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1
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Homan M, Jones NL, Bontems P, Carroll MW, Czinn SJ, Gold BD, Goodman K, Harris PR, Jerris R, Kalach N, Kori M, Megraud F, Rowland M, Tavares M. Updated joint ESPGHAN/NASPGHAN guidelines for management of Helicobacter pylori infection in children and adolescents (2023). J Pediatr Gastroenterol Nutr 2024; 79:758-785. [PMID: 39148213 DOI: 10.1002/jpn3.12314] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/22/2023] [Revised: 04/25/2024] [Accepted: 05/10/2024] [Indexed: 08/17/2024]
Abstract
BACKGROUND Evolving epidemiological data and increasing antibiotic resistance mandate an update of the European and North American Societies of Pediatric Gastroenterology, Hepatology and Nutrition guidelines. METHODS Certainty of evidence and strength of recommendations were rated by experts according to the Grading of Recommendation Assessment, Development, and Evaluation approach. PICO (patient population, intervention, comparator, and outcome) questions were developed and voted on by the group. Recommendations were formulated using the Evidence to Decision framework. RESULTS The current literature supports many of the previous recommendations and several new recommendations. Invasive testing with strain antimicrobial susceptibility analysis is recommended for the diagnosis and selection of eradication therapy for H. pylori infection. Molecular methods are acceptable for detection of infection and of antibiotic resistance in gastric biopsy specimens. Reliable, noninvasive tests can be used as a screening method for children with history of gastric cancer in a first-degree relative. When investigating causes of chronic immune thrombocytopenic purpura, testing for H. pylori is no longer recommended. When investigating other diseases such as inflammatory bowel disease, celiac disease, or eosinophilic esophagitis, specific diagnostic biopsies for H. pylori infection are not indicated. However, if H. pylori is an incidental finding, treatment may be considered after discussing the risks and benefits. Treatment should be based on antibiotic antimicrobial susceptibility testing and, if unavailable, regimens containing clarithromycin should be avoided. CONCLUSIONS Due to decreasing prevalence of infection, increasing challenges with antibiotic resistance, and emerging evidence regarding complications of infection, clinicians must be aware of these recommended changes to appropriately manage H. pylori infection and its clinical sequelae in children.
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Affiliation(s)
- Matjaž Homan
- Faculty of Medicine, Children's Hospital in Ljubljana, University of Ljubljana, Ljubljana, Slovenia
| | - Nicola L Jones
- Division of Gastroenterology Hepatology and Nutrition, SickKids, University of Toronto, Toronto, Ontario, Canada
| | - Patrick Bontems
- Université Libre de Bruxelles, Hôpital Unversitaire des Enfants Reine Fabiola, Hôpitaux Universitaires de Bruxelles, Brussels, Belgium
| | - Matthew W Carroll
- Division of Gastroenterology and Nutrition, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada
| | - Steven J Czinn
- University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - Benjamin D Gold
- GI Care for Kids, LLC, Children's Center for Digestive Healthcare LLC, Children's Healthcare of Atlanta, Atlanta, Georgia, USA
| | - Karen Goodman
- Faculty of Medicine and Dentistry, Division of Gastroenterology, University of Alberta, Edmonton, Alberta, Canada
| | - Paul R Harris
- Department of Pediatric Gastroenterology and Nutrition, Pontificia Universidad Catolica de Chile, Santiago, Chile
| | - Robert Jerris
- Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, Georgia, USA
| | - Nicolas Kalach
- Department of Pediatrics, Saint Vincent de Paul Hospital, Groupement des Hôpitaux de l'Institut Catholique de Lille (GHICL), Catholic University of Lille, Lille, France
| | - Michal Kori
- Pediatric Gastroenterology, Kaplan Medical Center, Rehovot, and Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel
| | | | - Marion Rowland
- School of Medicine and Medical Science, University College Dublin, Dublin, Ireland
| | - Marta Tavares
- Unidade de Gastroenterologia, Centro Materno Infantil do Norte, Centro Hospitalar Universitário de Santo António, Porto, Portugal
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Caradec T, Plé C, Sicoli G, Petrov R, Pradel E, Sobieski C, Antoine R, Orio M, Herledan A, Willand N, Hartkoorn RC. Small molecule MarR modulators potentiate metronidazole antibiotic activity in aerobic E. coli by inducing activation by the nitroreductase NfsA. J Biol Chem 2024; 300:107431. [PMID: 38825006 PMCID: PMC11259696 DOI: 10.1016/j.jbc.2024.107431] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2024] [Revised: 05/07/2024] [Accepted: 05/22/2024] [Indexed: 06/04/2024] Open
Abstract
Antibiotic-resistant Enterobacterales pose a major threat to healthcare systems worldwide, necessitating the development of novel strategies to fight such hard-to-kill bacteria. One potential approach is to develop molecules that force bacteria to hyper-activate prodrug antibiotics, thus rendering them more effective. In the present work, we aimed to obtain proof-of-concept data to support that small molecules targeting transcriptional regulators can potentiate the antibiotic activity of the prodrug metronidazole (MTZ) against Escherichia coli under aerobic conditions. By screening a chemical library of small molecules, a series of structurally related molecules were identified that had little inherent antibiotic activity but showed substantial activity in combination with ineffective concentrations of MTZ. Transcriptome analyses, functional genetics, thermal shift assays, and electrophoretic mobility shift assays were then used to demonstrate that these MTZ boosters target the transcriptional repressor MarR, resulting in the upregulation of the marRAB operon and its downstream MarA regulon. The associated upregulation of the flavin-containing nitroreductase, NfsA, was then shown to be critical for the booster-mediated potentiation of MTZ antibiotic activity. Transcriptomic studies, biochemical assays, and electron paramagnetic resonance measurements were then used to show that under aerobic conditions, NfsA catalyzed 1-electron reduction of MTZ to the MTZ radical anion which in turn induced lethal DNA damage in E. coli. This work reports the first example of prodrug boosting in Enterobacterales by transcriptional modulators and highlights that MTZ antibiotic activity can be chemically induced under anaerobic growth conditions.
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Affiliation(s)
- Thibault Caradec
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur Lille, U1019 - UMR 9017 - CIIL - Center for Infection and Immunity of Lille, Lille, France
| | - Coline Plé
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur Lille, U1019 - UMR 9017 - CIIL - Center for Infection and Immunity of Lille, Lille, France
| | - Giuseppe Sicoli
- CNRS UMR 8516, Univ. Lille, LASIRE - Laboratory of Advanced Spectroscopy on Interactions, Reactivity and Environment, Villeneuve d'Ascq, France
| | - Ravil Petrov
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur Lille, U1019 - UMR 9017 - CIIL - Center for Infection and Immunity of Lille, Lille, France
| | - Elizabeth Pradel
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur Lille, U1019 - UMR 9017 - CIIL - Center for Infection and Immunity of Lille, Lille, France
| | - Cécilia Sobieski
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur Lille, U1019 - UMR 9017 - CIIL - Center for Infection and Immunity of Lille, Lille, France
| | - Rudy Antoine
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur Lille, U1019 - UMR 9017 - CIIL - Center for Infection and Immunity of Lille, Lille, France
| | - Maylis Orio
- Aix Marseille Univ., CNRS, Centrale Marseille, iSm2, Marseille, France
| | - Adrien Herledan
- Univ. Lille, Inserm, Institut Pasteur de Lille, U1177 - Drugs and Molecules for Living Systems, Lille, France
| | - Nicolas Willand
- Univ. Lille, Inserm, Institut Pasteur de Lille, U1177 - Drugs and Molecules for Living Systems, Lille, France
| | - Ruben Christiaan Hartkoorn
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur Lille, U1019 - UMR 9017 - CIIL - Center for Infection and Immunity of Lille, Lille, France.
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Hamid A, Mäser P, Mahmoud AB. Drug Repurposing in the Chemotherapy of Infectious Diseases. Molecules 2024; 29:635. [PMID: 38338378 PMCID: PMC10856722 DOI: 10.3390/molecules29030635] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2023] [Revised: 01/18/2024] [Accepted: 01/24/2024] [Indexed: 02/12/2024] Open
Abstract
Repurposing is a universal mechanism for innovation, from the evolution of feathers to the invention of Velcro tape. Repurposing is particularly attractive for drug development, given that it costs more than a billion dollars and takes longer than ten years to make a new drug from scratch. The COVID-19 pandemic has triggered a large number of drug repurposing activities. At the same time, it has highlighted potential pitfalls, in particular when concessions are made to the target product profile. Here, we discuss the pros and cons of drug repurposing for infectious diseases and analyze different ways of repurposing. We distinguish between opportunistic and rational approaches, i.e., just saving time and money by screening compounds that are already approved versus repurposing based on a particular target that is common to different pathogens. The latter can be further distinguished into divergent and convergent: points of attack that are divergent share common ancestry (e.g., prokaryotic targets in the apicoplast of malaria parasites), whereas those that are convergent arise from a shared lifestyle (e.g., the susceptibility of bacteria, parasites, and tumor cells to antifolates due to their high rate of DNA synthesis). We illustrate how such different scenarios can be capitalized on by using examples of drugs that have been repurposed to, from, or within the field of anti-infective chemotherapy.
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Affiliation(s)
- Amal Hamid
- Faculty of Pharmacy, University of Khartoum, Khartoum 11111, Sudan;
| | - Pascal Mäser
- Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Allschwil, 4123 Basel, Switzerland
- Faculty of Science, University of Basel, 4001 Basel, Switzerland
| | - Abdelhalim Babiker Mahmoud
- Faculty of Pharmacy, University of Khartoum, Khartoum 11111, Sudan;
- Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Saarland, 66123 Saarbruecken, Germany
- Department of Microbial Drugs, Helmholtz Centre for Infection Research (HZI), 38124 Braunschweig, Germany
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Mokrousov I, Slavchev I, Solovieva N, Dogonadze M, Vyazovaya A, Valcheva V, Masharsky A, Belopolskaya O, Dimitrov S, Zhuravlev V, Portugal I, Perdigão J, Dobrikov GM. Molecular Insight into Mycobacterium tuberculosis Resistance to Nitrofuranyl Amides Gained through Metagenomics-like Analysis of Spontaneous Mutants. Pharmaceuticals (Basel) 2022; 15:ph15091136. [PMID: 36145357 PMCID: PMC9504009 DOI: 10.3390/ph15091136] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2022] [Revised: 09/04/2022] [Accepted: 09/08/2022] [Indexed: 11/16/2022] Open
Abstract
We performed synthesis of new nitrofuranyl amides and investigated their anti-TB activity and primary genetic response of mycobacteria through whole-genome sequencing (WGS) of spontaneous resistant mutants. The in vitro activity was assessed on reference strain Mycobacterium tuberculosis H37Rv. The most active compound 11 was used for in vitro selection of spontaneous resistant mutants. The same mutations in six genes were detected in bacterial cultures grown under increased concentrations of 11 (2×, 4×, 8× MIC). The mutant positions were presented as mixed wild type and mutant alleles while increasing the concentration of the compound led to the semi-proportional and significant increase in mutant alleles. The identified genes belong to different categories and pathways. Some of them were previously reported as mediating drug resistance or drug tolerance, and counteracting oxidative and nitrosative stress, in particular: Rv0224c, fbiC, iniA, and Rv1592c. Gene-set interaction analysis revealed a certain weak interaction for gene pairs Rv1592–Rv1639c and Rv1592–Rv0224c. To conclude, this study experimentally demonstrated a multifaceted primary genetic response of M. tuberculosis to the action of nitrofurans. All three 11-treated subcultures independently presented the same six SNPs, which suggests their non-random occurrence and likely causative relationship between compound action and possible resistance mechanism.
