Briony Elliott

Clostridium difficile-associated diarrhoea.

Clostridium difficile is an important nosocomial pathogen and the most frequently diagnosed cause of infectious hospital‐acquired diarrhoea. Toxigenic strains usually produce toxin A and toxin B, which are the primary virulence factors of C. difficile. Some recently described strains produce an additional toxin, an adenosine‐diphosphate ribosyltransferase known as binary toxin, the role of which in pathogenicity is unknown. There has been concern about the emergence of a hypervirulent fluoroquinolone‐resistant strain of C. difficile in North America and Europe. The use of fluoroquinolone antimicrobials appears to be acting as a selective pressure in the emergence of this strain. In this review, we describe the current state of knowledge about C. difficile as a cause of diarrhoeal illness.

Emergence of a Ribotype 244 Strain of Clostridium difficile Associated With Severe Disease and Related to the Epidemic Ribotype 027 Strain

BACKGROUND We identified 12 patients with Clostridium difficile infection between July 2011 and March 2012 from whom an unusual C. difficile strain was isolated. This strain had a single-nucleotide deletion of the tcdC gene at position 117 and binary toxin genes, which are characteristic of the hypervirulent ribotype (RT) 027 strain. METHODS A retrospective cohort study of 12 patients infected with C. difficile RT244 and 24 patients infected with non-RT244/non-RT027 strains matched for place of diagnosis and time of collection of specimen was performed. We performed whole-genome sequencing to understand the relationship of the RT244 strain to other C. difficile strains and further understand its virulence potential. RESULTS Clostridium difficile RT244 was associated with more severe disease and a higher mortality rate. Phylogenomic analysis using core genome single-nucleotide polymorphisms showed that RT244 is in the same genetic clade (clade 2) as RT027 but is distinct from all RT027 strains. The pathogenicity locus of the RT244 strain encodes a variant toxin B, and this was confirmed by demonstration of Clostridium sordellii-like cytopathic effect on Vero cells. Toxin B production in culture supernatants was lower than that seen with a RT027 strain. CONCLUSIONS Our findings demonstrate the pathogenic potential of this RT244 C. difficile strain and emphasize the importance of ongoing surveillance for emergent strains.

Diversity and Evolution in the Genome of Clostridium difficile

SUMMARY Clostridium difficile infection (CDI) is the leading cause of antimicrobial and health care-associated diarrhea in humans, presenting a significant burden to global health care systems. In the last 2 decades, PCR- and sequence-based techniques, particularly whole-genome sequencing (WGS), have significantly furthered our knowledge of the genetic diversity, evolution, epidemiology, and pathogenicity of this once enigmatic pathogen. C. difficile is taxonomically distinct from many other well-known clostridia, with a diverse population structure comprising hundreds of strain types spread across at least 6 phylogenetic clades. The C. difficile species is defined by a large diverse pangenome with extreme levels of evolutionary plasticity that has been shaped over long time periods by gene flux and recombination, often between divergent lineages. These evolutionary events are in response to environmental and anthropogenic activities and have led to the rapid emergence and worldwide dissemination of virulent clonal lineages. Moreover, genome analysis of large clinically relevant data sets has improved our understanding of CDI outbreaks, transmission, and recurrence. The epidemiology of CDI has changed dramatically over the last 15 years, and CDI may have a foodborne or zoonotic etiology. The WGS era promises to continue to redefine our view of this significant pathogen.

Evolutionary History of the Clostridium difficile Pathogenicity Locus

The symptoms of Clostridium difficile infection are caused by toxins expressed from its 19 kb pathogenicity locus (PaLoc). Stable integration of the PaLoc is suggested by its single chromosomal location and the clade specificity of its different genetic variants. However, the PaLoc is variably present, even among closely related strains, and thus resembles a mobile genetic element. Our aim was to explain these apparently conflicting observations by reconstructing the evolutionary history of the PaLoc. Phylogenetic analyses and annotation of the regions spanning the PaLoc were performed using C. difficile population-representative genomes chosen from a collection of 1,693 toxigenic (PaLoc present) and nontoxigenic (PaLoc absent) isolates. Comparison of the core genome and PaLoc phylogenies demonstrated an eventful evolutionary history, with distinct PaLoc variants acquired clade specifically after divergence. In particular, our data suggest a relatively recent PaLoc acquisition in clade 4. Exchanges and losses of the PaLoc DNA have also occurred, via long homologous recombination events involving flanking chromosomal sequences. The most recent loss event occurred ∼30 years ago within a clade 1 genotype. The genetic organization of the clade 3 PaLoc was unique in containing a stably integrated novel transposon (designated Tn6218), variants of which were found at multiple chromosomal locations. Tn6218 elements were Tn916-related but nonconjugative and occasionally contained genes conferring resistance to clinically relevant antibiotics. The evolutionary histories of two contrasting but clinically important genetic elements were thus characterized: the PaLoc, mobilized rarely via homologous recombination, and Tn6218, mobilized frequently through transposition.

New types of toxin A-negative, toxin B-positive strains among clinical isolates of Clostridium difficile in Australia

A total of 817 human clinical isolates of Clostridium difficile from all Australian states were screened for A(-)B(+) strains by toxin gene PCR assays. Nine (1.1 %) strains were confirmed to be A(-)B(+) by enzyme immunoassay for toxin production. Of these, six (66.7 %) were binary toxin-positive by PCR. Using PCR ribotyping and toxinotyping, the A(-)B(+) strains could be grouped into seven ribotypes and three toxinotypes. Only one of the ribotypes had been reported previously (017). The prevalence of ribotype 017 was low in this study with only two strains detected. Two new A(-)B(+) toxinotypes were also defined (XXX, XXXI). Toxinotype XXX had a toxin B gene similar to that of toxinotype IV (A(+)B(+)) but with a novel cytopathic region. Toxinotype XXXI was similar to other A(-)B(+) types (X, XVII), but had a larger deletion to the toxin A gene than in either of those types. The types of A(-)B(+) strains identified in this study differed markedly from those described in other regions.

