Differential gene expression in a Bacteroides fragilis metronidazole-resistant mutant

Objectives: The current work focused on molecular changes in a spontaneous Bacteroides fragilismutant selected by low concentrations of metronidazole as an adaptive response to the drug. Methods: A metronidazole-resistant strain derived from B. fragilis ATCC 25285 was selected by passage in the presence of drug using 0-4 mg/L gradient plates. Using a combination of proteomics for identification of differentially expressed proteins by two-dimensional electrophoresis and selected mutational analyses by single cross-over insertion and an allelic exchange, we have identified genes involved in the adaptive response to metronidazole. Results: There are significant changes in the protein profiles of resistant strains. These changes appeared to affect a wide range of metabolic proteins including lactate dehydrogenase (up-regulated) and flavodoxin (down-regulated), which may be involved in electron transfer reactions. Also, the enzymic activity of the pyruvate-ferredoxin oxidoreductase (PorA) complex was impaired. Mutant strains lacking the genes for flavodoxin and PorA were less susceptible to metronidazole than the sensitive parent, and a double flavodoxin/PorA mutant had even less susceptibility but none of the mutants were as resistant as the spontaneous metronidazole-resistant strain. Conclusions: Overall, the data indicated that there were global changes in the regulation of the physiology of the metronidazole-resistant strain. In addition, flavodoxin was identified as an important contributor to metronidazole sensitivity in B. fragilis.