Citation:

Donohue, M J., J A. Shoemaker, A W. Smallwood, AND M R. Rodgers. ANALYSIS OF AEROMONAS BY MASS SPECTROMETRY: SPECIATION AND VIRULENCE FACTORS. Presented at 52nd ASMS Conference, Nashville, TN, May 22-27, 2004.

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:

Introduction:

A number of bacteria, including Aeromonas hydrophila, are listed on the Environmental Protection Agency's 1998 Contaminant Candidate List (CCL) as research needs. One research priority designated by the CCL is the identification of virulence activity factor relationships (VFARs). VFARs may provide a more rapid means of identifying waterborne pathogens than the current process which relies on exposure and health effects as the two primary categories for screening potential microbial drinking water contaminants. The first step in the research project is to obtain the MALDI-MS spectra of the available virulent and avirulent species/strains of Aeromonas, and identify the masses which are unique to virulent Aeromonas species and strains.

Methods and Instrumentation:

Chosen ATCC strains and field samples of the microorganism were plated onto 5% sheep blood agar plate and incubated for 16 hr at 35 C. The cells were subsequently harvested, and the optical density of the solutions were then adjusted to an O.D.600 of 0.5. The harvested cells were then washed three times with water.
A 1 L aliquot of the bacterial sample was applied to the target and allowed to air dry before an 1.5 L aliquot of sinapinic acid matrix was overlaid on top. All spectra were generated on a Bruker BiFlex III MALDI-MS, operated in positive linear mode. The spectra for all samples was obtained by summing four scans of a 100 shots.

Preliminary Data:

The long range goal of this project is to identify novel virulence factors for Aeromonas using ESI-MS and MALDI-MS. In the process, the mass spectral fingerprints obtained can be used to determine if rapid species/strain differentiation is possible using MALDI-MS. Nine species of Aeromonas have been analyzed by MALDI-MS. The procedure provides a unique mass spectral fingerprint for each of the chosen isolates. The spectra obtained allow identification of the species differentiation in the genus Aeromonas. Unique masses were observed in each of the Aeromonas isolates providing a mean to differentiate between Aeromonas species by MALDI-MS. To test the method, a blind study was done using field samples. The MALDI analysis of the field samples was able to correctly identify all the Aeromonas species/isolates previously identified by PCR analysis. Further research is being conducted on identifying the proteins unique to virulent Aeromonas using these MALDI-MS mass spectral fingerprints.

Record Details: