Ebselen Exhibits Antimicrobial Activity Against Clostridioides difficile By Disrupting Redox Associated Metabolism

ABSTRACTHigh recurrence rates and spread of antibiotic-resistant strains necessitate the need for alternative therapeutics for Clostridioides difficile infections (CDIs). Ebselen, a reactive organoselenium compound inhibits C. difficile virulence toxins TcdA and TcdB, by covalently binding to their cysteine protease domains. Ebselen is thought to lack antibacterial activity against C. difficile cells and its anti-toxin action is suggested to be solely responsible for its efficacy. However, C. difficile has several essential cysteine-containing enzymes that could be potential sites for covalent modification by ebselen; hence, we re-evaluated its anti-C. difficile properties. In BHI agar, ebselen inhibited almost all C. difficile strains (MICs of 2-8 µg/ml), with ribotype 078 being intrinsically resistant (MIC>64 µg/ml). Wilkins-Chalgren and Brucella agars are recommended media for anaerobic susceptibility testing. Ebselen was either slightly attenuated by pyruvate found in Wilkins-Chalgren agar or obliterated by blood in Brucella agars. Transcriptome analysis showed ebselen altered redox-associated processes, cysteine metabolism and significantly enhanced the expression of Stickland proline metabolism to likely regenerate NAD+ from NADH. Intracellularly cells increased the uptake of cysteines, depleted non-protein thiols and disrupted NAD+/NADH redox ratio. Growth inhibitory concentrations of ebselen also reduced toxin and spore production. Taken together, ebselen has bactericidal activity against C. difficile, with multiple mechanisms of action that negatively impacts toxin production and sporulation. To harness the polypharmacological properties of ebselen, chemical optimization is warranted, especially to obtain derivatives that could be effective in severe CDI, where intestinal bleeding could occur.

Isolation and Characterization of the Novel Phage JD032 and Global Transcriptomic Response during JD032 Infection of Clostridioides difficile Ribotype 078

ABSTRACT Insights into the interaction between phages and their bacterial hosts are crucial for the development of phage therapy. However, only one study has investigated global gene expression of Clostridioides (formerly Clostridium) difficile carrying prophage, and transcriptional reprogramming during lytic infection has not been studied. Here, we presented the isolation, propagation, and characterization of a newly discovered 35,109-bp phage, JD032, and investigated the global transcriptomes of both JD032 and C. difficile ribotype 078 (RT078) strain TW11 during JD032 infection. Transcriptome sequencing (RNA-seq) revealed the progressive replacement of bacterial host mRNA with phage transcripts. The expressed genes of JD032 were clustered into early, middle, and late temporal categories that were functionally similar. Specifically, a gene (JD032_orf016) involved in the lysis-lysogeny decision was identified as an early expression gene. Only 17.7% (668/3,781) of the host genes were differentially expressed, and more genes were downregulated than upregulated. The expression of genes involved in host macromolecular synthesis (DNA/RNA/proteins) was altered by JD032 at the level of transcription. In particular, the expression of the ropA operon was downregulated. Most noteworthy is that the gene expression of some antiphage systems, including CRISPR-Cas, restriction-modification, and toxin-antitoxin systems, was suppressed by JD032 during infection. In addition, bacterial sporulation, adhesion, and virulence factor genes were significantly downregulated. This study provides the first description of the interaction between anaerobic spore-forming bacteria and phages during lytic infection and highlights new aspects of C. difficile phage-host interactions. IMPORTANCE C. difficile is one of the most clinically significant intestinal pathogens. Although phages have been shown to effectively control C. difficile infection, the host responses to phage predation have not been fully studied. In this study, we reported the isolation and characterization of a new phage, JD032, and analyzed the global transcriptomic changes in the hypervirulent RT078 C. difficile strain, TW11, during phage JD032 infection. We found that bacterial host mRNA was progressively replaced with phage transcripts, three temporal categories of JD032 gene expression, the extensive interplay between phage-bacterium, antiphage-like responses of the host and phage evasion, and decreased expression of sporulation- and virulence-related genes of the host after phage infection. These findings confirmed the complexity of interactions between C. difficile and phages and suggest that phages undergoing a lytic cycle may also cause different phenotypes in hosts, similar to prophages, which may inspire phage therapy for the control of C. difficile.

Clostridioides (Clostridium) Difficile in Food-Producing Animals, Horses and Household Pets: A Comprehensive Review

Clostridioides (Clostridium) difficile is ubiquitous in the environment and is also considered as a bacterium of great importance in diarrhea-associated disease for humans and different animal species. Food animals and household pets are frequently found positive for toxigenic C. difficile without exposing clinical signs of infection. Humans and animals share common C. difficile ribotypes (RTs) suggesting potential zoonotic transmission. However, the role of animals for the development of human infection due to C. difficile remains unclear. One major public health issue is the existence of asymptomatic animals that carry and shed the bacterium to the environment, and infect individuals or populations, directly or through the food chain. C. difficile ribotype 078 is frequently isolated from food animals and household pets as well as from their environment. Nevertheless, direct evidence for the transmission of this particular ribotype from animals to humans has never been established. This review will summarize the current available data on epidemiology, clinical presentations, risk factors and laboratory diagnosis of C. difficile infection in food animals and household pets, outline potential prevention and control strategies, and also describe the current evidence towards a zoonotic potential of C. difficile infection.

