation of the colibactin‐DNA adduct. The Cu(I) generated as a result could then also play a role in inducing double strand breaks in DNA. [ABSTRACT FROM AUTHOR]

Colibactin is a genotoxic natural product produced by select commensal bacteria in the human gut microbiota. The compound is a bis‐electrophile that is predicted to form interstrand DNA cross‐links in target cells, leading to double‐strand DNA breaks. The biosynthesis of colibactin is carried out by a mixed NRPS–PKS assembly line with several noncanonical features. An amidase, ClbL, plays a key role in the pathway, catalyzing the final step in the formation of the pseudodimeric scaffold. ClbL couples α‐aminoketone and β‐ketothioester intermediates attached to separate carrier domains on the NRPS–PKS assembly. Here, the 1.9 Å resolution structure of ClbL is reported, providing a structural basis for this key step in the colibactin biosynthetic pathway. The structure reveals an open hydrophobic active site surrounded by flexible loops, and comparison with homologous amidases supports its unusual function and predicts macromolecular interactions with pathway carrier‐protein substrates. Modeling protein–protein interactions supports a predicted molecular basis for enzyme–carrier domain interactions. Overall, the work provides structural insight into this unique enzyme that is central to the biosynthesis of colibactin. [ABSTRACT FROM AUTHOR]

Some Escherichia coli strains harbour the pks island, a 54 kb genomic island en-coding the biosynthesis genes for a genotoxic compound named colibactin. In eukaryotic cells, colibactin can induce DNA damage, cell cycle arrest and chro-mosomal instability. Production of colibactin has been implicated in the devel-opment of colorectal cancer (CRC). In this study, we demonstrate the inhibito-ry effect of D-Serine on the expression of the pks island in both prototypic and clinically-associated colibactin-producing strains and determine the implications for cytopathic effects on host cells. We also tested a comprehensive panel of proteinogenic L-amino acids and corresponding D-enantiomers for their ability to modulate clbB transcription. Whilst several D-amino acids exhibited the abil-ity to inhibit expression of clbB, D-Serine exerted the strongest repressing ac-tivity (>3.8-fold) and thus, we focussed additional experiments on D-Serine. To investigate the cellular effect, we investigated if repression of colibactin by D-Serine could reduce the cytopathic responses normally observed during infec-tion of HeLa cells with pks+ strains. Levels of γ-H2AX (a marker of DNA double strand breaks) were reduced 2.75-fold in cells infected with D-Serine treat-ment. Moreover, exposure of pks+ E. coli to D-Serine during infection caused a reduction in cellular senescence that was observable at 72 h post infection. The recent finding of an association between pks-carrying commensal E. coli and CRC, highlights the necessity for the development of colibactin targeting thera-peutics. Here we show that D-Serine can reduce expression of colibactin, and inhibit downstream cellular cytopathy, illuminating its potential to prevent colibactin-associated disease.

Abstract Background Colibactin is a genotoxin produced by Escherichia coli and other Enterobacteriaceae that is believed to increase the risk of colorectal cancer (CRC) of their symbiosis hosts, including human. A peptidase ClbP is the key enzyme for activation of colibactin. Inhibition of ClbP is considered to impede maturation of precolibactin into genotoxic colibactin. Therefore, ClbP-specific inhibitors could potentially prevent the onset of CRC, one of the leading causes of cancer-related deaths in the world. This study intends to establish an efficient screening system for identifying inhibitors that are specific to ClbP. Methods Two types of assays were applied in the screening procedure: a probe assay and an LC–MS assay. For the probe assay, we employed the synthesized probe which we described in our previous report. This probe can be hydrolyzed efficiently by ClbP to release a fluorophore. Hence it was applied here for detection of inhibition of ClbP. For the LC–MS assay, formation of the byproduct of precolibactin maturation process, N-myristoyl-D-asparagine, was quantified using a liquid chromatography–mass spectrometry (LC–MS) technique. The probe assay can be performed much faster, while the LC–MS assay is more accurate. Therefore, our method employed the two assays in sequence to screen a large number of compounds for inhibition of ClbP. Results A library of 67,965 standard compounds was evaluated by the screening method established in the current study, and one compound was found to show a moderate inhibitory activity against ClbP. Conclusion A simple screening method for ClbP-specific inhibitors was established. It was proven to be reliable and is believed to be useful in developing potential prophylactic agents for CRC.

Certain Enterobacteriaceae strains contain a 54-kb biosynthetic gene cluster referred to as “pks” encoding the biosynthesis of a secondary metabolite, colibactin. Colibactin-producing E. coli promote colorectal cancer (CRC) in preclinical models, and in vitro induce a specific mutational signature that is also detected in human CRC genomes. Yet, how colibactin exposure affects the mutational landscape of CRC in vivo remains unclear. Here we show that colibactin-producing E. coli-driven colonic tumors in mice have a significantly higher SBS burden and a larger percentage of these mutations can be attributed to a signature associated with mismatch repair deficiency (MMRd; SBS15), compared to tumors developed in the presence of colibactin-deficient E. coli. We found that the synthetic colibactin 742 but not an inactive analog 746 causes DNA damage and induces transcriptional activation of p53 and senescence signaling pathways in non-transformed human colonic epithelial cells. In MMRd colon cancer cells (HCT 116), chronic exposure to 742 resulted in the upregulation of BRCA1, Fanconi anemia, and MMR signaling pathways as revealed by global transcriptomic analysis. This was accompanied by increased T>N single-base substitutions (SBS) attributed to the proposed pks+ E. coli signature (SBS88), reactive oxygen species (SBS17), and mismatch-repair deficiency (SBS44). A significant co-occurrence between MMRd SBS44 and pks-associated SBS88 signature was observed in a large cohort of human CRC patients (n=2,945), and significantly more SBS44 mutations were found when SBS88 was also detected. Collectively, these findings reveal the host response mechanisms underlying colibactin genotoxic activity and suggest that colibactin may exacerbate MMRd-associated mutations.