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Affiliation(s)
- Igor Mokrousov
- Laboratory of Molecular Epidemiology and Evolutionary Genetics, St. Petersburg Pasteur Institute, 197101 St. Petersburg, Russia
- Henan International Joint Laboratory of Children’s Infectious Diseases, Children’s Hospital Affiliated to Zhengzhou University, Zhengzhou 450018, China
- Correspondence: (I.M.); (G.M.D.)
| | - Ivaylo Slavchev
- Institute of Organic Chemistry with Centre of Phytochemistry, Acad. G. Bonchev Street, bl. 9, 1113 Sofia, Bulgaria
| | - Natalia Solovieva
- St. Petersburg Research Institute of Phthisiopulmonology, 191036 St. Petersburg, Russia
| | - Marine Dogonadze
- St. Petersburg Research Institute of Phthisiopulmonology, 191036 St. Petersburg, Russia
| | - Anna Vyazovaya
- Laboratory of Molecular Epidemiology and Evolutionary Genetics, St. Petersburg Pasteur Institute, 197101 St. Petersburg, Russia
| | - Violeta Valcheva
- The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Street, bl. 26, 1113 Sofia, Bulgaria
| | - Aleksey Masharsky
- Resource Center “Bio-bank Center”, Research Park of St. Petersburg State University, 198504 St. Petersburg, Russia
| | - Olesya Belopolskaya
- Resource Center “Bio-bank Center”, Research Park of St. Petersburg State University, 198504 St. Petersburg, Russia
| | - Simeon Dimitrov
- The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Street, bl. 26, 1113 Sofia, Bulgaria
| | - Viacheslav Zhuravlev
- St. Petersburg Research Institute of Phthisiopulmonology, 191036 St. Petersburg, Russia
| | - Isabel Portugal
- iMed.ULisboa–Instituto de Investigação do Medicamento, Faculdade de Farmácia, Universidade de Lisboa, 1649004 Lisbon, Portugal
| | - João Perdigão
- iMed.ULisboa–Instituto de Investigação do Medicamento, Faculdade de Farmácia, Universidade de Lisboa, 1649004 Lisbon, Portugal
| | - Georgi M. Dobrikov
- Institute of Organic Chemistry with Centre of Phytochemistry, Acad. G. Bonchev Street, bl. 9, 1113 Sofia, Bulgaria
- Correspondence: (I.M.); (G.M.D.)
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Caraene ID, Gruchlik Y, Busetti F, Linge KL, Joll CA. Degradation of selected pharmaceuticals detected in wastewater systems using an enzyme-mediator system and identification of resulting transformation products. BIOCATAL BIOTRANSFOR 2021. [DOI: 10.1080/10242422.2021.2003344] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/07/2022]
Affiliation(s)
- Ionut Daniel Caraene
- Curtin Water Quality Research Group, School of Molecular and Life Sciences, Curtin University, Perth, Australia
| | - Yolanta Gruchlik
- Curtin Water Quality Research Group, School of Molecular and Life Sciences, Curtin University, Perth, Australia
| | | | - Kathryn L. Linge
- Curtin Water Quality Research Group, School of Molecular and Life Sciences, Curtin University, Perth, Australia
- ChemCentre, Perth, Australia
| | - Cynthia A. Joll
- Curtin Water Quality Research Group, School of Molecular and Life Sciences, Curtin University, Perth, Australia
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Hu B, Song N, Cao Y, Li M, Liu X, Zhou Z, Shi L, Yu Z. Noncanonical Amino Acids for Hypoxia-Responsive Peptide Self-Assembly and Fluorescence. J Am Chem Soc 2021; 143:13854-13864. [PMID: 34410694 DOI: 10.1021/jacs.1c06435] [Citation(s) in RCA: 51] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Design of endogenous stimuli-responsive amino acids allows for precisely modulating proteins or peptides under a biological microenvironment and thereby regulating their performance. Herein we report a noncanonical amino acid 2-nitroimidazol-1-yl alanine and explore its functions in creation of the nitroreductase (NTR)-responsive peptide-based supramolecular probes for efficient hypoxia imaging. On the basis of the reduction potential of the nitroimidazole unit, the amino acid was synthesized via the Mitsunobu reaction between 2-nitroimidazole and a serine derivate. We elucidated the relationship between the NTR-responsiveness of the amino acid and the structural feature of peptides involving a series of peptides. This eventually facilitates development of aromatic peptides undergoing NTR-responsive self-assembly by rationally optimizing the sequences. Due to the intrinsic role of 2-nitroimidazole in the fluorescence quench, we created a morphology-transformable supramolecular probe for imaging hypoxic tumor cells based on NTR reduction. We found that the resulting supramolecular probes penetrated into solid tumors, thus allowing for efficient fluorescence imaging of tumor cells in hypoxic regions. Our findings demonstrate development of a readily synthesized and versatile amino acid with exemplified properties in creating fluorescent peptide nanostructures responsive to a biological microenvironment, thus providing a powerful toolkit for synthetic biology and development of novel biomaterials.
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Affiliation(s)
- Binbin Hu
- Key Laboratory of Functional Polymer Materials, Ministry of Education, State Key Laboratory of Medicinal Chemical Biology, Institute of Polymer Chemistry, College of Chemistry, Nankai University, 94 Weijin Road, Tianjin 300071, China
| | - Na Song
- Key Laboratory of Functional Polymer Materials, Ministry of Education, State Key Laboratory of Medicinal Chemical Biology, Institute of Polymer Chemistry, College of Chemistry, Nankai University, 94 Weijin Road, Tianjin 300071, China
| | - Yawei Cao
- Key Laboratory of Functional Polymer Materials, Ministry of Education, State Key Laboratory of Medicinal Chemical Biology, Institute of Polymer Chemistry, College of Chemistry, Nankai University, 94 Weijin Road, Tianjin 300071, China
| | - Mingming Li
- Key Laboratory of Functional Polymer Materials, Ministry of Education, State Key Laboratory of Medicinal Chemical Biology, Institute of Polymer Chemistry, College of Chemistry, Nankai University, 94 Weijin Road, Tianjin 300071, China
| | - Xin Liu
- Key Laboratory of Functional Polymer Materials, Ministry of Education, State Key Laboratory of Medicinal Chemical Biology, Institute of Polymer Chemistry, College of Chemistry, Nankai University, 94 Weijin Road, Tianjin 300071, China
| | - Zhifei Zhou
- Key Laboratory of Functional Polymer Materials, Ministry of Education, State Key Laboratory of Medicinal Chemical Biology, Institute of Polymer Chemistry, College of Chemistry, Nankai University, 94 Weijin Road, Tianjin 300071, China
| | - Linqi Shi
- Key Laboratory of Functional Polymer Materials, Ministry of Education, State Key Laboratory of Medicinal Chemical Biology, Institute of Polymer Chemistry, College of Chemistry, Nankai University, 94 Weijin Road, Tianjin 300071, China
| | - Zhilin Yu
- Key Laboratory of Functional Polymer Materials, Ministry of Education, State Key Laboratory of Medicinal Chemical Biology, Institute of Polymer Chemistry, College of Chemistry, Nankai University, 94 Weijin Road, Tianjin 300071, China
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Current development of 5-nitrofuran-2-yl derivatives as antitubercular agents. Bioorg Chem 2019; 88:102969. [PMID: 31077910 DOI: 10.1016/j.bioorg.2019.102969] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2019] [Revised: 04/29/2019] [Accepted: 04/30/2019] [Indexed: 11/22/2022]
Abstract
Pulmonary tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB) and still remains one of the foremost fatal infectious diseases, infecting nearly a third of the worldwide population. The emergencies of multidrug-resistant and extensively drug-resistant tuberculosis (MDR and XDR-TB) prompt the efforts to deliver potent and novel anti-TB drugs. Research aimed at the development of new anti-TB drugs based on nitrofuran scaffold led to the identification of several candidates that were effective against actively growing as well as latent mycobacteria with unique modes of action. This review focuses on the recent advances in nitrofurans that could provide intriguing potential leads in the area of anti-TB drug discovery.
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8
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Siavoshi F, Saniee P, Malekzadeh R. Effective antimicrobial activity of rifabutin against multidrug-resistant Helicobacter pylori. Helicobacter 2018; 23:e12531. [PMID: 30230637 DOI: 10.1111/hel.12531] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/02/2018] [Revised: 07/21/2018] [Accepted: 07/21/2018] [Indexed: 12/23/2022]
Abstract
BACKGROUND Helicobacter pylori resistance to more than one antibiotic is the main reason for failure in bacterial eradication in a considerable number of patients. Rifabutin (RFB) with a broad-spectrum of antimicrobial therapy has been suggested for treatment of refractory multidrug-resistant infections. METHODS Helicobacter pylori isolates from 104 patients were examined for resistance to 5 currently used antibiotics and RFB, using agar dilution method. Twofold serial dilutions of antibiotics were used and MICs (μg/mL) determined as metronidazole (MTZ 8), clarithromycin (CLR 2), amoxicillin (AMX 1), tetracycline (TET 0.5), furazolidone (FRZ 0.5), and RFB (0.06). RESULTS Of 104 H. pylori isolates, only 7 (6.7%) were sensitive to all the 6 antibiotics. However, 30 (28.8%) were resistant to one antibiotic, 28 (26.9%) to two, 19 (18.2%) to three, 14 (13.4%) to four, and 6 (5.7%) to five currently used antibiotics. Overall, 67(64.4%) of isolates were resistant to 2-5 currently used antibiotics and considered as multidrug-resistant (MDR), with 59 (88.1%) showing sensitivity to RFB and 8 (11.9%) resistance (P < 0.05). Of 33 isolates resistant to both MTZ and CLR, 25 (75.7%) were sensitive to RFB and 8 (24.3%) resistant (P < 0.05). DISCUSSION In vitro antimicrobial effectiveness of RFB on MDR H. pylori including those with resistance to both MTZ and CLR was demonstrated. However, RFB efficacy decreased as the number of antibiotics responsible for MDR increased. Considering that RFB inhibits both extra- and intracellular H. pylori, it can be suggested as an effective antibiotic against of MDR H. pylori.
