ABSTRACT
Clostridioides difficile is a Gram-positive, anaerobic, spore forming pathogen of both humans and animals and is the most common identifiable infectious agent of nosocomial antibiotic-associated diarrhea. Infection can occur following the ingestion and germination of spores, often concurrently with a disruption to the gastrointestinal microbiota, with the resulting disease presenting as a spectrum, ranging from mild and self-limiting diarrhea to severe diarrhea that may progress to life-threating syndromes that include toxic megacolon and pseudomembranous colitis. Disease is induced through the activity of the C. difficile toxins TcdA and TcdB, both of which disrupt the Rho family of GTPases in host cells, causing cell rounding and death and leading to fluid loss and diarrhea. These toxins, despite their functional and structural similarity, do not contribute to disease equally. C. difficile infection (CDI) is made more complex by a high level of strain diversity and the emergence of epidemic strains, including ribotype 027-strains which induce more severe disease in patients. With the changing epidemiology of CDI, our understanding of C. difficile disease, diagnosis, and pathogenesis continues to evolve. This article provides an overview of the current diagnostic tests available for CDI, strain typing, the major toxins C. difficile produces and their mode of action, the host immune response to each toxin and during infection, animal models of disease, and the current treatment and prevention strategies for CDI.
INTRODUCTION
Clostridioides difficile is a Gram-positive anaerobic, spore-forming bacterium and the most common identifiable infectious agent of nosocomial antibiotic-associated diarrhea (AAD) (1, 2). This bacterium is also linked to several life-threatening syndromes in humans, including pseudomembranous colitis and toxic megacolon (1, 2). Disease symptoms associated with C. difficile infection (CDI), including diarrhea, fluid loss, and inflammation, result from the production and activity of two exotoxins, toxin A (TcdA) and toxin B (TcdB) (2). These toxins disrupt the Rho family of GTPases within the host cell, which eventually results in cell rounding and death (3, 4). C. difficile was originally discovered by Hall and O’Toole as Bacillus difficilis during their 1935 study of neonatal fecal microbiota and was named “difficilis” due to the difficulty of its cultivation and isolation (5). At the time of its initial isolation, C. difficile was not considered pathogenic, with recognition of C. difficile as a human pathogen occurring roughly 40 years after its initial discovery (5). With time, identification of the obligately anaerobic nature of this organism ultimately resulted in reclassification from B. difficilis to Clostridium difficile. More recently, with an update to the classification of the clostridia, it was first suggested that C. difficile be reclassified as Peptoclostridium difficile (6) and, more recently, to Clostridioides difficile (7). However, as recently highlighted in Smits et al. (8), there is reluctance for reclassification, because there currently exists a large knowledge base surrounding Clostridium difficile, in addition to a strong public awareness which could be lost upon reclassification. Despite these reservations, Clostridioides difficile has now been officially approved by the International Journal of Systematic and Evolutionary Microbiology and International Committee on Systematics of Prokaryotes and has begun to be used more commonly (9–11).
An increase in clinical CDI rates has been occurring for over a decade, coinciding with the emergence of new epidemic strains, including the BI/NAP1/027 group (referred to as ribotype 027 strains from here on), which appear to have enhanced virulence properties in comparison to historical isolates (8, 12, 13). The emergence of these epidemic C. difficile strains has resulted in increased disease severity in infected patients which has been recorded globally and has placed a great financial and administrative burden on hospitals worldwide (14–18).
BACTERIOLOGY, DIAGNOSIS, AND TYPING
Bacteriology and Diagnosis
As a strict anaerobe, the ability of C. difficile to form spores is important for disease transmission and recurrence (2). Spores facilitate the survival of C. difficile in harsh conditions, including in aerobic environments or in the acidic stomach during transit to the large intestine. Sporulation and spore persistence are major challenges, particularly in nosocomial environments, resulting from the resistance of spores to many commonly used disinfection regimens (19). Spores of C. difficile can be induced to germinate at a higher frequency when cultured in media supplemented with bile salts that are usually found in the intestine, which also greatly improves organism recovery from clinical samples (20).
Although C. difficile has been established as the leading identifiable cause of infectious AAD in the hospital setting, disease diagnosis remains a contentious issue (21, 22). CDI is defined as acute diarrhea accompanied by the presence of either toxigenic C. difficile or its toxins in stool samples (23). However, the lack of a clear distinction between specific clinical CDI and more general non-CDI symptoms in all patients prevents the accurate diagnosis of C. difficile disease. Current diagnostic methods for C. difficile disease include bacterial culture, enzyme immunoassays (EIAs), cell cytotoxicity neutralization assays (CCNAs), toxin detection tests, and nucleic acid amplification tests (NAATs).
Isolation of C. difficile from stool samples requires bacterial culture in an anaerobic environment on selective media such as cycloserine-cefoxitin fructose agar (CCFA) (24). The addition of sodium taurocholate and horse blood to CCFA results in enhanced spore germination and enables colony fluorescence, respectively, allowing for accurate identification (25, 26). However, nontoxigenic C. difficile strains also grow in these conditions, and culturing on CCFA can take up to 48 hours, delaying the reporting of results.
Toxigenic and nontoxigenic strains of C. difficile constitutively produce the cell wall-associated enzyme glutamate dehydrogenase (GDH), which is highly conserved across different ribotypes (27). A recent large-scale meta-analysis indicated that GDH EIAs are highly sensitive, accurate, and relatively cheap to conduct as screening tests, returning positive results for all strains of C. difficile, regardless of the presence or absence of toxin in stool samples (28). These assays are often utilized as initial screening tools for the presence of C. difficile because they are quicker and more sensitive than toxin EIAs (29). However, GDH EIAs are not recommended as the sole form of diagnosis (30, 31).
CCNAs have historically been viewed as the “gold standard” of CDI detection because they can detect the presence of TcdA and TcdB at picogram levels (31, 32). Cell lines such as Vero (African green monkey kidney epithelial cells) and HT29 (human colorectal adenocarcinoma cells) are cultured in the presence of fecal filtrates, with and without toxin-neutralizing antibodies, and observed for characteristic toxin-induced cell rounding over 24 to 48 hours (33, 34). Although the sensitivity of these assays is very high, CCNAs are more expensive than EIAs and bacterial culture, can take between 72 and 96 hours to complete, and require a high level of technical expertise to perform, making them impractical for routine diagnostic use or during CDI outbreaks (35).
NAATs detect the presence of C. difficile-specific nucleic acid sequences in fecal samples, primarily through PCR amplification of conserved regions within the genes that produce TcdA and TcdB (35). In recent years, NAATs have been commonly accepted as the most sensitive form of diagnosis relative to the other methods discussed above and are increasingly used in hospitals despite being more expensive than bacterial culture and GDH EIAs (36). However, NAATs are less specific for CDI compared to CCNAs, because they detect toxin genes instead of active toxin within fecal samples and thus cannot differentiate between asymptomatic carriage of C. difficile and CDI, potentially leading to overdiagnosis and overtreatment (36, 37).
Due to the differing degrees of sensitivity and specificity between these individual diagnostic tests, a multistep algorithm is likely to be the most effective strategy for accurate CDI diagnosis and case surveillance. Guidelines released in 2016 for Europe and the United Kingdom recommend the use of the more specific TcdA or TcdB EIAs to confirm CDI in patients that initially yield a positive result for the more sensitive NAATs or GDH EIAs and suggest the consideration of other factors such as the onset of disease symptoms and progression prior to diagnosis and subsequent treatment of CDI (38). Correlating disease symptoms with positive molecular tests could prove vital for accurate CDI diagnosis, because asymptomatic C. difficile colonization in otherwise healthy individuals has been reported to be between 4 and 15% via PCR ribotyping and stool culture in geographically distinct regions (39–41). The use of such algorithms is proposed to provide a quicker and more accurate diagnosis of positive CDI cases and to decrease false-positive results. Importantly, reducing false-positive cases decreases the risk of susceptible patients being physically grouped together with true CDI patients in hospitals, thereby exposing them to C. difficile (36). This also prevents unnecessary antibiotic treatment for CDI, which may lead to the rise of multidrug-resistant organisms such as vancomycin-resistant enterococci (42). Overall, algorithm-based diagnoses provide a more accurate representation of C. difficile-induced diarrhea, although the composition of each algorithm may vary between hospitals (22).
