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Clostridium difficile infections (CDI) are currently recognized as the leading cause of hospital-acquired diarrhea worldwide. CDIs begin with the ingestion of highly resistant Clostridium difficile spores. Bacterial communities found naturally in the gastrointestinal tract can provide a barrier against C. difficile colonization, thereby preventing CDI. With the current pharmaceutical advancements, exposure to several new broad-spectrum antibiotics can effectively disrupt and remove the protective barrier. As C. difficile spores travel through the gastrointestinal tract, the spores are able to germinate into actively growing cells, which can ultimately colonize a susceptible host. Due to the C. difficile’s lifecycle and its ability to form resistant spores, CDI is often difficult to treat, and the use of antimicrobials frequently results in relapse. The emergence of hypervirulent strains further highlights the need to address C. difficile infections. Research is currently geared toward developing novel antimicrobials that would specifically target C. difficile, as well as preventing C. difficile colonization.

Clostridium difficile infection (CDI) is a nosocomial disease mainly correlated with antibiotic-associated diarrhea. These infections are caused by Clostridium difficile, an anaerobic, rod shaped, gram-positive bacterium that is normally found in the gastrointestinal tract (Figure 1.1).1 

Figure 1.1

Gram stain of C. difficile. Vegetative C. difficile cells are rod shaped and stain purple in the presence of crystal violet, displaying a gram-positive phenotype. Scale = 10 µm.

Figure 1.1

Gram stain of C. difficile. Vegetative C. difficile cells are rod shaped and stain purple in the presence of crystal violet, displaying a gram-positive phenotype. Scale = 10 µm.

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Approximately 5% of healthy adults, and 50% of newborns are asymptomatic carriers of C. difficile.2 C. difficile was originally thought to be a commensal bacterium, but due to the recent boom of antibiotic therapies and advancements, it was quickly recognized that C. difficile is the leading cause of hospital-acquired diarrhea worldwide.3  In the United States alone, there are roughly 500 000 cases of CDI annually, with associated costs estimated to be approximately $4.8 billion.4 

C. difficile has a unique lifecycle such that it can form metabolically dormant, non-reproductive spores when stressed (Figure 1.2).5 

Figure 1.2

Schaeffer-Fulton stain of C. difficile. Upon environmental stress and starvation, vegetative C. difficile cells (pink) begin to produce highly resistant, non-reproductive spores (blue). Scale = 10 µm.

Figure 1.2

Schaeffer-Fulton stain of C. difficile. Upon environmental stress and starvation, vegetative C. difficile cells (pink) begin to produce highly resistant, non-reproductive spores (blue). Scale = 10 µm.

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These stressors include, but not limited to, nutrient limitation and desiccation. The resulting spores are highly resistant to harsh environmental factors such as stomach acid, extreme temperatures, and pharmaceutically relevant antibiotics.6  Spores can persist over prolonged periods of time, while constantly monitoring the environment for favorable conditions. Upon reintroduction to nutrient rich environments, the spores are able to revert back into actively growing cells through a process known as germination.6  When C. difficile has completed its lifecycle, transitioning from a spore to an actively growing cell, the newly germinated cells can now colonize the local environment.

CDI begins with the ingestion of the highly resistant C. difficile spores. As these spores travel through the gastrointestinal tract, various endogenous bile salts stimulate the spores to germinate into actively growing, vegetative cells.7  While the spores are metabolically dormant and act solely as a vehicle for infection, the vegetative cells are metabolically active, can produce toxins, and elicit disease.

The diversity of the endogenous bile salts depends heavily on the intestinal gut flora. Under normal circumstances, the bacteria found naturally in the gastrointestinal tract provide a barrier against C. difficile colonization by occupying nutrient-rich niches and by metabolizing specific bile salts required for C. difficile germination.8  With the current pharmaceutical advancements, several new broad-spectrum antibiotics have been developed. Exposure to antibiotics, such as clindamycin, 2nd and 3rd generation cephalosporins, and fluoroquinolones, can disrupt the natural gut flora.2,9,10  Disruption of the gut flora effectively removes the protective barrier, as the change in the bile salt diversity becomes more favorable for C. difficile (Figure 1.3).