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Affiliation(s)
- Farideh Siavoshi
- Department of Microbiology, School of Biology, University College of Sciences, University of Tehran, Tehran, Iran
| | - Parastoo Saniee
- Faculty of Life Sciences and Biotechnology, Department of Microbiology, Shahid Beheshti University G.C, Tehran, Iran
| | - Reza Malekzadeh
- Digestive Oncology Research Center, Digestive Disease Research Institute, Tehran University of Medical Sciences, Tehran, Iran
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Abstract
The 5-nitroimidazole drug metronidazole has remained the drug of choice in the treatment of anaerobic infections, parasitic as well as bacterial, ever since its development in 1959. In contrast to most other antimicrobials, it has a pleiotropic mode of action and reacts with a large number of molecules. Importantly, metronidazole, which is strictly speaking a prodrug, needs to be reduced at its nitro group in order to become toxic. Reduction of metronidazole, however, only takes place under very low concentrations of oxygen, explaining why metronidazole is exclusively toxic to microaerophilic and anaerobic microorganisms. In general, resistance rates amongst the pathogens treated with metronidazole have remained low until the present day. Nevertheless, metronidazole resistance does occur, and for the treatment of some pathogens, especially Helicobacter pylori, metronidazole has become almost useless in some parts of the world. This review will give an account on the current status of research on metronidazole's mode of action, metronidazole resistance in eukaryotes and prokaryotes, and on other 5-nitroimidazoles in use.
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Ang CW, Jarrad AM, Cooper MA, Blaskovich MAT. Nitroimidazoles: Molecular Fireworks That Combat a Broad Spectrum of Infectious Diseases. J Med Chem 2017; 60:7636-7657. [PMID: 28463485 DOI: 10.1021/acs.jmedchem.7b00143] [Citation(s) in RCA: 111] [Impact Index Per Article: 13.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Infectious diseases claim millions of lives every year, but with the advent of drug resistance, therapeutic options to treat infections are inadequate. There is now an urgent need to develop new and effective treatments. Nitroimidazoles are a class of antimicrobial drugs that have remarkable broad spectrum activity against parasites, mycobacteria, and anaerobic Gram-positive and Gram-negative bacteria. While nitroimidazoles were discovered in the 1950s, there has been renewed interest in their therapeutic potential, particularly for the treatment of parasitic infections and tuberculosis. In this review, we summarize different classes of nitroimidazoles that have been described in the literature in the past five years, from approved drugs and clinical candidates to examples undergoing preclinical or early stage development. The relatively "nonspecific" mode of action and resistance mechanisms of nitromidazoles are discussed, and contemporary strategies to facilitate nitroimidazole drug development are highlighted.
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Affiliation(s)
- Chee Wei Ang
- The Institute for Molecular Bioscience, The University of Queensland , St Lucia, Queensland 4072, Australia
| | - Angie M Jarrad
- The Institute for Molecular Bioscience, The University of Queensland , St Lucia, Queensland 4072, Australia
| | - Matthew A Cooper
- The Institute for Molecular Bioscience, The University of Queensland , St Lucia, Queensland 4072, Australia
| | - Mark A T Blaskovich
- The Institute for Molecular Bioscience, The University of Queensland , St Lucia, Queensland 4072, Australia
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11
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Hu Y, Zhang M, Lu B, Dai J. Helicobacter pylori and Antibiotic Resistance, A Continuing and Intractable Problem. Helicobacter 2016; 21:349-63. [PMID: 26822340 DOI: 10.1111/hel.12299] [Citation(s) in RCA: 102] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Helicobacter pylori, a human pathogen with a high global prevalence, is the causative pathogen for multiple gastrointestinal diseases, especially chronic gastritis, peptic ulcers, gastric mucosa-associated lymphoid tissue lymphoma, and gastric malignancies. Antibiotic therapies remain the mainstay for H. pylori eradication; however, this strategy is hampered by the emergence and spread of H. pylori antibiotic resistance. Exploring the mechanistic basis of this resistance is becoming one of the major research questions in contemporary biomedical research, as such knowledge could be exploited to devise novel rational avenues for counteracting the existing resistance and devising strategies to avoid the development of a novel anti-H. pylori medication. Encouragingly, important progress in this field has been made recently. Here, we attempt to review the current state and progress with respect to the molecular mechanism of antibiotic resistance for H. pylori. A picture is emerging in which mutations of various genes in H. pylori, resulting in decreased membrane permeability, altered oxidation-reduction potential, and a more efficient efflux pump system. The increased knowledge on these mechanisms produces hope that antibiotic resistance in H. pylori can ultimately be countered.
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Affiliation(s)
- Yue Hu
- Department of Gastroenterology, First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China
| | - Meng Zhang
- Department of Gastroenterology, First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China
| | - Bin Lu
- Department of Gastroenterology, First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China.
| | - Jinfeng Dai
- Department of Gastroenterology, First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China
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12
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Penuliar GM, Nakada-Tsukui K, Nozaki T. Phenotypic and transcriptional profiling in Entamoeba histolytica reveal costs to fitness and adaptive responses associated with metronidazole resistance. Front Microbiol 2015; 6:354. [PMID: 25999919 PMCID: PMC4419850 DOI: 10.3389/fmicb.2015.00354] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2015] [Accepted: 04/08/2015] [Indexed: 12/11/2022] Open
Abstract
Antimicrobial chemotherapy is critical in the fight against infectious diseases caused by Entamoeba histolytica. Among the drugs available for the treatment of amebiasis, metronidazole (MTZ) is considered the drug of choice. Recently, in vitro studies have described MTZ resistance and the potential mechanisms involved. Costs to fitness and adaptive responses associated with resistance, however, have not been investigated. In this study we generated an HM-1 derived strain resistant to 12 μM MTZ (MTZR). We examined its phenotypic and transcriptional profile to determine the consequences and mRNA level changes associated with MTZ resistance. Our results indicated increased cell size and granularity, and decreased rates in cell division, adhesion, phagocytosis, cytopathogenicity, and glucose consumption. Transcriptome analysis revealed 142 differentially expressed genes in MTZR. In contrast to other MTZ resistant parasites, MTZR did not down-regulate pyruvate:ferredoxin oxidoreductase, but showed increased expression of genes for a hypothetical protein (HP1) and several iron-sulfur flavoproteins, and downregulation of genes for leucine-rich proteins. Fisher's exact test showed 24 significantly enriched GO terms in MTZR, and a 3-way comparison of modulated genes in MTZR against those of MTZR cultured without MTZ and HM-1 cultured with MTZ, showed that 88 genes were specific to MTZR. Overall, our findings suggested that MTZ resistance is associated with specific transcriptional changes and decreased parasite virulence.
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Affiliation(s)
- Gil M Penuliar
- Department of Parasitology, National Institute of Infectious Diseases Tokyo, Japan ; Department of Parasitology, Gunma University Graduate School of Medicine Maebashi, Japan
| | - Kumiko Nakada-Tsukui
- Department of Parasitology, National Institute of Infectious Diseases Tokyo, Japan
| | - Tomoyoshi Nozaki
- Department of Parasitology, National Institute of Infectious Diseases Tokyo, Japan ; Graduate School of Life and Environmental Sciences, University of Tsukuba Tsukuba, Japan
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Pentacyclic nitrofurans with in vivo efficacy and activity against nonreplicating Mycobacterium tuberculosis. PLoS One 2014; 9:e87909. [PMID: 24505329 PMCID: PMC3914891 DOI: 10.1371/journal.pone.0087909] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2013] [Accepted: 12/30/2013] [Indexed: 11/19/2022] Open
Abstract
The reductively activated nitroaromatic class of antimicrobials, which include nitroimidazole and the more metabolically labile nitrofuran antitubercular agents, have demonstrated some potential for development as therapeutics against dormant TB bacilli. In previous studies, the pharmacokinetic properties of nitrofuranyl isoxazolines were improved by incorporation of the outer ring elements of the antitubercular nitroimidazole OPC-67683. This successfully increased stability of the resulting pentacyclic nitrofuran lead compound Lee1106 (referred to herein as 9a). In the current study, we report the synthesis and antimicrobial properties of 9a and panel of 9a analogs, which were developed to increase oral bioavailability. These hybrid nitrofurans remained potent inhibitors of Mycobacterium tuberculosis with favorable selectivity indices (>150) and a narrow spectrum of activity. In vivo, the pentacyclic nitrofuran compounds showed long half-lives and high volumes of distribution. Based on pharmacokinetic testing and lack of toxicity in vivo,9a remained the series lead. 9a exerted a lengthy post antibiotic effect and was highly active against nonreplicating M. tuberculosis grown under hypoxia. 9a showed a low potential for cross resistance to current antitubercular agents, and a mechanism of activation distinct from pre-clinical tuberculosis candidates PA-824 and OPC-67683. Together these studies show that 9a is a nanomolar inhibitor of actively growing as well as nonreplicating M. tuberculosis.
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Abstract
Mycobacterium tuberculosis (Mtb) is a metabolically flexible pathogen
that has the extraordinary ability to sense and adapt to the continuously changing host
environment experienced during decades of persistent infection. Mtb is
continually exposed to endogenous reactive oxygen species (ROS) as part of normal aerobic
respiration, as well as exogenous ROS and reactive nitrogen species (RNS) generated by the
host immune system in response to infection. The magnitude of tuberculosis (TB) disease is
further amplified by exposure to xenobiotics from the environment such as cigarette smoke
and air pollution, causing disruption of the intracellular
prooxidant–antioxidant balance. Both oxidative and reductive stresses induce
redox cascades that alter Mtb signal transduction, DNA and RNA synthesis,
protein synthesis and antimycobacterial drug resistance. As reviewed in this article,
Mtb has evolved specific mechanisms to protect itself against
endogenously produced oxidants, as well as defend against host and environmental oxidants
and reductants found specifically within the microenvironments of the lung. Maintaining an
appropriate redox balance is critical to the clinical outcome because several
antimycobacterial prodrugs are only effective upon bioreductive activation. Proper
homeostasis of oxido-reductive systems is essential for Mtb survival,
persistence and subsequent reactivation. The progress and remaining deficiencies in
understanding Mtb redox homeostasis are also discussed.
