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By: Mary N. Wessling, Ph.D. ELS
List Biological Laboratories, Inc.’s products are being used to confront one of the most pressing problems in health care today: stopping the worldwide spread of illness caused by Clostridium difficile (CD, C Difficile Toxin), a gram-positive, spore-forming anaerobic bacillus. The spores are highly resistant to adverse environmental conditions and are frequently among the contaminants in food products, where they germinate . The CD pathogen causes severe diarrhea and pseudomembranous colitis, among other dangerous gastrointestinal ailments. At present, the estimate is 500,000 cases in hospitals and long-term care facilities, with an annual mortality rate of 15,000 to 30,000 in the US and worldwide . The estimated annual cost of treating CD infections ranges from $436 million to $3.2 billion per year in the US alone .
C Difficile Toxin in Hospitals
Historically, CD was mainly a problem for hospitals and long-term care facilities, but infections may spread rapidly into the community, especially among persons who have required antibiotic treatments that kill off competing strains in the intestines and allow CD to multiply. CD is a rapidly evolving bacterium, with hypervirulent strains contributing to the increase in mortality. At present, 234 unique genomes have been identified that cause most of the hospital outbreaks in the US and Europe. The current testing of stool samples to confirm diagnosis requires up to 3 days to differentiate between dangerous CD infection (CDI) and less harmful causes of diarrhea. A delay in diagnosis of CD presents a difficulty in treatment, making the development of more rapid diagnostic techniques a high priority.
C Difficile Toxin Types A & B
Clostridium difficile produces two major toxins, C Difficile Toxin A aka TcdA (Product #152) and C Difficile Toxin B aka TcdB (Product #155), the latter being the more virulent. These toxins inactivate the Rho-GTPase through glycosylation, and the structural bases for their activities have been clarified by X-ray diffraction, biochemical assays, and molecular dynamics . The List Labs Toxin A and B products are playing an important role in the search for a more rapid and accurate methods to diagnose CDI. To meet the demands of those more sensitive and exacting methods, List’s difficile toxins are of higher purity than previously available products. The purity of List Labs’ toxins enables diverse creative and fascinating scientific approaches that adapt and modify known analytical techniques to CDI testing. What follows (in chronological order of publication) are recent studies that relied on List Labs’ products for their results.
C Difficile Infection Studies
Molecular diagnostic techniques are increasingly being used to quantify the seriousness of infection and to distinguish CDI from other causes. The study by Moura et al  used List Labs’ products (purified TcdA and TcdB) for a proteomic analysis that identified and quantified the protein factors involved in CD toxin production through an enhanced mass spectrometric (MS) method. This method provided a basis for development of improved MS methods that demand only small samples and contribute to a better understanding of toxin-mediated illnesses, their prevention and therapy.
In the same year, Lei and Bochner  used List Labs’ Toxins A and B in phenotype microarrays (PMs) under different culture conditions. Noting that with the evolution of the CD genome, multiple lineages evolved independently; they examined how the Toxin B cytopathic effect caused cell rounding and used that to measure the virulence of CD under different conditions. Using the PMs, they developed a 1-day test that compared the pure List Labs’ toxins and unpurified toxins to see parallels, which could be measured by colorimetry. This provided a more rapid test than the 3-day diagnostic tests currently in use.
In 2014, Huang et al  used CD toxin B to approach the problem of distinguishing between infection, colonization, and live and dead CD organisms. Their real-time microelectronic sensor-based analysis had high sensitivity and relied on reading the impedance of cells applied to microelectrodes to detect specific cellular processes through quantification over time. The method is rapid: the authors reported that 80% positive results were obtained within 24 hours. Concentration of the CD in stool measured by their method correlated with clinical severity, providing a method to monitor the progress of CDI in patients.
In 2014, an innovative study by Leslie et al  used human intestinal organoids (HOIs) derived from stem cells to model the disruption of barrier functions in the human intestine by CD. The HOIs were generated by directed differentiation of human pluripotent stem cells, which were then further differentiated into intestinal tissue. These HOI’s were subjected to the C. difficile strain, TcdA or TcdB. Increased toxicity under conditions favorable to production of toxins by CD was measured by presence of cell rounding using fluorescent dextran. Injection of TcdA replicates the disruption of the epithelial barrier function and structure observed in HIOs colonized with viable CD.