Emergence of a non-sporulating secondary phenotype in Clostridium (Clostridioides) difficile ribotype 078 isolated from humans and animals

Clostridium (Clostridioides) difficile is a Gram positive, spore forming anaerobic bacterium that is a leading cause of antibiotic associated diarrhoea in the developed world. C. difficile is a genetically diverse species that can be divided into 8 phylogenetically distinct clades with clade 5 found to be genetically distant from all others. Isolates with the PCR ribotype 078 belong to clade 5, and are often associated with C. difficile infection in both humans and animals. Colonisation of animals and humans by ribotype 078 raises questions about possible zoonotic transmission, and also the diversity of reservoirs for ribotype 078 strains within the environment. One of the key factors which enables C. difficile to be a successful, highly transmissible pathogen is its ability to produce oxygen resistant spores capable of surviving harsh conditions. Here we describe the existence of a non-sporulating variant of C. difficile ribotype 078 harbouring mutations leading to premature stop codons within the master regulator, Spo0A. As sporulation is imperative to the successful transmission of C. difficile this study was undertaken to investigate phenotypic characteristics of this asporogenous phenotype with regards to growth rate, antibiotic susceptibility, toxin production and biofilm formation.

A Role for Tetracycline Selection in Recent Evolution of Agriculture-Associated Clostridium difficile PCR Ribotype 078

ABSTRACT The increasing clinical importance of human infections (frequently severe) caused by Clostridium difficile PCR ribotype 078 (RT078) was first reported in 2008. The severity of symptoms (mortality of ≤30%) and the higher proportion of infections among community and younger patients raised concerns. Farm animals, especially pigs, have been identified as RT078 reservoirs. We aimed to understand the recent changes in RT078 epidemiology by investigating a possible role for antimicrobial selection in its recent evolutionary history. Phylogenetic analysis of international RT078 genomes (isolates from 2006 to 2014, n = 400), using time-scaled, recombination-corrected, maximum likelihood phylogenies, revealed several recent clonal expansions. A common ancestor of each expansion had independently acquired a different allele of the tetracycline resistance gene tetM. Consequently, an unusually high proportion (76.5%) of RT078 genomes were tetM positive. Multiple additional tetracycline resistance determinants were also identified (including efflux pump tet40), frequently sharing a high level of nucleotide sequence identity (up to 100%) with sequences found in the pig pathogen Streptococcus suis and in other zoonotic pathogens such as Campylobacter jejuni and Campylobacter coli. Each RT078 tetM clonal expansion lacked geographic structure, indicating rapid, recent international spread. Resistance determinants for C. difficile infection-triggering antimicrobials, including fluoroquinolones and clindamycin, were comparatively rare in RT078. Tetracyclines are used intensively in agriculture; this selective pressure, plus rapid, international spread via the food chain, may explain the increased RT078 prevalence in humans. Our work indicates that the use of antimicrobials outside the health care environment has selected for resistant organisms, and in the case of RT078, has contributed to the emergence of a human pathogen. IMPORTANCE Clostridium difficile PCR ribotype 078 (RT078) has multiple reservoirs; many are agricultural. Since 2005, this genotype has been increasingly associated with human infections in both clinical settings and the community. Investigations of RT078 whole-genome sequences revealed that tetracycline resistance had been acquired on multiple independent occasions. Phylogenetic analysis revealed a rapid, recent increase in numbers of closely related tetracycline-resistant RT078 (clonal expansions), suggesting that tetracycline selection has strongly influenced its recent evolutionary history. We demonstrate recent international spread of emergent, tetracycline-resistant RT078. A similar tetracycline-positive clonal expansion was also identified in unrelated nontoxigenic C. difficile, suggesting that this process may be widespread and may be independent of disease-causing ability. Resistance to typical C. difficile infection-associated antimicrobials (e.g., fluoroquinolones, clindamycin) occurred only sporadically within RT078. Selective pressure from tetracycline appears to be a key factor in the emergence of this human pathogen and the rapid international dissemination that followed, plausibly via the food chain.

Clostridium difficile in Asia: Opportunities for One Health Management

Clostridium difficile is a ubiquitous spore-forming bacterium which causes toxin-mediated diarrhoea and colitis in people whose gut microflora has been depleted by antimicrobial use, so it is a predominantly healthcare-associated disease. However, there are many One Health implications to C. difficile, given high colonisation rates in food production animals, contamination of outdoor environments by use of contaminated animal manure, increasing incidence of community-associated C. difficile infection (CDI), and demonstration of clonal groups of C. difficile shared between human clinical cases and food animals. In Asia, the epidemiology of CDI is not well understood given poor testing practices in many countries. The growing middle-class populations of Asia are presenting increasing demands for meat, thus production farming, particularly of pigs, chicken and cattle, is rapidly expanding in Asian countries. Few reports on C. difficile colonisation among production animals in Asia exist, but those that do show high prevalence rates, and possible importation of European strains of C. difficile like ribotype 078. This review summarises our current understanding of the One Health aspects of the epidemiology of CDI in Asia.

Complete Genome Sequences of Historic Clostridioides difficile Food-Dwelling Ribotype 078 Strains in Canada Identical to That of the Historic Human Clinical Strain M120 in the United Kingdom

Clostridioides (Clostridium) difficile is a spore-forming anaerobic bacterium that causes severe intestinal diseases in humans. Here, we report the complete genome sequence of the first C. difficile foodborne type strain (PCR ribotype 078) isolated from food animals in Canada in 2004, which has 100% similarity to the genome sequence of the historic human clinical strain M120.