As C. difficile spores germinate and outgrow, the resulting vegetative cells begin to produce and release two major toxins, TcdA and TcdB. The C-terminal region of both TcdA and TcdB contain a binding domain, which is able to interact with different carbohydrate and protein structures found on the surface of host cell membranes.11  TcdB binds to chondroitin sulfate proteoglycan 4 (CSPG4) and the poliovirus receptor-like 3 (PVRL3) found on the surface of intestinal epithelial cells.12  In contrast, TcdA can bind to glycoprotein 96 and sucrase isomaltase, both of which are found on the surface of human colonocytes. Once these toxins interact with the host cell receptors, the toxins are internalized by endocytosis. Acidification of the endosome causes the toxins to undergo conformational changes resulting in translocation into the host cell cytoplasm. Upon entry into the cytoplasm, the toxins undergo autocatalytic cleavage.11  Cleavage of the toxins allow for the release and activation of their glycosyltransferase domain (GTD) into the host cell.

Figure 1.3

(a) Germination of C. difficile spores and the use of anti-germinants. Upon antibiotic therapy the diversity of bile salts (yellow stars) shift to a population more favorable for C. difficile spore germination. As C. difficile spores (blue circles) are ingested, the spores travel through the gastrointestinal tract, recognize the specific bile salts, and are able to germinate into toxin producing cells (red rods). (b) In the presence of various bile salt analogs (red moons), as C. difficile spores travel through the gastrointestinal tract, the bile salt analogs compete with bile salts that would normally trigger germination, effectively blocking C. difficile germination and the progression of CDI.

Figure 1.3

(a) Germination of C. difficile spores and the use of anti-germinants. Upon antibiotic therapy the diversity of bile salts (yellow stars) shift to a population more favorable for C. difficile spore germination. As C. difficile spores (blue circles) are ingested, the spores travel through the gastrointestinal tract, recognize the specific bile salts, and are able to germinate into toxin producing cells (red rods). (b) In the presence of various bile salt analogs (red moons), as C. difficile spores travel through the gastrointestinal tract, the bile salt analogs compete with bile salts that would normally trigger germination, effectively blocking C. difficile germination and the progression of CDI.

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The GTD can transfer glucose from UDP-glucose to several crucial Rho proteins.11,13  Glucosylation of Rho proteins result in their inactivation.11  Since Rho proteins play an essential role in regulating the cell cytoskeleton, inactivation of these proteins can have several cytopathic effects including the disruption of cell-to-cell contacts and tight junctions, as well as increased epithelial permeability.14  Glucosylation of RhoA also activates the inflammasome and upregulates a pro-apoptotic protein, RhoB.

Expression of tcdA and tcdB is heavily regulated and dependent on resource availability. When carbon sources and other nutrients are readily available, toxin expression is inhibited.11  Conversely, toxin production is upregulated during stationary phase when resources are low. This type of regulation suggests that C. difficile virulence is a killing strategy used to improve resource availability by scavenging the host cell for resources. A combination of several factors contributes to C. difficile’s virulence. TcdA and TcdB are undoubtedly major contributors.11  Due to genetic variability between C. difficile strains, the extensive genotypic variances in the pathogenicity locus (PaLoc), which houses tcdA and tcdB, currently give rise to at least 31 different toxinotypes.11,15  These different toxinotypes result from mutations in tcdA and tcdB, as well as the regulatory factors that ultimately lead to the overexpression or repression of the toxin genes.15  Several other factors, such as rates of sporulation and toxin release, motility and host cell adherence, can contribute to C. difficile virulence.

