Roles of FrxA and RdxA Nitroreductases of Helicobacter pylori in Susceptibility and Resistance to Metronidazole

Abstract

had interpreted that resistance resulted from inactivation either of frxA or rdxA. These two interpretations were tested here. Resistance was defined as efficient colony formation by single cells from diluted cultures rather than as growth responses of more dense inocula on MTZ-containing medium. Tests of three of Kwon's Mtz s strains showed that each was type II, requiring inactivation of both rdxA and frxA to become resistant. In additional tests, derivatives of frxA mutant strains recovered from MTZ-containing medium were found to contain new mutations in rdxA, and frxA inactivation slowed MTZ-induced killing of Mtz s strains. Northern blot analyses indicated that frxA mRNA, and perhaps also rdxA mRNA, were more abundant in type II than in type I strains. We conclude that development of MTZ resistance in H. pylori requires inactivation of rdxA alone or of both rdxA and frxA, depending on bacterial genotype, but rarely, if ever, inactivation of frxA alone, and that H. pylori strains differ in regulation of nitroreductase gene expression. We suggest that such regulatory differences may be significant functionally during human infection.Helicobacter pylori is a genetically diverse bacterial species that chronically infects the stomachs of more than half of all people worldwide. Its long-term carriage is a major cause of chronic gastritis and peptic ulcer disease and is an early risk factor for gastric cancer (for reviews see references 5, 8, 28, and 32). Resistance to metronidazole (MTZ) is common and is important clinically as a primary cause of failure of MTZ-based anti-Helicobacter therapies (for reviews see references 10, 15, and 24). Frequencies of clinical isolates that are MTZ resistant range from only 10% in Japan (25) to 90% or more in India (26), and up to 50% or more of strains in the United States and Western Europe also are resistant (frequency varies among countries) (8, 23). These geographic differences probably reflect frequencies of MTZ use against other, mostly parasitic and anaerobic, infections and thus inadvertent MTZ exposure of resident H. pylori strains. Recent studies have implicated mutations in the chromosomal genes rdxA (HP0954) and frxA (HP0642) in the development of resistance (7,9,14,16,30, 35). These genes encode related nitroreductases that can convert MTZ from a harmless prodrug to products such as hydroxylamine that are both bactericidal and mutagenic (9, 29).There has been disagreement about the quantitative contributions of rdxA and frxA to MTZ susceptibility and resistance. On the one hand, Kwon and associates had concluded that inactivation of either gene by itself could make any typical H. pylori strain resistant to MTZ (Mtz r ) (21), and that following frxA inactivation, growth on MTZ-containing agar was not associated with mutation of rdxA (20). In contrast, we had concluded that rdxA inactivation is usually or always needed for a Mtz s strain to become Mtz r (16,17). Two types of Mtz s strains were distinguished, however, based on relative levels of FrxA nitroreductase...

Expanded therapeutic potential in activity space of next-generation 5-nitroimidazole antimicrobials with broad structural diversity

“…As a further test of the ability to overcome nitro drug resistance, we used a double mutant of H. pylori for two reductases, NADPH flavin oxidoreductase (FrxA) and oxygen-insensitive NADPH nitroreductase (RdxA), that activate nitro drugs and are involved in clinical drug resistance (18). Screening of the library revealed that 98.6% of compounds lost activity against the double mutant ( Fig.…”

Helicobacter pylori tissue tropism: mouse-colonizing strains can target different gastric niches

“…AF233683). X47 had been used primarily for studies of immune responses and for vaccine development (Ermak et al, 1998;Kleanthous et al, 2001;Londono-Arcila et al, 2002), but is becoming increasingly popular for mutational analyses (Jeong et al, 2001;O'Rourke et al, 2003), in part because it is easier than SS1 to transform with mutated DNAs. Strain X47 lacks the cag pathogenicity island entirely, but contains an s1 allele of vacA (GenBank accession no.…”

“…Growth under mildly acidic conditions, which depends on different genetic factors (Bijlsma et al, 2000), was scored by spotting 10 ml aliquots of a series of dilutions of equivalent cell suspensions on Brucella Agar (1?5 %) whose pH had been adjusted before autoclaving. Viability was scored quantitatively, as the ability of single cells to form colonies after appropriate dilution, essentially as in Jeong et al (2001).…”

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.…”