The rapid increase in microbial antibiotic resistance in Clostridioides difficile (C. difficile) strains and the formation of hypervirulent strains have been associated with a global increase in the incidence of C. difficile infection (CDI) and subsequently, an increase in the rate of recurrence. These consequences have led to an urgent need to develop new and promising alternative strategies to control this pathogen. Engineered probiotics are exciting new bacterial strains produced by editing the genome of the original probiotics. Recently, engineered probiotics have been used to develop delivery vehicles for vaccines, diagnostics, and therapeutics. Recent studies have demonstrated engineered probiotics may potentially be an effective approach to control or treat CDI. This review provides a brief overview of the considerations for engineered probiotics for medicinal use, with a focus on recent preclinical research using engineered probiotics to prevent or treat CDI. We also address the challenges faced in the production of engineered strains and how they may be overcome in the application of these agents to meet patient needs in the future.

Clostridioides difficile (C. difficile) infection (CDI) is common after antibiotic exposure and presents significant morbidity, mortality and healthcare costs worldwide. The rising incidence of recurrent CDI, driven by hypervirulent strains, widespread antibiotic use and increased community transmission, has led to an urgent need for novel therapeutic strategies. Conventional antibiotic treatments, although effective, face limitations due to rising antibiotic resistance and high recurrence rates, which can reach up to 60% after multiple infections. This has prompted exploration of alternative therapies such as fecal microbiota-based therapies, including fecal microbiota transplantation (FMT) and live biotherapeutics (LBPs), which demonstrate superior efficacy in preventing recurrence. They are aimed at restoring the gut microbiota. Fecal microbiota, live-jslm and fecal microbiota spores, live-brpk have been approved by the U.S. Food and Drug Administration in individuals aged 18 years or older for recurrent CDI after standard antimicrobial treatment. They have demonstrated high efficacy and a favorable safety profile in clinical trials. Another LBP under study includes VE-303, which is not derived from human donor stool. This review provides a comprehensive overview of the current therapeutic landscape for CDI, including its epidemiology, pathophysiology, risk factors, diagnostic modalities and treatment strategies. The review delves into the emerging role of live biotherapeutics, with a particular focus on fecal microbiota-based therapies. We explore their development, mechanisms of action, clinical applications and potential to revolutionize CDI management.

The diagnosis, pharmacologic management, and surgical options for Clostridioides difficile infection (CDI) are rapidly evolving, which presents a challenge for the busy surgeon to remain up to date on the latest clinical guidelines. This review provides an evidence-based practical guide for CDI management tailored to the needs of surgeons and surgical intensivists. Historically, the diagnosis of CDI relied on slow cell culture cytotoxicity neutralization assays, but now, the rapidly resulting nucleic acid amplification tests and enzyme immunoassays have become mainstream. In terms of antibiotic therapy, metronidazole and oral vancomycin were the main "workhorse" antibiotics in the early 2000s, but large randomized controlled trials have now demonstrated that fidaxomicin produces superior results. Regarding surgical intervention, total abdominal colectomy was once the only procedure of choice; however, diverting loop ileostomy with colonic lavage is emerging as a viable alternative. Finally, novel adjuncts such as fecal microbiota transplantation and targeted therapy against toxin B (bezlotoxumab) are playing an increasingly important role in the management of CDI.

Clostridioides difficile (C. difficile) remains the leading cause of healthcare-associated diarrhoea, posing treatment challenges because of antibiotic resistance and high relapse rates. Faecal microbiota transplantation is a novel treatment strategy to prevent relapses of C. difficile infection (CDI), however, the exact components conferring colonization resistance are unknown, hampering its translation to a medicinal product. The development of novel products independent of antibiotics, which increase colonization resistance or induce protective immune mechanisms is urgently needed.

To establish a framework for a Controlled Human Infection Model (CHIM) of C. difficile, in which healthy volunteers are exposed to toxigenic C. difficile spores, offering the possibility to test novel approaches and identify microbiota and immunological targets. Whereas experimental exposure to non-toxigenic C. difficile has been done before, a toxigenic C. difficile CHIM faces ethical, scientific, logistical, and biosafety challenges.

Specific challenges in developing a C. difficile CHIM were discussed by a group of international experts during a workshop organized by Inno4Vac, an Innovative Health Initiative-funded consortium.

The experts agreed that the main challenges are: developing a clinically relevant CHIM that induces mild to moderate CDI symptoms but not severe CDI, determining the optimal C. difficile inoculum dose, and understanding the timing and duration of antibiotic pretreatment in inducing susceptibility to CDI in healthy volunteers.

Should these challenges be tackled, a C. difficile CHIM will not only provide a way forward for the testing of novel products but also offer a framework for a better understanding of the pathophysiology, pathogenesis, and immunology of C. difficile colonization and infection.

The primary purposes of this review are to provide a brief overview of the microbiome, discuss the most relevant outcome data and key characteristics of each live microbiome agent, and pose questions for consideration going forward as these agents are integrated into clinical practice.

The management of Clostridiodes difficile infection (CDI) remains a difficult clinical conundrum, with recurrent CDI occurring in 15% to 35% of patients and causing significant morbidity and decreased quality of life. For patients with frequent CDI recurrences, fecal microbiota transplantation (FMT) has been demonstrated to have significant benefit but also significant risks, and FMT is not approved by the US Food and Drug Administration (FDA) for that indication. FDA has established a new therapeutic class for agents known as live biotherapeutic products (LBPs) that offer significant advantages over FMT, including standardized screening, testing, and manufacturing as well as known quantities of organisms contained within. Two new live microbiome products within this class were recently approved by FDA for prevention of CDI recurrences in adult patients following treatment for recurrent CDI with standard antimicrobial therapy. Both agents had demonstrated efficacy in registry trials in preventing CDI recurrence but differ significantly in a number of characteristics, such as route of administration. Cost as well as logistics are current obstacles to use of these therapies.

Live microbiome therapy is a promising solution for patients with recurrent CDI. Future studies should provide further evidence within yet-to-be-evaluated populations not included in registry studies. This along with real-world evidence will inform future use and clinical guideline placement.

