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

Loreen R. Stromberg, Zachary R. Stromberg, Afsheen Banisadr, Steven W. Graves, Rodney A. Moxley, Harshini Mukundan

Research output: Contribution to journalArticle

9 Citations (Scopus)

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 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.

Original languageEnglish (US)
Pages (from-to)1-7
Number of pages7
JournalJournal of Microbiological Methods
Volume116
DOIs
StatePublished - Sep 1 2015

Fingerprint

Shiga-Toxigenic Escherichia coli
O Antigens
Lipopolysaccharides
Antigens
Food Safety
Antibodies
Food Inspection
United States Department of Agriculture
Lipid A
Phenol
Complex Mixtures
Immunoblotting
Nucleic Acids
Antibody Formation
Electrophoresis
Western Blotting
Gels
Serogroup
Escherichia coli
Ligands

Keywords

  • Antibody specificity
  • E. coli
  • Lipopolysaccharide (LPS)
  • O-antigen (O-ag)
  • Serotyping
  • Shiga toxin-producing E. coli (STEC)

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Microbiology (medical)

Cite this

Purification and characterization of lipopolysaccharides from six strains of non-O157 Shiga toxin-producing Escherichia coli. / Stromberg, Loreen R.; Stromberg, Zachary R.; Banisadr, Afsheen; Graves, Steven W.; Moxley, Rodney A.; Mukundan, Harshini.

In: Journal of Microbiological Methods, Vol. 116, 01.09.2015, p. 1-7.

Research output: Contribution to journalArticle

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. In: Journal of Microbiological Methods. 2015 ; Vol. 116. pp. 1-7.
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