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Art and Science of Osteopathic Medicine  |   May 2019
Why Does Clostridium difficile Infection Recur?
Author Notes
  • From the Division of Infectious Diseases at Mayo Clinic Arizona in Phoenix. 
  • Disclaimer: Dr Orenstein is the editor in chief of the American Osteopathic Association. He was not involved in the editorial review or decision to publish this article. 
  • Financial Disclosures: None reported. 
  • Support: None reported. 
  •  *Address correspondence to Robert Orenstein, DO, 5777 E Mayo Blvd, Phoenix, AZ 85054. Email: orenstein.robert@mayo.edu
     
Article Information
Cardiovascular Disorders / Disaster Medicine / Gastroenterology / Geriatric Medicine / Preventive Medicine / Professional Issues / Urological Disorders / Being a DO
Art and Science of Osteopathic Medicine   |   May 2019
Why Does Clostridium difficile Infection Recur?
The Journal of the American Osteopathic Association, May 2019, Vol. 119, 322-326. doi:10.7556/jaoa.2019.054
The Journal of the American Osteopathic Association, May 2019, Vol. 119, 322-326. doi:10.7556/jaoa.2019.054
Abstract

Clostridium (or Clostridioides) difficile infection affects more than 500,000 people annually in the United States, one-third of whom have recurrent symptoms. The evolution of C difficile as a resilient pathogen has to do with its ability to persist in the environment and in the host, leading to recurrence and environmental spread. Understanding the mechanisms by which this microbe interacts with the environment, the host, and the gut microbiota are critical to solving this problem. This article presents a brief clinical vignette; discusses the current state of understanding of colonization, transmission, and recurrence; and considers the role the host plays in eliminating this infection. The understanding of these mechanisms and application of osteopathic principles has the potential to improve patient outcomes.

Clinical Vignette
A 72-year-old woman with recurrent urinary tract infections and mitral valve prolapse is seen by her dentist for a tooth extraction. She is given oral clindamycin, 300 mg, 1 hour before the procedure. Three days after the procedure, she develops watery and foul-smelling diarrhea 10 to 12 times per day, fecal incontinence, and abdominal cramps. Her primary care physician orders a Clostridium difficile toxin assay, and the result is positive. She is treated with 14 days of oral vancomycin and improves. Four days after stopping the oral vancomycin, her diarrhea recurs; another 2 weeks of oral vancomycin is prescribed, and she improves again. Five days later, symptoms recur, and she is given a 2-week vancomycin course followed by a 6-week tapered course. Again, she is well on treatment, but 5 days after completing the treatment course, all of her symptoms recur. After 4 months of diarrhea and treatment, she is frustrated, socially isolated, and economically devastated by the costs of her treatments. She comes to see you for a procedure called fecal microbiota transplantation because she has read that it is extremely effective for recurrent C difficile infection (CDI). She asks you why she keeps getting this infection and why this unusual treatment would likely cure her infection. 
Introduction
Clostridium (now called Clostridioides) difficile is a gram-positive, anaerobic, spore-forming bacteria that accounts for more than half a million infections each year in the United States.1 Nearly one-third of patients improve but then have symptoms recur, leading to significant morbidity, expense, and disruption in their quality of life.2 Recurrent disease in turn leads to the shedding of more spores into the environment, which may spread the infection to others. Osteopathic physicians understand that humans possess self-regulatory and self-healing functions and that rational treatment addresses alterations to normal structure and function to restore health. The human gut microbiota is a diverse microbial ecosystem that guides human energy extraction and helps regulate our adaptive and innate immune function. Disruptions in the microbial structure can lead to loss of these functions and the development of human disease. The best example of this disruptive process is CDI. It rarely causes disease in people with a diverse intestinal microbiota. However, loss of microbial diversity due to antibiotics, aging, and other stressors provides fertile ground for its growth and the spread of disease. 
C difficile exists in 2 forms: an environmentally hardy spore, responsible for transmission, and a vegetative form, which produces the toxins that cause disease. Its spectrum of illness ranges from asymptomatic colonization to mild diarrhea to sepsis and death. Currently used antimicrobial treatments for CDI, including oral vancomycin, fidaxomicin, and metronidazole, eliminate symptoms by targeting the toxin-producing vegetative form of the organism within the colon. These treatments play a limited role in the recurrence of CDI. By themselves, they may further alter the protective gut microbiota. New approaches, such as the use of probiotics to replenish the microbiota, are promising.3 One strategy for preventing recurrent CDI, fecal microbiota transplantation, has been highly effective.4 By transplanting healthy human donor fecal microbiota into the colon of an affected patient, this technique restores the host's defense against C difficile and stops recurrence in most patients. But what is the mechanism of this host and microbiota defense? The human body has a least 2 defenses against C difficile: (1) the local immune response to the toxins and (2) colonization resistance through the community of microbes that normally inhabit the human colon. 
