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The Continually Evolving Clostridium Difficile Species

Abstract and Introduction
Abstract
Clostridium difficile is a spore-forming Gram-positive bacterium that causes chronic diarrhea and sometimes life-threatening disease mainly in elderly and hospitalized patients. The reported incidence of C. difficile infection has changed dramatically over the last decade and has been related to the emergence of distinct clonal lineages that appear more transmissible and cause more severe infection. These include PCR ribotypes 027, 017 and more recently 078. Population biology studies using multilocus sequence typing and whole-genome comparisons has helped to define the C. difficilespecies into four clonal complexes that include PCR ribotypes 027, 017, 078 and 023, as well as a general grouping of most other PCR ribotypes. Further analysis of strains from diverse sources and geographical origins reveal significant microdiversity of clonal complexes and confirms that C. difficileis continuing to evolve. The study of C. difficile represents a real-time global evolutionary experiment where the pathogen is responding to a range of selective pressures created by human activity and practices in healthcare settings. The advent of whole-genome sequencing coupled with phylogeny (phylogeography and phylohistory) will provide unprecedented detail on the local and global emergence and disappearance of C. difficile clones, and facilitate more rational approaches to disease control. This review will highlight the emergence of virulent C. difficileclones and our current understanding of molecular epidemiology of the species

Clostridium difficile Infection

Clostridium difficile is a spore-forming, Gram-positive, anaerobic bacillus found as part of the normal gut commensal flora in up to 3% of healthy adults in the community and 24% of hospitalized patients.[1] C. difficile was first recognized as a cause of human gastrointestinal infection in the late 1970s,[2,3]and it is now the most frequent cause of healthcare-acquired infectious diarrhea in developed

countries.[4,5] Clinical presentation of C. difficile infection (CDI) is highly variable, ranging from uncomplicated asymptomatic carriage and mild diarrhea to lifethreatening toxic megacolon and pseudomembranous colitis (PMC) requiring surgical intervention.[4] Typical features of CDI include; watery diarrhea, abdominal pain and cramps, lower quadrant tenderness, fever, leukocytosis and hypoalbuminemia. Re-occurrences are common with a rate of up to 20% after the first episode of CDI and 50% after the second episode even after treatment. [4,6] Infection occurs with the ingestion of C. difficile spores by a susceptible host; these are highly resistant and are excreted from feces of infected patients.[6] Spores are easily transmitted via persons (usually hands of healthcare staff), fomites and air, and are thought to be the main vectors of environmental persistence and host transmission.[6] CDI is an important nosocomial pathogen in part due to the healthcare facility environment containing a high number of spores from infected patients; the hospital environment and patients undergoing antibiotic treatment provide a discrete ecosystem where C. difficile persists and where certain virulent clones thrive. A major risk factor for CDI is advancing age (65 years); this is generally thought to be due to a failure in the immune system, known as senescence of the immune response, resulting from a combination of comorbidities, immune-related changes in the fecal flora and normal age-related changes.[7]Additionally, it is generally accepted that alterations of the intestinal microflora permit overgrowth of C. difficile, and it is well described that the major risk factor for this is the consumption of broad-spectrum antibiotics, with clindamycin, penicillins, cephalosporin and the flouroquinolones being the most frequently implicated. [8 10] Other contributory factors for CDI include: gastrointestinal surgery; chemotherapeutic agents for cancer; that is, methotrexate; and proton-pump inhibitors, all of which affect the gastrointestinal microflora allowing C. difficile to proliferate and subsequently produce high levels of toxin(s). Several virulence factors have been described in C. difficile such as flagella,[11] fimbriae and proteolytic enzymes,[12] surface layer proteins[13] and paracreosol production,[14] though toxins A and B are recognized as the major virulence factors for CDI.[5,15] Their genes tcdA and tcdB are chromosomally located along with three accessory genes forming the 19.6-kb pathogenicity locus and are: tdcR, which encodes an alternative RNA polymerase sigma factor that is a positive regulator of toxin production;tcdC, a negative regulator of toxin production that interferes with the RNA polymerase formed with TcdR; and tcdE, a gene encoding a holin-like protein.[15] It has been postulated that tcdC is