Clostridioides difficile is the predominant pathogen in hospital-acquired infections and antibiotic-associated diarrhea. Dedicated networks and annual reports for C. difficile surveillance have been established in Europe and North America, however the extensive investigation on the prevalence of C. difficile infection (CDI) in China is limited. In this study, 1528 patients with diarrhea were recruited from seven geographically representative regions of China between July 2021 and July 2022. The positivity rate of toxigenic C. difficile using real-time fluorescence quantitative PCR test of feces was 10.2% (156/1528), and 125 (8.2%, 125/1528) strains were successfully isolated. The isolates from different geographical areas had divergent characteristics after multilocus sequence typing, toxin gene profiling, and antimicrobial susceptibility testing. No isolate from clade 2 were found, and clade 1 was still the main clade for these clinical isolates. Interestingly, clade 4, especially ST37, previously known as the characteristic type of China, showed a strong geographical divergence. Clade 3, although rare in China, has been detected in Hainan and Sichuan provinces. Most C. difficile isolates (76.8%, 96/125) were toxigenic. Clindamycin, erythromycin, and moxifloxacin were the top three antibiotics to which resistance was observed, with resistance rates of 81.3%, 63.6%, and 24.0%, respectively. Furthermore, 34 (27.2%, 34/125) multidrug-resistant (MDR) strains were identified. All the strains were sensitive to metronidazole, vancomycin, and meropenem. The genotype of C. difficile varies greatly among the different geographical regions in China, and new types are constantly emerging. Therefore, comprehensive, longitudinal, and standardized surveillance of C. difficile infections is needed in China, covering typical geographical areas.

Microbiome science has evolved rapidly in the past decade, with high-profile publications suggesting that the gut microbiome is a causal determinant of human health. This has led to the emergence of microbiome-focused biotechnology companies and pharmaceutical company investment in the research and development of gut-derived therapeutics. Despite the early promise of this field, the first generation of microbiome-derived therapeutics (faecal microbiota products) have only recently been approved for clinical use. Next-generation therapies based on readily culturable and as-yet-unculturable colonic bacterial species (with the latter estimated to comprise 63% of all detected species) have not yet progressed to pivotal phase 3 trials. This reflects the many challenges involved in developing a new class of drugs in an evolving field. Here we discuss the evolution of the live biotherapeutics field over the past decade, from the development of first-generation products to the emergence of rationally designed second- and third-generation live biotherapeutics. Finally, we present our outlook for the future of this field.

Clostridioides difficile is a leading cause of healthcare infections. Gut dysbiosis promotes C. difficile infection (CDI) and CDIs promote gut dysbiosis, leading to frequent CDI recurrence. Although therapies preventing recurrent CDI have been developed, including live biotherapeutic products, existing therapies are costly and do not prevent primary infections. Here, we show that an avirulent C. difficile isolate, ST1-75, protects mice from developing colitis induced by a virulent R20291 strain when coinfected at a 1:1 ratio. In metabolic analyses, avirulent ST1-75 depletes amino acids more rapidly than virulent R20291 and supplementation with amino acids ablates this competitive advantage, indicating that ST1-75 limits the growth of virulent R20291 through amino acid depletion. Overall, our study identifies inter-strain nutrient depletion as a potentially exploitable mechanism to reduce the incidence of CDI and reveals that the ST1-75 strain may be a biotherapeutic agent that can prevent CDI in high-risk patients.

Clostridioides difficile species lacking toxin genes (non-toxigenic C. difficile or NTCD) may confer protection against CDI. However, current diagnostic tests detect either toxin proteins or toxin genes and cannot detect NTCD. This study developed a molecular testing method that uniquely identified NTCD and assessed its prevalence in a clinical cohort. A quantitative PCR (qPCR) assay was developed and validated using reference strains. Analytic sensitivity was determined using DNA from reference NTCD strains, and qPCR efficiency was assessed using the slope of the standard curves of DNA dilutions. A random selection of 95 clinical stool samples, tested using the GDH enzyme and toxin enzyme immunoassay (EIA), was used to evaluate the prevalence of NTCD in hospitalized patients tested for CDI. The KB-1/KB-2 primers and probe designed were specific for NTCD strains and did not amplify with toxigenic C. difficile or other related strains. The NTCD qPCR assay analytical sensitivity was linear between 3 × 101 and 3 × 106 gDNA (R2 = 0.999; P < 0.0001). No NTCD was found in 25 GDH-EIA -/- samples compared to 5 of 25 (20%) GDH-EIA +/- samples and 2 of 23 (8.7%) GDH-EIA +/+ samples. Of samples detected with NTCD, median NTCD DNA was 33,039 (IQR: 22.449-45.688) in GDH-EIA +/- samples and 370 [IQR: 159-583] in GDH-EIA +/+ samples. The new qPCR NTCD assay identified NTCD colonization in 7% of hospitalized patients tested for CDI. This NTCD assay may have important implications for diagnostic and antimicrobial stewardship as colonization with NTCD strains may offer protection against CDI.IMPORTANCECurrent diagnostic strategies do not detect non-toxigenic Clostridioides difficile (NTCD) strains, which may provide protection against C. difficile infection (CDI). Detecting these strains is critical as it underscores the importance of avoiding unnecessary antibiotic treatment in patients colonized with NTCD. To better guide clinical decisions and enhance the understanding of NTCD epidemiology, molecular assays that specifically target non-coding regions unique to NTCD strains are needed. In this study, we developed and validated a qPCR assay capable of uniquely identifying NTCD strains. This innovative assay holds significant potential for applications in public health, infection control, diagnostic, and therapeutic strategies related to CDI.

The genomic revolution has fueled rapid progress in synthetic and systems biology, opening up new possibilities for using live biotherapeutic products (LBP) to treat, attenuate or prevent human diseases. Among LBP, bacteria-based therapies are particularly promising due to their ability to colonize diverse human tissues, modulate the immune system and secrete or deliver complex biological products. These bacterial LBP include engineered pathogenic species designed to target specific diseases, and microbiota species that promote microbial balance and immune system homeostasis, either through local administration or the gut-body axes. This review focuses on recent advancements in preclinical and clinical trials of bacteria-based LBP, highlighting both on-site and long-reaching strategies.

