No abstract

Helicobacter pylori is a human-pathogenic bacterial species that is subdivided geographically, with different genotypes predominating in different parts of the world. Here we test and extend an earlier conclusion that metronidazole (Mtz) resistance is due to mutation in rdxA (HP0954), which encodes a nitroreductase that converts Mtz from prodrug to bactericidal agent. We found that (i) rdxA genes PCR amplified from 50 representative Mtz r strains from previously unstudied populations in Asia, South Africa, Europe, and the Americas could, in each case, transform Mtz s H. pylori to Mtz r ; (ii) Mtz r mutant derivatives of a cultured Mtz s strain resulted from mutation in rdxA; and (iii) transformation of Mtz s strains with rdxA-null alleles usually resulted in moderate level Mtz resistance (16 g/ml). However, resistance to higher Mtz levels was common among clinical isolates, a result that implicates at least one additional gene. Expression in Escherichia coli of frxA (HP0642; flavin oxidoreductase), an rdxA paralog, made this normally resistant species Mtz s , and frxA inactivation enhanced Mtz resistance in rdxA-deficient cells but had little effect on the Mtz susceptibility of rdxA ؉ cells. Strains carrying frxA-null and rdxA-null alleles could mutate to even higher resistance, a result implicating one or more additional genes in residual Mtz susceptibility and hyperresistance. We conclude that most Mtz resistance in H. pylori depends on rdxA inactivation, that mutations in frxA can enhance resistance, and that genes that confer Mtz resistance without rdxA inactivation are rare or nonexistent in H. pylori populations.Helicobacter pylori is a gram-negative microaerophilic bacterium that chronically infects human gastric epithelial cell surfaces and the overlying gastric mucin, a niche that few if any other microbes can occupy. It is carried by more than half of all people worldwide and is an important human pathogen: a major cause of peptic ulcer disease, and a contributor to other illnesses, ranging from childhood malnutrition to gastric cancer, and to increased susceptibility to other food-and waterborne pathogens (7,8,32,38,47). There is great intrinsic and public health interest in fully elucidating H. pylori's metabolic pathways and how H. pylori maintains its redox balance during microaerobic growth. Such knowledge should help us to understand the extraordinary chronicity of H. pylori infection and factors that determine whether a given infection will be benign or virulent, elucidate mechanisms of drug susceptibility and resistance, and identify potential targets for new effective antimicrobial agents.Here we focus on mechanisms of susceptibility and resistance of H. pylori to metronidazole (Mtz), a synthetic nitroimidazole that is a key component of popular and affordable anti-H. pylori therapies worldwide and that is also widely used against various anaerobic and parasitic infections (13,36,45). Resistance to Mtz is common among H. pylori strains, with frequencies among clinical isolates ranging from 10 ...

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 nitroreductasedeficient 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.Prodrugs are particularly appealing as therapeutic agents because of their selective toxicity for microorganisms possessing specific activating enzymes (7). Metronidazole (MTZ) is a nitroimidazole prodrug that is often used in combination therapies against Helicobacter pylori, the microaerobic bacterium that causes gastritis and peptic ulcer disease and that is an early risk factor for gastric cancer (6,19,30). However, resistance to MTZ is very common and reduces the efficacy of MTZ-containing regimes (10). MTZ is also highly mutagenic, and hence its use may accelerate development of resistance to other clinically important antibiotics, such as clarithromycin (24,29). These considerations have stimulated interest in alternative redox-active prodrugs in either first-line or salvage (failed eradication) therapies, especially those against which resistance may be uncommon in H. pylori. Alternative prodrugs include the nitrofurans and nitrothiazoles, which exhibit antimicrobial activity against H. pylori and also against facultative anaerobes, anaerobic bacterial and protozoan species, and helminths (12,19,21,30,32,33). For these drugs, the spectrum of bioactivity and the mechanisms of activation are largely a function of the redox potential (EЈ 7 ) of the 5-nitro groups. For example, nitrofurans (nitrofurazone, nitrofurantoin, and furazolidone) display relatively high redox potentials (Ϫ250 to Ϫ270 mV) and are reductively activated by a...

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

Much of the normal high sensitivity of wild-type Helicobacter pylori to metronidazole (Mtz) depends on rdxA (HP0954), a gene encoding a novel nitroreductase that catalyzes the conversion of Mtz from a harmless prodrug to a bactericidal agent. Here we report that levels of Mtz that partially inhibit growth stimulate forward mutation to rifampin resistance in rdxA ؉ (Mtz s ) and also in rdxA (Mtz r ) H. pylori strains, and that expression of rdxA in Escherichia coli results in equivalent Mtz-induced mutation. A reversion test using defined lac tester strains of E. coli carrying rdxA ؉ indicated that CG-to-GC transversions and AT-to-GC transitions are induced more frequently than other base substitutions. Alkaline gel electrophoretic tests showed that Mtz concentrations near or higher than the MIC for growth also caused DNA breakage in H. pylori and in E. coli carrying rdxA ؉ , suggesting that this damage may account for most of the bactericidal action of Mtz. Coculture of Mtz s H. pylori with E. coli (highly resistant to Mtz) in the presence of Mtz did not stimulate forward mutation in E. coli, indicating that the mutagenic and bactericidal products of Mtz metabolism do not diffuse significantly to neighboring (bystander) cells. Our results suggest that the widespread use of Mtz against other pathogens in people chronically infected with H. pylori may stimulate mutation and recombination in H. pylori, thereby speeding host-specific adaptation, the evolution of virulence, and the emergence of resistance against Mtz and other clinically useful antimicrobials.Metronidazole (Mtz) [1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole] and related 5-nitroimidazoles are redox-active prodrugs that are often used to treat infections caused by anaerobic bacteria and protozoa (7,18,28). They are also a key component of combination therapies that are used to eradicate Helicobacter pylori, the microaerophilic bacterium which chronically infects the stomachs of more than half of all people worldwide and is the major cause of peptic ulcer disease and an early risk factor for gastric cancer (3,14,20,30). In anaerobes, redox-active enzymes such as pyruvate/ketoacid oxidoreductases and hydrogenase, active with the low-redox carriers (ferredoxin and flavodoxin), reduce 5-nitroimidazoles to mutagenic products that also cause DNA helix destabilization and singleand double-strand DNA breakage (7,18,19,28). High-level Mtz resistance is rare in anaerobes, because the activating enzymes are essential components of core metabolic pathways and because these microbes generally contain cytoplasmic components of very low redox potential that can spontaneously activate the drug (7,19,23,28). In contrast, moderate to high-level resistance to nitroimidazoles is common among H. pylori clinical isolates, with frequencies ranging from ϳ10 to 90% or more of strains, depending on geographic region (12). These frequencies generally reflect the incidence of Mtz usage against other (parasitic or anaerobic bacterial) infections in particular societies (6, 10).We ...