associated with severe infection though there is conflicting evidence for this.[15,16] In addition, not all strains produce toxins A and B; those that do not produce either are nonpathogenic, and although the majority of pathogenic strains produce both toxins, clinically relevant toxin A-negative toxin B-positive (AB+) strains have been well described in humans and animals. Strains have also been shown to produce an unrelated C. difficile binary toxin (CDT)[17,18] and a study has suggested that CDT has a role in adherence and colonization of C. difficile by stimulating microtubule-based protrusions from host cells[19] though its exact role in CDI has not yet been fully elucidated. Treatment of CDI is limited to a few tried and tested agents such as metronidazole and oral vancomycin.[20] Newer agents such as fidaxomicin have recently been licensed, though experience in clinical practice is limited.[21] The use of fecal transplantation from healthy patients to patients suffering CDI is an additional method which has been practiced in refractory cases of CDI with the aim of restoring normal microbiota, and a review of the literature by Guo et al. describes the casecontrol studies with promising results,[22] though as yet, no controlled studies have been performed and the usefulness of this treatment has yet to be confirmed. A recent review describing the current evidence for use of selected monoclonal antibodies directed against C. difficile toxins and vaccination brings hope of improving the current treatment options.[23] The use of monoclonal antibodies is a new and promising approach for the treatment of recurrent cases, and vaccines have also been considered, though robust data are yet to be produced.[23] Healthcare professionals face the task of balancing the risk of CDI in their patients with the administration of broad-spectrum antibiotics for severe infections. The continued use of antibiotics and other chemotherapeutic agents in an increasingly vulnerable, compromised and elderly patient group will remain a challenge for healthcare delivery for the foreseeable future, and more robust data are required before the use of metronidazole and vancomycin are abandoned and replaced with other treatment options.

Genotyping & Population Genetic Studies of C. difficile

A variety of molecular typing approaches have been developed to study C. difficile and CDI including: multilocus sequence typing (MLST), multilocus variable-number tandem-repeat analysis (MLVA), surface layer protein A gene sequence typing, amplified fragment length polymorphism, pulsed-field gel

electrophoresis (PFGE), restriction endonuclease analysis (REA), toxinotyping (based on sequence data of toxins A and B) and PCR ribotyping.[24] Generally, most of the methods are congruent,[24] though it is PCR ribotyping that is most widely accepted in Europe with the Anaerobe Reference Laboratory in Cardiff (UK) holding the collection of strains and assigning PCR ribotypes; more than 427 PCR ribotypes have been identified [Hall V, Pers. Comm.]. In North America, PFGE is the preferred method of typing. PCR ribotyping, PFGE and MLVA are useful for outbreak investigation. For many pathogens, MLST has become the gold standard for assessing the population structure and genetic relatedness of a given bacterial species providing the species is not strictly clonal or too recombinational, and an appropriate breadth of diversity of strains across the species can be tested.[25] In addition, MLST can be performed in high throughput, it is relatively inexpensive and allows for portability between different research and reference laboratories. The relative genetic uniformity of C. difficile means that it is an ideal species for MLST analysis to trace and monitor the emergence of different sequence types (STs). Lemee et al. first described a C. difficile MLST scheme based on seven housekeeping genes which was used to analyze a group of 72 C. difficile strains of limited genetic diversity.[26] In this study, profiles were used to define 34 different STs and all AB+ PCR ribotype 017 strains formed a separate lineage. A further study by Lemee et al. also found animal and human isolates to cluster.[27] A second MLST scheme for C. difficile was further developed which was validated using 152 isolates representing a wide range of PCR ribotypes and isolates from recent clinical samples.[28] The study found that there was relatively low overall genetic diversity detected within the housekeeping genes; however, the study identified four phylogenetic groups plus an outlier group. The majority of STs clustered into group one, but the four additional groups represented four emergent virulent lineages and close relatives; group two contained ST-1/PCR ribotype 027, group three contained ST22/PCR ribotype 023 and group four contained ST-37/PCR ribotype 017. A ST single outlier (ST-11) was associated with PCR ribotype 078. A subsequent study by the same group tested a representative collection of 1290 CDI patient isolates from between September 2006 and December 2009 (both hospital and community) and confirmed the clonal population structure and presence of four lineages (one containing mixed STs, ST-1/PCR ribotype 027, ST22/PCR ribotype 023, ST-37/PCR ribotype 017) found by other studies and also the additional lineage (ST-11/PCR ribotype 078).[29] The study identified 69 STs among the 1290 clinical isolates, 36 of which were new and which fell into