ABSTRACT The pks gene cluster encodes colibactin, which can cause DNA damage and enhance the virulence in Escherichia coli. However, the role of the pks gene in Klebsiella pneumoniae has not been fully discussed. The aim of this study was to analyze the relationship between the pks gene cluster and virulence factors, as well as to assess antibiotic resistance and biofilm formation capacity in clinical isolates of Klebsiella pneumoniae. Thirty-eight of 95 clinical K. pneumoniae strains were pks positive. pks-positive strains usually infected emergency department patients, and pks-negative strains often infected hospitalized patients. The positive rates of K1 capsular serotype and hypervirulence genes (peg-344, rmpA, rmpA2, iucA, and iroB) were significantly higher in the pks-positive isolates than the pks-negative isolates (P < 0.05). The biofilm formation ability of pks-positive isolates was stronger than that of pks-negative isolates. Antibacterial drug susceptibility test showed the resistance of pks-positive isolates was weaker than that of pks-negative isolates. In conclusion, patients with pks-positive K. pneumoniae infection might have worse treatment outcomes and prognosis. pks-positive K. pneumoniae might have stronger virulence and pathogenicity. Clinical infection with pks-positive K. pneumoniae needs further attention. IMPORTANCE The infection rate with pks-positive K. pneumoniae has been increasing in recent years. Two previous surveys in Taiwan reported 25.6% pks gene islands and 16.7% pks-positive K. pneumoniae strains in bloodstream infections, and Chinese scholars also did a survey of K. pneumoniae bloodstream infections in Changsha, China, and found 26.8% pks-positive K. pneumoniae. In addition, it was found that the pks gene cluster might encode colibactin, which could be related to the virulence of K. pneumoniae. Studies confirmed that the prevalence of colibactin-producing K. pneumoniae was increasing. It is necessary to consider the clear relationship between the pks gene cluster and high pathogenicity in K. pneumoniae.

Abstract 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 (

In silico genome mining provides easy access to secondary metabolite biosynthetic gene clusters (BGCs) encoding the biosynthesis of many bioactive compounds, which are the basis for many important drugs used in human medicine. However, the association between BGCs and other functions encoded in the genomes of producers have remained elusive. Here, we present a systems biology workflow that integrates genome mining with a detailed pangenome analysis for detecting genes associated with a particular BGC. We analyzed 3,889 enterobacterial genomes and found 13,266 BGCs, represented by 252 distinct BGC families and 347 additional singletons. A pangenome analysis revealed 88 genes putatively associated with a specific BGC coding for the colon cancer-related colibactin that code for diverse metabolic and regulatory functions. The presented workflow opens up the possibility to discover novel secondary metabolites, better understand their physiological roles, and provides a guide to identify and analyze BGC associated gene sets.

Background: Colibactin is a genotoxin produced by Escherichia coli and other Enterobacteriaceae that is believed to increase the risk of colorectal cancer (CRC) of their symbiosis hosts, including human. A peptidase ClbP is the key enzyme for activation of colibactin. Inhibition of ClbP is considered to impede maturation of precolibactin into genotoxic colibactin. Therefore, ClbP-specific inhibitors could potentially prevent the onset of CRC, one of the leading causes of cancer-related deaths in the world. This study intends to establish an efficient screening system for identifying inhibitors that are specific to ClbP. Methods: Two types of assays were applied in the screening procedure: a probe assay and an LC–MS assay. For the probe assay, we employed the synthesized probe which we described in our previous report. This probe can be hydrolyzed efficiently by ClbP to release a fluorophore. Hence it was applied here for detection of inhibition of ClbP. For the LC–MS assay, formation of the byproduct of precolibactin maturation process, N-myristoyl-D-asparagine, was quantified using a liquid chromatography–mass spectrometry (LC–MS) technique. The probe assay can be performed much faster, while the LC–MS assay is more accurate. Therefore, our method employed the two assays in sequence to screen a large number of compounds for inhibition of ClbP. Results: A library of 67,965 standard compounds was evaluated by the screening method established in the current study, and one compound was found to show a moderate inhibitory activity against ClbP. Conclusion: A simple screening method for ClbP-specific inhibitors was established. It was proven to be reliable and is believed to be useful in developing potential prophylactic agents for CRC. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae (K. pneumoniae) colonizes the human gut and is a causative factor of pyogenic liver abscess (PLA). Retrospective studies conducted on K. pneumoniae PLA patients revealed subsequent CRC development in later years of their life with increasing prevalence of these strains harbouring polyketide synthase (PKS) genes. To our knowledge there are no known studies directly implicating K. pneumoniae with CRC to date. Our aims are to characterize K. pneumoniae isolates from CRC patients and investigate its effects on cell proliferation in vitro. K. pneumoniae isolates were characterized by screening virulence genes including polyketide synthase (PKS), biofilm assay, antibiotic susceptibility, and string test to determine hypervirulent (hvKp) strains. Solubilised antigens of selected K. pneumoniae isolates were co-cultured with primary colon cell lines and CRC cell lines (Stage I-IV) for 48 h. The enhancement of proliferation was measured through MTT and ECIS assay. Twenty-five percent of K. pneumoniae isolates were PKS-positive out of which 50% were hvKp strains. The majority of the isolates were from the more virulent serotype of K1 (30%) and K2 (50%). PKS-positive K. pneumoniae isolates did not possess genes to confer carbapenem resistance but instead were more highly associated with siderophore genes (aerobactin, enterobactin, and yersiniabactin) and allantoin metabolism genes (allS, allS2). Cell proliferation in primary colon, SW1116 (Stage I), and SW480 (Stage II) CRC cell lines were enhanced when co-cultured with PKS-positive K. pneumoniae antigens. ECIS revealed enhanced cell proliferation upon recurrent antigen exposure. This demonstrates the possible role that PKS-positive K. pneumoniae has in exacerbating CRC progression. [ABSTRACT FROM AUTHOR]