Clostridioides difficile is a toxin-producing bacteria that is a main cause of antibiotic-associated diarrhea. Clostridioides difficile infections (CDI) are associated with disruptions within the gastrointestinal (GI) microbiota which can be further exacerbated by CDI-targeted antibiotic treatment thereby causing recurrent CDI (rCDI) and compounding the burden placed on patients and the healthcare system. Treatment of rCDI consists of antibiotics which can be paired with preventative therapeutics, such as bezlotoxumab or fecal microbiota transplants (FMTs), if sustained clinical response is not obtained. Newer preventative strategies have been recently approved to assist in restoring balance within the GI system with the goal of preventing recurrent infections.

Since the publication of the recent North American and European guidelines on management of Clostridioides difficile infection (CDI), new evidence describing the epidemiology, testing and treatment of CDI has emerged. Despite all advances in infection control and antibiotic stewardship, the incidence and burden of CDI in the hospitals and the community remains at a stable high. Coupled with the incidence of primary CDI, there is a stable high incidence of recurrent CDI. Testing for primary and recurrent CDI remains a clinical challenge owing to high sensitivity of the PCR (leading to false positives) and somewhat limited sensitivity of EIA for toxin. The pathophysiology of recurrent CDI involves an ongoing disruption of the microbiota owing to the infection and the treatment of CDI employed. Broad spectrum antibiotics such as vancomycin leads to further disruption of microbiota compared to fidaxomicin which has a lower disruption of the microbiota and leads to fewer recurrences. Owing to these data fidaxomicin is considered as the first line antibiotic for recurrent CDI. Intravenous bezlotoxumab is a monoclonal antibody that reduces the risk of recurrence in high-risk patients but does not restore the microbiota. Experimental fecal microbiota transplantation (FMT) has been available for more than a decade. Owing to the success of FMT, two new non-invasive donor dependent Food and Drug Administration (FDA) approved therapies have been available since late 2022. This review summarizes all these conundrums regarding CDI and provides clinical pearls to use in day-to-day practice.

Hepatobiliary cancers, including hepatocellular carcinoma and cancers of the biliary tract, share high mortality and rising incidence rates. They may also share several risk factors related to unhealthy western-type dietary and lifestyle patterns as well as increasing body weights and rates of obesity. Recent data also suggest a role for the gut microbiome in the development of hepatobiliary cancer and other liver pathologies. The gut microbiome and the liver interact bidirectionally through the "gut-liver axis," which describes the interactive relationship between the gut, its microbiota, and the liver. Here, we review the gut-liver interactions within the context of hepatobiliary carcinogenesis by outlining the experimental and observational evidence for the roles of gut microbiome dysbiosis, reduced gut barrier function, and exposure to inflammatory compounds as well as metabolic dysfunction as contributors to hepatobiliary cancer development. We also outline the latest findings regarding the impact of dietary and lifestyle factors on liver pathologies as mediated by the gut microbiome. Finally, we highlight some emerging gut microbiome editing techniques currently being investigated in the context of hepatobiliary diseases. Although much work remains to be done in determining the relationships between the gut microbiome and hepatobiliary cancers, emerging mechanistic insights are informing treatments, such as potential microbiota manipulation strategies and guiding public health advice on dietary/lifestyle patterns for the prevention of these lethal tumors.

SUMMARYHealthcare-associated infections (HAIs) represent a burden for public health with a high prevalence and high death rates associated with them. Pathogens with a high potential for antimicrobial resistance, such as ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) and Clostridioides difficile, are responsible for most HAIs. Despite the implementation of infection prevention and control intervention, globally, HAIs prevalence is stable and they are mainly due to endogenous pathogens. It is undeniable that complementary to infection prevention and control measures, prophylactic approaches by active or passive immunization are needed. Specific groups at-risk (elderly people, chronic condition as immunocompromised) and also healthcare workers are key targets. Medical procedures and specific interventions are known to be at risk of HAIs, in addition to hospital environmental exposure. Vaccines or monoclonal antibodies can be seen as attractive preventive approaches for HAIs. In this review, we present an overview of the vaccines and monoclonal antibodies in clinical development for prevention of the major bacterial HAIs pathogens. Based on the current state of knowledge, we look at the challenges and future perspectives to improve prevention by these means.

Composed of an elaborate ecosystem of bacteria, fungi, viruses and protozoa residing in the human digestive tract, the gut microbiome influences metabolism, immune modulation, bile acid homeostasis and host defence. Through observational and preclinical data, the gut microbiome has been implicated in the pathogenesis of a spectrum of chronic diseases ranging from psychiatric to gastrointestinal in nature. Until recently, the lack of unequivocal evidence supporting a causal link between gut microbiome and human health outcomes incited controversy regarding its significance. However, recent randomised controlled trial (RCT) evidence in conditions, such as Clostridioides difficile infection, cancer immunotherapy and ulcerative colitis, has supported a causal relationship and has underscored the potential of the microbiome as a therapeutic target. This review delineates the RCT evidence substantiating the potential for a causal relationship between the gut microbiome and human health outcomes, the seminal observational evidence that preceded these RCTs and the remaining knowledge gaps.

The emergence of antimicrobic-resistant infectious pathogens and the consequent rising in the incidence and prevalence of demises caused by or associated to infections which are not sensitive to drug treatments is one of today's major global health challenges. Antimicrobial resistance (AMR) can bring to therapeutic failure, infection's persistence and risk of serious illness, in particular in vulnerable populations such as the elderly, patients with neoplastic diseases or the immunocompromised. It is assessed that AMR will induce until 10 million deaths per year by 2050, becoming the leading cause of disease-related deaths. The World Health Organisation (WHO) and the United Nations General Assembly urgently call for new measures to combat the phenomenon. Research and development of new antimicrobial agents has decreased due to market failure. However, promising results are coming from new alternative therapeutic strategies such as monoclonal antibodies, microbiome modulators, nanomaterial-based therapeutics, vaccines, and phages. This narrative review aimed to analyse the benefits and weaknesses of alternative therapeutic strategies to antibiotics which treat multidrug-resistant bacterial infections.

Clostridioides difficile is the main causative agent of antibiotic-associated diarrhea (AAD) in hospitals in the developed world. Both infected patients and asymptomatic colonized individuals represent important transmission sources of C. difficile. C. difficile infection (CDI) shows a large range of symptoms, from mild diarrhea to severe manifestations such as pseudomembranous colitis. Epidemiological changes in CDIs have been observed in the last two decades, with the emergence of highly virulent types and more numerous and severe CDI cases in the community. C. difficile interacts with the gut microbiota throughout its entire life cycle, and the C. difficile's role as colonizer or invader largely depends on alterations in the gut microbiota, which C. difficile itself can promote and maintain. The restoration of the gut microbiota to a healthy state is considered potentially effective for the prevention and treatment of CDI. Besides a fecal microbiota transplantation (FMT), many other approaches to re-establishing intestinal eubiosis are currently under investigation. This review aims to explore current data on C. difficile and gut microbiota changes in colonized individuals and infected patients with a consideration of the recent emergence of highly virulent C. difficile types, with an overview of the microbial interventions used to restore the human gut microbiota.