the five lineages. Nontoxigenic strains appeared not to represent a separate lineage.[29] Another study investigated the population structure of 386 strains isolated from diverse human, animal and food sources, three continents and 68 PCR ribotypes.[30] The data was combined with 80 previously published STs and 43 unpublished STs and confirmed the five lineages associated with outbreaks of CDI in humans and demonstrated significant heterogeneity within the two lineages which contain hypervirulent ST-11 (PCR ribotype 078) and ST-1 (PCR ribotype 027) (Figure 1).

Emergent C. difficile lineages

Global Emergence of PCR Ribotype 027 & its Implications
Alarmingly, in the past decade a new group of highly virulent C. difficile strains were reported to be emerging that caused outbreaks of increased disease severity in North America and Europe. In 2003 an epidemic of CDI was reported in southern Quebec and the Montreal district of Canada.[31] Concurrently, a similar strain had also been isolated in several states of North America, and in March 2004, a report was made by the Stoke Mandeville Hospital in the UK describing a major outbreak including severe cases of CDI: 334 cases and 38 deaths.[32] By 2005, an excessive number of hospitals were reporting rates of CDI at five times their baseline rate with an increase in patient 30-day mortality.[33] Outbreak strains from the UK, North America and Canada were found to be due to the dissemination of an epidemic strain[4,34] and subsequently identified as BI by REA, NAP1 by PFGE and PCR ribotype 027 by PCR ribotyping and referred to as BI/NAP1/027.[24] In late 2005, other European countries documented outbreaks of this strain[3538] and similar reports from many other countries across the globe including Australia,[39] Korea,[40] Japan,[41]Hong Kong[42] and Costa Rica[43] were subsequently documented. Patients infected with PCR ribotype 027 were found to have more severe diarrhea, higher mortality and more reoccurrences of symptoms.[44] It has been thought that this increase in virulence and increase in reoccurrence rate were due to one or more of the following characteristics: exposure to fluoroquinolone antibiotics prior to CDI; increased production of toxin; prolonged production of toxin; and increased sporulation, which in turn increases the risk of transmission.