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Mukherjee T, Boshoff H. Nitroimidazoles for the treatment of TB: past, present and future. Future Med Chem 2011; 3:1427-54. [PMID: 21879846 PMCID: PMC3225966 DOI: 10.4155/fmc.11.90] [Citation(s) in RCA: 92] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
Tuberculosis remains a leading cause of death resulting from an infectious agent, and the spread of multi- and extensively drug-resistant strains of Mycobacterium tuberculosis poses a threat to management of global health. New drugs that effectively shorten the duration of treatment and are active against drug-resistant strains of this pathogen are urgently required to develop effective chemotherapies to combat this disease. Two nitroimidazoles, PA-824 and OPC-67683, are currently in Phase II clinical trials for the treatment of TB and the outcome of these may determine the future directions of drug development for anti-tubercular nitroimidazoles. In this review we summarize the development of these nitroimidazoles and alternative analogs in these series that may offer attractive alternatives to PA-824 and OPC-67683 for further development in the drug-discovery pipeline. Lastly, the potential pitfalls in the development of nitroimidazoles as drugs for TB are discussed.
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Affiliation(s)
- Tathagata Mukherjee
- Tuberculosis Research Section, LCID, NIAID, NIH, Room 2W20G, Building 33, 9000 Rockville Pike, Bethesda, MD 20892, USA
| | - Helena Boshoff
- Tuberculosis Research Section, LCID, NIAID, NIH, Room 2W20G, Building 33, 9000 Rockville Pike, Bethesda, MD 20892, USA
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Francesco VD, Zullo A, Hassan C, Giorgio F, Rosania R, Ierardi E. Mechanisms of Helicobacter pylori antibiotic resistance: An updated appraisal. World J Gastrointest Pathophysiol 2011; 2:35-41. [PMID: 21860834 PMCID: PMC3158889 DOI: 10.4291/wjgp.v2.i3.35] [Citation(s) in RCA: 100] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/18/2011] [Revised: 05/29/2011] [Accepted: 06/05/2011] [Indexed: 02/06/2023] Open
Abstract
Helicobacter pylori (H. pylori) antibiotic resistance is the main factor affecting the efficacy of the current eradicating therapies. The aim of this editorial is to report on the recent information about the mechanisms accounting for the resistance to the different antibiotics currently utilized in H. pylori eradicating treatments. Different mechanisms of resistance to clarithromycin, metronidazole, quinolones, amoxicillin and tetracycline are accurately detailed (point mutations, redox intracellular potential, pump efflux systems, membrane permeability) on the basis of the most recent data available from the literature. The next hope for the future is that by improving the knowledge of resistance mechanisms, the elaboration of rational and efficacious associations for the treatment of the infection will be possible. Another auspicious progress might be the possibility of a cheap, feasible and reliable laboratory test to predict the outcome of a therapeutic scheme.
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Leitsch D, Kolarich D, Duchêne M. The flavin inhibitor diphenyleneiodonium renders Trichomonas vaginalis resistant to metronidazole, inhibits thioredoxin reductase and flavin reductase, and shuts off hydrogenosomal enzymatic pathways. Mol Biochem Parasitol 2010; 171:17-24. [PMID: 20093143 DOI: 10.1016/j.molbiopara.2010.01.001] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2009] [Revised: 01/10/2010] [Accepted: 01/12/2010] [Indexed: 11/25/2022]
Abstract
Infections with the microaerophilic protozoan parasite Trichomonas vaginalis are commonly treated with metronidazole, a 5-nitroimidazole drug. Metronidazole is selectively toxic to microaerophiles and anaerobes because reduction at the drug's nitro group, which is a precondition for toxicity, occurs only quantitatively in these organisms. In our previous work we identified the flavin enzyme thioredoxin reductase as an electron donor to 5-nitroimidazole drugs in T. vaginalis and observed that highly metronidazole-resistant cell lines lack thioredoxin reductase and flavin reductase activities. In this study we added the flavin inhibitor diphenyleneiodonium (DPI) to T. vaginalis cultures in order to test our hypothesis that metronidazole reduction is catalyzed by flavin enzymes, e.g. thioredoxin reductase, and intracellular free flavins. Indeed, within hours, DPI rendered T. vaginalis insensitive to metronidazole concentrations as high as 1mM and prevented the formation of metronidazole adducts with proteins. Thioredoxin reductase activity was absent from DPI-treated cells and flavin reductase activity was sharply decreased. In addition, DPI-treated cells also upregulated the expression of antioxidant enzymes, i.e. thioredoxin peroxidases and superoxide dismutases, and displayed a fundamentally altered metabolism caused by inactivation of pyruvate:ferredoxin oxidoreductase (PFOR) and concomitant upregulation of lactate dehydrogenase (LDH) activity. Thus, the disruption of the cellular flavin metabolism by DPI mediated metabolic steps which are similar to that of cells with metronidazole resistance induced in vitro. Finally, we present direct evidence that the increased expression of antioxidant enzymes is dispensable for acquiring resistance to metronidazole.
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Affiliation(s)
- David Leitsch
- Department of Specific Prophylaxis and Tropical Medicine at the Center for Physiology, Pathophysiology and Immunology, Medical University of Vienna, A-1090 Vienna, Austria.
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Olekhnovich IN, Goodwin A, Hoffman PS. Characterization of the NAD(P)H oxidase and metronidazole reductase activities of the RdxA nitroreductase of Helicobacter pylori. FEBS J 2009; 276:3354-64. [PMID: 19438716 PMCID: PMC2751797 DOI: 10.1111/j.1742-4658.2009.07060.x] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
Metronidazole (MTZ) is widely used in combination therapies against the human gastric pathogen Helicobacter pylori. Resistance to this drug is common among clinical isolates and results from loss-of-function mutations in rdxA, which encodes an oxygen-insensitive nitroreductase. The RdxA-associated MTZ-reductase activity of H. pylori is lost upon cell disruption. Here we provide a mechanistic explanation for this phenomenon. Under aerobic conditions, His6-tagged RdxA protein (purified from Escherichia coli), catalyzed NAD(P)H-dependent reductions of nitroaromatic and quinone substrates including nitrofurazone, nitrofurantoin, furazolidone, CB1954 and 1,4-benzoquinone, but not MTZ. Unlike other nitroreductases, His6-RdxA exhibited potent NAD(P)H-oxidase activity (k(cat) = 2.8 s(-1)) which suggested two possible explanations for the role of oxygen in MTZ reduction: (a) NAD(P)H-oxidase activity promotes cellular hypoxia (nonspecific reduction of MTZ), and (b) molecular oxygen out-competes MTZ for reducing equivalents. The first hypothesis was eliminated upon finding that rdxA expression, although increasing MTZ toxicity in both E. coli and H. pylori constructs, did not increase paraquat toxicity, even though both are of similar redox potential. The second hypothesis was confirmed by demonstrating NAD(P)H-dependent MTZ-reductase activity (apparent K(m) = 122 +/- 58 microM, k(cat) = 0.24 s(-1)) under strictly anaerobic conditions. The MTZ-reductase activity of RdxA was 60 times greater than for NfsB (E. coli NTR), but 10 times lower than the NADPH-oxidase activity. Whether molecular oxygen directly competes with MTZ or alters the redox state of the FMN cofactors is discussed.
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Affiliation(s)
- Igor N. Olekhnovich
- Departments of Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22908-0734
- Division of Infectious Diseases and International Health and Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia 22908-0734
| | - Avery Goodwin
- Food and Drug Administration, Center for Drug Evaluation and Research, Silver Spring, Maryland 20993
| | - Paul S. Hoffman
- Departments of Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22908-0734
- Division of Infectious Diseases and International Health and Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia 22908-0734
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Leitsch D, Kolarich D, Binder M, Stadlmann J, Altmann F, Duchêne M. Trichomonas vaginalis: metronidazole and other nitroimidazole drugs are reduced by the flavin enzyme thioredoxin reductase and disrupt the cellular redox system. Implications for nitroimidazole toxicity and resistance. Mol Microbiol 2009; 72:518-36. [PMID: 19415801 DOI: 10.1111/j.1365-2958.2009.06675.x] [Citation(s) in RCA: 106] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Infections with the microaerophilic parasite Trichomonas vaginalis are treated with the 5-nitroimidazole drug metronidazole, which is also in use against Entamoeba histolytica, Giardia intestinalis and microaerophilic/anaerobic bacteria. Here we report that in T. vaginalis the flavin enzyme thioredoxin reductase displays nitroreductase activity with nitroimidazoles, including metronidazole, and with the nitrofuran drug furazolidone. Reactive metabolites of metronidazole and other nitroimidazoles form covalent adducts with several proteins that are known or assumed to be associated with thioredoxin-mediated redox regulation, including thioredoxin reductase itself, ribonucleotide reductase, thioredoxin peroxidase and cytosolic malate dehydrogenase. Disulphide reducing activity of thioredoxin reductase was greatly diminished in extracts of metronidazole-treated cells and intracellular non-protein thiol levels were sharply decreased. We generated a highly metronidazole-resistant cell line that displayed only minimal thioredoxin reductase activity, not due to diminished expression of the enzyme but due to the lack of its FAD cofactor. Reduction of free flavins, readily observed in metronidazole-susceptible cells, was also absent in the resistant cells. On the other hand, iron-depleted T. vaginalis cells, expressing only minimal amounts of PFOR and hydrogenosomal malate dehydrogenase, remained fully susceptible to metronidazole. Thus, taken together, our data suggest a flavin-based mechanism of metronidazole activation and thereby challenge the current model of hydrogenosomal activation of nitroimidazole drugs.
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Affiliation(s)
- David Leitsch
- Department of Specific Prophylaxis and Tropical Medicine at the Center for Physiology, Pathophysiology and Immunology, Medical University of Vienna, Vienna, Austria.
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A redox basis for metronidazole resistance in Helicobacter pylori. Antimicrob Agents Chemother 2009; 53:1884-91. [PMID: 19223619 DOI: 10.1128/aac.01449-08] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Metronidazole resistance in Helicobacter pylori has been attributed to mutations in rdxA or frxA. Insufficient data correlating RdxA and/or FrxA with the resistant phenotype, and the emergence of resistant strains with no mutations in either rdxA or frxA, indicated that the molecular basis of H. pylori resistance to metronidazole required further characterization. The rdxA and frxA genes of four matched pairs of metronidazole-susceptible and -resistant strains were sequenced. The resistant strains had mutations in either rdxA, frxA, neither gene, or both genes. The reduction rates of five substrates suggested that metabolic differences between susceptible and resistant strains cannot be explained only by mutations in rdxA and/or frxA. A more global approach to understanding the resistance phenotype was taken by employing two-dimensional gel electrophoresis combined with tandem mass spectrometry analyses to identify proteins differentially expressed by the matched pair of strains with no mutations in rdxA or frxA. Proteins involved in the oxireduction of ferredoxin were downregulated in the resistant strain. Other redox enzymes, such as thioredoxin reductase, alkyl hydroperoxide reductase, and superoxide dismutase, showed a pI change in the resistant strain. The data suggested that metronidazole resistance involved more complex metabolic changes than specific gene mutations, and they provided evidence of a role for the intracellular redox potential in the development of resistance.
