Investigation into the mobile genetic elements of Clostridium difficile

Clostridium difficile is a pathogenic bacterium that can colonise both humans and various animals. Toxin production leads to clinical symptoms ranging from mild to severe diarrhoea and can result in potentially fatal pseudomembranous colitis. These symptoms are caused by the disruption of the cytoskeleton and tight junctions of gut epithelial cells by the toxins. Genomic sequencing of C. difficile has indicated the chromosome carries a number of mobile genetic elements including conjugative transposons, which can encode antibiotic resistance genes. Analysing the sequence of a number of C. difficile strains indicated that each genome carries at least one and often multiple conjugative transposons. For many of the genes on these elements, functions were predicted using various bioinformatic tools. The study of conjugative transposons in C. difficile has been limited by the lack of resistance genes encoded by the elements. Therefore, an antibiotic resistance gene was inserted into six of the elements in strains 630 and R20291 and filter-matings performed. Conjugative transfer was shown for all elements from strain 630 but not for Tn6103 from R20291. The study of transconjugants of these matings showed the pathogenicity locus, encoding the two major toxins of C. difficile, to transfer at a low frequency into a non-toxigenic recipient strain. Whole genome sequencing of transconjugants determined that the transfer is not limited to the pathogenicity locus but includes varying sizes of chromosomal DNA flanking the pathogenicity locus. RNA-seq was used for the comparison of mutants for transcriptional regulators of conjugative transposons CTn2 and CTn4, however no significant differential expression was detected. Furthermore, strain 630Δerm, a commonly used laboratory strain for the generation of knockout mutants, was compared to the wildtype strain 630. A predicted oxidative stress operon was upregulated in 630Δerm which raises the question of the biological impact of these results on the knockout model

A role for ColV plasmids in the evolution of pathogenic Escherichia coli ST58.

Escherichia coli ST58 has recently emerged as a globally disseminated uropathogen that often progresses to sepsis. Unlike most pandemic extra-intestinal pathogenic E. coli (ExPEC), which belong to pathogenic phylogroup B2, ST58 belongs to the environmental/commensal phylogroup B1. Here, we present a pan-genomic analysis of a global collection of 752 ST58 isolates from diverse sources. We identify a large ST58 sub-lineage characterized by near ubiquitous carriage of ColV plasmids, which carry genes encoding virulence factors, and by a distinct accessory genome including genes typical of the Yersiniabactin High Pathogenicity Island. This sub-lineage includes three-quarters of all ExPEC sequences in our study and has a broad host range, although poultry and porcine sources predominate. By contrast, strains isolated from cattle often lack ColV plasmids. Our data indicate that ColV plasmid acquisition contributed to the divergence of the major ST58 sub-lineage, and different sub-lineages inhabit poultry, swine and cattle

Susceptibility of hamsters to clostridium difficile isolates of differing toxinotype

Clostridium difficile is the most commonly associated cause of antibiotic associated disease (AAD), which caused ~21,000 cases of AAD in 2011 in the U.K. alone. The golden Syrian hamster model of CDI is an acute model displaying many of the clinical features of C. difficile disease. Using this model we characterised three clinical strains of C. difficile, all differing in toxinotype; CD1342 (PaLoc negative), M68 (toxinotype VIII) and BI-7 (toxinotype III). The naturally occurring non-toxic strain colonised all hamsters within 1-day post challenge (d.p.c.) with high-levels of spores being shed in the faeces of animals that appeared well throughout the entire experiment. However, some changes including increased neutrophil influx and unclotted red blood cells were observed at early time points despite the fact that the known C. difficile toxins (TcdA, TcdB and CDT) are absent from the genome. In contrast, hamsters challenged with strain M68 resulted in a 45% mortality rate, with those that survived challenge remaining highly colonised. It is currently unclear why some hamsters survive infection, as bacterial and toxin levels and histology scores were similar to those culled at a similar time-point. Hamsters challenged with strain BI-7 resulted in a rapid fatal infection in 100% of the hamsters approximately 26 hr post challenge. Severe caecal pathology, including transmural neutrophil infiltrates and extensive submucosal damage correlated with high levels of toxin measured in gut filtrates ex vivo. These data describes the infection kinetics and disease outcomes of 3 clinical C. difficile isolates differing in toxin carriage and provides additional insights to the role of each toxin in disease progression

Differential expression of the EGF-TM7 family members CD97 and EMR2 in lipid-laden macrophages in atherosclerosis, multiple sclerosis and Gaucher disease

The members of the epidermal growth factor (EGF)-transmembrane (TM)7 family of adhesion class G-protein coupled receptors are abundantly expressed by cells of the myeloid lineage. A detailed investigation of their expression by functional subsets of activated macrophages is still lacking. Therefore, we determined the expression of CD97, EGF module-containing mucin-like receptor (EMR)2 and EMR3 by monocyte-derived macrophages experimentally polarized in vitro. This was compared to three types of disease-associated lipid-laden macrophages displaying an alternatively activated phenotype in situ. Polarization in vitro towards classically activated M1 versus alternatively activated M2 extremes of macrophage activation did not result in a congruent regulation of EGF-TM7 receptor mRNA and protein except for a down-regulation of CD97 by IL-10. In contrast, macrophages handling lipid overload in vivo displayed differences in the expression of CD97 and EMR2. While foamy macrophages in atherosclerotic vessels expressed both CD97 and EMR2, foam cells in multiple sclerosis brain expressed CD97, but only little EMR2. Foam cell formation in vitro by oxidized LDL and myelin did not affect CD97 or EMR2 expression. Gaucher spleen cells accumulating glucosylceramide expressed very high levels of CD97 and EMR2. These findings indicate that complex cellular expression programmes rather than activation modes regulate the expression of EGF-TM7 receptors in macrophages. (C) 2010 Elsevier B.V. All rights reserved

Emergence and global spread of epidemic healthcare-associated clostridium difficile

Epidemic C. difficile (027/BI/NAP1) has rapidly emerged in the past decade as the leading cause of antibiotic-associated diarrhea worldwide. However, the key events in evolutionary history leading to its emergence and the subsequent patterns of global spread remain unknown. Here, we define the global population structure of C. difficile 027/BI/NAP1 using whole-genome sequencing and phylogenetic analysis. We show that two distinct epidemic lineages, FQR1 and FQR2, not one as previously thought, emerged in North America within a relatively short period after acquiring the same fluoroquinolone resistance–conferring mutation and a highly related conjugative transposon. The two epidemic lineages showed distinct patterns of global spread, and the FQR2 lineage spread more widely, leading to healthcare-associated outbreaks in the UK, continental Europe and Australia. Our analysis identifies key genetic changes linked to the rapid transcontinental dissemination of epidemic C. difficile 027/BI/NAP1 and highlights the routes by which it spreads through the global healthcare system