Clostridium difficile-Associated Colitis: Role of the Immune Response and Approach to Treatment

Clostridium difficile infection (CDI) is the most common infectious cause of healthcareacquired diarrhoea. Approximately 15%–25% of all cases of antibiotic-associated colitis (AAC) are caused by C. difficile and this likelihood increases with the severity of disease, reaching 95%–100% among patients with documented antibiotic-associated pseudomembraneous colitis (PMC) (Bartlett, 1994). Since the initial report of C. difficile as the cause of AAC in 1978 (Larson et al., 1978), subsequent work has provided important information regarding risk factors, diagnosis and effective therapy. More recently, significant challenges have arisen due to increases in frequency and severity of disease, limitations of standard therapy and propensity for recurrence of infection. There has been an unanticipated increase in morbidity and mortality attributed to this disease, linked in part to the emergence of antimicrobial-resistance and the epidemic bacterial strain, BI/NAP1/ribotype 027 leading to a resurgence of CDI as a major cause of hospital-acquired infection.


Introduction
Clostridium difficile infection (CDI) is the most common infectious cause of healthcareacquired diarrhoea.Approximately 15%-25% of all cases of antibiotic-associated colitis (AAC) are caused by C. difficile and this likelihood increases with the severity of disease, reaching 95%-100% among patients with documented antibiotic-associated pseudomembraneous colitis (PMC) (Bartlett, 1994).Since the initial report of C. difficile as the cause of AAC in 1978 (Larson et al., 1978), subsequent work has provided important information regarding risk factors, diagnosis and effective therapy.More recently, significant challenges have arisen due to increases in frequency and severity of disease, limitations of standard therapy and propensity for recurrence of infection.There has been an unanticipated increase in morbidity and mortality attributed to this disease, linked in part to the emergence of antimicrobial-resistance and the epidemic bacterial strain, BI/NAP-1/ribotype 027 leading to a resurgence of CDI as a major cause of hospital-acquired infection.

Clinical presentation of CDI
Infection with C. difficile can result in clinical manifestations ranging from asymptomatic carriage to fulminant colitis and death (Bartlett, 1994).The pathogenesis of symptomatic CDI is characterised by an acute intestinal inflammatory response, prominent neutrophil infiltration and associated tissue injury (Savidge et al., 2003).Features of severe CDI include pseudomembranes visible on endoscopy, abdominal cramps, fever, marked increase in white cell count, rise in serum creatinine level and hypoalbuminemia (Cloud et al., 2009;Pepin et al., 2009).Systemic symptoms may be absent in mild disease but are common in moderate or severe disease.Accurate diagnosis early in the disease course is important for the successful management of CDI.

Pseudomembraneous colitis
Pseudomembranes are characterised as discrete plaques of yellow-white exudate <10mm diameter separated by normal or mildly hyperaemic mucosa usually restricted to the colon, with occasional cases involving the small intestine (Figure 1A &B).These may progress to form a membraneous exudate covering a large degree of the colonic epithelial surface.Ulceration into the sub-mucosa can result in severe cases.The lesions are characterised histologically as discrete regions of necrotic surface epithelium with accumulation of fibrin, mucous and cell debris (Pothoulakis, 1996).Polymorphonuclear cells and neutrophils infiltrate into the lesion exudate and the underlying lamina propria from the systemic bloodstream (Kelly et al., 1994a).Later stage lesions involve the superficial mucosa and crypts and further infiltration of polymorphonuclear cells and eosinophils.Lesions coalesce to create plaques covering larger areas of the mucosa.Rare complications involve deep necrosis and ulceration of the colon sub-mucosa which can lead to perforation, septicaemia and death.

