Identification and engraftment of new bacterial strains by shotgun metagenomic sequence analysis in patients with recurrent Clostridioides difficile infection before and after fecal microbiota transplantation and in healthy human subjects.

BACKGROUND: Recurrent Clostridioides diffícile infection (RCDI) is associated with major bacterial dysbiosis and colitis. Fecal microbiota transplantation (FMT) is a highly effective therapeutic modality for RCDI. While several studies have identified bacterial species associated with resolution of symptoms in patients, characterization of the fecal microbiome at the bacterial strain level in RCDI patients before and after FMT and healthy donors, has been lacking. The aim of this study was to examine the ability of bacterial strains from healthy donors to engraft in the gastrointestinal tract of patients with RCDI following FMT. METHODS: Fecal samples were collected from 22 patients with RCDI before and after FMT and their corresponding healthy donors. Total DNA was extracted from each sample and analyzed by shotgun metagenomic sequencing. The Cosmos-ID analysis platform was used for taxonomic assignment of sequences and calculation of the relative abundance (RA) of bacterial species and strains. From these data, the total number of bacterial strains (BSI), Shannon diversity index, dysbiosis index (DI), and bacterial engraftment factor, were calculated for each strain. FINDINGS: A marked reduction (p<0·0001) in the RA of total and specific bacterial strains, especially from phylum Firmicutes, was observed in RCDI patients prior to FMT. This change was associated with an increase in the DI (p<0·0001) and in pathobiont bacterial strains from phylum Proteobacteria, such as Escherichia coli O157:H7 and Klebsiella pneumoniae UCI 34. BSI was significantly lower in this group of patients as compared to healthy donors and correlated with the Shannon Index. (p<0·0001). Identification and engraftment of bacterial strains from healthy donors revealed a greater diversity and higher relative abundance of short-chain fatty acid (SCFA)-producing bacterial strains, including Lachnospiraceae bacterium 5_1_63FAA_u_t, Dorea formicigenerans ATCC 27755, Anaerostipes hadrusand others, in RCDI patients after FMT. INTERPRETATION: These observations identify a group of SCFA-producing bacterial strains from healthy donors that engraft well in patients with RCDI following FMT and are associated with complete resolution of clinical symptoms and bacterial dysbiosis.

Identification of RNAs bound by Hfq reveals widespread RNA partners and a sporulation regulator in the human pathogen Clostridioides difficile.

Noncoding RNAs (ncRNA) have emerged as important components of regulatory networks governing bacterial physiology and virulence. Previous deep-sequencing analysis identified a large diversity of ncRNAs in the human enteropathogen Clostridioides (Clostridium) difficile. Some of them are trans-encoded RNAs that could require the RNA chaperone protein Hfq for their action. Recent analysis suggested a pleiotropic role of Hfq in C. difficile with the most pronounced effect on sporulation, a key process during the infectious cycle of this pathogen. However, a global view of RNAs interacting with C. difficile Hfq is missing. In the present study, we performed RNA immunoprecipitation high-throughput sequencing (RIP-Seq) to identify Hfq-associated RNAs in C. difficile. Our work revealed a large set of Hfq-interacting mRNAs and ncRNAs, including mRNA leaders and coding regions, known and potential new ncRNAs. In addition to trans-encoded RNAs, new categories of Hfq ligands were found including cis-antisense RNAs, riboswitches and CRISPR RNAs. ncRNA-mRNA and ncRNA-ncRNA pairings were postulated through computational predictions. Investigation of one of the Hfq-associated ncRNAs, RCd1, suggests that this RNA contributes to the control of late stages of sporulation in C. difficile. Altogether, these data provide essential molecular basis for further studies of post-transcriptional regulatory network in this enteropathogen.

Intestinal Clostridioides difficile Can Cause Liver Injury through the Occurrence of Inflammation and Damage to Hepatocytes.

This study investigated if intestinal Clostridioides difficile (CD) causes liver injury. Four-week-old male C3H/HeN mice were treated with phosphate-buffered solution (control), CD, diethylnitrosamine (DEN) to induce liver injury with PBS (DEN+PBS), and DEN with CD (DEN+CD) for nine weeks. After sacrifice, livers and mesenteric lymph nodes (MLNs) were removed and bacterial translocation, transcriptomes, and proteins were analysed. CD was found in 20% of MLNs from the control and DEN+PBS groups, in 30% of MLNs from the CD group, and in 75% of MLNs from the DEN+CD groups, which had injured livers. Also, CD was detected in 50% of the livers in the DEN+CD group with CD-positive MLNs. Elevated IL-1ß, HB-EGF, EGFR, TGF-α, PCNA, DES, HMGB1, and CRP expressions were observed in the CD and DEN+CD groups as compared to the control and DEN+PBS groups. Protein levels of IL-6 and HMGB1 were higher in the CD and DEN+CD groups than in the control and DEN+PBS groups. These results indicate that intestinal CD can initiate and aggravate liver injury, and the mechanism of pathogenesis for liver injury should be investigated in further studies.