Strain Typing
Typing of C. difficile isolates is essential for identifying the strains responsible for CDI outbreaks and for monitoring the spread and emergence of new strain types. Several typing schemes have been developed for C. difficile, utilizing the molecular and serological differences that exist between strains (43, 44). Although several schemes have been developed, in recent years molecular techniques, specifically PCR-based methodologies, have been favored due to their reproducibility and relative low cost (44).
With respect to the recent epidemic ribotype 027 strains, three molecular techniques have primarily been used in their typing (44). The first technique, restriction endonuclease analysis (REA), identifies ribotype 027 strains as the REA type BI (44). In this typing scheme, whole genomic DNA extracts are fragmented by the restriction enzyme HindIII, followed by agarose gel electrophoresis to separate the resulting DNA fragments (45). The banding pattern obtained is then compared to that of a reference strain, and the strain under question is typed accordingly, utilizing the results obtained through other typing methods to further confirm the strain type (45). The second technique uses pulsed-field gel electrophoresis and results in the ribotype 027 strain belonging to the NAP1 (North American pulsed-field type 1) pulsotype (44). This technique works in a similar way to REA, but it uses the SmaI or SacII restriction enzymes to fragment whole genomic DNA, after which pulsed-field gel electrophoresis is performed (44). As with REA, analysis of the resulting banding pattern occurs through comparison to known reference strains, which is then used to classify each isolate (46). The third technique is ribotyping, which has become the typing method of choice in recent times because it is quick, sensitive, and reproducible, effectively distinguishing between different strain types (44, 47). Ribotyping relies on differences in the number of copies and overall length of the rRNA genes within the genome of C. difficile strains (47, 48). Due to these differences, amplification by PCR between the 16S and 23S rRNA intergenic spaces results in the production of distinct and characteristic banding or fingerprint patterns which are compared to those produced by reference strains to identify the ribotype to which each isolate belongs (48, 49). Over 400 ribotypes have been identified and, as with the other two typing schemes just described, comparative analysis between the typing schemes can be used to confirm each strain type (47–49).
In addition to the three major schemes used to type C. difficile strains, toxinotyping may be used to differentiate between strains (47). Toxinotyping assesses the genetic differences within a discrete 19.6-kb genomic region defined as the pathogenicity locus (PaLoc) (Fig. 1a), with a focus on the tcdA and tcdB genes, and a comparison is generally made to the PaLoc region of the reference strain VPI10463, which is classed as toxinotype 0 (50, 51). Toxinotypes range from I to XXXI, and new toxinotypes are frequently identified; some of these toxinotypes contain subsets which serve to subdivide toxinotypes with a small number of differences between them (52).
FIGURE 1.
Schematic representation of the C. difficile pathogenicity locus (PaLoc) and C. difficile transferase locus (CDTLoc) and flanking genes. (a) The PaLoc chromosomal region is responsible for both the production and regulation of the major toxins TcdA and TcdB (encoded by tcdA and tcdB), which are critical for pathogenesis, virulence, and the disease symptoms of CDI. PaLoc also encodes genes involved in the positive (tcdR) and negative (tcdC) regulation of toxin production, as well as a gene (tcdE) encoding a holin-like protein, TcdE, which may be involved in the release of toxins from the cell. Nontoxigenic strains lack this region and are consequently avirulent. (b) The CDTLoc chromosomal region is responsible for both the production and regulation of CDT/binary toxin (encoded by cdtA and cdtB). CDTLoc also encodes a gene involved in the positive regulation of CDT (cdtR). Modified from Nature Reviews Disease Primers (8).
Another technique, multilocus sequence typing, analyzes several C. difficile housekeeping and toxin genes to define strains based on their sequence type, which characterizes C. difficile strains into 6 distinct phylogenetic clades: clades 1 to 5 and clade C-I (53–58). Each clade represents a variety of both toxigenic and nontoxigenic C. difficile isolates, from many different ribotypes (53–58). Clade 1 is the most diverse of the 6 clades, with over 100 sequence types, including several ribotypes of clinical significance, such as ribotypes 001, 012, and 014, which are frequently associated with CDI throughout Europe (59) and throughout Asia and the United States (60, 61). Clade 2 includes the epidemic ribotype 027 strains associated with recent outbreaks and the related ribotype 244, which has a variant TcdB that shows cytopathic effects more similar to Paeniclostridium sordellii (previously Clostridium sordellii) TcsL, and is associated with more severe disease (62, 63). Analysis of clade 3 is less comprehensive, and it appears to be less diverse (62, 63). Clade 4 includes strains from ribotype 017, a TcdA–TcdB+CDT– variant strain that has caused significant disease throughout Asia, as well as being a predominant strain throughout Europe (64–68). Like the ribotype 244 TcdB variant, TcdB from ribotype 017 strains also has similarity to TcsL from P. sordellii, specifically in the enzymatic domain of the variant TcdB, which alters the cytopathic effects of the toxin (69). Clade 5 contains C. difficile strains from ribotype 078, a ribotype commonly associated with disease in animals, particularly livestock, including pigs and cows; however, in recent times ribotype 078 strains have also been associated with community-acquired cases of human CDI (59, 70, 71).
Clade C-I is a more significantly divergent clade than the other 5 clades and was originally thought to encompass several nontoxigenic strains; however, some recent variant toxigenic strains have been found to cluster with other clade C-I strains (56, 72). Recent work analyzing environmental C. difficile isolates has suggested a need for further classification of the clades, with the considerable divergence of clade C-I resulting in 3 distinct clades, C-I, C-II, and C-III (73). Strains belonging to clade C-I/II/III also show considerable divergence from the remaining 5 clades, which has brought into question if these strains are indeed C. difficile or if they represent a new species that is distinct from the other 5 clades (74).
Other typing schemes for C. difficile include multilocus variable-number tandem repeat analysis, whole-genome sequencing, and single nucleotide polymorphism analysis, all of which provide different genetic perspectives and degrees of sensitivity for strain typing (48). Use of the different typing schemes available for C. difficile has shown that a broad diversity of strains exists, and this is true for human clinical and animal isolates. In particular, whole-genome sequencing has provided new insights into the epidemiology and spread of C. difficile strains and has changed our understanding of the dissemination and transmission of this bacterium, allowing new and more effective infection control strategies to be developed (75).
EPIDEMIOLOGY
Over the last 2 decades the incidence of CDI has increased dramatically, correlating with the emergence of epidemic isolates of C. difficile in hospitals worldwide, in particular, ribotype 027 strains which have gained fluoroquinolone resistance (76). Nosocomial outbreaks of ribotype 027 C. difficile strains have undoubtedly contributed to the global increase in CDI rates (13, 77). In late 2002, an outbreak of C. difficile occurred in Quebec, Canada, which resulted in a dramatic increase in CDI rates (78). In Quebec, the overall incidence of CDI had increased nearly 4-fold from 1991 to 2003 (78). Furthermore, rates of infection in those above the age of 65 increased roughly 4-fold from 2002 to 2003 during the Quebec outbreak, which resulted in CDI rates increasing 8-fold from 1991 to 2003 in this age group (78). Complications from infection and mortality also increased during this time (78). The ribotype 027 strain responsible for the outbreak spread quickly, leading to several outbreaks across the United States, the United Kingdom, and Europe and highlighting the difficulty in containing CDIs and preventing the spread of disease (17, 77, 79). Interestingly, the increasing use of dietary trehalose has contributed to the dissemination of ribotype 027 strains because their ability to metabolize this sugar may be an additional factor contributing to the changing epidemiology of this strain type (80).
REA and pulsed-field gel electrophoresis analysis of isolates collected from clinical outbreaks across the United States from 2000 to 2003 highlighted the prevalence of BI/NAP1/027 strains, with an average of 51% of isolates classified as belonging to this class (13). Although the current incidence of ribotype 027 strains appears to be declining in the United States, indicated by surveillance of 10 of the Centers for Disease Control and Prevention (CDC) Emerging Infections Program sites in 2011, the prevalence of this strain type remains high (81). In this study, ribotype 027 strains accounted for 20.6% of community-associated CDI and 33.9% of hospital-acquired CDI in the United States, with ribotype 002, 020, and 106 strains also contributing considerably to CDI, albeit at lower rates of 10 to 10.7%, 10.3 to 11.4%, and 4.3 to 7.6%, respectively (81). Nevertheless, infection rates and mortality resulting from CDI still remain high in the United States. In 2013, the CDC estimated that C. difficile caused 250,000 infections per year and 14,000 deaths in the United States, but these numbers are likely underestimates (18). Estimates from CDC data in 2015 by Lessa et al. suggest that over 450,000 CDIs occurred in the United States, accounting for over 29,000 deaths annually (81). The disparity between the two estimates highlights an ever-growing need for more stringent detection of infections and subsequent analysis of CDI rates.