Clostridioides difficile (formerly Clostridium difficile) has been regarded as an 'urgent threat' and a significant global health problem, as life-threatening diarrhoea and refractory recurrence are common in patients with C. difficile infection (CDI). Unfortunately, the available anti-CDI drugs are limited. Recent guidelines recommend fidaxomicin and vancomycin as first-line drugs to treat CDI, bezlotoxumab to prevent recurrence, and faecal microbiota transplantation for rescue treatment. Currently, researchers are investigating therapeutic antibacterial drugs (e.g. teicoplanin, ridinilazole, ibezapolstat, surotomycin, cadazolid, and LFF571), preventive medications against recurrence (e.g. Rebyota, Vowst, VP20621, VE303, RBX7455, and MET-2), primary prevention strategies (e.g. vaccine, ribaxamase, and DAV132) and other anti-CDI medications in the preclinical stage (e.g. Raja 42, Myxopyronin B, and bacteriophage). This narrative review summarises current medications, including newly marketed drugs and products in development against CDI, to help clinicians treat CDI appropriately and to call for more research on innovation.

Clostridioides difficile (C. difficile) strains belonging to the epidemic BI/NAP1/027 (RT027) group have been associated with increased transmissibility and disease severity. In addition to the major toxin A and toxin B virulence factors, RT027 strains also encode the CDT binary toxin. Our lab previously identified a toxigenic RT027 isolate, ST1-75, that is avirulent in mice despite densely colonizing the colon. Here, we show that coinfecting mice with the avirulent ST1-75 and virulent R20291 strains protects mice from colitis due to rapid clearance of the virulent strain and persistence of the avirulent strain. Although avirulence of ST1-75 is due to a mutation in the cdtR gene, which encodes a response regulator that modulates the production of all three C. difficile toxins, the ability of ST1-75 to protect against acute colitis is not directly attributable to the cdtR mutation. Metabolomic analyses indicate that the ST1-75 strain depletes amino acids more rapidly than the R20291 strain and supplementation with amino acids ablates ST1-75's competitive advantage, suggesting that the ST1-75 strain limits the growth of virulent R20291 bacteria by amino acid depletion. Since the germination kinetics and sensitivity to the co-germinant glycine are similar for the ST1-75 and R20291 strains, our results identify the rapidity of in vivo nutrient depletion as a mechanism providing strain-specific, virulence-independent competitive advantages to different BI/NAP1/027 strains. They also suggest that the ST1-75 strain may, as a biotherapeutic agent, enhance resistance to CDI in high-risk patients.

Strong clinical data demonstrate that inflammatory bowel disease (IBD) is an independent risk factor for Clostridiodes difficile infection (CDI) and suggest a globally increased prevalence and severity of C. difficile coinfection in IBD patients (CDI-IBD). In addition to elderly individuals, children are also at higher risk of CDI-IBD. Rapid diagnosis is essential since the clinical manifestations of active IBD and CDI-IBD are indistinguishable. Antibiotics have been well established in the treatment of CDI-IBD, but they do not prevent recurrence.

Herein, the authors focus on reviewing recent research advances on the new therapies of CDI-IBD. The novel therapies include gut microbiota restoration therapies (such as prebiotics, probiotics and FMT), immunotherapy (such as vaccines and monoclonal antibodies) and diet strategies (such as groningen anti-inflammatory diet and mediterranean diet). Future extensive prospective and placebo-controlled studies are required to evaluate their efficacy and long-term safety.

Available studies show that the prevalence of CDI-IBD is not optimistic. Currently, potential treatment options for CDI-IBD include a number of probiotics and novel antibiotics. This review updates the knowledge on the management of CDI in IBD patients, which is timely and important for GI doctors and scientists.

Faecal microbiota transplantation (FMT) is the standard treatment for patients with multiple recurrent Clostridioides difficile infection (rCDI). Recently, new commercially developed human microbiota-derived medicinal products have been evaluated and Food and Drug Administration-approved with considerable differences in terms of composition, administration, and targeted populations.

To review available data on the different microbiota-derived treatments at the stage of advanced clinical evaluation and research in rCDI in comparison with FMT.

Phase II or III trials evaluating a microbiota-derived medicinal product to prevent rCDI.

Two commercial microbiota-derived medicinal products are approved by the Food and Drug Administration: Rebyota (RBX2660 Ferring Pharmaceuticals, marketed in the United States) and VOWST (SER-109 -Seres Therapeutics, marketed in the United States), whereas VE303 (Vedanta Biosciences Inc) will be studied in phase III trial. RBX2660 and SER-109 are based on the processing of stools from healthy donors, whereas VE303 consists of a defined bacterial consortium originating from human stools and produced from clonal cell banks. All have proven efficacy to prevent rCDI compared with placebo in patients considered at high risk of recurrence. However, the heterogeneity of the inclusion criteria, and the time between each episode and CDI diagnostics makes direct comparison between trials difficult. The differences regarding the risk of recurrence between the treatment and placebo arms were lower than previously described for FMT (FMT: Δ = 50.5%; RBX2660-phase III: Δ = 13.1%; SER-109-phase III: Δ = 28%; high-dose VE303-phase-II: Δ = 31.7%). All treatments presented a good overall safety profile with mainly mild gastrointestinal symptoms.

Stool-derived products and bacterial consortia need to be clearly distinguished in terms of product characterization and their associated risks with specific long-term post-marketing evaluation similar to registries used for FMT. Their place in the therapeutic strategy for patients with rCDI requires further studies to determine the most appropriate patient population and administration route to prevent rCDI.

