Nim-mediated Resistance

The anaerobic bacterium Bacteroides fragilis and other species of the genus Bacteroides (Bacteroides spp.) are generally harmless, even beneficial commensals of the human gut. They can, however, also constitute dangerous pathogens, especially if they leave their usual niche, e.g. in the course of abdominal surgery, and reach other parts of the body. There, Bacteroides spp. can cause life-threatening abscesses and sepsis. Bacteroides infections can be treated with antibiotics but, unfortunately, Bacteroides spp. quickly become resistant against a large number of antibiotics. Currently, carbapenems and metronidazole are the most widely used antibiotics against Bacteroides spp. Despite the usually high efficacy of metronidazole in the treatment of Bacteroides spp. infections, treatment failures can occur. Certain resistance genes, the so-called nim genes or their encoded gene products, the Nim proteins, respectively, render Bacteroides spp. less susceptible or even resistant to metronidazole. The mode of action of the Nim proteins has remained unresolved and previously held notions of their function have proved incorrect. It is the major aim of the present project to study the effects of Nim proteins on Bacteroides physiology, to identify the interaction partners of the Nim proteins in Bacteroides and, thereby, elucidate the mode of their action. To this end, two Vienna-based research groups (Vienna, Austria) and one research group from Szeged, Hungary, will cooperate in a binational research enterprise to resolve the underlying molecular mechanisms of Nim-mediated metronidazole resistance. We expect that a better understanding of the resistance mechanisms against metronidazole in Bacteroides spp. will enable the development of suitable counter-strategies in the future.

The anaerobic gut bacteria of the genus Bacteroides are important commensals but they can also cause serious infections when they leave their intestinal niche and reach other body sites, e.g. in the course of abdominal surgery or colon cancer. Bacteroides fragilis stands out as the Bacteroides species with the greatest pathogenic potential. It causes bacteraemia and abscesses in multiple body sites, often in mixed infections with aerobic bacteria, resulting in a death rate of up to 40%. B. fragilis infections are commonly treated with antibiotics such as amoxicillin and clavulanic acid, carbapenems, and metronidazole. Especially the latter two have retained their effectiveness against Bacteroides spp. but resistance does occur. Metronidazole resistance is associated with nim genes which are believed to encode for nitroreductases (Nim proteins) which deactivate metronidazole. In earlier studies, however, it was shown that the mode of action of Nim proteins is likely to be more complex. We wanted to elucidate the mechanism by which Nim proteins confer protection against metronidazole and performed biochemical comparisons and differential gene expression analyses with B. fragilis strains with and without nim genes. We found that nim genes prime B. fragilis for a development of high-level metronidazole resistance which is compatible with the competitive lifestyle in the host gut by allowing the retention of central metabolic pathways. In B. fragilis strains without a nim gene, however, metronidazole resistance is accompanied by the loss of important metabolic pathways because the uptake of iron, an essential component of many enzymes, is reduced. B. fragilis commonly obtains iron by importing haem, i.e. the oxygen binding component in haemoglobin which carries a centrally complexed iron atom. In metronidazole-resistant B. fragilis without a nim gene haem import is inhibited through downregulation of a haem binding protein, HmuY. In B. fragilis with a nim gene this is not the case. Further, after becoming highly resistant to metronidazole, B. fragilis strains without a nim gene become vulnerable to oxygen, whereas - again - strains with a nim gene do not. Surprisingly, however, the omission of haem in growth media did not enhance tolerance to metronidazole but rather rendered B. fragilis hypersensitive to metronidazole, even those strains which are highly resistant under normal conditions. Thus, concentrations of haem constitute a double-edged sword for B. fragilis when encountering metronidazole. These findings are of potential importance when testing for metronidazole resistance in the clinic because haem concentrations have to be monitored carefully. We also found that expression levels of nim genes are important for enabling the development of metronidazole resistance as described above. Also this finding is potentially important for clinical practice because expression levels of nim genes have so far not been considered in the assessment of metronidazole resistance.