Clostridioides difficile Spores: Bile Acid Sensors and Trojan Horses of Transmission

Abstract

The Gram-positive, spore-forming bacterium, Clostridioides difficile is the leading cause of healthcare-associated infections in the United States, although it also causes a significant number of community-acquired infections. C. difficile infections, which range in severity from mild diarrhea to toxic megacolon, cost more to treat than matched infections, with an annual treatment cost of approximately $6 billion for almost half-a-million infections. These high–treatment costs are due to the high rates of C. difficile disease recurrence (>20%) and necessity for special disinfection measures. These complications arise in part because C. difficile makes metabolically dormant spores, which are the major infectious particle of this obligate anaerobe. These seemingly inanimate life forms are inert to antibiotics, resistant to commonly used disinfectants, readily disseminated, and capable of surviving in the environment for a long period of time. However, upon sensing specific bile salts in the vertebrate gut, C. difficile spores transform back into the vegetative cells that are responsible for causing disease. This review discusses how spores are ideal vectors for disease transmission and how antibiotics modulate this process. We also describe the resistance properties of spores and how they create challenges eradicating spores, as well as promote their spread. Lastly, environmental reservoirs of C. difficile spores and strategies for destroying them particularly in health care environments will be discussed.

Clostridioides difficile (more commonly known as Clostridium difficile ) is the major cause of healthcare-associated diarrhea and identifiable cause of healthcare-associated infections in the United States. ^{1 2} However, community-acquired cases of C. difficile now represent approximately 30% of documented infections, ^{1 3} and genetic analyses of C. difficile isolates suggest that there are diverse environmental reservoirs for this important pathogen, ^{4 5} underscoring the importance of recognizing C. difficile as more than a nosocomial pathogen. Regardless, the burden of C. difficile infections is significant, with approximately 500,000 infections occurring each year in the United States that contribute to approximately 30,000 deaths at an estimated cost of approximately $6 billion/year in the United States. ⁶ The high costs associated with treating C. difficile infections are largely due to the high rate of their recurrence (>20%, ^{1 7} ) and necessity for special disinfection measures. ² Both these complications are a function of C. difficile 's ability to make metabolically dormant, aerotolerant spores which are the major transmissive form of this obligate anaerobe.

C. difficile , spores are resistant to harsh environmental conditions and commonly used disinfectants, ^{8 9} inert to antibiotics, readily disseminated due their ability to adhere to many surfaces, ¹⁰ and survive for a long period of time. These properties make these dormant life forms ideal vectors for transmission. Accordingly, C. difficile spores contaminate diverse and widespread environments ranging from soil to dog paws to fomites to hospital hand rails. ^{4 11} This review focuses on the spore form of C. difficile and its importance in disease transmission. We first describe the infection cycle of C. difficile and how its spores germinate in response to specific signals sensed in the gut and how antibiotics appear to modulate this process. We then discuss how the resistance properties of spores make them difficult to eradicate and promotes their spread. Lastly, we describe known environmental reservoirs of C. difficile spores, as well as strategies for eradicating them and reducing their transmission in health care environments.

The Spore Form of C. difficile Is Its Major Infectious Particle

Since C. difficile is an obligate anaerobe, its aerotolerant, metabolically spore form is its major transmissive form. ¹² C. difficile spores are produced as the bacterium grows in the gut of infected individuals; the spores are then excreted into the environment where they can go on to infect the same individual or additional individuals. C. difficile mutants defective in forming spores fail to transmit disease ^{12 13} due to the rapid killing of vegetative C. difficile cells upon exposure to atmospheric oxygen. ^{14 15} When C. difficile spores in the environment are ingested by susceptible individuals, the spores will germinate in response to specific signals sensed in the gut. The germinating spores will then outgrow into toxin-producing vegetative cells that secrete the toxins responsible for causing disease symptoms. ¹⁶ Accordingly, C. difficile mutants with defects in germination exhibit virulence defects due to an inability to initiate infection. ^{17 18 19}

C. difficile Spores Germinate in Response to Vertebrate Gut-Specific Signals, Namely, Bile Acids

The spore form facilitates C. difficile disease transmission because the structure of spores allows them to survive passage through the harsh, acidic environment of the stomach (discussed here). When C. difficile spores enter the small intestine, they will sense specific bile acids within the gut that induce them to exit dormancy and transform back into vegetative cells. ^{20 21 22} This process takes approximately 60 to 90 minutes in in vitro studies. ²³

