Experimental Induction of Paromomycin Resistance in Antimony-Resistant Strains of L. donovani: Outcome Dependent on In Vitro Selection Protocol

Abstract

Paromomycin (PMM) has recently been introduced for treatment of visceral leishmaniasis in India. Although no clinical resistance has yet been reported, proactive vigilance should be warranted. The present in vitro study compared the outcome and stability of experimental PMM-resistance induction on promastigotes and intracellular amastigotes. Cloned antimony-resistant L. donovani field isolates from India and Nepal were exposed to stepwise increasing concentrations of PMM (up to 500 µM), either as promastigotes or intracellular amastigotes. One resulting resistant strain was cloned and checked for stability of resistance by drug-free in vitro passage as promastigotes for 20 weeks or a single in vivo passage in the golden hamster. Resistance selection in promastigotes took about 25 weeks to reach the maximal 97 µM inclusion level that did not affect normal growth. Comparison of the IC₅₀ values between the parent and the selected strains revealed a 9 to 11-fold resistance for the Indian and 3 to 5-fold for the Nepalese strains whereby the resistant phenotype was also maintained at the level of the amastigote. Applying PMM pressure to intracellular amastigotes produced resistance after just two selection cycles (IC₅₀ = 199 µM) compared to the parent strain (IC₅₀ = 45 µM). In the amastigote-induced strains/clones, lower PMM susceptibilities were seen only in amastigotes and not at all in promastigotes. This resistance phenotype remained stable after serial in vitro passage as promastigote for 20 weeks and after a single in vivo passage in the hamster. This study clearly demonstrates that a different PMM-resistance phenotype is obtained whether drug selection is applied to promastigotes or intracellular amastigotes. These findings may have important relevance to resistance mechanism investigations and the likelihood of resistance development and detection in the field. Leishmaniasis is caused by protozoan parasites of the genus Leishmania and is transmitted by inoculation of infective promastigotes by the female sand fly. In the mammalian host, amastigotes live inside macrophage cells which may lead to various clinical symptoms. First-line treatment relies mainly on antimonials and miltefosine; however, drug resistance is a growing problem. The antibiotic paromomycin (PMM) has recently been added as treatment option, but it is now essential to proactively assess the likelihood of resistance development to safeguard its long term effectiveness. Since ‘resistant’ patient isolates are not yet available, we artificially selected for PMM resistance using two different in vitro protocols with drug pressure on either the extracellular promastigote or on the intracellular amastigote stage. Resistance in promastigotes was obtained after about 25 weeks and persisted in the intracellular amastigote. High levels of resistance were obtained within two selection cycles on amastigotes, but with the unexpected observation that the promastigotes remained fully susceptible. In addition, the resistance proved to be stable. We could clearly demonstrate that a different PMM-resistance is obtained dependent on the ‘stage-selection’ protocol. These findings have important relevance to resistance mechanism investigations and the likelihood of resistance development and detection in the field.