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 (H2O2; 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 H2O2 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 H2O2 or ASN. The last step in the assay was to determine the combined effectiveness of RIF and TET plus H2O2 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 degrees C for 48 hours and colonies were counted. While RIF alone or in combination with H2O2 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.