ABSTRACT The pks island is one of the most prevalent pathogenicity islands among the Escherichia coli strains that colonize the colon of colorectal carcinoma (CRC) patients. This pathogenic island encodes the production of a nonribosomal polyketide-peptide named colibactin, which induces double-strand breaks in DNA molecules. Detection or even depletion of this pks-producing bacteria could help to understand the role of these strains in the context of CRC. In this work, we performed a large-scale in silico screening of the pks cluster in more than 6,000 isolates of E. coli. The results obtained reveal that not all the pks-detected strains could produce a functional genotoxin and, using antibodies against pks-specific peptides from surface cell proteins, a methodology for detection and depletion of pks+ bacteria in gut microbiotas was proposed. With our method, we were able to deplete a human gut microbiota of this pks+ strains, opening the door to strain-directed microbiota modification and intervention studies that allow us to understand the relation between these genotoxic strains and some gastrointestinal diseases. IMPORTANCE The human gut microbiome has also been hypothesized to play a crucial role in the development and progression of colorectal carcinoma (CRC). Between the microorganisms of this community, the Escherichia coli strains carrying the pks genomic island were shown to be capable of promoting colon tumorigenesis in a colorectal cancer mouse model, and their presence seems to be directly related to a distinct mutational signature in patients suffering CRC. This work proposes a novel method for the detection and depletion of pks-carrying bacteria in human gut microbiotas. In contrast to methods based on probes, this methodology allows the depletion of low-abundance bacterial strains maintaining the viability of both targeted and non-targeted fractions of the microbiota, allowing the study of the contribution of these pks-carrying strains to different diseases, such as CRC, and their role in other physiological, metabolic or immune processes.

Intestinal inflammation is a risk factor for colorectal cancer formation, but the underlying mechanisms remain unknown. Here, we investigated whether colitis alters the colonic microbiota to enhance its cancer-inducing activity. Colitis increased epithelial oxygenation in the colon of mice and drove an expansion of Escherichia coli within the gut-associated microbial community through aerobic respiration. An aerobic expansion of colibactin-producing E. coli was required for the cancer-inducing activity of this pathobiont in a mouse model of colitis-associated colorectal cancer formation. We conclude that increased epithelial oxygenation in the colon is associated with an expansion of a prooncogenic driver species, thereby increasing the cancer-inducing activity of the microbiota.IMPORTANCE One of the environmental factors important for colorectal cancer formation is the gut microbiota, but the habitat filters that control its cancer-inducing activity remain unknown. Here, we show that chemically induced colitis elevates epithelial oxygenation in the colon, thereby driving an expansion of colibactin-producing Escherichia coli, a prooncogenic driver species. These data suggest that elevated epithelial oxygenation is a potential risk factor for colorectal cancer formation because the consequent changes in the gut habitat escalate the cancer-inducing activity of the microbiota.

IntroductionThe 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.MethodsMice 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.ResultsInulin 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.DiscussionOur 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.

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. [ABSTRACT FROM AUTHOR]

The structure and mode of action of colibactin with its potential involvement in cancer have been extensively studied but little is known about the intrinsic function of the biosynthetic gene cluster, coding for colibactin, as a bacterial genotoxin. Paradoxically, this pathogenicity island is also found in commensal and probiotic strains of Escherichia coli and in bacterial species colonizing olive trees and the digestive tract of bees. In this review, we summarize the available literature to address the following key questions. What does this genomic island really encode? What explains the extensive dissemination of this genetically mobile element? What do we really know about the biosynthetic and secretory pathways of colibactin? What is its inherent target/function? Colibactin synthesis is severely constrained: it needs to be produced efficiently and targeted to the nucleus of eukaryotic cells or to the genome of competitive bacterial species without damaging the DNA of the pks + bacteria. The energy-intensive synthesis of colibactin is finely regulated by environmental and primary metabolic compounds. The pks island interacts with other biosynthetic pathways to produce secondary metabolites that affect fitness, virulence, and interbacterial competition. The pks island expresses a repertoire of metabolites beyond colibactin, which impact host–pathogen and intermicrobial interactions. The high degree of sequence conservation between pks islands may be explained by the extensive selective benefits of expressing colibactin and its secondary metabolites that ultimately confer an advantage to pks + bacteria, in terms of their interactions with both the microbiota and the host, to effectively enable adaptation to specific ecological niches. [ABSTRACT FROM AUTHOR]

