Differentiation of Metronidazole-Sensitive and -Resistant Clinical Isolates of Helicobacter pylori by Immunoblotting with Antisera to the RdxA Protein

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Published inJournal of Clinical Microbiology Vol. 39; no. 9; pp. 3052 - 3055
Main Authors LATHAM, Stephanie R, OWEN, Robert J, ELVISS, Nicola C, LABIGNE, Agnés, JENKS, Peter J
Format Journal Article
LanguageEnglish
Published Washington, DC American Society for Microbiology 01.09.2001
Subjects
Animals
Anti-Bacterial Agents - pharmacology
Antibodies, Bacterial - immunology
Bacterial diseases
Bacterial diseases of the digestive system and abdomen
Bacterial Proteins - immunology
Bacteriology
Biological and medical sciences
Drug Resistance, Bacterial
Epidemiology
Fundamental and applied biological sciences. Psychology
Helicobacter Infections - microbiology
Helicobacter pylori
Helicobacter pylori - classification
Helicobacter pylori - drug effects
Human bacterial diseases
Humans
Immunoblotting
Infectious diseases
Medical sciences
Membrane Proteins - immunology
Metronidazole - pharmacology
Microbiology
Rabbits
rdxA gene
RdxA protein
Nitro compound
Imidazole derivatives
Sensitivity
Spirillales
Helicobacter pylori
Spirillaceae
Immunoblotting assay
Bacteria
Metronidazole
Differentiation
Clinical isolate
Protein
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Abstract Article Usage Stats Services JCM Citing Articles Google Scholar PubMed Related Content Social Bookmarking CiteULike Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter current issue JCM About JCM Subscribers Authors Reviewers Advertisers Inquiries from the Press Permissions & Commercial Reprints ASM Journals Public Access Policy JCM RSS Feeds 1752 N Street N.W. • Washington DC 20036 202.737.3600 • 202.942.9355 fax • journals@asmusa.org Print ISSN: 0095-1137 Online ISSN: 1098-660X Copyright © 2014 by the American Society for Microbiology.   For an alternate route to JCM .asm.org, visit: JCM       
AbstractList Antimicrobial resistance in Helicobacter pylori is a serious and increasing problem, and the development of rapid, reliable methods for detecting resistance would greatly improve the selection of antibiotics used to treat gastric infection with this organism. We assessed whether detection of the RdxA protein could provide the basis for determining the susceptibility of H. pylori to metronidazole. In order to raise polyclonal antisera to RdxA, we cloned the rdxA gene from H. pylori strain 26695 into the commercial expression vector pMAL-c2, purified the resultant fusion protein by affinity chromatography, and used this recombinant RdxA preparation to immunize rabbits. We then used this specific anti-RdxA antibody to perform immunoblotting on whole bacterial cell lysates of 17 metronidazole-sensitive and 27 metronidazole-resistant clinical isolates of H. pylori. While a 24-kDa immunoreactive band corresponding to the RdxA protein was observed in all metronidazole-sensitive strains, this band was absent in 25 of 27 resistant isolates. Our results indicate that testing for the absence of the RdxA protein would identify the majority of clinical isolates that will respond poorly to metronidazole-containing eradication regimens and have implications for the development of assays capable of detecting metronidazole resistance in H. pylori.
Antimicrobial resistance in Helicobacter pylori is a serious and increasing problem, and the development of rapid, reliable methods for detecting resistance would greatly improve the selection of antibiotics used to treat gastric infection with this organism. We assessed whether detection of the RdxA protein could provide the basis for determining the susceptibility of H. pylori to metronidazole. In order to raise polyclonal antisera to RdxA, we cloned the rdxA gene from H. pylori strain 26695 into the commercial expression vector pMAL-c2, purified the resultant fusion protein by affinity chromatography, and used this recombinant RdxA preparation to immunize rabbits. We then used this specific anti-RdxA antibody to perform immunoblotting on whole bacterial cell lysates of 17 metronidazole-sensitive and 27 metronidazole-resistant clinical isolates of H. pylori . While a 24-kDa immunoreactive band corresponding to the RdxA protein was observed in all metronidazole-sensitive strains, this band was absent in 25 of 27 resistant isolates. Our results indicate that testing for the absence of the RdxA protein would identify the majority of clinical isolates that will respond poorly to metronidazole-containing eradication regimens and have implications for the development of assays capable of detecting metronidazole resistance in H. pylori .