Risk factors
The major risk factors for CDI are increasing age, prolonged hospital stay and recent or current antimicrobial use.The most important risk factor is alteration of bowel microflora and subsequent loss of colonisation resistance associated with antimicrobial usage within the previous 2-3 months prior to infection (Bignardi, 1998;Dial et al., 2008).There are particular classes of antimicrobials that are associated with the highest risk of C. difficile acquisition, including clindamycin, cephalosporins and -lactam antimicrobials and more recently fluoroquinolones (Owens et al., 2008).Recent outbreaks involving a particular epidemic strain of C. difficile have been predominantly associated with fluoroquinolone usage (Loo et al., 2005;Pepin et al., 2005).The risk of developing CDI increases with the use of multiple antibiotics and prolonged duration of therapy.
In addition to these traditional risk factors, other factors associated with CDI include; underlying comorbidities, including inflammatory bowel disease (IBD), gastrointestinal procedures and exposure to an environment contaminated with toxigenic C. difficile, usually via hospitalisation (Johnson & Gerding, 1998).Proton pump inhibitors have been associated with CDI risk but their use is often a marker of the severity of underlying disease, which is in itself a strong risk factor for CDI (Kyne et al., 2000;Kyne et al., 2001).
The host immune status is also important in determining the outcome of colonisation with C. difficile.Immuno-compromised patients are at increased risk for CDI (Yolken et al., 1982) as are patients with a poor immune response to C. difficile toxins (Kyne et al., 2000;Mulligan et al., 1993).The role of protective host immunity in determining progression and severity of CDI, specifically the inability to mount an adequate colonic IgA and or serum IgG response to C. difficile toxins will be discussed further.
The incidence of CDI acquired in the community appears to be increasing.Cases have also been reported in which there has been no recorded exposure to antibiotics or recent hospitalisation.Similarly an increasing number of cases of CDI are occurring in lower age groups and children.This may be due to the emergence of a new strain that is more commonly associated with animal and non-hospital sources of infection, but this has yet to be fully substantiated (Goorhuis et al., 2008).

Toxins A and B
The major virulence factors of toxigenic Clostridium difficile are two large protein toxins A (TcdA) and B (TcdB) that have both been shown to be responsible for the profound intestinal inflammatory response seen in CDI (Kuehne et al., 2010;Thelestam & Chaves-Olarte, 2000).Toxins A and B are very similar in structure, with three functional domains: a receptor binding domain, a translocation domain and a catalytic domain involved in binding to the host cell, entry into the cytoplasm and inactivation of Rho GTPases respectively (von Eichel-Streiber et al., 1996).
Most disease-causing C. difficile strains produce both toxins, encoded by the pathogenicity locus (PaLoc) (Braun et al., 1996;Rupnik et al., 2005).Genetic variations in the toxin genes leads to strains of different 'toxinotypes' (Rupnik, 2008).Most variation across toxinotypes is seen in the binding domain of toxin A, which can lead to so called TcdA-TcdB+ strains which have been found to demonstrate comparable cytotoxicity to normal toxin-producing strains and have been involved in clinical outbreaks (Drudy et al., 2007).Up to 31 different toxinotypes have been identified so far (Rupnik, Feb 2011), highlighting the possibility of a wide variety of C. difficile toxin protein structures and cytotoxic activities.Infection with non-toxigenic strains that do not possess the pathogenicity locus can occur and is usually thought to result in asymptomatic colonisation.
The PaLoc also contains genes involved in regulation of transcription of the toxins (Braun et al., 1996;Rupnik et al., 2005).The epidemic strain BI/NAP-1/ribotype 027 has been shown to contain an 18bp deletion in one of these regulatory genes, tcdC, which results in increased toxin production (Dupuy et al., 2008).This is thought to contribute to the virulence of this strain and its involvement in recent epidemics (Warny et al., 2005).
Toxins A and B mediate their cytopathic effect by disrupting the cytoskeleton of intestinal epithelial cells and causing tight junctions to open, resulting in loss of integrity of the protective monolayer (Figure 2).
Once the intestinal epithelium is breached, the toxins are able to access the underlying lamina propria and come into contact with resident macrophages and circulating peripheral blood mononuclear cells (Pothoulakis, 2000;Thelestam & Chaves-Olarte, 2000).
C. difficile toxins are also capable of inducing the release of several classes of cytokines and neuroimmune pro-inflammatory mediators such as interleukin-8 (Il-8) and tumour necrosis factor-(TNF-) from intestinal epithelial cells, macrophages and mast cells.These recruit circulating inflammatory cells such as neutrophils into the site of infection and perpetuate the inflammation and fluid secretion associated with CDI (Pothoulakis, 2000).In addition to the indirect effect of the toxins on cells of the immune system, C. difficile toxin A has also been shown to directly bind to peripheral blood mononuclear cells, most notably monocytes and induce cell death by apoptosis (Modi et al., 2011;Solomon et al., 2005).This highlights the essential role that toxins play in initiating and prolonging bacterial infection by inactivating key elements of the host protective immune response.

