Characterization of Chicken IgY Specific to Clostridium difficile R20291 Spores and the Effect of Oral Administration in Mouse Models of Initiation and Recurrent Disease

Indexación: Web of Science; Scopus.Clostridium difficile infection (CDI) are the leading cause of world-wide nosocomial acquired diarrhea. The current main clinical challenge in CDI is the elevated rate of infection recurrence that may reach up to 30% of the patients, which has been associated to the formation of dormant spores during the infection. We sought to characterize the effects of oral administration of specific anti-spore IgY in mouse models of CDI and recurrent CDI. The specificity of anti-spore IgY was evaluated in vitro. In both, initiation mouse model and recurrence mouse model, we evaluated the prophylactic and therapeutic effect of anti-spore IgY, respectively. Our results demonstrate that anti-spore IgY exhibited high specificity and titers against C. difficile spores and reduced spore adherence to intestinal cells in vitro. Administration of anti-spore IgY to C57BL/6 mice prior and during CDI delayed the appearance of the diarrhea by 1.5 day, and spore adherence to the colonic mucosa by 90%. Notably, in the recurrence model, co-administration of anti-spore IgY coupled with vancomycin delayed the appearance of recurrent diarrhea by a median of 2 days. Collectively, these observations suggest that anti-spore IgY antibodies may be used as a novel prophylactic treatment for CDI, or in combination with antibiotics to treat CDI and prevent recurrence of the infection.https://www.frontiersin.org/articles/10.3389/fcimb.2017.00365/ful

Transcriptional analysis of temporal gene expression in germinating Clostridium difficile 630 endospores.

Clostridium difficile is the leading cause of hospital acquired diarrhoea in industrialised countries. Under conditions that are not favourable for growth, the pathogen produces metabolically dormant endospores via asymmetric cell division. These are extremely resistant to both chemical and physical stress and provide the mechanism by which C. difficile can evade the potentially fatal consequences of exposure to heat, oxygen, alcohol, and certain disinfectants. Spores are the primary infective agent and must germinate to allow for vegetative cell growth and toxin production. While spore germination in Bacillus is well understood, little is known about C. difficile germination and outgrowth. Here we use genome-wide transcriptional analysis to elucidate the temporal gene expression patterns in C. difficile 630 endospore germination. We have optimized methods for large scale production and purification of spores. The germination characteristics of purified spores have been characterized and RNA extraction protocols have been optimized. Gene expression was highly dynamic during germination and outgrowth, and was found to involve a large number of genes. Using this genome-wide, microarray approach we have identified 511 genes that are significantly up- or down-regulated during C. difficile germination (p≤0.01). A number of functional groups of genes appeared to be co-regulated. These included transport, protein synthesis and secretion, motility and chemotaxis as well as cell wall biogenesis. These data give insight into how C. difficile re-establishes its metabolism, re-builds the basic structures of the vegetative cell and resumes growth

Nasal immunization with the c-terminal domain of bcla3 induced specific igg production and attenuated disease symptoms in mice infected with clostridioides difficile spores

Clostridioides difficile is a Gram-positive, spore-forming bacterium that causes a severe intestinal infection. Spores of this pathogen enter in the human body through the oral route, interact with intestinal epithelial cells and persist in the gut. Once germinated, the vegetative cells colonize the intestine and produce toxins that enhance an immune response that perpetuate the disease. Therefore, spores are major players of the infection and ideal targets for new therapies. In this context, spore surface proteins of C. difficile, are potential antigens for the development of vaccines targeting C. difficile spores. Here, we report that the C-terminal domain of the spore surface protein BclA3, BclA3CTD, was identified as an antigenic epitope, over-produced in Escherichia coli and tested as an immunogen in mice. To increase antigen stability and efficiency, BclA3CTD was also exposed on the surface of B. subtilis spores, a mucosal vaccine delivery system. In the experimental conditions used in this study, free BclA3CTD induced antibody production in mice and attenuated some C. difficile infection symptoms after a challenge with the pathogen, while the spore-displayed antigen resulted less effective. Although dose regimen and immunization routes need to be optimized, our results suggest BclA3CTD as a potentially effective antigen to develop a new vaccination strategy targeting C. difficile spores

