Novel Culture Medium Developed for Pathogenic Free-Living Amoeba

Balamuthia mandrillaris is an opportunistic protist pathogen and the causative agent of necrotizing hemorrhagic granulomatous encephalitis among other infections, with an estimated fatality rate approaching 98%.

B. mandrillaris, unlike most of other free-living amoebae, does not feed on Gram-negative bacteria and therefore the use of non-nutrient agar coated with bacterial cultures has resulted to be ineffective for its growth. Other special medium have been recommended, but they have some disadvantages, as they require many components and their preparations are laborious.

Microbiologists at the Sonora Technological Institute (Mexico) developed an easy-to-prepare culture medium for axenic growth of B. mandrillaris using Cerva's medium as basal component. Ten strains of B. mandrillaris including a well-characterized US Centers for Disease and Prevention (CDC; Atlanta, GA, USA) reference stain and nine environmental isolates from water and soil were used during the study.

Each one of the 11 components of BM-3 culture medium was combined with Cerva's medium as basal culture medium. The cultures were observed daily for a four-week period under an inverted microscope Axiovert 135 (Zeiss; Gottingen, Germany). The number of trophozoites/mL was determined using a hemocytometer, and the doubling time was calculated by linear regression. The yields in each culture were determined at the end of the logarithmic growth phase.

After the first week of daily observations of the tested combinations of BM-3 components and Cerva's medium, amoebae numbers diminished in every flask. At the end of the second week of incubation, neither trophozoites nor cysts were seen in any modification, except for the one consisting of the basal medium complemented with 10× Hank's balanced salt solution (34.0 mL/500 mL) where growth was recovered. Hank's balanced salt solution was the only one that supported confluent growth of B. mandrillaris and where cell shape and motility of trophozoites were normal.

The authors concluded that nutrimental necessity of B. mandrillaris depends on adequate salt solution, protein extracts, and other nutrients obtained from the fetal bovine serum. When using axenic cultures, the development of a cheaper and easy-to-prepare medium for B. mandrillaris opens the possibility of increasing our knowledge on this lethal pathogen. (The study was published in the August 2015 issue of the journal Diagnostic Microbiology and Infectious Disease.

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Sonora Technological Institute 
Zeiss