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 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.

Comparison of immunomagnetic separation beads for detection of six non-O157 Shiga toxin-producing Escherichia coli serogroups in different matrices

“…Alternatively, it has also been observed in another study that antibodies targeting the core polysaccharide, rather than the O‐antigen, of STEC can cross‐react with other serogroups leading to non‐specificity (Stromberg et al . ).…”

“…Many sample types will have competing microbiota that are similar enough to the target STEC that the selectivity of the enrichment media is less impactful and could lead to competing interactions amongst the IMS bead antibodies and the non-target bacteria. Alternatively, it has also been observed in another study that antibodies targeting the core polysaccharide, rather than the Oantigen, of STEC can cross-react with other serogroups leading to non-specificity (Stromberg et al 2015a).…”

Membrane Insertion for the Detection of Lipopolysaccharides: Exploring the Dynamics of Amphiphile-in-Lipid Assays

“…coli (LPS O104:H4 and LPS O111:H11) using af647 labeled detection antibodies targeted against the specific O-ag ( Fig 4 ), Sensitive detection is demonstrated in both cases with LPS O104 demonstrating a significantly higher (s:n~39) response than LPS O111 (s:n~6). This difference can largely be attributed to the sensitivity of the respective antibodies [ 36 ]. Due to the large difference in s:n ratios in these assays, the limits of detection also demonstrate the same pattern (0.77 and 7.36 μg/mL respectively).…”

“…Lipopolysaccharides from six strains of non-O157 STEC (DEC10B [O26:H11], B8227-C8 [O45:H2], MT#80 [O103:H2], 0201 9611 [O111:H11], MDCH-4 [O113:H21], DA-37 [O121:H21], GS G5578620 [O145:NM], and TY-2482 [O104:H4]) were selected and prepared by hot phenol extraction and tested for antigen activity as we have previously described [ 36 ]. LPS O157:H7 was purchased from List Biological Labs (Campbell, CA), and LPS O111:B4, bovine serum albumin (BSA), Dulbecco’s phosphate buffered saline (PBS), Ethylenediaminetetraacetic acid (EDTA), and potassium chloride were from Sigma Aldrich (St. Louis, MO).…”

“…Factors such as conserved hydrophobic regions, micelle aggregation, poor binding affinity of antibodies, and association with serum lipoproteins have made detection of LPS and similar lipoglycans difficult targets for antigen specific assays [ 19 , 28 , 30 , 31 ]. Detection of the O-ag with classical methods such as latex agglutination or immunomagnetic separation utilize cross reactive polyclonal antibodies, which can lead to misidentification of the serogroup [ 32 – 36 ]. Enzyme-linked immunosorbent assays for detection of both LPS and O-ag serogroup identification have also been developed, but require extensive sample preparation, multiple antibodies, and yet suffer from non-specific interactions of the antibodies [ 30 , 37 – 41 ].…”