DNA detection of gut microbiota advancing routine characterization of microbial populations.
The population of the microbiota Microbiota (human)
Microbial flora harbored by normal, healthy individuals. A number of microorganisms have become adapted to a particular site or ecologic niche in or on their host. 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 microbial
pertaining to or emanating from a microbe.
the breakdown of organic material, especially feedstuffs, by microbial organisms. 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 overgrowth
Rapid growth in the sales of a mutual fund's shares to the extent that the fund has difficulty finding promising new investments or it must take such large positions in individual investments that its trading flexibility is reduced. 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 Lactobacilli,
n a type of bacteria that may play an important role in tooth decay. It is usually found in small amounts in dental plaque. Its concentration increases with high sugar intake. and streptococci Streptococcus (plural, streptococci)
A genus of spherical-shaped anaerobic bacteria occurring in pairs or chains. Sydenham's chorea is considered a complication of a streptococcal throat infection. 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 redox (rē`dŏks): see oxidation and reduction. 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 villi: see digestive system. , 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 bac·te·ri·ol·o·gy
The study of bacteria, especially in relation to medicine and agriculture.
bac·te 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 in situ hybridization A method for localizing a sequence of DNA, mRNA, or protein in a cell or tissue; the use of a DNA or RNA probe to detect a cDNA sequence in chromosome spreads or in interphase nuclei or an RNA sequence of cloned bacterial or cultured , (13-17) denaturing gradient gel electrophoresis, (18,20) and temperature gradient gel electrophoresis Temperature gradient gel electrophoresis (TGGE) is a form of electrophoresis where there is a temperature gradient across the gel. TGGE is useful for analyzing nucleic acids such as DNA and RNA, and sometimes for proteins. . 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 formalin /for·ma·lin/ (for´mah-lin) formaldehyde solution.
An aqueous solution of formaldehyde that is 37 percent by weight. vials for transport. This immediately kills all organisms, freezing the exact balance present at the time of collection. Since PCR PCR polymerase chain reaction.
polymerase chain reaction
Polymerase chain reaction (PCR) identification is only looking for the genes of the microbiota, living specimens are not necessary. (The DNA DNA: see nucleic acid.
or deoxyribonucleic acid
One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. 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 serological
pertaining to or emanating from serology.
one involving examination of blood serum usually for antibody. 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 hybridization /hy·brid·iza·tion/ (hi?brid-i-za´shun)
1. crossbreeding; the act or process of producing hybrids.
2. molecular hybridization
3. 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 inoculum /in·oc·u·lum/ (-ok´u-lum) pl. inoc´ula material used in inoculation.
n. pl. 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 quan·ti·tate
tr.v. quan·ti·tat·ed, quan·ti·tat·ing, quan·ti·tates
To determine or measure the quantity of.
[Back-formation from quantitative (analysis). 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.
The scientific study of parasites and of parasitism. Parasitism is a subdivision of symbiosis and is defined as an intimate association between an organism (parasite) and another, larger species of organism (host) upon which the parasite is
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 Giardia /Gi·ar·dia/ (je-ahr´de-ah) a genus of flagellate protozoa parasitic in the intestinal tract of humans and other animals, which may cause giardiasis; G. lam´blia (G. intestina´lis) is the species found in humans. 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]
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.
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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 University of Florida is the third-largest university in the United States, with 50,912 students (as of Fall 2006) and has the eighth-largest budget (nearly $1.9 billion per year). UF is home to 16 colleges and more than 150 research centers and institutes. 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
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.
Richard S. Lord, PhD
Dr. Lord received his BS in Chemistry from Georgia State University History
Georgia State University was founded in 1913 as the Georgia School of Technology's "School of Commerce." The school focused on what was called "the new science of business. 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 (body, education) University of Arizona - The University was founded in 1885 as a Land Grant institution with a three-fold mission of teaching, research and public service. , 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 MINDD Minimum Due Date Foundation Think Tank, based in Sydney, Australia.