By: Karen Crawford, Ph.D.
President, List Biological Laboratories, Inc.
You hear about Clostridium difficile at your doctor’s office and in news articles, but what does it mean and how does it affect the world around us?
C. Difficile Statisitcs
- C. Difficile Mortality Rates: Complications from C. difficile infection (CDI) have become an increasing patient safety concern worldwide (CDC). Mortality rates from C. difficile-associated disease have been increasing, from 5.7 deaths per million (1999) to 23.7 per million (2004) in the USA, which is higher than deaths caused from all other intestinal infectious diseases combined.
- Cost of Treating C. Diff: The cost of treating CDIs in the USA is estimated at over $3 billion per year.
- Increased Virulence of C. Difficile Strains: Hypervirulent C.difficile strains, toxinotype III and ribotype 027 (B1/NAP1) cause C. difficile infections with high rates of pseudomembranous colitis and mortality. Increased virulence of these strains relative to the classical strains has been attributed to a natural resistance to fluoroquinoline (McDonald, 2005), higher sporulation rates(Akerlund, 2008), production of a third toxin, binary toxin or C. difficile transferase (CDT) which adds to pathogenicity and enhances colonization(Schwan, 2009), and more efficient translocation of the toxin(Lanis, 2010).
- Widespread in All Types of Facilities: In 2012, the Centers for Disease Control and Prevention reported that CDI now affects all types of medical care facilities, including community care facilities such as retirement homes.
How C. Difficile Affects the Intestine
C. difficile enterotoxins A and B are the key to pathogenesis of CDI. C. difficile toxin A (TcdA) and toxin B (TcdB) are both cytotoxic and cause inflammation in intestine, but they have slightly different activities (Theriot, 2013). Toxin B is an extremely potent cytotoxin, that glycosylates small GTPase of the Rho family (Cdc42 and Rac) which control the actin cytoskeleton in eukaryotic host cells; this glycosylation disrupts signaling pathways of the cell cycle and lead to apoptosis. TcdA has an activity like TcdB, but it is much less potent as a cytotoxin, but more commonly noted for its enterotoxic activity and large size (308 kDa vs 270 kDa for TcdB). These toxins are the major virulence factors for C. difficile and cause inflammation and damage to cells in the intestine when the normal gut microflora are disrupted, such as after a round of treatment with antibiotics (Theriot, 2013; Carter, 2010).
Earlier studies using animal models of CDI had suggested that the toxins act synergistically because purified TcdA alone was able to induce C. difficile disease pathology and TcdB was not effective unless it was co-administered with TcdA. However, the isolation of some new, clinically relevant toxin A-negative, toxin B-positive (A−B+) strains of Clostridium difficile from humans (Drudy 2010), indicated that toxin B may be the key to its virulence as a pathogen (Lyras 2009, Carter 2010). The emergence of these new strains has prompted researchers to evaluate current C. difficile diagnostic methods (Alder 2014, Brown 2011, Garamella 2012, Grein 2014) and recommend ensuring that medical laboratories can detect both TcdA and TcdB in specimens.
List Labs Offers TcdA and TcdB for Purchase
List Biological Laboratories has been producing TcdA and TcdB since 2000. These toxins are purified proteins that are tested to ensure that the activity is preserved. Along with chicken antibodies to each toxin, TcdA and TcdB can be used in disease modeling as well as the development of diagnostic tools for CDI detection and diagnosis.