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21
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Stanley KN, Jones K. High frequency of metronidazole resistance among strains ofCampylobacter jejuniisolated from birds. Lett Appl Microbiol 2008. [DOI: 10.1046/j.1472-765x.1998.00442.x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Affiliation(s)
- K. N. Stanley
- Department of Biological Sciences, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster, UK
| | - K. Jones
- Department of Biological Sciences, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster, UK
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Khan A, Sarkar S, Sarkar D. Bactericidal activity of 2-nitroimidazole against the active replicating stage of Mycobacterium bovis BCG and Mycobacterium tuberculosis with intracellular efficacy in THP-1 macrophages. Int J Antimicrob Agents 2008; 32:40-5. [PMID: 18538548 DOI: 10.1016/j.ijantimicag.2008.02.022] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2007] [Revised: 02/27/2008] [Accepted: 02/29/2008] [Indexed: 11/19/2022]
Abstract
This study evaluated the antituberculous potential of 2-nitroimidazole under in vitro conditions. Minimal bactericidal concentrations of the compound against actively replicating Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra were found to be 0.226 microg/mL and 0.556 microg/mL in enriched and minimal medium, respectively. Minimal inhibitory concentrations were >100 times lower than reported antituberculous nitroimidazoles such as nitrofurantoin and furaltadone, indicating the greater potential of 2-nitroimidazole. No discernible effect of 2-nitroimidazole was seen on saprophytic Mycobacterium smegmatis and the representative bacterial strain Escherichia coli DH5alpha, indicating the specificity of the molecule against tuberculous mycobacteria. The compound was also found to be effective against M. tuberculosis in the intracellular environment of the human monocytic cell line THP-1, with a reduction in viability of bacilli by 2.5 log after 144 h of incubation at a concentration of 0.113 microg/mL. A five-fold higher concentration (0.565 microg/mL) of 2-nitroimidazole sterilised the macrophages of intracellular pathogens within 192 h, without affecting the host. However, 2-nitroimidazole was unable to affect significantly the viability of dormant non-replicating bacilli of M. bovis BCG and M. tuberculosis in Wayne's in vitro model. Overall, the results indicate that 2-nitroimidazole is a potent antituberculous agent active against the organism's active replicating stage, with promising intracellular efficacy as well.
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Affiliation(s)
- Arshad Khan
- Combi Chem-Bio Resource Center, National Chemical Laboratory, Dr Homi Bhabha Road, Pune, India
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23
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Leitsch D, Kolarich D, Wilson IBH, Altmann F, Duchêne M. Nitroimidazole action in Entamoeba histolytica: a central role for thioredoxin reductase. PLoS Biol 2008; 5:e211. [PMID: 17676992 PMCID: PMC1933457 DOI: 10.1371/journal.pbio.0050211] [Citation(s) in RCA: 119] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2007] [Accepted: 06/07/2007] [Indexed: 02/06/2023] Open
Abstract
Metronidazole, a 5-nitroimidazole drug, has been the gold standard for several decades in the treatment of infections with microaerophilic protist parasites, including Entamoeba histolytica. For activation, the drug must be chemically reduced, but little is known about the targets of the active metabolites. Applying two-dimensional gel electrophoresis and mass spectrometry, we searched for protein targets in E. histolytica. Of all proteins visualized, only five were found to form adducts with metronidazole metabolites: thioredoxin, thioredoxin reductase, superoxide dismutase, purine nucleoside phosphorylase, and a previously unknown protein. Recombinant thioredoxin reductase carrying the modification displayed reduced enzymatic activity. In treated cells, essential non-protein thiols such as free cysteine were also affected by covalent adduct formation, their levels being drastically reduced. Accordingly, addition of cysteine allowed E. histolytica to survive in the presence of otherwise lethal metronidazole concentrations and reduced protein adduct formation. Finally, we discovered that thioredoxin reductase reduces metronidazole and other nitro compounds, suggesting a new model of metronidazole activation in E. histolytica with a central role for thioredoxin reductase. By reducing metronidazole, the enzyme renders itself and associated thiol-containing proteins vulnerable to adduct formation. Because thioredoxin reductase is a ubiquitous enzyme, similar processes could occur in other eukaryotic or prokaryotic organisms. The protist parasites Entamoeba histolytica, Trichomonas vaginalis, and Giardia intestinalis grow in environments with low oxygen concentration. Infections with these parasites are commonly treated with metronidazole, a nitroimidazole drug that must be reduced for activation, resulting in several toxic metabolites. We examined the soluble proteome of metronidazole-treated E. histolytica cells for target proteins of these metabolites, applying two-dimensional gel electrophoresis and mass spectrometry. Of about 1,500 proteins visualized, only five formed covalent adducts with metronidazole metabolites, including thioredoxin, thioredoxin reductase, and superoxide dismutase. Metronidazole-bound thioredoxin reductase displayed diminished activity. In addition to these proteins, small thiol molecules, including cysteine, formed adducts with metronidazole. Supplementation with cysteine allowed the cells to survive otherwise lethal metronidazole concentrations. Finally, we discovered that one of the modified proteins, thioredoxin reductase, reduces metronidazole, suggesting a central role for this enzyme with regard to metronidazole toxicity. Taken together, our work reveals a new area of molecular interactions of activated metronidazole with cellular components. Because thioredoxin reductase is a ubiquitous enzyme, similar processes could also occur in other eukaryotic or prokaryotic organisms. Metronidazole is used for treatment of infections with microaerophilic protist parasites. Here, a new model of metronidazole activation is proposed, with a central role for thioredoxin reductase.
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Affiliation(s)
- David Leitsch
- Department of Specific Prophylaxis and Tropical Medicine, Center for Physiology and Pathophysiology, Medical University of Vienna, Vienna, Austria
| | - Daniel Kolarich
- Department of Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, Austria
| | - Iain B. H Wilson
- Department of Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, Austria
| | - Friedrich Altmann
- Department of Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, Austria
| | - Michael Duchêne
- Department of Specific Prophylaxis and Tropical Medicine, Center for Physiology and Pathophysiology, Medical University of Vienna, Vienna, Austria
- * To whom correspondence should be addressed. E-mail:
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P Moder K. Review Pulmonary- Allergy, Dermatological, Gastrointestinal & Arthritis: Current therapies for peptic ulcer disease. Expert Opin Ther Pat 2008. [DOI: 10.1517/13543776.6.1.29] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
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Rossouw DD, Lötter MG, du Raan H, Jansen SE, Höhn A, Burger BV. Radiosynthesis and evaluation of two novel 123I-labeled 2-methyl-4-nitroimidazole derivatives as potential infection imaging agents. Nucl Med Biol 2005; 32:385-94. [PMID: 15878508 DOI: 10.1016/j.nucmedbio.2005.02.001] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2004] [Revised: 01/17/2005] [Accepted: 02/04/2005] [Indexed: 11/21/2022]
Abstract
INTRODUCTION The inflammation- and infection-seeking properties of (131)I-labeled ornidazole, a 5-nitroimidazole derivative, have recently been reported. Whole-body images in rabbits showed a more rapid uptake in inflamed areas compared to (67)Ga. In the present study, two novel (123)I-labeled 2-methyl-4-nitroimidazole derivatives were synthesized and their infection-seeking properties compared with those of (67)Ga and (123)I-labeled ornidazole. METHODS Radiolabeling was carried out by means of iodide-for-tosylate, triflate or halogen exchange. Various methods were utilized in order to synthesize the labeling precursors for the (123)I-labeled novel compounds. Serum stability studies on all of the (123)I-labeled tracers were followed by gamma camera imaging studies on rabbits artificially infected with Escherichia coli bacteria. RESULTS AND CONCLUSIONS The (123)I-labeled tracers were obtained in moderate to good radiochemical yields (34-80%) and acceptable radiochemical purities (93-99%). In contrast to (123)I-labeled ornidazole, 1-[(1-hydroxy-3-[(123)I]iodoprop-2-yloxy)methyl]-2-methyl-4-nitroimidazole (2) and 1-[(1-[(123)I]iodoprop-2-yloxy)methyl]-2-methyl-4-nitroimidazole (3) showed high serum stability. Compared to noninfected controls, all of the (123)I-labeled tracers showed increased uptake at the area of induced infection after 6 and 24 h, but the uptake was significantly lower than in the case of (67)Ga over the same period. Tracer 3 showed a slightly superior uptake after 6 h than the other (123)I-labeled tracers over the same period. The advantage of the initially slightly faster rate at which nitroimidazole tracers appear to accumulate in the infection area in comparison to (67)Ga might not outweigh the advantage of the eventual higher target to nontarget ratio displayed by (67)Ga.
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Affiliation(s)
- Daniel D Rossouw
- iThemba Laboratory for Accelerator-Based Sciences, PO Box 722, Somerset-West 7129, South Africa.
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Kaakoush NO, Mendz GL. Helicobacter pyloridisulphide reductases: role in metronidazole reduction. ACTA ACUST UNITED AC 2005; 44:137-42. [PMID: 15866207 DOI: 10.1016/j.femsim.2004.11.007] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2004] [Revised: 10/27/2004] [Accepted: 11/22/2004] [Indexed: 11/28/2022]
Abstract
Disulphide reductases play an important role in maintaining intracellular redox potential. Three disulphide reductase activities were identified in Helicobacter pylori, which used dithiobis-2-nitrobenzoic acid, glutathione or l-cystine and ferredoxin as substrates. The kinetic parameters of these activities were determined and it was demonstrated that the reductase activities were inhibited by the presence of metronidazole. Substrate competition experiments served to show inhibition of metronidazole reduction by dithiobis-2-nitrobenzoic acid, glutathione and ferredoxin in lysates from metronidazole susceptible and resistant matched pairs of strains. The study demonstrated that the activities of three disulphide reductases were modulated by the presence of metronidazole, and that metronidazole reduction was inhibited by the presence of disulphide reductase substrates.
