Benefits and concerns of probiotics: an overview of the potential genotoxicity of the colibactin-producing Escherichia coli Nissle 1917 strain

ABSTRACT 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.


Introduction
The human microbiota, a diverse community of microorganisms residing in different sites of the human body, plays a central role in maintaining human health.This complex ecosystem influences various physiological processes, including digestion, immune function, metabolism, and even neurological health. 1 The composition and diversity of the microbiota have been linked to numerous disorders ranging from gastrointestinal diseases to obesity and mental health issues. 2Consequently, understanding the factors influencing microbiota composition and promoting a balanced microbiota is critical to well-being. 3 In this context, several studies have shown the beneficial effects of administering specific microorganisms that promote microbiota homeostasis and, consequently, human health. 4,5Such microorganisms are commonly referred to as probiotics, which are live bacteria or yeasts that, when administered in sufficient quantities, provide health benefits to the host.Probiotics are commonly found in certain foods and dietary supplements and include a variety of bacterial strains, such as Lacticaseibacillus, Lactobacillus, and Bifidobacterium genera. 6robiotics can improve the diversity and stability of gut microbiota, contributing to host homeostasis.These beneficial effects are mainly achieved by outcompeting harmful bacteria, creating a more favorable environment for beneficial species, and influencing the gut's immune response. 7,8s living microorganisms, probiotics metabolize and produce substances that help the host in detoxifying harmful compounds or counteract pathogen adhesion.The products of probiotic metabolism are defined as postbiotics and include all bioactive compounds with a biological function.These compounds include short-chain fatty acids (SCFAs), peptides, enzymes, and various metabolites.Of note, postbiotics may contribute significantly to the health benefits attributed to probiotic consumption.They modulate immune responses, strengthen the intestinal epithelial barrier (IEB), and have anti-inflammatory and antioxidant effects. 9,10Although probiotics and postbiotics offer numerous health benefits, they can have potential adverse effects that may occur in certain situations.
Indeed, it has been reported that the administration of probiotics in immunocompromised individuals, or those with preexisting health conditions, can lead to infections.This is particularly true for people with weakened immune systems, such as pediatric patients, transplant recipients, or patients undergoing chemotherapy. 113][14] Less serious negative effects include bloating, indigestion, and gas production.However, these symptoms are generally transient and diminish when the gut microbiota adapts. 15n rare cases, the administration of probiotics can lead to hypersensitive reactions manifested by different symptoms, such as rashes, itching, and swelling. 16,17In addition, probiotics may alter the effects of concomitant treatments through interactions with the host, especially those based on the administration of immunosuppressive drugs or antibiotics, thus interfering with their efficacy. 18,19espite these rare adverse effects, there is a general consensus on the safety of using well-known and widely tested probiotics such as Lacticaseibacillus rhamnosus GG, Lactobacillus acidophilus, Ligilactobacillus salivarius, Saccharomyces boulardii, Bifidobacterium longum, etc. [20][21][22] Of note, postbiotics also showed negative effects on human health.In particular, some postbiotics may also modulate the immune system with unfavorable effects in individuals with autoimmune diseases by either enhancing or dampening immune responses. 23More importantly, bacteria and probiotics can be involved in the production of harmful metabolites and metabolism byproducts.5][26] In this context, certain probiotics can be involved in the production and metabolism of histamine reducing intestinal inflammation through the activation of the histamine H2 receptor. 27][30] It is important to point out that most people can safely include probiotics and postbiotics into their diet without adverse effects.However, specific risks should be considered, especially for vulnerable populations, including individuals with weakened immune systems, preexisting health conditions, allergies, or young children. 31espite their potential benefits, the safety of probiotics and postbiotics is a growing concern.In addition, probiotics are not classified as drugs by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA), or other agencies. 32,33This classification raises challenges in evaluating and regulating the safety and efficacy of probiotics.Case reports of adverse events have highlighted the need for standardized testing and robust quality controls to ensure that probiotics are safe to consume, particularly in vulnerable populations.Therefore, the evolving landscape of probiotics underscores the need for rigorous safety assessments and regulatory frameworks to address their unique nature as living microorganisms rather than traditional pharmaceuticals.
