Diversity and Evolution in the Genome of Clostridium difficile.

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.

Evaluation of the Cepheid Xpert C. difficile/Epi and meridian bioscience illumigene C. difficile assays for detecting Clostridium difficile ribotype 033 strains.

Clostridium difficile PCR ribotype 033 (RT033) is found in the gastrointestinal tracts of production animals and, occasionally, humans. The illumigene C. difficile assay (Meridian Bioscience, Inc.) failed to detect any of 52 C. difficile RT033 isolates, while all strains signaled positive for the binary toxin genes but were reported as negative for C. difficile by the Xpert C. difficile/Epi assay (Cepheid).

Molecular characterization and antimicrobial susceptibilities of Clostridium difficile clinical isolates from Victoria, Australia.

Some Australian strain types of Clostridium difficile appear unique, highlighting the global diversity of this bacterium. We examined recent and historic local isolates, finding predominantly toxinotype 0 strains, but also toxinotypes V and VIII. All isolates tested were susceptible to vancomycin and metronidazole, while moxifloxacin resistance was only detected in recent strains.

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.

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.

Severe infection with Clostridium difficile PCR ribotype 027 acquired in Melbourne, Australia.

We report the first recognised case of infection with Clostridium difficile PCR ribotype 027 acquired in Australia. This pathogen has caused significant morbidity and mortality in widespread hospital-based outbreaks in the northern hemisphere. Clinicians need to be aware of the clinical picture, limitations of diagnostic tests, availability of further testing for epidemic strains, new therapeutic approaches, and in-hospital control strategies for this infection.

Clostridioides difficile infection in the Asia-Pacific region.

Clostridioides difficile causes healthcare-related diarrhoea in high-income countries. Highly resistant spores persist in healthcare facilities, primarily infecting patients who have recently received antimicrobials. C. difficile infection (CDI) has been studied in detail in North America and Europe; however, the epidemiology of CDI elsewhere, including the Asia-Pacific region, is largely unknown. A survey of CDI was performed in 13 Asia-Pacific countries. Epidemiological data on 600 cases were collected and molecular typing undertaken on 414 C. difficile isolates. Healthcare facility-associated CDI comprised 53.6% of cases, while community-associated CDI was 16.5%. The median age of cases was 63.0 years and 45.3% were female, 77.5% had used antibiotics in the previous 8 weeks, most frequently third-generation cephalosporins (31.7%), and 47.3% had used proton pump inhibitors. Recurrence (9.1%) and mortality (5.2%) rates were low, while complications including colitis or pseudomembranous colitis (13.8%), colectomy (0.4%), and toxic megacolon (0.2%) were uncommon. Common C. difficile strains were ribotypes 017 (16.7%), 014/020 (11.1%) and 018 (9.9%), with wide variation between countries. Binary toxin-positive strains of C. difficile were detected rarely. Overall, disease severity appeared mild, and mortality and recurrence were low. Continued education about, and surveillance of, CDI in Asia are required to reduce the burden of disease.

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.

Laboratory-based surveillance of Clostridium difficile strains circulating in the Australian healthcare setting in 2012.

Clostridium difficile infection (CDI) has risen in prominence in Australia recently. We conducted laboratory-based surveillance of CDI to examine C. difficile circulating in Australia in October/November 2012. We collected 542 isolates from all States and Territories of Australia except the Northern Territory. The most common ribotypes (RTs) were RTs 014/020 (25.5%), 002 (10.5%), 056 (5.9%) and 070 (4.2%). The survey results were compared with results from a similar Australian survey conducted in 2010. Proportions of RTs 014/020 and 002 remained similar, while RTs 056 (5.9%), 015 (4.1%), 017 (3.3%) and 244 (2.4%) increased in prevalence. Basic clinical and demographic data were available for 338 cases. The majority were healthcare facility-associated (HCFA-CDI, 51.5%) while 17.5% were community-associated (CA-CDI). While no RTs were associated with CA-CDI, RTs 056 and 126 were recently found in Australian production animals, indicating a possible community health threat in Australia.

Clostridium difficile infection: Evolution, phylogeny and molecular epidemiology.

Over the recent decades, Clostridium difficile infection (CDI) has emerged as a global public health threat. Despite growing attention, C. difficile remains a poorly understood pathogen, however, the exquisite sensitivity offered by next generation sequencing (NGS) technology has enabled analysis of the genome of C. difficile, giving us access to massive genomic data on factors such as virulence, evolution, and genetic relatedness within C. difficile groups. NGS has also demonstrated excellence in investigations of outbreaks and disease transmission, in both small and large-scale applications. This review summarizes the molecular epidemiology, evolution, and phylogeny of C. difficile, one of the most important pathogens worldwide in the current antibiotic resistance era.

Prevalence of binary toxin positive Clostridium difficile in diarrhoeal humans in the absence of epidemic ribotype 027.

Virulence of Clostridium difficile is primarily attributed to the large clostridial toxins A and B while the role of binary toxin (CDT) remains unclear. The prevalence of human strains of C. difficile possessing only CDT genes (A-B-CDT+) is generally low (< 5%), however, this genotype is commonly found in neonatal livestock both in Australia and elsewhere. Zoonotic transmission of C. difficile has been suggested previously. Most human diagnostic tests will not detect A-B-CDT+ strains of C. difficile because they focus on detection of toxin A and/or B. We performed a prospective investigation into the prevalence and genetic characteristics of A-B-CDT+ C. difficile in symptomatic humans. All glutamate dehydrogenase or toxin B gene positive faecal specimens from symptomatic inpatients over 30 days (n = 43) were cultured by enrichment, and C. difficile PCR ribotypes (RTs) and toxin gene profiles determined. From 39 culture-positive specimens, 43 C. difficile isolates were recovered, including two A-B-CDT+ isolates. This corresponded to an A-B-CDT+ prevalence of 2/35 (5.7%) isolates possessing at least one toxin, 2/10 (20%) A-B- isolates, 2/3 CDT+ isolates and 1/28 (3.6%) presumed true CDI cases. No link to Australian livestock-associated C. difficile was found. Neither A-B-CDT+ isolate was the predominant A-B-CDT+ strain found in Australia, RT 033, nor did they belong to toxinotype XI. Previous reports infrequently describe A-B-CDT+ C. difficile in patients and strain collections but the prevalence of human A-B-CDT+ C. difficile is rarely investigated. This study highlights the occurrence of A-B-CDT+ strains of C. difficile in symptomatic patients, warranting further investigations of its role in human infection.