Exposure to the fluoroquinolone class of antibiotics has been thought to be the strongest risk factor for CDI with PCR ribotype 027.[45] The high rates of outbreaks caused by this PCR ribotype may in part be attributed to the acquisition of highlevel fluoroquinolone resistance and higher patient exposure rates for this class of drug[46] providing the epidemic strain a selective advantage allowing it to flourish. However, the available data linking C. difficile and flouroquinolones is suggestive and inconclusive.[47] Epidemic strains of PCR ribotype 027 have been reported to produce significantly higher levels of toxins A and B compared with other strains;[48] one study found PCR ribotype 027 to produce 16-fold more toxin A and 23-fold more toxin B then other PCR ribotypes.[49] PCR ribotype 027 was also found to have an 18-bp deletion and a frameshift mutation due to a single base pair deletion at position 117 in thetcdC gene.[15,50] The frameshift mutation results in a truncated protein lacking in function, and it is hypothesized that this leads to the deregulated expression of toxins A and B. Strains with the deletion have been shown to produce more toxin in the logarithmic phase of growth and subsequent association with more severe disease.[51] Another study found an increased duration of toxin production,[52] though these findings remain to be confirmed. In addition, a study found sporulation rates of epidemic strains were significantly greater than that of the nonepidemic strains,[53] though recent studies have shown that sporulation rates are not PCR ribotype specific.[54,55] PCR ribotype 027 strains are not new, the PCR ribotyping banding pattern was first assigned in 1998 to an isolate from a young patient suffering PMC in Paris (France) in 1985.[17] Additionally, historical BI strains dating back to 1984 that caused sporadic CDI in North America have been documented.[48] A three-way whole-genome comparison between the original PCR ribotype 027 Paris strain (CD196), the index PCR ribotype 027 UK (Stoke Mandeville outbreak) strain (R20291) and the previously published multidrug-resistant PCR ribotype 012 strain (630) found PCR ribotype 027 genomes to have lineage-specific genes along with distinct phenotypic differences observed with motility, antibiotic resistance and toxicity.[56] The modern epidemic PCR ribotype 027 strain was found to have five unique genetic regions absent from both the nonepidemic PCR ribotype 027 strain and the 012 strain. Forty-seven coding sequences were present in the modern PCR ribotype 027 strain but absent from the Paris strain and, in addition, three coding sequences were present in the Paris strain that appeared to have been lost in the modern PCR ribotype 027 strain. Furthermore, three coding sequences were found to be truncated by sequence loss in both the modern and historic PCR ribotype 027 strains, but were intact in the PCR ribotype

012 strain, and ten coding sequences were truncated in the PCR ribotype 012 strain but not in the PCR ribotype 027 strains. This raises many questions as to the reasons why only some strains appear highly virulent. Are there unique genetic regions or gene losses that provide the modern strain with increased virulence and/or transmissibility? Are there genetic change(s) that account for the explosive global outbreak of the BI/NAP1/027 strain? Rates of CDI, most notably PCR ribotype 027 in the UK and other parts of Europe, appear to have declined in the last 4 years and between 2007/2008 and 2010/2011, there was a 42.9% decrease in the number of PCR ribotype 027 strains isolated by the C. difficile Ribotype Network (CDRN) in the UK [CDRN, Unpublished Data]. This has occurred simultaneously with an increase in a variety of other PCR ribotypes (Figure 2).

(Enlarge Image)

Figure 2. Prevalence of Clostridium difficile ribotypes in England by quarter (April 2007March 2011). Clostridium difficile Ribotyping Network (CDRN) for England and Northern Ireland 2010/11 Annual Report.201

The Emergence of Toxin AB+ Strains (PCR Ribotype 017)

The rapid spread of the PCR ribotype 027 strain has detracted attention from other virulent strains of C. difficile. For example, toxin AB+ strains have emerged in the past decade in parts of Asia and Europe. The first documented outbreak due to a toxin AB+ C. difficile strain was reported in a Canadian hospital in 1998.[57] Outbreaks have since been documented to have occurred in the Netherlands between 1997 and 1998,[58] and hospitals in both Japan[59] and Dublin.[60] A study compared AB+ strains from the UK, Belgium and the USA using three typing methodologies, and 20 out of 23 isolates were found to be PCR ribotype 017 (others being PCR ribotype 110, 036 and 047).[61] There have been increasing reports of PCR ribotype 017 in recent years. A Polish study found 36 out of 79 (45.6%) isolates collected from hospitalized patients suffering CDI over a 2-year period to be PCR ribotype 017; this was identified to be their