To shed light on all these relevant aspects, in the present narrative review, a thorough search of the literature was performed utilizing various databases including PubMed/MedLine, Web of Science, Scopus, Google Scholar, and Cochrane Library.Specific keywords were employed to refine the search and select only relevant studies of three-year-old children overlaps with that of adults. 39,40Conversely, the elderly showed a significant reduction of Bifidobacterium and Firmicutes, whereas the relative abundance of Enterobacteriaceae and Clostridia increases triggering intestinal inflammation, which is strictly related to the onset of several pathological conditions. 41,42ut microbiota is involved in several biological functions, including metabolism, nutrient absorption, synthesis of bioactive compounds, and fermentation. 43,44Moreover, gut microbiota prevents the colonization of harmful pathogens by competing for resources and producing antimicrobial compounds thus contributing to the maintenance of IEB integrity and functionality.Interestingly, the gut microbiota is also involved in immune system regulation, influencing its responsiveness to pathogens and contributing to immune homeostasis. 45,46nder physiological conditions, gut microbiota is characterized by a stable core microbiota, high αdiversity, and microbial gene richness (eubiosis) contributing to well-being.However, any imbalance in the composition and diversity of the microbial community within the gastrointestinal tract (dysbiosis) may lead to several pathological conditions, including obesity, diabetes, IBD, and cancer (Figure 1). 47,48Notably, gut dysbiosis is due to an overgrowth of pathogens compared to beneficial microbes, whose activity is related to genomic instability and tumor development.For instance, it has been reported that colibactin, a genotoxic secondary metabolite produced by different Escherichia coli strains, could play a critical role in colorectal cancer (CRC) initiation inducing DNA double-strand breaks (DSBs) in host cells and leading to genomic mutations. 49,50][53] In this context, microbiota-based therapy has recently emerged as an innovative approach to restore microbial eubiosis and maintain intestinal bacteria balance.8][59] However, several challenges are associated with microbiota-based therapeutics and their effective application in clinical practice.Therefore, further studies are mandatory to understand the interplay between the gut microbiota community and the host.

Probiotics in prevention and complementary therapy
As widely described in the previous subsection, human health is closely related to the composition of the gut microbiota. 36Among these microorganisms, a particular group is spotlighted for its potential to positively influence human health: probiotics.As widely known, probiotics are defined as living microorganisms that, when administered in appropriate amounts, provide health benefits to the host. 60,61These positive effects are achieved through some distinctive features that enable probiotics to contribute to human health.These include their ability to survive the acidic environment of the stomach (also defined as resilience), the immunomodulatory and anti-inflammatory effects, and the ability to adhere to the intestinal lumen and exert beneficial effects. 7n addition to these physical properties, probiotics influence the molecular and cellular functions of the surrounding cells and tissues through several mechanisms. 62Specifically, they compete with harmful microorganisms for resources through competitive exclusion, inhibit their growth, and decrease their gastrointestinal adhesion. 8dditionally, probiotics help strengthen IEB function and effectively prevent toxic substances or metabolites from entering the bloodstream. 63he term probiotics encompasses a variety of microorganisms, each with specific healthpromoting properties.The first microorganisms to be recognized as probiotics were lactic acid bacteria (LAB), including well-known genera such as Lacticaseibacillus and Streptococcus. 64These bacteria, known for their ability to ferment sugars into lactic acid, are found in various fermented foods and dairy products and contribute to the maintenance of gastrointestinal homeostasis.Other known probiotics include the genera Bifidobacterium and Enterobacterium, which are known for their prevalence in the intestines of breastfed infants and their potential contribution to digestive health. 65,66ome probiotic strains can be introduced through specific foods, including breast milk or fermented foods (e.g.yogurt, kefir, sauerkraut, kimchi, and miso).][69] As keepers of normal gastrointestinal functions, probiotics strengthen the gut barrier by enhancing the integrity of tight junctions between gut epithelial cells. 702][73] On the contrary, probiotics can promote the expression of proteins involved in the cell-cell interaction.In this context, zonulin was recently identified as a major component responsible for the regulation of intestinal cell tight junctions and its degradation was associated with a pathological condition defined as "leaky gut" or "leaky gut syndrome" characterized by chronic systemic inflammation and intestinal permeation.A growing body of research has also demonstrated the positive effects of integral gut microbiota and probiotics in inducing the expression and correct function of zonulin, suggesting how probiotics may promote health by regulating the homeostasis of the gut barrier. 63,74losely connected to the integrity of the mucosa and IEB, there are also the effects of probiotics in favoring mucosal immunity.Indeed, probiotics orchestrate a variety of immune responses, modulating the activity of immune cells and influencing the production of immunoglobulins through the modulation of bacterial antigens. 75,76These multifaceted interactions strengthen the host's defenses against pathogens or other pro-inflammatory stimuli.In particular, probiotics are able to modulate the production of pro-inflammatory factors and enhance anti-inflammatory responses mainly through the regulation of the toll-like receptors (e.g.TLR2 and TLR4) and, in turn, the inactivation of the pro-inflammatory triggers produced by dendritic cells and macrophages. 77oncomitantly, probiotics may reduce the protein levels of interleukins and other pro-inflammatory biomarkers, limiting the immune response when unnecessary.Such effects are mediated by the regulation of T-cell differentiation, the activation of T helper cell type 2 (Th2) and the production of anti-inflammatory interleukin (IL)-4 and IL-10. 78Overall, probiotics and balanced gut microbiota favor a correct immune state, which prevents chronic inflammation preserving the host's ability to respond to pathogens and stimuli.
