Fecal microbiota transplantation: current challenges and future landscapes.

SUMMARYGiven the importance of gut microbial homeostasis in maintaining health, there has been considerable interest in developing innovative therapeutic strategies for restoring gut microbiota. One such approach, fecal microbiota transplantation (FMT), is the main "whole gut microbiome replacement" strategy and has been integrated into clinical practice guidelines for treating recurrent Clostridioides difficile infection (rCDI). Furthermore, the potential application of FMT in other indications such as inflammatory bowel disease (IBD), metabolic syndrome, and solid tumor malignancies is an area of intense interest and active research. However, the complex and variable nature of FMT makes it challenging to address its precise functionality and to assess clinical efficacy and safety in different disease contexts. In this review, we outline clinical applications, efficacy, durability, and safety of FMT and provide a comprehensive assessment of its procedural and administration aspects. The clinical applications of FMT in children and cancer immunotherapy are also described. We focus on data from human studies in IBD in contrast with rCDI to delineate the putative mechanisms of this treatment in IBD as a model, including colonization resistance and functional restoration through bacterial engraftment, modulating effects of virome/phageome, gut metabolome and host interactions, and immunoregulatory actions of FMT. Furthermore, we comprehensively review omics technologies, metagenomic approaches, and bioinformatics pipelines to characterize complex microbial communities and discuss their limitations. FMT regulatory challenges, ethical considerations, and pharmacomicrobiomics are also highlighted to shed light on future development of tailored microbiome-based therapeutics.

Proteolytic activity of surface-exposed HtrA determines its expression level and is needed to survive acidic conditions in Clostridioides difficile.

To survive in the host, pathogenic bacteria need to be able to react to the unfavorable conditions that they encounter, like low pH, elevated temperatures, antimicrobial peptides and many more. These conditions may lead to unfolding of envelope proteins and this may be lethal. One of the mechanisms through which bacteria are able to survive these conditions is through the protease/foldase activity of the high temperature requirement A (HtrA) protein. The gut pathogen Clostridioides difficile encodes one HtrA homolog that is predicted to contain a membrane anchor and a single PDZ domain. The function of HtrA in C. difficile is hitherto unknown but previous work has shown that an insertional mutant of htrA displayed elevated toxin levels, less sporulation and decreased binding to target cells. Here, we show that HtrA is membrane associated and localized on the surface of C. difficile and characterize the requirements for proteolytic activity of recombinant soluble HtrA. In addition, we show that the level of HtrA in the bacteria heavily depends on its proteolytic activity. Finally, we show that proteolytic activity of HtrA is required for survival under acidic conditions.

In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates.

Clostridioides difficile is the leading cause of postantibiotic diarrhea in adults. During infection, the bacterium must rapidly adapt to the host environment by using survival strategies. Protein phosphorylation is a reversible post-translational modification employed ubiquitously for signal transduction and cellular regulation. Hanks-type serine/threonine kinases (STKs) and serine/threonine phosphatases have emerged as important players in bacterial cell signaling and pathogenicity. C. difficile encodes two STKs (PrkC and CD2148) and one phosphatase. We optimized a titanium dioxide phosphopeptide enrichment approach to determine the phosphoproteome of C. difficile. We identified and quantified 2500 proteins representing 63% of the theoretical proteome. To identify STK and serine/threonine phosphatase targets, we then performed comparative large-scale phosphoproteomics of the WT strain and isogenic ΔprkC, CD2148, Δstp, and prkC CD2148 mutants. We detected 635 proteins containing phosphorylated peptides. We showed that PrkC is phosphorylated on multiple sites in vivo and autophosphorylates in vitro. We were unable to detect a phosphorylation for CD2148 in vivo, whereas this kinase was phosphorylated in vitro only in the presence of PrkC. Forty-one phosphoproteins were identified as phosphorylated under the control of CD2148, whereas 114 proteins were phosphorylated under the control of PrkC including 27 phosphoproteins more phosphorylated in the ∆stp mutant. We also observed enrichment for phosphothreonine among the phosphopeptides more phosphorylated in the Δstp mutant. Both kinases targeted pathways required for metabolism, translation, and stress response, whereas cell division and peptidoglycan metabolism were more specifically controlled by PrkC-dependent phosphorylation in agreement with the phenotypes of the ΔprkC mutant. Using a combination of approaches, we confirmed that FtsK was phosphorylated in vivo under the control of PrkC and that Spo0A was a substrate of PrkC in vitro. This study provides a detailed mapping of kinase-substrate relationships in C. difficile, paving the way for the identification of new biomarkers and therapeutic targets.

