The association of Blastocystis sp. ST3 with gram negative bacteria is altered by short-term purification strategies

Blastocystis sp. is a gut protozoan parasite that is associated with gastrointestinal symptoms. Over time, it has garnered controversial findings on having a beneficial or harmful role in the gut microbiota. To date, studies on the interaction between Blastocystis sp. and bacteria remain limited due to challenges in establishing axenic Blastocystis sp. cultures. Although axenization is imperative for Blastocystis sp.-bacteria interaction, the process is time-consuming. This study evaluates alternative approaches to study Blastocystis sp.-bacteria associations when axenization is unfeasible in a limited timeframe. Blastocystis sp. cells were purified using mechanical (Lymphopure-density gradient centrifugation) and chemical (antibiotic cocktail treatment) purification methods and subsequently assessed by establishing a growth profile of purified Blastocystis sp. cells co-cultured with Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa at varying concentrations. Our results demonstrated that both purification methods successfully reduced bacterial load. However, mechanical purification was able to reduce the bacterial load to a greater extent across both isolates compared to chemical purification. Growth of Blastocystis sp. in co-cultures varied according to the bacterial species and their respective concentrations. The symptomatic Blastocystis sp. isolate generally peaked at lower bacterial concentrations compared to the asymptomatic Blastocystis sp. isolate, implicating an isolate-specific association with bacteria. This observation is more apparent in mechanically purified co-cultures. Our findings suggest that mechanical purification can be a feasible time-saving alternative to axenization. Our preliminary data hypothesizes that Blastocystis sp.-bacteria association is influenced by the status of the protozoan isolate, bacterial concentration, and purification methods.