More complex interactions were also described between probiotics and host cells; however, these are not the focus of the present manuscript.What it is important to note is that probiotics establish an intricate interplay within the gut ecosystem and the host, fostering an environment where beneficial microorganisms proliferate and barrier function, mucosal immunity, and immune responses are effective in counteracting harmful pathogens.Therefore, probiotics are widely used for the maintenance of digestive health, to induce immune modulation, metabolic equilibrium, and, more recently, to maintain mental wellness by modulating the so-called "gut-brain axis". 79s regards the fields of application of probiotics, notably, these were first discovered for their pivotal role in promoting digestive health in patients suffering from different gastrointestinal disorders, including irritable bowel diseases, diarrhea, constipation, etc.Several studies also demonstrated that probiotics can modulate gut motility, alleviate abdominal discomfort, and recalibrate stool consistency through their potential to restore gut microbiota imbalances and revert dysbiosis. 80ome authors have also proposed probiotic administration as a treatment to limit or mitigate other microbial infections.Accordingly, the regulation of immunoglobulins, cytokines, and immune cells mediated by probiotics contributes to immune resilience and the mitigation of infections. 81urthermore, metabolic disorders emerge as pivotal frontiers where probiotics have been proposed for their potential to mitigate food intolerances and weight gain.Specifically, probiotics actively participate in the degradation of foods by modulating energy extraction from food, promoting fat oxidation, and influencing adipose tissue homeostasis.All these probiotic functions encouraged novel potential strategies to treat metabolic syndrome, obesity and other food-related disorders.In addition, the SCFAs and other metabolites obtained from probiotics further support the adoption of probiotics for the treatment of metabolic disorders. 82ecently, neuronal and neurodegenerative disorders have emerged as other fields of application of probiotics.As mentioned above, probiotics actively interact with the gut-brain axis, an intricate bidirectional communication channel that interconnects gut physiology with cognitive functions.As probiotics are effective in maintaining gut eubiosis, researchers have proposed their application to induce mood regulation and improve cognitive functions. 83,84Such intricated relationship existing among gut microbiota, probiotics, and cognitive functions seems to be mediated by several factors, including microbial metabolites, neurotransmitters, and neuroinflammatory molecules actively modulated by probiotics. 85Certain probiotics ameliorate symptoms of anxiety, depression, and cognitive decline; however, further studies are necessary to unveil the underlying molecular mechanisms through which microorganisms influence neurocognitive functions beyond the gut confines.
Some researchers have reported adverse effects associated with probiotic administration.Although very rare, some probiotic strains may cause systemic infections, particularly in immunocompromised individuals. 12Moreover, other findings have demonstrated that some probiotics or postbiotics may lead to allergic reactions in some cases, highlighting how the administration of probiotics should be considered according to the individual response to probiotics or their excipients, as well as the composition of gut microbiota and the clinical conditions of patients. 86r these reasons, it is necessary to claim that probiotics are not currently considered as drugs; therefore, they are not supervised by regulatory agencies like the EMA and the FDA.This suggests how standardized guidelines for probiotic use are needed to establish the safety and efficacy of each microorganism in specific clinical settings.
Currently, several probiotics are commercialized alone or in different combinations and brands.Table 1 reports the most widely used probiotics, indicating their primary effects and, eventually, adverse events observed following their administration as reviewed by Zommiti M et al. (2020) and Sanders ME et al. (2010). 60,87

Postbiotics, metabolites, and their potential applications
Postbiotics, also known as metabiotics, have recently attracted growing interest for their potential application as a promising adjuvant strategy in preventing and treating several diseases.According to The International Scientific Association of Probiotics and Prebiotics (ISAPP), a postbiotic is defined as "a preparation of inanimate microorganisms and/or their components that confers a health benefit on the host". 112Therefore, postbiotics refer to non-viable microbial cells and their components, as well as metabolites released after microbial cell lysis or secreted by gut microbes during fermentation processes. 113Since they are contained in different fermented products, postbiotics may be naturally assumed with diet.Moreover, postbiotics may be also obtained in vitro and commercialized as dietary supplements by using well-defined probiotic bacteria strains processed with specific biotechnological approaches, including biomass production, radiations, high pressure and temperature, and inactivation. 114,115Among LAB, Lacticaseibacillus and Bifidobacterium are the most used for postbiotic production. 116Starting from these bacteria strains, it is possible to obtain both postbiotics and probiotic metabolites with beneficial properties, including cell-free supernatant, SCFAs (e.g.acetate, propionate, and butyrate), bacteriocins (e.g.nisin, lactocyclicin, bovicin, and lacticin), non-viable microbial cells or cell envelope components, bacterial lysates, exo-and endo- polysaccharides, vitamins, biosurfactants, cell surface proteins, organic acids, and teichoic acids. 117,118everal studies recently showed that postbiotics and probiotic metabolites exert antioxidant, antiinflammatory, immunomodulatory, and antitumoral activity, highlighting their beneficial effects on host cells. 9,10,20,119,120][123] Compared to viable bacteria, postbiotics represent a safer alternative strategy for children and immunocompromised patients.5][126] Interestingly, postbiotics or purified probiotic metabolites provide a standardized and consistent dose of bioactive compounds, whereas the effectiveness of probiotics can vary based on several factors, including storage conditions and individual response.Other advantages characterizing postbiotics are represented by higher stability and longer shelf-life than probiotic supplements. 127In addition, postbiotics offer benefits without the need for colonization and may be easily incorporated into various products (e.g.functional foods and cosmetics), making them effective for several applications. 128,129urrently, the clinical application of postbiotics and probiotic metabolites is still in its infancy and little data are available from in vivo studies and clinical trials.Therefore, advanced research focusing on the postbiotics-host interactions is needed to corroborate their employment in clinical and non-clinical settings.

