Research Results
BioRAD CFX96™ Real-Time PCR Detection System
THERMOCYCLER
Provided by a Research Grant from the
Palm Beach Infectious Disease Institute
Lisa Plano, M.D., Ph.D.
Associate Professor
Departments of Pediatrics and Microbiology & Immunology
Miller School of Medicine
University of Miami
Through the support of the PBIDI, our laboratory was able to acquire a BioRAD CFX96™ Real-Time PCR Detection System, a multi-channeled real time Thermocyler. This state of the art piece of equipment is designed to allow up to 96 real time polymerase chain reactions (PCR) to be performed and accurately interpreted in a matter in minutes using a fraction of the necessary reagents required for conventional PCR. This technology/machine allows the investigator to watch these reactions proceed in real-time along with all the appropriate positive and negative controls, and ultimately confirms its own reactions by providing a definitive test of the end products of the reactions. All of this is accomplished for up to 5 gene targets from 44 unknown samples at a time in approximately 90 minutes. This is in comparison to, and contrasted with, performing these analyses utilizing conventional polymerase chain reactions. These reactions are done in slower machines that require significantly more reagents, followed by additional steps, reagents and equipment to visualize the product of PCR and determine the experimental results. Conventional PCR and PCR product direct visualization can lead to difficult to interpret results and the need to re-do and/or confirm these experimental results. The complete analysis can take days to weeks, not minutes and ultimately costs more in time and reagents than the reactions completed in the CFX96 format. Having and using this machine has allowed us to more precisely determine the results for the presence or absence of the genes of interest in the bacterial samples obtained from serious MRSA infection. These are the genes that are thought to be responsible for contributing to the infecting bacteria’s ability to cause these serious conditions. Although the work could have been accomplished using conventional methods, this platform gave us the ability to generate more reliable data, in a fraction of the time that it would have taken to accomplished the task otherwise, with less cost for reagents and a built in system to store and initially analyze our results. The tasks certainly could not have been accomplished within the same time frame using conventional methods and we are exceedingly grateful to be able to use such an excellent instrument.
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The Palm Beach Infectious Disease Institute
Research Grant Progress Report: Characteristics of Invasive Methicillin-resistant Staphylococcus aureus
Gordon Dickinson, M.D. Professor of Medicine
Division of Infectious Diseases and Chief
Infectious Disease Section, Medical Service
Miami VA Medical Center
Lisa R. W. Plano, M.D., Ph.D.
University of Miami, Miller School of Medicine
Concept:
Serious infections caused by Staphylococcus aureus (staph) have been recognized throughout the antibiotic era, and although much has been learned about the epidemiology of this bacterial pathogen, efforts to prevent and treat the conditions caused by staph are not always successful. It is a complex bacterium that has established a niche among humans, typically the nose, but also the gastrointestinal tract and moist folds of the skin. Most staph colonizes these sites without causing any trouble, but it can invade and cause severe infections of skin and soft tissues, sinuses, lungs, joints, bones and heart valves. Why does this bacterium, that has established itself as a component of harmless human flora, periodically turn pathogen and cause disease? The process that culminates in invasive infection is governed by three factors:
- First, contact must be made. Approximately one third of healthy humans will be colonized with these bacteria; will have S. aureus present on their skin or in their nose, at any given time. This presence is clearly a prerequisite for disease.
- Second, the host’s natural defenses – other “normal” bacterial flora, intact skin or specific immunity may prevent or curtail invasion of S. aureus.
- Third, not all S. aureus are alike and some are more likely to cause infection than others. These bacteria have the potential to carry a wide array of virulence factors, greater than 40 different proteins, which the bacteria can use to enhance colonization or infection. Some of these factors have been identified and others have been implicated in the direct cause of invasive disease or serious diseases like toxic shock syndrome and necrotizing (flesh eating) infections.
We think that the opportunity for contact with S. aureus is similar for essentially all members of the general population and that considered in mass, their host defenses or immunity to infection will be equal. We hypothesized that the armamentarium of the individual strain of S. aureus, the array of virulence factors an individual bacterium can produce, plays an important role in what diseases it will cause and how serious an infection will be. We also believe that we may be able to make assumptions about which organisms are more likely to actually cause infections, as opposed to just colonizing a healthy person, also based upon which of these virulence factors the bacterium could make. To begin to examine our hypotheses, we proposed to compare the characteristics of infecting MRSA strains, collected from persons with serious invasive staphylococcal infections to strains collected with persons colonized without invasive disease.
Findings:
This remains an ongoing project. Our work began with collecting MRSA strains from hospitalized and seriously infected individuals and performing sophisticated genetic analyses to determine which of these virulence factors the particular bacteria could make. To date, 134 MRSA from serious infections along with supporting clinical data about these infections and 6 MRSA from colonized individuals have been collected and stored. Of the infecting MRSA, 84 have been completely analyzed for the presence or absence of 20 different virulence factors and had additional genetic characterizations done for determination of types or characteristics associated with particular populations of MRSA. These clinical data and virulence factor results have been further analyzed for associations with the actual types of infections the individual bacteria caused. These analyses have allowed us to begin to link new factors with specific infections not previously known to have an association. These investigations are ongoing, as more bacteria, both harmless colonizing as well as infecting organisms are identified and isolated and the remainder of the collected organisms are at various stages of analysis.
Future directions:
Our plans going forward are to complete the analyses as outlined, to determine the special and unique characteristics of these invasive/serious infection associated MRSA. In addition, we would like to initiate comparisons of this population of aggressive bacteria with other MRSA populations currently stored in Dr. Plano’s laboratory. Currently Dr. Plano has clinical isolates that were associated with superficial skin infections that did not require hospitalization, has environmental isolates collected at a recreational beach and numerous colonizing organisms not associated with infection or the current study. Comparisons of the virulence factor profiles and additional genetic characteristics of these populations with the invasive MRSA from serious infections will give great insight into what is required for a S. aureus to be an aggressive MRSA and what might be a simple marker for a harmless colonizing bacterium. Lastly, in collaboration with international investigators, we plan to compare the invasive organisms collected from these studies with MRSA currently being collected in China by a former research associate of Dr. Plano, who is currently managing her own laboratory. We are excited by the potential strength of analyzing these diverse populations of MRSA and look forward to continuing these important investigations.