Binary toxin
In addition to toxins A and B, a further toxigenic component, binary toxin, is expressed in a subset of toxigenic C. difficile strains, most notably the epidemic BI/NAP-1/ribotype 027 strain (Rupnik, 2008;Stubbs et al., 2000).Although possessing an ADP-ribosylation function and cytotoxic activity against mammalian cell lines, the role of binary toxin in disease is unclear (Geric et al., 2006).

Surface layer proteins
In addition to the major secreted toxins, the vegetative C. difficile bacterium produces other potential virulence factors.Surface layer proteins are considered to play a vital role in colonisation of the gut and bacterial adherence to the mucosa.They are also important immunogens and can induce the host inflammatory and antibody response (Ausiello et al., 2006;Calabi et al., 2002;Drudy et al., 2004;Pechine et al., 2005).As seen with the toxin proteins, surface layer proteins can be highly variable, particularly surface layer protein A (SlpA) resulting in varying degrees of bacterial adherence ability.

Flagella
Flagella are also major bacterial virulence factors that enable chemotaxis and penetration of the mucus layer and direct adherence of the bacterium to the epithelial cell surface for localised secretion of toxins.Pathogenic C. difficile strains display a range of flagella proteins and subsequent adhesion capabilities (Tasteyre et al., 2001).The immunogenic potential of flagellin is widely appreciated and specific Toll-like receptors (TLRs) are present on the epithelial cell surface to detect flagellin as part of the host cell sensing of pathogens.

Host factors that influence outcome of infection
The host's ability to respond to C. difficile virulence factors plays a major role in determining the outcome of colonisation with this organism.Infection with the same toxigenic strain can result in a variety of outcomes, including asymptomatic carriage through to symptomatic or severe disease.Certain intrinsic host features, including toxin receptor density (Eglow et al., 1992) and presence or absence of barrier flora (Borriello, 1990) are important in the initial stages of colonisation, however early host detection of infection and initiation of an appropriate immune response likely plays a role in preventing prolonged and severe disease and in providing protection from recurrence of infection.

Host protective microflora
The most important risk factor for the development of CDI is antimicrobial use, which leads to alteration of the commensal gut microflora and loss of colonisation resistance, enabling unrestricted growth of toxigenic C. difficile.It is proposed that the healthy gut microflora affords resistance by physical inhibition of pathogen adhesion to the mucosa, blocking production of microbial toxins, competition for nutrients and stimulation and development of the mucosal immune system (Mazmanian et al., 2005;Weinstein & Cebra, 1991).
Recent metagenomic studies have provided a detailed insight into the various bacterial species groups involved in host protection against C. difficile and have identified an important role for members of the Bifidobacterium and Bacteroides species (Hopkins & Macfarlane, 2002;Hopkins et al., 2002) Bacterial species diversity was markedly lower in CDI patients and consisted of a higher number of facultative anaerobes than in healthy controls (Hopkins & Macfarlane, 2002).
Healthy resident faecal microflora have been shown to directly suppress the growth of C. difficile introduced in vivo, possibly through production of volatile fatty acids and bacterial metabolic by-products (Rolfe, 1984).This may explain how approximately 5% of asymptomatic healthy adults carry low concentrations of C. difficile in their colon.They have been shown in vitro to be held in check by normal gut flora (Fekety & Shah, 1993).Nontoxigenic strains of C. difficile may colonise the gut and prevent infection with toxigenic strains (Borriello & Barclay, 1985;Sambol et al., 2002) and even asymptomatic colonisation with toxigenic strains has been associated with a decreased risk of CDI (Shim et al., 1998).
The precise mechanisms by which asymptomatic colonisation is able to protect against CDI have yet to be defined, however continuous immunological challenge with C. difficile bacterial peptides and/or low levels of toxins may prime the immune system to act efficiently in response to a subsequent infection (Viscidi et al., 1983).The commensal microflora has also been shown to profoundly influence the development of the humoral components of the gut immune system, influencing IgA production by B cells (Weinstein & Cebra, 1991).