Probiotic safety
Over the years, several types of microbes have been proposed as probiotics not only for the maintenance of gut homeostasis but also in clinical settings as an adjuvant treatment.Despite the positive effects of probiotics on host health status have been widely reported, the expanding consumption of probiotic products may also be associated with different adverse events, including gastrointestinal symptoms (e.g.nausea, flatulence, and soft stools), systemic infections, horizontal gene transfer, production of secondary metabolites with genotoxic effects, and excessive immune stimulation in vulnerable subjects. 130,131In this field, regulatory requirements for probiotic safety may vary depending on the country and it is difficult to provide globally accepted guidelines (Figure 2).
The first step of the probiotic safety assessment is represented by whole genome sequencing (WGS) of the probiotic candidate to establish bacterial identity at the strain level and retrieve genomic data. 132Secondly, a well-documented history of safe use is an important requirement to corroborate probiotic-safe consumption.Notably, the analytic revision of the literature may be a useful benchmark to obtain information on the route of administration, impact on host health, intake levels, as well as any potential side effects.Nevertheless, a history of safe use alone is inadequate for this purpose and a more comprehensive analyses are necessary to confirm probiotic safety. 133,134The evaluation of the antibiotic resistance of probiotics is relevant for the use of the candidate strain as a supplement since it could be directly or indirectly transferred to pathogenic bacteria, enhancing their colonization capacity and resistance to treatment.Specifically, the assessment of antibiotic resistance is based on both genotyping and phenotyping approaches, including WGS to detect antibiotic resistance genes and estimation of the minimal inhibitory concentration (MIC) for each clinically relevant antibiotic, respectively. 30,135,136Another critical feature that must be considered in probiotic safety is the virulence and toxigenic potential, whose assessment should be based not only on taxonomic identification and evidence of virulence or toxin production but also supported by both short-term and longterm in vitro and in vivo studies to investigate the mechanisms of action and potential side effects of the strain under investigation. 31,137A typical example is represented by the Escherichia genus, especially Escherichia coli species, which is characterized by several potential pathogenic strains expressing genotoxins and secondary metabolites that have been recently described to have detrimental effects on host cells. 49,138,139These strains and other probiotic candidates should be tested for biogenic amines production (e.g.histamine and tyramine) and for mucin metabolism capability, whose excessive degradation is related to chronic diseases like obesity, diabetes, IBD, and CRC. 140,1413][144] Finally, the production process of probiotic supplements must be conducted in facilities that comply with Good Manufacturing Practice (GMP), ensuring the quality, potency, and purity of these products including the absence of microbial and chemical contaminants. 145,146t present, several guidelines and recommendations to establish probiotic safety have been published by International Associations and Agencies including the ISAPP, the Food and Agriculture Organization of the United Nations (FAO), and the World Health Organization (WHO).The first guidelines for the evaluation of probiotic safety in food were produced in 2002 by two joint FAO/ WHO working group meetings where two independent reports were obtained.Both reports were then adopted to clearly define "probiotics" and to establish specific recommendations (11 and 6 recommendations established in the Cordoba and London Joint Meeting, respectively) necessary to assess probiotic safety and effects. 147,148In brief, these preliminary recommendations include the adoption of a precise definition for probiotics and the necessity to adhere to established guidelines for labeling strains as probiotics.They stressed the importance of establishing a regulatory framework on probiotics, promoting the implementation of good manufacturing practices for probiotic foods, and the establishment of surveillance systems for adverse events associated with probiotics. 147,148ore recently, the ISAPP elaborated the expert consensus document titled "The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic", a document produced at the end of the ISAPP consensus meeting on the appropriate use and scope of the term probiotics which contains also guidelines and recommendations to clearly establish probiotics. 149More in detail, this document provided some recommendations including: i) corrections on the old definition of probiotics provided by the FAO/WHO (the new definition is "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host"); ii) considering probiotics only microbial species that have been shown to confer health benefits in properly controlled studies; iii) including more details for products whose label contains the claim "contains probiotics"; iv-v) keep live culture in fermented foods and fecal microbial transplants outside the probiotic frameworks; vi) including as probiotics new bacteria and consortia with adequate evidence of safety and efficacy. 