Notably, the responsiveness of C. difficile spores to bile acids, which are only made in vertebrates and secreted into the gastrointestinal tract, ²⁴ highlights how C. difficile has evolved to ensure that it germinates and grows exclusively in the vertebrate gut. Over 30 years ago, Wilson et al showed that taurocholate promotes C. difficile spore recovery. ²⁵ Twenty years later, Sorg and Sonenshein showed that cholate derivatives, such as taurocholate, deoxycholate, and glycolate, can induce spore germination and determined that taurocholate is the most potent germinant. ²² Interestingly, they also showed that chenodeoxycholate, which differs from cholate only by a single hydroxyl group at the 12th carbon of the sterol ring, is a potent competitive inhibitor of taurocholate-induced germination. ^{26 27} Subsequent studies have shown that many chenodeoxycholate derivatives prevent spore germination just as many cholate derivatives induce spore germination. ^{28 29} However, germination activators and inhibitors do not neatly segregate into cholate- and chenodeoxycholate-derivatives, respectively. ^{22 28 30}

Bile Acids Can Be Toxic to C. difficile Vegetative Cell Growth

Both cholate and chenodeoxycholate are produced in the liver as the end products of cholesterol metabolism. These bile acids can also be conjugated to the amino acids, glycine, or taurine, in the liver; this modification lowers their pK _a so that they can be termed bile salts. ²⁴ By definition, these four bile molecules are termed primary bile acids because they are made in the liver. Primary bile acids are secreted into the small intestine (duodenum) following a meal where they aid in digestion. While most of these bile acids (approximately 95%) are reabsorbed by bile salt transporters in the terminal ileum via the enterohepatic recirculation system, ^{24 31} the bile acids that are not recycled will undergo a wide variety of bacterial-mediated transformations as they move through the gastrointestinal tract. These enzymatic modifications will generate over approximately 50 different bile acids, ³² which are termed secondary bile acids. The major class of microbiota-mediated transformations can be grouped into the following: (i) the removal of the conjugated amino acids by dedicated bile acid hydrolases, which are produced by many members of the microbiota in both the small intestine and large intestine, and (ii) the epimerization/dehydroxylation of bile acids in the large intestine. While a wide range of bacteria in the gut can perform α-dehydroxylation of bile acids, only a tiny fraction (0.0001%) can dehydroxylate bile acids at the seventh carbon position to generate deoxycholate from cholate and lithocholate from chenodeoxyholate.

Notably, the 7-dehydroxylated secondary bile acids, deoxycholate, and lithocholate are toxic to C. difficile vegetative growth, ^{22 27 33 34 35 36} with lithocholate being a particularly potent growth inhibitor. ^{33 34} Accordingly, secondary bile acids are thought to be important determinants of the colonization resistance conferred by a healthy gut microbiota. ^{31 34 37}

Antibiotic Treatment Decreases the Levels of the Secondary Bile Acids that Are Toxic to C. difficile Vegetative Cell Growth

The role of secondary bile acids in suppressing C. difficile growth has been suggested by several studies demonstrating that antibiotic administration decreases the numbers of bile acid-converting commensals, thus reducing growth-inhibitory secondary bile acids while increasing primary bile acids. ^{34 36 38} Vegetative C. difficile grows poorly in cecal extracts from naïve (nonantibiotic treated mice), whereas C. difficile readily grows in cecal extracts from antibiotic-treated mice. ^{34 36} Furthermore, treatment of cecal extracts from naïve (nonantibiotic treated) mice with cholestyramine, a bile acid chelator, makes these extracts permissible to C. difficile growth. ³⁴ Conversely, preincubation of cecal extracts from antibiotic-treated mice with Clostridium scindens , one of the few bacteria known to generate secondary 7-dehydroxylated bile acids, ²⁴ converts these extracts into a nonpermissible growth medium for C. difficile . ³⁴ Notably, C. difficile strains that grow better in the presence of lithocholate are more frequently associated with causing severe disease, ³³ and successful fecal microbiota transplantation correlates with an increase in secondary bile acids to levels found prior to antibiotic treatment. ^{37 39} Taken together, these studies strongly suggest that secondary bile acids help suppress C. difficile vegetative cell growth.