Similar to the United States, in the United Kingdom C. difficile disease rates were reported to be four times greater than those seen in the late 1990s in both England and Wales, reaching a peak between 2007 and 2010 and coinciding with outbreaks of ribotype 027 strains (82–84). From late 2003 to 2005, two severe and devastating outbreaks of C. difficile were documented in the Stoke Mandeville Hospital in England, in which a total of 334 cases of CDI were recorded, resulting in 38 fatalities (85). Strain R20291 was isolated during this outbreak and was identified as a ribotype 027 strain (86). These outbreaks were associated with several contributing factors, including insufficient isolation of infected individuals and poor implementation of infection control measures and practices by staff, which would have minimized and prevented C. difficile spread and contamination (85). In the years following this outbreak, rates of CDI appeared to be declining in the United Kingdom, from 164.8 deaths per million people in 2008 to 34.3 deaths per million people in 2015, as a result of increased awareness and infection control management; however, infections and mortalities resulting from CDI still occur at high rates in the United Kingdom (82).
Several other European countries, including France, Germany, and the Netherlands, have experienced similar recent outbreaks of C. difficile, with ribotype 027 strains found to be prevalent during these outbreaks (87, 88). Although ribotype 027 isolates are still highly prevalent throughout Europe, accounting for 19% of the reported CDIs, a wide diversity of C. difficile ribotypes also contribute to these infections, highlighting the diverse global epidemiology of C. difficile (89). The diversity of C. difficile throughout Asia is dramatically different and is dominated by a high number of variant C. difficile strains, in particular, ribotype 017 C. difficile, a TcdA–TcdB+CDT– variant strain which, for example, comprises up to 40% of all strains isolated in Korea and China (65, 67, 68, 90).
Although C. difficile is an important nosocomial pathogen, it can also cause community-acquired infections, which occur without recent exposure to C. difficile during hospitalization or during a hospital stay. Interestingly, community-acquired CDIs often occur without the presence of risk factors commonly associated with nosocomial CDI, such as advanced age or prior antibiotic use (8, 70). Furthermore, community-acquired CDI rates appear to be on the rise, accounting for 20 to 27% of all CDI cases, with an increasing number of cases detected in the United States and Europe (70). Although ribotype 027 strains do contribute to community-acquired CDI, other isolates, including ribotype 078 strains which are often isolated from pigs and cows, also contribute to community-acquired CDI in high numbers (59, 70, 71). This may be linked to the shedding of spores within feces of livestock and domesticated animals, which contributes to the transmission and spread of disease from animal to animal (91) and may also aid in potential zoonotic transmission from animals to humans (92). This potential zoonotic transmission probably occurs via the ingestion of spore-contaminated meat products derived from C. difficile-infected livestock (92). In the United States, C. difficile spores have been detected in up to 42.4% and 41.3% of tested beef and pork products, respectively, with ribotype 078 and 027 strains being isolated in high frequencies from these samples. Similar C. difficile spore contamination of meat products occurs in Canada, albeit at lower levels, with levels of detection ranging from 6.1 to 20% for beef and 1.8 to 12.2% for pork products (93). Detection of C. difficile spores in meat products throughout Europe is considerably lower, with less than 5% of tested samples yielding a positive C. difficile culture (93). Nevertheless, detection of C. difficile spores within retail meat products has the potential to aid in transmission from animal to human host and may account for the emergence of typically animal-associated C. difficile strains in humans (70, 71).
PATHOGENESIS
Susceptibility to CDI
Following the ingestion of C. difficile spores, infection may be established within the host upon spore germination and vegetative cell colonization of the colon (94). However, susceptibility to colonization and disease is generally not seen in healthy individuals who have an established and diverse gastrointestinal microbiota, which provides a physical barrier to colonization and contributes to the induction and maintenance of a protective immune response to invading pathogens (95–97). The commensal microbiota provides colonization resistance by occupying and interacting with the colonic niche but becomes disrupted in individuals undergoing antibiotic treatment (98).
A number of factors may contribute to reduced colonization resistance in antibiotic-treated hosts. Disruption to the microbiota may change the nutrient profile within the gastrointestinal tract, which may create a niche that is more suitable for C. difficile (99). Furthermore, with reduced microbiota-mediated priming of the innate immune system following antibiotic disruption, recognition of invading pathogens such as C. difficile may be compromised, thereby increasing host susceptibility to opportunistic infection (98). Under normal conditions when the microbiota is intact, immune responses occur within the host which promote the production of several gastric and immune molecules, including mucus/mucin and antimicrobial peptides such as defensins and immunoglobulin A (IgA), which help to prevent gut colonization by pathogens (98). Although significant disruption of the host microbiota may occur following multiple doses of antibiotics, a single dose is sufficient to “shift” the microbiota and promote CDI (98, 100). For example, depletion of the microbiota can change the cleavage of sugar substrates within the gut, resulting in higher levels of sialic acid and succinate, promoting the growth of C. difficile (99). Antibiotic resistance of C. difficile strains exacerbates this problem, as has been seen with fluoroquinolone resistance in the epidemic ribotype 027 strains following the overuse of this group of antibiotics (101).
Colonization and infection with C. difficile occurs in people from a range of age groups, but increased risk of infection, infection rates, and disease severity occurs in elderly and in immune-compromised patients (23, 76, 102). C. difficile often presents as a nosocomial infection and is the leading cause of infectious AAD, accounting for about 25% of all cases (21). More recently, substantial increases in CDI rates have been recorded, with the persistence of C. difficile in hospitals and spore-mediated transmission likely to play a fundamental role in this phenomenon (13, 76). In addition to this, rates of community-acquired CDI are also increasing and often occur in patients that are not typically at risk of infection such as children, pregnant women, and individuals with no recent antibiotic use (8, 70).
Disease Associated with CDI
CDI is associated with a variety of disease symptoms, ranging from asymptomatic carriage of C. difficile to severe and life-threatening disease complications (2). The spectrum of disease that is observed in CDI patients begins as mild diarrhea and may increase in severity with the progression of infection. Mild diarrhea associated with CDI often resolves following the cessation of antibiotic therapy; however, it may progress to severe diarrhea, leading to dehydration and mild inflammation of the colon with the possible development of fever (103). The progression of disease in severe cases of CDI may lead to the development of pseudomembranous colitis, with toxin-mediated damage resulting in the formation of plaques throughout the colon, consisting of cellular debris, fibrin, and neutrophils (104). Patients may also present with fever, abdominal pain, and elevated white blood cell counts (104).
A rare yet significant complication of severe disease is toxic megacolon, which results in detrimental inflammation and swelling of the colon, colonic perforation, sepsis, and in many individuals, death (105). Disease complications may be compounded by the onset of multiple organ dysfunction syndrome, promoting patient fatality (106). This syndrome can be defined as “the development of potentially reversible physiologic derangement involving two or more organ systems not involved in the disorder that resulted in ICU admission,” which includes damage to the lungs, kidneys, heart, liver, or brain (107). Extraintestinal infections may occur following CDI, presenting as abdominal abscess and wounds, ascites, bacteremia, brain abscess, catheter infection, perianal abscess, peritonitis, and toxemia (106, 108–110). Recent work by Carter et al. (111) has shown that damage and architectural disorganization occurs in the kidneys, lymph nodes, spleen, and thymus during murine CDI. Toxemia, or escape of toxins into the blood, in C. difficile-infected piglets has also been shown to result in systemic pathologies of the lungs, which seemingly occurs via TcdB-mediated damage of the colonic epithelium, because neutralizing antibodies to TcdB, but not TcdA, were able to block colonic damage and systemic pathology progression (112). Although this level of disease severity is considered rare in humans, the increasing incidence of CDI and the more severe disease symptoms associated with ribotype 027 strain infections has resulted in more cases involving life-threatening complications (105).
Disease in C. difficile-infected animals shares many of the characteristics seen in human disease. One of the most well-characterized CDI-associated diseases in animals is C. difficile-associated enteritis in piglets (113). As in humans, CDI often leads to diarrhea in piglets, which may be yellow and pus-like, and may progress into severe life-threatening disease (113). As a result of these disease symptoms, weight-loss, difficulties digesting food, dehydration, and weakness often occur, which over time may result in piglet death (114). On examination of the colon tissue, swelling and edema of the epithelium may also be observed, with colonic dilation and hemorrhage also common (114). As seen in humans, neutrophil infiltration and pseudomembranous formations throughout the colon are common, particularly in severe enteritis (114). Disease in animals is not restricted to pigs, with infection and disease occurring in other animals including horses, cows, and domesticated dogs, as well as exotic and wild animals including penguins and elephants (115–117).