149Besides the update provided by the ISAPP in 2014, clear guidelines and recommendations on probiotics safety were already published in 2010 by Sanders ME and collaborators (2010), who actively participated in ISAPP and other relevant Organizations.The authors elaborated a comprehensive manuscript evaluating the effects of probiotics in the context of the potential vulnerability of the consumer or patient, dose and duration of consumption, and both the manner and frequency of administration. 87This document was recently updated (2023) still maintaining the main concepts expressed in 2010. 92Briefly, these guidelines describe the correct use of probiotics in individuals with immunological defects, critically ill (e.g.hospitalized patients), IBDs, infants, and premature infants.In addition, the authors provided a comprehensive description of microbiological and metabolic issues related to probiotic safety discerning the precise microbial identification, evaluating the colonization properties of probiotics, their antibiotic resistance and the transferability of the genome, the genetic stability/genetic transfer, their pathogenicity and toxigenicity.The documents also described key initiatives in probiotic safety promoted by different consortia, including the European Food Safety Authority (EFSA), the already mentioned FAO/WHO recommendations, the Qualified Presumption of Safety (QPS) document and the PROSAFE European Project. 87,92In particular, the QPS is a document introduced by the European Union Scientific Committee on Animal Nutrition in 2002 for the safety assessment of microorganisms used in feed and food applications consisting of four evaluation steps: i) defining the taxonomy of the microbe; ii) gathering sufficient information on its safety in industrial or human environments; iii) excluding pathogenicity; iv) defining its end use.In the case of no safety concerns or if any concerns have been addressed, QPS status may be granted, exempting the microbial strain from further safety assessment.This strategy was first adopted for LAB, including some probiotic species.Compared to the American GRAS system, the QPS is a more flexible tool to assess probiotic safety. 150imilarly, the PROSAFE project aims to establish evidence-based safety assessment guidelines for probiotic LAB for human consumption.The project includes five recommendations: i) using molecular methods for taxonomic classification and depositing probiotic strains in public collections; ii) avoiding the development of microbes with abnormal resistance levels to antimicrobials without proper risk assessment; iii) avoiding the use of LAB with known virulence genes, while other properties like bile acid deconjugation were deemed irrelevant for safety assessment; iv) conducting human colonization studies following European guidelines; v) using animal models for safety assessment, specifically rat models of experimental endocarditis. 151verall, the safety and quality assessment of probiotics definitely represents a public concern, which should investigate the risk-to-benefit ratio to guide the medical community on the proper use of probiotics.

The "curious" case of Escherichia coli Nissle 1917
EcN, an intestinal Gram-negative bacillus belonging to the B2 phylogenetic group of Escherichia coli, was first isolated by Alfred Nissle (serovar O6:K5:H1) in 1917 from the feces of a German soldier which did not develop any gastrointestinal disorder during a severe outbreak of shigellosis in contrast to his comrades.Since then, EcN has been formulated as a probiotic supplement, better known as Mutaflor®, and commercialized in Germany and other European countries. 1524][155][156] In particular, EcN is characterized by a high colonization and adhesion capabilities to epithelial cells due to the presence of K5 capsule, mobile flagella, and fimbriae.Interestingly, several studies highlighted that the antimicrobial activity of this strain is mainly related to the production of siderophores and cytotoxic necrosis factors. 157,158It has also been reported that EcN protects IEB functionality, enhancing the expression levels of tight junction proteins and mucin secretion by epithelial cells.][161] Table 2 summarizes the ongoing and completed clinical trials focusing on the efficacy and safety of the probiotic EcN for the treatment of different pathological conditions.
Among the reported clinical trials, the NCT05816577 study entitled "Safety and Viability of an E. Coli Nissle Colibactin Knockout in Healthy Volunteers" investigated the safety and properties of a colibactin-knockout EcN strain (EcN ΔClbP) through a randomized, controlled intervention trial in healthy subjects (https://clinicaltrials.gov).In this regard, previous studies demonstrated that EcN genome harbors the pks (polyketide synthase) pathogenicity island encoding for a hybrid peptide-polyketide genotoxin, known as colibactin, for which several reports showed a critical role in the pathogenesis of CRC due to the induction of DNA DSBs and interstrand cross-links (ICLs) responsible for chromosomal abnormalities and gene mutations. 169,170ecently, Huber AR and colleagues (2024) observed that intestinal organoids co-cultured with a colibactin-producing EcN presented mutational patterns similar to those observed in CRC.In addition, the authors demonstrated that colibactinmediated mutations are responsible for the development of CRC diagnosed at a younger age. 171These findings are in line with those obtained by other research groups independently.Pleguezuelos-Manzano C et al. (2020), tested human intestinal organoids with both genotoxic pks + EcN and pks − mutant EcN showing a unique mutational signature in organoids treated with colibactin-producing bacteria.This colibactin-related signature was also found in a subset of CRC genomes, suggesting a direct link between exposure to colibactin-producing bacteria and CRC mutagenesis. 