Genetic Identification of Microorganisms

The advent of molecular technologies and the application of genetic identification in clinical and forensic microbiology have greatly improved the capability of laboratories to detect and specifically identify an organism quickly and accurately.

In the wake of the 2001 anthrax attacks utilizing the United States mail, a great deal of investigative attention turned to identification of the source of the anthrax used in the attacks. Scientists continue to track the source of the anthrax utilizing genetic identification principles, techniques, and technologies.

The genetic identification of microorganisms utilizes molecular technologies to evaluate specific regions of the genome and uniquely determine to which genus, species, or strain a microorganism belongs. This work grew out of the similar, highly successful applications in human identification using the same basic techniques. Thus, the genetic identification of microorganisms has also been referred to a microbial fingerprinting.

Genetic identification of microorganisms is basically a comparison study. To identify an unknown organism, appropriate sequences from the unknown are compared to documented sequences from known organisms. Homology between the sequences results in a positive test. An exact match will occur when 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 sequences 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. The sequences utilized fall into two different categories, 1) fragments derived from the transcriptionally active, coding regions of the genome, and, 2) fragments present in inactive, noncoding regions. Of the two, the noncoding genomic material is more susceptible to mutation and will therefore show a higher degree of variability.

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 entirely different drugs, 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. Since the late 1980s, there has been a resurgence of this disease accompanied by the appearance of several new strains with antimicrobial resistance. 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 can be used, but has been replaced by newer technologies in most laboratories. 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 (polymerase chain reaction) and LCR (ligase change reaction) 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. Multiplex PCR has made it possible to discriminate between a number of different species in a single amplification reaction. For viruses with a RNA genome, RT-PCR (reverse transcriptase PCR) is widely utilized for identification and quantitation.

The anthrax outbreak in the Unites States in the fall of 2001 illustrated the significance of these technologies. 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 are quickly informed of their status, alleviating unnecessary anxiety.

The second stage of the investigation was to locate the origin of the anthrax cells. 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 clearly showed that all samples were derived from the same strain of anthrax, known as the "Ames strain" since the cell line was established in Iowa. 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. Total genome sequencing of these fifteen strains and a one-to-one base comparison with the lethal anthrax genome may detect further variation that will allow a unique identification to be made.