In 2015, Hong et al  used an extension of a method they had developed previously combined with ELISA, eventually applying single-stranded DNA molecular recognition elements (MRE) to microchips. They used List Labs’ lyophilized toxin B, reconstituted and immobilized on magnetic beads; after incubation with an ssDNA library, 80 randomly selected clones were sequenced and analyzed. The goal was to identify ssDNA MREs that bind to toxin B; eventually, fluorescence was used to examine the structure of one selected MRE bound to toxin B. This very complex process yielded an MRE bound with high specificity with toxin B in human fecal matter; it demonstrated a proof-of-concept diagnostic application.
In quite another approach to preventing the morbidity and mortality attributed to CD, a study by Zilbermintz et al  showed that the antimalarial drug amodiaquine has a protective effect against CD. The drug was one of a group of existing FDA-approved compounds screened to extend their use to as broad-spectrum, host-oriented therapies. Amodiaquine interferes with the functioning of the host protein cathepsin B targeted by CD and other pathogens as well. The aim of their approach is to find therapies that circumvent the effect of pathogen mutations that lead to drug resistance. All toxins used in the study were purchased from List Biological Laboratories. Though not a new diagnostic technique, this discovery promised an approach to CDI using existing pharmacology, which then could reduce the immense cost of treating CD patients .
By: Suzanne Canada, Ph.D.
Tanager Medical Writing
While you go about your day, you are surrounded by micro-organisms. Although most of us spend a lot of time washing up and trying not to think about the propensity of creatures that share our personal space, scientists have been studying them. Due to their great progress, we are reaching an understanding of how these bacteria and fungi affect our bodies’ functions . The evidence indicates that this inner-ecosystem can not only cause disease if perturbed, but also influence our overall health! Organisms including Escherichia coli, Helicobacter pylori, Streptococcus thermophilus, and species of Clostridia, Lactobacillus, and Bacterioides inhabit our gut. Corynebacterium jeikeium as well as Staphylococcus species live on our skin, and other Streptococci as well as Neisseria and Candida albicans inhabit our mouth and upper respiratory system . The makeup and diversity of organisms has been found to be strongly influenced, not only by what you eat , but also by who you live with [4, 5]. With greater understanding of this rich soup of life that we carry with us, the microbiome has become the new frontier in cutting-edge drug development .
In the last three years, research into the molecular basis of microbial influence has blossomed. The first and most obvious application for this information was in treating C. difficile infections; which result from overgrowth of the opportunistic pathogen after an antibiotic regimen or hospital stay. Researchers found that fecal transplants from a healthy individual were an effective way to treat this potentially fatal infection [7, 8]. The role of intestinal microbes in Inflammatory Bowel Disease (IBD) has been established  in the last year or two. Based on this knowledge, possible treatments for IBD, such as ulcerative colitis and Crohn’s disease, are in development. Other publications point to microbes’ role in inflammation of the skin and respiratory tract, including acne and asthma. More excitement has been generated as investigators have found links to other chronic diseases including diabetes [12, 13], hypertension [14, 15], and chronic liver disease . Preliminary investigations suggest a connection between overall gut microbial composition and obesity . Some studies in mouse models have even linked the microbiome to the neurological conditions of Alzheimer’s  and autism [19, 20].
With all this research going on, you need a great resource like LIST Biological Laboratories, with experience and expertise with microbial products spanning over 25 years. LIST has several products available that can serve as positive controls for your microbial research. Potent toxins from C. difficile are available (LIST products #157, #158), as well as antibodies that aid in their detection (LIST products #753, #754). Lipopolysaccharides are also available, which cause inflammation and activation of immune signaling cascades, and are extracted from bacterial cell walls of E. coli O111:B4, O55:B5, O157:H7, J5 and K12; Salmonella typhimurium, Salmonella minnesota and Bordetella pertussis. Other acute immune system activators such as Staphylococcal toxins (LIST products #120, #122) and Shiga toxins (LIST products #161 & #162) are also available.
