Mycobacterium tuberculosis strain with deletions in menT3 and menT4 is attenuated and confers protection in mice and guinea pigs.

The genome of Mycobacterium tuberculosis encodes for a large repertoire of toxin-antitoxin systems. In the present study, MenT3 and MenT4 toxins belonging to MenAT subfamily of TA systems have been functionally characterized. We demonstrate that ectopic expression of these toxins inhibits bacterial growth and this is rescued upon co-expression of their cognate antitoxins. Here, we show that simultaneous deletion of menT3 and menT4 results in enhanced susceptibility of M. tuberculosis upon exposure to oxidative stress and attenuated growth in guinea pigs and mice. We observed reduced expression of transcripts encoding for proteins that are essential or required for intracellular growth in mid-log phase cultures of ΔmenT4ΔT3 compared to parental strain. Further, the transcript levels of proteins involved in efficient bacterial clearance were increased in lung tissues of ΔmenT4ΔT3 infected mice relative to parental strain infected mice. We show that immunization of mice and guinea pigs with ΔmenT4ΔT3 confers significant protection against M. tuberculosis infection. Remarkably, immunization of mice with ΔmenT4ΔT3 results in increased antigen-specific TH1 bias and activated memory T cell response. We conclude that MenT3 and MenT4 are important for M. tuberculosis pathogenicity and strains lacking menT3 and menT4 have the potential to be explored further as vaccine candidates.

Mechanisms of bacterial immunity, protection, and survival during interbacterial warfare.

Most bacteria live in communities, often with closely related strains and species with whom they must compete for space and resources. Consequently, bacteria have acquired or evolved mechanisms to antagonize competitors through the production of antibacterial toxins. Similar to bacterial systems that combat phage infection and mechanisms to thwart antibiotics, bacteria have also acquired and evolved features to protect themselves from antibacterial toxins. Just as there is a large body of research identifying and characterizing antibacterial proteins and toxin delivery systems, studies of bacterial mechanisms to resist and survive assault from competitors' weapons have also expanded tremendously. Emerging data are beginning to reveal protective processes and mechanisms that are as diverse as the toxins themselves. Protection against antibacterial toxins can be acquired by horizontal gene transfer, receptor or target alteration, induction of protective functions, physical barriers, and other diverse processes. Here, we review recent studies in this rapidly expanding field.

Fit-for-purpose heterodivalent single-domain antibody for gastrointestinal targeting of toxin B from Clostridium difficile.

Single-domain antibodies (sdAbs), such as VHHs, are increasingly being developed for gastrointestinal (GI) applications against pathogens to strengthen gut health. However, what constitutes a suitable developability profile for applying these proteins in a gastrointestinal setting remains poorly explored. Here, we describe an in vitro methodology for the identification of sdAb derivatives, more specifically divalent VHH constructs, that display extraordinary developability properties for oral delivery and functionality in the GI environment. We showcase this by developing a heterodivalent VHH construct that cross-inhibits the toxic activity of the glycosyltransferase domains (GTDs) from three different toxinotypes of cytotoxin B (TcdB) from lineages of Clostridium difficile. We show that the VHH construct possesses high stability and binding activity under gastric conditions, in the presence of bile salts, and at high temperatures. We suggest that the incorporation of early developability assessment could significantly aid in the efficient discovery of VHHs and related constructs fit for oral delivery and GI applications.

Clostridioides difficile Toxins: Host Cell Interactions and Their Role in Disease Pathogenesis.

Clostridioides difficile, a Gram-positive anaerobic bacterium, is the leading cause of hospital-acquired antibiotic-associated diarrhea worldwide. The severity of C. difficile infection (CDI) varies, ranging from mild diarrhea to life-threatening conditions such as pseudomembranous colitis and toxic megacolon. Central to the pathogenesis of the infection are toxins produced by C. difficile, with toxin A (TcdA) and toxin B (TcdB) as the main virulence factors. Additionally, some strains produce a third toxin known as C. difficile transferase (CDT). Toxins damage the colonic epithelium, initiating a cascade of cellular events that lead to inflammation, fluid secretion, and further tissue damage within the colon. Mechanistically, the toxins bind to cell surface receptors, internalize, and then inactivate GTPase proteins, disrupting the organization of the cytoskeleton and affecting various Rho-dependent cellular processes. This results in a loss of epithelial barrier functions and the induction of cell death. The third toxin, CDT, however, functions as a binary actin-ADP-ribosylating toxin, causing actin depolymerization and inducing the formation of microtubule-based protrusions. In this review, we summarize our current understanding of the interaction between C. difficile toxins and host cells, elucidating the functional consequences of their actions. Furthermore, we will outline how this knowledge forms the basis for developing innovative, toxin-based strategies for treating and preventing CDI.