C. difficile Spore Germination Occurs in the Small Intestine and Is Promoted upon Antibiotic Treatment

Antibiotic treatment not only promotes C. difficile vegetative growth in intestinal extracts, it also enhances C. difficile spore germination ^{20 21} presumably by increasing the levels of the bile acid germinants, taurocholate, and cholate. ^{21 36 38} As mentioned earlier, C. difficile spore germination occurs in the small intestine, ²¹ with the ileum supporting the highest levels of spore germination in mice. ⁴⁰ Although germinant levels are highest in the duodenum because bile salts enter the small intestine at this site, germination is lower in duodenal extracts compared with ileal extracts ⁴⁰ presumably because the lower pH of the duodenum prevents C. difficile spore germination. ⁴⁰

Oversimplification of the Effects of Primary and Secondary Bile Acids on C. difficile Spore Germination and Vegetative Cell Growth

A common but inaccurate simplification in the literature is that primary bile acids activate C. difficile spore germination, while secondary bile acids inhibit C. difficile vegetative cell growth. While the latter statement is true, both primary and secondary bile acids, namely, (tauro)cholate and deoxycholate, respectively, induce C. difficile spore germination. ^{22 28 29} Notably, even though deoxycholate is a better germinant than cholate, ^{28 29} deoxycholate prevents C. difficile growth, while cholate does not. ^{22 36} Furthermore, the primary bile acid, chenodeoxycholate, is a potent inhibitor of C. difficile spore germination and vegetative cell growth. ²⁷ Thus, germination properties cannot be inferred based on the site of production of the bile acids.

The TLDR (“Too Long, Didn't Read”) of Bile Acids and C. difficile Physiology

The current understanding of how bile acids affect C. difficile pathogenesis is that gut dysbiosis, for example, caused by antibiotics or recurrent CDI, decreases secondary bile acids and creates a more permissive environment for C. difficile vegetative cell growth. Thus, the generation of secondary bile acids by the gut microbiota is an important contributor to colonization resistance against CDI. Changes in conjugated bile acid levels during these dysbiotic conditions likely also promotes C. difficile spore germination, since germinant levels in the small intestine increase after antibiotic treatment. ^{21 36 38} However, the functional importance of the more permissive germination environment created by antibiotic treatment remains unknown because there is conflicting evidence linking variation in C. difficile germinant sensitivity to disease severity. ^{41 42 43 44 45} Since these correlative studies are complicated by the high genetic variability observed between C. difficile strains, studies using isogenic C. difficile mutants with differential germinant sensitivity are needed to directly address this question.

C. difficile Spores also Sense Amino Acid and Calcium Cogerminants

While bile acid germinants are essential for inducing C. difficile spore germination, these vertebrate-specific small molecules must act in concert with at least one class of cogerminant in vitro. ^{22 29 46 47} There are two classes of cogerminants, amino acids ²² and calcium ions. ⁴⁶ While a wide range of amino acids can potentiate taurocholate-induced C. difficile spore germination, glycine is the most potent amino acid cogerminant. ^{28 47} Likewise, calcium ions are more potent at potentiating taurocholate-induced spore germination than magnesium and zinc ions, ⁴⁶ and depletion of calcium ions from mouse ileal contents is sufficient to prevent C. difficile spore germination. ⁴⁶ Interestingly, proton pump inhibitors appear to sensitize individuals to CDI, ⁴⁸ and these gastric acid-suppressing agents also decrease calcium absorption from the intestine, leading to the hypothesis that PPIs promote C. difficile spore germination in the gut and thus increase susceptibility to CDI. ⁴⁶ Regardless, in the complex environment of the small intestine, a combination of amino acids and calcium ions likely synergize to trigger germination in response to cholate-derived germinants, since subactivating concentrations of glycine and calcium chloride strongly potentiate C. difficile spore germination. ⁴⁰