The Lachnospiraceae family holds promise as a source of next-generation probiotics, yet a comprehensive delineation of its diversity is lacking, hampering the identification of suitable strains for future applications. To address this knowledge gap, we conducted an in-depth genomic and functional analysis of 1868 high-quality genomes, combining data from public databases with our new isolates. This data set represented 387 colonization-selective species-level clusters, of which eight genera represented multilineage clusters. Pan-genome analysis, single-nucleotide polymorphism (SNP) identification, and probiotic functional predictions revealed that species taxonomy, habitats, and geography together shape the functional diversity of Lachnospiraceae. Moreover, analyses of associations with atherosclerotic cardiovascular disease (ACVD) and inflammatory bowel disease (IBD) indicated that several strains of potentially novel Lachnospiraceae species possess the capacity to reduce the abundance of opportunistic pathogens, thereby imparting potential health benefits. Our findings shed light on the untapped potential of novel species enabling knowledge-based selection of strains for the development of next-generation probiotics holding promise for improving human health and disease management.

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated diarrhea, despite the widespread implementation of contact precautions for patients with CDI. Here, we investigate strain contamination in a hospital setting and the genomic determinants of disease outcomes. Across two wards over 6 months, we selectively cultured C. difficile from patients (n = 384) and their environments. Whole-genome sequencing (WGS) of 146 isolates revealed that most C. difficile isolates were from clade 1 (131/146, 89.7%), while only one isolate of the hypervirulent ST1 was recovered. Of culture-positive admissions (n = 79), 19 (24%) patients were colonized with toxigenic C. difficile on admission to the hospital. We defined 25 strain networks at ≤2 core gene single nucleotide polymorphisms; two of these networks contain strains from different patients. Strain networks were temporally linked (P < 0.0001). To understand the genomic correlates of the disease, we conducted WGS on an additional cohort of C. difficile (n = 102 isolates) from the same hospital and confirmed that clade 1 isolates are responsible for most CDI cases. We found that while toxigenic C. difficile isolates are associated with the presence of cdtR, nontoxigenic isolates have an increased abundance of prophages. Our pangenomic analysis of clade 1 isolates suggests that while toxin genes (tcdABER and cdtR) were associated with CDI symptoms, they are dispensable for patient colonization. These data indicate that toxigenic and nontoxigenic C. difficile contamination persist in a hospital setting and highlight further investigation into how accessory genomic repertoires contribute to C. difficile colonization and disease.

Clostridioides difficile infection remains a leading cause of hospital-associated diarrhea, despite increased antibiotic stewardship and transmission prevention strategies. This suggests a changing genomic landscape of C. difficile. Our study provides insight into the nature of prevalent C. difficile strains in a hospital setting and transmission patterns among carriers. Longitudinal sampling of surfaces and patient stool revealed that both toxigenic and nontoxigenic strains of C. difficile clade 1 dominate these two wards. Moreover, quantification of transmission in carriers of these clade 1 isolates underscores the need to revisit infection prevention measures in this patient group. We identified unique genetic signatures associated with virulence in this clade. Our data highlight the complexities of preventing transmission of this pathogen in a hospital setting and the need to investigate the mechanisms of in vivo persistence and virulence of prevalent lineages in the host gut microbiome.

Clostridioides difficile infection (CDI) remains a significant contributor to healthcare costs and morbidity due to high rates of recurrence. Currently, available antibiotic treatment strategies further disrupt the fecal microbiome and do not address the alterations in commensal flora (dysbiosis) that set the stage for CDI. Advances in microbiome-based research have resulted in the development of new agents, classified as live biotherapeutic products (LBPs), for preventing recurrent CDI (rCDI) by restoring eubiosis. Prior to the LBPs, fecal microbiota transplantation (FMT) was available for this purpose; however, lack of large-scale availability and safety concerns have remained barriers to its widespread use. The LBPs are an exciting development, but questions remain. Some are derived directly from human stool while other developmental products contain a defined microbial consortium manufactured ex vivo, and they may be composed of either living bacteria or their spores, making it difficult to compare members of this heterogenous drug class to one another. None have been studied head-to head or against FMT in preventing rCDI. As a class, they have considerable variability in their biologic composition, biopharmaceutic science, route of administration, stages of development, and clinical trial data. This review will start by explaining the role of dysbiosis in CDI, then give the details of the biopharmaceutical components for the LBPs which are approved or in development including how they differ from FMT and from one another. We then discuss the clinical trials of the LBPs currently approved for rCDI and end with the future clinical directions of LBPs beyond C. difficile.

Within the Innovative Health Initiative (IHI) Inno4Vac CHIMICHURRI project, a regulatory workshop was organised on the development and manufacture of challenge agent strains for Controlled Human Infection Model (CHIM) studies. Developers are often uncertain about which GMP requirements or regulatory guidelines apply but should be guided by the 2022 technical white paper "Considerations on the Principles of Development and Manufacturing Qualities of Challenge Agents for Use in Human Infection Models" (published by hVIVO, Wellcome Trust, HIC-Vac consortium members). Where those recommendations cannot be met, regulators advise following the "Principles of GMP" until definitive guidelines are available. Sourcing wild-type virus isolates is a significant challenge for developers. Still, it is preferred over reverse genetics challenge strains for several reasons, including implications and regulations around genetically modified organisms (GMOs). Official informed consent guidelines for collecting isolates are needed, and the characterisation of these isolates still presents risks and uncertainty. Workshop topics included ethics, liability, standardised clinical endpoints, selection criteria, sharing of challenge agents, and addressing population heterogeneity concerning vaccine response and clinical course. The organisers are confident that the workshop discussions will contribute to advancing ethical, safe, and high-quality CHIM studies of influenza, RSV and C. difficile, including adequate regulatory frameworks.

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in premature infants. Evidence indicates that bile acid homeostasis is disrupted during NEC: ileal bile acid levels are elevated in animals with experimental NEC, as is expression of the apical sodium-dependent bile acid transporter (Asbt). In addition, bile acids, which are synthesized in the liver, are extensively modified by the gut microbiome, including via the conversion of primary bile acids to more cytotoxic secondary forms. We hypothesized that the addition of bile acid-modifying bacteria would increase susceptibility to NEC in a neonatal rat model of the disease. The secondary bile acid-producing species Clostridium scindens exacerbated both incidence and severity of NEC. C. scindens upregulated the bile acid transporter Asbt and increased levels of intraenterocyte bile acids. Treatment with C. scindens also altered bile acid profiles and increased hydrophobicity of the ileal intracellular bile acid pool. The ability of C. scindens to enhance NEC requires bile acids, as pharmacological sequestration of ileal bile acids protects animals from developing disease. These findings indicate that bile acid-modifying bacteria can contribute to NEC pathology and provide additional evidence for the role of bile acids in the pathophysiology of experimental NEC.NEW & NOTEWORTHY Necrotizing enterocolitis (NEC), a life-threatening gastrointestinal emergency in premature infants, is characterized by dysregulation of bile acid homeostasis. We demonstrate that administering the secondary bile acid-producing bacterium Clostridium scindens enhances NEC in a neonatal rat model of the disease. C. scindens-enhanced NEC is dependent on bile acids and driven by upregulation of the ileal bile acid transporter Asbt. This is the first report of bile acid-modifying bacteria exacerbating experimental NEC pathology.