Supplementation with organic yeast-derived selenium provides immune protection against experimental necrotic enteritis in broiler chickens.

Necrotic enteritis (NE) is a potentially fatal poultry disease that causes enormous economic losses in the poultry industry worldwide. The study aimed to evaluate the effects of dietary organic yeast-derived selenium (Se) on immune protection against experimental necrotic enteritis (NE) in commercial broilers. Chickens were fed basal diets supplemented with different Se levels (0.25, 0.50, and 1.00 Se mg/kg). To induce NE, Clostridium perfringens (C. perfringens) was orally administered at 14 days of age post hatch. The results showed that birds fed 0.25 Se mg/kg exhibited significantly increased body weight gain compared with the non-supplemented/infected birds. There were no significant differences in gut lesions between the Se-supplemented groups and the non-supplemented group. The antibody levels against α-toxin and NetB toxin increased with the increase between 0.25 Se mg/kg and 0.50 Se mg/kg. In the jejunal scrapings and spleen, the Se-supplementation groups up-regulated the transcripts for pro-inflammatory cytokines IL-1ß, IL-6, IL-8, iNOS, and LITAF and avian ß-defensin 6, 8, and 13 (AvBD6, 8 and 13). In conclusion, supplementation with organic yeast-derived Se alleviates the negative consequences and provides beneficial protection against experimental NE.

Clostridioides difficile toxin B subverts germinal center and antibody recall responses by stimulating a drug-treatable CXCR4-dependent mechanism.

Recurrent Clostridioides difficile infection (CDI) results in significant morbidity and mortality. We previously established that CDI in mice does not protect against reinfection and is associated with poor pathogen-specific B cell memory (Bmem), recapitulating our observations with human Bmem. Here, we demonstrate that the secreted toxin TcdB2 is responsible for subversion of Bmem responses. TcdB2 from an endemic C. difficile strain delayed immunoglobulin G (IgG) class switch following vaccination, attenuated IgG recall to a vaccine booster, and prevented germinal center formation. The mechanism of TcdB2 action included increased B cell CXCR4 expression and responsiveness to its ligand CXCL12, accounting for altered cell migration and a failure of germinal center-dependent Bmem. These results were reproduced in a C. difficile infection model, and a US Food and Drug Administration (FDA)-approved CXCR4-blocking drug rescued germinal center formation. We therefore provide mechanistic insights into C. difficile-associated pathogenesis and illuminate a target for clinical intervention to limit recurrent disease.

An in vitro assay for toxicity testing of Clostridium perfringens type C ß-toxin.

Introduction: Veterinary vaccines against Clostridium perfringens type C need to be tested for absence of toxicity, as mandated by pharmacopoeias worldwide. This toxicity testing is required at multiple manufacturing steps and relies on outdated mouse tests that involve severe animal suffering. Clostridium perfringens type C produces several toxins of which the ß-toxin is the primary component responsible for causing disease. Here, we describe the successful development of a new cell-based in vitro assay that can address the specific toxicity of the ß-toxin. Methods: Development of the cell-based assay followed the principle of in vitro testing developed for Cl. septicum vaccines, which is based on Vero cells. We screened four cell lines and selected the THP-1 cell line, which was shown to be the most specific and sensitive for ß-toxin activity, in combination with a commercially available method to determine cell viability (MTS assay) as a readout. Results: The current animal test is estimated to detect 100 - 1000-fold dilutions of the Cl. perfringens type C non-inactivated antigen. When tested with an active Cl. perfringens type C antigen preparation, derived from a commercial vaccine manufacturing process, our THP-1 cell-based assay was able to detect toxin activity from undiluted to over 10000-fold dilution, showing a linear range between approximately 1000- and 10000-fold dilutions. Assay specificity for the ß-toxin was confirmed with neutralizing antibodies and lack of reaction to Cl. perfringens culture medium. In addition, assay parameters demonstrated good repeatability. Conclusions: Here, we have shown proof of concept for a THP-1 cell-based assay for toxicity testing of veterinary Cl. perfringens type C vaccines that is suitable for all vaccine production steps. This result represents a significant step towards the replacement of animal-based toxicity testing of this veterinary clostridial antigen. As a next step, assessment of the assay's sensitivity and repeatability and validation of the method will have to be performed in a commercial manufacturing context in order to formally implement the assay in vaccine quality control.