Interestingly, so called “hypervirulent” strains of C. difficile have begun to reveal themselves in the healthcare setting worldwide. Hypervirulent strains are highly variable—some strains may have higher rates of sporulation and toxin production.13  TcdC, an anti-sigma factor that acts as a negative regulator of toxin production, is upregulated during exponential growth.15  Several hypervirulent strains contain mutations in tcdC that result in the constant, unregulated production of C. difficile toxins.15  Toxins produced by hypervirulent strains can also undergo necessary conformational changes at higher pH ranges, allowing the toxins to enter the host cell cytoplasm earlier and at a faster rate, relative to toxins produced by non-hypervirulent strains.11 Clostridium difficile transferase (CDT), a ribosyltransferase, is a binary toxin produced only by several strains of C. difficile. Similar to TcdA and TcdB, CDT binds to a host-cell by interacting with a surface molecule, specifically lipolysis-stimulated lipoprotein receptor (LSR).16,17  CDT eventually localizes into the host cell cytoplasm where it begins to ribosylate G actin.15  At low concentrations of the toxin, CDT induces microtubules to form protrusions from the host-cell membrane, facilitating C. difficile adherence to the surface of the intestinal epithelial cells.15,18  At high concentrations of CDT, actin polymerization is inhibited and actin depolymerization is induced ultimately causing the collapse of the host cell cytoskeleton.17 

Symptom severity in CDI patients can range from mild diarrhea, to life-threatening pseudomembranous colitis, a condition which causes exudative plaques on the intestinal mucosa. Mild CDI is defined solely by the presence of diarrhea. Other symptoms that can indicate moderate disease include abdominal pain, loss of appetite, fever, nausea, vomiting, gastrointestinal bleeding, bloody stools, and weight loss.

Severe disease can include some or all of the symptoms associated with mild-to-moderate disease plus additional indicative symptoms. Severe CDI is indicated by a serum albumin < 3 g dl−1 with an elevated white blood cell (WBC) count of ≥15 000 cells/mm3 and/or abdominal tenderness.19 

In more severe cases of CDI, patients can develop several complications. If symptoms progress, they can lead to hypotension, fever above 38.5 °C, ileus (a condition in which peristaltic activity to move stool through the gastrointestinal tract is diminished) or abdominal distention, altered mental state, WBC count ≥35 000 cells/mm3, serum lactate levels >2.2 mmol L−1, and ultimately organ failure.19 

Another complication called pseudomembranous colitis is unique to CDI. Pseudomembranous colitis occurs when toxins produced by C. difficile cells damage the walls of the colon causing inflammation and thickening of the colonic mucosa producing yellowish exudates called pseudomembranes to form along the colon. This can lead to other complications such as perforated colon and toxic megacolon. Toxic megacolon can render the colon incapable of expelling gas and stool contents, potentially causing the colon to rupture.

CDI relapse is characterized by the return of symptoms within eight weeks of primary diagnosis after initial symptoms have previously been resolved.20  CDI recurrence after initial treatment can reach up to 25% in treated patients.21  Chances of subsequent recurrences nearly doubles to 45% after the first recurrence.22  One explanation for CDI relapse is that resident C. difficile spores may have survived in the gut after completion or discontinuation of antibiotic treatment.21 C. difficile spores may also be picked up via contamination of the local environment.21  Therefore, relapse and reinfection may be difficult to distinguish. However, reinfections can be identified by the diagnosis with a different C. difficile strain.23,24  Other possible reasons for relapse include poor immune response leading to inadequate production of antibodies against to C. difficile toxins and frequent disruption of normal gut flora.22  Moreover, epidemiologic factors such as advanced age, use of other antibiotics, and prolonged hospital stay may also contribute to increased risk for recurrent CDI.22  Emergence of resistant strains and hypervirulent strains over the past decade has made treatment of recurrent CDI increasingly difficult.25 

CDI may mimic flu-like symptoms or flare-ups of other gastrointestinal diseases. Therefore, early and accurate diagnosis of CDI is important to the successful management of the disease. In 2010, the Society for Healthcare Epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA) recommended a two-step algorithm for CDI diagnosis.26  First, an initial immunoassay screen of stool samples for the presence of C. difficile is performed. If C. difficile is found to be absent in the initial screen, C. difficile should be ruled out as the cause of diarrhea. Following a positive result from the initial screen, detection of toxins from the stool is tested. Positive indications for C. difficile toxins along with moderate-to-severe symptoms can warrant the need for additional and more invasive testing.

Initial screening of stools for the presence of C. difficile can be performed by a common and relatively inexpensive enzyme immunoassay (EIA) test called the common antigen test or glutamate dehydrogenase (GDH) test. The GDH test looks for the GDH enzyme that is produced in relatively large amounts by C. difficile and can be readily detected the stools of CDI patients.27  Although this EIA can give results within 15–45 min, it tends to have lower sensitivities than other tests since it only test for the presence of the C. difficile organisms rather than the production of toxins.

