Genetic Identification of Microorganisms
Genetic Identification of Microorganisms
History and Scientific Foundations
Introduction
Genetic identification of microorganisms utilizes molecular technologies to evaluate specific regions of a microbial genome and uniquely determine to which genus, species, or strain that microorganism belongs. The techniques used were adapted from the DNA fingerprinting technology originally developed for human identification, which has led some individuals to refer to the genetic identification of microorganisms as a “microbial fingerprinting.” Having these technologies available has resulted in a great improvement in the ability of clinical and forensic microbiology laboratories to detect and specifically identify an organism quickly and accurately.
History and Scientific Foundations
The process of genetic identification of microorganisms is basically a comparison study. In order to identify an unknown organism, its key DNA sequences (the order of structural units, called nucleotides, that make up a strand of DNA) are compared to DNA sequences from known organisms. An exact match will occur when the DNA sequences from the two organisms are the same. Related individuals have genetic material that is identical for some regions and dissimilar for others. Unrelated individuals will have significant differences in the DNA regions being evaluated. Developing a database of key sequences that are unique to and characteristic of a series of known organisms facilitates this type of analysis.
Applications and Research
Depending on the level of specificity required, an assay can provide information on the genus, species, and/or strain of a microorganism. The most basic type of identification is classification to a genus. Although this general identification does not discriminate between the related species that comprise the genus, it can be useful in a variety of situations. For example, if a person is thought to have tuberculosis, a test to determine if Mycobacterium cells (the genus that includes the tuberculosis causing organism) are present in a sputum sample will most likely confirm the diagnosis. However, if there are several species within a genus that cause similar diseases but that respond to different drug therapy, it would then be critical to know exactly which species is present for proper treatment. A more specific test using genomic sequences unique to each species would be needed for this type of discrimination.
In some instances, it is important to take the analysis one step further to detect genetically distinct subspecies or strains. Variant strains usually arise as a result of physical separation and evolution of the genome. If one homogeneous sample of cells is split and sent to two different locations, over time, changes (mutations) may occur that will distinguish the two populations as unique entities. The importance of this issue can be appreciated when considering tuberculosis (TB). Since the late 1980s, there has been a resurgence of this disease accompanied by the appearance of several new strains that are resistant to the standard antibiotic treatments (known as MDR-TB or multi-drug resistant TB). The use of genetic identification for rapid determination of which strain is present has been essential to protect health care workers and provide appropriate therapy for affected individuals.
The tools used for genetic studies include standard molecular technologies. Total sequencing of an organism's genome is one approach, but this method is time consuming and expensive. Southern blot analysis was used originally, but, in most laboratories, this has now been supplanted by newer technologies such as PCR (polymerase chain reaction). Solution-phase hybridization using DNA probes has proven effective for many organisms. In this procedure, probes labeled with a reporter molecule are combined with cells in solution and upon hybridization with target cells, a chemiluminescent signal that can be quantitated by a luminometer is emitted. A variation of this scheme is to capture the target cells by hybridization to a probe followed by a second hybridization that results in precipitation of the cells for quantitation. These assays are rapid, relatively inexpensive and highly sensitive. However, they require the presence of a relatively large number of organisms to be effective. Amplification technologies such as PCR, LCR (ligase change reaction), and, for viruses with a RNA genome, RT-PCR (reverse transcriptase PCR) allow detection of very low concentrations of organisms from cultures or patient specimens such as blood or body tissues. Primers are designed to selectively amplify genomic sequences unique to each species, and, by screening unknowns for the presence or absence these regions, the unknown is identified. To speed the process up, multiplex PCR can be used to discriminate between several different species in a single amplification reaction. Going one step further, microarray technology will allow comparisons among much larger numbers of microorganisms and may be more successful at identifying specimens that contain more than one species.
WORDS TO KNOW
ASSAY: A determination of an amount of a particular compound in a sample (e.g., to make chemical tests to determine the relative amount of a particular substance in a sample). A method used to quantify a biological compound.
CHEMILUMINESCENT SIGNAL: A chemiluminescent signal is the production of light that results from a chemical reaction. A variety of tests to detect infectious organisms or target components of the organisms rely on the binding of a chemical-containing probe to the target and the subsequent development of light following the addition of a reactive compound.
DNA FINGERPRINTING: DNA fingerprinting is the term applied to a range of techniques that are used to show similarities and dissimilarities between the DNA present in different individuals (or organisms).
