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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. https://doi.org/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. https://doi.org/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. https://doi.org/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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Works referenced in this record:

Structure of the O-chain of the lipopolysaccharide of Haemophilus pleuropneumoniae serotype 1
journal, December 1986

  • Altman, Eleonora; Brisson, Jean-Robert; Perry, Malcolm B.
  • Biochemistry and Cell Biology, Vol. 64, Issue 12
  • DOI: 10.1139/o86-173

Targeting Bacterial Endotoxin: Two Sides of a Coin
journal, January 2007

  • Bosshart, H.; Heinzelmann, M.
  • Annals of the New York Academy of Sciences, Vol. 1096, Issue 1
  • DOI: 10.1196/annals.1397.064

Comparison of a Commercial Reversed Passive Latex Agglutination Assay to an Enzyme Immunoassay for the Detection of Shiga Toxin-Producing Escherichia coli
journal, November 2003

  • Carroll, K. C.; Adamson, K.; Korgenski, K.
  • European Journal of Clinical Microbiology & Infectious Diseases, Vol. 22, Issue 11
  • DOI: 10.1007/s10096-003-1053-9

Anti-capsular antibodies activate killing of Escherichia coli O8:K87 by the alternate complement pathway in porcine serum
journal, November 2006


Evaluation of different methods for the detection of outer membrane proteins and lipopolysaccharides of Pasteurella haemolytica by immunoblotting
journal, January 1994


The detection of non-O157 E. coli in food by immunomagnetic separation
journal, July 2004


Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real-time PCR
journal, March 2005


The structure of the sialic acid-containing Escherichia coli O104 O-specific polysaccharide and its linkage to the core region in lipopolysaccharide
journal, December 1992


A PCR–ELISA for detecting Shiga toxin-producing Escherichia coli
journal, March 2002


Detection of the Top Six Non-O157 Shiga Toxin–Producing Escherichia coli O Groups by ELISA
journal, November 2012

  • Hegde, Narasimha V.; Cote, Rebecca; Jayarao, Bhushan M.
  • Foodborne Pathogens and Disease, Vol. 9, Issue 11
  • DOI: 10.1089/fpd.2012.1231

Growing Concerns and Recent Outbreaks Involving Non-O157:H7 Serotypes of Verotoxigenic Escherichia coli
journal, October 1996


Pathogenic Escherichia coli
journal, February 2004

  • Kaper, James B.; Nataro, James P.; Mobley, Harry L. T.
  • Nature Reviews Microbiology, Vol. 2, Issue 2
  • DOI: 10.1038/nrmicro818

Evaluation of a Microplate Latex Agglutination Method (Verotox-F Assay) for Detecting and Characterizing Verotoxins (Shiga Toxins) in Escherichia coli
journal, February 1999

  • Karmali, Mohamed A.; Petric, Martin; Bielaszewska, Martina
  • Journal of Clinical Microbiology, Vol. 37, Issue 2
  • DOI: 10.1128/JCM.37.2.396-399.1999

Separation of bacterial capsular and lipopolysaccharides by preparative electrophoresis
journal, January 1996

  • Kim, John S.; Reuhs, Bradley L.; Rahman, M. Mahburbur
  • Glycobiology, Vol. 6, Issue 4
  • DOI: 10.1093/glycob/6.4.433

Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4
journal, August 1970


Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis
journal, January 1990

  • Lesse, Alan J.; Campagnari, Anthony A.; Bittner, William E.
  • Journal of Immunological Methods, Vol. 126, Issue 1
  • DOI: 10.1016/0022-1759(90)90018-Q

Redefining the Requisite Lipopolysaccharide Structure in Escherichia coli
journal, January 2006

  • Meredith, Timothy C.; Aggarwal, Parag; Mamat, Uwe
  • ACS Chemical Biology, Vol. 1, Issue 1
  • DOI: 10.1021/cb0500015

Rapid detection of Mycobacterium tuberculosis biomarkers in a sandwich immunoassay format using a waveguide-based optical biosensor
journal, September 2012

  • Mukundan, Harshini; Kumar, Sandeep; Price, Dominique N.
  • Tuberculosis, Vol. 92, Issue 5, p. 407-416
  • DOI: 10.1016/j.tube.2012.05.009

Shiga Toxin-Producing Escherichia coli O104:H4: a New Challenge for Microbiology
journal, June 2012

  • Muniesa, Maite; Hammerl, Jens A.; Hertwig, Stefan
  • Applied and Environmental Microbiology, Vol. 78, Issue 12
  • DOI: 10.1128/AEM.00217-12

Serology, chemistry, and genetics of O and K antigens of Escherichia coli.
journal, January 1977


Lipopolysaccharide Heterogeneity in Salmonella typhimurium Analyzed by Sodium Dodecyl Sulfate/Polyacrylamide Gel Electrophoresis
journal, June 1980


Structural studies on the Shigella-like Escherichia coli O121 O-specific polysaccharide
journal, September 1997


Rapid serological analysis of bacterial lipopolysaccharides by electrotransfer to nitrocellulose
journal, December 1985


Lipopolysaccharide Endotoxins
journal, June 2002


Discovery of new biosynthetic pathways: the lipid A story
journal, October 2008

  • Raetz, Christian R. H.; Guan, Ziqiang; Ingram, Brian O.
  • Journal of Lipid Research, Vol. 50, Issue Supplement
  • DOI: 10.1194/jlr.R800060-JLR200

Detection of stealthy small amphiphilic biomarkers
journal, August 2014

  • Sakamuri, Rama Murthy; Capek, Petr; Dickerson, Tobin J.
  • Journal of Microbiological Methods, Vol. 103
  • DOI: 10.1016/j.mimet.2014.05.012

The use of multiplex PCR to detect and differentiate food- and beverage-associated microorganisms: A review
journal, April 2007


Detection of five Shiga toxin-producing Escherichia coli genes with multiplex PCR
journal, June 2014


The structures of Escherichia coli O-polysaccharide antigens
journal, May 2006


Isolation and characterization of Shiga toxin-producing Escherichia coli O157:H7 and non-O157 from beef carcasses at a slaughter plant in Mexico
journal, January 2007

  • Varela-Hernández, J. J.; Cabrera-Diaz, E.; Cardona-López, M. A.
  • International Journal of Food Microbiology, Vol. 113, Issue 2
  • DOI: 10.1016/j.ijfoodmicro.2006.06.028

Production and characterization of monoclonal antibodies specific for the lipopolysaccharide of Escherichia coli O157
journal, March 1997


Biosynthesis of lipopolysaccharide O antigens
journal, May 1995


Rapid isolation method for lipopolysaccharide and lipid A from Gram-negative bacteria
journal, January 2000

  • Yi, Eugene C.; Hackett, Murray
  • The Analyst, Vol. 125, Issue 4
  • DOI: 10.1039/b000368i

Negative staining of lipopolysaccharides on polyacrylamide gels by using eosin B
journal, July 2012


    Works referencing / citing this record:

    Comparison of immunomagnetic separation beads for detection of six non-O157 Shiga toxin-producing Escherichia coli serogroups in different matrices
    journal, July 2017

    • Kraft, A. L.; Lacher, D. W.; Shelver, W. L.
    • Letters in Applied Microbiology, Vol. 65, Issue 3
    • DOI: 10.1111/lam.12771