Clostridioides difficile (syn. Clostridium difficile) is a bacterium known for causing serious diarrheal infections, and may also cause colon cancer. It is known also as C. difficile, or C. diff (), and is a Gram-positive species of spore-forming bacteria. Clostridioides spp. are anaerobic, motile bacteria, ubiquitous in nature and especially prevalent in soil. Its vegetative cells are rod-shaped, pleomorphic, and occur in pairs or short chains. Under the microscope, they appear as long, irregular (often drumstick- or spindle-shaped) cells with a bulge at their terminal ends (forms subterminal spores). C. difficile cells show optimum growth on blood agar at human body temperatures in the absence of oxygen. C. difficile is catalase- and superoxide dismutase-negative, and produces up to three types of toxins: enterotoxin A, cytotoxin B and Clostridioides difficile transferase. Under stress conditions, the bacteria produce spores that tolerate extreme conditions that the active bacteria cannot tolerate.
Clostridioides difficile is an important human pathogen; according to the CDC, in 2017 there were 223,900 cases in hospitalized patients and 12,800 deaths in the United States. Although C. difficile is known as a hospital- and antibiotic-associated pathogen, at most one third of infections can be traced to transmission from an infected person in hospitals, and only a small number of antibiotics are directly associated with an elevated risk of developing a C. difficile infection (CDI), namely clindamycin, fluoroquinolones and cephalosporins. Most infections are acquired outside of hospitals, and most antibiotics have similar elevated risk of infection on par with many non-antibiotic risk factors, such as using stool softeners and receiving an enema.
Clostridioides difficile can become established in the human colon without causing disease. Although early estimates indicated that C. difficile was present in 2–5% of the adult population, later research indicated that colonization is closely associated with a history of unrelated diarrheal illnesses, such as food poisoning or laxative abuse. Individuals with no history of gastrointestinal disturbances appear unlikely to become asymptomatic carriers. These carriers are thought to be a major infection reservoir.
Taxonomy The species was transferred from the genus Clostridium to Clostridioides in 2016, thus giving it the binomial Clostridioides difficile. This new name reflects the taxonomic differences between this species and members of the genus Clostridium, while maintaining the common name as C. difficile. , no other species are in this new genus.
Human pathogen Pathogenic C. difficile strains produce multiple toxins. The best-characterized are enterotoxin (C. difficile toxin A) and cytotoxin (C. difficile toxin B), both of which may produce diarrhea and inflammation in infected patients (C. difficile colitis), although their relative contributions have been debated. The diarrhea may range from a few days of intestinal fluid loss to life-threatening pseudomembranous colitis, which is associated with intense colon inflammation and pseudomembrane formation on the intestinal mucosal surface. This may progress to toxic megacolon, a severe form of colonic distention that can put a patient at risk for colon perforation, sepsis and shock. Toxins A and B are glucosyltransferases that target and inactivate the Rho family of GTPases. Toxin B (cytotoxin) induces actin depolymerization by a mechanism correlated with a decrease in the ADP-ribosylation of the low molecular mass GTP-binding Rho proteins. A binary toxin (AB toxin), but its role in disease is not fully understood.
Additional virulence factors include an adhesion factor that mediates the binding to human colonic cells and a hyaluronidase.[Medical Micriobiology, Fifth Edition, Patrick Murray, Elsevier Mosby, 2005, page 412] The bacterium also produces the chemical para-cresol, which inhibits the growth of other microbes in its vicinity and allows it to outcompete normal human gut flora.
The production of these toxins is not constitutive but is instead tightly controlled by metabolic and environmental signals. CodY is a global nutrient-sensing repressor that suppresses toxin gene expression when preferred amino acids such as proline, glycine, leucine, and valine are abundant. As these nutrients become limiting, CodY-mediated repression is relieved, permitting toxin production. CcpA links carbon availability to toxin regulation, with high glycolytic flux repressing tcdA and tcdB expression. The redox-sensing regulator Rex further connects the NADH/NAD⁺ ratio to virulence gene expression. Nitrosative stress generated by host immune cells in the form of reactive nitrogen species (RNS) represents an additional environmental signal shaping toxin production. The transcriptional regulator HcpR mediates C. difficiles defense against RNS, and disruption of this defense is associated with elevated TcdA and TcdB toxin production.
Antibiotic resistance Antibiotic treatment of C. difficile infections may be difficult, due both to antibiotic resistance and physiological factors of the bacterium (spore formation, protective effects of the pseudomembrane). A highly toxic strain of C. difficile, resistant to fluoroquinolone antibiotics, such as ciprofloxacin and levofloxacin, said to be causing geographically dispersed outbreaks in North America, was reported in 2005. In 2005 the US Centers for Disease Control in Atlanta warned of an epidemic strain with increased virulence, antibiotic resistance, or both. In 2018, resistance to other antibiotics such as metronidazole, the first choice of antimicrobial drug when treating CDI, was observed in up to 12% of clinical isolates. While treatment with various antibiotics continues, more diverse and stronger resistance will continue in C. difficile populations.