Immunization of turkeys with Clostridium septicum alpha toxin-based recombinant subunit proteins can confer protection against experimental Clostridial dermatitis.

Clostridial dermatitis (CD), caused by Clostridium septicum, is an emerging disease of increasing economic importance in turkeys. Currently, there are no effective vaccines for CD control. Here, two non-toxic domains of C. septicum alpha toxin, namely ntATX-D1 and ntATX-D2, were identified, cloned, and expressed in Escherichia coli as recombinant subunit proteins to investigate their use as potential vaccine candidates. Experimental groups consisted of a Negative control (NCx) that did not receive C. septicum challenge, while the adjuvant-only Positive control (PCx), ntATX-D1 immunization (D1) and ntATX-D2 immunization (D2) groups received C. septicum challenge. Turkeys were immunized subcutaneously with 100 µg of protein at 7, 8 and 9 weeks of age along with an oil-in-water nano-emulsion adjuvant, followed by C. septicum challenge at 11 weeks of age. Results showed that while 46.2% of birds in the PCx group died post-challenge, the rate of mortality in D1- or D2-immunization groups was 13.3%. The gross and histopathological lesions in the skin, muscle and spleen showed that the disease severity was highest in PCx group, while the D2-immunized birds had significantly lower lesion scores when compared to PCx. Gene expression analysis revealed that PCx birds had significantly higher expression of pro-inflammatory cytokine genes in the skin, muscle and spleen than the NCx group, while the D2 group had significantly lower expression of these genes compared to PCx. Peripheral blood cellular analysis showed increased frequencies of activated CD4+ and/or CD8+ cells in the D1 and D2-immunized groups. Additionally, the immunized turkeys developed antigen-specific serum IgY antibodies. Collectively, these findings indicate that ntATX proteins, specifically the ntATX-D2 can be a promising vaccine candidate for protecting turkeys against CD and that the protection mechanisms may include downregulation of C. septicum-induced inflammation and increased CD4+ and CD8+ cellular activation.

Facile construction of sandwich ELISA based on double-nanobody for specific detection of α-hemolysin in food samples.

α-hemolysin (Hla), a toxin secreted by Staphylococcus aureus (S. aureus), has been proved to be involved in the occurrence and aggravation of food poisoning. Hence, it is quite essential to establish its rapid detection methods to guarantee food safety. Sandwich ELISA based on nanobody is well known to be viable for toxins, but there is absence of nanobody against Hla, let alone a pair for it. Therefore, in this paper, we screened specific nanobodies by bio-panning and obtained the optimal nanobody pair for sandwich ELISA firstly. Then, RANbody, a novel nanobody owning both recognition and catalytic capability, is generated in a single step and at low cost through molecular recombination technology. Subsequently, sandwich ELISA was developed to detect Hla based on the nanobody and RANbody, that not only eliminated the use of secondary antibodies and animal-derived antibody, but also reduced detection time and cost, compared with traditional sandwich ELISA. Lastly, the performance has been evaluated, especially for specificity which showed no response to other hemolysins and a low limit of detection of 10 ng/mL. Besides, the proposed sandwich ELISA exhibits favorable feasibility and was successfully employed for the detection of Hla in milk and pork samples.

The impact of existing total anti-toxin B IgG immunity in outcomes of recurrent Clostridioides difficile infection.

Late anti-toxin-B humoral immunity acquired after treatment is important for preventing recurrent Clostridioides difficile infection. We prospectively-measured anti-toxin-B IgG and neutralization titers at diagnosis as potential early predictors of recurrence. High anti-toxin-B-IgG/neutralizing antibodies were associated with short-lasting protection within 6-weeks, however, no difference in recurrence risk was observed by 90-days post-infection.

Neutralization of the Staphylococcus aureus Panton-Valentine leukocidin by African and Caucasian sera.