DNA PROBES: Substances (agents) that bind directly to a predefined specific sequence of nucleic acids in DNA.
GENOME: All of the genetic information for a cell or organism. The complete sequence of genes within a cell or virus.
HYBRIDIZATION: A process of combining two or more different molecules or organisms to create a new molecule or organism (oftentimes called a hybrid organism).
PCR (POLYMERASE CHAIN REACTION): The Polymerase Chain Reaction, or PCR, refers to a widely used technique in molecular biology involving the amplification of specific sequences of genomic DNA.
QUANTITATED: An act of determining the quantity of something, such as the number or concentration of bacteria in an infectious disease.
REVERSE TRANSCRIPTASE: An enzyme that makes it possible for a retrovirus to produce DNA (deoxyribonucleic acid) from RNA (ribonucleic acid).
SOUTHERN BLOT ANALYSIS: Southern blot refers to an electrophoresis technique where pieces of deoxyribonucleic acid (DNA) that have resulted from enzyme digestion are separated from one another on the basis of size, followed by the transfer of the DNA fragments to a flexible membrane. The membrane can then be exposed to various probes to identify target regions of the genetic material.
Impacts and Issues
Microorganism identification technologies were important during the investigation of the anthrax outbreak in the United States in the fall of 2001. Because an anthrax infection can mimic cold or flu symptoms, the earliest victims did not realize they were harboring a deadly bacterium. After confirmation that anthrax was the causative agent in the first death, genetic technologies were utilized to confirm the presence of anthrax in other locations and for other potential victims. Results were available more rapidly than would have been possible using standard microbiological methodology and appropriate treatment regimens could be established immediately. Furthermore, unaffected individuals were quickly informed of their status, alleviating unnecessary anxiety.
The attention then turned to identification of the source of the anthrax used in the attacks. The evidence indicated that this event was not a random, natural phenomenon, and that an individual or individuals had most likely dispersed the cells as an act of bioterrorism. In response to this threat, government agencies collected samples from all sites for analysis. A key element in the search was the genetic identification of the cells found in patients and mail from Florida, New York, and Washington, D.C. The PCR studies suggested that the samples were derived from the same strain of anthrax, known as the “Ames strain”. Although this strain has been distributed to many different research laboratories around the world, careful analysis revealed minor changes in the genome that allowed investigators to narrow the search to about fifteen United States laboratories. Unfortunately, despite further extensive genetic studies of these fifteen strains and comparison to the lethal anthrax genome, a final confirmation of the source of the anthrax used in the bioterrorism attacks still eludes investigators. This is due to the overall similarity between the strains and the lack of unique characters in the strain used in the attacks that could provide a definitive identification.
IN CONTEXT: TERRORISM AND BIOLOGICAL WARFARE
The capability for detecting and identifying microorganisms quickly and accurately is required to protect both troops on the battlefields and civilians confronted with terrorist attacks using biological agents. Because the systems currently available for sensing biological molecules rely on technologies that require several steps to identify biological weapons, the procedures are both labor and time intensive. The Defense Advanced Research Projects Agency (DARPA) initiated the Biosensor Technologies program in 2002 to develop fast, sensitive, automatic technologies for the detection and identification of biological warfare agents. The program focuses on a variety of technologies including surface receptor properties, nucleic acid sequences, identification of molecules found in the breath and mass spectrometry.
BIBLIOGRAPHY
Books
Dale, Jeremy W., and Simon F. Park. Molecular Genetics. New York: John Wiley & Sons, 2004.
James, Jenny Lynd. Microbial Hazard Indentification in Fresh Fruits and Vegetables. New York: Wiley-Interscience, 2006.
Persing, David H., et al, eds. Molecular Microbiology: Diagnostic Principles and Practice. Seattle: Corixa Corp, 2003.
Periodicals
Jernigan, D.B., et al. “Investigation of Bioterrorism-Related Anthrax, United States, 2001: Epidemiologic Findings.” Emerging Infectious Diseases. 8 (2002): 1019–1028.
Peplies, Jorg, Frank Oliver Glockner, and Rudolf Amann. “Optimization Strategies for DNA Microarray-Based Detection of Bacteria with 16S rRNA-Targeting Oligonucleotide Probes.” Applied and Environmental Microbiology. 69 (2003): 1397–1407.
Read, Timothy R., et al. “Comparative Genome Sequencing for Discovery of Novel Polymorphisms in Bacillus anthracis.” Science. 296 (2002): 2028–2033.
Constance Stein