The Klebsiella species present a remarkable genetic and ecological diversity, being ubiquitous in nature. In particular, the Klebsiella pneumoniae species complex (KpSC) has emerged as a major public health threat in the world, being an interesting model to assess the risk posed by strains recovered from animals and the environment to humans. We therefore performed a genomic surveillance analysis of the KpSC using every public genome in Brazil, aiming to show their local and global relationships, and the connectivity of antibiotic resistance and virulence considering human, animal, and environmental sources. The 390 genomes from distinct sources encompassed the K. pneumoniae, Klebsiella quasipneumoniae subsp. quasipneumoniae, Klebsiella quasipneumoniae subsp. similipneumoniae, Klebsiella variicola subsp. variicola, Klebsiella variicola subsp. tropica, and Klebsiella grimontii species and subspecies. K. pneumoniae harbored dozens of antibiotic resistance genes, while most of the genomes belong to the high-risk pandemic CC258 occurring in humans, animals, and the environment. In K. pneumoniae ST11, a high prevalence of the virulence determinants yersiniabactin, colibactin, and T6SS was revealed in association with multi-drug resistance (MDR), including carbapenem resistance. A diversity of resistance genes is carried by plasmids, some shared between strains from different STs, regions, and sources. Therefore, here were revealed some factors driving the success of KpSC as a pathogen. [ABSTRACT FROM AUTHOR]

Appropriate screening of early asymptomatic cases can reduce the disease burden and mortality rate of sporadic colorectal cancer (CRC) significantly. Currently, fecal occult blood testing (FOBT) is able to detect up to 80% of ­asymptomatic cases in the population aged 50+. Therefore, there is still a demand for new screening tests that would complement FOBT, mainly by detecting at least a part of the FOBT-negative CRC and adenoma cases, or possibly by identifying person at increased risk of sporadic CRC in order to offer them tailored follow-up. Among the potential markers studied, our knowledge has advanced at most in toxigenic gram-negative bacteria. In this review, we assess their potential critically and recommend those best suited for prospective evaluation of their true ability to increase the sensitivity of FOBT when combined during general population screening. In our opinion, colibactin and Bacteroides fragilis toxin are the best candidates, possibly complemented by the cytotoxic necrotizing factor (CNF).

The pks genotoxic K. pneumoniae has recently triggered a widespread alarm. DNA damage and higher virulence have been linked to colibactin, a genotoxin expressed by the pks genomic island. Little is known about its molecular epidemiology in clinical isolates from Egypt. Therefore, this study was conducted to determine the prevalence and the microbiological and clinical features of pks harboring hospital-acquired K. pneumoniae isolates from Egypt. Eighty-seven hospital-acquired K. pneumoniae isolates from various specimen types were screened for pks colibactin island markers clbB, clbQ, clbA, and clbN by PCR. The pks-positive hvKp isolates were classified to one of the capsular types K1 and K2 using multiplex-PCR targeting K-serotype wzi and rmpA genes. The prevalence of pks+ strains was 27.6% (24/87). K1 capsular type, phenotypic, and genotypic hypervirulent isolates were significantly higher among pks+ strains than pks− strains (P < 0.001), while pks+ K. pneumoniae strains were found to be significantly less resistant to 8 of the antibiotic compounds tested than pks− strains. Carriage of K1 capsular type and mucoviscosity-associated rmp A gene and diabetes mellitus were identified to remain independent risk factors having a substantial association to pks-positivity by multivariate regression analysis. In conclusion, Hospital-acquired K. pneumoniae isolates in Egypt had an increased prevalence of the pks colibactin genotoxin. The significant occurrence of hypervirulent determinants in pks+ K. pneumoniae highlighted the genotoxin's possible pathogenicity combined with its distribution in several specimen types, which necessitates clinical attention and epidemic tracking.

Diet provides macronutrients (carbohydrates, proteins, and fats), micronutrients (vitamins and minerals), and phytochemicals (non-nutrient bioactive compounds). Emerging evidence suggests that above dietary components can directly impact the composition and metabolic activity of the mammalian gut microbiota and in turn, affect both physical and mental health. There is a growing recognition that rise in chronic disease burden in Western countries may due to progressive loss of beneficial bacteria and microbial diversity. This perspective explores the possibility of using Indian thali, an ancient approach to diet that provides both fiber and different phytochemicals by incorporating a variety of plant foods in different colors. This variety helps to restore diversity in the gut bacteria and may potentially prevent or reverse chronic disease, such as colon cancer or type 2 diabetes.

Colibactin is synthesized by a 54-kb genomic island, leads to toxicity in eukaryotic cells, and plays a vital role in many diseases, including neonatal sepsis and meningitis. Avian pathogenic Escherichia coli (APEC) is speculated to be an armory of extraintestinal pathogenic Escherichia coli and can be a potential zoonotic bacterium that threatens human and animal health. In this study, the APEC XM meningitis mouse model was successfully established to investigate the effect of colibactin in in vivo infection. The clbH-deletion mutant strain induced lower γ-H2AX expression, no megalocytosis, and no cell cycle arrest in bEnd.3 cells, which showed that the deletion of clbH decreased the production of colibactin in the APEC XM strain. The deletion of clbH did not affect the APEC XM strain’s ability of adhering to and invading bEnd.3 cells. In vitro, the non-colibactin-producing strain displayed significantly lower serum resistance and it also induced a lower level of cytokine mRNA and few disruptions of tight junction proteins in infected bEnd.3 cells. Meningitis did not occur in APEC ΔclbH-infected mice in vivo, who showed fewer clinical symptoms and fewer lesions on radiological and histopathological analyses. Compared with the APEX XM strain, APEC ΔclbH induced lower bacterial colonization in tissues, lower mRNA expression of cytokines in brain tissues, and slight destruction of the brain blood barrier. These results indicate that clbH is a necessary component for the synthesis of genotoxic colibactin, and colibactin is related to the development of meningitis induced by APEC XM.