Clostridioides difficile is a major nosocomial pathogen, causing significant morbidity and mortality worldwide. Antibiotic usage, a major risk factor for Clostridioides difficile infection (CDI), disrupts the gut microbiota, allowing C. difficile to proliferate and cause infection, and can often lead to recurrent CDI (rCDI). Fecal microbiota transplantation (FMT) and live biotherapeutic products (LBPs) have emerged as effective treatments for rCDI and aim to restore colonization resistance provided by a healthy gut microbiota. However, much is still unknown about the mechanisms mediating their success. Bile acids, extensively modified by gut microbes, affect C. difficile's germination, growth, and toxin production while also shaping the gut microbiota and influencing host immune responses. Additionally, microbial interactions, such as nutrient competition and cross-feeding, contribute to colonization resistance against C. difficile and may contribute to the success of microbiota-focused therapeutics. Bile acids as well as other microbial mediated interactions could have implications for other diseases being treated with microbiota-focused therapeutics. This review focuses on the intricate interplay between bile acid modifications, microbial ecology, and host responses with a focus on C. difficile, hoping to shed light on how to move forward with the development of new microbiota mediated therapeutic strategies to combat rCDI and other intestinal diseases.

Repeated exposure to antibiotics and changes in the diet and environment shift the gut microbial diversity and composition, making the host susceptible to pathogenic infection. The emergence and ongoing spread of AMR pathogens is a challenging public health issue. Recent evidence showed that probiotics and prebiotics may play a role in decolonizing drug-resistant pathogens by enhancing the colonization resistance in the gut. This review aims to analyze available evidence from human-controlled trials to determine the effect size of probiotic interventions in decolonizing AMR pathogenic bacteria from the gut. We further studied the effects of prebiotics in human and animal studies. PubMed, Embase, Web of Science, Scopus, and CINAHL were used to collect articles. The random-effects model meta-analysis was used to pool the data. GRADE Pro and Cochrane collaboration tools were used to assess the bias and quality of evidence. Out of 1395 citations, 29 RCTs were eligible, involving 2871 subjects who underwent either probiotics or placebo treatment to decolonize AMR pathogens. The persistence of pathogenic bacteria after treatment was 22%(probiotics) and 30.8%(placebo). The pooled odds ratio was 0.59(95% CI:0.43-0.81), favoring probiotics with moderate certainty (p = 0.0001) and low heterogeneity (I2 = 49.2%, p = 0.0001). The funnel plot showed no asymmetry in the study distribution (Kendall'sTau = -1.06, p = 0.445). In subgroup, C. difficile showed the highest decolonization (82.4%) in probiotics group. Lactobacillus-based probiotics and Saccharomyces boulardii decolonize 71% and 77% of pathogens effectively. The types of probiotics (p < 0.018) and pathogens (p < 0.02) significantly moderate the outcome of decolonization, whereas the dosages and regions of the studies were insignificant (p < 0.05). Prebiotics reduced the pathogens from 30% to 80% of initial challenges. Moderate certainty of evidence suggests that probiotics and prebiotics may decolonize pathogens through modulation of gut diversity. However, more clinical outcomes are required on particular strains to confirm the decolonization of the pathogens. Protocol registration: PROSPERO (ID = CRD42021276045).

Multidrug-resistant microorganisms have become a major public health concern around the world. The gut microbiome is a gold mine for bioactive compounds that protect the human body from pathogens. We used a multi-omics approach that integrated whole-genome sequencing (WGS) of 74 commensal gut microbiome isolates with metabolome analysis to discover their metabolic interaction with Salmonella and other antibiotic-resistant pathogens. We evaluated differences in the functional potential of these selected isolates based on WGS annotation profiles. Furthermore, the top altered metabolites in co-culture supernatants of selected commensal gut microbiome isolates were identified including a series of dipeptides and examined for their ability to prevent the growth of various antibiotic-resistant bacteria. Our results provide compelling evidence that the gut microbiome produces metabolites, including the compound class of dipeptides that can potentially be applied for anti-infection medication, especially against antibiotic-resistant pathogens. Our established pipeline for the discovery and validation of bioactive metabolites from the gut microbiome as novel candidates for multidrug-resistant infections represents a new avenue for the discovery of antimicrobial lead structures.

Clostridioides difficile infection (CDI) remains a significant cause of morbidity and mortality worldwide. Historically, two antibiotics (metronidazole and vancomycin) and a recent third (fidaxomicin) have been used for CDI treatment; convincing data are now available showing that metronidazole is the least efficacious agent. The European Society of Clinical Microbiology and Infectious Diseases (ESCMID) management guidance for CDI were updated in 2021. This guidance document outlines the treatment options for a variety of CDI clinical scenarios and for non-antimicrobial management (e.g., faecal microbiota transplantation, FMT). One of the main changes is that metronidazole is no longer recommended as first-line CDI treatment. Rather, fidaxomicin is preferred on the basis of reduced recurrence rates with vancomycin as an acceptable alternative. Recommended options for recurrent CDI now include bezlotoxumab as well as FMT.A 2017 survey of 20 European countries highlighted variation internationally in CDI management strategies. A variety of restrictions were in place in 65% countries prior to use of new anti-CDI treatments, including committee/infection specialist approval or economic review/restrictions. This survey was repeated in November 2022 to assess the current landscape of CDI management practices in Europe. Of 64 respondents from 17 countries, national CDI guidelines existed in 14 countries, and 11 have already/plan to incorporate the ESCMID 2021 CDI guidance, though implementation has not been surveyed in 6. Vancomycin is the most commonly used first-line agent for the treatment of CDI (n = 42, 66%), followed by fidaxomicin (n = 30, 47%). Six (9%) respondents use metronidazole as first-line agent for CDI treatment, whereas 22 (34%) only in selected low-risk patient groups. Fidaxomicin is more likely to be used in high-risk patient groups. Availability of anti-CDI therapy influenced prescribing in six respondents (9%). Approval pre-prescription was required before vancomycin (n = 3, 5%), fidaxomicin (n = 10, 6%), bezlotoxumab (n = 11, 17%) and FMT (n = 10, 6%). Implementation of CDI guidelines is rarely audited.Novel anti-CDI agents are being evaluated; it is not yet clear what will be the roles of these agents. The treatment of recurrent CDI is particularly troublesome, and several different live biotherapeutics are being developed, in addition to FMT.