Pathogenesis
Humans acquire CDI by ingesting bacterial spores from their environment. These spores are widespread and can be found in foods, on pets, on lawns, and on many of the surfaces that humans come into contact with daily. These spores are created by Clostridia when nutritional resources for their growth are limited, or through quorum sensing, a bacterial communication mechanism that regulates genes in response to population density.5,6 In the environment, spores are hardy, evading destruction by common solvents such as alcohol, heat, and oxygen, allowing them to persist for prolonged periods. When ingested, these spores evade the host defenses of gastric acid and motility and pass into the jejunum where they interact with bile acids. The interaction of the C difficile spores within the gut lumen plays a key role in whether infection develops. If the spores cannot germinate into the form of the organism that produces the C difficile toxins, then disease cannot occur. Thus, the germination and sporulation pathways of C difficile may be the key to understanding how to prevent disease. These pathways are complex and incompletely understood—current knowledge derives primarily from animal models and studies of spore biology of other spore-forming organisms. 
Role of Gut Microbiota in Colonization, Disease, or Eradication
A permissible environment for spore germination exists after the colonic microbiota is disrupted by antimicrobial therapy. This allows C difficile to enter the colonic niche with several potential outcomes—colonization, disease, sporulation, or, in the absence of germination, eradication. 
Colonization
C difficile can be a transient or persistent colonizer depending on the host's colonic microbiota and immune response. Thus, diagnostic testing results of feces that identify the organism (culture), its substrates (C difficile antigen tests), or its DNA (polymerase chain reaction vs toxin B) may be positive in the absence of disease. Hence, testing patients for a cure after a bout of diarrhea is not recommended. The mechanism by which transient colonization progresses to disease is regulated by the host's colonic microbiota and the production of IgG antibodies against the toxins of C difficile. Alterations of the colonic microbiota by antimicrobial agents may lead to diminished microbial diversity. 
The 4 predominant bacterial phyla of the human gut are Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria, with the greatest abundance being the anaerobes, Bacteroidetes. Antimicrobials frequently lead to reductions in Bacteroidetes and increases in Proteobacteria. Fewer Proteobacteria and an increased prevalence of Firmicutes and Bacteroidetes were found in carriers of C difficile as compared with those with CDI.13 Decreases in butyrate-producing bacteria—in particular, the groups Ruminococcus and Lachnospiraceae—may lead to disease rather than colonization. The specificity of these microbiota alterations may regulate the difference between colonization and disease. Human and mouse studies suggest that reduced diversity and richness of species leads to colonization, but the absence of particular bacterial taxa leads from colonization to disease.13 For example, administration of Clostridium scindens to antibiotic-treated mice protected them from progression to CDI after spore ingestion.13 
Another potential mechanism for the proliferation of C difficile is the availability of nutrients in the presence of altered microbial structure. The ready availability of nutrients in the gut lumen following elimination of competitive bacterial species may allow it to thrive. Sialidase-producing bacteria cleave sugars from glycosylated proteins bound to the colonic epithelial cell membrane. The release of this free sialic acid provides an energy substrate for C difficile, allowing its persistence in the vegetative state where it can produce its toxins.14 However, when nutritional resources are less abundant, C difficile may enter a sporulation pathway to allow for its survival. 
Germination—The Trigger for Disease
Germination is the process by which the C difficile spore uncoats and releases the vegetative form of the organism, which can produce toxins and cause disease. If germination does not occur, the host will not develop disease, though they may shed the spores asymptomatically and infect others. 
Normally, spores pass through the ileum and into the cecum. C difficile spores interact with the amino acid glycine and taurocholic acid synergistically to lead to germination of the spore and release of the vegetative form of the organism. This pathway is highly regulated by several bacterial genes. The critical regulator of this process is the CspBAC gene locus.8-11 Activation of CspC triggers a proteolytic cascade ending with release of the vegetative form of C difficile, which may then go on to produce its toxins, which in turn lead to the manifestations we recognize as C difficile–associated disease. The deletion of CspC renders C difficile unresponsive to taurocholic acid, thus inhibiting germination and preventing disease. 
Although most bile acids are reabsorbed in the small intestine via enterohepatic circulation, some are transported into the colon. In the cecum, bile salt hydrolases on the surface of microbiota remove conjugated amino acids forming primary bile acids, which are then further metabolized to the cholate derivatives, deoxycholate and chenodeoxycholate. Normally, secondary bile acids provide protection against C difficile. One microbe in particular, C scindens, transports the unconjugated bile acids into the cytoplasm where 7 alpha-dehydroxylation transforms cholate to deoxycholate and chenodeoxycholate to lithocholate.12 These secondary bile salts inhibit spore germination and vegetative growth. Elimination of these beneficial microbes prevents the formation of secondary bile acids, providing a favorable environment for C difficile to grow and cause disease. 