Clostridioides difficile spores resist many disinfectants, including high concentrations of bleach.
A recent study published by researchers in Nature Communications found that a chain mail-like armor, called "The S-layer" may be responsible for C. difficile's apparent immunity to antibiotic treatments, human saliva, as well as an enzyme human host cells normally use to fight viruses.
The study also found that removing a region of the S-layer called D2 made C. diff cells susceptible to lysozyme, an enzyme typically found in saliva that tears open microbes' exteriors. Without the protection layer, the bacteria would also be vulnerable to the means of attacking the virus mentioned above.
Persistence in the gut Beyond spore formation and toxin secretion, an important virulence factor of Clostridioides difficile is its set of cell-surface proteins. These proteins are located on or beyond the bacterial peptidoglycan layer and include the surface (S-) layer, flagella, pili, and approximately 28 accessory cell-wall proteins (CWPs). Cell-surface proteins in C. difficile have been associated with several functions, including adhesion to host cells, biofilm formation, surface interactions, and motility.
The S-layer is the most abundant and functionally significant cell-surface component. Unlike in many other prokaryotes, the C. difficile S-layer is heterodimeric, consisting of low- and high-molecular-weight S-layer proteins. The S-layer contributes to maintaining cell-envelope stability and has been implicated in several virulence-related processes, including spore and toxin production, as well as resistance to antibiotics and environmental stress.
Research Deletion of the slpA gene, which encodes the S-layer protein in Clostridioides difficile, results in marked changes and impairments in both cell and colony morphology. The mutant strain also exhibits reduced ability to withstand stress during the late stationary phase and shows approximately double the level of biofilm formation compared to the wild type. However, loss of the S-layer protein increases bacterial susceptibility to the antibiotic vancomycin and to environmental stress induced by Triton X-100. The absence of the S-layer further decreases sporulation efficiency, toxin release, and adherence capacity.
Transmission
Clostridioides difficile is transmitted from person or animal to person by the fecal-oral route, shed in faeces. The organism forms heat-resistant aero-tolerant spores that are resistant to alcohol-based hand cleansers or routine surface cleaning. Spores survive in clinical environments for long periods. Any surface, device, or material (e.g., toilets, bathing tubs, or rectal thermometers) that becomes contaminated with feces may serve as a reservoir for C. difficile spores, which can live for long periods of time on surfaces. Because of this, the bacterium may be cultured from almost any surface. Once spores are ingested, their acid resistance allows them to pass through the stomach unscathed. They germinate and multiply into vegetative cells in the colon upon exposure to bile acids. Consequently, the World Health Organization advocates the use of soap in addition to alcohol solutions to limit the spread of the spores. Sporulation was shown to be significantly reduced after inactivation of C. difficile's DNA methyltransferase CamA,''' raising the prospect of developing a drug that may inhibit this bacterium in a specific manner.
Susceptibility to colonization appears to be triggered by diarrheal illnesses, such as food poisoning, Crohn's disease or laxative abuse; people without a history of diarrheal illnesses who are exposed to C. difficile spores will not necessarily become carriers. Once people are colonized by C. difficile, they remain carriers for year-long time scales, but the day-to-day abundance of C. difficile fluctuates considerably – fluctuating from being below the limit of detection to high levels of shedding from one day to the next. GI disturbances in carriers appear to trigger periods of increased shedding which may be an important factor for transmission.
Host range Clostridioides difficile infects pigs, calves, and humans, and inhabits a natural reservoir of soil, faeces of domestic animals and humans, sewage, the human intestinal tract, and retail meat.
A 2015 CDC study estimated that C. difficile afflicted almost half a million Americans and caused 29,000 deaths in 2011. The study estimated that 40% of cases began in nursing homes or community health-care settings, while 24% occurred in hospitals.Clostridioides difficile is common in the human digestive system. However, it is often outcompeted for nutrients by other bacteria, limiting its spread. If the sudden introduction of an antibiotic disrupts the microbiome, C. difficile may be able to grow as many of its competitors die. The incubation period is 5–10 days, with a range of 1 day to weeks following antibiotic treatment. Additionally, carriage of C. difficile with high levels of toxins is common in young children, while disease is rare. The production of one or even both toxins is not always sufficient for producing symptoms.
Signs and symptoms Symptoms of C. difficile infection include diarrhea (at least three loose bowel movements a day), dehydration, abdominal pain that can be severe, loss of appetite, and nausea.