Following a positive GDH test, the second step of the two-step algorithm includes a cell cytotoxicity neutralization assay (CCNA) which tests for the presence of toxin B in stool filtrate. CCNA has long been considered to be the traditional “gold standard” for the detection of C. difficile toxins.28  In a CCNA, filtrate of the collected stool sample is added to mammalian cultures (e.g. human fibroblast). If C. difficile TcdB is present in the filtrate, the mammalian cells will round up and necrotize.29  To verify that the cytopathic effect is caused by C. difficile toxin, the cell cultures are supplemented with an antitoxin (monoclonal antibodies). If the cytopathic effect is reversed, a test is positive for TcdB. While CCNA is highly sensitive and specific, it has a slow turnaround of 24 to 48 h and requires technical expertise.

Alternatively, a three-step algorithm for CDI diagnosis may be used. The three-step algorithm includes the steps of the two-step algorithm with the addition of an intermediate EIA test that detects the presence of free TcdA and TcdB (TOX-A/BII) in stool.30  If results from the TOX-A/BII EIA test is positive, the stool is said to be positive for C. difficile toxins. If the test is negative, CCNA will be performed in the third step.

Alternately, the third step of the three-step algorithm may employ a molecular diagnostic test instead of CCNA.31  Nucleic acid amplification tests (NAATs) allow for the detection of C. difficile toxin gene fragments via real-time quantitative polymerase chain reaction (RT-PCR).19  Through molecular methods, Toxin B (tcdB) and binary toxin (cdtA and cdtB) can be detected.31  NAATs have better sensitivity than CCNA to test for non-free toxins. Although this method tests for the presence of the toxin, it does not indicate the expression of the toxins genes. NAATs can be costly and must be interpreted with caution as they may detect toxigenic strains in asymptomatic carriers who may have diarrhea caused by other pathologies.32 

The three-step algorithm provides an effective and reliable method to diagnosing CDI and may eliminate the need to perform a CCNA test if the TOX-A/BII EIA test is positive. However, due to a lack in sensitivity, it has been recently widely accepted that the TOX-A/BII EIA is not well-suited to be a stand-alone test to diagnose CDI.26  Therefore, the two-step algorithm is usually preferred over the three-step algorithm as it more practical, cost effective, and requires less workload in comparison to the three-step algorithm.30 

Colonoscopy and Computed Tomography (CT scan) may also be used to diagnose conditions caused by CDI such as pseudomembranous colitis. These imaging methods are used less often than laboratory tests as they can be more costly, unpleasant to the patient, and less sensitive.33 

Although CDI can occasionally occur in healthy individuals, CDI is most prevalent among elderly and immunocompromised patients.19,34  Patients are more likely to get CDI in healthcare-acquired settings (e.g. hospitals and long-term care facilities) than in community-acquired settings. Because C. difficile spores can survive for long periods of time on hospital surfaces and patient beds, proper precautions must be taken in these settings to prevent CDI from spreading.

The Association for Professionals in Infection Control and Epidemiology (APIC) suggests that hospitals implement infection control programs.19  Three recommendations include a criteria index for patients who have risk factors for CDI (e.g. malignancy, advanced age, and recent hospitalization or stay at a long-term care facility), advocacy for physicians to use proper C. difficile diagnostic testing with rapid turn-around times and high sensitivity for toxin detection, and appropriate notification to staff members of positive C. difficile test results to ensure that proper precautions and treatments be taken.19  When a C. difficile infection control protocol was implemented at the University of Pittsburgh Medical Center-Presbyterian, a decrease from 7.2 cases per 1000 discharges to 4.8 cases per 1000 discharges was obtained in a 5 year-period.35 

Proper hand hygiene is critical in the prevention of CDI. Although alcohol antiseptics can be used to kill most vegetative bacteria and viruses, they do not affect C. difficile spores due to their intrinsic resistance.19  Therefore, healthcare providers and visitors should be required to wash their hands with antimicrobial hand soap and water. Since a person can easily contaminate their hands with C. difficile spores by contacting an infected patient, healthcare personnel and visitors must also use gloves and gowns upon entry into a CDI patient’s room. An intervention study incorporated an infection prevention education program with vinyl gloving wearing surveillance for six months. The study showed a significant decline in CDI rates from 7.7 cases per 1000 patients to 1.5 cases per 1000 patients six months after intervention.36  Other patient contact precautions include the use of single-use disposable equipment and the limit of patient contact until resolution of diarrhea.

