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DNA detection of gut microbiota advancing routine characterization of microbial populations.

Introduction

The population of the microbiota of the human gastrointestinal (GI) tract is widely diverse and complex with a high population density. All major groups of organisms are represented in relative amounts that change enormously from mouth to anus (see Figure 1). The populations are disrupted by infections, antibiotics, inadequate digestion, and immune system stress. (1) While the microbial mass is comprised predominately of bacteria, a variety of fungi and protozoa are also present. In the colon, there are over [10.sup.11] bacterial cells per gram and over 400 different species. These bacterial cells outnumber host cells by at least a factor of ten. This microbial population has important influences on host physiological, nutritional, and immunological processes. In fact, this biomass should more rightly be considered a rapidly adapting, renewable organ with considerable metabolic activity and significant influence on human health. Consequently, there is renewed and growing interest in routine clinical identification of the types and activities of gut microbes. (2)

The normal, healthy balance in microbiota provides colonization resistance to pathogens. Since anaerobes comprise over 95% of the bacterial population, their analysis is of prime importance. Gut microbes might also stimulate immune responses to prevent conditions such as intestinal dysbiosis (a state of disordered microbial ecology that causes disease). Specifically, the concept of dysbiosis rests on the assumption that patterns of intestinal flora, specifically overgrowth of some microorganisms found commonly in intestinal flora, have an impact on human health. Symptoms and conditions thought to be caused or complicated by dysbiosis include inflammatory bowel diseases, inflammatory or autoimmune disorders, food allergy, atopic eczema, unexplained fatigue, arthritis, mental/emotional disorders in both children and adults, malnutrition, and breast and colon cancers. (3, 4)

Difficulties in Accurately Assessing Microbiota Content

Most studies of microbiota in the human GI tract have used fecal samples. These do not necessarily represent the populations along the entire GI tract from stomach to rectum. Conditions and species can alter greatly along this tract and generally increase from lower to higher population densities as shown in Figure 1. The stomach and proximal small intestine with highly acid conditions and rapid flow contain [10.sup.3] to [10.sup.5] bacteria per gram of content. These are predominated by acid tolerant lactobacilli and streptococci bacteria. The distal small intestine to the ileocecal valve usually ranges to [10.sup.8] bacteria per gram. The large intestine generates the highest growth due to longer residence time, and population densities range from [10.sup.10] to [10.sup.11] bacteria per gram. The bacterial metabolism of this region generates a low redox potential and high amount of short-chain fatty acids.

Not only does the total microbiota change throughout the length of the GI tract, but there are different microenvironments where these organisms grow. At least four microhabitats exist: the intestinal lumen, the unstirred mucus layer that covers the epithelium, the deeper mucus layer in the crypts between villi, and the surface mucosa of the epithelial cells. (5,6) Given this diverse ecological community, the question arises as to how to sample the various environments to identify populations of microbes and ultimately understand the host-microbe interactions. This problem is an extremely difficult one, since any intervention to obtain a sample potentially disrupts the population. The practice of fecal sampling should be understood primarily to represent organisms growing in the colon. Since more than 98% of fecal bacteria will not grow in oxygen, (5) standard culture techniques miss the majority of organisms present.

Conventional Techniques Vs. New Technologies

Conventional bacteriological methods like microscopy, culture, and identification may be used for the analysis and/or quantification of the intestinal microbiota. (7-9) Limitations of conventional methods include low sensitivities, (10) inability to detect noncultivatable bacteria and unknown species, time-consuming aspects, and low levels of reproducibility due to the multitude of species to be identified and quantified. In addition, the large differences in growth rates and growth requirements of the different species present in the human gut indicate that quantification by culture is bound to be inaccurate due to restrictions of growth media choices. To overcome the problems of culture, techniques based on 16S ribosomal DNA (rDNA) genes were developed. (11,12) These include fluorescent in situ hybridization, (13-17) denaturing gradient gel electrophoresis, (18,20) and temperature gradient gel electrophoresis. These techniques have high sensitivities, although they are laborious and technically demanding.

Another problematic issue with present stool analysis procedures is that of specimen transport. Since growth in culture media requires living organisms, sample collection must be done using nutrient broth containers to maintain microbial viability. This allows continued growth of species during transport and until the sample is actually plated out for culture in the laboratory. This growth allows for a significant change in the balance of microbes from that which was present in the patient. Some species will more actively grow at the expense of others. DNA analysis eliminates this problem by placing the specimen in formalin vials for transport. This immediately kills all organisms, freezing the exact balance present at the time of collection. Since PCR identification is only looking for the genes of the microbiota, living specimens are not necessary. (The DNA of ingested bacteria is generally degraded, so it is not detected in the stool specimen.) The more accurate assessment of populations in the patient's colon allows the clinician to develop the most appropriate therapy based on the patient's true gut microbiota, resulting in better clinical results.