Antigermination Strategies for Preventing C. difficile Infection

The observation that specific bile acids can potently inhibit C. difficile spore germination, combined with the finding that C. difficile germination mutants are impaired in their ability to colonize hamsters and cause disease ^{17 18 19} and have led to the proposal that bile salt analogs could be used to prevent C. difficile -associated disease. Consistent with this hypothesis, synthetic bile acid analogs have been developed that prevent C. difficile spore germination both in vitro ^{28 30 49} and during infection of mice, ⁵⁰ suggesting that these antigermination agents could be developed for clinical use. Indeed, even subinhibitory doses of these agents reduce disease severity. ⁵⁰ Importantly, these synthetic molecules cause minimal changes to the gut microbiota, ⁵¹ suggesting that they could also help to prevent recurrent infection. Furthermore, combining these inhibitors with suboptimal concentrations of vancomycin effectively prevented the onset of C. difficile disease in hamsters, ⁵¹ which are highly sensitive to CDI, highlighting the potential for developing these bile salt analogs in combination with existing therapies.

Further supporting the potential of antigermination therapies, oral dosing of a patient with the endogenous bile acid ursodeoxycholate (UDCA), a weak inhibitor of spore germination, nevertheless resolved the patient's case of C. difficile ileal pouchitis. ³⁹ This treatment strategy was possible because the patient had part of their colon removed so that they could not reabsorb the administered UDCA. However, since UDCA can also inhibit vegetative C. difficile growth, ³⁵ it is difficult to distinguish whether UDCA's inhibition of C. difficile spore germination versus vegetative cell growth or both were critical to the success of this treatment. While antigermination compounds based on bile acid derivatives show promise in animal models, it will be important to determine the long-term effects of exposure to the synthetic bile acid analogs and their breakdown products, since some bile acids have been implicated in colon carcinogenesis. ²⁴

The Perfect Reservoir: C. difficile Spores Are Found in a Wide Variety of Environments

Although the vegetative form of C. difficile grows exclusively within the vertebrate gut, C. difficile spores can be found in almost every environment imaginable because C. difficile grows in a wide range of vertebrate hosts, from elephants to ostriches. ⁵² Since C. difficile strongly induces sporulation inside the gut, fecal matter from infected hosts is a major source of environmental contamination. Consistent with the idea that infected individuals are key vectors of transmission, spores are detectable in the feces of infected mice within approximately 24 hours after inoculation, with approximately 20% of the C. difficile population inducing sporulation at this time. ²¹ Components within the gut may specifically promote entry into sporulation, since a recent study showed that fecal water potently induces C. difficile genes required for spore formation. ⁵³ Below we discuss the many different environments that C. difficile spores are either produced in or can persist within to seed infections in hospitals and the wider community.

Infected Individuals Are Important Reservoirs of C. difficile Infection

Health Care Workers Can Facilitate the Spread of C. difficile in Outbreak Settings

The importance of infected individuals in spreading CDIs is evident in outbreaks where the same strain of C. difficile is isolated from multiple patients. ^{54 55 56} C. difficile spores are readily isolated from the skin of infected patients, from multiple sites like the chest and abdomen ⁵⁷ and their hospital rooms. ^{58 59} The extent of environmental contamination has been found to correlate with contamination of health care worker hands and C. difficile transmission to patients, ^{58 60} suggesting that health care workers are also important vectors for transmitting CDI particularly in outbreak settings. ⁶¹

The efficacy of isolating infected patients in reducing C. difficile transmission in hospital settings also highlights the importance of these individuals in spreading disease. ^{62 63} Notably, infected patients have been shown to shed spores for 1 to 4 weeks even after they have resolved their infection, particularly if they continue to take antibiotics for non-CDI related reasons. ⁵⁹ As will be discussed below, these asymptomatic patients may be important vectors of C. difficile disease transmission.

Antibiotic use promotes asymptomatic carriage of C. difficile in multiple studies in humans and mice. A small, prospective study of asymptomatic carriage in a long-term care facility revealed that prior CDI, as well as prior antibiotic use, increases the prevalence of asymptomatic carriage. ⁶⁴ Furthermore, asymptomatic carriers were more than twice to thrice more likely to have C. difficile spores isolated from their skin and surrounding environment than noncarriers. ⁶⁴ Even inhabiting a bed previously occupied by a patient taking antibiotics can increase a hospital patient's risk for CDI. ⁶⁵ Studies in mice also indicate that antibiotics promote C. difficile transmission, ⁶⁶ since mice asymptomatically colonized with C. difficile excreted high levels of C. difficile spores following antibiotic treatment. Notably, this super-shedder state persisted as long as mice were treated with antibiotics, and mice continued shedding spores for approximately 2 weeks following antibiotic treatment even though they were asymptomatic.