Because the environmental surfaces are common sources for nosocomial infective agents such as C. difficile, the use of disinfectants is recommended. The Environmental Protection Agency (EPA) advocates for disinfectants that are sporicidal.19  Other chlorine-containing agents also have the potential to decontaminate C. difficile infected surfaces, but are recommended to have a minimum of 5000 ppm of chlorine.

Another C. difficile prevention intervention includes placing CDI patients into private rooms. A cohort study showed that patients in double rooms had a higher acquisition of CDI than patients placed in single rooms.37  Moreover, hospitalized patients who do not have diarrhea are not recommended to be screened for C. difficile as some patients may be asymptomatic carriers of C. difficile.38  In a study about asymptomatic carriers, metronidazole was shown to be ineffective at eliminating C. difficile carriage, and vancomycin, though initially clearing up C. difficile from stools, was unsuccessful at preventing recolonization of C. difficile.38  The high rate of recolonization often resulted in emergence of new strains and an asymptomatic carrier even developed CDI after vancomycin treatment.38 

Managing and minimizing the type, frequency, and variety of antibiotics taken for other illness can also reduce the susceptibility of patients to CDI. Although most antibiotics can cause patients to become susceptible to CDI, clindamycin, cephalosporins, and fluoroquinolones pose a higher risk for CDI.39  The duration of antibiotics and use of multiple antibiotics can also increase susceptibility. One study showed that an antimicrobial stewardship program decreased CDI incidence by 60%.40 

CDI recurrences can be treated with repeated regimens of metronidazole and vancomycin, though this method may not be successful in preventing future recurrences. Furthermore, metronidazole is usually avoided in recurrent CDI treatment as prolonged use may result in neurotoxicity and hepatic toxicity.25  Tapered doses followed by pulsed doses of vancomycin may also be used to manage subsequent episodes.22  For more severe recurrences or in the case of multiple recurrences, novel methods or more drastic measures such as rifamixin “chaser” therapy, nitazoxanide, intravenous immunoglobulin (antibodies), fecal microbiota transplant (FMT), and probiotics may be considered.22,25 

Rifamixin “chaser” therapy is effective in decreasing recurrent diarrhea among CDI patients who have already undergone conventional treatments. Rifamixin is a nonabsorbed antibiotic that targets C. difficile without killing enteric gut bacteria, allowing the gut flora to regrow and potential reduce CDI recurrence.41  Rifaxmixin, however, has been linked with high resistance levels caused by an amino acid substitution in the β-subunit of RNA polymerase.41,42  Therefore, rifamixin is used in recurrent CDI treatment after vancomycin treatment as a “chaser” and should be used with caution due to the possibility of resistance.43 

Nitazoxanide is a nitrothiazolide antiparasitic agent that has been used following standard CDI treatments in a few studies. Due to the small sample size of one study, noninferiority or superiority of nitazoxanide to vancomycin could not be made.44  Another small study showed 54% clinical cure of patients given a 10 day-course of nitazoxanide after treatment failure with 14 days of metronidazole. However, 20% of patients relapsed and 27% failed to resolve symptoms (succumbing to the disease at various points in the trial).45  Larger studies are necessary to compare the nitazoxanide with standard CDI treatments.

Treatments for CDI require a rigorous regimen, which normally combines decontamination of the local environment, together with a choice of treatment with metronidazole, vancomycin, and/or fidaxomicin. However, due to C. difficile’s lifecycle and its ability to form spores, complete decontamination is often difficult and the use of antimicrobials frequently leads to relapse.46  Due to the high rates of relapse, new forms of treatment and prevention have been evaluated such as the use of anti-toxin, vaccines, and fecal transplants.