BACKGROUND: The prevalence of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin (PVL) gene is higher in Africa (≈50%) compared to Europe (< 5%). The study aimed to measure anti-PVL-antibodies in Africans and Germans in a multi-center study and to test whether detected antibodies can neutralize the cytotoxic effect of PVL on polymorphonuclear leukocytes (PMNs). METHODS: Sera from asymptomatic Africans (n = 22, Nigeria, Gabon) and Caucasians (n = 22, Germany) were used to quantify antibody titers against PVL and α-hemolysin (in arbitrary units [AU]) by ELISA. PMNs from one African and German donor were exposed to 5 nM recombinant PVL to measure the neutralizing effect of serial dilutions of pooled sera from African and Caucasian participants, or donor sera at 0.625 and 2.5% (v/v). RESULTS: Anti-PVL-antibodies were significantly higher in Africans than in Germans (1.9 vs. 0.7 AU, p < 0.0001). The pooled sera from the study participants neutralized the cytotoxic effect of PVL on African and German PMNs in a dose dependent manner. Also, neutralization of PVL on PMNs from the African and German donors had a stronger effect with African sera (half-maximal inhibitory concentration (IC50) = 0.27 and 0.47%, respectively) compared to Caucasian sera (IC50 = 3.51 and 3.59% respectively). CONCLUSION: Africans have higher levels of neutralizing anti-PVL-antibodies. It remains unclear if or at what level these antibodies protect against PVL-related diseases.

Lymphostatin, a virulence factor of attaching and effacing Escherichia coli, inhibits proliferation and cytokine responses of human T cells in a manner associated with cell cycle arrest but not apoptosis or necrosis.

Lymphostatin is a virulence factor of enteropathogenic E. coli (EPEC) and non-O157 serogroup enterohaemorrhagic E. coli. Previous studies using whole-cell lysates of EPEC showed that lymphostatin inhibits the mitogen-activated proliferation of bulk human peripheral blood mononuclear cells (PBMCs) and the production of cytokines IL-2, IL-4, IL-5, and IFN-γ. Here, we used highly purified lymphostatin and PBMC-derived T cells to show that lymphostatin inhibits anti-CD3/anti-CD28-activated proliferation of human CD4+ and CD8+ T cells and blocks the synthesis of IL-2, IL-4, IL-10 and IFN-γ without affecting cell viability and in a manner dependent on an N-terminal DTD glycosyltransferase motif. Such inhibition was not observed with T cells activated by phorbol 12-myristate 13-acetate and ionomycin, implying that lymphostatin targets T cell receptor signaling. Analysis of the expression of CD69 indicated that lymphostatin suppresses T cell activation at an early stage and no impacts on apoptosis or necrosis were observed. Flow cytometric analysis of the DNA content of lymphostatin-treated CD4+ and CD8+ T cells showed a concentration- and DTD-dependent accumulation of the cells in the G0/G1 phase of the cell cycle, and corresponding reduction of the percentage of cells in S phase. Consistent with this, we found a marked reduction in the abundance of cyclins D3, E and A and loss of phosphorylated Rb over time in activated T cells from 8 donors treated with lymphostatin. Moreover, the cyclin-dependent kinase (cdk) inhibitor p27kip1, which inhibits progression of the cell cycle at G1 by acting on cyclin E-cdk2 or cyclin D-cdk4 complexes, was found to be accumulated in lymphostatin-treated T cells. Analysis of the abundance of phosphorylated kinases involved in signal transduction found that 30 of 39 were reduced in abundance following lymphostatin treatment of T cells from 5 donors, albeit not significantly so. Our data provide novel insights into the mode of action of lymphostatin on human T lymphocytes.

Human OTULIN haploinsufficiency impairs cell-intrinsic immunity to staphylococcal α-toxin.

The molecular basis of interindividual clinical variability upon infection with Staphylococcus aureus is unclear. We describe patients with haploinsufficiency for the linear deubiquitinase OTULIN, encoded by a gene on chromosome 5p. Patients suffer from episodes of life-threatening necrosis, typically triggered by S. aureus infection. The disorder is phenocopied in patients with the 5p- (Cri-du-Chat) chromosomal deletion syndrome. OTULIN haploinsufficiency causes an accumulation of linear ubiquitin in dermal fibroblasts, but tumor necrosis factor receptor-mediated nuclear factor κB signaling remains intact. Blood leukocyte subsets are unaffected. The OTULIN-dependent accumulation of caveolin-1 in dermal fibroblasts, but not leukocytes, facilitates the cytotoxic damage inflicted by the staphylococcal virulence factor α-toxin. Naturally elicited antibodies against α-toxin contribute to incomplete clinical penetrance. Human OTULIN haploinsufficiency underlies life-threatening staphylococcal disease by disrupting cell-intrinsic immunity to α-toxin in nonleukocytic cells.