Animal Models of CDI
Studies of C. difficile and CDI have used specific animal models to provide key insights into the role played by virulence factors in disease pathogenesis (33, 118, 119). The hamster model of C. difficile-associated disease was used extensively in early research because of the sensitivity of hamsters to the C. difficile toxins, with infections almost always resulting in hamster death (120). Most other animals (including humans) are not as sensitive to the effects of the toxins (120); thus, the extreme sensitivity of hamsters to the C. difficile toxins has prevented the more subtle aspects of infection and disease from being teased out. The use of hamsters in infection studies is also limiting because of a lack of reagents such as specific antibodies, or hamster genetic variant strains, making more detailed research using this animal model difficult (120).
The more recent development of a murine model of CDI by Chen et al. in 2008 (120), which mimics C. difficile susceptibility and disease in humans, has overcome many of the limitations associated with the hamster model of CDI (121). The murine CDI model provides the opportunity to analyze C. difficile disease from a new and more detailed perspective (120). In the model by Chen et al. mice are pretreated with an antibiotic cocktail prior to infection challenge and, following infection, animals develop many of the characteristic disease pathologies associated with clinical CDI, including diarrhea and weight loss (120). Infection also induces severe colonic pathology, with epithelial damage, submucosal inflammation, and neutrophil influx seen, similar to human disease, further supporting the utility of this model in developing a better understanding of C. difficile disease (120). C. difficile-infected mice also succumb to disease quickly and exhibit observable signs of disease 2 to 3 days postinfection. This model of CDI and the availability of reagents and genetically manipulated mice has facilitated recent studies focused on defining the contribution of key immune pathways to C. difficile disease, which are described in more detail below, providing important new insights into the progression of CDI and the host response (120, 122–125).
Following the development of the Chen et al. model, additional murine models of CDI were developed which utilize different pretreatment regimes and result in different disease pathology and outcomes (126). In 2009, Lawley et al. developed a less severe CDI model through disruption of the intestinal microbiota via oral administration of clindamycin for four consecutive days prior to infection (122). Infection in these mice was established following exposure to “carrier state” mice, which were induced to carry C. difficile in their microbiota through the administration of neomycin 24 hours prior to challenge. Mice infected with C. difficile in this model show some weight loss, diarrhea, and damage to the intestinal epithelium, albeit at lower levels than in the Chen et al. model (122). In 2012, Buffie et al. showed that a single dosage of clindamycin by intraperitoneal injection was sufficient to induce CDI with disease pathology and outcome similar to the other models (127). The third murine CDI model, developed by Theriot et al. in 2011, induces C. difficile susceptibility through the oral administration of cefoperazone for 10 days, 12 days prior to infection (128). Dependent on the C. difficile strain and dosage used for infection, mice developed severe disease within 2 days of infection, characterized by weight loss, diarrhea, and damage/inflammation of the intestinal epithelium (128). More recently, the oral administration of either metronidazole or streptomycin to mice has been shown to induce susceptibility to CDI, allowing for high levels of C. difficile colonization (129). Gnotobiotic mice, which are void of colonic microbiota, have also been used to model CDI, presenting similar disease profiles to the aforementioned models; however, since a complete lack of colonic microbiota is unlikely in human CDI, this model does not accurately represent human CDI (130). More recently, a mouse model of CDI has been established in mice with humanized microbiota, allowing for the analysis of disease under conditions more similar to human CDI (131). Together, these different murine models of CDI have provided valuable insights into CDI and disease progression under different circumstances and conditions.
Other C. difficile disease models include the porcine model, which emulates human disease symptoms and severity and which has provided new insights into the understanding of C. difficile disease (114). Through the infection of gnotobiotic piglets with C. difficile spores, Steele et al. (114) were able to replicate natural C. difficile disease, with infected pigs developing diarrhea 48 hours after infection. Severe colonic pathologies and lesions were also observed, leading to the formation of pseudomembranes in some animals, much like those seen during severe CDI in humans. With these similarities to human disease, this model provides a valuable resource for the development of our understanding of CDI and provides an appropriate model for the testing of potential therapeutics and treatments for CDI (114).
C. difficile Toxins
TcdA and TcdB
Several C. difficile virulence factors have been identified, the most important of which are TcdA and TcdB (33, 111, 118, 132). Typically, the 19.6-kb PaLoc encodes both the tcdA and tcdB genes, producing TcdA (308 kD) and TcdB (270 kD), respectively, and also encodes genes involved in the positive (tcdR) and negative (tcdC) regulation of toxin production (Fig. 1a) (133, 134). TcdR is an alternative RNA polymerase sigma factor, which shares similarity to the alternative sigma factors BotR in Clostridium botulinum (135), TetR in Clostridium tetani (136), TpeR and UviA in Clostridium perfringens (137–139), and TcsR in P. sordellii (140). TcdR positively regulates the transcription of tcdA, tcdB, and tcdR; however, this process is influenced by various environmental stimuli, including changes in the metabolism of amino acids via CodY (141) and carbon sources such as glucose via CcpA (142). Other regulatory proteins involved in aspects of the C. difficile life cycle, including Spo0A, a master regulator of sporulation, and SigD, a regulator of flagella, also regulate toxin transcription (143, 144). TcdC is an anti-sigma factor, which is thought to negatively regulate toxin transcription by disrupting the function of TcdR, but this occurs with considerable variability across different strain backgrounds (145–147). PaLoc also carries a gene (tcdE) encoding a holin-like protein, TcdE, which may be involved in the release of toxins from the cell, but this hypothesis is contentious (148–150). Variants of the C. difficile PaLoc exist, which contain mutations or deletions within the locus, altering the toxin profile of these strains (72). More recently, the PaLoc has been found in alternate genomic locations in specific strains, although such strains do not appear to be common (72).
TcdA and TcdB are members of the large clostridial glucosylating toxin family, members of which are also found in several other pathogenic clostridia (132). These include the hemorrhagic (TcsH) and lethal (TcsL) toxins produced by P. sordellii (151), α-toxin (TcnA) produced by Clostridium novyi (152), and TpeL produced by C. perfringens (153), all of which function in a similar way to the C. difficile toxins and inactivate Rho, Rac, or Ras small GTPases. These small GTPases play a vital role in maintaining cellular architecture and are involved in the maintenance of the actin cytoskeleton, cellular migration, and division (154). Inactivation of these small GTPases via the covalent transfer of a glucose moiety by one of the large clostridial toxins, or an acetyl-glucosamine moiety only by TcnA and TpeL, leads to the disruption of their normal function and consequently results in cell rounding and cell death (152, 153, 155, 156).
TcdA and TcdB, which have 63% amino acid similarity, share an overall conserved structure (Fig. 2), with four functional domains that are involved in cellular binding, entry, and enzymatic activity of the toxins (157–159). The C-terminal ends of TcdA and TcdB, consisting of the combined repetitive oligo-peptides (CROPS) domain, are thought to facilitate receptor binding for each toxin (157); however, toxins which lack this domain can still induce cell rounding in tissue culture, suggesting that there are CROPS-dependent and -independent receptor binding regions within each toxin (160–165). CROPS-mediated receptor binding has been shown to increase TcdA-mediated cytotoxicity compared to a CROPS deletion mutant (165), while recombinant C-terminal-TcdA (REP321) blocks cytotoxicity in F9 cells when used to saturate the cell surface prior to intoxication (166). Immunization of mice with REP231 also protected mice against subcutaneous lethal dosing with TcdA (166), highlighting the importance of the CROPS in receptor binding and cell entry. Several receptors have been proposed for TcdA, including the α-Gal-(1,3)-β-Gal-(1,4)-β-GlcNAcO(CH2)8CO2CH3 carbohydrate moiety (167) and glycoprotein 96 (gp96) (160) on human cells, but the exact mechanism of binding still remains unclear. Three potential host receptors have been identified for TcdB: poliovirus receptor-like protein 3 (162), chondroitin sulfate proteoglycan 4 (161), and members of the frizzled family receptors (163). Binding to these receptors occurs in a CROPS-independent manner at regions adjacent to the CROPS domain; however, further in vivo characterization of the role for these receptors in CDI is required (161–164). Thus, it appears that receptor-mediated binding of TcdA and TcdB is a complex process involving multiple binding regions within the C-terminal portion of each toxin, which interact with an array of receptors that are not yet fully characterized.