Metronidazole (Figure 1.4) is a nitroaromatic, broad-spectrum antibiotic that is highly active against anaerobic bacteria, such as C. difficile.47  Due to metronidazole’s low molecular weight, it can enter C. difficile’s cytoplasm via passive diffusion. In anaerobic bacteria, flavodoxin and ferredoxin act as electron acceptors as pyruvate becomes oxidatively decarboxylated.2  In the presence of metronidazole, electrons are instead donated to the 5-nitro group on the imidazole ring.47  The resulting nitroso free radical is reactive and will target DNA.2  While the exact mechanism has not yet been fully elucidated, it is believed that DNA experiences oxidative damage—this damage produces numerous single stranded and double stranded breaks, ultimately leading to DNA degradation and cell death.47 

Figure 1.4

Chemical structures of clinically relevant antimicrobials in the treatment of CDI.

Figure 1.4

Chemical structures of clinically relevant antimicrobials in the treatment of CDI.

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While metronidazole is a promising treatment due to its ability to specifically damage anaerobic bacterial DNA, some clinically relevant C. difficile strains have shown resistance against metronidazole.47  Metronidazole resistance is currently believed to result from several different mechanisms, including reduced influx, slow activation of metronidazole, increased DNA repair, inactivation of reduced metronidazole, or increased efflux of the drug.47  Resistance to metronidazole can also arise from the horizontal transfer of genes conferring resistance of nitroimidazoles.47 

Vancomycin (Figure 1.4) is a second antibiotic of choice that works relatively well for severe cases of CDI.48  Vancomycin is a large, hydrophilic, broad-spectrum antibiotic that inhibits the maturation of the peptidoglycan in gram-positive bacteria.2  In addition to preventing peptidoglycan biosynthesis in vegetative cells, vancomycin also prevents the outgrowth of C. difficile spores.46  Due to the large molecular weight, vancomycin can be administered orally as it is minimally absorbed and can be found at high concentrations in the gut and feces.48  In addition to having favorable pharmacokinetics, vancomycin resistance has not yet been reported in C. difficile.2 

Because metronidazole broadly targets anaerobic bacteria and vancomycin targets gram-positive organisms, they suppress the growth of the gut microbiota and thereby removing the natural protective barrier against C. difficile.21  Therefore, treatment with metronidazole or vancomycin frequently results in CDI relapse.46  Fidaxomicin (Figure 1.4), a newer anti-CDI treatment, has been shown to be active against C. difficile, and not target gram-negative bacteria.3,49  Originally isolated from Dactylosporangium aurantiacum, fidaxomicin is an 18-membered macrocyclic antimicrobial that inhibits bacterial RNA polymerase, ultimately halting transcription.3,50  While other RNA synthesis inhibitors prevent the initiation and elongation steps, fidaxomicin halts transcription by binding to RNA polymerase prior to DNA separation.2,48 

Hydrolysis of an isobutyryl ester by an unknown esterase located at the 4′ position of fidaxomicin yields OP-1118, the major byproduct of fidaxomicin metabolism.48  Similar to fidaxomicin, OP-1118 also displays potent bactericidal activity against C. difficile.50  Fidaxomicin has also been shown to inhibit spore production and, similar to vancomycin, also prevent the outgrowth of C. difficile spores.3,46,50  Fidaxomicin and OP-1118, like vancomycin, demonstrate low systematic absorption when taken orally, and can be found at high concentrations in the gut and feces.48  Fidaxomicin and OP-1118 also seem to decrease C. difficile toxins.50  Finally while compared to both vancomycin and metronidazole, fidaxomicin was associated with a lower rate of recurrence.21 

TcdA and TcdB are the main determinants of CDI and the recent overexpression of these toxins by hypervirulent strains has brought about the idea of using anti-toxins to control the course of the disease. The use of anti-toxins could potentially neutralize toxins, while simultaneously allowing the re-colonization of the natural gut flora and therefore prevent relapse. The use of two human monoclonal antitoxins, actoxumab and bezlotoxumab, have been shown to neutralize both TcdA and TcdB, respectively.51  Actoxumab and bezlotoxumab bind to the C-terminal regions of TcdA and TcdB, thereby inhibiting the toxins’ ability to bind to their host substrates. Indeed, actoxumab and bezlotoxumab have been shown to prevent epithelial damage and inflammation in mouse models.51  The use of antitoxins in combination with vancomycin has been reported to decrease the rate of recurrence, compared with the use of vancomycin alone.2,52  While advances have been made to produce antitoxins against TcdA and TcdB, antitoxins against CDT have not yet been fully addressed. Additionally, while these antitoxins are able to effectively reduce the morbidity and mortality of CDI, the antitoxins play no role in inhibiting or reducing C. difficile colonization.53 

