Zooming in on the intracellular microbiome composition of bacterivorous Acanthamoeba isolates

Abstract

Acanthamoeba, a free-living amoeba (FLA) in water and soil, is an emerging pathogen causing severe eye infections known as Acanthamoeba keratitis (AK). 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 (LEfSe) 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 to 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.

First report on the infection of Acanthamoeba and Entamoeba in the endangered Chinese Milu deer (Elaphurus davidianus) in China

“…It is noteworthy that recent studies have demonstrated that certain microorganisms can parasitize Acanthamoeba [ 59 , 60 ], consistent with previous research describing Acanthamoeba as a “Trojan horse.” However, whether Acanthamoeba can act as a “Trojan horse” to facilitate the invasion of pathogenic microorganisms into the moose remains an unanswered question. Although this study did not explore this phenomenon in greater depth, we believe that this represents a compelling and highly valuable avenue for future research.…”

First report on the infection of Acanthamoeba and Entamoeba in the endangered Chinese Milu deer (Elaphurus davidianus) in China

“…It is noteworthy that recent studies have demonstrated that certain microorganisms can parasitize Acanthamoeba [ 59 , 60 ], consistent with previous research describing Acanthamoeba as a “Trojan horse.” However, whether Acanthamoeba can act as a “Trojan horse” to facilitate the invasion of pathogenic microorganisms into the moose remains an unanswered question. Although this study did not explore this phenomenon in greater depth, we believe that this represents a compelling and highly valuable avenue for future research.…”

Drug susceptibility of a clinical isolate of Balamuthia mandrillaris , a pathogenic free-living amoeba

“…There is growing evidence that the environmental and clinical isolates of the keratitis-causing Acanthamoeba spp. also harbor endosymbiotic bacteria ( 38 , 39 ). Using the same primer sequences specific to bacteria 16s rRNA, the PCR and DNA sequencing showed amplification of the 16s rRNA gene, which is located in the mitochondrial genome of the B. mandrillaris KM-20 strain ( Data S10 ).…”

Isolation of Acanthamoeba Species and Bacterial Symbiont Variability in Puna Salt Plains, Argentina

“… 2021 ; Rayamajhee, Willcox, Sharma et al. 2024 ). Despite the shared taxa, it should be noted that the amoebic isolation procedure has the potential to influence the dynamics of the bacterial microbiome, and more research is required to develop a more comprehensive approach to detecting hidden microbiomes.…”

First report on the infection of Acanthamoeba and Entamoeba in the endangered Chinese Milu deer (Elaphurus davidianus) in China

“…It is noteworthy that recent studies have demonstrated that certain microorganisms can parasitize Acanthamoeba [ 59 , 60 ], consistent with previous research describing Acanthamoeba as a “Trojan horse.” However, whether Acanthamoeba can act as a “Trojan horse” to facilitate the invasion of pathogenic microorganisms into the moose remains an unanswered question. Although this study did not explore this phenomenon in greater depth, we believe that this represents a compelling and highly valuable avenue for future research.…”

Drug susceptibility of a clinical isolate of Balamuthia mandrillaris , a pathogenic free-living amoeba

“…There is growing evidence that the environmental and clinical isolates of the keratitis-causing Acanthamoeba spp. also harbor endosymbiotic bacteria ( 38 , 39 ). Using the same primer sequences specific to bacteria 16s rRNA, the PCR and DNA sequencing showed amplification of the 16s rRNA gene, which is located in the mitochondrial genome of the B. mandrillaris KM-20 strain ( Data S10 ).…”

Isolation of Acanthamoeba Species and Bacterial Symbiont Variability in Puna Salt Plains, Argentina

“… 2021 ; Rayamajhee, Willcox, Sharma et al. 2024 ). Despite the shared taxa, it should be noted that the amoebic isolation procedure has the potential to influence the dynamics of the bacterial microbiome, and more research is required to develop a more comprehensive approach to detecting hidden microbiomes.…”

First report on the infection of Acanthamoeba and Entamoeba in the endangered Chinese Milu deer (Elaphurus davidianus) in China

“…It is noteworthy that recent studies have demonstrated that certain microorganisms can parasitize Acanthamoeba [ 59 , 60 ], consistent with previous research describing Acanthamoeba as a “Trojan horse.” However, whether Acanthamoeba can act as a “Trojan horse” to facilitate the invasion of pathogenic microorganisms into the moose remains an unanswered question. Although this study did not explore this phenomenon in greater depth, we believe that this represents a compelling and highly valuable avenue for future research.…”

Drug susceptibility of a clinical isolate of Balamuthia mandrillaris , a pathogenic free-living amoeba

“…There is growing evidence that the environmental and clinical isolates of the keratitis-causing Acanthamoeba spp. also harbor endosymbiotic bacteria ( 38 , 39 ). Using the same primer sequences specific to bacteria 16s rRNA, the PCR and DNA sequencing showed amplification of the 16s rRNA gene, which is located in the mitochondrial genome of the B. mandrillaris KM-20 strain ( Data S10 ).…”

Isolation of Acanthamoeba Species and Bacterial Symbiont Variability in Puna Salt Plains, Argentina

“… 2021 ; Rayamajhee, Willcox, Sharma et al. 2024 ). Despite the shared taxa, it should be noted that the amoebic isolation procedure has the potential to influence the dynamics of the bacterial microbiome, and more research is required to develop a more comprehensive approach to detecting hidden microbiomes.…”