FIGURE 2.
Schematic representation of C. difficile TcdA and TcdB. (a) Comparison of TcdA and TcdB functional domains. Comparison of the four functional domains of the toxins, including the glucosyltransferase domain (GTD) (red), auto-protease domain (APD) (blue), delivery domain (yellow), and combined repetitive oligo-peptides (CROPS) domain (gray). (b) The crystal structure of the TcdA GTD (red), APD (blue), and delivery domains (yellow) in the absence of the binding domain. (c) Three-dimensional structure of TcdA, superimposed with the crystal structure of TcdA. Reprinted from Annual Review of Microbiology (254).
Following receptor binding, both TcdA and TcdB enter the cell via clathrin-mediated endocytosis, with acidification of the endosome resulting in a conformational change in the pore-forming domain of the toxin, allowing insertion of the toxin into the membrane and facilitating toxin entry into the cytosol (Fig. 3) (157, 168). Once translocated across the endosomal membrane, the glucosyltransferase domain (GTD) of each toxin is cleaved off by the auto-protease domain, following activation of this domain by host inositol hexakisphosphate (InsP6), which releases the enzymatic GTD into the cytosol (169, 170). Once in the cytosol, the GTD then inactivates host cell Rho family GTPases. A conserved tryptophan residue at amino acid position 102 and a DXD motif at position 286 to 288 are located in the GTD of TcdA and TcdB and are essential for binding to UDP-glucose and for the catalytic function of the monoglucosyltransferase domain (171). Inactivation of Rho family GTPases induces cell rounding and death (172). These outcomes occur because of a lack of regulation in actin polymerization within the intoxicated cell, resulting in the disruption of a number of essential host cell features, including the actin cytoskeleton, stress fibers, and tight junctions (173). TcdB has also been shown to promote the formation of NADPH oxidase complexes, releasing reactive oxygen and leading to necrotic cell death (174). TcdB from ribotype 027 strains appears dissimilar to historical TcdB in some aspects, with variations in the CROPS domain (175). The differences between the binding domains of ribotype 027 TcdB and historical TcdB are thought to provide an increase in tissue tropism, because ribotype 027 TcdB induced more severe and extensive pathologies when used to treat embryonic zebra fish (175). Specifically, ribotype 027 TcdB displayed increased cardiotoxicity, resulting in severe cardiac damage and necrosis after 24 hours compared to historical TcdB in this model, which induced swelling and bleeding of the cardiac tissue at a slower rate (175).
FIGURE 3.
Binding and entry of C. difficile toxins into colonic epithelial cells. TcdA and TcdB bind to the cell surface via interaction of the binding region and one or more cell surface receptors, including the α-Gal-(1,3)-β-Gal-(1,4)-β-GlcNAcO(CH2)8CO2CH3 carbohydrate moiety and glycoprotein 96 (gp96) or surface receptors including frizzled (FZD), chondroitin sulfate proteoglycan 4 (CSPG4), and poliovirus-like receptor 3 (PVLR3) respectively, resulting in receptor-mediated endocytosis. Binding can occur via CROPS (green)-dependent and -independent mechanisms. Once internalized, the endosome acidifies, resulting in a conformational change and membrane insertion of the delivery domain and translocation of the auto-protease domain (APD; gray) and glucosyltransferase (GTD; red) into the cytosol. The APD then interacts with host inositol hexakisphosphate (InsP6; blue), cleaving the GTD, which can interact with the Rho family of GTPases. Reprinted from Annual Review of Microbiology (254).
Recent work by Chumbler et al. (176) has also shown that TcdA and TcdB induce cell damage in distinct ways, with TcdA inducing apoptosis via glucosyltransferase activity and TcdB via two modes of action that are dependent on toxin concentration. At low concentrations, TcdB induced apoptosis in young adult mouse colonic cells, in a similar way to TcdA (176). Unlike TcdA, which only induced apoptosis, high concentrations of TcdB (≥100 pM) induced necrosis, which, from analysis using an enzymatically inactive TcdB, occurs independently of glucosyltransferase activity (176). Despite these insights, the exact mechanism for the vastly different roles of TcdA and TcdB in disease still remains unclear. Notably, TcdB has a substantially smaller CROPS domain than TcdA, which may confer a largely different three-dimensional structure, as depicted in modeling of the CROPS region from smaller crystalized fragments (Fig. 4) (157, 177). The difference in physical size between these domains may account for some of the disparity in disease associated with the two toxins, either altering the binding of each toxin to its receptors or resulting in steric hindrance differences which may influence functional capacity within the complex gastrointestinal compartment.
FIGURE 4.
Comparison of the TcdA and TcdB combined repetitive oligopeptides (CROPS) domains. Three-dimensional models of the TcdA and TcdB CROPS domains based on the crystal structure of a 127-amino acid fragment from the C-terminal region of TcdA characterized in reference 177. Long repeats are in green; short repeats are in blue. N, N terminus; C, C terminus. Modified from Frontiers in Cellular and Infection Microbiology (157).
The role of TcdA and TcdB in the development and progression of C. difficile disease has been contentious. Early research in this area, involving the intragastric administration of TcdA and TcdB to hamsters, suggested that TcdA was the major virulence factor in the development of severe C. difficile disease, with TcdB thought to be unable to induce disease in the absence of TcdA despite being a potent cytotoxin in cell-based in vitro assays (119). In hamsters, toxin coadministration, using sublethal doses of TcdA together with TcdB, was able to induce severe disease symptoms, indicating possible synergy between TcdA and TcdB (119). Similar results were seen in a rabbit ileal loop model, where exposure to TcdA but not TcdB was able to induce intestinal hemorrhage, fluid secretion, and inflammation (4). However, the isolation of clinical C. difficile variant strains that encoded only TcdB suggested that, at least in humans, TcdB alone is sufficient to induce disease, raising questions about the clinical relevance of previous studies using purified toxins (178, 179). Early studies did not assess the role of the toxins in the context of infection but instead used purified toxins and the intragastric administration of the toxins to animals to a site that is not where colonization and infection occur. A true representation of CDI and disease was therefore not achieved in these studies.
More recently, multiple laboratories have used various CDI animal models together with genetically engineered isogenic C. difficile toxin mutants to elucidate the role of TcdA and TcdB in disease. Each of these studies, which utilized hamsters and mice together with historical or epidemic C. difficile isolates and their isogenic toxin gene mutants, showed that TcdB induced severe disease in both the absence and presence of TcdA, highlighting the important role that TcdB plays in disease (33, 111, 118, 180). Although these studies have redefined TcdB as a key mediator of C. difficile disease, the role of TcdA in disease remains somewhat unclear. In two of the studies, TcdA was shown to have a minor role in disease, with hamsters or mice infected with TcdA-producing (TcdA+B–) strains not progressing to severe diarrhea or mortality and with mild pathology and damage seen in mouse colonic tissue (111, 118). In the other two studies, TcdA appeared to have a more intermediate role in disease, with TcdA+B– C. difficile strains inducing some disease or mortality but at a slower rate in comparison to the wild-type (WT) and TcdA–B+ strains (33, 180). Nevertheless, a key finding from these studies was that TcdB plays a key role in disease, which is supported by the high number of clinical TcdA–B+ C. difficile strains that are isolated from patients with CDI, which currently comprise between 0.2 to 40% of all strains isolated, dependent on geographical location (64–68, 90). The recent emergence of a single TcdA+B– C. difficile isolate is interesting since historically no such strains have been isolated; the clinical importance of this strain type remains to be determined, but future epidemiological tracking will be important to determine if such a strain type disseminates further (72).
C. difficile transferase
Some C. difficile strains, including ribotype 027 strains, produce a third toxin known as binary toxin or C. difficile transferase (CDT) (77, 181). CDT is an ADP-ribosyltransferase encoded by a 6.2-kb chromosomal locus, defined as the CdtLoc, which is independent of the PaLoc region (Fig. 1b) (77, 182, 183). CDT shares similarity with other clostridial binary toxins including C. botulinum C2 toxin, C. perfringens iota toxin and the Clostridium spiroforme CST toxin (184, 185). The locus encodes the cdtA and cdtB genes, which produce the enzymatic (CDTa) and binding (CDTb) components of the toxin, respectively. Similar to other clostridial binary toxins, CDTb is proteolytically activated prior to oligomerization into a predicted heptamer, which may occur prior to attachment to the cell surface via lipolysis-stimulated lipoprotein receptor or upon monomer binding (185). Once oligomerized, CDTa may bind, allowing for entry into the cell via endocytosis (185). Upon endosome acidification, CDTa is translocated into the cytosol, where it ADP-ribosylates actin, disrupting the cell cytoskeleton and inducing microtubule protrusions in intoxicated cells (181).
