A novel clonal and axenic isolation pipeline for the human gut protist Blastocystis reveals within-subtype strain coexistence
Abstract
Blastocystis is the most prevalent protist in the human gut microbiome, but its study is impeded by limitations in existing cultivation methods that hinder routine axenic and clonal isolation. These barriers have left open the question of whether multiple strains of a protist species can coexist within a single host, as observed within the bacterial gut microbiota. To address this, we introduce egg-based monophasic (EBM) medium, a novel optically clear liquid medium that supports exponential growth of Blastocystis from low inoculum densities in standard tissue-culture plates and flasks, a capability not supported by existing media formulations tested here. Using EBM medium, we established a modular axenization and clonal isolation pipeline and recovered axenic and clonally isolated cultures representing Blastocystis subtypes 1–4 from human and non-human primate hosts. To investigate within-host strain diversity, we sequenced a subtype 1 culture before and after clonal isolation. The non-clonal culture yielded a 21 Mb assembly with gene copy distribution and allelic polymorphism patterns consistent with multiple strains, whereas its clonal derivative yielded a 15 Mb assembly with patterns consistent with a single strain. These results indicate that the original culture contained multiple closely related strains co-colonizing one host. We identified the same genomic signature in published xenic cultures established from single human donors, supporting the presence of strain-level diversity within Blastocystis subtypes. Our pipeline provides a scalable platform for Blastocystis cultivation, imaging, and clonal isolation, enabling investigation of strain-level diversity that may be obscured in non-clonal cultures.
Importance: Blastocystis is the most prevalent protist in the human gut, yet experimental investigation remains limited by a difficulty in obtaining single-microbe and genetically defined cultures. Existing methods make it difficult to separate the protist from associated bacteria or recover individual lineages from mixed cultures. We developed a liquid growth medium compatible with standard tissue culture plates, live-cell imaging, and a modular workflow for producing axenic and clonal isolates. We use this pipeline to create axenic and clonal isolates across four common subtypes. Applying this workflow exposed a mixture of closely related strains in a single-subtype culture, a finding supported by analyses of independent published cultures. This culturing pipeline enables controlled investigation of strain-level diversity, and provides a new experimental framework for the study of Blastocystis biology and its interactions with the gut microbiome.
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BibTeX
@misc{leon2026novel,
title = {{A novel clonal and axenic isolation pipeline for the human gut protist Blastocystis reveals within-subtype strain coexistence}},
author = {Fredrick Leon and Johnathan B. Shih and Lydia E. Keller and Shobini Palaniappan and Lucie Degaetano and Gabriela A. Nine and Abigail L. Lind},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.19.760120},
url = {https://www.biorxiv.org/content/10.64898/2026.10.19.760120v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - A novel clonal and axenic isolation pipeline for the human gut protist Blastocystis reveals within-subtype strain coexistence
AU - Fredrick Leon
AU - Johnathan B. Shih
AU - Lydia E. Keller
AU - Shobini Palaniappan
AU - Lucie Degaetano
AU - Gabriela A. Nine
AU - Abigail L. Lind
PY - 2026
DA - 2026/10/19
PB - bioRxiv
DO - 10.64898/2026.10.19.760120
UR - https://www.biorxiv.org/content/10.64898/2026.10.19.760120v1
AB - Blastocystis is the most prevalent protist in the human gut microbiome, but its study is impeded by limitations in existing cultivation methods that hinder routine axenic and clonal isolation. These barriers have left open the question of whether multiple strains of a protist species can coexist within a single host, as observed within the bacterial gut microbiota. To address this, we introduce egg-based monophasic (EBM) medium, a novel optically clear liquid medium that supports exponential growth of Blastocystis from low inoculum densities in standard tissue-culture plates and flasks, a capability not supported by existing media formulations tested here. Using EBM medium, we established a modular axenization and clonal isolation pipeline and recovered axenic and clonally isolated cultures representing Blastocystis subtypes 1–4 from human and non-human primate hosts. To investigate within-host strain diversity, we sequenced a subtype 1 culture before and after clonal isolation. The non-clonal culture yielded a 21 Mb assembly with gene copy distribution and allelic polymorphism patterns consistent with multiple strains, whereas its clonal derivative yielded a 15 Mb assembly with patterns consistent with a single strain. These results indicate that the original culture contained multiple closely related strains co-colonizing one host. We identified the same genomic signature in published xenic cultures established from single human donors, supporting the presence of strain-level diversity within Blastocystis subtypes. Our pipeline provides a scalable platform for Blastocystis cultivation, imaging, and clonal isolation, enabling investigation of strain-level diversity that may be obscured in non-clonal cultures. Importance: Blastocystis is the most prevalent protist in the human gut, yet experimental investigation remains limited by a difficulty in obtaining single-microbe and genetically defined cultures. Existing methods make it difficult to separate the protist from associated bacteria or recover individual lineages from mixed cultures. We developed a liquid growth medium compatible with standard tissue culture plates, live-cell imaging, and a modular workflow for producing axenic and clonal isolates. We use this pipeline to create axenic and clonal isolates across four common subtypes. Applying this workflow exposed a mixture of closely related strains in a single-subtype culture, a finding supported by analyses of independent published cultures. This culturing pipeline enables controlled investigation of strain-level diversity, and provides a new experimental framework for the study of Blastocystis biology and its interactions with the gut microbiome.
SN - 2692-8205
ER -