predominant PCR ribotype.[62] The outbreak report described by Drudy et al. found 85 out of 90 (95%) isolates from two university hospitals in Dublin (Ireland) to be PCR ribotype 017 and were invariably fluoroquinolone resistant.[60] In China, a 6month study found 41 out of 110 (37.3%) of hospital isolates to be PCR ribotype 017,[63] and 105 out of 408 (25.7%) of isolates collected from 12 Korean hospitals over 2 years were also found to be PCR ribotype 017 with identification of a hospital clone.[64,65] A retrospective study published by Goorhuis et al. conducted in a general hospital in Buenos Aires (Argentina) found 119 out of 131 (91%) to be PCR ribotype 017.[66] Similar to PCR ribotype 027 isolates, toxin AB+ strains have been associated with severe CDI[67,68] and there is evidence that the prevalence of clinically relevant cases of CDI due to toxin AB+ strains are increasing globally.[66] With this, there is concern that the observed increase in prevalence is a significant underestimate, and that there are still some laboratories under-reporting CDI caused by toxin AB+ strains due to the use of laboratory tests that only detect toxin A. Additionally, the epidemiology of C. difficile in the Asia/Pacific regions and eastern parts of Europe appears to differ from elsewhere where the prevalence of toxin AB+ strains appears higher. An Israeli study found 56.5% of the isolates from hospitalized patients to be toxin AB+.[69] Komatsu et al. found 39% of isolates from a Japanese hospital in 2002 to be toxin AB+,[59] whilst Rupnik et al. report an increased incidence of PCR ribotype 017 strains from both Japan and Korea. [70] A multicenter study of the prevalence of toxigenic C. difficile in Korea found 50.3% of all reported cases in 2004 to be toxin AB+.[71] A study by Kim et al. noted an increasing prevalence in Korea with 99 out of 462 (21.4%) cases of C. difficile being toxin AB+.[72] A further study by Shin et al. revealed 21 out of 29 (72.4%) PMC cases to be due to a toxin AB+ strain.[67] These data suggest that there should be heightened vigilance with toxin AB+ strains, which have shown to cause equally severe infection like with PCR ribotype 027.

Emergence of the Animal-associated PCR Ribotype 078

C. difficile is recognized as both a gut colonizer and a cause of CDI in domestic animals and livestock.[7380] PCR ribotype 078 isolates have been found to be the most predominant from animal species with CDI, most notably pigs, calves and horses;[78,8082] it was identified in 119 out of 144 (83%) neonatal pig and 31 out of 33 (94%) calf strains.[78] C. difficile has also been found in contaminated food for human consumption with PCR ribotype 078 being the most frequently implicated.[77,8385] Recently, a study

found C. difficile in 4.8% (five out of 119) of seafood and fish samples from a grocery store and all toxin positive isolates were found to be PCR ribotype 078.[86] Between 2005 and 2008, the incidence of PCR ribotype 078 among patients in Holland increased from 3 to 13% with both PCR ribotype 027 and 078 strains presenting with similar disease severity.[81] Of concern is that although CDI is primarily associated with exposure to a healthcare-associated environment, patients from the community do develop CDI. A population-based cohort study found that community-acquired CDI affected those who lack the traditional risk factors such as hospitalization or antibiotic exposure, and patients with community-acquired CDI were also found to be younger.[87] The documented cases of community-acquired CDI also appear to be on the rise, and the increased incidence of community acquired infection with PCR ribotype 078 could be linked to the fact that this strain is found in both humans and animals. In addition, with the increase in documented cases of isolation of C. difficile from food products for human consumption, concerns have been raised about possible transmission between animals and humans in the community. Although C. difficile is not a proven foodborne pathogen, there is evidence that the same strain can cause symptomatic disease in both pigs and humans,[88] and studies from several countries have found certain strains to be indistinguishable between humans and other mammals.[80,81,88] In addition, we can also speculate that the changes in antibiotic prescribing in humans, veterinary medicine and animal production over the years have altered the prevalence of both C. difficile and specific ribotypes. As well as forming a distinct, distantly related evolutionary lineage and being associated with CDI in animals and contaminated foodstuffs, like PCR ribotype 027, PCR ribotype 078 has been described as an emerging pathogen. There has been a 4.4% increase in its prevalence between 2007/08 (1.8%) and 2010/11 (6.2%) in the UK.[201] There are also other PCR ribotypes that share a similar banding pattern with PCR ribotype 078; these being 126, 033, 045, 066 and 127[89,90] which, with the exception of PCR ribotype 045, have been shown to be identical by MLST (ST11). Interestingly, PCR ribotype 126[78,91] and 127[30] have also all been associated with animals. However, it is currently unknown how and when these PCR ribotypes evolved or coevolved.

