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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.

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., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: The association between the intestinal microbiota and psychiatric disorders is becoming increasingly apparent. The gut microbiota contributes to colorectal carcinogenesis (CRC), as demonstrated with colibactin-producing Escherichia coli (CoPEC)., Aim: To evaluate the association between CoPEC prevalence and anxiety- and depressive-like behaviors with both preclinical and clinical approaches., Methods: Patients followed after a CRC surgery and for whom the prevalence of CoPEC has been investigated underwent a psychiatric interview. Results were compared according to the CoPEC colonization. In parallel C57BL6/J wild type mice and mice with a CRC susceptibility were chronically infected with a CoPEC strain. Their behavior was assessed using the Elevated Plus Maze test, the Forced Swimming Test and the Behavior recognition system PhenoTyper ® ., Results: In a limited cohort, all patients with CoPEC colonization presented with psychiatric disorders several years before cancer diagnosis, whereas only one patient (17%) without CoPEC did. This result was confirmed in C57BL6/J wild-type mice and in a CRC susceptibility mouse model (adenomatous polyposis coli multiple intestinal neoplasia/+ ). Mice exhibited a significant increase in anxiety- and depressive-like behaviors after chronic infection with a CoPEC strain., Conclusion: This finding provides the first evidence that CoPEC infection can induce microbiota-gut-brain axis disturbances in addition to its procarcinogenic properties., Competing Interests: Conflict-of-interest statement: Pr. Bonnet has nothing to disclose., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

Co-culture of intestinal organoids with a colibactin-producing pks + E. coli strain (EcC) revealed mutational signatures also found in colorectal cancer (CRC). E. coli Nissle 1917 (EcN) remains a commonly used probiotic, despite harboring the pks operon and inducing double strand DNA breaks. We determine the mutagenicity of EcN and three CRC-derived pks + E. coli strains with an analytical framework based on sequence characteristic of colibactin-induced mutations. All strains, including EcN, display varying levels of mutagenic activity. Furthermore, a machine learning approach attributing individual mutations to colibactin reveals that patients with colibactin-induced mutations are diagnosed at a younger age and that colibactin can induce a specific APC mutation. These approaches allow the sensitive detection of colibactin-induced mutations in ∼12% of CRC genomes and even in whole exome sequencing data, representing a crucial step toward pinpointing the mutagenic activity of distinct pks + E. coli strains., Competing Interests: Declaration of interests H.C. is a full-time member of the executive board of F. Hoffmann-La Roche Ltd. as head of Pharma, Research and Early Development (pRED) in Basel, Switzerland. Additionally, H.C. is inventor on multiple organoid patents, licensed by the KNAW to the Foundation HUB in Utrecht., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2024. The Author(s), under exclusive licence to Microbiological Society of Korea.)

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 +/EC blm+ 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 +/EC blm+ 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., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

The bacterial genotoxin colibactin promotes colorectal cancer (CRC) tumorigenesis, but systematic assessment of its impact on DNA repair is lacking, and its effect on response to DNA-damaging chemotherapeutics is unknown. We find that CRC cell lines display differential response to colibactin on the basis of homologous recombination (HR) proficiency. Sensitivity to colibactin is induced by inhibition of ATM, which regulates DNA double-strand break repair, and blunted by HR reconstitution. Conversely, CRC cells chronically infected with colibactin develop a tolerant phenotype characterized by restored HR activity. Notably, sensitivity to colibactin correlates with response to irinotecan active metabolite SN38, in both cell lines and patient-derived organoids. Moreover, CRC cells that acquire colibactin tolerance develop cross-resistance to SN38, and a trend toward poorer response to irinotecan is observed in a retrospective cohort of CRCs harboring colibactin genomic island. Our results shed insight into colibactin activity and provide translational evidence on its chemoresistance-promoting role in CRC., Competing Interests: Declaration of interests The authors declare the following competing interests, which are unrelated to the results of the study. A.B. reports receiving commercial research grants from Neophore, AstraZeneca, and Boehringer; he is an advisory board member/unpaid consultant for Inivata and Neophore, holds ownership interest in Neophore, and is an advisory board member/consultant for Illumina, Guardant Health, Inivata, and Roche/Genentech Global CRC. F.D.N. received honoraria from Pierre Fabre. L.T. has received research grants from Menarini, Merck KGaA, Merus, Pfizer, Servier, and Symphogen. S.A. acted as consultant for MSD Italia outside the submitted work and has a patent (102022000007535) pending. A.S.-B. is an advisory board member for Amgen, Bayer, Novartis, Pierre Fabre, and Servier. S.S. is an advisory board member for Agenus, AstraZeneca, Bayer, Bristol Myers Squibb, CheckmAb, Daiichi Sankyo, Guardant Health, Menarini, Merck, Novartis, Roche-Genentech, and Seagen., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The biosynthetic machinery for the production of colibactin is encoded by 19 genes ( clbA - S ) within the pks pathogenicity island harboured by many E. coli of the B2-phylogroup. Colibactin is a potent genotoxic metabolite which causes DNA-damage and which has potential roles in microbial competition and fitness of pks + bacteria. Colibactin has also been strongly implicated in the development of colorectal cancer. Given the genotoxicity of colibactin and the metabolic cost of its synthesis, the regulatory system governing the clb cluster is accordingly highly complex, and many of the mechanisms remain to be elucidated. In this review we summarise the current understanding of regulation of colibactin biosynthesis by internal molecular components and how these factors are modulated by signals from the external environment.

