Comprehensive characterization of extracellular vesicles produced by environmental (Neff) and clinical (T4) strains of Acanthamoeba castellanii.

We conducted a comprehensive comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains, Neff (environmental) and T4 (clinical). Morphological analysis via transmission electron microscopy revealed slightly larger Neff EVs (average = 194.5 nm) compared to more polydisperse T4 EVs (average = 168.4 nm). Nanoparticle tracking analysis (NTA) and dynamic light scattering validated these differences. Proteomic analysis of the EVs identified 1,352 proteins, with 1,107 common, 161 exclusive in Neff, and 84 exclusively in T4 EVs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping revealed distinct molecular functions and biological processes and notably, the T4 EVs enrichment in serine proteases, aligned with its pathogenicity. Lipidomic analysis revealed a prevalence of unsaturated lipid species in Neff EVs, particularly triacylglycerols, phosphatidylethanolamines (PEs), and phosphatidylserine, while T4 EVs were enriched in diacylglycerols and diacylglyceryl trimethylhomoserine, phosphatidylcholine and less unsaturated PEs, suggesting differences in lipid metabolism and membrane permeability. Metabolomic analysis indicated Neff EVs enrichment in glycerolipid metabolism, glycolysis, and nucleotide synthesis, while T4 EVs, methionine metabolism. Furthermore, RNA-seq of EVs revealed differential transcript between the strains, with Neff EVs enriched in transcripts related to gluconeogenesis and translation, suggesting gene regulation and metabolic shift, while in the T4 EVs transcripts were associated with signal transduction and protein kinase activity, indicating rapid responses to environmental changes. In this novel study, data integration highlighted the differences in enzyme profiles, metabolic processes, and potential origins of EVs in the two strains shedding light on the diversity and complexity of A. castellanii EVs and having implications for understanding host-pathogen interactions and developing targeted interventions for Acanthamoeba-related diseases.IMPORTANCEA comprehensive and fully comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains of distinct virulence, a Neff (environmental) and T4 (clinical), revealed striking differences in their morphology and protein, lipid, metabolites, and transcripts levels. Data integration highlighted the differences in enzyme profiles, metabolic processes, and potential distinct origin of EVs from both strains, shedding light on the diversity and complexity of A. castellanii EVs, with direct implications for understanding host-pathogen interactions, disease mechanisms, and developing new therapies for the clinical intervention of Acanthamoeba-related diseases.

Validation and redescription of Acanthamoeba terricola Pussard, 1964 (Amoebozoa: Acanthamoebidae).

Acanthamoeba castellanii (Douglas, 1930) Page, 1967 is the type species of a widespread genus of free-living amoebae, potentially pathogenic for humans and animals. The Neff strain is one of the most widely used in biological research, serving as a model for both A. castellanii and the whole genus in general. The Neff strain, isolated in California, closely resembles another strain found in France and originally described as a separate species, Acanthamoeba terricola Pussard, 1964, but both were successively synonymized with A. castellanii. Molecular sequence analysis has largely replaced morphological diagnosis for species identification in Acanthamoeba, and rDNA phylogenies show that the Neff strain forms a distinct lineage from that of the type strain of A. castellanii. In this study, we compared the type strain of A. terricola with the Neff strain and A. castellanii, and analysed the available molecular data including new sequences obtained from A. terricola. Here we provide molecular evidence to validate the species A. terricola. The Neff strain is therefore transferred to A. terricola and should no longer be considered as belonging to A. castellanii.

Isolation, characterization, and pathogenicity assay of Acanthamoeba and its endosymbionts in respiratory disorders and COVID-19 hospitalized patients, northern Iran.

Acanthamoeba spp., are common free-living amoebae found in nature that can serve as reservoirs for certain microorganisms. The SARS-CoV-2 virus is a newly emerged respiratory infection, and the investigation of parasitic infections remains an area of limited research. Given that Acanthamoeba can act as a host for various endosymbiotic microbial pathogens and its pathogenicity assay is not fully understood, this study aimed to identify Acanthamoeba and its bacterial and fungal endosymbionts in patients with chronic respiratory disorders and hospitalized COVID-19 patients in northern Iran. Additionally, a pathogenicity assay was conducted on Acanthamoeba isolates. Urine, nasopharyngeal swab, and respiratory specimens were collected from two groups, and each sample was cultured on 1.5% non-nutrient agar medium. The cultures were then incubated at room temperature and monitored daily for a period of two weeks. Eight Acanthamoeba isolates were identified, and PCR was performed to confirm the presence of amoebae and identify their endosymbionts. Four isolates were found to have bacterial endosymbionts, including Stenotrophomonas maltophilia and Achromobacter sp., while two isolates harbored fungal endosymbionts, including an uncultured fungus and Gloeotinia sp. In the pathogenicity assay, five isolates exhibited a higher degree of pathogenicity compared to the other three. This study provides significant insights into the comorbidity of acanthamoebiasis and COVID-19 on a global scale, and presents the first evidence of Gloeotinia sp. as a fungal endosymbiont. Nevertheless, further research is required to fully comprehend the symbiotic patterns and establish effective treatment protocols.

