Project description:Clostridioides difficile BI/NAP1/ribotype 027 is an epidemic hypervirulent strain found worldwide, including in Latin America. We examined the genomes and exoproteomes of two multilocus sequence type (MLST) clade 2 C. difficile strains considered hypervirulent: ICC-45 (ribotype SLO231/UK[CE]821), isolated in Brazil, and NAP1/027/ST01 (LIBA5756), isolated during a 2010 outbreak in Costa Rica. C. difficile isolates were cultured and extracellular proteins were analyzed using high-performance liquid chromatography-tandem mass spectrometry. Genomic analysis revealed that these isolates shared most of the gene composition. Only 83 and 290 NAP1/027 genes were considered singletons in ICC-45 and NAP1/027, respectively. Exoproteome analysis revealed 197 proteins, of which 192 were similar in both strains. Only five proteins were exclusive to the ICC-45 strain. These proteins were involved with catalytic and binding functions and indirectly interacted with proteins related to pathogenicity. Most proteins, including TcdA, TcdB, flagellin subunit, and cell surface protein, were overrepresented in the ICC-45 strain; 14 proteins, including mature S-layer protein, were present in higher proportions in LIBA5756. These data show close similarity between the genome and proteins in the supernatant of two strains with hypervirulent features isolated in Latin America and underscore the importance of epidemiological surveillance of the transmission and emergence of new strains.
Project description:Clostridium difficile (C. difficile) strains belonging to PCR ribotype 027, PFGE type NAP1, REA type B1 and toxinotype III, termed NAP1/027, have been implicated in the increased frequency of outbreaks of Clostridium difficile-associated diarrhoea (CDAD) in North America and Europe. The NAP1/027 strains appears to be more virulent with an increased mortality and frequency of relapse. Current European C. difficile microarrays are designed to the first sequenced and annotated C. difficile complete genome - strain 630 (ribotype 12). A high density oligonucleotide microarray was designed to C. difficile 630 (CD630) sequence and extra probes corresponding to two PCR ribotypes O27 strains C. difficile R20291 and QCD-32g58 were also included. Comparative genomic hybridisation was used to identify markers of ribotype 027 strains and markers to identify CD630. Strains hybridised to the array included the most prevalent ribotypes found in the UK and Europe (106 and 001) as well as the emerging hypervirulent ribotype 078.
Project description:Gene expression level of Clostridioides difficile (C. difficile) strain R20291 comparing control C. difficile carring pMTL84151 as vector plasmid with C. difficile conjugated with a pMTL84151-03890 gene. Goal was to determine the effects of 03890 gene conjugation on C. difficile strain R20291 gene expression.
Project description:The intestines house a diverse microbiota that must compete for nutrients to survive, but the specific limiting nutrients that control pathogen colonization are not clearly defined. Clostridioides difficile colonization typically requires prior disruption of the microbiota, suggesting that outcompeting commensals for resources is key in establishing C. difficile infection (CDI). The immune protein calprotectin (CP) is released into the gut lumen during CDI to chelate zinc (Zn) and other essential nutrient metals. Yet, the impact of Zn limitation on C. difficile colonization is unknown. To define C. difficile responses to Zn limitation, we performed RNA sequencing on C. difficile exposed to CP. In media with CP, C. difficile upregulated genes involved in metal homeostasis and amino acid metabolism.