The Potential Emergence of Other PCR Ribotypes

Population genetics and MLST analyses revealed a distinct lineage of C. difficile that invariably contained PCR ribotype 023 strains (Figure 1).[30] Although this PCR ribotype is not well documented, two studies have reported its prevalence in Europe,[92,93] and retrospective analysis in the UK has revealed that these strains have been associated with equally severe CDI similar to PCR ribotype 027 (e.g., association with toxic megacolon).[94] Given these observations, PCR ribotype 023 strains are clearly worth monitoring. Other common PCR ribotypes including 001, 002, 014, 015 and 106, which, by phylogenomic analysis, do not form a distinct lineage, but occur within the heterologous group and may form progenitors for distinct sublineages to emerge from. With the decline in the prevalence of the epidemic PCR ribotype 027, there has been a simultaneous increase in the numbers of other PCR ribotypes and emerging PCR ribotypes causing CDI (Figure 2). Two previously assigned BI/NAP1/027 strains have since been found not to be PCR ribotype 027 but instead closely related PCR ribotypes 176 and 198.[95] Both have extremely similar banding patterns to that of PCR ribotype 027 (plus or minus one band) meaning that the different typing methodologies do not correspond as well as previously thought (Figure 3). PCR ribotypes 027, 176 and 198 were also previously placed in the same clade using comparative genome microarray analyses,[91] and all clustered by MLST analysis,[30] suggesting that they have coevolved and possibly evolved from each other.

(Enlarge Image)

Figure 3. PCR ribotyping of BI-6, R20291 and BI-11 strains.

Previous international studies have demonstrated a high degree of similarity amongst toxin AB+ strains and a dominant clone has been identified; however, a study of Australian toxin AB+ strains were found to be heterogenous with only two out of nine isolates being PCR ribotype 017 and the rest being recent (PCR ribotypes 237, 280, 281 and 291) or unassigned PCR ribotypes.[96] Some of the

recent and unassigned PCR ribotypes described in the Australian study do appear to have very similar PCR ribotyping banding patterns (although not similar to PCR ribotype 017); it is possible that there has been spontaneous loss of toxin A in an existing A+B+ strain; however, this has yet to be confirmed. It is also thought that this is possibly the result of niche adaption, though the evolutionary timescales are not yet known.

Future Studies & the Application of Whole-Genome Sequencing

PCR ribotyping and MLST analyses have proven very useful in studying the epidemiology and population structure of C. difficile. However, neither technique provides information on the phenotypic differences between strains that would allow for identification of increased virulence and transmissibility. Comparative phylogenomics using DNA microarrays employs a genome-wide approach that can be used to identify specific gene differences which can be related to specific groups, for example, source or disease severity, providing a diverse and appropriate selection of strains are sampled. Whole-genome analysis using DNA microarrays was the first technique to identify the varied lineages of C. difficile;[91] however, the most definitive typing system in terms of genetic differences and most informative in terms of gene content is whole-genome sequencing. The advent of next-generation sequencing has seen a rapid increase in bacterial genomes sequenced, which can be used for phylogenetic and evolutionary analyses. Application of whole-genome sequencing comparisons for epidemiology and evolution studies have been published for several bacterial pathogens.[97] Vibrio cholerae has recently been studied using third-generation single-molecule real-time DNA sequencing.[98] Rapid analysis of the V. cholerae genome sequences of the 2011 Haitian outbreak identified the epidemic to likely be a result of the introduction of a strain from a distant geographic source through human activity. Whole-genome analysis has also been applied to an epidemic clonal lineage of the notorious hospital-acquired methicillinresistantStaphylococcus aureus (MRSA).[99] The study based on single-nucleotide polymorphism (SNP) analysis from whole-genome sequence data analyzed 63 geographically diverse isolates from a clonal lineage spanning four decades, which revealed clear evidence of geographical grouping and intercontinental spread over time. The SNP-based study also revealed the microevolution of MRSA within a Thai hospital over a seven month period which could differentiate