How Important Are Asymptomatic Carriers of C. difficile to Disease Transmission?

Despite these observations, the importance of asymptomatic carriage of C. difficile in transmitting infection is a topic of active debate. ⁶⁷ Two recent studies using either advanced typing or sequencing methods to track C. difficile isolates suggest that asymptomatic carriers are indeed important sources of C. difficile transmission. In a large-scale analysis of C. difficile cases in the United Kingdom, 45% of hospital-onset CDI cases were genetically dissimilar to all previous cases, while approximately 40% were linked to another symptomatic case. ⁵ Another large study in the United States observed that approximately 30% of hospital-acquired CDIs could be attributed to asymptomatic carriers, ⁶⁸ while another associated asymptomatic carriers with an approximately two-fold increased risk of CDI in a hospital setting. ⁶⁹ In a quasiexperimental study in which carriers of C. difficile were identified upon admission and handled with modified contact precautions, CDI rates decreased by approximately 60% decrease in, whereas no change in CDI rates was observed in nonintervention comparator hospitals. ⁷⁰ While the observed decrease in CDI in the intervention hospital could be due to changes in antibiotic prescription due to knowledge of a patient's C. difficile carrier status, this study nevertheless suggests that active screening for CDI could reduce a hospital's disease burden. Regardless, additional controlled intervention trials are needed to establish whether interventions targeting asymptomatic carriers will be effective at preventing CDI. ^{67 71}

Interestingly, the so-called “hypervirulent,” epidemic ribotype NAP1/027 is less frequently isolated from asymptomatic carriers of C. difficile that have no history of CDI relative to patients with CDI. ⁷² This observation further supports the notion that asymptomatic carriers of C. difficile may be important reservoirs for transmitting CDI particularly in the community.

Environmental Reservoirs of C. difficile Infection

The observation that over 40% of C. difficile cases in a large-scale study were genetically unrelated to other cases observed within a hospital network in the United Kingdom suggested that there is a large environmental reservoir for C. difficile outside of the hospital setting. While the exact reservoirs for C. difficile outside of hospital settings (defined as “community”) are unknown, young infants (who are frequent asymptomatic carriers of C. difficile ), pet dogs, and farm animals have been proposed as sources of CDI.

Babies as Vectors for C. difficile Infection?

Children less than 1 year old frequently carry toxigenic and nontoxigenic C. difficile (approximately 35% carriage); this carriage rate decreases to approximately 3% in children by approximately 8 years. ⁷³ A recent large scale analysis of healthy infants (<2 years) without significant health care exposure in the United Kingdom reported similar carriage rates. ⁷⁴ Stoesser et al further determined that strains isolated from infants were associated with 13% of CDI C. difficile strains isolated during the same time period, suggesting that there is a common community reservoir for infant colonization, as well as CDI. ⁷⁴

C. difficile Can Be Transmitted between Animals and Humans but Likely Not from Food

Pets have also been proposed as an important reservoir for C. difficile disease transmission, ⁴ and colonization of pets has been correlated with asymptomatic carriage of C. difficile in infants. ⁷⁴ Approximately 10 to 50% of dogs and 20% of cats carry C. difficile , ^{75 76} with C. difficile being isolated in 31% of households with a dog. ⁷⁶ In a survey of sandboxes for children and dogs in Spain, C. difficile could be isolated from approximately 50% of these sandboxes, ⁷⁷ and C. difficile was isolated from shoes and dog paws in approximately 20 to 40% of households surveyed in Slovenia. ¹¹

The link between C. difficile colonization of farm animals with transmission to humans has been explored more fully. C. difficile is an important cause of neonatal enteritis in piglets, ⁵² and the location of pig farms and C. difficile ribotype 078 infections has been shown to overlap significantly in the Netherlands. ^{4 52 78 79} The 078 is the primary ribotype found in piglets, and a recent large-scale genome-wide analysis of 078 strains isolated from 22 countries in Europe revealed that human and animal 078 strains are essentially indistinguishable, suggesting that there is bidirectional, intercontinental transmission between farm animals and humans. ⁸⁰ The 014 ribotype is highly prevalent in neonatal pigs in Australia, and, notably, it is the most frequent ribotype associated with CDI. ⁸¹ Analogous to the 078 ribotype, whole genome analyses of 014 strains indicates that many strains isolated from humans are genetically identical to those isolated from pigs despite being separated by large distances. ⁸¹ C. difficile is also frequently found in approximately 50% of <7-day-old calves despite being present in only 2% of adult cattle, ⁸² so young cows may be another important source of C. difficile spores.