Development and evaluation of C. difficile vaccines have been ongoing for the past two decades. Formalin-inactivated whole toxins have shown to be an effective vaccine in protecting hamsters against CDI.53  Truncated forms of TcdA, TcdB, as well as hybridized versions of the two toxins have been considered to be used as vaccines. Inoculation of Golden Syrian hamsters with TcdB fragments, in combination with TcdA, was shown to generate a strong immunogenic response, ultimately producing antibodies that neutralized the toxins.52  Interestingly, the C-terminal binding domains are able to produce a greater immunogenic response compared to the N-terminal GTD.3  As a result, the C-terminal regions of TcdA and TcdB have been hybridized together to form an effective recombinant vaccine without the presence of the glycosyltransferase domains.3 

While the use of toxoids and recombinant vaccines are able to effectively reduce the symptoms of CDI, asymptomatic colonization by C. difficile still occurs. A vaccine that targets surface antigens could potentially reduce C. difficile colonization and transmission, rather than directly decreasing the lethal outcome. The surfaces of C. difficile vegetative cells contain three highly intricate carbohydrate structures termed PSI, PSII, and PSIII.5  PSI and PSIII are found only in some C. difficile ribotpes and they seem to be expressed stochastically.54  PSII is an ideal target for vaccine development since it is expressed at higher levels relative to PSI and PSIII, and can be found more abundantly across the C. difficile ribotypes.54  PSII is a polysaccharide composed of repeating hexaglycosyl units, connected by a phosphodiester group. Interestingly, antibodies have been raised against phosphorylated hexaglycosyl units, and these hexaglycosyl units can be found in the biofilms produced by C. difficile.53  In previous studies mice and rabbits were inoculated with conjugated forms of PSII and in both cases, immune responses were raised against the native forms of PSII.5  Lipoteicholic acid (LTA), another surface antigen that is highly conserved on the surface of C. difficile cells, can also stimulate an immune response in mouse and rabbit models.3  Although antibodies have been raised in response to PSII and LTA, no evidence has yet been reported about the protective efficacy of these antibodies and vaccines.53 

As the transition from a metabolically dormant spore to a toxin-producing cell is required for disease, the germination process may be a target for CDI treatment and prevention. Upon recognition of taurocholate, a primary bile salt found in the gastrointestinal tract, germination is stimulated in C. difficile spores.1  Gut microbiota, found during normal circumstances, are able to break down and metabolize taurocholate into chenodeoxycholate (CDCA), a secondary bile salt.8  While taurocholate normally induces germination, CDCA inhibits C. difficile spore germination and therefore blocks the onset of disease.55  All existing treatments of CDI currently focus on combating the established disease. The use of anti-germinants would prevent spore germination, ultimately playing a role in disease prophylaxis (Figure 1.3B).56 

Indeed, the use of several synthetic bile salts to block C. difficile’s spore germination has been evaluated. CamSA, a synthetic analog of taurocholate, has been shown to inhibit C. difficile spore germination in vitro at micromolar concentrations.1  The use of CamSA has also been shown to protect mice when challenged with C. difficile after antibiotic treatments.56  While the use of anti-germinants shows great potential CDI prophylaxis, different strains of C. difficile display different germination phenotypes, and therefore a singular anti-germinant may not be sufficient in completely preventing disease.57 

Fecal Microbiota Transplant (FMT) is a non-traditional method to treating multiply-recurrent CDI.58  FMT is the introduction of stools from a tested healthy donor into the colon of a patient.59  Fecal transplantation has been used in veterinary medicine for over 100 years and was first performed in humans in 1958. FMT has been studied widely since then and is currently being tested in various other gastrointestinal diseases including Crohn’s Disease, Irritable Bowel Syndrome (IBS), and Ulcerative Colitis.