New insights into the type A glycan modification of Clostridioides difficile flagellar protein flagellin C by phosphoproteomics analysis.

The type A glycan modification found in human pathogen Clostridioides difficile consists of a monosaccharide (GlcNAc) that is linked to an N-methylated threonine through a phosphodiester bond. This structure has previously been described on the flagellar protein flagellin C of several C. difficile strains and is important for bacterial motility. The study of post-translational modifications often relies on some type of enrichment strategy; however, a procedure for enrichment of this modification has not yet been demonstrated. In this study, we show that an approach that is commonly used in phosphoproteomics, Fe3+-immobilized metal affinity chromatography, also enriches for peptides with this unique post-translational modification. Using LC-MS/MS analyses of immobilized metal affinity chromatography-captured tryptic peptides, we observed not only type A-modified C. difficile flagellin peptides but also a variety of truncated/modified type A structures on these peptides. Using an elaborate set of mass spectrometry analyses, we demonstrate that one of these modifications consists of a type A structure containing a phosphonate (2-aminoethylphosphonate), a modification that is rarely observed and has hitherto not been described in C. difficile. In conclusion, we show that a common enrichment strategy results in reliable identification of peptides carrying a type A glycan modification, and that the results obtained can be used to advance models about its biosynthesis.

Carriage of three plasmids in a single human clinical isolate of Clostridioides difficile.

A subset of clinical isolates of Clostridioides difficile contains one or more plasmids and these plasmids can harbor virulence and antimicrobial resistance determinants. Despite their potential importance, C. difficile plasmids remain poorly characterized. Here, we provide the complete genome sequence of a human clinical isolate that carries three high-copy number plasmids from three different plasmid families that are therefore compatible. For two of these, we identify a region capable of sustaining plasmid replication in C. difficile that is also compatible with the plasmid pCD630 that is found in many laboratory strains. Together, our data advance our understanding of C. difficile plasmid biology.

Prevalence of Clostridioides difficile in dogs (Canis familiaris) with gastrointestinal disorders in Rio de Janeiro.

Clostridioides difficile infections (CDI) have a high morbidity and mortality rate and have always been considered a nosocomial disease. Nonetheless, the number of cases of community-acquired CDI is increasing, and new evidence suggests additional C. difficile reservoirs exist. Pathogenic C. difficile strains have been found in livestock, domestic animals, and meat, so a zoonotic transmission has been proposed. OBJECTIVE: The goal of this study was to isolate C. difficile strains in dogs at a veterinary clinic in Rio de Janeiro, Brazil, and characterize clinical and pathological findings associated with lower gastrointestinal tract disorders. METHODS: Fifty stool samples and biopsy fragments from dogs were obtained and cultured in the CDBA selective medium. All suggestive C. difficile colonies were confirmed by MALDI-TOF MS and PCR (tpi gene). Vancomycin, metronidazole, moxifloxacin, erythromycin, and rifampicin were tested for antibiotic susceptibility. Biofilm, motility assays, and a PCR for the toxins (tcdA, tcdB, and cdtB), as well as ribotyping, were also performed. RESULTS: Blood samples and colonic biopsy fragments were examined in C. difficile positive dogs. Ten animals (20%) tested positive for C. difficile by using stool samples, but not from biopsy fragments. Most C. difficile strains were toxigenic: six were A+B+ belonging to RT106; two were A+B+ belonging to RT014/020; and two were A-B- belonging to RT010. All strains were biofilm producers. In the motility test, 40% of strains were as motile as the positive control, CD630 (RT012). In the disc diffusion test, two strains (RT010) were resistant to erythromycin and metronidazole; and another to metronidazole (RT014/020). In terms of C. difficile clinicopathological correlations, no statistically significant morphological changes, such as pseudomembranous and "volcano" lesions, were observed. Regarding hematological data, dogs positive for C. difficile had leucopenia (p = 0.02) and lymphopenia (p = 0.03). There was a significant correlation between senility and the presence of C. difficile in the dogs studied (p = 0,02). CONCLUSIONS: Although C. difficile has not been linked to canine diarrheal disorders, it appears to be more common in dogs with intestinal dysfunctions. The isolation of ribotypes frequently involved in human CDI outbreaks around the world supports the theory of C. difficile zoonotic transmission.