Hydrogen-Deuterium Exchange Mass Spectrometry Reveals a Novel Binding Region of a Neutralizing Fully Human Monoclonal Antibody to Anthrax Protective Antigen.

Anthrax vaccine adsorbed (AVA) containing protective antigen (PA) is the only FDA-approved anthrax vaccine in the United States. Characterization of the binding of AVA-induced anti-PA human antibodies against the PA antigen after vaccination is crucial to understanding mechanisms of the AVA-elicited humoral immune response. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is often coupled with a short liquid chromatography gradient (e.g., 5-10 min) for the characterization of protein interactions. We recently developed a long-gradient (e.g., 90 min), sub-zero temperature, ultra-high performance liquid chromatography HDX-MS (UPLC-HDX-MS) platform that has significantly increased separation power and limited back-exchange for the analysis of protein samples with high complexity. In this study, we demonstrated the utility of this platform for mapping antibody-antigen epitopes by examining four fully human monoclonal antibodies to anthrax PA. Antibody p1C03, with limited neutralizing activity in vivo, bound to a region on domain 1A of PA. p6C04 and p1A06, with no neutralizing activities, bound to the same helix on domain 3 to prevent oligomerization of PA. We found p6C01 strongly bound to domain 3 on a different helix region. We also identified a secondary epitope for p6C01, which likely leads to the blocking of furin cleavage of PA after p6C01 binding. This novel binding of p6C01 results in highly neutralizing activity. This is the first report of this distinct binding mechanism for a highly neutralizing fully human antibody to anthrax protective antigen. Studying such epitopes can facilitate the development of novel therapeutics against anthrax.

Aerosolized Intratracheal Inoculation of Recombinant Protective Antigen (rPA) Vaccine Provides Protection Against Inhalational Anthrax in B10.D2-Hc0 Mice.

Anthrax caused by Bacillus anthracis is a fatal zoonotic disease with a high lethality and poor prognosis. Inhalational anthrax is the most severe of the three forms of anthrax. The currently licensed commercial human anthrax vaccines require a complex immunization procedure for efficacy and have side effects that limit its use in emergent situations. Thus, development of a better anthrax vaccine is necessary. In this study, we evaluate the potency and efficacy of aerosolized intratracheal (i.t.) inoculation with recombinant protective antigen (rPA) subunit vaccines against aerosolized B. anthracis Pasteur II spores (an attenuated strain) challenge in a B10.D2-Hc0 mouse (deficient in complement component C5) model. Immunization of rPA in liquid, powder or powder reconstituted formulations via i.t. route conferred 100% protection against a 20× LD50 aerosolized Pasteur II spore challenge in mice, compared with only 50% of subcutaneous (s.c.) injection with liquid rPA. Consistently, i.t. inoculation of rPA vaccines induced a higher lethal toxin (LeTx) neutralizing antibody titer, a stronger lung mucosal immune response and a greater cellular immune response than s.c. injection. Our results demonstrate that immunization with rPA dry powder vaccine via i.t. route may provide a stable and effective strategy to improve currently available anthrax vaccines and B10.D2-Hc0 mice challenged with B. anthracis attenuated strains might be an alternative model for anthrax vaccine candidate screening.

Vaccination of mice with hybrid protein containing Exotoxin S and PcrV with adjuvants alum and MPL protects Pseudomonas aeruginosa infections.

