Contamination Source Identification (CSI): Tools of discovery
As the need for contamination detection increases, so can the ability to understand a microorganism through DNA sequencing
By Lisa Suneus
Explicitly described, direct comparisons of cultivation-based and genetic-based identification methods have obvious deficiencies within each. Nonetheless, it can be assured that microbial detection will be revealed by one, if not the other. The question remains as to which tools are more sensitive, economically feasible, easily validated, and time-saving.
DNA sequencing has become a reliable source in recognizing the root of an unknown microbial contaminant. As the need for contamination discovery and detection increases, we should progressively rely more on our ability to understand a microorganism from its profound genetic sequence (molecules of all life forms) and less on our dependence on unreliable, time-consuming cultivation.
Those most concerned about microbial contamination include hospital staff, pharmaceutical manufacturers, researchers, food preparers, food packagers, homeowners, human disaster relief workers, and anyone concerned about reducing morbidity worldwide due to controllable microbial infestation.
The first step in solving contamination issues is identifying the problem and formulating a plan of defense. Traditional cultivation tools have been hindered by a lack of microbial speciation and by inaccurate characterization. Using genetic material as a tool for understanding the capacity of an organism to thrive is pertinent to knowing why it inhabits a particular environment. A relevant example is the elucidation of the genetic trigger in sporulating organisms such as Bacillus anthracis (causative agent of anthrax) when it is forced to flourish under harsh and barren growth conditions.
Current microbial identification methodologies
Conventional culture-dependent methods used to determine microbial burdens are time-consuming and can only enumerate organisms that are prompted to grow on the appropriate laboratory media. This procedure promotes microbiologists’ guesswork in culturing the unknown organism on various media until it demonstrates a notable reaction observable by the eye. Other current methods of microbial identification focus on the immune response of the host (e.g., human) to the unknown substance, visual observation of the organism using a microscope, or by-product recognition (e.g., adenosine triphosphate, respiration by-product) using specialized systems requiring expensive dyes (e.g., luminometer).
A microbiology analyst places a sample in the genetic analyzer to perform a thirty-minute microbial identification.
Basic microbiological techniques have yielded an array of diverse microbial cultivation methods, and the use of specific reagents has created a variety of useful biochemical systems for microbial classification and identification. Inoculation and incubation of organisms on growth media in the laboratory is the classic means of colony purification amplification based on previously studied and documented growth requirements. However, it should be presumed that there are microorganisms in our external environment that have been completely overlooked. This is either due to their resistance to cultivation on artificial media or because we simply have not been affected yet by their presence. Recent explorations of microbial diversity and investigation via DNA sequencing within our external environment (e.g., oceans) have yielded bacteria that had not been previously recognized or ever cultivated in the laboratory.
An analyst, preparing for another run, performs routine maintenance on the genetic analyzer.
Sequence-based molecular methods, such as polymerase chain reaction (PCR), are culture independent. PCR is a technique used to increase the number of copies of a specific region of DNA in order to produce enough DNA to be tested. This technique can be used to identify pathogenic bacteria and/or viruses.
PCR is based on amplification of the hyper-variable region of the 16 subunit ribosomal RNA (16S rDNA) gene. Currently more than 7,000 bacterial 16S rDNA sequences are available. Although this molecular method reveals the DNA sequence of a variable region within a bacterial genome, there is still room for human error in making a final interpretation. Experienced microbiologists and biochemists would prove to be excellent resources for these interpretation tasks.
An analyst prepares a sample for PCR (polymerase chain reaction). Directly in front of the analyst is the thermocycler, which performs the PCR process.
Cultivation-based methods can be laborious and ultimately expensive with the use of plates, tubes, growth supplements, incubators, refrigerators, freezers, inoculating loops, microscopes, proficient staff, and the most precious commodity, time. Most significantly, these methods are limited to pathogens with known growth requirements. Unknown growth requirements yield failures in detecting infections or source contamination caused by newly emerging organisms. Many organisms that fail to bring forth a detectable host immune response can thrive unnoticed, and the visual appearance of microorganisms and by-products is too nonspecific.
