Chloramphenicol accumulation by Haemophilus influenzae

Abstract

The mechanism of chloramphenicol transport into susceptible strains of Haemophilus influenzae cells has not been reported previously. We examined apparent uptake of chloramphenicol by bacterial cells by using high-pressure liquid chromatography to quantitate drug disappearance from liquid media. Cell-associated chloramphenicol concentration is 1,000-fold greater than the extracellular drug concentration. Under incubation conditions associated with chloramphenicol disappearance from media, cellular protein synthesis was inhibited; however, if accumulation was inhibited, protein synthesis occurred in the presence of the drug. Chloramphenicol uptake appeared saturable (Km = 0.96 mM, Vmax = 0.9 mumol/min per mg of protein) and energy dependent: disappearance from media was markedly decreased by 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone, compounds which disrupt the proton motive force. Uptake occurred only in median which can support growth and was dependent upon temperature and pH. Drug accumulation was minimally affected by inhibitors of electron transport or by gentamicin and puromycin, both inhibitors of protein synthesis. The rate of disappearance was inhibited by SCH24893, a fluorinated chloramphenicol analog which also inhibits protein synthesis. We conclude that chloramphenicol accumulation by H. influenzae occurs by energy-dependent transport.