Sequence-Based Identification of Metronidazole-Resistant Clostridioides difficile Isolates.

The plasmid pCD-METRO confers metronidazole resistance in Clostridioides difficile. We showed high sequence similarity among pCD-METRO plasmids from different isolates and identified pCD-METRO and associated metronidazole-resistant isolates in clinical and veterinary reservoirs in the Americas. We recommend using PCR or genomic assays to detect pCD-METRO in metronidazole-resistant C. difficile.

Fecal Microbiota Transplantation Influences Procarcinogenic Escherichia coli in Recipient Recurrent Clostridioides difficile Patients.

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

Practical observations on the use of fluorescent reporter systems in Clostridioides difficile.

Fluorescence microscopy is a valuable tool to study a broad variety of bacterial cell components and dynamics thereof. For Clostridioides difficile, the fluorescent proteins CFPopt, mCherryOpt and phiLOV2.1, and the self-labelling tags SNAPCd and HaloTag, hereafter collectively referred as fluorescent systems, have been described to explore different cellular pathways. In this study, we sought to characterize previously used fluorescent systems in C. difficile cells. We performed single cell analyses using fluorescence microscopy of exponentially growing C. difficile cells harbouring different fluorescent systems, either expressing these separately in the cytosol or fused to the C-terminus of HupA, under defined conditions. We show that the intrinsic fluorescence of C. difficile cells increases during growth, independent of sigB or spo0A. However, when C. difficile cells are exposed to environmental oxygen autofluorescence is enhanced. Cytosolic overexpression of the different fluorescent systems alone, using the same expression signals, showed heterogeneous expression of the fluorescent systems. High levels of mCherryOpt were toxic for C. difficile cells limiting the applicability of this fluorophore as a transcriptional reporter. When fused to HupA, a C. difficile histone-like protein, the fluorescent systems behaved similarly and did not affect the HupA overproduction phenotype. The present study compares several commonly used fluorescent systems for application as transcriptional or translational reporters in microscopy and summarizes the limitations and key challenges for live-cell imaging of C. difficile. Due to independence of molecular oxygen and fluorescent signal, SNAPCd appears the most suitable candidate for live-cell imaging in C. difficile to date.

Haem is crucial for medium-dependent metronidazole resistance in clinical isolates of Clostridioides difficile.

BACKGROUND: Until recently, metronidazole was the first-line treatment for Clostridioides difficile infection and it is still commonly used. Though resistance has been reported due to the plasmid pCD-METRO, this does not explain all cases. OBJECTIVES: To identify factors that contribute to plasmid-independent metronidazole resistance of C. difficile. METHODS: Here, we investigate resistance to metronidazole in a collection of clinical isolates of C. difficile using a combination of antimicrobial susceptibility testing on different solid agar media and WGS of selected isolates. RESULTS: We find that nearly all isolates demonstrate a haem-dependent increase in the MIC of metronidazole, which in some cases leads to isolates qualifying as resistant (MIC >2 mg/L). Moreover, we find an SNP in the haem-responsive gene hsmA, which defines a metronidazole-resistant lineage of PCR ribotype 010/MLST ST15 isolates that also includes pCD-METRO-containing strains. CONCLUSIONS: Our data demonstrate that haem is crucial for medium-dependent metronidazole resistance in C. difficile.