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Title: Purification and characterization of lipopolysaccharides from six strains of non-O157 Shiga toxin-producing Escherichia coli

Abstract

Certain Shiga toxin-producing Escherichia coli (STEC) are virulent human pathogens that are most often acquired through contaminated food. The United States Department of Agriculture, Food Safety and Inspection Service has declared several serogroups of STEC as adulterants in non-intact raw beef products. Hence, sensitive and specific tests for the detection of these STEC are a necessity for implementation in food safety programs. E. coli serogroups are identified by their respective O-antigen moiety on the lipopolysaccharide (LPS) macromolecule. We propose that the development of O-antigen-specific immunological assays can facilitate simple and rapid discriminatory detection of STEC in beef. However, the resources (antigens and antibodies) required for such development are not readily available. To overcome this, we extracted and characterized LPS and O-antigen from six STEC strains. Using hot phenol extraction, we isolated the LPS component from each strain and purified it using a series of steps to eliminate proteins, nucleic acids, and lipid A antigens. Antigens and crude LPS extracts were characterized using gel electrophoresis, immunoblotting, and modified Western blotting with commercially available antibodies, thus assessing the serogroup specificity and sensitivity of available ligands as well. The results indicate that, while many commercially available antibodies bind LPS, their activities and specificitiesmore » are highly variable, and often not as specific as those required for serogroup discrimination. This variability could be minimized by the production of antibodies specific for the O-antigen. Additionally, the antigens generated from this study provide a source of characterized LPS and O-antigen standards for six serogroups of STEC.« less

Authors:
 [1];  [2];  [3];  [4];  [2];  [5]
  1. Los Alamos National Lab. (LANL), Los Alamos, NM (United States); The New Mexico Consortium, Los Alamos, NM (United States); Univ. of New Mexico, Albuquerque, NM (United States)
  2. Univ. of Nebraska, Lincoln, NE (United States)
  3. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
  4. The New Mexico Consortium, Los Alamos, NM (United States); Univ. of New Mexico, Albuquerque, NM (United States)
  5. Los Alamos National Lab. (LANL), Los Alamos, NM (United States); The New Mexico Consortium, Los Alamos, NM (United States)
Publication Date:
Research Org.:
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Sponsoring Org.:
USDA; USDOE
OSTI Identifier:
1492572
Report Number(s):
LA-UR-15-21477
Journal ID: ISSN 0167-7012
Grant/Contract Number:  
89233218CNA000001
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Microbiological Methods
Additional Journal Information:
Journal Volume: 116; Journal Issue: C; Journal ID: ISSN 0167-7012
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Biological Science; E. coli, LPS, antibody specificity, O-Antigen, serotyping, Shiga Toxin-Producing E. coli, STEC

Citation Formats

Stromberg, Loreen R., Stromberg, Zachary R., Banisadr, Afsheen, Graves, Steven W., Moxley, Rodney A., and Mukundan, Harshini. Purification and characterization of lipopolysaccharides from six strains of non-O157 Shiga toxin-producing Escherichia coli. United States: N. p., 2015. Web. doi:10.1016/j.mimet.2015.06.008.
Stromberg, Loreen R., Stromberg, Zachary R., Banisadr, Afsheen, Graves, Steven W., Moxley, Rodney A., & Mukundan, Harshini. Purification and characterization of lipopolysaccharides from six strains of non-O157 Shiga toxin-producing Escherichia coli. United States. doi:10.1016/j.mimet.2015.06.008.
Stromberg, Loreen R., Stromberg, Zachary R., Banisadr, Afsheen, Graves, Steven W., Moxley, Rodney A., and Mukundan, Harshini. Thu . "Purification and characterization of lipopolysaccharides from six strains of non-O157 Shiga toxin-producing Escherichia coli". United States. doi:10.1016/j.mimet.2015.06.008. https://www.osti.gov/servlets/purl/1492572.
@article{osti_1492572,
title = {Purification and characterization of lipopolysaccharides from six strains of non-O157 Shiga toxin-producing Escherichia coli},
author = {Stromberg, Loreen R. and Stromberg, Zachary R. and Banisadr, Afsheen and Graves, Steven W. and Moxley, Rodney A. and Mukundan, Harshini},
abstractNote = {Certain Shiga toxin-producing Escherichia coli (STEC) are virulent human pathogens that are most often acquired through contaminated food. The United States Department of Agriculture, Food Safety and Inspection Service has declared several serogroups of STEC as adulterants in non-intact raw beef products. Hence, sensitive and specific tests for the detection of these STEC are a necessity for implementation in food safety programs. E. coli serogroups are identified by their respective O-antigen moiety on the lipopolysaccharide (LPS) macromolecule. We propose that the development of O-antigen-specific immunological assays can facilitate simple and rapid discriminatory detection of STEC in beef. However, the resources (antigens and antibodies) required for such development are not readily available. To overcome this, we extracted and characterized LPS and O-antigen from six STEC strains. Using hot phenol extraction, we isolated the LPS component from each strain and purified it using a series of steps to eliminate proteins, nucleic acids, and lipid A antigens. Antigens and crude LPS extracts were characterized using gel electrophoresis, immunoblotting, and modified Western blotting with commercially available antibodies, thus assessing the serogroup specificity and sensitivity of available ligands as well. The results indicate that, while many commercially available antibodies bind LPS, their activities and specificities are highly variable, and often not as specific as those required for serogroup discrimination. This variability could be minimized by the production of antibodies specific for the O-antigen. Additionally, the antigens generated from this study provide a source of characterized LPS and O-antigen standards for six serogroups of STEC.},
doi = {10.1016/j.mimet.2015.06.008},
journal = {Journal of Microbiological Methods},
number = C,
volume = 116,
place = {United States},
year = {2015},
month = {6}
}

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