In case the assortment of purified bacterial products on hand are insufficient for your research needs, LIST also provides contract manufacturing for biotherapeutics, as well as microbial purification services.
What is Clostridium Difficile?
Clostridium difficile is the causative agent of antibiotic-associated diarrhea and pseudomembranous colitis. It produces two major exotoxins, Toxin A and Toxin B; however, about 10% of strains isolated from patients with colitis also contain genes, coding for a unique ADP-ribosylating toxin, CDT Binary Toxin. Additionally, Clostridium difficile produces and secretes a glutamate dehydrogenase (cdGDH).
List Labs offers four new products in the C. difficile family
New product offerings from List Labs cover other proteins produced concurrently with the exotoxins. These proteins are valuable as alternate markers allowing more sensitive or more accurate determination of C. difficile infections (CDI).
These new products, all related to C. difficile, will be of interest to diagnostic developers, vaccine manufacturers, as well as, to those doing research in infectious diseases. List Labs is notably offering antigens and antibodies for two C. difficile proteins which are present in C. difficile infections. These products add to our C. difficile reagents which also include the main virulence factors Toxin A and Toxin B, and the antibodies: Goat Anti-Toxin A, Chicken Anti-Toxin B and Chicken Anti-Toxin B.
The first two products are components of CDT Binary Toxin, an ADP-ribosyltransferase. This toxin is composed of two independently produced components, the enzymatic subunit A, CdtA, and the binding and translocation subunit B, CdtB, which mediates cell entry of CdtA. CDT Binary Toxin causes depolymerization of the actin cytoskeleton and formation of microtubule-based membrane protrusions,resulting in cell rounding and cell death, and it is suggested to be involved in enhanced bacterial adhesion and colonization of hypervirulent C. difficile strains. The cell surface receptor has been identified as lipolysis stimulated lipoprotein receptor (LSR).
CDT Binary Toxin, A Subunit (CDTa), Product # 157, is recombinantly expressed in E. coli and purified using affinity chromatography. The affinity tag has subsequently been cleaved from the protein prior to packaging. Binary toxin A subunit has been tested in an in vitro ADP-ribosylation assay. It is non-toxic and unable to penetrate cells in the absence of the B subunit binding and translocation domain. Expression and purification of the A subunit from a recombinant setting ensures that there is no possible contamination with the B subunit.
1) #157A, Binary Toxin from Clostridium difficile A Subunit 20 ug, price $350
CDT Binary Toxin, B Subunit (CDTb), Product # 158, is recombinantly expressed in E. coli, purified using affinity chromatography and the affinity tag cleaved. Prior to packaging, the B subunit is nicked with trypsin for activation. The B subunit of the Binary Toxin is non-toxic, and does not contain any enzymatic activity.
2) #158A, Binary Toxin from Clostridium difficile B Subunit 40 ug, price $350
The next products are chicken antibodies: Chicken Anti-CDT Binary B subunit antibodies, with and without biotin. Antibodies have been raised against C. difficile Binary Toxin B Subunit and affinity purified on an antigen column, Product # 758. These antibodies are suitable for use in Western Blot assays and ELISAs as an effective probe for C. difficile Binary Toxin B Subunit. Additionally, purified antibody has been labeled with biotin, Product # 759, providing antibodies for both capture and detection.
3) #758A, Anti – C. difficile Binary Toxin B Subunit (Chicken IgY) 0.1 mg, price $290
4) #759A, Biotinylated Anti – C. difficile Binary Toxin B Subunit (Chicken IgY) 0.1 mg, price $315
Use of CDT on subconfluent Caco-2 cells is described by Schwan et al, 2009. Toxin –induced cellular processes were observed on these cells after one hour treatment with CDT Binary Toxin, a mixture of 20 ng/ml of CDTa and 40 ng/ml of CDTb.
Use our new Citations Finder to see additional citations of how C. Difficile from List Labs has been used in research.
What Are Toxin Neutralization Assays & How Do They Work?