Importantly, environments that contact animal waste products frequently harbor C. difficile . Roll-out lawns that are frequently produced using manure have been found to contain C. difficile spores with high frequency, ⁸³ and numerous studies have identified C. difficile spores in water and soil samples in Europe. ⁴ Compost can be manufactured from pig feces and piggery effluent pond water, and treated effluent pond water can be applied to land used for agriculture or pasture. Since water treatment and composting does not necessarily destroy C. difficile spores, C. difficile can be readily isolated from vegetables grown in these environments. ⁸⁴

The extent to which C. difficile spores contaminate our food supply remains unclear. Numerous studies have isolated C. difficile from raw meat, lettuce, and shellfish ⁸⁵ but no instances of food-borne transmission of C. difficile have been reported. ⁴ While these observations highlight the ubiquity of C. difficile spores in the surrounding environment, the likelihood of acquiring an infection from these many sources remains unclear, since the infective dose and level of contamination needed to transmit infection remains unknown and may be highly host dependent. ⁴

C. difficile Ribotypes Are Associated with Different Environmental Reservoirs

Analyses of C. difficile strains isolated from different hosts indicate that certain ribotypes are more frequently associated with specific environmental reservoirs. While the NAP1/027 strain is one of the most commonly identified strains in the United States in healthcare-associated CDIs (although its frequency has diminished markedly in Europe), ² asymptomatic carriers of C. difficile are two-fold less likely to carry this strain ⁷² as mentioned earlier. Notably, NAP1/027 strains were not observed in at least two studies of infants in the United Kingdom, ⁷⁴ suggesting that distinct environmental reservoirs lead to infant colonization. Furthermore, the 078 ribotype is often isolated from calves and pigs and is more frequently associated with community-associated CDIs than the 027 ribotype. ⁷⁸

C. difficile Spores Resist Commonly Used Disinfectants but Are Sensitive to Bleach

The apparent ready dissemination of C. difficile spores throughout the environment depends upon their ability to persist in the environment for a long period of time. This persistence is a function of the metabolic dormancy of bacterial spores and the fact that C. difficile spores only germinate in the presence of vertebrate animal-derived bile salt signals. ²² Spore stocks in laboratory do not lose viability over the course of at least several years, and bacterial spores are thought to be able to survive for hundreds if not thousands of years. ⁹ The partially dehydrated nature of the bacterial spore cytosol and the protection of their DNA by specialized DNA binding proteins contributes to their dormancy and longevity, as well as their ability, to resist ultraviolet (UV) light. ^{9 86}

Like other bacterial spores, C. difficile spores have a central, partially dehydrated core (cytosol) that is surrounded by a thick layer of modified peptidoglycan cell wall known as the cortex, followed by a series of proteinaceous shells known as the coat. ⁸⁷ The coat helps to protect spores from oxidative insults and enzymatic digestion, ⁸⁸ while the cortex plays a critical role in maintaining the dormancy of C. difficile spores by preventing additional water from entering the spore core. The cortex also allows C. difficile and other bacterial spores to survive extremes of temperature, as well as ethanol-based sanitizers. ⁹ C. difficile spores can survive being heated to 80°C for 15 minutes ^{89 90} and are fully viable if resuspended in 100% ethanol, highlighting the inability of ethanol-based sanitizers to inactivate C. difficile spores. ^{8 91}

Not only are C. difficile spores unaffected by ethanol-based sanitizers, they are also fully resistant to the chlorhexidine-based sanitizers typically used to eradicate vancomycin-resistant enterococci (VRE), and C. difficile spores are also partially resistant to isothiazolinone-based disinfecting detergents. ^{8 91} Thus, disinfection agents commonly used to eradicate other healthcare-associated pathogens are ineffective at killing C. difficile spores.