As a significant cause of global morbidity and mortality, Clostridioides difficile infections (CDIs) are listed by the Centres for Disease Control and prevention as one of the top 5 urgent threats in the USA. CDI occurs from gut microbiome dysbiosis, typically through antibiotic-mediated disruption; however, antibiotics are the treatment of choice, which can result in recurrent infections. Here, we highlight new treatments available and provide a perspective on different classes of future treatments.

Due to the reduced risk of disease recurrence, the microbiome-sparing antibiotic Fidaxomicin has been recommended as the first-line treatment for C. difficile infection. Based on the success of faecal microbiota transplantations (FMT) in treating CDI recurrence, defined microbiome biotherapeutics offer a safer and more tightly controlled alterative as an adjunct to antibiotic therapy. Given the association between antibiotic-mediated dysbiosis of the intestinal microbiota and the recurrence of CDI, future prospective therapies aim to reduce the dependence on antibiotics for the treatment of CDI.

With current first-in-line antibiotic therapy options associated with high levels of recurrent CDI, the availability of new generation targeted therapeutics can really impact treatment success. There are still unknowns about the long-term implications of these new CDI therapeutics, but efforts to expand the CDI treatment toolbox can offer multiple solutions for clinicians to treat this multifaceted infectious disease to reduce patient suffering.

Clostridioides difficile causes a range of debilitating intestinal symptoms that may be fatal. It is particularly problematic as a hospital-acquired infection, causing significant costs to the health care system. Antibiotics, such as vancomycin and fidaxomicin, are still the drugs of choice for C. difficile infections, but their effectiveness is limited, and microbial interventions are emerging as a new treatment option. This paper focuses on alternative treatment approaches, which are currently in various stages of development and can be divided into four therapeutic strategies. Direct killing of C. difficile (i) includes beside established antibiotics, less studied bacteriophages, and their derivatives, such as endolysins and tailocins. Restoration of microbiota composition and function (ii) is achieved with fecal microbiota transplantation, which has recently been approved, with standardized defined microbial mixtures, and with probiotics, which have been administered with moderate success. Prevention of deleterious effects of antibiotics on microbiota is achieved with agents for the neutralization of antibiotics that act in the gut and are nearing regulatory approval. Neutralization of C. difficile toxins (iii) which are crucial virulence factors is achieved with antibodies/antibody fragments or alternative binding proteins. Of these, the monoclonal antibody bezlotoxumab is already in clinical use. Immunomodulation (iv) can help eliminate or prevent C. difficile infection by interfering with cytokine signaling. Small-molecule agents without bacteriolytic activity are usually selected by drug repurposing and can act via a variety of mechanisms. The multiple treatment options described in this article provide optimism for the future treatment of C. difficile infection.

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated diarrhea, despite the widespread implementation of contact precautions for patients with CDI. Here, we investigate strain contamination in a hospital setting and genomic determinants of disease outcomes. Across two wards over six months, we selectively cultured C. difficile from patients (n=384) and their environments. Whole-genome sequencing (WGS) of 146 isolates revealed that most C. difficile isolates were from clade 1 (131/146, 89.7%), while only one isolate of the hypervirulent ST1 was recovered. Of culture-positive admissions (n=79), 19 (24%) of patients were colonized with toxigenic C. difficile on admission to the hospital. We defined 25 strain networks at ≤ 2 core gene SNPs; 2 of these networks contain strains from different patients. Strain networks were temporally linked (p<0.0001). To understand genomic correlates of disease, we conducted WGS on an additional cohort of C. difficile (n=102 isolates) from the same hospital and confirmed that clade 1 isolates are responsible for most CDI cases. We found that while toxigenic C. difficile isolates are associated with the presence of cdtR , nontoxigenic isolates have an increased abundance of prophages. Our pangenomic analysis of clade 1 isolates suggests that while toxin genes ( tcdABER and cdtR ) were associated with CDI symptoms, they are dispensable for patient colonization. These data indicate toxigenic and nontoxigenic C. difficile contamination persists in a hospital setting and highlight further investigation into how accessory genomic repertoires contribute to C. difficile colonization and disease.

Fecal microbiota transplantation (FMT) has emerged as a promising therapeutic approach for dysbiosis-related diseases. However, the clinical practice of crude fecal transplants presents limitations in terms of acceptability and reproductivity. Consequently, two alternative solutions to FMT are developed: transplanting bacteria communities or virome. Advanced methods for transplanting bacteria mainly include washed microbiota transplantation and bacteria spores treatment. Transplanting the virome is also explored, with the development of fecal virome transplantation, which involves filtering the virome from feces. These approaches provide more palatable options for patients and healthcare providers while minimizing research heterogeneity. In general, the evolution of the next generation of FMT in global trends is fecal microbiota components transplantation which mainly focuses on transplanting bacteria or virome.

Clostridioides difficile infection (CDI) is a leading nosocomial infection, posing a substantial public health challenge within the United States and globally. CDI typically occurs in hospitalized elderly patients who have been administered antibiotics; however, there has been a rise in the occurrence of CDI in the community among young adults who have not been exposed to antibiotics. C. difficile releases toxins, which damage large intestinal epithelium, leading to toxic megacolon, sepsis, and even death. Unfortunately, existing antibiotic therapies do not always prevent these consequences, with up to one-third of treated patients experiencing a recurrence of the infection. Host factors play a crucial role in the pathogenesis of CDI, and accumulating evidence shows that modulation of host immune responses may potentially alter the disease outcome. In this review, we provide an overview of our current knowledge regarding the role of innate and adaptive immune responses on CDI outcomes. Moreover, we present a summary of non-antibiotic microbiome-based therapies that can effectively influence host immune responses, along with immunization strategies that are intended to tackle both the treatment and prevention of CDI.

