Clostridia Detection and Comparison of Organic Acid Detection Versus Stool Testing

William Shaw, Ph.D.

Continued research at The Great Plains Laboratory has resulted in new information on Clostridia bacteria markers that will soon be available for the urine organic acid test. New information will soon be available for the organic acid interpretations of 3 (3 hydroxyphenyl)-3 hydroxypropionic acid (HPHPA), 4-hydroxyphenylacetic acid, phenyllactic acid, and 3-indoleacetic acid at the beginning of 2015.

In addition, this article will help to clarify information about the increased value of organic acid testing compared to stool testing for assessing Clostridia species.


First, the species that are the major producers of the precursors of HPHPA have been identified and include C. botulinum, C. sporogenes, and C.caloritolerans. (It is common to use the abbreviation for the Clostridia genus "C" when giving the genus and species designation.)

C. botulinum is a gram-positive, rod-shaped, anaerobic, spore-forming, motile bacterium with the ability to produce the neurotoxin botulinum. The botulinum toxin can cause a severe flaccid paralytic disease in humans and animals and is the most potent toxin known to humankind (natural or synthetic) with a lethal dose of less than 1 μg (microgram) in humans. Symptoms of botulism include weakness, trouble seeing, feeling tired, and trouble speaking. This may then be followed by weakness of the arms, chest muscles, and legs. In food borne botulism, symptoms generally begin 18 to 36 hours after eating a contaminated food, but they can occur as early as 6 hours or as late as 10 days after eating the food.

It is interesting that the symptoms of botulism vary widely from a mild illness for which the patient may seek no medical treatment to a fulminant disease, killing within 24 hours (1). Since laboratory testing for this organism is only available at state health departments, it seems likely that many cases of botulism, especially the mild cases, may be undiagnosed. I suspect that some children with autistic behavior,with extremely high urine HPHPA, little or no speech, and extremely severe low muscle tone might actually have undiagnosed botulism, and further research on this possibility is warranted.

C. sporogenes is virtually identical to C. botulinum except it is lacking the gene for the botulinum neurotoxin. Like C. botulinum, it is an anaerobic gram-positive, rod-shaped bacterium that produces oval, subterminal endospores, and is commonly found in soil.

C. caloritolerans is named after its extreme heat (calor) resistance (tolerans). It can survive at the boiling point for 8 hours (2); its ability to resist heat may allow transmission even in well-cooked food. No scientific papers on any disease associations (other than my own articles dealing with its production of HPHPA) were found, which means there is still a great deal of research opportunity for microbiologists in the future.


4-Hydroxyphenylacetic Acid

High 4-hydroxyphenylacetic acid is associated with small intestinal bacteria overgrowth due to its production by the following Clostridia bacteria: C. diificile, C. stricklandii, C. lituseburense, C. subterminale, C. putrefaciens, and C. propionicum. C. difficile can be distinguished from the other species by its production of 4-cresol; none of the other species produce 4-cresol. No information on the pathogenicity of the other species producing 4-hydroxyphenylacetic acid is available. However, it is likely that 4-hydroxyphenylacetic is also an inhibitor of dopamine-beta-hydroxylase and appropriate treatment with probiotics or antibiotics may be clinically useful. 4-hydroxyphenylacetic acid is associated with bacterial overgrowth of the small intestine (3). Elevated values are common in celiac disease and cystic fibrosis, and have also been reported in jejuna web, transient lactose intolerance, Giardia infection, ileal resection, ileo-colic intersusseception, septicemia, and projectile vomiting. The elevations of 4-hydroxyphenylacetic acid in celiac disease and cystic fibrosis are so prevalent that involvement of these Clostridia bacteria may play a role in these illnesses. In C. difficileinfections 4-hydroxyphenylacetic acid is utilized by this bacteria to produce 4-cresol.


Phenyllactic Acid

Very high amounts of phenyllactic acid are found in the rare genetic disease phenylketonuria (PKU). Moderate amounts of phenyllactic acid may be due to gastrointestinal overgrowth of the intestine of the following Clostridia bacteria: C. sordellii, C. stricklandii, C. mangenoti, C. ghoni, and C. bifermentans. C sordellii is usually considered a nonpathogen except in immunocompromised people, but has been implicated in catastrophic infectious gynecologic illnesses among women of childbearing age. The other species have rarely or never been reported to be pathogenic.