CDTLoc also encodes cdtR, producing CdtR, an orphan LytTR family response regulator responsible for regulation of CDT production (Fig. 1b) (183, 186). Interestingly, CdtR has recently been shown to also regulate the production of TcdA and TcdB in some strains of C. difficile, because insertional inactivation of cdtR resulted in reduced levels of TcdA/TcdB production and significantly reduced virulence in a mouse model of CDI (187). Although CdtR appears to regulate toxin production and virulence in some CDT-producing strains, the exact role of CDT in disease remains unclear.
Overall, CDT was found to be present in approximately 5% of historical isolates, but the prevalence of this toxin in C. difficile has steadily increased over the last 2 decades, with CDT detected in clinical strains at levels ranging from 17 to 45% (185). CDT was found to be highly prevalent in animal strains and is encoded by 23 to 100% of such isolates (92, 183). Importantly, CDT is also now found in approximately one-third of human clinical isolates (187, 188). CDT alone was thought to be unable to induce disease since clinical isolates producing only CDT, but not TcdA or TcdB, were unable to induce diarrhea or death in hamsters (189). However, the isolation of clinical strains that only produce CDT has raised questions about the role of this toxin in disease.
Since infection with strains producing only CDT and not TcdA or TcdB appears able to induce diarrhea in some patients, the role of this toxin in disease is still unclear (190). It may be that CDT acts synergistically with TcdA and TcdB to promote colonization and induce severe disease (181, 189, 191). Work by Schwan et al. (181) suggests that CDT may act as a colonization factor by inducing the formation of microtubule structures at the host cell surface, aiding in the adherence of the bacterium to the host cell. CDT also appears to increase the virulence of a TcdB mutant of strain R20291 in a hamster model of disease, but the mechanism by which this occurs is unknown (180). Recent work by Cowardin et al. (191) has also shown that CDT can enhance virulence to some degree, with CDTa mutants of two C. difficile isolates and a single CDTb mutant inducing less severe disease than the WT strains in a mouse model of CDI. This difference in disease profile associated with the CDT mutants was linked with increased levels of eosinophils in the colons of mice infected with the CDTb mutant of strain R20291 and suggests that eosinophils may play a protective role in CDI (191). Thus, it appears that CDT may play a synergistic role with TcdA and TcdB to enhance virulence by decreasing eosinophil recruitment. Analysis of eosinophil involvement in disease progression in naturally occurring CDT-negative strains has yet to be investigated, and may provide further insights into this phenomenon. With the prevalence of CDT-positive animal isolates and the increasing emergence of these CDT-positive strains in humans (185), the enhanced virulence associated with CDT could have severe consequences on disease profiles and severity.
Immune Response to C. difficile
Our current understanding of the role of the host immune system during CDI is limited, particularly with respect to the cytokine response and the changes that occur to immune cell populations in gut tissues during infection in response to each toxin. Clinical observations show that humoral immunity is induced during CDI, specifically, increased levels of IgG and IgM are detected in response to infection with strains producing TcdA and TcdB and in response to nontoxin antigens (192, 193). An additional hallmark of CDI seen in humans, mice, and pigs is an abundant influx of polymorphonuclear leukocytes into infected gut tissues (114, 120).
Cytokine and Chemokine Responses to Toxins
The immune response to C. difficile and its toxins has been previously investigated, but most of this work was performed through the application of purified toxins or C. difficile antigens to cell lines or rabbit and mouse ileal loop models (194–200). In several studies, purified TcdA was shown to induce the release of the key neutrophil and monocyte chemoattractants interleukin 8 (IL-8) and monocyte-chemoattractant protein 1 from HT-29 cells, a human colonic adenocarcinoma cell line (195, 201, 202), and T84 cells, a human intestinal epithelial cell line (197, 201). TcdA also induced tumor necrosis factor α (TNF-α) release in murine and human monocytes (198, 203), and TcdA stimulation of human monocytes induced IL-1 and IL-6 production (203). Purified TcdA has also been shown to induce the synthesis and release of IL-8 in a human xenograft ileal loop intoxication model (199). Exposure of tissues in a mouse ileal loop intoxication model to purified TcdA also promotes the transcription of proinflammatory cytokines and chemokines, including interferon-γ, macrophage inflammatory protein 1α (MIP-1α), MIP-2, and TNF-α (200), and in a rat ileal loop intoxication model, MIP-2, within 2 to 6 hours of toxin exposure (194). When injected directly into the cecum, TcdA also alters the expression of several chemokines involved in neutrophil chemotaxis, including chemokine (C-X-C motif) ligand 1 (CXCL1), IL-1β, and IL-6 (204, 205). Taken together, these data suggest that the production of TcdA during infection will alter the cytokine milieu to induce a highly inflammatory environment, most likely contributing to the inflammatory pathology observed during CDI. However, infection studies of the immune response that occurs with strains that only produce TcdA have not been performed.
Less is known about the immunomodulatory role of TcdB compared to knowledge of this role for TcdA. TcdB can induce IL-8 release in a human xenograft ileal loop model (199), IL-1β release in a mouse ileal loop model (196), TNF-α in rat macrophages (206), and TNF-α, IL-1, and IL-6 production in human macrophages (203). TcdB has also been shown to induce IL-1β via the pyrin inflammasome in mouse bone marrow-derived macrophages (207). As for TcdA, infection studies on the immune response that occurs with TcdB-producing strains in the absence of the other toxins have not been performed.
Cytokine and Chemokine Responses to Nontoxin Antigens
Nontoxin antigens of C. difficile have also been investigated for their role in the induction of the host immune response. A recent investigation into the interaction between nontoxin C. difficile antigens, including surface layer proteins (SLPs), flagella, and heat shock proteins 42 and 60, isolated from several C. difficile isolates, and the human macrophage cell line THP-1 showed that a number of proinflammatory cytokines were induced (208). These nontoxin antigens were isolated from a diverse group of strains belonging to ribotypes 012, 087, 027, 011, and 106, and antigens from all isolates promoted IL-1β, IL-6, IL-8, IL-10, IL-12p70, and TNF-α synthesis in THP-1 cells, to similar levels (208). However, a comparison to an untreated control was not included in this study (208); these results therefore require validation through additional research. SLPs isolated from C. difficile strains have also been shown to induce the release of several cytokines, including IL-1β, IL-6, IL-10, IL-12, IL-23, monocyte-chemoattractant protein, MIP-2, and TNF-α (125, 209–211). C. difficile-mediated cytokine induction during infection is not the result of toxin action alone and results from stimulation of the host immune response to numerous C. difficile factors. Further investigation of these effects is clearly needed.
Immune Response During CDI
The role of myeloid differentiation factor 88 (MyD88), an adaptor molecule critical for Toll-like receptor (TLR) signaling, in CDI was assessed using MyD88–/– mice (122). When compared to WT (MyD88+/+) mice, MyD88–/– mice infected with C. difficile strain M68, a TcdA- and TcdB-producing virulent human isolate, developed more severe disease with pronounced weight loss and a moribund appearance, together with edema and epithelial hyperplasia detected upon microscopic examination of cecal tissues. Furthermore, the survival rate of MyD88–/– infected mice was significantly less than WT infected mice, with 48% of the MyD88–/– infected mice not surviving the infection, compared to 100% survival in the WT mice. (122). Considering the critical role that MyD88 plays in the downstream signaling of several TLRs and nuclear factor-κB activation, it is not surprising that infected MyD88–/– mice developed more severe disease pathology (212). This important study confirmed the critical role that the innate immune system plays in CDI, providing an appropriate platform on which to base other studies of the immune response to CDI.
