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THE APPLICATION OF WHOLE CELL BACTERIAL ANALYSIS TO ENVIRONMENTAL CHALLENGES
Citation:
SHOEMAKER, J. A. THE APPLICATION OF WHOLE CELL BACTERIAL ANALYSIS TO ENVIRONMENTAL CHALLENGES. Presented at ASMS Fall Workshop, San Diego, CA, December 08 - 09, 2005.
Impact/Purpose:
This particular task is comprised of 4 subtasks: 1.) Characterization of Potential Viral Biomarkers by Mass Spectrometry; 2.) Characterization of Parasites by Mass Spectrometric Techniques;
3.) Rapid Discrimination of Bacterial Indicators of Fecal Contamination and Bacterial Pathogens by Mass Spectrometric Techniques; and 4.) Investigation of Aeromonas Virulence Factors Using Mass Spectrometry.
The purpose of this research project is to use mass spectrometric techniques, such as electrospray ionization (ESI), capillary electrophoresis (CE) and matrix assisted laser desorption ionization (MALDI) mass spectrometry, to provide "protein mass fingerprinting" and protein sequencing information for viruses, bacteria and protozoa that cause waterborne disease. These protein mass fingerprinting libraries will be evaluated to determine whether mass spectrometric techniques can identify protein fingerprints related to the infectivity/viability of selected microorganisms and whether they can differentiate between infective / non-infective genus and strains of the selected microorganisms. The characteristic proteins identified by mass spectrometry as markers of infectivity/viability or strain differentiation can then be used to develop more sensitive microbiological drinking water methods.
Description:
The U.S. EPA National Exposure Research Laboratory's proteomic research efforts was presented at the American Society for Mass Spectrometry's 2005 Fall Workshop on Mass Spectrometry of Microorganisms. The U.S. EPA is investigating the potential of using this technique as a way to rapidly identify Aeromonas species in drinking water. A number of bacteria, including Aeromonas hydrophila, are listed on EPA's 1998 Contaminant Candidate List (CCL) as research needs. To assess if matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) can be used to identify species of Aeromonas, as well as predict the strain's possible pathogenicity, the project is broken down into three major objectives: 1) to create a mass spectral library of known Aeromonas strains, 2) identify unknowns isolates, and 3) identify m/z ions that may be used as biomarkers of human pathogenicity.
A library of mass spectral fingerprints of a number of Aeromonas strains was generated using MALDI-MS. In addition to investigating virulence factors, this mass spectral library was used to rapidly species/strain differentiate using MALDI-MS. Unique masses were observed in each of the Aeromonas isolates providing a means to differentiate between Aeromonas species. Mass lists were created from each strain's spectrum and cluster analysis performed using Phylogenetic Analysis Using Parsimony* (PAUP*).
A blind study was done using 51 isolates from a collection of 205 isolates obtained from drinking water distribution systems. Of the 51 isolates used in this study, traditional biochemical analyses were only able to confidently identify 25 isolates, and tentatively identify the other 26 isolates. Species identification for the isolates was accomplished by comparing mass spectral fingerprints of the isolates against well characterized Aeromonas ATCC strains using the PAUP software. The MALDI-MS analysis of the water distribution samples correlated 100% with the biochemical identification of the 25 confirmed isolates. Of the 26 isolates that were only tentatively identified, MALDI-MS analysis was able to identify, with a 95% confidence, 15 of the 26 isolates.
Animal studies are being conducted to identify virulent and avirulent strains of the isolates obtained from drinking water distribution systems. This research, along with the MALDI-MS analysis of the isolates, will be used to identify potential biomarkers of human pathogenicity. Once these biomarkers are identified, further research will be conducted to identify the proteins unique to virulent Aeromonas using MALDI-MS mass spectral fingerprints. Electrospray (ESI)-MS/MS will be used then to sequence the virulent proteins.