Human Clostridium difficile infection caused by a livestock-associated PCR ribotype 237 strain in Western Australia.

INTRODUCTION: Clostridium difficile infection (CDI) is a significant gastrointestinal disease in the developed world and increasingly recognised as a zoonotic infection. In North America and Europe, the PCR ribotype (RT) 078 strain of C. difficile is commonly found in production animals and as a cause of disease in humans although proof of transmission from animals is lacking. This strain is absent in Australian livestock. We report a case of human CDI caused by a strain of C. difficile belonging to known Australian livestock-associated RT 237. CASE PRESENTATION: A young male was admitted for multiple trauma following a motor vehicle accident and placed on piperacillin/tazobactam for pneumonia. After 4 days of treatment, he developed symptoms of CDI, which was confirmed in the laboratory. His symptoms resolved after 6 days of intravenous metronidazole. The strain of C. difficile isolated was identified as RT 237, an unusual RT previously found in with several Western Australia piggeries. CONCLUSION: This case of CDI caused by an unusual livestock-associated C. difficile RT 237 supports the hypothesis of zoonotic transmission. The case highlights the potential of livestock to act as reservoir for C. difficile and the need for continued surveillance of CDI in both human and animal populations.

Laboratory-based surveillance of Clostridium difficile circulating in Australia, September - November 2010.

Clostridium difficile rose in prominence in the early 2000s with large-scale outbreaks of a particular binary toxin-positive strain, ribotype 027, in North America and Europe. In Australia outbreaks of the same scale had not and have not been seen. A survey of C. difficile across Australia was performed for 1 month in 2010. A collection of 330 C. difficile isolates from all States and Territories except Victoria and the Northern Territory was amassed. PCR ribotyping revealed a diverse array of strains. Ribotypes 014/020 (30.0%) and 002 (11.8%) were most common, followed by 054 (4.2%), 056 (3.9%), 070 (3.6%) and 005 (3.3%). The collection also contained few binary toxin positive strains, namely 027 (0.9%), 078 (0.3%), 244 (0.3%), 251 (0.3%) and 127 (0.3%). The survey highlights the need for vigilance for emerging strains in Australia, and gives an overview of the molecular epidemiology of C. difficile in Australia prior to an increase in incidence noted from mid-2011.

Molecular methods for detecting and typing of Clostridium difficile.

Since the early 2000s, Clostridium difficile has emerged as a major international pathogen. Recently, strains of C. difficile in circulation appear to be changing, with greater diversity, leading to challenges for diagnostics and surveillance. Currently molecular diagnostic methods are favoured for their high sensitivity and rapid processing times; however, a number of issues still exist with molecular tests, in particular high cost, low clinical specificity and failure to detect some variant C. difficile strains. Molecular typing methods are used to determine the continually evolving epidemiology of C. difficile infection. Typing methods including PCR ribotyping and pulsed field gel electrophoresis are currently popular in Europe and North America, respectively, while high-throughput next-generation sequencing is likely to become more widely used in years to come. This review discusses current molecular detection and typing techniques for C. difficile.

Molecular Epidemiology of Clostridium difficile Infection in a Large Teaching Hospital in Thailand.

Clostridium difficile infection (CDI) is a leading cause of healthcare-associated morbidity and mortality worldwide. In Thailand, CDI exhibits low recurrence and mortality and its molecular epidemiology is unknown. CDI surveillance was conducted in a tertiary facility (Siriraj Hospital, Bangkok). A total of 53 toxigenic C. difficile strains from Thai patients were analyzed by multi-locus sequence typing (MLST), PCR ribotyping, and pulse-field gel electrophoresis (PFGE). The mean age of the cohort was 64 years and 62.3% were female; 37.7% of patients were exposed to > two antibiotics prior to a diagnosis of CDI, with beta-lactams the most commonly used drug (56.3%). Metronidazole was used most commonly (77.5%; success rate 83.9%), and non-responders were treated with vancomycin (success rate 100%). None of the isolates carried binary toxin genes. Most isolates (98.2-100%) were susceptible to metronidazole, vancomycin, tigecycline and daptomycin. There were 11 sequence types (STs), 13 ribotypes (RTs) and four PFGE types. Six previously identified STs (ST12, ST13, ST14, ST33, ST41 and ST45) and five novel STs unique to Thailand (ST66, ST67, ST68, ST69 and ST70) were identified. PCR RTs UK 017 (ST45) (45.3%) and UK 014/020 (ST33) (24.5%) were the most common. High concordance was observed between the MLST and ribotyping results (p<0.001). C. difficile isolates from Thai patients were highly susceptible to standard antimicrobial agents. In conclusion, the five STs indicate the high genetic diversity and unique polymorphisms in Thailand. Moreover, the emergence of antimicrobial resistance to vancomycin warranted continuous surveillance to prevent further spread of the toxigenic C. difficile isolates.

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.

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.

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.

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.