In Vitro Effect of Reactive Nitrogen and Oxygen Intermediates Alone and in Combination with Some Antibiotics Against Brucella Melitensis Clinical Isolates

Abstract

Brucella spp. replicate and survive in lympho-proliferative tissues and cells, thus effective treatment of brucellosis requires the combined and long term use of intracellularly active antibiotics. Elimination of the microorganism largely depends on the reactive oxygen and nitrogen intermediates released by activated macrophages. In this study we aimed to determine the in vitro activity of hydrogen peroxide (H<inf>2</inf>O<inf>2</inf>; reactive oxygen intermediate) and acidified sodium nitrite (ASN; reactive nitrogen intermediate) alone and in combination with rifampicin (RIF) and tetracycline (TET) against four clinical isolates of Brucella melitensis. Initially minimal inhibitory concentrations of RIF and TET were determined by microbroth dilution susceptibility test. The activity of 2 and 5 mM H<inf>2</inf>O<inf>2</inf> and 3 and 6 mM ASN was tested against each isolate by direct colony count from the agar plates inoculated with bacterial suspensions treated with H<inf>2</inf>O<inf>2</inf> or ASN. The last step in the assay was to determine the combined effectiveness of RIF and TET plus H<inf>2</inf>O<inf>2</inf> and ASN. From each three rolls of assay apparatus samples were taken at 0., 1., 6. and 24. hours and inoculated on Brucella agar. The plates were incubated at 37°C for 48 hours and colonies were counted. While RIF alone or in combination with H<inf>2</inf>O<inf>2</inf> supressed the growth of bacteria even in the first hour, TET alone did not show any effect in 24 hours. However, in combination with reactive oxygen and nitrogen intermediates TET affected bacterial growth starting from six hours. In conclusion, further explanation of the interactions between antibiotics and the substances produced by the immune system of the host during the infections caused by intracellular pathogens, might have an important impact on the determination of the treatment protocols and the measures to prevent relapses.