Article Usage Stats Services JCM Citing Articles Google Scholar PubMed Related Content Social Bookmarking CiteULike Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter current issue JCM About JCM Subscribers Authors Reviewers Advertisers Inquiries from the Press Permissions & Commercial Reprints ASM Journals Public Access Policy JCM RSS Feeds 1752 N Street N.W. • Washington DC 20036 202.737.3600 • 202.942.9355 fax • journals@asmusa.org Print ISSN: 0095-1137 Online ISSN: 1098-660X Copyright © 2014 by the American Society for Microbiology.   For an alternate route to JCM .asm.org, visit: JCM       
ABSTRACT Antimicrobial resistance in Helicobacter pylori is a serious and increasing problem, and the development of rapid, reliable methods for detecting resistance would greatly improve the selection of antibiotics used to treat gastric infection with this organism. We assessed whether detection of the RdxA protein could provide the basis for determining the susceptibility of H. pylori to metronidazole. In order to raise polyclonal antisera to RdxA, we cloned the rdxA gene from H. pylori strain 26695 into the commercial expression vector pMAL-c2, purified the resultant fusion protein by affinity chromatography, and used this recombinant RdxA preparation to immunize rabbits. We then used this specific anti-RdxA antibody to perform immunoblotting on whole bacterial cell lysates of 17 metronidazole-sensitive and 27 metronidazole-resistant clinical isolates of H. pylori . While a 24-kDa immunoreactive band corresponding to the RdxA protein was observed in all metronidazole-sensitive strains, this band was absent in 25 of 27 resistant isolates. Our results indicate that testing for the absence of the RdxA protein would identify the majority of clinical isolates that will respond poorly to metronidazole-containing eradication regimens and have implications for the development of assays capable of detecting metronidazole resistance in H. pylori .
Author Robert J. Owen
Nicola C. Elviss
Peter J. Jenks
Stephanie R. Latham
Agnès Labigne
AuthorAffiliation Department of Medical Microbiology, Royal Free and University College Medical School, 1 and Helicobacter Reference Unit, Central Public Health Laboratory, 2 London, and Institute of Infections and Immunity, University of Nottingham, Nottingham, 4 United Kingdom, and Unité de Pathogénie Bactérienne des Muqueuses, Institut Pasteur, Paris, France 3
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Issue 9
Keywords Nitro compound
Imidazole derivatives
Sensitivity
Spirillales
Helicobacter pylori
Spirillaceae
Immunoblotting assay
Bacteria
Metronidazole
Differentiation
Clinical isolate
Protein
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Corresponding author. Mailing address: Institute of Infections and Immunity, Floor C, West Block, University Hospital, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom. Phone: 44-115-9249924, ext. 42457. Fax: 44-115-9709923. E-mail: Peter.Jenks@nottingham.ac.uk.
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Snippet Article Usage Stats Services JCM Citing Articles Google Scholar PubMed Related Content Social Bookmarking CiteULike Delicious Digg Facebook Google+ Mendeley...
Antimicrobial resistance in Helicobacter pylori is a serious and increasing problem, and the development of rapid, reliable methods for detecting resistance...
ABSTRACT Antimicrobial resistance in Helicobacter pylori is a serious and increasing problem, and the development of rapid, reliable methods for detecting...
Antimicrobial resistance in Helicobacter pylori is a serious and increasing problem, and the development of rapid, reliable methods for detecting resistance...
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SubjectTerms Animals
Anti-Bacterial Agents - pharmacology
Antibodies, Bacterial - immunology
Bacterial diseases
Bacterial diseases of the digestive system and abdomen
Bacterial Proteins - immunology
Bacteriology
Biological and medical sciences
Drug Resistance, Bacterial
Epidemiology
Fundamental and applied biological sciences. Psychology
Helicobacter Infections - microbiology
Helicobacter pylori
Helicobacter pylori - classification
Helicobacter pylori - drug effects
Human bacterial diseases
Humans
Immunoblotting
Infectious diseases
Medical sciences
Membrane Proteins - immunology
Metronidazole - pharmacology
Microbiology
Rabbits
rdxA gene
RdxA protein
Title Differentiation of Metronidazole-Sensitive and -Resistant Clinical Isolates of Helicobacter pylori by Immunoblotting with Antisera to the RdxA Protein
URI http://jcm.asm.org/content/39/9/3052.abstract
https://www.ncbi.nlm.nih.gov/pubmed/11526127
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Volume 39
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