-12 hours post-infection
C. difficile toxins act quickly to breach the protective mucosal barrier in order to release nutrients for bacterial growth in to the lumen.The host needs to respond rapidly to circumvent further cellular damage and possible dissemination of toxins into the submucosa and bloodstream.The early host pro-inflammatory response or inducible innate immune response is stimulated initially by the epithelial cells, the first and major cell type encountered by microorganisms in the mucosa.
The intestinal epithelium has developed a wide array of protective mechanisms to prevent bacterial adherence and maintain the integrity of the monolayer, including mucous secretion and tight junctions.Epithelial cells are also able to sense pathogen-associated molecules such as LPS, peptidoglycan and flagellin via specific cell surface TLR receptors.Epithelial cells intoxicated by C. difficile release pro-inflammatory mediators including Interleukin-8 (Il-8) and macrophage inflammatory protein-2 (MIP-2) into the underlying lamina propria (Flegel et al., 1991;Mahida et al., 1998).
The sub-mucosal macrophages, monocytes and dendritic cells then disseminate the inflammatory cascade, via the further release of pro-inflammatory cytokines and neuropeptides that recruit peripheral blood cells to the site of infection (Pothoulakis, 1996).
The combined action of cytokines and histamine increases permeability of the vascular endothelium causing fluid leakage and the symptomatic profuse watery diarrhoea associated with C. difficile infection.The expression of specific monocyte and leucocyteadhesion molecules, including CD18 integrins and selectins are upregulated and enable migration of leukocytes and monocytes towards the site of cytokine release (Kelly et al., 1994a).Phagocytosis and digestion of the bacteria with lysozyme, collagenase and peroxidise promotes clearance of the infection, but contributes to the extensive cellular necrosis characterised by the pseudomembraneous plaques.

-12 days post infection
The importance of the adaptive immune response in influencing the outcome of C. difficile colonisation has been appreciated for many years (Aronsson et al., 1985;Johnson et al., 1992;Warny et al., 1994), however the contributions that individual immunoglobulin classes play in protection and prevention have taken longer to elucidate.
An initial challenge with C. difficile (irrespective whether this is associated with symptoms or not) will stimulate the maturation and multiplication of naive B-cells, generating antibody-producing plasma cells and specific memory B-cells.The process of immunoglobulin class switching is then induced by specific cytokines, to ensure that the full range of immunoglobulin classes are generated in response to specific C. difficile antigens.It is likely that the initial immune challenge occurs in infancy, as approximately 60% of healthy non-colonised or asymptomatic adults have detectable serum IgG and IgA antibodies to C. diffiicle toxins (Kelly et al., 1992;Sanchez-Hurtado et al., 2008;Viscidi et al., 1983).
Certain immunoglobulin classes are more important than others in mediating immunity to C. difficile.IgA is mainly associated with the gut mucosa and prevention of pathogen colonisation, whereas IgM is released first in infection, followed by IgG, which provides the major protection against pathogens (Underdown & Schiff, 1986).A selective reduction in mucosal IgA has been shown to be associated with severe CDI and reduction in colonic IgA producing cells may predispose to recurrence of infection (Johal et al., 2004).
Serum anti-toxin antibody levels have been found to play an important role in determining the outcome of colonisation and protection against CDI recurrence.Patients who are asymptomatically colonised with C. difficile have higher serum anti-toxin A IgG levels than colonised patients who develop diarrhoea (Kyne et al., 2000).This suggests that high antitoxin A IgG levels at the time of colonistion protect against CDI.
Once colonisation has progressed to CDI, serum IgM and IgG antibody responses to C. difficile toxins A, B and non-toxin antigens have been shown to be higher in patients who experience a single episode of CDI compared to patients with recurrent CDI (Kyne et al., 2001).The importance of varying antibody levels during the course of infection was also highlighted, as early IgM responses and later IgG responses (day 3 and day 12 post onset of diarrhoea respectively) were significantly higher in those patients who did not experience recurrence of CDI (Kyne et al., 2001).Deficiency in certain subclasses of IgG (IgG2 and IgG3) has also been found to be related to recurrence of disease (Katchar et al., 2007).A high natural anti-toxin antibody response does not always however protect from symptomatic CDI.Patients who are critically ill are less likely to be protected than those who are less severely ill, despite similar antibody levels (Kyne et al., 2000).This suggests that other host factors are also important in immune protection and should be considered when predicting outcome of colonisation on host immune status alone.
Immunity to C. difficile surface layer proteins (SLP's) would be expected to provide important protection during bacterial colonisation and prevention of symptomatic CDI.IgM, IgA and IgG antibody levels to SLP's were shown to be similar in control patients, asymptomatic carriers and CDI patients, however a lower anti-SLP IgM response was observed in patients with recurrent CDI compared to patients with a single episode of CDI (Drudy et al., 2004).A separate study showed that anti-SLP IgG levels in patients with CDI and asymptomatic carriers were higher compared to levels in control patients without CDI (Sanchez-Hurtado et al., 2008).
C. difficile bacterial flagellar proteins FliC and FliD, and the other surface-associated proteins expressed during the course of infection, have also been shown to be immunogenic.Antibodies against these proteins may be detected after CDI diagnosis and for at least 2 weeks following diagnosis (Pechine et al., 2005).