FMT is a method that is used to restore the diverse gut microbiota that have been killed by the use to antibiotics. Patients with recurring CDI have been shown to have abnormally disproportionate gut microbiota. By reintroducing normal gut bacteria back into the patient via donor feces, it is suggested that the rich and diverse gut flora and colonization resistance can be restored to correct the imbalance.

Patients who receive FMT usually have had multiple bouts of recurrences and have failed conventional treatment methods. On average, studies have shown a cure rate of 91–93% after 90 days of FMT following a course of vancomycin treatment.59 

FMT is most commonly done via a colonoscopy, endoscopy, or through an enema, but can also be taken as a frozen oral capsule.60  Administration of fecal samples into the colon via colonoscopy or fecal enema show high success rates of 86–100%.59  Other endoscopic procedures such as fecal sample introduction into the gastric cavity or small intestine yielded slightly lower success rates of 77–94%.59  A lower success rate of 80% was reported for FMT administration via oral capsules.59 

Although the cost of FMT is less expensive than other CDI treatments, the unappealing aesthetics of the procedure is often a concern of patients.61,62  Although FMT has been deemed relatively safe, a potential risk is the transmission of infectious agents from the donor feces to the patient. Nevertheless, donor stools undergo rigorous screening for common bacterial and viral enteropathogens.59  Long-term follow-up studies of FMT are also limited. One study showed that 4 out of 77 patients developed autoimmune diseases such as rheumatoid arthritis after FMT treatment. However, no clear relationship between the autoimmune disease and FMT have been linked. Exclusion of patients from FMT may include contraindications for colonoscopy, need for continuous antibiotic treatment for another diseases, and failure to understand FMT treatment due to conditions such as dementia.58 

Similarly to FMT, probiotics aim to introduce “good” microorganisms into the GI tract of CDI patient. Probiotics can include combinations of bacteria and yeast. Unlike FMT, probiotics typically include only a limited number of microbial species. Common microorganisms used in probiotic mixtures include Saccharomyces boulardii, Lactobacilli, Clostridia, Streptococci, and Bifidobacteria. Lactobacilli are commonly found in yogurt and other fermented foods. Bifidobacteria are found in dairy products and can be used to ease symptoms of Irritable Bowel Syndrome (IBS).63 

Although the use of probiotic therapy is theoretically useful, there is insufficient data to support the efficacy of probiotic use in the treatment of both primary and secondary CDI. S. boulardii has been shown to be well-suited for CDI prevention. In a study of adjunct probiotic use with antibiotics in recurrent CDI, patients showed 35% fewer recurrences than the control group of 65%. However, due to inadequate randomization of antibiotics in the study, a clear link to probiotic effectiveness is questionable. Another criticism of probiotics is the lack of regulation in their manufacturing process. While pharmaceutical drugs must include conditions that the drug is proven to treat along with side effects, contraindications, and adverse drug interactions, probiotics are regulated as dietary supplements. Thus, these products may not contain what is indicated on the labels and are not evaluated for safety.64  Because dietary supplements are usually self-prescribed, there is no controlled method for reporting adverse reactions and side effects.65  In general, drugs are considered unsafe until they can be proven safe, whereas probiotics and dietary supplements considered safe until they can be proven unsafe. Another potential risk, as noted in some sporadic case reports, include bloodstream infections due to bacteremia and/or fungemia from the use of live S. boulardii and Lactobacillus species-based probiotics in immunocompromised patients such as those with HIV and malignancy.66 

Incidences of CDI both in healthcare-acquired and community-acquired settings have increased significantly over last several years.67,68  CDI has surpassed methicillin-resistant Staphylococcus aureus (MRSA) as the most common hospital-acquired infection (HAI).69  Although elderly individuals in healthcare-related settings are traditionally the targets of CDI, younger individuals are now becoming increasingly susceptible to CDI in the community.69  The rise in CDI is due in part to the emergence of antibiotic-resistant and hypervirulent strains of C. difficile contributing to high rates of relapse and virulence.68,70  CDI is also a multi-faceted problem involving many variables ranging from strains characteristics to patient risk factors to environmental control. Therefore, much is still to be learned about C. difficile and new avenues of CDI treatment to be explored.

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