Algal species as substrates for cultivating potentially pathogenic free-living amoebae: a novel in vitro strategy

Abstract

Free-living amoebae (FLA) are ubiquitous protists of ecological and clinical importance whose in vitro cultivation traditionally relies on bacterial food sources, most commonly Escherichia coli on non-nutrient agar. Although this approach is widely used, bacteria-based substrates may introduce experimental and biosafety-related limitations because some bacteria can survive phagocytosis and persist within amoebae, which may act as intracellular reservoirs that protect endocytobionts from environmental stress and antimicrobial treatments and may also promote bacterial interactions associated with horizontal gene transfer and the dissemination of virulence- or resistance-related traits. To evaluate a non-bacterial alternative for laboratory cultivation, this study assessed three green algal species — Scenedesmus obliquus , Chlamydomonas reinhardtii , and Chlorella vulgaris — as trophic substrates for three potentially pathogenic FLA on non-nutrient agar, using E. coli as the conventional positive control. Among the tested amoebae, Acanthamoeba genotype T3 (accession no. OR568985) exhibited the greatest growth across all tested substrates, with the highest cell densities recorded on S. obliquus , followed by C. reinhardtii and E. coli by day 7. Naegleria clarki (accession no. OR568992) and Vermamoeba vermiformis (accession no. OR568993) also proliferated on algal substrates, although growth was generally higher on E. coli . Strong positive correlations between algal- and bacterial-based growth patterns indicated broadly similar temporal trends in amoebal proliferation. Overall, these findings demonstrate that selected green algae, particularly S. obliquus and C. reinhardtii , can support measurable growth of potentially pathogenic FLA and represent promising non-bacterial substrates for laboratory cultivation.