The Clostridioides difficile Cysteine-Rich Exosporium Morphogenetic Protein, CdeC, Exhibits Self-Assembly Properties That Lead to Organized Inclusion Bodies in Escherichia coli

Indexación: Scopus.Clostridioides difficile is an obligately anaerobic, spore-forming, Grampositive pathogenic bacterium that is considered the leading cause of nosocomial diarrhea worldwide. Recent studies have attempted to understand the biology of the outermost layer of C. difficile spores, the exosporium, which is believed to contribute to early interactions with the host. The fundamental role of the cysteine-rich proteins CdeC and CdeM has been described. However, the molecular details behind the mechanism of exosporium assembly are missing. The underlying mechanisms that govern exosporium assembly in C. difficile remain poorly studied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. In this work, we observed that CdeC was able to form organized inclusion bodies (IBs) in Escherichia coli filled with lamella-like structures separated by an interspace of 5 to 15 nm; however, CdeC expression in an E. coli strain with a more oxidative environment led to the loss of the lamella-like organization of CdeC IBs. Additionally, dithiothreitol (DTT) treatment of CdeC inclusion bodies released monomeric soluble forms of CdeC. Deletions in different portions of CdeC did not affect CdeC's ability to aggregate and form oligomers stable under denaturation conditions but affected CdeC's self-assembly properties. Overall, these observations have important implications in further studies elucidating the role of CdeC in the exosporium assembly of C. difficile spores. IMPORTANCE The endospore of Clostridioides difficile is the vehicle for transmission and persistence of the pathogen, and, specifically, the exosporium is the first contact between the host and the spore. The underlying mechanisms that govern exosporium assembly in C. difficile remain understudied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. Understanding the exosporium assembly's molecular bases may be essential to developing new therapies against C. difficile infection.https://journals.asm.org/doi/epub/10.1128/mSphere.01065-2

Characterization of Exosporium Layer Variability of Clostridioides difficile Spores in the Epidemically Relevant Strain R20291.

Indexación: Scopus.Clostridioides difficile is a Gram-positive anaerobic intestinal pathogenic bacterium and the causative agent of antibiotic-associated diarrhea. C. difficile spore is a dormant state which acts as a vehicle of transmission and infection. In C. difficile spores, the outermost exosporium layer is the first barrier of interaction with the host and should carry spore ligands involved in spore-host interactions. C. difficile forms two types of spores (i.e., thin and thick exosporium layers). In this communication, we contribute to understand several biological aspects of these two exosporium morphotypes. By transmission electron microscopy, we demonstrate that both exosporium morphotypes appear simultaneously during sporulation and that spore-coat laminations are formed under anaerobic conditions. Nycodenz density-gradient allows enrichment of spores with a thick-exosporium layer morphotype and presence of polar appendage. Using translational fluorescent fusions with exosporium proteins BclA3, CdeA, CdeC, and CdeM as well as with several spore coat proteins, we observed that expression intensity and distribution of SNAP-translational fusions in R20291 strain is highly heterogeneous. Electron micrographs demonstrate that multicopy expression of CdeC, but not CdeM, SNAP translational fusion, increases the abundance of the thick exosporium morphotype. Collectively, these results raise further questions on how these distinctive exosporium morphotypes are made during spore formation. © Copyright © 2020 Pizarro-Guajardo, Calderón-Romero, Romero-Rodríguez and Paredes-Sabja.https://www.frontiersin.org/articles/10.3389/fmicb.2020.01345/ful

Nasal immunization with the c-terminal domain of bcla3 induced specific igg production and attenuated disease symptoms in mice infected with clostridioides difficile spores

Indexación: Scopus.Clostridioides difficile is a Gram-positive, spore-forming bacterium that causes a severe intestinal infection. Spores of this pathogen enter in the human body through the oral route, interact with intestinal epithelial cells and persist in the gut. Once germinated, the vegetative cells colonize the intestine and produce toxins that enhance an immune response that perpetuate the disease. Therefore, spores are major players of the infection and ideal targets for new therapies. In this context, spore surface proteins of C. difficile, are potential antigens for the development of vaccines targeting C. difficile spores. Here, we report that the C-terminal domain of the spore surface protein BclA3, BclA3CTD, was identified as an antigenic epitope, over-produced in Escherichia coli and tested as an immunogen in mice. To increase antigen stability and efficiency, BclA3CTD was also exposed on the surface of B. subtilis spores, a mucosal vaccine delivery system. In the experimental conditions used in this study, free BclA3CTD induced antibody production in mice and attenuated some C. difficile infection symptoms after a challenge with the pathogen, while the spore-displayed antigen resulted less effective. Although dose regimen and immunization routes need to be optimized, our results suggest BclA3CTD as a potentially effective antigen to develop a new vaccination strategy targeting C. difficile spores. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.https://www.mdpi.com/1422-0067/21/18/669