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Affiliation(s)
- Nadeem O Kaakoush
- School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Biological Science Builiding, Sydney, NSW 2052, Australia
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Gerrits MM, van der Wouden EJ, Bax DA, van Zwet AA, van Vliet AH, de Jong A, Kusters JG, Thijs JC, Kuipers EJ. Role of the rdxA and frxA genes in oxygen-dependent metronidazole resistance of Helicobacter pylori. J Med Microbiol 2004; 53:1123-1128. [PMID: 15496391 DOI: 10.1099/jmm.0.45701-0] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Almost 50 % of all Helicobacter pylori isolates are resistant to metronidazole, which reduces the efficacy of metronidazole-containing regimens, but does not make them completely ineffective. This discrepancy between in vitro metronidazole resistance and treatment outcome may partially be explained by changes in oxygen pressure in the gastric environment, as metronidazole-resistant (MtzR) H. pylori isolates become metronidazole-susceptible (MtzS) under low oxygen conditions in vitro. In H. pylori the rdxA and frxA genes encode reductases which are required for the activation of metronidazole, and inactivation of these genes results in metronidazole resistance. Here the role of inactivating mutations in these genes on the reversibility of metronidazole resistance under low oxygen conditions is established. Clinical H. pylori isolates containing mutations resulting in a truncated RdxA and/or FrxA protein were selected and incubated under anaerobic conditions, and the effect of these conditions on the MICs of metronidazole, amoxycillin, clarithromycin and tetracycline, and cell viability were determined. While anaerobiosis had no effect on amoxycillin, clarithromycin and tetracycline resistance, all isolates lost their metronidazole resistance when cultured under anaerobic conditions. This loss of metronidazole resistance also occurred in the presence of the protein synthesis inhibitor chloramphenicol. Thus, factor(s) that activate metronidazole under low oxygen tension are not specifically induced by low oxygen conditions, but are already present under microaerophilic conditions. As there were no significant differences in cell viability between the clinical isolates, it is likely that neither the rdxA nor the frxA gene participates in the reversibility of metronidazole resistance.
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Affiliation(s)
- Monique M Gerrits
- Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands 2Department of Internal Medicine, Bethesda Hospital, Hoogeveen, The Netherlands 3Regional Public Health Laboratory Groningen/Drenthe, Groningen, The Netherlands
| | - Egbert-Jan van der Wouden
- Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands 2Department of Internal Medicine, Bethesda Hospital, Hoogeveen, The Netherlands 3Regional Public Health Laboratory Groningen/Drenthe, Groningen, The Netherlands
| | - Dorine A Bax
- Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands 2Department of Internal Medicine, Bethesda Hospital, Hoogeveen, The Netherlands 3Regional Public Health Laboratory Groningen/Drenthe, Groningen, The Netherlands
| | - Anton A van Zwet
- Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands 2Department of Internal Medicine, Bethesda Hospital, Hoogeveen, The Netherlands 3Regional Public Health Laboratory Groningen/Drenthe, Groningen, The Netherlands
| | - Arnoud Hm van Vliet
- Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands 2Department of Internal Medicine, Bethesda Hospital, Hoogeveen, The Netherlands 3Regional Public Health Laboratory Groningen/Drenthe, Groningen, The Netherlands
| | - Albertine de Jong
- Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands 2Department of Internal Medicine, Bethesda Hospital, Hoogeveen, The Netherlands 3Regional Public Health Laboratory Groningen/Drenthe, Groningen, The Netherlands
| | - Johannes G Kusters
- Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands 2Department of Internal Medicine, Bethesda Hospital, Hoogeveen, The Netherlands 3Regional Public Health Laboratory Groningen/Drenthe, Groningen, The Netherlands
| | - Jaap C Thijs
- Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands 2Department of Internal Medicine, Bethesda Hospital, Hoogeveen, The Netherlands 3Regional Public Health Laboratory Groningen/Drenthe, Groningen, The Netherlands
| | - Ernst J Kuipers
- Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands 2Department of Internal Medicine, Bethesda Hospital, Hoogeveen, The Netherlands 3Regional Public Health Laboratory Groningen/Drenthe, Groningen, The Netherlands
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Evidence of self-protonation on the electrodic reduction mechanism of an anti-Helicobacter pylori metronidazole isotere. J Electroanal Chem (Lausanne) 2004. [DOI: 10.1016/j.jelechem.2004.05.007] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
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Sisson G, Goodwin A, Raudonikiene A, Hughes NJ, Mukhopadhyay AK, Berg DE, Hoffman PS. Enzymes associated with reductive activation and action of nitazoxanide, nitrofurans, and metronidazole in Helicobacter pylori. Antimicrob Agents Chemother 2002; 46:2116-23. [PMID: 12069963 PMCID: PMC127316 DOI: 10.1128/aac.46.7.2116-2123.2002] [Citation(s) in RCA: 137] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Nitazoxanide (NTZ) is a redox-active nitrothiazolyl-salicylamide prodrug that kills Helicobacter pylori and also many anaerobic bacterial, protozoan, and helminthic species. Here we describe development and use of a spectrophotometric assay, based on nitroreduction of NTZ at 412 nm, to identify H. pylori enzymes responsible for its activation and mode of action. Three enzymes that reduce NTZ were identified: two related NADPH nitroreductases, which also mediate susceptibility to metronidazole (MTZ) (RdxA and FrxA), and pyruvate oxidoreductase (POR). Recombinant His-tagged RdxA, FrxA, and POR, overexpressed in nitroreductase-deficient Escherichia coli, each rapidly reduced NTZ, whereas only FrxA and to a lesser extent POR reduced nitrofuran substrates (furazolidone, nitrofurantoin, and nitrofurazone). POR exhibited no MTZ reductase activity either in extracts of H. pylori or following overexpression in E. coli; RdxA exhibited no nitrofuran reductase activity, and FrxA exhibited no MTZ reductase activity. Analysis of mutation to rifampin resistance (Rif(r)) indicated that NTZ was not mutagenic and that nitrofurans were only weakly mutagenic. Alkaline gel DNA electrophoresis indicated that none of these prodrugs caused DNA breakage. In contrast, MTZ caused DNA damage and was strongly mutagenic. We conclude that POR, an essential enzyme, is responsible for most or all of the bactericidal effects of NTZ against H. pylori. While loss-of-function mutations in rdxA and frxA produce a Mtz(r) phenotype, they do not contribute much to the innate susceptibility of H. pylori to NTZ or nitrofurans.
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Affiliation(s)
- Gary Sisson
- Department of Microbiology and ImmunologyFaculty of Medicine, Dalhousie University, HaliFax, Nova Scotia B3H 4H7, Canada
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van der Wouden EJ, Thijs JC, Kusters JG, van Zwet AA, Kleibeuker JH. Mechanism and clinical significance of metronidazole resistance in Helicobacter pylori. SCANDINAVIAN JOURNAL OF GASTROENTEROLOGY. SUPPLEMENT 2002:10-4. [PMID: 11768554 DOI: 10.1080/003655201753265055] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/23/2023]
Abstract
Metronidazole was introduced in 1959 for the treatment of Trichomonas vaginalis, but was subsequently shown to be active against anaerobic and some micro-aerophilic bacteria as well. In anaerobic microorganisms with their low redox potential, metronidazole is reduced to its active metabolite by a one-electron transfer step. Metronidazole is often used in treatment regimens for Helicobacter pylori, a microaerophilic bacterium, but resistance to this drug is frequently encountered. The metabolism of metronidazole in H. pylori must differ from that in anaerobic bacteria as metabolites formed by a one-electron transfer are readily re-oxidized in the micro-aerophilic environment of H. pylori. This process is called 'futile cycling' and is accompanied by the formation of toxic oxygen radicals that are neutralized by an active scavenger system. Recently, it has been shown that in H. pylori, in contrast to the situation in anaerobes, an oxygen-insensitive nitroreductase. encoded by the rdxA gene, is responsible for the activation of metronidazole. Activation by this enzyme is by a two-electron transfer step, preventing futile cycling' and thereby enabling the activation of metronidazole in a micro-aerophilic environment. Metronidazole resistance has been shown to be associated with null mutations in the rdxA gene in most clinical isolates. However, there may be some 'background metronidazole susceptibility' in metronidazole-resistant strains caused by other (oxygen-sensitive) nitroreductases. Recently, three meta-analyses of the impact of metronidazole resistance on treatment efficacy have all shown a significant reduction in efficacy of metronidazole containing regimens in patients infected with a resistant strain. The impact of resistance proved to be dependent on the other components of the regimen and on treatment duration.
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Abstract
Modern triple drug regimens are highly effective for treating Helicobacter pylori infection, but bacterial resistance to one of the most effective antibiotics, metronidazole, is a serious and increasing problem. The activity of metronidazole in H. pylori is dependent on reduction of its nitro moiety to highly reactive compounds that cause DNA strand breakage. The acquisition of resistance is highly associated with mutational inactivation of the rdxA gene, which encodes an oxygen-insensitive NADPH nitroreductase. Recent evidence has suggested that inactivation of frxA (NADPH flavin oxidoreductase), fdxB (ferredoxin-like protein) and possibly other reductase-encoding genes may also contribute to the resistant phenotype. Improved understanding of the mechanisms of metronidazole resistance in H. pylori is essential for the development and validation of biopsy-based tests for detection of resistance in clinical practice.
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Affiliation(s)
- Peter J Jenks
- Institute of Infections and Immunity, Queen's Medical Centre, University Hospital, Floor C, West Block, NG7 2UH, Nottingham, UK.
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Kusters JG, Kuipers EJ. Antibiotic resistance of Helicobacter pylori. SYMPOSIUM SERIES (SOCIETY FOR APPLIED MICROBIOLOGY) 2001:134S-44S. [PMID: 11422569 DOI: 10.1046/j.1365-2672.2001.01362.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Affiliation(s)
- J G Kusters
- Department of Medical Microbiology, Vrije Universiteit, Amsterdam, the Netherlands.
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Abstract
Helicobacter pylori (Hp) is a Gram-negative bacteria able to live in the human stomach, a very surprising fact considering the acid environment of gastric mucosa. Identified by Marshall and Warren in 1982 [1,2], this bacterium seems aetiologically related to many gastric diseases, previously known as 'acid related diseases'. Compelling evidence demonstrates that Hp is the most important aetiological agent of gastritis [3], the principal causal factor in peptic ulcer [4], contributes to the genesis of gastric cancer [5] and has a critical role in the development of many mucosa-associated lymphoid tissue (MALT) lymphomas [6]. Although experimental data have recently provided hard evidence to support the role of Hp in the genesis of gastritis, ulcer and carcinoma [7], a critical argument for Hp generating peptic ulcer disease has been, in fact, the change in the natural history of peptic ulcer that follows the cure of the infection.
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Affiliation(s)
- F Gomollón
- Digestive Disease Service, Hospital Miguel Servet, Paseo de Isabel la Católica, s/n, Zaragoza 50009, Spain.