Building on the MyD88 studies, recent investigations have explored the mechanism by which MyD88 deficiency increased disease severity and mortality during CDI (213). A detailed analysis of cecal and colonic histopathology of WT (MyD88+/+) and MyD88–/– mice following C. difficile challenge was performed, which showed a significant reduction in neutrophil and monocyte infiltration into the colonic lamina propria of MyD88–/– mice (213). Furthermore, MyD88–/– mice had impaired CXCL1 transcription, which is an important observation since CXCL1 is a mouse homologue of the human chemokine IL-8, which is responsible for neutrophil trafficking (213). To study the role of neutrophils in CDI, neutrophils were depleted from mice by injection of a neutrophil-specific, Ly6G monoclonal antibody 1A8 (213). Mice depleted of neutrophils showed a 70% mortality rate following C. difficile challenge compared to 25% mortality in mice with intact neutrophils (213). By contrast, little mortality was observed in C-C chemokine receptor type 2-deficient mice (CCR2–/–, which are deficient in monocyte chemotaxis) infected with C. difficile, suggesting that CCR2 and, in turn, monocyte recruitment, are not important for protection from fulminant C. difficile disease progression (213). Collectively, these results suggest that neutrophils, and neutrophil chemotaxis mediated by CXCL1, play an important role in preventing severe CDI (213, 214). The increased disease severity and mortality associated with neutrophil depletion during CDI was speculated to be linked with the translocation of microbiota to extraintestinal sites. Depletion of neutrophils and monocytes via monoclonal antibody blockage of their receptor, Gr-1, resulted in substantial increases in microbiota translocation to the mesenteric lymph nodes of C. difficile-infected mice compared to untreated infected mice (213). Together, these data suggested that neutrophil, and to a lesser extent, monocyte, recruitment to the colon plays a crucial role in preventing excessive microbial dissemination, thereby reducing the broader host effects of CDI and highlighting the complex nature of this infectious disease (213).
An earlier study also showed a link between reduced neutrophil infiltration and the dissemination of the commensal microbiota during severe CDI, under conditions in which the host was immunocompromised (124). In this study, the involvement of the intracellular pattern recognition receptor nucleotide-binding oligomerization domain 1 (NOD1) in C. difficile disease was assessed (124). NOD1 detects meso-diaminopimelic acid peptidoglycans common to Gram-negative bacteria, while a second receptor, NOD2, detects muramyl dipeptide, a component of Gram-positive and Gram-negative peptidoglycans (215). Strikingly, NOD1 is critical in protection from life-threatening CDI, with NOD1–/– mice succumbing to infection at a more rapid rate than WT mice (124). By contrast, NOD2–/– mice were not more susceptible to C. difficile disease despite the association between Gram-positive bacteria and NOD2 (124). Increased mortality in C. difficile-infected NOD1–/– mice was associated with reduced neutrophil trafficking to the intestine as well as reduced CXCL1 expression in comparison to infected WT (NOD1+/+) mice (124). The reduction in CXCL1 expression coincided with an increase in commensal microbiota contamination in the lungs, kidneys, mesenteric lymph nodes, and spleen of NOD1–/– mice, suggesting that neutrophilic infiltration of gut tissues plays an important role in preventing the dissemination of the commensal microbiota when C. difficile-mediated tissue damage occurs (124). The association between NOD1 and C. difficile defined in this study was unexpected since, at the time, Gram-positive bacteria were not believed to produce meso-diaminopimelic acid peptidoglycans. However, it now appears that Bacillus and many other clostridia, including C. difficile, do produce meso-diaminopimelic acid peptidoglycans, so this phenotype now appears not to be exclusive to Gram-negative bacteria and may provide an explanation for the link between NOD1 and CDI (124, 215, 216).
The association between severe C. difficile disease, reduced neutrophil infiltration, and commensal microbiota dissemination has also been shown in infection studies involving apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD) (ASC)–/– mice, which lack a key component of the inflammasome, an immune factor which is involved in CDI (123, 196). The inflammasome is an intracellular caspase-activating complex often consisting of the pattern recognition receptor NOD-like receptor family, pyrin domain-containing 1 (NLRP1) or NLRP3, the adaptor protein ASC, and caspase-1, which is responsible for the cleavage of pro-IL-1β and pro-IL-18 into their active forms (196, 217). ASC–/– mice infected with C. difficile display impaired neutrophil recruitment to the cecum and colon in addition to higher levels of commensal microbiota dissemination to the spleen, liver, and lungs upon development of fulminant disease compared to WT (ASC+/+) infected mice (123). These differences in pathology resulted in an increased mortality in ASC–/– mice, with 54% of infected mice succumbing to disease, compared to mortality in 3% of ASC+/+ infected mice (123). The reduction in neutrophil recruitment in this study was linked to a deficiency in CXCL1 production in ASC–/– mice following C. difficile challenge (123). Work performed prior to these studies had shown that the NLRP3 inflammasome was involved in TcdA- and TcdB-mediated IL-1β release in a mouse ileal loop intoxication model (196). It was therefore not surprising that ASC–/– mice infected with C. difficile had reduced IL-1β levels compared to WT (ASC+/+) infected mice, and it has been suggested that the reduction in CXCL1 expression in ASC–/– infected mice results from the disruption of an IL-1β-mediated positive feedback loop for CXCL1 release (123).
In addition to NOD1 and NLRP3, TLRs also appear to be important in the host response to CDI. TLR4 interacts with C. difficile SLPs and induces the release of several cytokines, including IL-23, IL-1β, and TNF-α (125). TLR4–/– mice develop severe C. difficile disease, with dramatic weight loss and increased mortality occurring following CDI, compared to WT (TLR4+/+) infected mice, which showed moderate weight loss and pathology (125). TLR5 also plays an important role in the prevention of severe CDI, with stimulation of TLR5 via exogenous flagellin, a TLR5 ligand, promoting increased survival following C. difficile challenge (218). More recently, infection of mice with an R20291 fliC mutant, which lacks flagella, resulted in less severe disease than WT (fliC+/+) CDI, characterized by less goblet cell loss and epithelial damage, and a 50% increase in survival (219). Furthermore, infection of TLR5–/– mice with WT R20291 resulted in little epithelial damage, further supporting the role of TLR5 in CDI and in the induction of the inflammatory response (219). This was confirmed on cytokine gene analysis, which showed that WT infection induced significant upregulation of CXCL1, IL-6, IL-1β, IL-22, CXCL10, and TNF-α expression compared to infection with the fliC mutant, which only induced a significant change in CXCL1 expression (219).
Several immune cells, including eosinophils (220), neutrophils (221), and innate lymphoid cells (222), have also been shown to play a key role in modulating disease severity during CDI. Reduced levels of eosinophil infiltration to the colon in mice (linked to reduced IL-25 signaling) during CDI were shown to promote severe disease, resulting in greater than 50% mortality, compared to mice pretreated with recombinant IL-25 (to induce eosinophil infiltration), which showed ∼20% mortality following infection (220). Neutrophil recruitment to the site of infection during CDI has been shown to have various effects on disease severity during CDI in different studies. Chronic neutrophil inflammation exacerbates disease by inducing nonspecific damage to the colon (221), but neutropenia patients have a higher incidence of severe and potentially fatal CDI (223). Furthermore, blocking neutrophil recruitment to the colon promotes systemic dissemination of the gut microbiota and increased mortality during CDI (213). The role of neutrophils in disease is therefore complex. With respect to other immune cell types, C. difficile-infection of Ragγc–/– mice, which lack innate lymphoid cells, results in ∼90% mortality, compared to an ∼30% survival rate in WT mice, suggesting that innate lymphoid cells play a critical role in protecting mice from severe C. difficile disease (222).
Recent clinical data have also provided insights into the human host response during CDI. Diarrheal specimens from CDI patients were compared to those collected from non-CDI AAD patients and healthy people and were found to have higher levels of complement component 5a, granulocyte-colony stimulating factor, IL-8, IL-13, IL-16, IL-27, monocyte-chemoattractant protein 1, and TNF-α (224), suggesting that the human host immune response to CDI involves many different pathways, most of which have not been examined in detail. In a separate study, a comparative analysis of serum cytokine levels in CDI and uninfected patients showed that CDI induced a significant increase in cytokine release into the blood system (225). Infection with C. difficile resulted in a greater than 2-fold increase in IL-1β, IL-2, IL-5, IL-6, IL-8, IL-10, IL-13, IL-15, IL-16, IL-17A, and TNF-α release into the blood when compared to uninfected donors, with elevated levels of IL-2 and IL-15 associated with increased disease severity (225). Collectively, these studies highlight the importance of the innate immune response in CDI and disease.