Polymerase Chain Reaction (PCR)

One of the most important and profound contributions to molecular biology is the advent of the polymerase chain reaction (PCR). It is a powerful tool, enabling us to detect a single genome of an infectious agent in any body fluid with high accuracy and sensitivity. Many infectious agents that are missed by routine cultures, serological assays, DNA probes, and Southern blot hybridizations can be detected by PCR. Therefore, PCR-based tests are best suited for the clinical and epidemiological investigation of pathogenic bacteria and viruses. The introduction of PCR in the late 1980s dominated the clinical market, because it was superior to all previously used culture techniques and the more recently developed DNA probes and kits. PCR-based tests are several orders of magnitude more sensitive than those based on direct hybridization with the DNA probe. PCR does not depend on the ability of an organism to grow in culture. Furthermore, PCR is fast, sensitive, and capable of copying a single DNA sequence of a viable or non-viable cell over a billion times within three to five hours. The sensitivity of the PCR test is also based on the fact that PCR methodology requires only one to five cells for detection, whereas a positive culture requires an inoculum equivalent to about 1000 to 5000 cells. This difference makes PCR the most sensitive detection method available by several orders of magnitude. (21)

Advantages of PCR Amplifications of Target Microbial DNA for Organism Detection:

Ability to detect non-viable organisms that are not retrievable by culture based methods

Ability to detect and identify organisms that cannot be cultured or are extremely difficult to grow (e.g., anaerobes)

More rapid detection and identification of organisms that grow slowly (e.g., mycobacteria and fungi)

Ability to detect entire classes or previously unknown organisms directly in clinical specimens by using broad range primers

Ability to quantitate infectious organism burden for better clinical responsiveness

Laboratories that make the transition to molecular diagnostics will become an integral part of hospital operations as they demonstrate the value of their improved services. The clinical microbiology laboratory is transitioning into the molecular age. Through rapid pathogen and antibiotic resistance identification and screening tests, rapid molecular diagnostics are playing an increasingly important role in diagnosing and preventing infections and improving overall hospital operations. As physicians, pharmacists, and even hospital administrators demand rapid microbiology results, many laboratories are focusing on being part of cross-functional implementation teams that not only assure the new tests are implemented efficiently, but that the results affect real change for patient management, hospital operations, and laboratory efficacy.

Parasitology

Parasitology is yet another field of microbiology to be greatly improved with molecular technologies. Parasite infections are a major cause of nonviral diarrhea even in developed countries. Classically, parasites have been identified by microscopy and enzyme immunoassays. (22) In recent studies, molecular techniques have proven to be more sensitive and specific than classic laboratory methods. (22-24) Because Giardia cysts are shed sporadically and the number may vary from day to day, laboratories have adopted multiple stool collections to help increase identification rates for all parasite examinations. (23) Even with the advent of antigen detection systems, there has long been uncertainty in diagnosis when no ova or parasites are found in the stool. Due to the nearly 100% sensitivity and specificity of DNA analysis, combined with the need for very low amounts of genomic DNA (as low as 2.5 cells per gram), (23) the multiple-day specimen collection, laborious and technically challenging microscopy, and resulting delays in reporting have been alleviated. With PCR technology, only one fecal sample is needed for 100% sensitivity and specificity in parasitology examinations.

Detection of Antibiotic-Resistance Genes

The development of bacterial resistance to antibiotic drugs involves an active change or mutation in the microbial genome that alters the microbe's metabolic or structural responsiveness to the mechanism of the drug's action. This genetic change is passed in the population as cells replicate. This genetic material can also be passed on to other strains of bacteria through plasmid sharing. The development of antibiotic resistance is becoming a serious public health issue, as overuse of antibiotics continually selects for mutated strains that have developed resistance.

The human intestinal microbiota represent over 400 species. All antibiotic resistance strategies that bacteria develop are encoded in one or more genes. These genes are readily shared among and across species and genera and even among distantly related bacteria. These genes confer resistance to different classes of drugs, and their sequences are known. Using PCR techniques, an antibiotic resistance gene in a single organism can be readily detected in large microbial populations like those found in fecal material.