The hypervirulent lineages of Klebsiella pneumoniae (HvKp) cause invasive infections such as Klebsiella-liver abscess. Invasive infection often occurs after initial colonization of the host gastrointestinal tract by HvKp. Over 80% of HvKp isolates belong to the clonal group 23 sublineage I that has acquired genomic islands (GIs) GIE492 and ICEKp10. Our analysis of 12 361 K. pneumoniae genomes revealed that GIs GIE492 and ICEKp10 are co-associated with the CG23-I and CG10118 HvKp lineages. GIE492 and ICEKp10 enable HvKp to make a functional bacteriocin microcin E492 (mccE492) and the genotoxin colibactin, respectively. We discovered that GIE492 and ICEKp10 play cooperative roles and enhance gastrointestinal colonization by HvKp. Colibactin is the primary driver of this effect, modifying gut microbiome diversity. Our in vitro assays demonstrate that colibactin and mccE492 kill or inhibit a range of Gram-negative Klebsiella species and Escherichia coli strains, including Gram-positive bacteria, sometimes cooperatively. Moreover, mccE492 and colibactin kill human anaerobic gut commensals that are similar to the taxa found altered by colibactin in the mouse intestines. Our findings suggest that GIs GIE492 and ICEKp10 enable HvKp to kill several commensal bacterial taxa during interspecies interactions in the gut. Thus, acquisition of GIE492 and ICEKp10 could enable better carriage in host populations and explain the dominance of the CG23-I HvKp lineage., (© The Author(s) [2024]. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Recently, the mounting integration of probiotics into human health strategies has gathered considerable attention. Although the benefits of probiotics have been widely recognized in patients with gastrointestinal disorders, immune system modulation, and chronic-degenerative diseases, there is a growing need to evaluate their potential risks. In this context, new concerns have arisen regarding the safety of probiotics as some strains may have adverse effects in humans. Among these strains, Escherichia coli Nissle 1917 ( EcN ) exhibited traits of concern due to a pathogenic locus in its genome that produces potentially genotoxic metabolites. As the use of probiotics for therapeutic purposes is increasing, the effects of potentially harmful probiotics must be carefully evaluated. To this end, in this narrative review article, we reported the findings of the most relevant in vitro and in vivo studies investigating the expanding applications of probiotics and their impact on human well-being addressing concerns arising from the presence of antibiotic resistance and pathogenic elements, with a focus on the polyketide synthase ( pks ) pathogenic island of EcN . In this context, the literature data here discussed encourages a thorough profiling of probiotics to identify potential harmful elements as done for EcN where potential genotoxic effects of colibactin, a secondary metabolite, were observed. Specifically, while some studies suggest EcN is safe for gastrointestinal health, conflicting findings highlight the need for further research to clarify its safety and optimize its use in therapy. Overall, the data here presented suggest that a comprehensive assessment of the evolving landscape of probiotics is essential to make evidence-based decisions and ensure their correct use in humans.

Human 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.

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.