Sporulation
When conditions are unfavorable to growth due to a hostile microbiota or external environment, C difficile goes into hiding and sporulates. 
Sporulation allows C difficile to survive both internally within the host and in the external environment. These spores then propagate the cycle of re-infection when ingested by a susceptible host. One trigger for sporulation is the organism's need for nutrients. In the absence of nutrients, C difficile sporulates via a complex sporulation program mediated by the regulator spo0A. For sporulation to occur, spo0A must be phosphorylated by a histidine kinase to become activated. It regulates sporulation-specific RNA polymerase sigma factors, which trigger a cascade for spore development. C difficile spores are then formed rapidly, within 12 hours. In antimicrobially treated mice, infection with either an epidemic C difficile strain or a spo0A mutant strain (lacking the spo0A gene and hence unable to form spores) led to the rapid development of CDI. Disease symptoms improved in both groups after treatment with vancomycin. However, the mice infected with spo0A mutants did not shed C difficile in the stool, whereas those infected with strains with the spo0A gene developed multiple C difficile recurrences.15 This mouse model suggests that control of the genetic regulation of sporulation may be a strategy to reduce recurrent infection. 
C difficile's nutritional state and its regulators may control sporulation. There are 2 nutritional regulators, CodY and CcpA, that help regulate sporulation. CodY is a repressor that binds to DNA in the presence of guanosine-triphosphate (GTP) and branched chain amino acids and represses C difficile sporulation.6 CodY is a transcriptional regulator and sensor of the metabolic state of the cell. When nutrients are abundant, such as during exponential growth, it represses spo0A. CcpA is a transcriptional regulator keenly sensitive to carbohydrate availability that represses spo0A and other genes involved in spore formation in the presence of glucose.12 In the absence of germination into the vegetative form, which can cause disease or sporulation which propogates further infection, C difficile is eradicated. 
Why Might Certain Strains Be More Likely to Cause Recurrence or Severe Disease?
The international epidemic of CDI beginning in the mid-1990s appeared to be driven primarily by a few virulent, fluoroquinolone-resistant, epidemic strains. Mouse models have demonstrated that some strains of C difficile may be associated with hypersporulation16 and excessive virulence in their hosts. Two human C difficile strains, ribotype 027 (RT027) and 078 (RT078) have been associated with the large epidemic of CDI internationally, with a 10-fold increase in cases from 1995 to 2007.18 Why these specific strains emerged is unclear. 
One speculation has been that changes in our diet and consumption of specific foods or food additives may have introduced these strains. A recent hypothesis is that the alpha disaccharide food additive, trehalose, might be one of the triggers.17 Because of its ability to survive dehydration, it has been used as an excipient during freeze drying of pharmaceutical products and as an ingredient for dried, baked, and processed foods, including nutritional drinks and energy products, frozen foods, dairy products, and fruits. This sugar is highly acid and heat stable and became increasingly used as a food additive beginning around the year 2000. All C difficile encode a phosphotrehalose (TreA), which is essential in the metabolism of trehalose-6-phosphate to glucose and glucose-6-phosphate (G6P). Compared with other C difficile strains, RT027 and RT078 strains have markedly enhanced growth in the presence of trehalose and can grow easily even with low trehalose concentrations.17 This characteristic gives these strains a competitive advantage in the setting of elevated trehalose levels in the distal ileum and colon from increased trehalose consumption.17 
In genetically modified mice with human microbiota, the mice that were challenged with trehalose and the RT027 strain produced greater amounts of toxin B and had higher mortality rates than those that received nonepidemic strains. These data suggest that trehalose may have contributed to the epidemic of CDI.17 The role of food additives and dietary changes in driving the C difficile epidemic requires further exploration. 
Conclusion
Understanding the pathogenesis of recurrent CDI allows you to advise the patient described in the clinical vignette that her recurrent illness is due to the persistence of bacterial spores that are not eliminated by oral vancomycin. The ability of these spores to persist occurred as a result of the disruption of the diverse ecosystem in her gut. An osteopathic approach to her problem begins with understanding the relationship of the disturbed host structure to its functional consequences—diarrhea and poor quality of life. Rational treatment requires restoration of normal structure to her gut microbiota, which will enhance her immune function and prevent against further spore germination. 
Future avenues for investigation include the role of specific foods (prebiotics) and additives on the gut microbial structure and function. Can the diet be manipulated to prevent recurrent disease or boost the immune response against C difficile? At present, the investigational procedure of fecal microbiota transplantation may be the most effective means of restoring her health both structurally and functionally. Repletion of her gut microbiota has a high likelihood of breaking the cycle of recurrence. 
Author Contributions
All authors provided substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; all authors drafted the article or revised it critically for important intellectual content; all authors gave final approval of the version of the article to be published; and all authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. 
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