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The Development of a Method for the Characterization Of Non-point Sources of Escherichia Coli Using Protein Biomarkers Presenters: Dr. Joe Emily; Dr. Mahtabuddin U. Ahmed; Dr. Fernande Mofo and Mr. Ronald Whitmore Research Site: BioAnalytical Support Laboratory (BASL), PO Box 7351, Department of Biological and Physical Sciences, South Carolina State University, Orangeburg, South Carolina 29117 Introduction Non-point source (NPS) microbial contamination is well recognized as a major threat to the safety of watersheds utilized for recreation and as sources of drinking water. The ability to identify the source of microbial pollution, Microbial source tracking (MST), is essential to any efforts directed toward eliminating NPS pollution. Although Coliform bacteria are commonly recognized indicators of fecal contamination of surface drinking water sources, it is very difficult to distinguish between contamination from human versus animal sources. Presently used methods of microbial source tracking (MST) such as antibiotic resistance, phenotype number ratios, DNA sequencing and ribotyping all have certain limitations such as cost, speed, accuracy and precision. Most of these techniques depend on the belief that certain strains of E. Coli tend to inhabit the gut of specific species of warm blooded mammals. Theory Although DNA and RNA store and transport messages from the genetic code, proteins are the actual agents which mediate the actions which are mandated by this nucleic acid message. Therefore if DNA and RNA typing can be used to distinguish between individuals of the same species then surely proteins can be utilized to identify different strains within a species. And if it is true that certain strains of E. Coli tend to inhabit the gut of specific host mammals species then there may exist proteins or peptide fragments within E. Coli strains that can be used to identify their host animal of origin. Objectives We propose to develop a method using electrospray ionization-liquid chromatography/mass spectrometry (ESI-LC/MS) which is capable of identifying the specific host sources of microbial contamination based on peptide biomarkers within the various strains of E. coli bacteria. By comparing the mass spectra of proteins and peptides extracted from different E. coli strains isolated from the intestines of humans, and various wild and domestic animals we hope to develop a method capable of accurately linking strains recovered from suspected contaminated water sources to their host animal source of origin. Design of Experiment • • • • • • • #1 isolate E. Coli strains from several different animal sources (hogs, cows, chickens, humans, horses) #2 extract proteins from lysed cells #3 enzymatically digesting these proteins #4 separate peptides via multidimensional nanospray liquid chromatography #5 optimize the measurement of the peptide fragments via electrospray MS. #6 search mass spectra for peptide fragments which are likely candidates to be used as strain specific biomarkers #7 test the efficiency of these biomarkers at identifying the specific animal host source of coliform contaminated waters Procedure/Method Water Sample • pour 100 ml of sample through .45 um filter • dilute samples with high bacteria counts if necessary Membrane Filter • add 2.0 ml of M-FC media to sterilepetri dishes • place filter pads in petri dishes Selective Media (M-FC) • Place petri dishes in waterproof plastic whirlpack bags • seal, invert and submerge bags in water bath • incubate for 24 hours at 44.5 C Fecal Coliform Colonies • Pick individual colonies and transfer each colony to culture tubes containing LT Broth • each colony should represents a single coliform bacteria strain from a potential animal host Lauryl Tryptose Broth • Incubate at 35 C for 24 hours •If gas (+) produced transfer to EC media • If no gas (-) produced wait another 24 hrs and re-evaluate EC Broth • Incubate at 35 C for 24 hours • If gas (+) produced in 24 hrs then transfer to LB broth • If no gas (-) produced then wait another 24 hours • and re-evaluate LB Broth (Mid-Log Phase Growth) • Shake flask at 225 rpm for 24 h at 37 C Harvested Whole Cells • centrifuge at 8,000 g for 10 min • wash 3x with water or 8.5g/L NaCl Reconstituted Cells • Resuspend (5mg/ml) in 50mN ammonium bicarbonate containing 8M urea • add internal std (bovine insulin, ubiquitin, equine cytochrome C) Lyse Cells • Sonicate for 2 min (1 pulse/s w/ 0.75 duration) Supernatant • Separate supernatant from cell debris Determine Protein Concentration • Determine Protein Concentration Tryptic Digest 2. 3. 4. reduce w/ 1mM Dithiothreitol (DTT) 45 min at 37 C alkylate w/ 10 mN idoacetamide for 1 hr at room temp digest w/ trypsin (1:50 w/w ratio) Desalt Peptide Mixture • Acidify digest to pH 3.0 w/ formic acid • desalt and concentrate with C18 Solid phase extraction columns • elute peptides from column with 75% acetonitrile, 0.1% formic acid Peptide Mixture 2D-Liquid Chromatograh Mass Spectrometer Peptide MS Data Intensity mass 1.Discriminant Analysis 2. Cluster Analysis Determine the Source of Coliform Contamination Data The initial phases of these experiments are currently in progress. Conclusion Developing a method for Microbial Source Tracking (MST) using protein biomarkers is an ambitious project which is very challenging. If successful, MST-Proteomics will prove to be a very useful tool in watershed management in the future. References 1. Meays, Cynthia L,; Broersma, Klass, Source tracking fecal bacteria in water: a critcal review, J. Of Env. Man., 73, 71-79, 2004 2. Simpson, Joyce; Santo Domingo, Jorge, Microbial Source Tracking: State of the Science, Env. Sci. & Tech, 36, 5279-5288, 2002 3. Whitlock, John E.; Jones, David T.; Identification of the sources of fecal coliforms in an urban watershed using antibiotic resistance analysis, Water Research, 36, 4273-4282, 2002 4. Albert, John M.; Munakaata-Marr, Junko; Statistical Evaluation of Baacterial Source Tracking Data Obtained by rep-PCR DNA Fingerpringting of Escherichia coli, Env. Sci & Tech, 37, 4554-4560, 2003 5. Xiang, Fan, Anderson, Anderson, Gordon A.; Smith, Richard D., Characterization of Microorganisms and Biomarkers Development from Global ESI-MS/MS Analyses of Cell Lysates, Anal. Chem., 72, 2475-2481 6. Ho, Yen-Pen; Hsu, Po-Hsi, Investigating the effects of protein patterns on microorganism identification by high performance liquid chromatography-mass spectrometry and protein database searches, J. Chrom. A., 976, 103-111 Acknowledgements Extramural Associates Program (NIH/NICHD) South Carolina Alliance for Minority Participation (NSF)