DNA Sequence Analysis of rdxA and frxA from 12 Pairs of Metronidazole-Sensitive and -Resistant Clinical Helicobacter pylori Isolates

Abstract

We previously reported that inactivation of rdxA and/or frxA converted Helicobacter pylori from metronidazole sensitive to metronidazole resistant. To examine the individual roles of rdxA and frxA in the development of metronidazole resistance in H. pylori, we examined the status of rdxA and frxA from 12 pairs of metronidazolesensitive and -resistant H. pylori isolates obtained following unsuccessful therapy containing metronidazole. Arbitrary primed fingerprinting analyses revealed that the genotypes of 11 sensitive and resistant pairs of strains were essentially identical. Amino acid sequence identities of RdxA and FrxA from the 14 metronidazolesensitive isolates ranged from 92 to 98% and 95 to 98%, respectively, compared to that of H. pylori J99 (MIC, 1 g/ml). All strains with high-level metronidazole resistance (MICs, 128 g/ml) contained premature truncation of both RdxA and FrxA caused by nonsense and/or frameshift mutations. Strains with intermediate resistance to metronidazole (MICs, 32 to 64 g/ml) contained a single premature truncation and/or altered RdxA and FrxA caused by nonsense, frameshift, and unique missense mutations. The low-level metronidazoleresistant strains (MICs, 8 g/ml) contained unique missense mutations in FrxA but no specific changes in RdxA. The results demonstrate that alterations in both the rdxA and frxA genes are required for moderate and high-level metronidazole resistance and that metronidazole resistance that develops during anti-H. pylori therapy containing metronidazole is most likely to involve a single sensitive strain infection rather than a coinfection with a metronidazole-resistant strain.Over 50% of the world population is infected with the important human pathogen Helicobacter pylori. The primary impediments to successful H. pylori treatment are lack of compliance with drug regimens and the presence of antibioticresistant H. pylori (10). Metronidazole was a critical ingredient of the first successful therapy for H. pylori and remains a major component of newer proton pump inhibitor-containing quadruple therapies (2, 12). The background prevalence of metronidazole resistance is high in the United States (20 to 45% of isolates) and is increased in those who have used the drug for other indications (23). Effective therapy for H. pylori infection requires two or more antimicrobial agents often given with antisecretory drugs to reduce gastric acidity (2, 12). Metronidazole resistance decreases the effectiveness of metronidazolecontaining anti-H. pylori therapies (2, 5).The antimicrobial action of metronidazole is dependent on reductive activation of metronidazole by the redox system of the target cell. Theoretically, any redox system possessing a reduction potential that is more negative than that of metronidazole in the cell would donate its electrons preferentially to metronidazole and lead to reductive activation (7, 17). Goodwin et al. reported that oxygen-insensitive NADPH nitroreductase (rdxA) was a putative metronidazole nitroreductase-encoding gene involved in metro...

Genetic analysis of Helicobacter pylori clinical isolates suggests resistance to metronidazole can occur without the loss of functional rdxA

“…These findings suggest that the amino-acid changes should be attributed to natural genetic diversity and are not associated with MTZ resistance. Recent studies revealed the RdxA amino-acid changes of Arg10-Lys, Arg16-His, Met21-Ala, His53-Arg, Met56-Ile, Leu62-Val, Ala68-Val, Gly98-Ser, Gly163-Asp and Ala206-Thr in MTZ-resistant strains, 2,31,32 suggesting that those substitutions may be important in MTZ resistance. However, in our study the replacement with the missense-mutated rdxA containing amino-acid substitutions (Met21-Ala, Leu62-Val, Gly98-Ser and Ala206-Thr) failed to transfer the MTZ-resistant phenotype, suggesting that the substitutions are not important for MTZ resistance.…”

“…Earlier, presumably important mutations of rdxA for MTZ resistance were simply identified by comparison of the RdxA sequence between MTZsensitive and -resistant H. pylori strains. 2,31,32 However, these mutations were not evaluated directly for their contribution to MTZ resistance. Comparative analysis of the RdxA sequences in our study predicted that the changes of Lys64-Asn and Gly98-Asn in the R3 strain, and Tyr26-Val and Ala40-Pro in the R5 strain might be responsible for MTZ resistance.…”

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

“…Las mutaciones que afectan las posiciones A68V, A118T, R90K, R131K, R16H, S88P y H97T, y las que introducen un codón de parada, como N73* y Q50*, se han correlacionado con la resistencia al metronidazol, especialmente cuando hay valores altos en las concentraciones inhibitorias mínimas (CIM) (13,20,27) y cuando la CIM por dilución en agar es igual o superior a 12 µg/ml (13,22,25).…”

“…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 usaron los iniciadores rdxA F 5'-GACAATTATTAAACGAGCGC-3' y rdxA R 5'-CCTCCAATAATGCAACTATC-3' correspondientes a las posiciones 1013697 a 1014157 de la cepa de referencia 26695 (acceso en Genbank: gb| CP003904.1) (19), reportados por Kwon, et al (20).…”