Genetic predisposition to infection: Il-8 promoter mutations
Mucosal Il-8 and neutrophil recruitment have been shown to be essential for the pathogenesis of CDI and subsequent amplification of the acute inflammatory response.In particular, the neutrophil response to C difficile toxins is likely to play a role in determining the severity of CDI.
C. difficile toxins induce monocytes and epithelial cells to produce Il-8, by increasing binding of nuclear factors to the Il-8 promoter and up-regulating transcription of the Il-8 gene (Linevsky et al., 1997).Some patients may have a genetic pre-disposition to severe CDI due to a single nucleotide polymorphism (SNP) in the Il-8 gene promoter.The presence of an AA (rather than AT or TT) genotype at position −251 has been shown to be associated with increased susceptibility to C. difficile toxins and increased fecal Il-8 levels (Jiang et al., 2006;Jiang et al., 2007).The Il-8 promoter AA genotype SNP is also associated with the lack of a protective adaptive antitoxin A antibody response in hospital patients with CDI (Jiang et al., 2006;Jiang et al., 2007).

Antimicrobial therapy
Non-severe CDI can be treated initially with metronidazole for 10-14 days (ASHP, 1998).For severe disease, vancomycin treatment for 10 -14 days is suggested.Reliance on antimicrobial agents for treatment of C. difficile infection would be expected to further damage the host microflora, increasing the risk of recurrence.Recurrence can occur in up to 25% of cases (Kelly et al., 1994b) and may be due to persistence of initial infection after treatment or reinfection.Treatment for recurrences may follow vancomycin administration in a pulsed regimen (Mc Farland et al., 2002) that allows the germination of spores in between antimicrobial administration to improve efficacy.Other antibiotics, nitazoxanide, teicoplanin and most recently fidaxomicin h a v e b e e n s h o w n t o b e a s e f f e c t i v e a s vancomycin treatment and have been indicated as possible alternative therapies ( de Lalla et al., 1992 ;Louie et al.;Musher et al., 2009).
Non-antimicrobial approaches to management of CDI and in preventing colonisation, such as immune-mediated therapy would limit the effect on the gut microflora, lessening the risk of replapse. Currently

Immune-mediated therapy
An effective antibody response to C. difficile toxins and non-toxin antigens is important in influencing the outcome of colonisation and symptomatic CDI and provides the basis for the development of antibody-mediated therapeutics and vaccines.An overview of immunisation strategies in CDI treatment and prevention has been examined in various studies and is reviewed below and illustrated in Figure 3.

Passive immunisation therapy
Passive immunotherapy, involving the direct transfer of antibodies has been studied in both humans and animals and has mainly focused on antibodies against C. difficile toxins A and B

Animal studies
Oral and parenteral administration of bovine anti-toxoid immunoglobulin concentrate (BIC) has been shown to prevent diarrhoea and death in both the hamster and mouse models of CDI (Kelly et al., 1996;Lyerly et al., 1991).Similarly, hen IgY antibodies to recombinant www.intechopen.comColitis 74 peptides of both toxins A an B have also been shown to prevent diarrhoea or death when administered orally to hamsters (Kink & Williams, 1998).
Parenteral immunisation with monoclonal antibodies against the binding region of toxin A was able to protect gnotobiotic mice from diarrhoea and death following toxigenic C. difficile challenge (Corthier et al., 1991).The best protection against CDI and recurrence is however afforded by combination therapy with neutralising antibodies against both toxins A and B. Human monoclonal antibodies (HuMabs) have been developed after immunisation of human immunoglobulin gene transgenic mice with inactivated toxins A and B (Babcock et al., 2006).Intraperitoneal administration of anti toxin A and B HuMabs protected hamsters from mortality and recurrence of CDI.

