Sequential Inactivation of rdxA (HP0954) and frxA (HP0642) Nitroreductase Genes Causes Moderate and High-Level Metronidazole Resistance in Helicobacter pylori

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 ...

Mutations frequency of RdxA a nitroreductase Helicobacter pylori for activation of metronidazole: a population study in the Department of Cauca

“…En contraposición a estos hallazgos, en otros estudios se ha reportado la presencia de aislamientos resistentes en los cuales las proteínas RdxA estaban intactas, lo cual sugiere que habría otros genes involucrados en la resistencia al metronidazol, como el gen frxA, el cual participa en su activación en el interior de la célula (20,26). Se ha postulado que las mutaciones en la proteína FrxA pueden aumentar los valores de la CIM en los aislamientos resistentes y que las que ocurren en los codones de parada reforzarían el fenotipo resistente (25).…”

“…Se ha postulado que las mutaciones en la proteína FrxA pueden aumentar los valores de la CIM en los aislamientos resistentes y que las que ocurren en los codones de parada reforzarían el fenotipo resistente (25). Por consiguiente, no hay que desconocer el papel que cumplen otras nitrorreductasas como la frxA, la fdxA y la rpsU en la resistencia al metronidazol (26,27,30).…”

“…En el cuadro 2 se presentan todas las mutaciones distribuidas en codones de parada y mutaciones puntuales desde la posición 46 hasta la posición 160 de la proteína RdxA. Por otra parte, el 78,2 % de las muestras presentó mutaciones relacionadas con la resistencia a metronidazol (49,10,13,22, 25), específicamente mutaciones puntuales tales como R131K (en 101 muestras), R90K (en 97 muestras), en A118T (en 42 muestras), H97T (en 26 muestras), E133K (en 11 muestras), y S108A (en 9 muestras), en tanto que el 21,8 % de las mutaciones puntuales no habían sido reportadas en aislamientos resistentes: la D59N (en 153 muestras), la G98S (en 27 muestras), la H97Y (en 18 muestras), la A118S (en 5 muestras) y la I160F (en 32 muestras) (4,6,12,22,26). Se encontraron, además, mutaciones relacionadas con los siguientes codones de parada: Q50*(en cinco muestras); I160* en seis muestras, E75* en tres, C159* en diez, y en la posición I62* en dos muestras.…”

Julian Davies and the discovery of kanamycin resistance transposon Tn5

“…In collaboration with Paul Hoffman, we found that resistance to metronidazole is due to inactivation of one or more nonessential nitroreductase genes that, when functional, cause conversion of metronidazole to bactericidal and mutagenic hydroxylamine type compounds. [67][68][69] In addition, we and others showed that resistance to erythromycin-related macrolides, which is also common, stems from a point mutation at any of several sites in 23 S ribosomal RNA genes; and that tetracycline resistance, which is rare, typically involves a cluster of substitution mutations in its 16 S ribosomal RNA target. [70][71][72] AR GENES IN DEVELOPING COUNTRY LOW-INCOME SETTINGS As a final illustration of Julian's impact on me, I was pleased to help Washington University colleague Gautam Dantas and his students set-up collaborations with my long-term collaborators in Peru (Bob Gilman) and El Salvador (Teresita Bertoli) to examine the 'resistomes' in a peri-urban low-income community in Lima, Peru and in a Salvadorean agricultural community.…”

Sterile and disposable fluidic subsystem suitable for clinical high speed fluorescence‐activated cell sorting

“…Samples were tested for the presence of Mycoplasma genomic DNA using a PCR Mycoplasma test kit (MD Biosciences, St. Paul, MN). Samples were tested for the presence of Helicobacter pylori genomic DNA by PCR amplification of the frxA reductase gene using the frxA-F and frxA-R primers described in Jeong et al (34). frxA PCR was done using 1.25 units Taq polymerase (New England Biolabs, Beverly, MA), standard PCR buffer, 2 mM MgCl 2 , 0.04 mM primers, and the following thermocycling program: 3 min at 958C, 35 cycles (958C for 30 s, 538C for 30 s, and 728C for 1 min), and 728C for 10 min.…”