Pseudomonas aeruginosa as a common pathogen causing urinary tract infections (UTIs) has been resistant to different antibiotics and developing an effective vaccine can be an alternative strategy. In the present study, the immunogenicity and protection efficacy of formulations composed of a hybrid protein composed of P. aeruginosa V-antigen (PcrV) and exoenzyme S (ExoS) with alum and MPL were evaluated. The hybrid protein could increase the specific systemic and mucosal immune responses, as well as cellular responses as compared with control groups. Combining of alum or MPL adjuvant with the hybrid protein significantly improved the levels of IgG1, serum IgA, mucosal IgG, and IL-17 as compared to the ExoS.PcrV alone. After bladder challenge with a P. aeruginosa strain, the bacterial loads of bladder and kidneys were significantly decreased in mice received ExoS.PcrV admixed with alum and ExoS.PcrV admixed with MPL than controls. The present study indicated that immunization of mice with a hybrid protein composed of ExoS and PcrV could induce multifactorial immune responses and opsonize the bacteria and decrease the viable bacterial cells. Because P. aeruginosa have caused therapeutic challenges worldwide, our study proposed ExoS.PcrV + alum and ExoS.PcrV + MPL as promising candidates for the prevention of infections caused by P. aeruginosa.

Rapid pathogen-specific recruitment of immune effector cells in the skin by secreted toxins.

Swift recruitment of phagocytic leucocytes is critical in preventing infection when bacteria breach through the protective layers of the skin. According to canonical models, this occurs via an indirect process that is initiated by contact of bacteria with resident skin cells and which is independent of the pathogenic potential of the invader. Here we describe a more rapid mechanism of leucocyte recruitment to the site of intrusion of the important skin pathogen Staphylococcus aureus that is based on direct recognition of specific bacterial toxins, the phenol-soluble modulins (PSMs), by circulating leucocytes. We used a combination of intravital imaging, ear infection and skin abscess models, and in vitro gene expression studies to demonstrate that this early recruitment was dependent on the transcription factor EGR1 and contributed to the prevention of infection. Our findings refine the classical notion of the non-specific and resident cell-dependent character of the innate immune response to bacterial infection by demonstrating a pathogen-specific high-alert mechanism involving direct recruitment of immune effector cells by secreted bacterial products.

Characterization of the adaptive immune response of donors receiving live anthrax vaccine.

Live anthrax vaccine containing spores from attenuated strains STI-1 of Bacillus anthracis is used in Russia and former CIS (Commonwealth of Independent States) to prevent anthrax. In this paper we studied the duration of circulation of antibodies specific to spore antigens, the protective antigen (PA), the lethal factor (LF) and their domains (D) in donors' blood at different times after their immunization with live anthrax vaccine. The relationship between the toxin neutralization activity level and the level of antibodies to PA, LF and their domains was tested. The effect of age, gender and number of vaccinations on the level of adaptive post-vaccination immune response has been studied. It was shown that antibodies against PA-D1 circulate in the blood of donors for 1 year or more after immunization with live anthrax vaccine. Antibodies against all domains of LF and PA-D4 were detected in 11 months after vaccination. Antibodies against the spores were detected in 8 months after vaccination. A moderate positive correlation was found between the titers of antibodies to PA, LF, or their domains, and the TNA of the samples of blood serum from the donors.

A Multi-Factorial Observational Study on Sequential Fecal Microbiota Transplant in Patients with Medically Refractory Clostridioides difficile Infection.

Fecal microbiota transplantation (FMT) is highly effective in recurrent Clostridioides difficile infection (CDI); increasing evidence supports FMT in severe or fulminant Clostridioides difficile infection (SFCDI). However, the multifactorial mechanisms that underpin the efficacy of FMT are not fully understood. Systems biology approaches using high-throughput technologies may help with mechanistic dissection of host-microbial interactions. Here, we have undertaken a deep phenomics study on four adults receiving sequential FMT for SFCDI, in which we performed a longitudinal, integrative analysis of multiple host factors and intestinal microbiome changes. Stool samples were profiled for changes in gut microbiota and metabolites and blood samples for alterations in targeted epigenomic, metabonomic, glycomic, immune proteomic, immunophenotyping, immune functional assays, and T-cell receptor (TCR) repertoires, respectively. We characterised temporal trajectories in gut microbial and host immunometabolic data sets in three responders and one non-responder to sequential FMT. A total of 562 features were used for analysis, of which 78 features were identified, which differed between the responders and the non-responder. The observed dynamic phenotypic changes may potentially suggest immunosenescent signals in the non-responder and may help to underpin the mechanisms accompanying successful FMT, although our study is limited by a small sample size and significant heterogeneity in patient baseline characteristics. Our multi-omics integrative longitudinal analytical approach extends the knowledge regarding mechanisms of efficacy of FMT and highlights preliminary novel signatures, which should be validated in larger studies.