The human microbiota produces molecules that are evolved to interact with the diverse cellular machinery of both the host and microbes, mediating health and diseases. One of the most puzzling microbiome molecules is colibactin, a genotoxin encoded in some commensal and extraintestinal microbes and is implicated in initiating colorectal cancer. The colibactin cluster was discovered more than 15 years ago, and most of the research studies have been focused on revealing the biosynthesis and precise structure of the cryptic encoded molecule(s) and the mechanism of carcinogenesis. In 2022, the Balskus group revealed that colibactin not only hits targets in the eukaryotic cell machinery but also in the prokaryotic cell. To that end, colibactin crosslinks the DNA resulting in activation of the SOS signaling pathway, leading to prophage induction from bacterial lysogens and modulation of virulence genes in pathogenic species. These unique activities of colibactin highlight its ecological role in shaping gut microbial communities and further consequences that impact human health. This review dives in-depth into the molecular mechanisms underpinning colibactin cellular targets in eukaryotic and prokaryotic cells, aiming to understand the fine details of the role of secreted microbiome chemistry in mediating host-microbe and microbe-microbe interactions. This understanding translates into a better realization of microbiome potential and how this could be advanced to future microbiome-based therapeutics or diagnostic biomarkers. [ABSTRACT FROM AUTHOR]

In silico genome mining provides easy access to secondary metabolite biosynthetic gene clusters (BGCs) encoding the biosynthesis of many bioactive compounds, which are the basis for many important drugs used in human medicine. However, the association between BGCs and other functions encoded in the genomes of producers have remained elusive. Here, we present a systems biology workflow that integrates genome mining with a detailed pangenome analysis for detecting genes associated with a particular BGC. We analyzed 3,889 enterobacterial genomes and found 13,266 BGCs, represented by 252 distinct BGC families and 347 additional singletons. A pangenome analysis revealed 88 genes putatively associated with a specific BGC coding for the colon cancer-related colibactin that code for diverse metabolic and regulatory functions. The presented workflow opens up the possibility to discover novel secondary metabolites, better understand their physiological roles, and provides a guide to identify and analyze BGC associated gene sets. [ABSTRACT FROM AUTHOR]

Engineered microbes are rapidly being developed for the delivery of therapeutic modalities to sites of disease. Escherichia coli Nissle 1917 (EcN), a genetically tractable probiotic with a well-established human safety record, is emerging as a favored chassis. Here, we summarize the latest progress in rationally engineered variants of EcN for the treatment of infectious diseases, metabolic disorders, and inflammatory bowel diseases (IBDs) when administered orally, as well as cancers when injected directly into tumors or the systemic circulation. We also discuss emerging studies that raise potential safety concerns regarding these EcN-based strains as therapeutics due to their secretion of a genotoxic colibactin that can promote the formation of DNA double-stranded breaks in mammalian DNA. Due to its genetic tractability; track record of safety in humans; and ability to home to, survive, and replicate in the gastrointestinal tract and tumors, Escherichia coli Nissle 1917 (EcN) has become a favored chassis for engineering 'smart microbes' that deliver therapeutic modalities to sites of disease. EcN variants engineered with new therapeutic properties via alterations in its metabolic pathways or the introduction of in situ drug delivery systems are showing promise for the treatment of bacterial infections, inflammatory bowel diseases, metabolic disorders, and cancer. Clinical trials are underway to test the safety and efficacy of EcN-based smart microbes for inborn diseases of metabolism and cancer. EcN synthesizes a genotoxin called colibactin capable of causing DNA double-strand breaks in mammalian cells, raising safety concerns about its use clinically. [ABSTRACT FROM AUTHOR]

We present the genome sequence of Escherichia coli 5C LMG S-33222, a non-pathogenic microorganism with potential probiotic features. The strain was isolated in 2021 from a fecal sample of a healthy Italian infant. The total genome size is 4,712,575 bp with a G + C content of 51%.

Colibactin, a nonribosomal peptide/polyketide produced by pks+ Enterobacteriaceae , is a virulence factor and putative carcinogen that damages DNA by interstrand crosslinking (ICL). While the clb genes for colibactin biosynthesis have been identified, studies are needed to elucidate the mechanisms regulating colibactin production and activity. Here we perform untargeted metabolomics of pks+ Escherichia coli cultures to identify L-tryptophan as a candidate repressor of colibactin activity. When pks+ E. coli is grown in a minimal medium supplemented with L-tryptophan in vitro ICL of plasmid DNA is reduced by >80%. L-tryptophan does not affect the transcription of clb genes but protects from copper toxicity and triggers the expression of genes to export copper to the periplasm where copper can directly inhibit the ClbP peptidase domain. Thus, L-tryptophan and copper interact and repress colibactin activity, potentially reducing its carcinogenic effects in the intestine., Importance: Colibactin is a small molecule produced by pks + Enterobacteriaceae that damages DNA, leading to oncogenic mutations in human genomes. Colibactin-producing Escherichia coli ( pks +) cells promote tumorigenesis in mouse models of colorectal cancer (CRC) and are elevated in abundance in CRC patient biopsies, making it important to identify the regulatory systems governing colibactin production. Here, we apply a systems biology approach to explore metabolite repression of colibactin production in pks + E. coli . We identify L-tryptophan as a repressor of colibactin genotoxicity that stimulates the expression of genes to export copper to the periplasm where it can inhibit ClbP, the colibactin-activating peptidase. These results work toward an antibiotic-sparing, prophylactic strategy to inhibit colibactin genotoxicity and its tumorigenic effects in the intestine.