For clinical detection or vaccine testing, it is hard to beat a toxin neutralization assay. Toxin neutralization assays (TNA) assess the ability of antibodies to protect cells in culture from the cytotoxic affect of the specific toxins. Interestingly, these assays may be used for sensitive and reliable testing for disease states where toxins are involved, as well as for development of vaccines to treat infectious disease. In TNA testing, potential sources of toxin and antibodies are combined and applied to cell culture in a series of dilutions. Excess toxin in the sample, not neutralized by the antibody, will kill the cells, the amount of excess toxin determined by the dilution of the sample which will cause a defined amount of cell death. The end point in such assays is cell viability, and this may be visualized by several different methods. A commonly used method is to visualize viable cells through metabolism of a staining reagent; the intensity of the developed color is directly proportional to the percent of remaining cell viability. TNA assays can also be used as a definitive identification of the causal agent of the disease.
TNA Assays for Clostridium Difficile Diagnosis
Cytotoxin neutralization (CTN or TNA) assays have great value in the specific diagnosis of C. difficile. Laboratory diagnosis is described by Alfa and Sepehri (Alfa, 2013). These assays can progress through a stepwise process starting with testing for glutamate dehydrogenase (GDH) in stool from potential C. difficile infected (CDI) patients. C. difficile GDH (cdGDH) is a highly active enzyme which can be readily detected and correlates well with C. difficile infections. Test results that are negative for GDH can identify samples in which C. difficile is highly unlikely, whereas tests positive for this enzyme can be used to identify potential C. difficile infections. However, since GDH is also produced by other inhabitants of the digestive tract, the presence of GDH is not conclusive evidence of C. difficile. To take diagnosis a step farther, immunological assays for C. difficile toxins A and B are used and when positive, identify C. difficile infections. Low sensitivity of these assays produce false negative results when only a small amount of toxin is present; this is when a TNA assay on highly sensitive cells comes into play. Depending on the type of cell culture, it is possible to detect C. difficile toxin B at a concentration of picograms per ml. Because cell cultures may be killed by a variety of components in a test sample, specific identity of the toxin relies on the use of standard neutralizing antibodies directed uniquely to C. difficile toxin A or B. When the antibody protects the cells from toxin directed death, the presence of, for example, the C. difficile toxin B is shown; this is a positive indication of a CDI patient. Toxin neutralization is a valuable assay in identifying patients infected with C. difficile and List Labs products are used in the development of these assays and subsequent testing to detect the toxins in samples. Products available from List Lab are C. difficile toxin A, C. difficile toxin B, our new product C. difficile GDH as well as antibodies directed to these three proteins, all of which are used to perform TNA.
TNA Assays Used to Evaluate Potential Vaccines
Another equally important use of toxin neutralization is in testing for the evaluation of potential vaccines. A paper published in 2013 by Xie et al describes a TNA developed for the evaluation of hyperimmune sera raised in animals against potential C. difficile toxin A (TcdA) and toxin B (TcdB) toxoid vaccine candidates. The authors optimized the assay using Vero cells for detection of neutralizing antibodies and for the determination of toxin potency.
TNA Assays for Anthrax Vaccines
Similar toxin neutralization assays have been developed and optimized for anthrax vaccines. These assays have been in use for over 10 years. A good review of these assays using different types of cultured cells to measure antibody levels created in response to different vaccines was provided by Ngundi et al, 2010. Anthrax toxin products available from List Labs are protective antigen (PA), lethal factor (LF), edema factor (EF), as well as the respective antibodies, all of which are used in these assays.
Alfa et al (2013) Combination of culture, antigen and toxin detection, and cytotoxin neutralization assay for optimal Clostridium difficile diagnostic testing. Can J Infect Dis Med Microbiol 24(2) 89-92. PMCID 3720004
Ngundi et al (2010) Comparison of Three Anthrax Toxin Neutralization Assays. Clinical and Vaccine Immunology 17(6) 895–903. PMID: 20375243
Xie et al (2013) Development and Optimization of a Novel Assay to Measure Neutralizing Antibodies Against Clostridium difficile Toxins. Clinical and Vaccine Immunology 20(4) 517-525. PMID: 23389929
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
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