Emergence and spread of predominantly community-onset Clostridium difficile PCR ribotype 244 infection in Australia, 2010 to 2012

We describe an Australia-wide Clostridium difficile outbreak in 2011 and 2012 involving the previously uncommon ribotype 244. In Western Australia, 14 of 25 cases were community-associated, 11 were detected in patients younger than 65 years, 14 presented to emergency/outpatient departments, and 14 to non-tertiary/community hospitals. Using whole genome sequencing, we confirm ribotype 244 is from the same C. difficile clade as the epidemic ribotype 027. Like ribotype 027, it produces toxins A, B, and binary toxin, however it is fluoroquinolone-susceptible and thousands of single nucleotide variants distinct from ribotype 027. Fifteen outbreak isolates from across Australia were sequenced. Despite their geographic separation, all were genetically highly related without evidence of geographic clustering, consistent with a point source, for example affecting the national food chain. Comparison with reference laboratory strains revealed the outbreak clone shared a common ancestor with isolates from the United States and United Kingdom (UK). A strain obtained in the UK was phylogenetically related to our outbreak. Follow-up of that case revealed the patient had recently returned from Australia. Our data demonstrate new C. difficile strains are an on-going threat, with potential for rapid spread. Active surveillance is needed to identify and control emerging lineages.

Bacteremia with a large clostridial toxin-negative, binary toxin-positive strain of Clostridium difficile

Bacteremia caused by Clostridium difficile is rare. In this report, we describe a case of C. difficile bacteremia caused by an unusual strain of C. difficile. The isolate contained neither toxin A nor B genes, however, binary toxin genes were present (tcdA(-), tcdB(-), cdtA(+), cdtB(+)) and a 7.2-kb element unrelated to the PaLoc was found inserted within the PaLoc integration site. The clinical relevance of the isolate could not be determined.

Novel Molecular Type of Clostridium difficile in Neonatal Pigs, Western Australia

Clostridium difficile causes neonatal enteritis in piglets; strains of PCR ribotype 078 are most commonly identified. We investigated C. difficile prevalence in piglets in Australia and isolated a novel strain with a unique pathogenicity locus. In a mouse infection model, this strain produced more weight loss than did a ribotype 078 strain.

Clostridium difficile in horses in Australia – a preliminary study

During a 24 month period from 2007 to 2009, 174 faecal specimens from horses in Australia (predominantly from Western Australia) were tested for Clostridium difficile. C. difficile was isolated from 14 (23 %) of 62 diarrhoeal animals (including 10 foals) and from none of 112 healthy adult horses. These isolates were toxin profiled by PCR for toxin A, toxin B and binary toxin, and ribotyped. Ten of the equine isolates were A(+)B(+)CDT(-). Other toxin profiles detected were A(-)B(-)CDT(+) (one isolate), A(+)B(+)CDT(+) (two isolates) and A(-)B(-)CDT(-) (three isolates). There were six different ribotypes detected in the horses, ribotype 012 being the most common with six isolates. Two horses (one adult and one foal) had two strains of C. difficile isolated on different days. These strains had the same toxin profile but different ribotypes. None of the equine isolates was ribotype 078, which is A(+)B(+)CDT(+) and a significant cause of animal disease overseas. All isolates were susceptible to metronidazole and vancomycin. These results suggest that the epidemiology of C. difficile in horses in Australia is currently similar to that in other parts of the world, but requires further surveillance to monitor changes.

The Complexity and Diversity of the Pathogenicity Locus in Clostridium difficile Clade 5

The symptoms of Clostridium difficile infection are caused by two closely related toxins, TcdA and TcdB, which are encoded by the 19.6 kb Pathogenicity Locus (PaLoc). The PaLoc is variably present among strains, and in this respect it resembles a mobile genetic element. The C. difficile population structure consists mainly of five phylogenetic clades designated 1–5. Certain genotypes of clade 5 are associated with recently emergent highly pathogenic strains causing human disease and animal infections. The aim of this study was to explore the evolutionary history of the PaLoc in C. difficile clade 5. Phylogenetic analyses and annotation of clade 5 PaLoc variants and adjoining genomic regions were undertaken using a representative collection of toxigenic and nontoxigenic strains. Comparison of the core genome and PaLoc phylogenies obtained for clade 5 and representatives of the other clades identified two distinct PaLoc acquisition events, one involving a toxin A+B+ PaLoc variant and the other an A−B+ variant. Although the exact mechanism of each PaLoc acquisition is unclear, evidence of possible homologous recombination with other clades and between clade 5 lineages was found within the PaLoc and adjacent regions. The generation of nontoxigenic variants by PaLoc loss via homologous recombination with PaLoc-negative members of other clades was suggested by analysis of cdu2, although none is likely to have occurred recently. A variant of the putative holin gene present in the clade 5 A−B+ PaLoc was likely acquired via allelic exchange with an unknown element. Fine-scale phylogenetic analysis of C. difficile clade 5 revealed the extent of its genetic diversity, consistent with ancient evolutionary origins and a complex evolutionary history for the PaLoc.