Polyketide synthase (pks) island harboring Escherichia coli are, under the right circumstances, able to produce the genotoxin colibactin. Colibactin is a risk factor for the development of colorectal cancer and associated with mutational signatures SBS88 and ID18. This study explores colibactin-associated mutational signatures in biallelic NTHL1 and MUTYH patients. Targeted Next Generation Sequencing (NGS) was performed on colorectal adenomas and carcinomas of one biallelic NTHL and 12 biallelic MUTYH patients. Additional fecal metagenomics and genome sequencing followed by mutational signature analysis was conducted for the NTHL1 patient. Targeted NGS of the NTHL1 patient showed somatic APC variants fitting SBS88 which was confirmed using WGS. Furthermore, fecal metagenomics revealed pks genes. Also, in 1 out of 11 MUTYH patient a somatic variant was detected fitting SBS88. This report shows that colibactin may influence development of colorectal neoplasms in predisposed patients., (© 2023 The Authors. Genes, Chromosomes and Cancer published by Wiley Periodicals LLC.)

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., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

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., (open access.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

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., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: Abelardo Margolles and Borja Sánchez are co-founders of Microviable Therapeutics SL. Abelardo Margolles is member of the scientific board of Microviable Therapeutics SL. Borja Sánchez is Minister of Science, Innovation and University of the Government of the Principality of Asturias. Results presented in this paper are protected under European Patent EP19383077 (WO2021110833A1 and US20230029322A1; Tools and methods to detect and isolate colibactin producing bacteria).

The genotoxin colibactin produced by Escherichia coli is involved in the development of colorectal cancers. This secondary metabolite is synthesized by a multi-protein machinery, mainly composed of non-ribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) enzymes. In order to decipher the function of a PKS-NRPS hybrid enzyme implicated in a key step of colibactin biosynthesis, we conducted an extensive structural characterization of the ClbK megaenzyme. Here we present the crystal structure of the complete trans-AT PKS module of ClbK showing structural specificities of hybrid enzymes. In addition, we report the SAXS solution structure of the full-length ClbK hybrid that reveals a dimeric organization as well as several catalytic chambers. These results provide a structural framework for the transfer of a colibactin precursor through a PKS-NRPS hybrid enzyme and can pave the way for re-engineering PKS-NRPS hybrid megaenzymes to generate diverse metabolites with many applications., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Some Escherichia coli strains harbour the pks island, a 54 kb genomic island encoding the biosynthesis genes for a genotoxic compound named colibactin. In eukaryotic cells, colibactin can induce DNA damage, cell cycle arrest and chromosomal instability. Production of colibactin has been implicated in the development of colorectal cancer (CRC). In this study, we demonstrate the inhibitory 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 ability to inhibit expression of clbB , D-Serine exerted the strongest repressing activity (>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 infection 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 treatment. Moreover, exposure of pks + and CRC, highlights the necessity for the development of colibactin targeting therapeutics. Here we show that D-Serine can reduce expression of colibactin, and inhibit downstream cellular cytopathy, illuminating its potential to prevent colibactin-associated disease.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 therapeutics. Here we show that D-Serine can reduce expression of colibactin, and inhibit downstream cellular cytopathy, illuminating its potential to prevent colibactin-associated disease., Competing Interests: Conflict of Interest: All authors declare that they have no conflicts of interest., (Copyright: © 2023 Hallam et al.)

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., (© 2023. The Author(s).)

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.

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 Apc Min/+ 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 Apc Min/+ mice. The tumorigenic effect of EcN101 in Apc Min/+ 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Oliero, Hajjar, Cuisiniere, Fragoso, Calvé and Santos.)

Trichothecenes (TCT) are very common mycotoxins. While the effects of DON, the most prevalent TCT, have been extensively studied, less is known about the effect of other trichothecenes. DON has ribotoxic, pro-inflammatory, and cytotoxic potential and induces multiple toxic effects in humans and animals. Although DON is not genotoxic by itself, it has recently been shown that this toxin exacerbates the genotoxicity induced by model or bacterial genotoxins. Here, we show that five TCT, namely T-2 toxin (T-2), diacetoxyscirpenol (DAS), nivalenol (NIV), fusarenon-X (FX), and the newly discovered NX toxin, also exacerbate the DNA damage inflicted by various genotoxins. The exacerbation was dose dependent and observed with phleomycin, a model genotoxin, captan, a pesticide with genotoxic potential, and colibactin, a bacterial genotoxin produced by the intestinal microbiota. For this newly described effect, the trichothecenes ranked in the following order: T-2>DAS > FX > NIV ≥ DON ≥ NX. The genotoxic exacerbating effect of TCT correlated with their ribotoxic potential, as measured by the inhibition of protein synthesis. In conclusion, our data demonstrate that TCT, which are not genotoxic by themselves, exacerbate DNA damage induced by various genotoxins. Therefore, foodborne TCT could enhance the carcinogenic potential of genotoxins present in the diet or produced by intestinal bacteria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Up 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.