172The genotoxic effects of EcN were also demonstrated by treating primary murine colon epithelial organoids with short-term exposure to colibactin-producing EcN.Specifically, the authors observed that organoids infected with pks + EcN presented CRC-like phenotype including increased proliferation, Wnt-independence, and impaired cell differentiation.NGS experiments also revealed an elevated mutational burden, chromosomal aberrations, and epigenetic alterations affecting the p53-signaling pathway, including the dysregulation of miR-34a. 173hese data suggest how the precise characterization of EcN safety is mandatory to classify it as a probiotic compared to other E. coli strains.In this regard, a recent study tested the safety and kinetics of EcN both in humans and nonhuman primates demonstrating the safety of EcN with a probiotic clearance obtained after 1 week of treatment cessation in humans and a longer period in cynomolgus monkeys. 174In addition, wet and in silico metagenomic studies confirmed the presence of several gene products associated with the production of potentially harmful secondary metabolites, including 40 associated with colibactin, 10 with salmochelin, 30 with aerobactin, and nine with yersiniabactin and enterobactin.The same results were obtained by comparing EcN with other E. coli probiotic strains (e.g.CEC15) where the presence of colibactin-producing genes was not found. 175,176Other studies compared the genomics and metabolomics profile of nonpathogenic EcN with other pathogenic E. coli strains revealing strong genetic similarities between EcN and UPEC strain CFT073; however, this latter possesses several virulence factors, like the toxin α-hemolysin and type 1, P, and F1C fimbrial adhesins, which lack in the probiotic strain EcN (serotype O6:K5: H1) that is thus considered nonpathogenic. 177,178lthough EcN has been administered to patients for over a century, the identification of colibactin raises a safety issue that must be further investigated to clarify the pathogenicity and/or probiotic effect of the EcN strain.

Colibactin biosynthesis and genotoxic activity
Colibactin, a secondary metabolite belonging to the heterogeneous bacterial toxin family of cyclomodulins, is synthesized by a genomic island termed pks.Interestingly, this pathogenic island has been identified in different bacteria of the Enterobacteriaceae family, especially Klebsiella pneumoniae and Escherichia coli strains of the B2 phylogenetic group, which may be found as commensal microbes in the gut microbiota and in a variety of infections as opportunistic pathogens. 179,180The pks island (54-kb) consists of a gene cluster of 19 genes (ClbA -ClbS), which have a functional role in colibactin biosynthesis.Specifically, ClbH, ClbJ, and ClbN encode for various NRPS (Non-Ribosomal Peptide Synthetase) enzymes, while ClbC, ClbI, and ClbO encode for PKS enzymes (Figure 3). 181,182The remaining members of this cluster are involved in the encoding of hybrid NRPS-PKS enzymes (ClbB and ClbK), proteins responsible for the synthesis and transfer of amminomalonyl unit (ClbD -ClbG), accessory proteins (ClbA, ClbM, ClbP, and ClbS), and other enzymes (ClbL, ClbQ, and ClbR) (Figure 3). 181,182he colibactin biosynthetic pathway is regulated by the transcriptional activator ClbR and the phosphopantetheinyl-transferase (PPTase) ClbA.Following transcriptional activation, the hybrid NRPS-PKS enzymes catalyze the production of the intermediate pre-colibactin, which is characterized by a structural motif at the N-terminus (N-myristoyl-D-Asn motif). 183,184Precolibactin is then translocated into the periplasm and converted into the genotoxin colibactin via the transporter ClbM and the peptidase ClbP, respectively.Notably, the production of the mature colibactin is due to the removal of the N-terminus structural motif, which induces cyclization of the linear intermediate.The bioactive molecule results in two symmetrical subunits containing cyclopropane, which show a high affinity for binding adenine residues on DNA (Figure 3). 183,184s widely reported in the literature, colibactinproducing bacteria showed detrimental effects on host cells inducing DNA DSBs and chromosome aberrations. 173,185,186Among the most frequently observed structural aberrations, colibactin induces single nucleotide variants (SNV), insertion and deletion (indel) mutations, and rearrangement breakpoints.In a model of epithelial colon cells, Iftekhar A and colleagues (2021) identified multiple copy number variants (CNV), including whole chromosomes or chromosome arm loss. 173Other groups identified a colibactin mutational signature consisting of the increase of T > N substitutions at ATA, ATT, and TTT trinucleotides repeats, the increase of small indels at A: T homopolymers, and the enrichment of structural variant breakpoints affecting the AAWWTT motif. 172,187As a general mechanism, colibactin acts as a DNA alkylating agent leading to DNA ICLs and DSBs.These breaks trigger cellcycle arrest and ultimately promote the neoplastic transformation of cells. 188oreover, a higher abundance of pks + Escherichia coli has been detected in CRC patients compared to healthy controls, highlighting that colibactin overexpression could play a critical role in tumor development and invasiveness. 49,172,187lthough previous studies have provided deep insights into the biosynthesis and activity of colibactin, further investigations are needed to clarify the genotoxic potential of bacterial strains producing this metabolite.

