Production and characterization of stable amphotericin-resistant amastigotes and promastigotes of Leishmania mexicana

Abstract

The sensitivities of Leishmania mexicana amastigote and promastigote forms to amphotericin B were investigated in vitro and found to be strongly influenced by the culture media used. When differences in culture media were minimized, there was no significant difference in the 50% inhibitory concentration values between the two life cycle stages. Stable amphotericin B-resistant amastigote and promastigote lines were produced by the application of increasing drug pressure to long-term cultures. Lines capable of growth in concentrations of amphotericin B lethal to normal parasites were produced. Compared to normal parasites, these amphotericin-resistant lines showed marked differences in membrane sterol compositions, with very high levels of 4,14,dimethyl-cholesta-8,24-dienol and other methyl sterols. They also showed a consistent morphological feature, the presence of multilamellar membrane-like material in the flagellar pocket, revealed by transmission electron microscopy. Amphotericin-resistant parasites were capable of infecting BALB/c mice, but the resulting lesion growth was slower than that after infection with normal parasites. However, unlike normal parasites, the amphotericin-resistant parasites were unaffected by experimental chemotherapy with amphotericin B. These results show that amphotericin B resistance could arise as a result of increased clinical use of amphotericin B therapy.

FIG. 1.

Transmission electron micrographs showing (A) a longitudinal section through an AMB-resistant L. mexicana amastigote and (B) a transverse section through an AMB-resistant promastigote. Multilamellar membrane-like material in the flagellar pocket is indicated by arrows; the flagellum is indicated by arrowheads.

FIG. 2.

Aflagellate AMB-resistant L. mexicana “promastigote” forms. (A) The culture shown was derived by an in vitro culture of AMB-resistant lesion amastigotes isolated from BALB/c mice under promastigote culture conditions in the presence of 4.0 μg/ml AMB. Bar, 10 μm. (B) Control, wild-type promastigotes.

FIG. 3.

Experimental chemotherapy of BALB/c mice infected with (A) wild-type and (B) AMB-resistant L. mexicana. Groups of eight to nine mice were either treated with 1 mg/kg AMB for a 2-week period (▪), beginning at the points indicated by the arrows, or left as untreated controls (▴). Lesion size is length multiplied by width, expressed in mm.