3-Indoleacetic Acid

High 3-indoleacetic acid in urine is a byproduct of C. stricklandii, C. lituseburense, C. subterminale, and C. putrefaciens. No information on the pathogenicity of these species producing indoleacetic acid is available. However, very high amounts of this metabolite derived from tryptophan might indicate a depletion of tryptophan needed for other physiological functions.



4-cresol is predominantly produced by C. difficile, a pathogenic bacteria, that is one of the most common pathogens spread in hospitals. Toxin-producing strains of C. difficile can cause illness ranging from mild or moderate diarrhea to pseudomembranous colitis, which can lead to toxic dilatation of the colon (megacolon), sepsis, and death (4). 4-cresol (para-cresol) has been used as a specific marker for Clostridium difficile (5). 4-Cresol, a phenolic compound, is classified as a type-B toxic agent and can cause rapid circulatory collapse and death in humans (6). Yokoyama et al. (7) have recently proposed that intestinal production of 4-cresol may be responsible for a growth-depressing effect on animals. Signs of acute toxicity in animals typically include hypoactivity, salivation, tremors and convulsions. High amounts of 4-cresol have been found in autism (8); the amount of 4- cresol in the urine has been found elevated in baseline samples and in replica samples of autistic children. Higher values of 4-cresol are found in girls with autism compared to boys with autism and higher values are associated with greater clinical severity of autistic symptoms and history of behavioral regression. 4-cresol is apparently produced by Clostridia difficile as an antimicrobial compound that kills other species of bacteria in the gastrointestinal tract, allowing the Clostridia difficile to proliferate and predominate.


Organic acid test superior to stool testing for Clostridia testing

C. difficile is the only species of 100 species of Clostridia from the gastrointestinal tract to be commonly tested in hospital laboratories throughout the world. However, this species is not commonly cultured, but rather is detected by its toxin formation. The gastrointestinal damage caused by C. difficile is thought to be due to exposure to two toxins produced by C. difficile, toxin A and toxin B, with toxin B considered to be more toxic (4). The toxins can be tested by immunoassay of stool samples which is a fairly rapid test. Toxigenic stool culture, which requires growing the bacteria in a culture and detecting the presence of the toxins, is the most sensitive test for C. difficile, and it is still considered to be the gold standard (4). However, it can take 2 to 3 days for results. Polymerase chain reaction (PCR) evaluation of the C. difficile toxins is also becoming more available. Virtually all of the research on C. difficile is related to the effects of this species of bacteria on the intestinal tract. Toxin-negative C. difficile strains are considered nonpathogenic for the infection of the intestine (4) but cresol producing strains that don't produce toxins and B may be pathogenic due to their effects on brain metabolism and for the inherent toxicity of 4-cresol itself.

In addition, urinary 4-cresol elevations associated with C. difficileovergrowth are much less common than urinary HPHPA elevations associated with other Clostridia species. In a survey of 1000 consecutive samples submitted for urine organic acids tests, The Great Plains Laboratory found that 15.2% were abnormally elevated for HPHPA, 6.8% were abnormally elevated for 4-cresol, and 1.6% were abnormally elevated for both HPHPA and 4-cresol for a total positive percentage of 23.6%. Thus, if only stool testing for Clostridium difficile is performed on a patient, at least 15.2/23.6 or 64.4% (nearly two-thirds) of patients with clinically significant infections with other types of Clostridia might be missed.

Sometimes total Clostridia are tested using culture methods or PCR (polymerase chain reaction) technology. In one case, a parent showed me the stool test results of their child with autism. They had done a stool test with a laboratory using PCR technology to determine both C. difficile and total Clostridia. The total Clostridia was reported as extremely low and the C. difficile negative, but The Great Plains Laboratory organic acid test found high levels of the HPHPA marker. If the parent had relied on the stool test alone, their child might have missed an important therapeutic intervention that can restore normal neurotransmitter balance. The advantage of The Great Plains Laboratory organic acid test is that it is not necessary to determine particular species of Clostridia because it is the HPHPA and/or 4-cresol that are neurotoxic.