The knowledge of the presence of a drug-resistant gene may be quite significant for the clinician when considering treatment of a patient for a pathogen infection. For example, suppose a pathogen is detected in a stool analysis. An analysis of the presence of antibiotic resistance genes is also performed on the sample, and drug sensitivities are then run on the pathogen. It is found to be sensitive to two antibiotics. But suppose there is also a drug-resistant gene present in the sample to one of the drugs (a very possible scenario). It would be imperative, then, that this drug not be used in treating the patient. Otherwise, even though the pathogen is killed, the other organisms that have the gene conferring resistance to the drug would thrive relative to other microbes present. This would set up a potentially dangerous situation where antibiotic resistance is propagated in the population, because that gene can be readily spread to other organisms present in the individual. (25-27) Knowledge of the presence of antibiotic resistance genes in fecal specimens, therefore, represents a significant advance in the treatment of patients and maintenance of health.

[FIGURE 1 OMITTED]

Conclusion

DNA analysis technology allows for a significant advancement in understanding of how GI tract microbiota affect human health. It improves patient care by giving clinicians greater options and more tools in treating patients. The increased speed of analysis and improved accuracy offers the potential for making this the standard method of stool analysis.

Notes

1. Fuller R, Perdigon G. Gut Flora, Nutrition, Immunity and Health. Oxford; Malden, MA: Blackwell Pub.; 2003.

2. Mackie RI, Sghir A, Gaskins HR. Developmental microbial ecology of the neonatal gastrointestinal tract. Am J Clin Nutr. May 1999;69(5):1035S-1045S.

3. Hawrelak JA, Myers SP. The causes of intestinal dysbiosis: a review. Altern Med Rev. Jun 2004;9(2):180-197.

4. Galland L, Barrie S. Intestinal dysbiosis and the causes of diseases. J. Advancement Med. 1993;6:67-82.

5. Savage DC. Microbial ecology of the gastrointestinal tract. Annu Rev Microbiol. 1977;31:107-133.

6. Berg RD. The indigenous gastrointestinal microflora. Trends Microbiol. Nov 1996;4(11):430-435.

7. O'Sullivan DJ. Methods of analysis of the intestinal microflora. In: Tannock GW, ed. Probiotics: A Critical Review. Wymondham: Horizon Scientific Press; 1999:23-44.

8. Tannock GW. Analysis of the intestinal microflora: a renaissance. Antonie Van Leeuwenhoek. Jul-Nov 1999;76(1-4):265-278.

9. Finegold S, Sutter V, Mathisen G. Normal indigenous intestinal flora. New York: Academic Press; 1983.

10. Dutta S, Chatterjee A, Dutta P, et al. Sensitivity and performance characteristics of a direct PCR with stool samples in comparison to conventional techniques for diagnosis of Shigella and enteroinvasive Escherichia coli infection in children with acute diarrhoea in Calcutta, India. J Med Microbiol. Aug 2001;50(8):667-674.

11. Amann RI, Ludwig W, Schleifer KH. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev. Mar 1995;59(1):143-169.

12. Wilson KH, Blitchington RB. Human colonic biota studied by ribosomal DNA sequence analysis. Appl Environ Microbiol. Jul 1996;62(7):2273-2278.

13. Franks AH, Harmsen HJ, Raangs GC, Jansen GJ, Schut F, Welling GW. Variations of bacterial populations in human feces measured by fluorescent in situ hybridization with group-specific 16S rRNA-targeted oligonucleotide probes. Appl Environ Microbiol. Sep 1998;64(9):3336-3345.

14. Jansen GJ, Mooibroek M, Idema J, Harmsen HJ, Welling GW, Degener JE. Rapid identification of bacteria in blood cultures by using fluorescently labeled oligonucleotide probes. J Clin Microbiol. Feb 2000;38(2):814-817.

15. Langendijk PS, Schut F, Jansen GJ, et al. Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in fecal samples. Appl Environ Microbiol. Aug 1995;61(8):3069-3075.

16. Muyzer G, Smalla K. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie Van Leeuwenhoek. Jan 1998;73(1):127-141.

17. Welling GW, Elfferich P, Raangs GC, Wildeboer-Veloo AC, Jansen GJ, Degener JE. 16S ribosomal RNA-targeted oligonucleotide probes for monitoring of intestinal tract bacteria. Scand J Gastroenterol Suppl. 1997;222:17-19.

18. Suau A, Bonnet R, Sutren M, et al. Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut. Appl Environ Microbiol. Nov 1999;65(11):4799-4807.

19. Simpson JM, McCracken VJ, White BA, Gaskins HR, Mackie RI. Application of denaturant gradient gel electrophoresis for the analysis of the porcine gastrointestinal microbiota. J Microbiol Methods. Jun 1999;36(3):167-179.