Clostridioides difficile infection (CDI) has become the most common healthcare-associated infection in the United States, with considerable morbidity, mortality, and healthcare costs. Assessing new preventive strategies is vital. We present a literature review of studies evaluating a strategy of screening and isolation of asymptomatic carriers in hospital settings. Asymptomatic detection of C. difficile is reported in ~ 10-20% of admitted patients. Risk factors for carriage include recent hospitalization, previous antibiotics, older age, lower functional capacity, immunosuppression, and others. Asymptomatic C. difficile carriers of toxigenic strains are at higher risk for progression to CDI. They are also shedders of C. difficile spores and may contribute to the persistence and transmission of this bacterium. Screening for asymptomatic carriers at hospital admission can theoretically reduce CDI by isolating carriers to reduce transmission, and implementing antibiotic stewardship measures targeting carriers to prevent progression to clinical illness. Several observational studies, summarized in this review, have reported implementing screening and isolation strategies, and found a reduction in CDI rates. Nevertheless, the data are still limited to a few observational studies, and this strategy is not commonly practiced. Studies supporting screening were performed in North America, coinciding with the period of dominance of the 027/BI/NAP1 strain. Additional studies evaluating screening, followed by infection control and antibiotic stewardship measures, are needed.

Clostridioides difficile is considered a nosocomial pathogen that flares up in patients exposed to antibiotic treatment. However, four out of ten patients diagnosed with C. difficile infection (CDI) acquired the infection from non-hospitalized individuals, many of whom have not been treated with antibiotics. Treatment of recurrent CDI (rCDI) with antibiotics, especially vancomycin (VAN) and metronidazole (MNZ), increases the risk of experiencing a relapse by as much as 70%. Fidaxomicin, on the other hand, proved more effective than VAN and MNZ by preventing the initial transcription of RNA toxin genes. Alternative forms of treatment include quorum quenching (QQ) that blocks toxin synthesis, binding of small anion molecules such as tolevamer to toxins, monoclonal antibodies, such as bezlotoxumab and actoxumab, bacteriophage therapy, probiotics, and fecal microbial transplants (FMTs). This review summarizes factors that affect the colonization of C. difficile and the pathogenicity of toxins TcdA and TcdB. The different approaches experimented with in the destruction of C. difficile and treatment of CDI are evaluated.

Non-toxigenic Clostridioides difficile (NTCD) has been shown to decrease the risk of recurrent C. difficile infection (CDI) in patients following metronidazole or vancomycin treatment for CDI. Limited data on the prevalence of NTCD strains in symptomatic patients and their clinical characteristics are available. We conducted this study to investigate the prevalence of NTCD in diarrhoea patients and their clinical characteristics. Between July 2017 and June 2018, unduplicated stool specimens were collected from patients with diarrhoea. The characteristics and episodes of C. difficile infection in patients with NTCD and toxigenic strains were compared. Among the 1182 stool specimens collected, 236 (18.5%) were identified as growing C. difficile, and 19.5% of the identified isolates were found to be NTCD. Multivariate analysis showed that community-onset diarrhoea (OR = 4.13, 95% CI 1.07-15.97; p = 0.040), underlying diabetes (OR = 3.64, 95% CI 1.46-9.25; p = 0.006), previous use of glycopeptides (OR = 4.75, 95% CI 1.37-16.42; p = 0.014), and the lack of use of proton pump inhibitors (PPIs) (OR = 3.57, 95% CI 1.39-9.09; p = 0.009) were independently associated with the NTCD group. Although there was no statistical significance, the number of CDI episodes occurring after 90 days tended to be lower in the NTCD group (2.2%) than in the toxigenic group (11.2%). A considerable portion of the C. difficile strains isolated from patients with diarrhoea showed NTCD. Further, more extensive studies are needed to clearly define the protective effects of NTCD strains in patients with diarrhoea.

The high prevalence of infectious diseases in Africa, combined with weak healthcare systems, poor antimicrobial stewardship, and an unchecked drug supply chain, is steadily reversing the trend in the fight against infectious diseases in this part of the world, posing severe threats to antimicrobial resistance (AMR). AMR continuously evolves and threatens to undermine antimicrobial efficacy and undo advances against infectious diseases. This brewing pandemic is now recognized as a significant worldwide health danger, implicated in several cases of morbidity, mortality, and increasing healthcare costs. Vaccine technology has been proven to be the principal remedy to this imminent danger since it prevents microbial infections. However, since Africa cannot produce its vaccines, it relies on external sources and, as a result, it is significantly affected by vaccine nationalism, hoarding, and instabilities in global supply chains. This has further adversely impacted the ability of African governments to regulate rollouts, protect their citizens, and ultimately rejoin the global economy. This dependency is a severe challenge to Africa's health resilience, as it is unsustainable. Given the inevitability of potential global pandemics and the alarming incidences of multi-drug resistance infections reported daily, Africa must develop the capability to produce its vaccines. The review utilized a systematic search of academic databases and grey literature, as well as a manual search of relevant reports and articles. In this review, we outline the public health threats and concerns that AMR poses to Africans, and the hurdles and advances achieved in vaccine development over the years. We also highlight possible strategies, particularly collaborative efforts, that will accelerate vaccine production and ease the strain of infectious diseases and antimicrobial resistance in Africa. Key findings indicate that Africa has significant gaps in its vaccine manufacturing and distribution capacity, with only a few countries having the ability to produce vaccines. Additionally, existing vaccine production facilities are often outdated and require significant investment to meet international standards. The review also highlights successful initiatives in Africa, such as the mRNA vaccine hub and the African Vaccine Manufacturing Initiative, which have demonstrated the potential for building local vaccine manufacturing capacity. The study concludes that Africa needs to prioritize investment in vaccine research and development, regulatory capacity, and infrastructure to build a sustainable vaccine manufacturing ecosystem. Overall, this review emphasizes the urgent need for Africa to develop its vaccine manufacturing capacity to improve vaccine access and strengthen its ability to respond to future pandemics. The findings underscore the importance of collaboration between African governments, international organizations, and the private sector to build a resilient vaccine ecosystem in Africa.