TREATMENT, PREVENTION, AND DISEASE RECCURENCE
Antibiotic Treatment of CDI
Depending on disease severity, CDI may require antibiotic treatment to ensure infection clearance. Antibiotic interventions for CDI include oral metronidazole, vancomycin, and more recently, fidaxomicin, in most cases for 10 days (226). For cases of mild and moderate CDI, the European Society of Clinical Microbiology and Infectious Diseases recommends oral metronidazole (either 250 mg four times a day or 500 mg three times a day), because it is less expensive than vancomycin and fidaxomicin while reducing the risk of developing antibiotic resistance in other pathogens associated with the use of the clinically important vancomycin (226). When treating severe CDI, vancomycin is preferred over metronidazole (226). Vancomycin is usually administered at >125 mg at least two to four times a day and has a reduced oral availability and colonic absorption compared to metronidazole, enabling retention in the gut lumen, which facilitates more efficient C. difficile clearance (226). Treatment of CDI with fidaxomicin is as effective as vancomycin treatment, with dosage between 50 mg to 200 mg twice a day being sufficient to clear infection (226). Importantly, treatment with fidaxomicin is associated with less recurrent disease compared to metronidazole and vancomycin treatment of CDI. This is because it has a high level of activity against C. difficile but a narrow spectrum of activity against other bacteria, particularly Gram-negative bacteria, which results in a reduced impact on the protective colonic microbiota (227, 228).
Prevention
Because there is currently no effective vaccine against C. difficile disease, prevention strategies are mainly focused on controlling spread in high-risk environments, including hospitals and nursing homes, and antibiotic stewardship. Although C. difficile spores are nearly ubiquitous in health care facilities, infection control guidelines suggested by Vonberg et al. (229) indicate that early diagnosis, extensive education of hospital staff, isolation of infected patients, proper hand hygiene, and hospital surface and equipment disinfection are measures that limit the spread of C. difficile. Improving true-positive diagnosis also reduces the unnecessary use of antibiotics, which has been successful in reducing CDI incidence in hospitalized patients (229).
The normal microbiota of the colon prevents overgrowth of C. difficile in colonized individuals, although this protective effect can be disrupted following treatment with antibiotics (230). C. difficile cell proliferation and toxin production follows, resulting in the symptoms associated with CDI (230). Probiotics have been investigated as a potential preventative therapy for cases of noninfectious AAD with some success, yet studies in the context of primary CDI have proven inconclusive (231). Patients age 65 and older that received a multistrain preparation of lactobacilli and bifidobacteria in a multicenter trial exhibited no reduction in AAD and C. difficile-associated diarrhea compared to a placebo group (232). Similarly, provision of the yeast Saccharomyces boulardii to hospitalized patients age 50 or over along with prophylactic antibiotic treatment resulted in C. difficile-associated diarrhea in 2.1% of patients, compared to 1.5% receiving the placebo, in a single-center trial (233).
Recurrent CDI and Alternative Therapeutics
Although antibiotic treatment of C. difficile can effectively clear a primary infection, recurrence of disease occurs in roughly 25% of patients (234). Antibiotic treatment, which depletes colonic microbiota, is a key risk factor for CDI (234). CDI recurrence may occur through infection relapse, either with the same infectious strain causing disease or through infection with a different strain not related to the first infection (235–237). In both cases recurrence is related to disruption of the host microbiota and probably also due to damage that has occurred to the colonic epithelium, the latter of which may contribute to patient susceptibility (238, 239).
The broad-spectrum microbial activity of both metronidazole and vancomycin, although efficient at treating CDI, also allows these antibiotics to continue to disrupt the protective microbiota (240), consequently resulting in patient relapse rates of 19 to 23% and 18 to 32%, respectively (234). Following an initial recurrence of CDI, the risk of a second recurrence increases to approximately 45%, which is also likely to be linked to treatment-mediated depletion of the colonic microbiota (234). The risk of a third CDI recurrence increases to roughly 65% when vancomycin or metronidazole are used to treat the earlier CDI episodes (234). With recurrence rates of roughly 13% and a narrower spectrum of activity on the microbiota compared to vancomycin and metronidazole, fidaxomicin has emerged as a more promising treatment for CDI. The high cost of this antibiotic has unfortunately prevented broad clinical implementation, with a cost more than two times that of vancomycin and over 100 times that of generic metronidazole (241).
The relatively high levels of relapse associated with antibiotic treatment of CDI have resulted in the investigation of alternative therapeutics which aim to treat CDI without disrupting the microbiota. One such therapy, fecal microbiota transplant (FMT), aims to replace or replenish the microbiota of the gastrointestinal tract in times of dysbiosis and is used almost exclusively to treat CDI, particularly after several relapse events (8). By reintroducing “healthy” microbiota from a donor to the colon of CDI patients, in most cases following a failure to treat CDI with antibiotics, the resolution of symptoms and eradication of C. difficile can be achieved in approximately 80 to 100% of patients (242). However, with a majority of previous FMT work being conducted without effective controls, its efficacy as a treatment for CDI has been a contentious subject. In a recent randomized controlled trial, FMT was highly superior to standard antibiotic treatment of CDI (243). However, because FMT is inherently difficult to standardize and control, due to variation in the microbiota of each individual, Food and Drug Administration (FDA) approval can be difficult to achieve (244). Despite the effectiveness of FMT at reducing recurrent disease, this treatment may not be suitable for all patients, including some immunocompromised individuals for whom organisms from another individual’s microbiota may present as pathogenic under these conditions. However, several recent cases have used FMT to treat recurrent CDI in immunocompromised individuals, including solid organ transplant patients, without substantial consequences (245), suggesting that the use of FMT may be a viable treatment for recurrent CDI in select cases. There is undoubtedly a need for alternative treatments for CDI with fewer secondary consequences on the microbiota, and FMT is one such treatment, which has provided a starting point for the development and optimization of microbiota therapy-based approaches (246).
Interestingly, patients with low serum antibody levels are more prone to severe CDI and disease recurrence, while asymptomatic carriers of C. difficile have elevated serum IgG levels (247, 248). This observation led to studies of the provision of passive immunization via intravenous immunoglobulin (IVIG) supplementation in the treatment of both severe primary CDI and recurrent disease. This potential therapeutic is prepared from pooled human serum and contains C. difficile toxin antibodies (249). Leung et al. (250) demonstrated that IVIG treatment of five children experiencing recurrent CDI who had low serum IgG levels against TcdA resulted in resolution of all clinical signs of colitis with treatment every 3 weeks for up to 6 months, with a rise in IgG TcdA antibodies following therapy. Furthermore, clinical symptoms of CDI were resolved in two adult patients with severe pseudomembranous colitis that did not respond to standard-of-care metronidazole and vancomycin treatment when treated with pooled normal human immunoglobulin (251). However, the mechanism of IVIG-mediated symptom relief is still unknown because only a handful of small-scale case studies have been conducted. IVIG could be beneficial as a therapeutic option for patients that do not respond to treatment with traditional antibiotics or those that cannot be given FMT, although there is an urgent need for randomized trials to understand dosage and timing of treatment, as well as possible side effects.
More recent alternative therapies include the TcdB-specific monoclonal antibody therapy bezlotoxumab, which has recently obtained FDA approval and has been suggested for use in patients with relapsing disease since it does not disrupt the protective microbiota like antibiotics do (238). More recently, work by Hutton et al. (252) has shown that hyper-immune bovine colostrum directed toward TcdB, as well as a combination of TcdB, SLP, and spore exosporium-directed colostrum, is effective at treating and preventing severe disease progression in primary CDI in approximately 70 to 75% of C. difficile-infected mice, compared to mice treated with nonimmune colostrum, of which >90% succumbed to disease. Furthermore, the combination therapy also prevented relapse of CDI in roughly 80% of mice, compared to the roughly 90% of mice that relapsed without secondary treatment (252). Both bezlotoxumab and colostrum antibody approaches represent viable alternative therapies for CDI that may help to minimize disease and the recurrence of CDI, which has become increasingly urgent since the emergence of the epidemic 027 strains. The clinical link between increased CDI recurrence and the emergence of ribotype 027 strains is supported by studies identifying higher recurrence rates in patients infected with ribotype 027 strains compared to infections with other strains (253). Disease recurrence has therefore become a focus of CDI treatment and prevention strategies in recent years.
CONCLUSION
Although there has been a substantial increase in our knowledge of C. difficile pathogenesis and treatment of infection which has resulted from the development of suitable animal disease models and advances in technology, several key aspects of disease and recurrent infection have yet to be investigated in detail. With the ever-changing epidemiology of CDI and the complexity of C. difficile-mediated disease, coupled with the recent listing of C. difficile as an urgent public health threat by the CDC, there is a need for an improvement of our understanding of disease pathogenesis and for the development of effective alternative therapeutics for the treatment of infection to provide a better standard-of-care for patients suffering from CDI.
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