Human studies
Initial studies examining the efficacy of oral administration of bovine immunoglobulin (BIC) in preventing or treating human CDI were promising, but have not progressed further (Kelly et al., 1996;Warny et al., 1999).Bovine anti-C.difficile IgA purified from the milk of immunised cows (Mucomilk) was also found to be highly effective at reducing recurrent human CDI, but studies were also not continued further (Mattila et al., 2008;van Dissel et al., 2005).Intravenous immunisation with pooled anti-toxin A and B IgG immunoglobulin (IVIG) has been the most widely studied mode of passive immunotherapy in humans (Abougergi et al., 2010;O'Horo & Safdar, 2009;Wilcox, 2004).Administration of IVIG to children with low antitoxin antibody levels and relapsing CDI resulted in an increase in antibody levels and subsequent resolution of CDI (Leung et al., 1991).IVIG has also been used for the treatment of severe or refractory CDI with varying success.There is a lack of consensus regarding the optimal dose (150 -400mg/kg) and dosing regimen (1-3 doses) of IVIG.The lack of randomised controlled clinical trials for IVIG and an association with increased mortality when administered to critically ill patients with CDI in observational studies has served to limit its attractiveness as a possible therapy (Abougergi et al., 2010).
Passive intravenous immunotherapy with HuMabs against toxins A and B has been shown to be well tolerated and effective in protecting patients from recurrence of CDI (Babcock et al., 2006;Leav et al., 2010;Lowy et al., 2010).When used in symptomatic CDI patients in conjunction with standard antimicrobial therapy, recurrence rates were reduced by 72%, although severity of symptoms was not reduced (Lowy et al., 2010).The circulating HuMabs provided protection from subsequent challenge or recurrence due to a half-life of up to 26 days.A role for HuMabs in reducing symptomatic CDI is less well defined however they may be useful as prophylaxis for patients at high risk of infection or in enabling clearance of persistent infection.

Active immunisation/vaccination
As soon as the importance of C. difficile toxins in initiating symptomatic infection was appreciated, studies into active immunisation with inactivated toxin protein toxoids as a means of protecting from CDI were carried out (Kim et al., 1987;Libby et al., 1982).

Animal studies
Hamsters vaccinated with formalin-inactivated C. difficile culture filtrate by combined parenteral and mucosal immunisation were found to be protected from CDI (Torres et al., 1995).Similarly, parenteral administration of a toxoid vaccine to hamsters induced high serum levels of anti-toxin A and B antibodies that mediated immune protection against diarrhoea and death (Giannasca et al., 1999).Anti-toxin antibodies were therefore confirmed as the major mediators of the protective response.
Vaccination of mice via transcutaneous administration of a toxin A toxoid derivative and cholera toxin as an immunoadjuvant also showed good induction of anti-toxin A IgG antibodies (Ghose et al., 2007).
Subsequent studies have focused on the use of recombinant polypeptides from toxin A, thought at the time to be the main toxin mediator of intestinal injury in CDI.Immunisation with recombinant toxin A binding domain polypeptides was shown to partially protect hamsters against CDI, and induced a systemic neutralizing immune response in mice, when administered with an E. coli toxin adjuvant (Lyerly et al., 1990;Ward et al., 1999).Oral immunisation with an attenuated Vibrio cholerae live vector modified to express a fusion protein of the binding domain of toxin A was also shown to induce effective immunity against toxin A in rabbits (Ryan et al., 1997).A DNA vaccine against the binding domain of toxin A has also been developed for human use.It was shown in a trial to be successfully expressed in host mice cells and induced the production of neutralising antibodies and protected from death (Gardiner et al., 2009).

Human studies
Only one candidate C. difficile vaccine has progressed to human trials.A formalininactivated toxin A and B toxoid vaccine was safely tolerated in healthy individuals after intramuscular administration and was shown to induce high serum anti-toxin antibody levels (Aboudola et al., 2003;Kotloff et al., 2001).When used to treat patients with recurrent CDI, the vaccine was also successful (Sougioultzis et al., 2005).Phase II clinical trials of a C. difficile toxoid vaccine are currently in progress.It is thought that this vaccine would be most cost effective when targeted at those at high-risk of developing CDI and in preventing recurrence thereby reducing the economic burden of C. difficile disease (Lee et al., 2010).