Colibactin, 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.

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., (© 2022. The Author(s).)

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?, (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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.

Chaperone proteins are essential in all living cells to ensure protein homeostasis. Hsp90 is a major adenosine triphosphate (ATP)-dependent chaperone highly conserved from bacteria to eukaryotes. Recent studies have shown that bacterial Hsp90 is essential in some bacteria in stress conditions and that it participates in the virulence of pathogenic bacteria. In vitro, bacterial Hsp90 directly interacts and collaborates with the Hsp70 chaperone DnaK to reactivate model substrate proteins; however, it is still unknown whether this collaboration is relevant in vivo with physiological substrates. Here, we used site-directed mutagenesis on Hsp90 to impair DnaK binding, thereby uncoupling the chaperone activities. We tested the mutants in vivo in two bacterial models in which Hsp90 has known physiological functions. We found that the Hsp90 point mutants were defective to support (1) growth under heat stress and activation of an essential Hsp90 client in the aquatic bacterium Shewanella oneidensis and (2) biosynthesis of the colibactin toxin involved in the virulence of pathogenic Escherichia coli . Our study therefore demonstrates the essentiality of the direct collaboration between Hsp90 and DnaK in vivo in bacteria to support client folding. It also suggests that this collaboration already functional in bacteria has served as an evolutionary basis for a more complex Hsp70-Hsp90 collaboration found in eukaryotes.

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., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mousa.)

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., Competing Interests: Declaration of interests C.F.L. is a member of the Scientific Advisory Board of Synlogic, Inc., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

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., Competing Interests: Conflict of interest: The authors declare no conflict of interest., (© 2022 the Author(s), licensee AIMS Press.)

SignificanceIn a polymicrobial battlefield where different species compete for nutrients and colonization niches, antimicrobial compounds are the sword and shield of commensal microbes in competition with invading pathogens and each other. The identification of an Escherichia coli -produced genotoxin, colibactin, and its specific targeted killing of enteric pathogens and commensals, including Vibrio cholerae and Bacteroides fragilis , sheds light on our understanding of intermicrobial interactions in the mammalian gut. Our findings elucidate the mechanisms through which genotoxins shape microbial communities and provide a platform for probing the larger role of enteric multibacterial interactions regarding infection and disease outcomes.

Escherichia coli strains encoding colibactin (pks), hemolysin-associated cytotoxic necrotizing factor (cnf), and cytolethal distending toxin (cdt) are associated with intestinal inflammation and cancer, urinary tract infection, and septicemia in susceptible hosts. Over a 2-year period, an inbred laboratory colony of specific-pathogen free (SPF) cats (∼25) presented with resorptions, stillbirths, and pyometras in >50 % of pregnancies. Hemolytic E. coli were cultured from vaginal and preputial swabs of clinically normal, intact males, healthy kittens, and placenta and fetal tissues of a dam with reproductive disorders. We hypothesized cats from this colony were colonized with cytotoxin-encoding E. coli. 27 E. coli isolates were cultured from 20 fresh feces representing the majority of cats with and without fertility failures. Two E. coli isolates were also cultured from vaginal swabs from the same cat. 22 isolates (75.9 %) demonstrated hemolysis on blood agar. Twelve isolates (41.4 %) were pks+, 14 (48.3 %) were cnf+, and 10 (34.5 %) were cdt+ by PCR. Serotypes and virulence factor profiles were consistent with the extraintestinal E. coli (ExPEC) pathotype. Antibiotic resistance to cephalothin was exhibited in 13/14 representative isolates. Whole genome sequence analysis of 3 representative isolates confirmed the hemolysin-associated cnf, cdt, and the pks gene island. Representative isolates were cytotoxic to cervical epithelial cells in vitro. This study indicated ExPEC were present in SPF cats with a history of reproductive failure. While causality cannot be established, it is probable ExPEC was associated with impaired reproductive health and breeding success. Since treatment of the colony with cefovecin, reproductive performance has appreciably improved., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background/aim: Among colorectal cancer-associated 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., (Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

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).

