The susceptibility of pathogenic free-living amebae to chemotherapeutic agents : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University

Abstract

The treatment of infections caused by pathogenic free-living amebae (PFLA) has, until only recently, been far from successful. The continued screening of chemotherapeutic agents against amebae of the genera Naegleria and Acanthamoeba is therefore of the utmost importance. Seven chemotherapeutic agents, amphotericin B, rifampicin, tetracycline, polymyxin B sulphate, 5-fluorocytosine, miconazole and R41,400 were screened for activity against a non-pathogenic and pathogenic species of Naegleria and a non-pathogenic and pathogenic species of Acanthamoeba in axenic culture. For the Naegleria spp. amphotericin B, miconazole and R41,400 were found to be active. Acanthamoebae spp. were found to be susceptible only to 5-fluorocytosine and R41,400. The possible use of combinations of drugs against the amebae was also investigated in axenic culture. For Naegleria fowleri (MsT) amphotericin B with either tetracycline or rifampicin showed a synergistic effect. Polymyxin B sulphate and 5-fluorocytosine showed synergistic activity against Acanthamoeba culbertsoni (A-1) but when polymyxin B was combined with tetracycline or rifampicin no significant additive effect was seen. After axenic culture testing the susceptibility of the pathogenic species, N. fowleri (MsT) and A. culbertsoni (A-1), to the agents which showed activity, was investigated in a Vero cell culture system. For N. fowleri (MsT) the results of axenic testing were confirmed with amphotericin B, miconazole and R41,400 protecting the monolayer from the destructive effects of the amebae. 5-Fluorocytosine inhibited the formation of cytopathic effect (CPE) when the cell cultures were inoculated with A. culbertsoni (A-1) but viable amebae were still present. R41,400 had no effect on A. culbertsoni (A-1) at concentrations at or above those which were cytotoxic to the Vero cells. The use of combinations of drugs was also investigated in Vero cell culture. Amphotericin B and rifampicin showed an antagonistic rather than a synergistic effect when used against N. fowleri (MsT) in cell culture but amphotericin B and tetracycline showed synergistic activity. For A. culbertsoni (A-1) the synergistic activity of polymyxin B and 5-fluorocytosine was confirmed. The lack of an additive effect between polymyxin B and either tetracycline or rifampicin was also shown in cell culture. The new imidazole derivative R41,400, which showed promise against N. fowleri (MsT) in in vitro tests was then tested in the in vivo situation. Mice experimentally infected with N. fowleri (MsT) were treated once or twice daily intraperitoneally with different doses of R41,400. At the higher dosage levels tested the drug appeared to have a deleterious effect, the average time for death being less than that for the controls