Zooming in on the intracellular microbiome composition of bacterivorous Acanthamoeba isolates

Binod Rayamajhee; Mark Willcox; Savitri Sharma; Ronnie Mooney; Constantinos Petsoglou; Paul R Badenoch; Samendra Sherchan; Fiona L Henriquez; Nicole Carnt

doi:10.1093/ismeco/ycae016

Zooming in on the intracellular microbiome composition of bacterivorous Acanthamoeba isolates

ISME Commun. 2024 Jan 23;4(1):ycae016. doi: 10.1093/ismeco/ycae016. eCollection 2024 Jan.

Authors

Binod Rayamajhee¹, Mark Willcox¹, Savitri Sharma², Ronnie Mooney³, Constantinos Petsoglou^{4

5}, Paul R Badenoch⁶, Samendra Sherchan⁷, Fiona L Henriquez³, Nicole Carnt¹

Affiliations

¹ School of Optometry and Vision Science, Faculty of Medicine and Health, UNSW, Sydney, NSW 2052, Australia.
² Jhaveri Microbiology Centre, Prof Brien Holden Eye Research Centre, Hyderabad Eye Research Foundation, L. V. Prasad Eye Institute (LVPEI), Hyderabad, 500034, India.
³ School of Health and Life Sciences, University of the West of Scotland, Blantyre, PA1 2BE, United Kingdom.
⁴ Sydney and Sydney Eye Hospital, South-Eastern Sydney Local Health District, Sydney, NSW 2000, Australia.
⁵ Save Sight Institute, University of Sydney, Sydney, NSW 2000, Australia.
⁶ College of Medicine and Public Health, Flinders University, Adelaide, 5042, Australia.
⁷ School of Public Health and Tropical Medicine, Tulane University, New Orleans, LA 70112, United States.

Abstract

Acanthamoeba, a free-living amoeba in water and soil, is an emerging pathogen causing severe eye infection known as Acanthamoeba keratitis. In its natural environment, Acanthamoeba performs a dual function as an environmental heterotrophic predator and host for a range of microorganisms that resist digestion. Our objective was to characterize the intracellular microorganisms of phylogenetically distinct Acanthamoeba spp. isolated in Australia and India through directly sequencing 16S rRNA amplicons from the amoebae. The presence of intracellular bacteria was further confirmed by in situ hybridization and electron microscopy. Among the 51 isolates assessed, 41% harboured intracellular bacteria which were clustered into four major phyla: Pseudomonadota (previously known as Proteobacteria), Bacteroidota (previously known as Bacteroidetes), Actinomycetota (previously known as Actinobacteria), and Bacillota (previously known as Firmicutes). The linear discriminate analysis effect size analysis identified distinct microbial abundance patterns among the sample types; Pseudomonas species was abundant in Australian corneal isolates (P < 0.007), Enterobacteriales showed higher abundance in Indian corneal isolates (P < 0.017), and Bacteroidota was abundant in Australian water isolates (P < 0.019). The bacterial beta diversity of Acanthamoeba isolates from keratitis patients in India and Australia significantly differed (P < 0.05), while alpha diversity did not vary based on the country of origin or source of isolation (P > 0.05). More diverse intracellular bacteria were identified in water isolates as compared with clinical isolates. Confocal and electron microscopy confirmed the bacterial cells undergoing binary fission within the amoebal host, indicating the presence of viable bacteria. This study sheds light on the possibility of a sympatric lifestyle within Acanthamoeba, thereby emphasizing its crucial role as a bunker and carrier of potential human pathogens.

Keywords: Acanthamoeba; environmental predator; eye infection; sympatric lifestyle; training ground.