A study on Nim expression in Bacteroides fragilis

Abstract

Members of the genus Bacteroides, mainly Bacteroides fragilis, can cause severe disease in man, especially after intestinal perforation in the course of abdominal surgery. Treatment is based on a small number of antibiotics, including metronidazole, which has proved to be highly reliable throughout the last 40 to 50 years. Nevertheless, metronidazole resistance does occur in Bacteroides and has been mainly attributed to Nim proteins, a class of proteins with a suggested nitroreductase function. Despite the potentially high importance of Nim proteins for human health, information on the expression of nim genes in B. fragilis is still lacking. It was the aim of this study to demonstrate expression of nim genes in B. fragilis at the protein level and, furthermore, to correlate Nim levels with the magnitude of metronidazole resistance. By the application of 2D gel electrophoresis, Nim proteins could be readily identified in nim-positive strains, but their levels were not elevated to a relevant extent after induction of resistance with high doses of metronidazole. Thus, the data herein do not provide evidence for Nim proteins acting as nitroreductases using metronidazole as a substrate, because no correlation between Nim levels and levels of metronidazole resistance could be observed. Furthermore, no evidence was found that Nim proteins protect B. fragilis from metronidazole by sequestering the activated antibiotic.

Figure 1

Representative images of 2D-gels (pH range 3-10 non-linear, 12.5% PAA) from strain 638R, either without nim gene (left image), with plasmid pIP417 (nimA, IS1186), nimA-positive, (middle), or with plasmid pIP421 (nimD, IS1169), nimD-positive, (right). The Nim proteins (encircled) are easily discernible as prominent spots in the lower molecular mass range of the gel. The theoretical molecular weights of NimA and NimD amount to 20.2 kDa and 18.5 kDa, respectively. Directions of increasing molecular mass (MW) and increasing pH are indicated by arrows.

Figure 2

Relative copy number determination of the nimA plasmid (pIP417) depending on metronidazole exposure/adaptation. Error bars indicate SEM with 95% confidence.

Figure 3

2DE analysis of metronidazole treated 638R. (a), integrity and relative abundance of NimA and the putative reductase (E1WVQ6_BACF6) were checked on 2D-gels after 2 h of incubation of equally divided 638R (nimA) stationary phase cultures either without drug or with 50 µM of metronidazole. The broken circle indicates the location of the metronidazole adduct of the isolated protein spot. Percentages refer to the relative abundance of the measured proteins as compared to total protein visualized. (b), validation of metronidazole adduct formation in 638R (nim-negative). Broken circles indicate adducts of the metronidazole adduct of the isolated protein spot with either metronidazole or tinidazole.