|Perera, Ann -|
|Bias, Preeti -|
|John, Manohar -|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 12, 2014
Publication Date: N/A
Technical Abstract: In the United States, Escherichia coli (E. coli) O157 infection, associated with the consumption of contaminated ground beef, has resulted in an unnecessary burden for both the meat industry and the health care system, with meat recalls and often fatal human disease. Cattle, the primary reservoirs for E. coli O157, remain disease-free and asymptomatic, making it difficult to identify carrier versus non-carrier animals. Global metabolite profiling of the bovine blood will provide an opportunity to distinguish between such animals and enable the development of metabolomics based biomarkers. Here we present the results of our comprehensive, proof-of-principle, metabolomics profiling of bovine blood samples, using a combination of GC-MS and LC-MS techniques including FT-ICR, for the determination of the E. coli infection status in cattle. Metabolites were extracted from whole blood with slight modification to Jiya et al (2005). Fifty microliter of extract was used for GC-MS. Methoxymation was carried out with 50 microliter of methoxyamine hydrochloride at 60 degree C for 90 minutes followed by silylation (50 microliter of BSTFA plus 1 percent TMS at 60 degree C for 30 minutes) prior to GC-MS. The Agilent GC-MS model 7890 A/5975C was used with HP-5MSI 30m x 0.250mm ID column. LC-MS was conducted with an YMC-Pack ODS-AM RP C18 (10cm x 2.1mm x 3 micrometer) analytical column. The LC mobile phase was ACN and water with 0.1 percent acetic acid. ESI-MS and MS/MS experiments were performed on a Bruker Daltonics SolariX FTICR equipped with a 7.0 tesla shielded superconducting magnet. The metabolic abundance data was analyzed using a combination of univariate and multivariate methods. Bovine whole blood samples were collected, from an animal experimentally colonized with E. coli O157, before-during-after the colonization. Each sample representing a single sampling time was analyzed in triplicate. Identification of metabolites was a difficult task due to it’s dynamic range of abundance. However, high-resolution mass spectrometric analysis with MS-MS capabilities enabled identification of most of unknown metabolites despite their low abundances. Our GC-MS analysis on blood extracts detected 242 metabolites. The hierarchical cluster analysis and the PCA analysis showed that the metabolic profiles at the three time points were clearly separable from each other and 66/242 metabolic feature significantly changed between the 3 time points with a p-value threshold of 0.05. Overall examination of the metabolites revealed a significant change in galactose, galactopyrosides, galactopyranosides, mannose, fructose, sorbitol and mannopyranosides metabolism indicating that E. coli O157 strain may have a unique ability to impact mannose, fructose and galactose metabolism for their selective benefit over other Escherichia coli strains and thus providing an opportunity to develop biomarkers for the unique identification of cattle colonized with E. coli O157 in their intestines. Novel Aspect: Comprehensive panel of blood metabolites was investigated using metabolite profiling that could lead to biomarker discovery for E. coli O157-colonization.