20. Zoetendal EG, Akkermans AD, De Vos WM. Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria. Appl Environ Microbiol. Oct 1998;64(10):3854-3859.

21. Forbes BA, Sahm DF, Weissfeld AS, Baron EJ. Bailey & Scott's Diagnostic Microbiology. 10th ed. St. Louis: Mosby; 1998.

22. Verweij JJ, Blange RA, Templeton K, et al. Simultaneous detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in fecal samples by using multiplex real-time PCR. J Clin Microbiol. Mar 2004;42(3):1220-1223.

23. Ghosh S, Debnath A, Sil A, De S, Chattopadhyay DJ, Das P. PCR detection of Giardia lamblia in stool: targeting intergenic spacer region of multicopy rRNA gene. Mol Cell Probes. Jun 2000;14(3):181-189.

24. Morgan UM, Pallant L, Dwyer BW, Forbes DA, Rich G, Thompson RC. Comparison of PCR and microscopy for detection of Cryptosporidium parvum in human fecal specimens: clinical trial. J Clin Microbiol. Apr 1998;36(4):995-998.

25. Bergeron MG, Ouellette M. Preventing antibiotic resistance through rapid genotypic identification of bacteria and of their antibiotic resistance genes in the clinical microbiology laboratory. J Clin Microbiol. Aug 1998;36(8):2169-2172.

26. Martineau F, Picard FJ, Grenier L, Roy PH, Ouellette M, Bergeron MG. Multiplex PCR assays for the detection of clinically relevant antibiotic resistance genes in staphylococci isolated from patients infected after cardiac surgery. The ESPRIT Trial. J Antimicrob Chemother. Oct 2000;46(4):527-534.

27. Martineau F, Picard FJ, Lansac N, et al. Correlation between the resistance genotype determined by multiplex PCR assays and the antibiotic susceptibility patterns of Staphylococcus aureus and Staphylococcus epidermidis. Antimicrob Agents Chemother. Feb 2000;44(2):231-238.

by J. Alexander Bralley, PhD, Robert M. David, PhD, and Richard S. Lord, PhD*

J. Alexander Bralley, PhD

Dr. Bralley received his doctorate in Medical Sciences from the University of Florida College of Medicine in 1980. As founder of Metametrix (1984), he has been instrumental in developing nutritional/metabolic analyses and application guidelines for 20 years. He is a member of several professional organizations for nutrition and laboratory science and sits on the editorial review boards for Alternative Medicine Review and Integrative Medicine--A Clinician's Journal. In conjunction with Dr. Richard Lord he wrote the landmark book, Laboratory Evaluations in Molecular Medicine. He has served on the executive board for the Clinical Nutrition Certification Board and on the certification exam review board for International and American Association for Clinical Nutrition. He is also a clinical laboratory director licensed by state and federal laboratory agencies

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Robert M. David, PhD

Dr. David received his doctorate in biochemistry from Creighton Medical School in Omaha and did his post-doctoral training at the Wake-Forrest Medical Center. He served as technical director for Damon/Metpath/Corning/Quest laboratories and for Quintiles, a pharmaceutical testing laboratory. He joined Metametrix in 1996 as Laboratory Director and is instrumental in developing new laboratory procedures for clinical tests, quality control, laboratory automation and the integration of new assays from research into the clinical laboratory.

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Richard S. Lord, PhD

Dr. Lord received his BS in Chemistry from Georgia State University and his PhD in Biochemistry from the University of Texas in 1970. He then went on to complete postdoctoral fellowships at the Clayton Foundation Biochemical Institute, the University of Arizona, and the National Institute of Health. He served as president for Doctor's Data and Horizon Laboratories from 1974-1981. For the next eight years, Dr. Lord was a Professor and Chairman in the Department of Chemistry for Life University where he was instrumental in the initiation and design of a BS degree in Nutritional Science. Dr. Lord joined Metametrix in 1989 and has been actively involved in the development of new testing methodologies and innovative laboratory report combinations. In addition to co-authoring the book, Laboratory Evaluations in Molecular Medicine, Dr. Lord also publishes technical articles, lectures, and consults with health professionals regarding the application of nutritional, metabolic assays to patient care. Over the past three years, Dr. Lord has also become an active participant in the Defeat Autism Now! Think Tank, and the MINDD Foundation Think Tank, based in Sydney, Australia.

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Author:Bralley, J. Alexander; David, Robert M.; Lord, Richard S.
Publication:Townsend Letter
Geographic Code:1USA
Date:Jan 1, 2008
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