Adler A, Schwartzberg Y, Samra Z, Schwartz O, Carmeli Y, et al. (2014) Trends and Changes in Clostridium difficile Diagnostic Policies and Their Impact on the Proportion of Positive Samples: a National Survey. Clin Microbiol Infect Mar 27. doi: 10.1111/1469-0691.12634. [Epub ahead of print]. PMID: 24674056
Akerlund T, Persson I, Unemo M, Noren T, Svenungsson B, Wullt M, Burman LG (2008) Increased sporulation rate of epidemic Clostridium difficile type 027/nap1. J Clin Microbiol 46: 1530–1533. PMID: 18287318
Brown NA, Lebar WD, Young CL, Hankerd RE, Newton DW (2011) Diagnosis of Clostridium difficile infection: comparison of four methods on specimens collected in Cary-Blair transport medium and tcdB PCR on fresh versus frozen samples. Infect Dis Rep 3(1):e5. PMID: 24470904
Carter GP, Rood JI, Lyras, D (2010) The role of toxin A and toxin B in Clostridium difficile-associated disease: Past and present perspectives. Gut Microbes 1(1):58-64. PMCID: PMC2906822
Drudy D, Fanning S, Kyne L (2010) Toxin A-negative, toxin B-positive Clostridium difficile. Int J Infect Dis 11(1):5-10. PMID: 16857405
Garimella PS, Agarwal R, Katz A (2012) The utility of repeat enzyme immunoassay testing for the diagnosis of Clostridium difficile infection: a systematic review of the literature. J Postgrad Med 58(3):194-8. PMID: 23023352
Grein JD, Ochner M, Hoang H, Jin A, Morgan MA, Murthy AR (2014) Comparison of testing approaches for Clostridium difficile infection at a large community hospital. Clin Microbiol Infect 20(1):65-9. PMID: 23521523
Lanis JM, Barua S, Ballard JD (2010) Variations in TcdB activity and the hypervirulence of emerging strains of Clostridium difficile . PLoS Pathog 6:e1001061. PMID: 20808849
Lyras D, O’Connor JR, Howarth PM, Sambol SP, Carter GP, et al. (2009) Toxin B is essential for virulence of Clostridium difficile. Nature 458(7242): 1176–1179. PMID: 19252482
McDonald LC, Killgore GE, Thompson A, Owens RC Jr, Kazakova SV, Sambol SP, Johnson S, Gerding DN (2005) An epidemic, toxin gene-variant strain of Clostridium difficile. N Engl J Med 353: 2433–2441. PMID: 16322603
Schwan C, Stecher B, Tzivelekidis T, van Ham M, Rohde M, et al. (2009) Clostridium difficile Toxin CDT Induces Formation of Microtubule-Based Protrusions and Increases Adherence of Bacteria. PLoS Pathog 5: e1000626. PMID: 19834554
Theriot CM, Young VB (2013) Microbial and metabolic interactions between the gastrointestinal tract and Clostridium difficile infection Gut Microbes 5(1). PMID: 24335555
Shiga Toxins May be Purchased without Government Approval
The CDC has removed Shiga toxins (Stx1 and Stx2) from the list of materials requiring oversight. As a result, Shiga Toxins are no longer classified as select agents and may be purchased without government approval for your research and investigative needs. While Shiga toxins carry fewer restrictions, the interest in them and their value for research has never been higher.
Usage of Shiga Toxins in Research
As tools, these cytotoxins are valuable in studying intracellular transport within the Golgi apparatus. They can be used to eliminate mammalian cell types with Gb3 receptors. Shiga toxins are potent virulence factors, important in human health. They are implicated in many cases of food borne illness, estimated to affect 76 million people and cause 5,000 deaths every year in the United States alone. Shiga toxin producing bacteria, usually Escherichia coli O157, enter the food chain through contamination, infect the gastrointestinal tract and cause diarrheal illness. The bacteria infect the large intestine and produce Shiga toxin which crosses the gastrointestinal epithelium entering the blood stream; ultimately the toxins are responsible for organ damage. These potent virulence factors are important targets for the development of therapies and for the detection of contamination.
Shiga toxins function by inhibiting eukaryotic protein synthesis by cleaving a specific adenine from the 28S RNA of the 60S subunit of the ribosome. Although Shiga toxin 1 and Shiga toxin 2 share only 56% amino acid homology, making them immunologically distinct, activities of these two forms of toxin, binding affinity to Gb3 and N-glycosidase activity, appear to be identical. In spite of these similarities, Shiga toxin 2 is more closely associated with human disease. Although endothelial cells are the primary cell type vulnerable to shiga toxin, several other types express Gb3 receptors and are therefore potential targets.
Get Shiga Toxins for Research from ListLabs
Both Shiga 1 and Shiga 2 and mouse antibodies to the toxins are available from List Labs. You can read more about them here. At this time we are evaluating polyclonal and monoclonal antibodies that recognize all seven subtypes of Stx2 and monoclonals that recognize all subtypes of Stx1. Look for these antibodies to appear in our future offerings.