The potential genotoxic effect of EcN
0][191][192] Despite a long history of safe use as a beneficial bacterial strain with no obvious side effects, the genomic identification of the pks locus encoding colibactin has recently raised a safety concern since this genotoxin could play a pivotal role in CRC development inducing DNA damage and gene mutations. 139,193,194In this field, a number of recent studies investigated the EcN biosafety to assess any genotoxic effect (Table 3).Janosch D et al. (2019) performed in vitro tests (Comet Assay and Ames test) to evaluate the activity of EcN strain against standard mutagens NQO (4-nitroquinoline-1-oxide), H 2 O 2 (hydrogen peroxide), and BaP (benzo[a]pyrene).Of note, preincubation with EcN preparation significantly reduced the genotoxic activity of such mutagenic compounds, indicating that the probiotic EcN could play a pivotal role in maintaining the health status of the host. 195Similarly, Dubbert S and colleagues (2020) reported that EcN cell-free supernatant showed no mutagenic activity.These results were further corroborated on animal models by testing the genotoxicity of the EcN product (1•10 11 colonyforming unit (CFU)/mL) orally administered for 28 consecutive days.Notably, no difference was detected when analyzing the intestinal tissue of treated and control rats, suggesting that EcN did not exert any genotoxic activity. 196However, it is important to note that the standard assays used by Dubbert S and colleagues were considered inappropriate by Nougayrède JP and colleagues.Indeed, they used an Ames test in which Salmonella enterica serovar Typhimurium reporter bacteria were exposed to EcN, and then Salmonella growth was expected upon mutagenesis.Moreover, Salmonella is readily killed by the siderophores/microcins produced by EcN; thus, the absence of growth of the reporter bacteria was incorrectly interpreted as an absence of the effect of colibactin.In addition, Dubbert   Thus, the assays they used cannot detect colibactin-associated mutagenic damage. 28In 2020, another study explored the pathogenic potential of EcN compared to enterohemorrhagic (EHEC) and uropathogenic (UPEC) Escherichia coli strains. 197Using human intestinal organoids, the authors observed that the EcN treatment did not affect the IEB structure and function, whereas EHEC and UPEC strains destroyed the intestinal tissues.Moreover, organoid co-cultures revealed that EcN guaranteed the IEB integrity, enhancing immune defenses and inhibiting the growth of pathogenic bacteria. 197lthough the aforementioned studies indicated that EcN may be considered a safe and efficient probiotic strain for host gastrointestinal health, heterogeneous findings are reported in the literature regarding the potential genotoxic effect of pks + EcN on host cells (Table 3).In this regard, Olier M and colleagues (2012) treated the IEC-6 cells with a non-genotoxic mutant of EcN (ΔclbA), observing no detrimental effect compared to wildtype EcN, which induced DNA DSBs to intestinal cells.Simultaneously, oral administration of the EcN ΔclbA (10 9 CFU/day) to animal models of colitis revealed that the isogenic mutant did not protect the colon, while the supplementation of wild-type EcN decreased the severity of the inflammatory status, suggesting that the probiotic effect of EcN is strictly associated to its genotoxic activity. 198Interestingly, Cañas MA et al. ( 2016) also evaluated the potential genotoxic/cytotoxic effects of outer membrane vesicles (OMVs) derived from the probiotic EcN.Notably, the authors demonstrated in vitro that EcN did not affect cell viability of the intestinal epithelial cell lines; however, the exposure to OMVs (5 μg/mL for 48 h) induced DNA DBSs in HT-29 cells, suggesting that the synthesis of colibactin could be implicated in the EcN genotoxic effect. 199Another study focused the attention on the beneficial effects of EcN-derived OMVs demonstrating a potential modulating effect on gut and liver metabolism and a better control of obesity and diabetes in mice models. 200Similarly, Han L et al. (2024)  recently tested the efficacy of EcN-OMVs in reducing intestinal inflammation by developing an innovative delivery system using aldehyde-silica microspheres.The engineered vesicles showed better stability to gastric juice both in vitro and in vivo as well as increased anti-inflammatory effects reducing the expression of TNF-α and IL-1β, and increasing the expression of zonula occludens-1. 201he close interaction between the probiotic activity and the genotoxic potential of EcN was also evaluated by Massip C et al. (2019), focusing on ClbP, an essential enzyme for the synthesis of mature colibactin.Interestingly, the authors reported that the antimicrobial activity was significantly reduced in ΔClbP mutant compared to ΔclbA mutant and wild-type EcN, highlighting that the pks genomic island could be responsible not only for the genotoxic activity of EcN, but also for its probiotic function. 202Similarly, the genotoxic and mutagenic properties of EcN were further confirmed by Nougayrède JP and colleagues (2021).Specifically, the authors noted that the infection of epithelial cells with EcN wild type caused the synthesis of colibactin leading to DNA ICLs and gene mutations, while no DNA damages were observed for the ΔclbA and ΔClbP mutants.Moreover, the genotoxic activity of colibactin-producing EcN was also demonstrated through the analyses of colon tissue samples derived from mice treated with EcN suspensions (10 8 CFU/mL). 28verall, the conflicting results of the abovementioned studies highlight a serious safety concern that must not be ignored.Therefore, deeper investigations are mandatory to provide novel insights into the long-term risk-to-benefit ratio of the probiotic EcN.Despite the safety concerns here reported, at present, there are no specific recommendations for the administration of EcN.Some authors have demonstrated in vivo that the administration of EcN to immunocompromised mice with altered gut microbiota is not recommended as they can develop severe adverse events, including septic episodes and impaired IEB functions.Since EcN is also used to reduce the incidence of necrotizing enterocolitis and death in very lowbirth-weight preterm infants, it should be noted that a case of severe sepsis in a preterm infant was observed after the administration of EcN. 203nterestingly, according to the European Crohn's and Colitis Organization (ECCO), EcN is the only recommended probiotic for the treatment of ulcerative colitis as demonstrated by multiple clinical studies. 163,192Beyond these isolated recommendations, to date there are no consensus documents on the correct use of EcN, therefore, efforts in this direction are necessary.

