Citation:

DONOHUE, M. J., W. SMALLWOOD, D. J. LYE, J. A. SHOEMAKER, AND M. R. RODGERS. THE IDENTIFICATION OF THE WATER-BORNE PATHOGEN AEROMONAS USING WHOLE CELL ANALYSIS BY MATRIX ASSISTED LASER DESORPTION/IONIZATION-MASS. Presented at 53rd American Society for Mass Spectrometry Conference, San Antonio, TX, June 05 - 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:

MALDI-MS has long been established as a tool by which microorganisms can be characterized and identified. The U.S. Environmental Protection Agency (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 the EPA's 2005 Contaminant Candidate List (CCL) as requiring research.

To assess if MALDI-MS can be used to identify species of Aeromonas, as well as predict the strains 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.

Methods and Instrumentation:

Bacteria growth

Aeromonas reference strains, representing the 17 recognized species, were obtained from several culture collections. The Aeromonas environmental isolates were collected from a pilot study of water distribution systems (WDS), between the years of 2000-2001, using EPA Method 1605. Over 205 were obtained from the surveyed distribution centers.

Both reference strains and WDS isolates (stored at -20 0C) were used to inoculate tryptic soy agar plates (Difco, Detroit, MI, USA). Individual colonies were carefully removed from the agar surface with a sterile loop to inoculate 5 mL of tryptic soy broth (TSB). The cultures were incubated overnight at 370C. One-hundred microliters of the TSB broth were lawned onto 5% sheeps blood agar plates and incubated overnight at 350C. Five milliliters of sterile saline were then used to harvest bacteria from the plates.

Sample Preparation

The harvested cells were centrifuged at 4,000 RPM. The supernatant was removed and the pellet was washed three times with water. The dried cells were then overlaid with a 1.0
MALDI-MS analysis

All mass spectra were generated on a Biflex III (Bruker Daltonics, Billerica, MA, USA) MALDI-TOF mass spectrometer, operated in positive linear mode. The spectra were obtained by summing four scans of 100 shots each. The summed spectra were smoothed and calibrated externally using the [M+H]+ ions of insulin and myoglobin. Blank controls were analyzed in parallel with the bacteria samples to verify that the protein signals were not from contamination introduced during sample preparation. Peak lists were created from each strains spectrum and later analyzed by Phylogenetic Analysis Using Parsimony* (PAUP*).

Results and Discussion:

The long range goal of this project is to identify novel virulence factors for Aeromonas using MS techniques. 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 can be used to determine if rapid species/strain differentiation is possible using MALDI-MS. Unique masses were observed in each of the Aeromonas isolates, providing a means to differentiate between Aeromonas species by MALDI-MS.

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 definitively identify 25 isolates, and tentatively identify the other 26 isolates. Species identification was accomplished by comparing mass spectral fingerprints of the isolates against the 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. In the cases where a clear identity was not determined, research is on-going to ascertain whether the isolate is a mixture of species.

Concurrently with the above research, animal studies were 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 then be used to sequence the virulent proteins.

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