Association of Fidaxomicin with C. difficile spores: Effects of Persistence on Subsequent Spore Recovery, Outgrowth and Toxin Production.

Background: We have previously shown that fidaxomicin instillation prevents spore recovery in an in-vitro gut model, whereas vancomycin does not. The reasons for this are unclear. Here, we have investigated persistence of fidaxomicin and vancomycin on C. difficile spores, and examined post-antibiotic exposure spore recovery, outgrowth and toxin production. Methods: Prevalent UK C. difficile ribotypes (n=10) were incubated with 200mg/L fidaxomicin, vancomycin or a non-antimicrobial containing control for 1 h in faecal filtrate or Phosphate Buffered Saline. Spores were washed three times with faecal filtrate or phosphate buffered saline, and residual spore-associated antimicrobial activity was determined by bioassay. For three ribotypes (027, 078, 015), antimicrobial-exposed, faecal filtrate-washed spores and controls were inoculated into broth. Viable vegetative and spore counts were enumerated on CCEYL agar. Percentage phase bright spores, phase dark spores and vegetative cells were enumerated by phase contrast microscopy at 0, 3, 6, 24 and 48 h post-inoculation. Toxin levels (24 and 48h) were determined by cell cytotoxicity assay. Results: Fidaxomicin, but not vancomycin persisted on spores of all ribotypes following washing in saline (mean=10.1mg/L; range= 4.0-14mg/L) and faecal filtrate (mean =17.4mg/L; 8.4-22.1mg/L). Outgrowth and proliferation rates of vancomycin-exposed spores were similar to controls, whereas fidaxomicin-exposed spores showed no vegetative cell growth after 24 and 48 h. At 48h, toxin levels averaged 3.7 and 3.3 relative units (RU) in control and vancomycin-exposed samples, respectively, but were undetectable in fidaxomicin-exposed samples. Conclusion: Fidaxomicin persists on C. difficile spores, whereas vancomycin does not. This persistence prevents subsequent growth and toxin production in vitro. This may have implications on spore viability, thereby impacting CDI recurrence and transmission rates

Origin, genomic diversity and microevolution of the clostridium difficile b1/nap1/rt027/st01 strain in costa rica, chile, honduras and mexico

Indexación: Scopus.Clostridium difficile B1/NAP1/RT027/ST01 has been responsible for outbreaks of antibiotic-associated diarrhoea in clinical settings worldwide and is associated with severe disease presentations and increased mortality rates. Two fluoroquinolone-resistant (FQR) lineages of the epidemic B1/NAP1/RT027/ST01 strain emerged in the USA in the early 1990s and disseminated trans continentally (FQR1 and FQR2). However, it is unclear when and from where they entered Latin America (LA) and whether isolates from LA exhibit unique genomic features when compared to B1/NAP1/RT027/ST01 isolates from other regions of the world. To answer the first issue we compared whole-genome sequences (WGS) of 25 clinical isolates typed as NAP1, RT027 or ST01 in Costa Rica (n=16), Chile (n=5), Honduras (n=3) and Mexico (n=1) to WGS of 129 global isolates from the same genotype using Bayesian phylogenomics. The second question was addressed through a detailed analysis of the number and type of mutations of the LA isolates and their mobile resistome. All but two B1/NAP1/RT027/ST01 isolates from LA belong to the FQR2 lineage (n=23, 92 %), confirming its widespread distribution. As indicated by analysis of a dataset composed of 154 WGS, the B1/NAP1/RT027/ST01 strain was introduced into the four LA countries analysed between 1998 and 2005 from North America (twice) and Europe (at least four times). These events occurred soon after the emergence of the FQR lineages and more than one decade before the first report of the detection of the B1/NAP1/RT027/ST01 in LA. A total of 552 SNPs were identified across all genomes examined (3.8–4.3 Mb) in pairwise comparisons to the R20291 reference genome. Moreover, pairwise SNP distances were among the smallest distances determined in this species so far (0 to 55). Despite this high level of genomic conservation, 39 unique SNPs (7 %) in genes that play roles in the infection process (i.e. slpA) or antibiotic resistance (i.e. rpoB, fusA) distinguished the LA isolates. In addition, isolates from Chile, Honduras and Mexico had twice as many antibiotic resistance genes (ARGs, n=4) than related isolates from other regions. Their unique set of ARGs includes a cfr-like gene and tetM, which were found as part of putative mobile genetic elements whose sequences resemble undescribed integrative and conjugative elements. These results show multiple, independent introductions of B1/NAP1/RT027/ ST01 isolates from the FQR1 and FQR2 lineages from different geographical sources into LA and a rather rapid accumulation of distinct mutations and acquired ARG by the LA isolates.https://www.microbiologyresearch.org/content/journal/mgen/10.1099/mgen.0.00035