Conclusions
As widely reported in the literature, gut microbiota eubiosis and host health status are strictly interconnected.However, any alteration of such equilibrium may lead to dysbiotic microbiota and a higher risk of acute and chronic gastrointestinal disorders associated with the overgrowth of pathogens and the production of toxic metabolites.In this context, both probiotics and postbiotics have been largely employed as adjuvant therapeutic strategies to restore the composition of the gut microbiota and ameliorate host health conditions by exerting antiinflammatory and immunomodulatory effects.EcN is a typical example of probiotic mainly used for the treatment of gastrointestinal disorders like IBD and UC.Despite a well-documented history of safe use, the identification of the pks genomic island encoding the genotoxin colibactin suspected to induce DNA damage and CRC has raised a safety concern regarding the use of EcN.In this field, a number of recent studies focused on EcN biosafety, investigating its genotoxic potential both in vitro and in vivo.Overall, these studies have all indicated that EcN may be considered a safe and efficient probiotic strain for host gastrointestinal health; however, some reports highlighted the genotoxic effect of colibactin produced by pks + EcN on host cells able to cause DNA damage and genetic mutations.
Therefore, the exact boundary between the probiotic and pathogenicity activity of the EcN strain has not been fully deciphered yet due to the conflicting findings of studies reported in the present review article.Therefore, since the EcN genotoxic potential remains an open question of paramount importance, further studies are mandatory to establish its safety and define the optimal use of this probiotic as an adjuvant therapy for gastrointestinal disorders.

Figure 2 .
Figure 2. Pipeline for the assessment of probiotic safety.

197 - 199 -- 200 -
In vitro (rat IEC-6 cells) -In vivo (Wistar rats and SCID mice) -In vitro: Treatment with infection dose of 20 or 100 bacteria per cell -In vivo: daily oral gavage of 10 9 CFU of EcN dissolved in PBS for 45 days P: EcN induces the reduction of colon inflammation, colitis, MPO activity and IL-1β levels G: EcN increases DNA DSBs, chromosomal abnormalities, and IL-10 levels 198 In vitro (Caco-2 and HT-29 cells) Treatment with 5 μg/ml of outer membrane vesicles (OMV) from EcN for 168 h P: EcN OMV reduces HT-29 cell proliferation and oxidative stress.No alteration of cell viability G: EcN OMV induces DNA DSBs In vitro (HeLa cells) -In vivo (C57BL/6 mice) In vitro: Treatment with 5 x 10 8 CFU/ml of EcN for 4h -In vivo: Treatment with 10 9 CFU of EcN for 4 days P: EcN showed antibacterial activity toward S. Typhimurium in vivo G: production of colibactin and increase of DNA damages In vitro (HeLa and CHO cells) -In vivo (SPF and BALB/c mice) -In vitro: Cells were treated with a dose of multiplicity of infection of 400 bacteria per cell for 4h -In vivo: SPF mice were treated with 10

Table 1 .
Main functions and reported safety concerns of the most used probiotics.

Table 2 .
Ongoing and completed clinical studies on EcN (Mutaflor®) deposited in the main registries (https://trialsearch.who.int;https://clinicaltrials.gov).EcN for 28 days was well tolerated and no safety concerns occurred.After EcN cessation a median of 7 days for clearing of the EcN.

Table 3 .
Recent in vitro and in vivo studies evaluating the probiotic and genotoxic potential of the EcN strain.
P: EcN improves IEB function, host defense, and E-cadherin levels in HIO.EcN also reduces ROS, apoptosis, EHEC and UPEC strains growth et al. used a standard in vitro Comet assay to analyze the exposed intestinal epithelial cells of rats; the Comet assay detected a variety of DNA lesions through electrophoresis of broken DNA but could not detect DNA cross-links that inhibit DNA electrophoretic mobility.