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Tolia V, Brown W, El-Baba M, Lin CH. Helicobacter pylori culture and antimicrobial susceptibility from pediatric patients in Michigan. Pediatr Infect Dis J 2000; 19:1167-71. [PMID: 11144378 DOI: 10.1097/00006454-200012000-00010] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
BACKGROUND We recently observed a high failure rate in the eradication of Helicobacter pylori infection in children with 2-week triple therapy using lansoprazole, amoxicillin and clarithromycin. We performed a prospective evaluation of antral biopsies of all children subsequently diagnosed with H. pylori gastritis for culture and antimicrobial susceptibility assessment. METHODS All children with antral nodularity and/or an elevated anti-H. pylori IgG titer underwent antral biopsies for histology, urease test and culture while undergoing an upper endoscopy for routine indications. All positive cultures were tested for antimicrobial susceptibility by E-test for clarithromycin, amoxicillin, tetracycline and metronidazole. RESULTS Thirty-one children (16 male, 15 female) between 2 and 19 years of age were diagnosed with H. pylori gastritis by histology. However, culture was positive in only 22 of 31 (71%) patients. The E-test in vitro antimicrobial susceptibility testing revealed that 95.6% of the isolates were susceptible to amoxicillin, 59% to clarithromycin and 54.6% to metronidazole. There was no resistance to tetracycline. CONCLUSION Evaluation of antibiotic resistance profiles from pediatric patients from different geographic areas can help in optimizing therapeutic regimen to prevent treatment failures. Metronidazole and clarithromycin resistance is much higher in our pediatric population than reported in adults and could be a major contributor to failure of H. pylori eradication.
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Affiliation(s)
- V Tolia
- Division of Pediatric Gastroenterology, Children's Hospital of Michigan, Wayne State University School of Medicine, Detroit 48201, USA
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36
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Lehmann FS, Drewe J, Terracciano L, Beglinger C. Effect of ornidazole and clarithromycin resistance on eradication of Helicobacter pylori in peptic ulcer disease. Aliment Pharmacol Ther 2000; 14:305-9. [PMID: 10735923 DOI: 10.1046/j.1365-2036.2000.00717.x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
BACKGROUND Clarithromycin and nitroimidazoles such as metronidazole and ornidazole are among the most frequently used antibiotics for curing Helicobacter pylori infection. However, controversial data exist on whether their in vitro resistance has a negative impact on treatment outcome. METHODS Patients with H. pylori positive active peptic ulcer disease were randomly assigned to receive lansoprazole 30 mg o.d., amoxycillin 1 g b.d. and ornidazole 500 mg b.d. (LAO) or lansoprazole 30 mg o.d., amoxycillin 1 g b.d. and clarithromycin 500 mg b.d. (LAC) for 2 weeks. Pre-treatment resistance to ornidazole and clarithromycin was assessed by Epsilometer (E-) test. Four weeks after completion of treatment, patients underwent a 13C urea breath test to assess H. pylori status. RESULTS Data from 80 patients with active peptic ulcer disease and positive H. pylori status were analysed. The prevalence of primary drug resistance was 25% for metronidazole and 7.5% for clarithromycin. In patients treated with LAO, effective treatment was achieved in 87% of metronidazole-susceptible, but only 30% of metronidazole-resistant strains (P < 0.01). In the LAC group, therapy was successful in 81% of clarithromycin-susceptible strains, whereas treatment failed in all patients with primary clarithromycin resistance (n = 3). CONCLUSION Resistance against nitroimidazoles significantly affects treatment outcome in H. pylori eradication therapy.
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Affiliation(s)
- F S Lehmann
- Division of Gastroenterology, University Hospital of Basel, Switzerland
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Abstract
This paper describes the purification of thioredoxin reductase (TR) and the characterization, purification, and cloning of thioredoxin (Trx) from Helicobacter pylori. Purification, amino acid sequence analysis, and molecular cloning of the gene encoding thioredoxin revealed that it is a 12-kDa protein which possesses the conserved redox active motif CGPC. The gene encoding Trx was amplified by polymerase chain reaction and inserted into a pET expression vector and used to transform Escherichia coli. Trx was overexpressed by induction with isopropyl-1-thio-beta-D-galactopyranoside as a decahistidine fusion protein and was recovered from the cytoplasm as a soluble and active protein. The redox activity of this protein was characterized using several mammalian proteins of different architecture but all containing disulfide bonds. H. pylori thioredoxin efficiently reduced insulin, human immunoglobulins (IgG/IgA/sIgA), and soluble mucin. Subcellular fractionation analysis of H. pylori revealed that thioredoxin was associated largely with the cytoplasm and inner membrane fractions of the cell in addition to being recovered in the phosphate-buffered saline-soluble fraction of freshly harvested cells. H. pylori TR was purified to homogeneity by chromatography on DEAE-52, Cibacron blue 3GA, and 2',5'-ADP-agarose. Gel filtration revealed that the native TR had a molecular mass of 70 kDa which represented a homodimer composed of two 35-kDa subunits, as determined by SDS-polyacrylamide gel electrophoresis. H. pylori TR (NADPH-dependent) efficiently catalyzed the reduction of 5,5'-dithiobis(nitrobenzoic acid) in the presence of either native or recombinant H. pylori Trx. H. pylori Trx behaved also as a stress response element as broth grown bacteria secreted Trx in response to chemical, biological, and environmental stresses. These observations suggest that Trx may conceivably assist H. pylori in the process of colonization by inducing focal disruption of the oligomeric structure of mucin while rendering host antibody inactive through catalytic reduction.
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Affiliation(s)
- H J Windle
- Department of Clinical Medicine, Trinity College Dublin, Trinity Centre for Health Sciences, St. James's Hospital, Dublin 8, Ireland.
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Debets-Ossenkopp YJ, Pot RG, van Westerloo DJ, Goodwin A, Vandenbroucke-Grauls CM, Berg DE, Hoffman PS, Kusters JG. Insertion of mini-IS605 and deletion of adjacent sequences in the nitroreductase (rdxA) gene cause metronidazole resistance in Helicobacter pylori NCTC11637. Antimicrob Agents Chemother 1999; 43:2657-62. [PMID: 10543743 PMCID: PMC89539 DOI: 10.1128/aac.43.11.2657] [Citation(s) in RCA: 59] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
We found that NCTC11637, the type strain of Helicobacter pylori, the causative agent of peptic ulcer disease and an early risk factor for gastric cancer, is metronidazole resistant. DNA transformation, PCR-based restriction analysis, and DNA sequencing collectively showed that the metronidazole resistance of this strain was due to mutation in rdxA (gene HP0954 in the full genome sequence of H. pylori 26695) and that resistance did not depend on mutation in any of the other genes that had previously been suggested: catalase (katA), ferredoxin (fdx), flavodoxin (fldA), pyruvate:flavodoxin oxidoreductase (porgammadeltaalphabeta), RecA (recA), or superoxide dismutase (sodB). This is in accord with another recent study that attributed metronidazole resistance to point mutations in rdxA. However, the mechanism of rdxA inactivation that we found in NCTC11637 is itself also novel: insertion of mini-IS605, one of the endogenous transposable elements of H. pylori, and deletion of adjacent DNA sequences including 462 bp of the 851-bp-long rdxA gene.
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Affiliation(s)
- Y J Debets-Ossenkopp
- Department of Medical Microbiology, Faculty of Medicine, Vrije Universiteit, Amsterdam, The Netherlands
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Chida-Sakata N, Baba M, Inagawa H, Wada A, Tanaka T, Hoshihara Y, Takemoto T. Significance of anaerobic preincubation of Helicobacter pylori for measuring metronidazole susceptibility by the Etest. Microbiol Immunol 1999; 43:397-401. [PMID: 10449245 DOI: 10.1111/j.1348-0421.1999.tb02422.x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
The Etest is widely used for measuring the susceptibility of Helicobacter pylori to metronidazole. By using 55 H. pylori isolates from 55 patients and a standard H. pylori strain, NCTC11637, we compared metronidazole susceptibility results obtained from the Etest with or without anaerobic preincubation to those obtained from the agar dilution method. Mueller Hinton agar plates supplemented with 5% horse blood were used for both methods. For the Etest, plates were incubated for 72 hr at 35 C under microaerophilic conditions after 0-, 4- or 24-hr periods of anaerobic preincubation. For the agar dilution method, the plates were incubated at the same microaerophilic conditions as those for the Etest. Without anaerobic preincubation for the Etest, 39 of the 56 (70%) H. pylori isolates were categorized as resistant to metronidazole (minimal inhibitory concentration>8 mg/liter), whereas only one of the 56 (1.8%) isolates was resistant according to the agar dilution method. The resistant and susceptible agreement rate was 32%. Four-hour anaerobic preincubation did not alter the readings of the Etest significantly. However, when the Etest was performed with 24-hr anaerobic preincubation, the number of isolates categorized as resistant was reduced to six (11%), improving the agreement rate to 91%. For measuring the metronidazole susceptibility of H. pylori by the Etest, 24-hr anaerobic preincubation is necessary to agree with the results obtained by the agar dilution test.
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Affiliation(s)
- N Chida-Sakata
- Department of Pathological, Toranomon Hospital, Tokyo, Japan
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Chatsuwan T, Amyes SG. Setting the standard for determining the in-vitro susceptibility of Helicobacter pylori to metronidazole. J Antimicrob Chemother 1999; 44:291-3. [PMID: 10473240 DOI: 10.1093/jac/44.2.291a] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
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Osato MS, Reddy R, Graham DY. Metronidazole and clarithromycin resistance amongst Helicobacter pylori isolates from a large metropolitan hospital in the United States. Int J Antimicrob Agents 1999; 12:341-7. [PMID: 10493611 DOI: 10.1016/s0924-8579(99)00079-5] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
BACKGROUND Metronidazole and clarithromycin-based therapies are among the most efficacious treatment regimens for H. pylori infection. Resistance to metronidazole or clarithromycin is associated with impaired therapy with these agents. We conducted a retrospective review of susceptibility data to determine the frequency of primary metronidazole and clarithromycin resistance among H. pylori isolates from a single metropolitan hospital in the United States. The database comprised 933 patients who presented at the Digestive Diseases Clinic at the Veterans Affairs Medical Center in Houston between September 1988 and January 1997 with complaints of dyspepsia, abdominal pain and peptic ulcer disease. One hundred and seventy-nine of these patients had both pharmaceutical records available for evaluation and culture and antimicrobial susceptibility data for analysis. The MICs were determined by both E-test and broth microdilution tests. The frequency of primary metronidazole resistance was 37.4% (67/179). The level of primary clarithromycin resistance was 6.1%. Dual metronidazole and clarithromycin resistance was present in approx. 3%. The high level of primary metronidazole and clarithromycin resistance in H. pylori isolates from this metropolitan hospital is such that antimicrobial susceptibility data should be available so that informed choice can be made for specific eradication therapies, especially in patients who fail treatment.