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ballén, Gabasa, Ratia, Sánchez and Soto.)

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., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Colorectal cancer (CRC) is one of the deadliest cancers in the world. Specific strains of intestinal Escherichia coli (E. coli) may influence the initiation and development of CRC by exploiting virulence factors and inflammatory pathways. Mucosa-associated E. coli strains are more prevalent in CRC biopsies in comparison to healthy controls. Moreover, these strains can survive and replicate within macrophages and induce a pro-inflammatory response. Chronic exposure to inflammatory mediators can lead to increased cell proliferation and cancer. Production of colobactin toxin by the majority of mucosa-associated E. coli isolated from CRC patients is another notable finding. Colibactin-producing E. coli strains, in particular, induce double-strand DNA breaks, stop the cell cycle, involve in chromosomal rearrangements of mammalian cells and are implicated in carcinogenic effects in animal models. Moreover, some enteropathogenic E. coli (EPEC) strains are able to survive and replicate in colon cells as chronic intracellular pathogens and may promote susceptibility to CRC by downregulation of DNA Mismatch Repair (MMR) proteins. In this review, we discuss current evidence and focus on the mechanisms by which E. coli can influence the development of CRC., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Colibactin is a genotoxin produced primarily by Escherichia coli harboring the genomic pks island ( pks + &nbsp; E. coli ). Pks cause host cell DNA damage, leading to chromosomal instability and gene mutations. The signature of colibactin-induced mutations has been described and found in human colorectal cancer (CRC) genomes. An inflamed intestinal environment drives the expansion of + &nbsp; E. coli cause host cell DNA damage, leading to chromosomal instability and gene mutations. The signature of colibactin-induced mutations has been described and found in human colorectal cancer (CRC) genomes. An inflamed intestinal environment drives the expansion of pks in the gut microbiota and diminishes the production of colibactin. Here, we describe the link between intestinal inflammation and colorectal cancer induced by + &nbsp; E. coli and promotes tumorigenesis. Mesalamine (i.e., 5-aminosalycilic acid), an effective anti-inflammatory drug, is an inhibitor of the bacterial polyphosphate kinase (PPK). This drug not only inhibits the production of intestinal inflammatory mediators and the proliferation of CRC cells, but also limits the abundance of E. coli in the gut microbiota and diminishes the production of colibactin. Here, we describe the link between intestinal inflammation and colorectal cancer induced by pks + &nbsp; E. coli . We discuss the potential mechanisms of the pleiotropic role of mesalamine in treating both inflammatory bowel diseases and reducing the risk of CRC due to pks + &nbsp; E. coli .

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 Δ clbH -infected mice in vivo , who showed fewer clinical symptoms and fewer lesions on radiological and histopathological analyses. Compared with the APEX XM strain, APEC Δ clbH 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 frequent occurrence of E. coli positive for cyclomodulins such as colibactin (CLB), the cytotoxic necrotizing factor (CNF), and the cytolethal distending factor (CDT) in colorectal cancer (CRC) patients published so far provides the opportunity to use them as CRC screening markers. We examined the practicability and performance of a low-cost detection approach that relied on culture followed by simplified DNA extraction and PCR in E. coli isolates recovered from 130 CRC patients and 111 controls. Our results showed a statistically significant association between CRC and the presence of colibactin genes clbB and clbN , the cnf gene, and newly, the hemolytic phenotype of E. coli isolates. We also observed a significant increase in the mean number of morphologically distinct E. coli isolates per patient in the CRC cohort compared to controls, indicating that the cyclomodulin-producing E. coli strains may represent potentially preventable harmful newcomers in CRC patients. A colibactin gene assay showed the highest detection rate (45.4%), and males would benefit from the screening more than females. However, because of the high number of false positives, practical use of this marker must be explored. In our opinion, it may serve as an auxiliary marker to increase the specificity and/or sensitivity of the well-established fecal immunochemical test (FIT) in CRC screening.