the introduction of community-acquired strains from person-to-person spread within the hospital environment. The next few years are likely to see an escalation of whole-genome sequencing studies for the comparative analysis of C. difficile strains. A SNP-based study of 21 PCR ribotype 027 strains revealed chronological microevolution and confirmed the toxin AB+ PCR ribotype 017, 027 and 078 lineages, with the latter appearing to be highly divergent.[100] Further phylogenetic analysis has demonstrated that C. difficile is a genetically diverse species, with estimates of the date of the most recent common ancestor varying from 1.1 to 85 million years.[97,100] By contrast, disease-causing isolates of this species have arisen from multiple lineages over short evolutionary timescales suggesting that virulence evolved independently in the multiple, highly epidemic 017, 027 and 078 lineages. The most recent common ancestor of the recent hypervirulent PCR ribotype 027 lineage was estimated to be approximately 30 years ago, which is consistent with the dates of its recorded global spread.[33,100,101]To date, the data generated using MLST, microarrays, shotgun sequencing and phenotypic assays has shown inadequate resolution and generated limited data which has left several questions and hypotheses. We do not yet know exactly how this species is evolving whether it be in a linear or divergent fashion and at what rate. What is the phylohistory and phylogeography of this species? Are strains emerging on one continent, spreading globally and then disappearing? Are strains mutating in real-time between patients in a hospital setting? Has the latter already been demonstrated with the PCR ribotypes 027, 176 and 198? And what is the driving force for these observations? We do not yet fully understand the species population structure and evolutionary history. In the future, it is envisaged that high-throughput genome sequencing will enable detailed epidemiological reconstructions at the hospital, national and global levels. Genetic epidemiological studies are likely to prove useful for tracing the routes of transmission and sources of infection for hospital-acquired infections. The most recent MLST study investigated wardbased transmission of 1276 C. difficile isolates collected over 2.5 years from a geographically large endemic setting. This study subdivided outbreaks into distinct lineages defined by MLST[102] and concluded that up to three-quarters of new cases of CDI are not easily explained by conventional assumptions of wardbased transmission from symptomatic patients. The MLST study was reasonably discriminatory to identify transmission, though not powerful enough to analyze the transmission of each ST individually. Whole-genome sequencing promises to provide further insights, and for C. difficile, it is envisaged that this approach alongside strong geographical, patient and epidemiological data, will be able to

monitor persistence, transmission and evolution within hospitals by spatial temporally mapping the genotypes to the hospital floor plans. The development of screening platforms based on high-throughput sequencing has the potential to shape current practices adopted for hospital infection control and the management of patients. They will provide a sophisticated level of information for the investigation of clonal epidemic strains and should be able to unequivocally differentiate between causes of recurrent disease such as relapse (same strains) and re-infection (different strains). In the near future, it is anticipated that the data provided by whole-genome sequencing of multiple global strains from multiple sources will allow for a detailed description of the population structure of C. difficile. It is envisaged that this will facilitate the identification of virulence determinants. It is hoped that by utilizing this knowledge in conjunction with same-day sequencing of C. difficile isolates in a routine diagnostic laboratory, clinicians will be alerted to potentially highly pathogenic variants, antibiotic-resistant strains and potential outbreaks, such that timely appropriate clinical care can be implemented.