One example of the setbacks imposed by traditional methods is the growth and detection of the tuberculosis-causing organism (e.g., Mycobacterium tuberculosis). Speciation of this slow-growing organism can take weeks. One example of an organism that could only be understood with the advent of PCR is Whipple bacillus. This microbe, like many others, cannot be cultivated, although it is microscopically visible. Molecular methods (e.g., genetic analysis) have exposed unknown contaminants that have proven deadly because of our prior lack of rapid detection means (e.g., hantaviruses, the origin of Kaposi’s sarcoma, and the cause of “cat scratch disease”).
When determining which genetic criteria are useful for identifying uncharacterized microorganisms, it is most important that the sequence be amenable to amplification. Second, the targeted sequence should be conserved, consistently stable, and uninfluenced by evolutionary pressures. Third, the target sequence should be a portion of the genome that is specifically variable among known organisms and capable of discernment in comparisons to other unrelated organisms. The bacterial DNA sequence of the small subunit ribosomal RNA (16S rDNA) and the fungal 28S rDNA sequence meet these criteria and are the basis of the majority of the current rapid microbial identification instrumentation. Broad-range PCR as a method for “pathogen discovery” is not limited to ribosomal DNA as a target because any genetically reliable family of gene sequences found among a coherent group of microorganisms can be targeted.
There is, unfortunately, a downside to the genetic means of identification. Genetic analysis, with the proper interpretation may help resolve contamination questions, but it can be difficult to determine whether a particular analysis is accurate. The interpretations are currently based on the compilation of publicly- and privately-owned databases validated by expert researchers. From a practical standpoint, sequence-based databases support investigations that require a bare understanding of an organism’s evolutionary status in respect to other similar microbes, but they are currently incomplete. The creation of a broad-spectrum, continuously growing database is paramount because molecular methods can be applied to several industries in which one might expect to find uncharacterized microbial pathogens.
Other methods of microbial ID
As acknowledged in well-documented sources, sequence-based approaches take the lead in speed, sensitivity and the specificity of genetic characterization. Other applications include bioassays for newly characterized biochemical reactions among microbes. The application that has shown the most potential is the use of DNA arrays. DNA arrays are based on a set of broad-spectrum probes designed to assess ribosomal RNA sequence diversity among various bacteria on an inert platform such as a microchip.
In addition to revolutionizing environmental microbiology, molecular methods may offer benefits for clinicians, cleanroom practitioners, and biotechnologists. Contamination control in the aseptic environments of biotechnology and pharmaceutical facilities is critical for the safe and effective manufacture of healthcare products. Regulated by current Good Manufacturing Practices (cGMP) and Good Lab Practices (GLPs), these facilities must provide documentation stating that products have been prepared and tested in an area that has been predetermined to be free of excessive levels of viable and nonviable particle contaminants.
Maintaining cleanroom environments is a costly, critical task. Most organizations cannot afford to shut down an entire cleanroom facility due to contamination issues. Enforcing an environmental program that utilizes rapid methods, such as genetic analysis, can decrease facility downtime and ensure that compliance is met. Rapid molecular methods can determine an unknown microbial identification from a viable or nonviable sample and can make use of microtubes and microplates that can hold from 96 to 384 samples at one time. Cultivation methods, conversely, can yield large waste loads, and only one isolated organism-which must be viable-can be speciated at a time.
Rapid molecular methods should be the primary tool for microbial ID for the cleanrooms and critical environments of every industry concerned with maintaining a clean workplace that ensures the integrity of products for human use. Characterization of an unknown organism is the first step to understanding its source and subsequently how to remove it from critical environments. Ultimately, pathogen detection and identification will only be clearly defined by a combination of traditional and novel tools of discovery and a multidisciplinary effort coordinated by microbiologists, researchers, and environmentalists.
Lisa Suneus is the microbiology laboratory manager for Boston Analytical, Inc. She holds a Master of Science degree in microbiology, immunology, and parasitology. She can be reached at LSuneus@bostonanalytical.com.