Background & Aims: Patients with multiple recurrent Clostridioides difficile infection (rCDI) have a disturbed gut microbiota that can be restored by fecal microbiota transplantation (FMT). Despite extensive screening, healthy feces donors may carry bacteria in their intestinal tract that could have long-term health effects, such as potentially procarcinogenic polyketide synthase-positive (pks + ) Escherichia coli. Here, we aim to determine whether the pks abundance and persistence of pks + E coli is influenced by pks status of the donor feces., Methods: In a cohort of 49 patients with rCDI treated with FMT and matching donor samples-the largest cohort of its kind, to our knowledge-we retrospectively screened fecal metagenomes for pks + E coli and compared the presence of pks in patients before and after treatment and to their respective donors., Results: The pks island was more prevalent (P = .026) and abundant (P < .001) in patients with rCDI (pre-FMT, 27 of 49 [55%]; median, 0.46 reads per kilobase per million [RPKM] pks) than in healthy donors (3 of 8 donors [37.5%], 11 of 38 samples [29%]; median, 0.01 RPKM pks). The pks status of patients post-FMT depended on the pks status of the donor suspension with which the patient was treated (P = .046). Particularly, persistence (8 of 9 cases) or clearance (13 of 18) of pks + E coli in pks + patients was correlated to pks in the donor (P = .004)., Conclusions: We conclude that FMT contributes to pks + E coli persistence or eradication in patients with rCDI but that donor-to-patient transmission of pks + E coli is unlikely., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Escherichia coli Nissle 1917 (EcN) is a Gram-negative bacterium that is used to treat inflammatory bowel diseases. The probiotic character of EcN is not well-understood, but its ability to produce secondary metabolites plays an important role in its activity. The EcN genome encodes for an aryl polyene (APE) biosynthetic gene cluster (BGC), and APE products have a role in biofilm formation. We show here that this unusual polyketide assembly line synthase produces four APE molecules which are likely cis/trans isomers. Within the APE BGC, two acyl carrier proteins are involved in biosynthesis. Acyl carrier proteins require activation by post-translational modification with a phosphopantetheinyl transferase (PPTase). Through analysis of single, double, and triple mutants of three PPTases, the PPTase-BGC crosstalk relationship in EcN was characterized. Understanding PPTase-BGC crosstalk is important for the engineering of secondary metabolite production hosts and for targeting of PPTases with new antibiotics. KEY POINTS: • Escherichia coli Nissle 1917 biosynthesizes four aryl polyene isoforms. • Phosphopantetheinyl transferase crosstalk is important for biosynthesis., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The probiotic Escherichia coli strain Nissle 1917 (DSM 6601, Mutaflor), generally considered beneficial and safe, has been used for a century to treat various intestinal diseases. However, Nissle 1917 hosts in its genome the pks pathogenicity island that codes for the biosynthesis of the genotoxin colibactin. Colibactin is a potent DNA alkylator, suspected to play a role in colorectal cancer development. We show in this study that Nissle 1917 is functionally capable of producing colibactin and inducing interstrand cross-links in the genomic DNA of epithelial cells exposed to the probiotic. This toxicity was even exacerbated with lower doses of the probiotic, when the exposed cells started to divide again but exhibited aberrant anaphases and increased gene mutation frequency. DNA damage was confirmed in vivo in mouse models of intestinal colonization, demonstrating that Nissle 1917 produces the genotoxin in the gut lumen. Although it is possible that daily treatment of adult humans with their microbiota does not produce the same effects, administration of Nissle 1917 as a probiotic or as a chassis to deliver therapeutics might exert long-term adverse effects and thus should be considered in a risk-versus-benefit evaluation. IMPORTANCE Nissle 1917 is sold as a probiotic and considered safe even though it has been known since 2006 that it harbors the genes for colibactin synthesis. Colibactin is a potent genotoxin that is now linked to causative mutations found in human colorectal cancer. Many papers concerning the use of this strain in clinical applications ignore or elude this fact or misleadingly suggest that Nissle 1917 does not induce DNA damage. Here, we demonstrate that Nissle 1917 produces colibactin in vitro and in vivo and induces mutagenic DNA damage. This is a serious safety concern that must not be ignored in the interests of patients, the general public, health care professionals, and ethical probiotic manufacturers.