People sometimes assume that a test using DNA is more accurate than other types of testing. However, DNA testing is fraught with complexities. The nucleic acids of Clostridia are extremely diverse. The content of the nucleic acid bases guanosine and cytosine (G+C) is used to classify bacteria species. The G+C content of DNA of Clostridia species ranges from 21-54 % (9). The majority of intestinal species have G+C contents in the lower half of this range. Ribosomal RNA cataloging confirms that Clostridia occupy six independent sublines with multiple branches including non-Clostridia species. The failure to offer documentation on which species are being detected and how validation was performed should lead to caution by the user of such testing, especially when such tests may be labeled "experimental". Similar complexities exist with traditional culture methods for Clostridia since results are commonly reported from 0 to 4+. Since many Clostridia are not pathogenic, what does a high Clostridia level of 4+ indicate since beneficial, neutral, and harmful species are lumped together in one category? In reality, the results of stool tests for total Clostridia are virtually meaningless and may lead to inappropriate patient treatment.

It is estimated that there are about 10 billion cells of Clostridia per gram of stool. Clostridium ramosum is the most common (53% of all subjects tested) with a mean count of about one billion per gram of stool (9). The prevalence of some Clostridia species is highly dependent on diet. Stool samples of vegetarians did not contain Clostridium perfringens whereas meat and fish eaters had high amounts (10).

Since HPHPA is associated with multiple species of Clostridia but not Clostridium difficile, there is really no available confirmation test for determining the specific species of Clostridium producing HPHPA. As mentioned above, stool testing for total Clostridia is useless since it cannot currently differentiate between harmful or beneficial species. Since HPHPA, in my experience, disappears after treatment with vancomycin or metronidazole, I always recommend treatment based on the HPHPA value with a follow-up test 30 days after completion of treatment.

Confirmation testing of Clostridium difficile could be performed when 4-cresol is elevated. However, the prevalent testing for Clostridium difficile toxins A and B are focused on strains that cause gastrointestinal damage. Strains that produce 4-cresol but not toxins A or B may still cause significant psychiatric disease, so performing these toxin tests may muddy the interpretation of the clinical situation if these tests are negative. I think that it is easier to treat based on the 4-cresol results and then do follow-up testing of the 4-cresol on the organic acid test 30 days after completion of treatment.

Clinical References

  • Beatty, H. Botulism. In: Harrison's Principles of Internal Medicine, 10th edition, ed. R. Petersdorf, et al. McGraw Hill. New York. 1983. Pages 1009-1013.
  • Meyer, K.F. and Lang, O.W. A highly heat-resistant sporulating anaerobic bacterium: Clostridium caloritolerans, N. SP. The Journal of Infectious Diseases Vol. 39, No. 4 (Oct., 1926), pp. 321-327
  • Chalmers, R.A., Valman. H.B., and Liberman, M.M., Measurement of 4-hydroxyphenylacetic aciduria as a screening test for small-bowel disease. Clin Chem 25:1791, 1979
  • Carrico, R.M. Association for Professionals in Infection Control and Epidemiology (APIC) Implementation Guide to Preventing Clostridium difficile Infections (accessed Oct 30,2014)
  • Sivsammye, G. and Sims, H.V. Presumptive identification of Clostridium difficile by detection of p-cresol (4-cresol) in prepared peptone yeast glucose broth supplemented with p-hydroxyphenylacetic acid. J Clin Microbiol. Aug 1990; 28(8): 1851–1853.
  • Phua, T.J., Rogers, T.R., and Pallett, A.P. Prospective study of Clostridium difficile colonization and paracresol detection in the stools of babies on a special care unit. J. Hyg., Camb. (1984). 93. 17-25 17
  • Yokoyama, M. T., Tabori, C., Miller, E. R. and Hogberg, M. G. (1982). The effects of antibiotics in the weanling pig diet on growth and the excretion of volatile phenolic and aromatic bacterial metabolites. The American Journal of Clinical Nutrition 35, 1417-1424.
  • Persico, A.M. and Napolioni, V. Urinary p-cresol (4-cresol) in autism spectrum disorder. Neurotoxicology and Teratology 36 (2012) 82–90
  • Wells, J.M. and Allison, C. Molecular genetics of intestinal anaerobes. In: Human Colonic Bacteria. Role in Nutrition, Physiology, and Pathology. Gibson and MacFarlane, ed. CRC Press. Ann Arbor. 1995. Page28
  • 10. Conway, P. Microbial ecology of the human large intestine. In: Human Colonic Bacteria. Role in Nutrition, Physiology, and Pathology. Gibson and MacFarlane, ed. CRC Press. Ann Arbor. 1995. Pages 1-24