Abstract Background The Escherichia coli strain that is known to produce the genotoxic secondary metabolite colibactin is linked to colorectal oncogenesis. Therefore, understanding the properties of such colibactin-positive E. coli and the molecular mechanism of oncogenesis by colibactin may provide us with opportunities for early diagnosis or prevention of colorectal oncogenesis. While there have been major advances in the characterization of colibactin-positive E. coli and the toxin it produces, the infection route of the clb + strain remains poorly characterized. Results We examined infants and their treatments during and post-birth periods to examine potential transmission of colibactin-positive E. coli to infants. Here, analysis of fecal samples of infants over the first month of birth for the presence of a colibactin biosynthetic gene revealed that the bacterium may be transmitted from mother to infant through intimate contacts, such as natural childbirth and breastfeeding, but not through food intake. Conclusions Our finding suggests that transmission of colibactin-positive E. coli appears to be occurring at the very early stage of life of the newborn and hints at the possibility of developing early preventive measures against colorectal cancer.

The human microbiota produces molecules that are evolved to interact with the diverse cellular machinery of both the host and microbes, mediating health and diseases. One of the most puzzling microbiome molecules is colibactin, a genotoxin encoded in some commensal and extraintestinal microbes and is implicated in initiating colorectal cancer. The colibactin cluster was discovered more than 15 years ago, and most of the research studies have been focused on revealing the biosynthesis and precise structure of the cryptic encoded molecule(s) and the mechanism of carcinogenesis. In 2022, the Balskus group revealed that colibactin not only hits targets in the eukaryotic cell machinery but also in the prokaryotic cell. To that end, colibactin crosslinks the DNA resulting in activation of the SOS signaling pathway, leading to prophage induction from bacterial lysogens and modulation of virulence genes in pathogenic species. These unique activities of colibactin highlight its ecological role in shaping gut microbial communities and further consequences that impact human health. This review dives in-depth into the molecular mechanisms underpinning colibactin cellular targets in eukaryotic and prokaryotic cells, aiming to understand the fine details of the role of secreted microbiome chemistry in mediating host–microbe and microbe–microbe interactions. This understanding translates into a better realization of microbiome potential and how this could be advanced to future microbiome-based therapeutics or diagnostic biomarkers.

Abstract 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.

Klebsiella pneumoniae (K. pneumoniae) colonizes the human gut and is a causative factor of pyogenic liver abscess (PLA). Retrospective studies conducted on K. pneumoniae PLA patients revealed subsequent CRC development in later years of their life with increasing prevalence of these strains harbouring polyketide synthase (PKS) genes. To our knowledge there are no known studies directly implicating K. pneumoniae with CRC to date. Our aims are to characterize K. pneumoniae isolates from CRC patients and investigate its effects on cell proliferation in vitro. K. pneumoniae isolates were characterized by screening virulence genes including polyketide synthase (PKS), biofilm assay, antibiotic susceptibility, and string test to determine hypervirulent (hvKp) strains. Solubilised antigens of selected K. pneumoniae isolates were co-cultured with primary colon cell lines and CRC cell lines (Stage I-IV) for 48 h. The enhancement of proliferation was measured through MTT and ECIS assay. Twenty-five percent of K. pneumoniae isolates were PKS-positive out of which 50% were hvKp strains. The majority of the isolates were from the more virulent serotype of K1 (30%) and K2 (50%). PKS-positive K. pneumoniae isolates did not possess genes to confer carbapenem resistance but instead were more highly associated with siderophore genes (aerobactin, enterobactin, and yersiniabactin) and allantoin metabolism genes (allS, allS2). Cell proliferation in primary colon, SW1116 (Stage I), and SW480 (Stage II) CRC cell lines were enhanced when co-cultured with PKS-positive K. pneumoniae antigens. ECIS revealed enhanced cell proliferation upon recurrent antigen exposure. This demonstrates the possible role that PKS-positive K. pneumoniae has in exacerbating CRC progression.

Members of complex microbial communities that reside in environments such as the mammalian gut have evolved mechanisms of interspecies competition, which may be directed at resident microbial and host cells. While previous work has focused mainly on metabolic or niche competition, few specific intermicrobial targeting mechanisms have been elucidated in the mammalian gut. Here, we show that a genotoxin produced by commensal Escherichia coli, colibactin, which was previously shown to induce DNA damage in host intestinal cells, is also able to target via a contact-dependent mechanism a variety of enteric pathogens and commensals, including the important human diarrheal pathogen Vibrio cholerae. We find that colibactin-mediated killing depends on accumulation of intracellular reactive oxygen species, leading to DNA damage and loss of target cell fitness. We also show that the presence of colibactin is associated with cholera outcomes in a large human metagenomic dataset and that colibactin can shape the microbiome by species-specific targeting of a common human gut-associated microbe Bacteroides fragilis, suggesting that genotoxin-mediated mechanisms may have broad effects in the complex polymicrobial interactions that shape commensal microbial communities and their effects on host health and disease. [ABSTRACT FROM AUTHOR]

Background/Aim: Among colorectal cancerassociated intestinal microbiota, colibactin-producing (clb+) bacteria are attracting attention. We aimed to clarify the interaction between clb+ Escherichia coli and normal colorectal epithelial cells in vivo and in vitro. Materials and Methods: Five-week-old female Balb/c mice were divided in an untreated group, a group treated with clb+ E. coli isolated from a Japanese patient with colorectal cancer (E. coli-50), and a group treated with non colibactin-producing E. coli (E. coli-50/ΔclbP). Mice were sacrificed at 18 weeks of treatment. Results: Treatment with clb+ E. coli increased positivity for H2A histone family member X phosphorylated at Ser-139 (γH2AX) in epithelial cells of the luminal surface of the mouse rectum but this did not occur in the E. coli- 50/ΔclbP and untreated groups. In an in vitro setting, the ratio of apoptotic cells was increased and cell counts were reduced by treatment with clb+ E. coli more than in untreated cells and normal rat colorectal epithelial cells. Conclusion: E. coli-50 induced DNA damage in the mouse rectum, possibly by direct interaction between clb+ E. coli and normal colorectal epithelial cells. Our findings imply that regulation of clb+ E. coli infection may be a useful strategy for colorectal cancer control. [ABSTRACT FROM AUTHOR]