Fortunately, oxidizing disinfectants like bleach, hydrogen peroxide, and Virkon are effective at killing C. difficile spores, ^{8 91} so bleach has been widely adopted in health care settings for cleaning the rooms of patients infected with C. difficile . In addition, while bacterial spores can resist UV irradiation, extended exposure times will kill C. difficile spores, since as will be discussed below, UV-C can effectively disinfect rooms of C. difficile patients. ⁹² Lastly, a recent report has shown that a nonaqueous gel of glycerol monolaureate can kill C. difficile spores, suggesting that this antimicrobial could be used as a method for decontaminating environmental surfaces as well as skin. ⁹³

Efficacy of Cleaning and Containment Measures in Preventing the Spread of C. difficile Infections

Considerable work has been done developing methods for reducing the spread of C. difficile in health care settings. Glove and gowns are strongly recommended by the Infectious Disease Society of America (IDSA) for limiting the spread of C. difficile , and hand washing with soap and water is the preferred method for hand disinfection, following glove removal. ² Daily disinfection of high-touch surfaces in the rooms of patients infected with C. difficile has been shown to reduce contamination of hands or gloves of health care workers. ⁹⁴ Using whole genome sequencing, a recent study showed that an 027 clone rapidly spread through a long-term care facility despite terminal cleaning of the originating patient's room with bleach. ⁵⁴ Environmental sampling of the facility revealed that the 027 isolate was widely distributed throughout the facility, not just in the patient's room. Guided by these results, targeted bleach-based cleaning was performed, and the 027 isolate was removed from this facility. Thus, careful disinfection measures can help to contain outbreaks.

The efficacy of different disinfection measures was recently evaluated by the Center for Disease Control (CDC). In a multicenter, cluster-randomized trial, three different strategies for terminal disinfection of rooms harboring patients with multidrug resistant infections like C. difficile were evaluated for their ability to eradicate these organisms and reduce infection frequency. ⁹⁵ The three strategies included 10% bleach, UV-C, and bleach and UV-C. Interestingly, of these methods, only the UV-C treatment led to a reduction in C. difficile , as well as VRE, cases within a hospital. UV-C disinfection of rooms of patients with CDI or on contact precautions was shown in a prospective study to reduce CDI incidence by approximately 25%, whereas the disease incidence increased by 16% in nonstudy units. ⁹²

A relatively new strategy for specifically eliminating C. difficile spores takes advantage of the responsiveness of C. difficile spores to bile salt germinants and the subsequent loss of resistance properties that occurs during germination. A variety of methods have been tested, but they all involve applying a solution of taurocholate bile salts to induce C. difficile spore germination and combining this treatment with either UV-C, ⁹⁶ ethanol or acid ⁹⁷ or nisin ⁹⁸ to kill the outgrowing spores. While these studies indicate that this combination strategy can effectively decrease spore numbers, a potential limiting factor of this strategy is the relatively high cost of taurocholate and the need for high concentrations of this bile salt to induce germination.

Conclusion

The spore form of C. difficile clearly creates unique challenges to containing the transmission of this medically important pathogen. Infected C. difficile patients are important disease vectors because they shed spores during and several weeks after infection, and these spores are readily spread in the hospital environment. However, even though C. difficile is often considered a nosocomial pathogen, community-acquired infections represent a significant amount of the C. difficile disease burden, and this likely reflects the many reservoirs of C. difficile spores that have been identified ranging from asymptomatic carriers, infants, farm animals, pets, lawns, and even food. However, it is important to note that the only environmental reservoirs that have experimental evidence suggesting them as vectors of transmission are asymptomatic carriers and farm animals.

The ubiquity of C. difficile spores in the surrounding environment strongly suggests that we are frequently exposed to, and ingesting, C. difficile spores, yet only a small fraction of individuals are susceptible to infection. Numerous studies have shown that a healthy microbiota protects against C. difficile infection, but the question is to what extent different prevention and/or containment measures can prevent C. difficile infection. The importance of asymptomatic carriers to hospital transmission of C. difficile needs to be rigorously studied as the impact of different disinfection and containment measures, since only one large-scale study has been performed. Numerous small, prospective studies have been conducted, but larger scale studies are needed. Finally, understanding the basic biology of C. difficile spores may lead to antisporulation therapies that could reduce disease transmission, and antigermination strategies that show promise in animal models have the potential to be translated in humans with further development.