The orphan germinant receptor protein GerXAO (but not GerX3b) is essential for L-alanine induced germination in Clostridium botulinum Group II

Clostridium botulinum is an anaerobic spore forming bacterium that produces the potent botulinum neurotoxin that causes a severe and fatal neuro-paralytic disease of humans and animals (botulism). C. botulinum Group II is a psychrotrophic saccharolytic bacterium that forms spores of moderate heat resistance and is a particular hazard in minimally heated chilled foods. Spore germination is a fundamental process that allows the spore to transition to a vegetative cell and typically involves a germinant receptor (GR) that responds to environmental signals. Analysis of C. botulinum Group II genomes shows they contain a single GR cluster (gerX3b), and an additional single gerA subunit (gerXAO). Spores of C. botulinum Group II strain Eklund 17B germinated in response to the addition of L-alanine, but did not germinate following the addition of exogenous Ca2+-DPA. Insertional inactivation experiments in this strain unexpectedly revealed that the orphan GR GerXAO is essential for L-alanine stimulated germination. GerX3bA and GerX3bC affected the germination rate but were unable to induce germination in the absence of GerXAO. No role could be identified for GerX3bB. This is the first study to identify the functional germination receptor of C. botulinum Group II

Visualization of fidaxomicin association with the exosporium layer of Clostridioides difficile spores

Indexación ScopusBackground: Fidaxomicin has novel pharmacologic effects on C. difficile spore formation including outgrowth inhibition and persistent spore attachment. However, the mechanism of fidaxomicin attachment on spores has not undergone rigorous microscopic studies. Materials & methods: Fidaxomicin attachment to C. difficile spores of three distinct ribotypes and C. difficile mutant spores with inactivation of exosporium or spore-coat protein-coding genes were visualized using confocal microscopy with a fidaxomicin-bodipy compound (green fluorescence). The pharmacologic effect of the fidaxomicin-bodipy compound was determined. Confocal microscopy experiments included direct effect on C. difficile wild-type and mutant spores, effect of exosporium removal, and direct attachment to a comparator spore forming organism, Bacillus subtilis. Results: The fidaxomicin-bodipy compound MIC was 1 mg/L compared to 0.06 mg/L for unlabeled fidaxomicin, a 16-fold increase. Using confocal microscopy, the intracellular localization of fidaxomicin into vegetative C. difficile cells was observed consistent with its RNA polymerase mechanism of action and inhibited spore outgrowth. The fidaxomicin-bodipy compound was visualized outside of the core of C. difficile spores with no co-localization with the membrane staining dye FM4-64. Exosporium removal reduced fidaxomicin-bodipy association with C. difficile spores. Reduced fidaxomicin-bodipy was observed in C. difficile mutant spores for the spore surface proteins CdeC and CotE. Conclusion: This study visualized a direct attachment of fidaxomicin to C. difficile spores that was diminished with mutants of specific exosporium and spore coat proteins. These data provide advanced insight regarding the anti-spore properties of fidaxomicin. © 2021 The Authorshttps://www-sciencedirect-com.recursosbiblioteca.unab.cl/science/article/pii/S1075996421000354?via%3Dihu