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Affiliation(s)
- M S Osato
- Department of Medicine, Veterans Affairs Medical Center, Houston, TX 77030, USA.
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Houben MH, van de Beek D, Hensen EF, de Craen AJ, Rauws EA, Tytgat GN. A systematic review of Helicobacter pylori eradication therapy--the impact of antimicrobial resistance on eradication rates. Aliment Pharmacol Ther 1999; 13:1047-55. [PMID: 10468680 DOI: 10.1046/j.1365-2036.1999.00555.x] [Citation(s) in RCA: 224] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
BACKGROUND We systematically reviewed all available data in the literature to determine the overall eradication rates of currently advised Helicobacter pylori eradication regimens and to resolve conflicting evidence on the impact of antimicrobial resistance on the eradication rates. METHODS A comprehensive search of all published trials on H. pylori eradication therapy was carried out via an electronic database search, hand-searching and checking reference lists of pharmaceutical companies and other reviews. Full papers and abstracts in the English language which study currently advised eradication regimes were included. RESULTS 770 study-arms were analysed. Mean eradication rates for bismuth based triple, proton pump inhibitor triple, quadruple and ranitidine bismuth citrate combination therapies vary from 65 to 92%. In case of nitroimidazole resistance, a drop in efficacy of up to 50% was found for bismuth-based triple and proton pump inhibitor-based triple therapies. For quadruple therapy, a significant difference in efficacy was found in the equal-effects analysis; however, this could not be confirmed in the random-effects analysis. In case of clarithromycin resistance, a mean drop in efficacy of 56% was found for one- and two-week clarithromycin containing proton pump inhibitor-triple therapies and of 58% for two-week ranitidine bismuth citrate combined with clarithromycin therapies. For ranitidine bismuth citrate combined with clarithromycin and nitroimidazole, no difference in efficacy was found in case of nitroimidazole or clarithromycin resistance, but data are still scarce. CONCLUSIONS The cure rate with most regimens dropped significantly, in case of nitroimidazole-resistant strains, compared to nitroimidazole-susceptible strains. In case of clarithromycin resistance, the efficacy of most regimens is also decreased; however, data are still scarce. These data should allow physicians to make a better choice of an appropriate therapy for their patients.
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Affiliation(s)
- M H Houben
- Department of Gastroenterology, Academic Medical Center, Amsterdam, The Netherlands.
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Malfertheiner P, Bayerdörffer E, Diete U, Gil J, Lind T, Misiuna P, O'Morain C, Sipponen P, Spiller RC, Stasiewicz J, Treichel H, Ujszászy L, Unge P, Zanten SJ, Zeijlon L. The GU-MACH study: the effect of 1-week omeprazole triple therapy on Helicobacter pylori infection in patients with gastric ulcer. Aliment Pharmacol Ther 1999; 13:703-712. [PMID: 10383498 DOI: 10.1046/j.1365-2036.1999.00535.x] [Citation(s) in RCA: 96] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
AIMS To study the efficacy of omeprazole triple therapy in the eradication of Helicobacter pylori in patients with active gastric ulcer, and to assess healing and relapse of gastric ulcer. METHODS A double-blind, randomized study was carried out in 18 centres in Germany, Hungary and Poland. Patients (n = 160) with gastric ulcer and a positive H. pylori screening test were randomized to a 7-day twice daily treatment with omeprazole 20 mg, clarithromycin 500 mg and amoxycillin 1000 mg (OAC) or omeprazole 20 mg, clarithromycin 250 mg and metronidazole 400 mg (OMC), or with omeprazole 20 mg once daily (O). After completion of this 1-week treatment, patients were treated with omeprazole until healing (maximum 12 weeks), and followed for 6 months. H. pylori was assessed by urea breath test (UBT) and histology. RESULTS Eradication rates ITT were OAC 79% (95% CI: 65-90%), OMC 86% (95% CI: 73-94%) and O 4% (95% CI: 0-14%). Eradication rates PP were OAC 83% (95% CI: 68-93%), OMC 93% (95% CI: 80-98%) and O 3% (95% CI: 0-13%). Gastric ulcer relapses occurred in 5, 0 and 11 patients in the groups, respectively. CONCLUSIONS The results from the study demonstrate that OMC and OAC 1-week regimens are safe and effective for eradication of H. pylori in gastric ulcer patients, and that ulcer relapse is infrequent after successful eradication.
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Abstract
Recent studies on antibacterials have focused on the development of antimycobacterial agents and antibacterial peptides, and on furthering the understanding of agents that have been available for several decades, including imidazoles, beta-lactams and quinolones. New areas of research include antisense oligonucleotides, antibacterial peptides and a new class of agents, oxazolidinones.
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Affiliation(s)
- L J Piddock
- Antimicrobial Agents Research Group, Department of Infection, University of Birmingham Medical School, Vincent Drive, Edgbaston, Birmingham B15 2TT UK.
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Goodwin A, Kersulyte D, Sisson G, Veldhuyzen van Zanten SJ, Berg DE, Hoffman PS. Metronidazole resistance in Helicobacter pylori is due to null mutations in a gene (rdxA) that encodes an oxygen-insensitive NADPH nitroreductase. Mol Microbiol 1998; 28:383-93. [PMID: 9622362 DOI: 10.1046/j.1365-2958.1998.00806.x] [Citation(s) in RCA: 260] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Metronidazole (Mtz) is a critical component of combination therapies that are used against Helicobacter pylori, the major cause of peptic ulcer disease. Many H. pylori strains are Mtz resistant (MtzR), however, and here we show that MtzR results from loss of oxygen-insensitive NADPH nitroreductase activity. The underlying gene (called 'rdxA') was identified in several steps: transformation of Mtz-susceptible (MtzS) H. pylori with cosmids from a MtzR strain, subcloning, polymerase chain reaction (PCR) and DNA sequencing. We also found that (i) E. coli (normally MtzR) was rendered MtzS by a functional H. pylori rdxA gene; (ii) introduction of rdxA on a shuttle vector plasmid into formerly MtzR H. pylori rendered it MtzS; and (iii) replacement of rdxA in MtzS H. pylori with an rdxA::camR null insertion allele resulted in a MtzR phenotype. The 630 bp rdxA genes of five pairs of H. pylori isolates from infections that were mixed (MtzR/MtzS), but uniform in overall genotype, were sequenced. In each case, the paired rdxA genes differed from one another by one to three base substitutions. Typical rdxA genes from unrelated isolates differ by 5% in DNA sequence. Therefore, the near identity of rdxA genes from paired MtzR and MtzS isolates implicates de novo mutation, rather than horizontal gene transfer in the development of MtzR. Horizontal gene transfer could readily be demonstrated under laboratory conditions with mutant rdxA alleles. RdxA is a homologue of the classical nitroreductases (CNRs) of the enteric bacteria, but differs in cysteine content (6 vs. 1 or 2 in CNRs) and isoelectric point (pI=7.99 vs. 5.4-5.6), which might account for its reduction of low redox drugs such as Mtz. We suggest that many rdxA (MtzR) mutations may have been selected by prior use of Mtz against other infections. H. pylori itself is an early risk factor for gastric cancer; the possibility that its carcinogenic effects are exacerbated by Mtz use, which is frequent in many societies, or the reduction of nitroaromatic compounds to toxic, mutagenic and carcinogenic products, may be of significant concern in public health.
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Affiliation(s)
- A Goodwin
- Department of Microbiology and Immunology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada
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Affiliation(s)
- G N Tytgat
- Department of Gastroenterology-Hepatology, Academic Medical Center, Amsterdam, The Netherlands
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Hoffman PS, Goodwin A, Johnsen J, Magee K, Veldhuyzen van Zanten SJ. Metabolic activities of metronidazole-sensitive and -resistant strains of Helicobacter pylori: repression of pyruvate oxidoreductase and expression of isocitrate lyase activity correlate with resistance. J Bacteriol 1996; 178:4822-9. [PMID: 8759844 PMCID: PMC178263 DOI: 10.1128/jb.178.16.4822-4829.1996] [Citation(s) in RCA: 95] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023] Open
Abstract
In this study, we compared metronidazole (Mtz)-sensitive and -resistant strains of Helicobacter pylori for metabolic differences that might correlate with drug resistance. Included in this study was an isogenic Mtz(r) strain, HP1107, that was constructed by transforming genomic DNA from Mtz(r) strain HP439 into Mtz(s) strain HP500. Enzyme activities were also measured for Mtz(r) strains grown in the presence or absence of 18 micrograms of metronidazole per ml (ca. one-half of the MIC). These studies confirmed the presence of the Embden-Meyerhof-Parnas, Entner-Doudoroff, and pentose pathways. H. pylori strains expressed enzymatic activities indicative of a complete and active Krebs cycle. All strains expressed pyruvate oxidoreductase (POR) and alpha-ketoglutarate oxidoreductase (KOR) as measured with the redox-active dye benzyl viologen (30 to 96 nmol/min/mg of protein for POR and 30 nmol/min/mg of protein for KOR). When grown in the presence of Mtz at > or = 3.5 micrograms/ml, Mtz(r) strains expressed no detectable POR or KOR activity. The apparent repression of POR and KOR activities by Mtz affected bacterial growth as manifest by extended lag periods and growth yield reductions of > 30%. A dose-dependent relationship was demonstrated between the metronidazole concentration in the growth medium and the specific activity of POR measured in bacterial cell extracts. The observed repression was not due to inactivation of POR by Mtz. In addition to repression of POR and KOR activities, growth in the presence of Mtz also led to decreases in the activities of various Krebs cycle enzymes, including aconitase, isocitrate dehydrogenase and succinate dehydrogenase. All of the Mtz(r) strains examined expressed isocitrate lyase and malate synthase activities indicative of the glyoxylate bypass. No isocitrate lyase activity was detected in Mtz(s) strain HP500. Isocitrate lyase activity was expressed by HP500 following transformation to Mtz resistance (Mtz(r) strain HP1107) with DNA from an Mtz(r) strain. The results of this study suggest that Mtz resistance may be a recessive trait, possibly involving inactivation of a regulatory gene, that results in constitutive expression of isocitrate lyase. Repression of POR and KOR activities in response to low levels of Mtz may be a general response of H. pylori strains to Mtz, but only resistant strains manage to survive via activation of compensatory metabolic pathways.
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Affiliation(s)
- P S Hoffman
- Department of Microbiology and Immunology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada.
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