Escherichia coli is a well-characterized bacterium highly prevalent in the human intestinal tract and the cause of many important infections. The aim of this study was to characterize 376 extraintestinal pathogenic E. coli strains collected from four hospitals in Catalonia (Spain) between 2016 and 2017 in terms of antimicrobial resistance, siderophore production, phylogroup classification, and the presence of selected virulence and antimicrobial resistance genes. In addition, the association between these characteristics and the ability to form biofilms was also analyzed. The strains studied were classified into four groups according to their biofilm formation ability: non-biofilm formers (15.7%), weak (23.1%), moderate (35.6%), and strong biofilm formers (25.6%). The strains were highly resistant to ciprofloxacin (48.7%), trimethoprim-sulfamethoxazole (47.9%), and ampicillin (38%), showing a correlation between higher resistance to ciprofloxacin and lower biofilm production. Seventy-three strains (19.4%) were ESBL-producers. However, no relationship between the presence of ESBL and biofilm formation was found. The virulence factor genes fimH (92%), pgaA (84.6%), and irp1 (77.1%) were the most prevalent in all the studied strains. A statistically significant correlation was found between biofilm formation and the presence of iroN , papA , fimH , sfa , cnf , hlyA , iutA , and colibactin-encoding genes clbA , clbB, clbN , and clbQ. Interestingly, a high prevalence of colibactin-encoding genes (19.9%) was observed. Colibactin is a virulence factor, which interferes with the eukaryotic cell cycle and has been associated with colorectal cancer in humans. Most colibactin-encoding E. coli isolates belonged to phylogroup B2, exhibited low antimicrobial resistance but moderate or high biofilm-forming ability, and were significantly associated with most of the virulence factor genes tested. Additionally, the analysis of their clonal relatedness by PFGE showed 48 different clusters, indicating a high clonal diversity among the colibactin-positive strains. Several studies have correlated the pathogenicity of E. coli and the presence of virulence factor genes; however, colibactin and its relationship to biofilm formation have been scarcely investigated. The increasing prevalence of colibactin in E. coli and other Enterobacteriaceae and the recently described correlation with biofilm formation, makes colibactin a promising therapeutic target to prevent biofilm formation and its associated adverse effects. [ABSTRACT FROM AUTHOR]

Appropriate screening of early asymptomatic cases can reduce the disease burden and mortality rate of sporadic colorectal cancer (CRC) significantly. Currently, fecal occult blood testing (FOBT) is able to detect up to 80% of asymptomatic cases in the population aged 50+. Therefore, there is still a demand for new screening tests that would complement FOBT, mainly by detecting at least a part of the FOBT-negative CRC and adenoma cases, or possibly by identifying person at increased risk of sporadic CRC in order to offer them tailored follow-up. Among the potential markers studied, our knowledge has advanced at most in toxigenic gram-negative bacteria. In this review, we assess their potential critically and recommend those best suited for prospective evaluation of their true ability to increase the sensitivity of FOBT when combined during general population screening. In our opinion, colibactin and Bacteroides fragilis toxin are the best candidates, possibly complemented by the cytotoxic necrotizing factor (CNF). [ABSTRACT FROM AUTHOR]

Escherichia coli containing polyketide synthase in the gut microbiota (pks+E coli) produce a polyketide‐peptide genotoxin, colibactin, and are suspected to play a role in the development of colorectal neoplasia. To clarify the role of pks+E coli in the early stage of tumorigenesis, we investigated whether the pks status of E coli was associated with the prevalence of colorectal neoplasia. This cross‐sectional analysis of data from a prospective cohort in Izu Oshima, Japan included asymptomatic residents aged 40‐79 years who underwent screening colonoscopy and provided a stool sample. We identified 543 participants with colorectal neoplasia (22 colorectal cancer and 521 adenoma) as cases and 425 participants with normal colon as controls. The pks status of E coli was assayed using stool DNA and specific primers that detected pks+E coli. The proportion of pks+E coli was 32.6% among cases and 30.8% among controls. Compared with those with pks−E coli, the odds ratio (OR) (95% confidence interval) for participants with pks+E coli was 1.04 (0.77‐1.41) after adjusting for potential confounders. No statistically significant associations were observed regardless of tumor site or number of colorectal adenoma lesions. However, stratified analyses revealed increased ORs among participants who consumed cereals over the median intake or vegetables under the median intake. Overall, we found no statistically significant association between pks+E coli and the prevalence of colorectal adenoma lesions among this Japanese cohort. However, positive associations were suggested under certain intake levels of cereals or vegetables. [ABSTRACT FROM AUTHOR]