Innate immunity-mediated therapy
The inflammatory response to CDI is mediated in the early stages by the innate immune response, most notably the release of Il-8 from intoxicated epithelial cells and the infiltration of neutrophils from the bloodstream.Pre-treatment of rabbits with monoclonal anti-CD18 antibodies was found to protect from severe inflammation and tissue necrosis, by preventing neutrophils from migrating across the vascular endothelium (Kelly et al., 1994a).C. difficile toxins would still be able to cause intestinal epithelial damage in anti-CD18treated patients via triggering infiltration of other inflammatory cells including monocytes and mast cells via Il-8 release from intoxicated intestinal epithelial cells.Amplification of the inflammatory response by neutrophils however would be greatly reduced.The symptoms of CDI in anti-CD18-treated patients would therefore be expected to be less severe and disease may resolve independently of treatment.
Inflammatory mediators have also been shown to reduce intestinal injury in CDI by blocking signals that perpetuate the inflammatory cascade (Kim et al., 2007;Kokkotou et al., 2009).Their use in conjunction with antibiotics or other therapies may be more effective in the most severe CDI cases.

Future strategies for prevention and treatment of CDI
Vaccination strategies currently under trial focus on inducing an immune response to C. difficile toxins A and B, however colonisation is the first step in pathogenesis.Other C. difficile targets, such as surface layer proteins and flagella antigens that have been developed as vaccine antigens to inhibit colonisation have proven to reduce C. difficile colonisation in mice and hamsters, but did not prevent it completely (Brun et al., 2008;Ni Eidhin et al., 2008;Pechine et al., 2007).Widespread vaccination against C. difficile would be expected to reduce bacterial carriage and therefore spread of the organism, but may serve to inadvertently reduce other protective members of the Clostridium spp. in the gut microflora leading to unanticipated side-effects.
Development of toxoid vaccines against both toxin A and B should also take into account the variety in toxin protein structures exhibited by the 31 toxinotypes currently identified.
All vaccines currently under trial are based on inactivated toxoids from a reference strain of C. difficile (Aboudola et al., 2003;Sougioultzis et al., 2005), therefore would in theory only protect immunised patients from a proportion of the possible toxin proteins that would be encountered in infection with multiple strain types.This limited cross-reactivity could be further reduced in vaccines where recombinant polypeptides from the cell-binding domain have been used.This would be most important in recurrent cases, where a small proportion of patients can be infected with a different strain type to their initial infection, rather than reinfection or lack of clearance of their existing colonising strain.
The impact of aging on the efficient functioning of the immune system is also underinvestigated with respect to CDI.Older patients are most at risk of developing CDI and therefore are the population most likely to be targeted for vaccination.Due to immune senescence many older patients may not mount an adequate antibody response to vaccination (Burns, 2004).

Conclusions
The outcome of C. difficile infection, whether it is asymptomatic colonisation or fulminant colitis, is mediated at every stage by the host immune system.The innate immune response plays the foremost role in CDI-induced inflammation and tissue injury, whilst the adaptive immune response mainly mediates protection, either from initial infection or subsequent recurrence.The most effective therapies against C. difficile would therefore augment a specific immunoglobulin response whilst suppressing the innate response that triggers neutrophil migration into the mucosa.Vaccine targets developed to protect the at-risk population have shown initial efficacy, however further trials are needed to determine whether they have the widespread cross-reactivity needed to protect patients from all possible strains of C. difficile.It also remains to be seen whether the majority of older patients will be able to mount an immune response of a sufficient magnitude to the vaccine, to provide adequate protection.

Fig. 1 .
Fig. 1. (A) Pseudomembraneous colitis as confirmed by sigmoidoscopy.Discrete yellow plaques indicate the ulcerated lesions.(B) Histopathological features of pseudomembraneous colitis include loss of crypt structure, infiltration of polymorphonuclear cells and surface accumulation of fibrin, mucous and necrotic cell debris.

Fig. 2 .
Fig. 2. The role of C. difficile toxins A and B in mediating the host inflammatory response

Fig. 3 .
Fig. 3.An overview of immunisation, both active and passive and vaccination against C. difficile.

Table 1 .
available antimicrobial and non-antimicrobial therapies in use for management of CDI and those under development are outlined in Table 1.Treatment strategies for the management of CDI