The pks genotoxic K. pneumoniae has recently triggered a widespread alarm. DNA damage and higher virulence have been linked to colibactin, a genotoxin expressed by the pks genomic island. Little is known about its molecular epidemiology in clinical isolates from Egypt. Therefore, this study was conducted to determine the prevalence and the microbiological and clinical features of pks harboring hospital-acquired K. pneumoniae isolates from Egypt. Eighty-seven hospital-acquired K. pneumoniae isolates from various specimen types were screened for pks colibactin island markers clbB, clbQ, clbA, and clbN by PCR. The pkspositive hvKp isolates were classified to one of the capsular types K1 and K2 using multiplex-PCR targeting K-serotype wzi and rmpA genes. The prevalence of pks+ strains was 27.6% (24/87). K1 capsular type, phenotypic, and genotypic hypervirulent isolates were significantly higher among pks+ strains than pksstrains (P < 0.001), while pks+ K. pneumoniae strains were found to be significantly less resistant to 8 of the antibiotic compounds tested than pks-strains. Carriage of K1 capsular type and mucoviscosity-associated rmp A gene and diabetes mellitus were identified to remain independent risk factors having a substantial association to pks-positivity by multivariate regression analysis. In conclusion, Hospital-acquired K. pneumoniae isolates in Egypt had an increased prevalence of the pks colibactin genotoxin. The significant occurrence of hypervirulent determinants in pks+ K. pneumoniae highlighted the genotoxin's possible pathogenicity combined with its distribution in several specimen types, which necessitates clinical attention and epidemic tracking. [ABSTRACT FROM AUTHOR]

Colibactin is synthesized by a 54-kb genomic island, leads to toxicity in eukaryotic cells, and plays a vital role in many diseases, including neonatal sepsis and meningitis. Avian pathogenic Escherichia coli (APEC) is speculated to be an armory of extraintestinal pathogenic Escherichia coli and can be a potential zoonotic bacterium that threatens human and animal health. In this study, the APEC XM meningitis mouse model was successfully established to investigate the effect of colibactin in in vivo infection. The clbH-deletion mutant strain induced lower γ-H2AX expression, no megalocytosis, and no cell cycle arrest in bEnd.3 cells, which showed that the deletion of clbH decreased the production of colibactin in the APEC XM strain. The deletion of clbH did not affect the APEC XM strain's ability of adhering to and invading bEnd.3 cells. In vitro, the non-colibactin-producing strain displayed significantly lower serum resistance and it also induced a lower level of cytokine mRNA and few disruptions of tight junction proteins in infected bEnd.3 cells. Meningitis did not occur in APEC ΔclbH-infected mice in vivo, who showed fewer clinical symptoms and fewer lesions on radiological and histopathological analyses. Compared with the APEX XM strain, APEC ΔclbH induced lower bacterial colonization in tissues, lower mRNA expression of cytokines in brain tissues, and slight destruction of the brain blood barrier. These results indicate that clbH is a necessary component for the synthesis of genotoxic colibactin, and colibactin is related to the development of meningitis induced by APEC XM. [ABSTRACT FROM AUTHOR]

Abstract Introduction Colibactin is a small genotoxic molecule produced by enteric bacteria, including certain Escherichia coli (E. coli) strains harbored in the human large intestine. This polyketide-peptide genotoxin is considered to contribute to the development of colorectal cancer. The colibactin-producing (clb +) microorganisms possess a 54-kilobase genomic island (clb gene cluster). In the present study, to assess the distribution of the clb gene cluster, genotyping analysis was carried out among E. coli strains randomly chosen from the Japan Collection of Microorganisms, RIKEN BRC, Japan. Findings The analysis revealed that two of six strains possessed a clb gene cluster. These clb + strains JCM5263 and JCM5491 induced genotoxicity in in vitro micronucleus (MN) tests using rodent CHO AA8 cells. Since the induction level of MN by JCM5263 was high, a bacterial umu test was carried out with a cell extract of the strain, revealing that the extract had SOS-inducing potency in the umu tester bacterium. Conclusion These results support the observations that the clb gene cluster is widely distributed in nature and clb + E. coli having genotoxic potencies is not rare among microorganisms.

Escherichia coli is a well-characterized bacterium highly prevalent in the human intestinal tract and the cause of many important infections. The aim of this study was to characterize 376 extraintestinal pathogenic E. coli strains collected from four hospitals in Catalonia (Spain) between 2016 and 2017 in terms of antimicrobial resistance, siderophore production, phylogroup classification, and the presence of selected virulence and antimicrobial resistance genes. In addition, the association between these characteristics and the ability to form biofilms was also analyzed. The strains studied were classified into four groups according to their biofilm formation ability: non-biofilm formers (15.7%), weak (23.1%), moderate (35.6%), and strong biofilm formers (25.6%). The strains were highly resistant to ciprofloxacin (48.7%), trimethoprim-sulfamethoxazole (47.9%), and ampicillin (38%), showing a correlation between higher resistance to ciprofloxacin and lower biofilm production. Seventy-three strains (19.4%) were ESBL-producers. However, no relationship between the presence of ESBL and biofilm formation was found. The virulence factor genes fimH (92%), pgaA (84.6%), and irp1 (77.1%) were the most prevalent in all the studied strains. A statistically significant correlation was found between biofilm formation and the presence of iroN, papA, fimH, sfa, cnf, hlyA, iutA, and colibactin-encoding genes clbA, clbB, clbN, and clbQ. Interestingly, a high prevalence of colibactin-encoding genes (19.9%) was observed. Colibactin is a virulence factor, which interferes with the eukaryotic cell cycle and has been associated with colorectal cancer in humans. Most colibactin-encoding E. coli isolates belonged to phylogroup B2, exhibited low antimicrobial resistance but moderate or high biofilm-forming ability, and were significantly associated with most of the virulence factor genes tested. Additionally, the analysis of their clonal relatedness by PFGE showed 48 different clusters, indicating a high clonal diversity among the colibactin-positive strains. Several studies have correlated the pathogenicity of E. coli and the presence of virulence factor genes; however, colibactin and its relationship to biofilm formation have been scarcely investigated. The increasing prevalence of colibactin in E. coli and other Enterobacteriaceae and the recently described correlation with biofilm formation, makes colibactin a promising therapeutic target to prevent biofilm formation and its associated adverse effects.