Occurrence of Free-Living Amoebae in Non-Human Primate Gut.

The gut microbiome reflects health and predicts possible disease in hosts. A holistic view of this community is needed, focusing on identifying species and dissecting how species interact with their host and each other, regardless of whether their presence is beneficial, inconsequential, or detrimental. The distribution of gut-associated eukaryotes within and across non-human primates is likely driven by host behavior and ecology. To ascertain the existence of free-living amoebae (FLA) in the gut of wild and captive non-human primates, 101 stool samples were collected and submitted to culture-dependent microscopy examination and DNA sequencing. Free-living amoebae were detected in 45.4% (46/101) of fecal samples analyzed, and their morphological characteristics matched those of Acanthamoeba spp., Vermamoeba spp., heterolobosean amoeboflagellates and fan-shaped amoebae of the family Vannellidae. Sequence analysis of the PCR products revealed that the suspected amoebae are highly homologous (99% identity and 100% query coverage) with Acanthamoeba T4 genotype and Vermamoeba vermiformis amoebae. The results showed a great diversity of amoebae in the non-human primate's microbiome, which may pose a potential risk to the health of NHPs. To our knowledge, this is the first report of free-living amoebae in non-human primates that are naturally infected. However, it is unknown whether gut-borne amoebae exploit a viable ecological niche or are simply transient residents in the gut.

Inhibitory and anti-adherent effects of Piper betle L. leaf extract against Acanthamoeba triangularis in co-infection with Staphylococcus aureus and Pseudomonas aeruginosa: A sustainable one-health approach.

Background and Aim: Keratitis is a serious ocular infection often caused by pathogenic microorganisms such as Acanthamoeba spp. Among other harmful microbes, Acanthamoeba keratitis presents a particular challenge due to its resistance to conventional antimicrobial agents. Piper betle Linn., commonly known as betel leaf, has been traditionally used for its medicinal properties. This study aimed to assess the potential of the leaf ethanol extract of P. betle Linn. in the treatment of Acanthamoeba triangularis in monoculture and co-culture with two prevalent pathogenic bacteria, Staphylococcus aureus and Pseudomonas aeruginosa, associated with keratitis. Materials and Methods: Minimum inhibitory concentrations (MICs) of A. triangularis, S. aureus, and P. aeruginosa extracts in monoculture and coinfected conditions were examined. In addition, this study explored the potential of the extract in preventing Acanthamoeba adherence in both monoculture and co-culture environments. Scanning electron microscopy (SEM) analysis confirmed the impact of the extract on Acanthamoeba cell membranes, including acanthopodia. Furthermore, a time-kill kinetic assay was used to validate the amoebicidal activity of the extract against A. triangularis and the tested bacteria. Results: MICs for trophozoites, cysts, P. aeruginosa, and S. aureus in the monoculture were 0.25, 0.25, 0.51, and 0.128 mg/mL, respectively, whereas the MICs for Acanthamoeba coinfected with bacteria were higher than those in the monoculture. This extract inhibited the growth of A. triangularis trophozoites and cysts for up to 72 h. Moreover, P. betle extract effectively prevented the adherence of Acanthamoeba to contact lenses under monoculture conditions. SEM analysis confirmed that P. betle extract affects the cell membrane of Acanthamoeba, including Acanthopodia. In addition, the time-kill kinetic assay confirmed that the extract contained amoebicidal activity against A. triangularis, including the tested bacteria. Notably, S. aureus was more susceptible than A. triangularis and P. aeruginosa to P. betle extract treatment. Unexpectedly, our study revealed that S. aureus negatively affected A. triangularis in the co-culture after 3 days of incubation, whereas P. aeruginosa facilitated the growth of A. triangularis in the presence of the extract. Conclusion: This study provides compelling evidence of the anti-adhesive and anti-Acanthamoeba properties of P. betle leaf extract against A. triangularis under monoculture and co-culture conditions. The observed impact on Acanthamoeba cell membranes, coupled with the time-kill kinetic assay results, underscores the potential of P. betle leaf extract as a promising agent for combating Acanthamoeba-related infections in humans and animals.

Development of ophthalmic nanoemulsions of ß-caryophyllene for the treatment of Acanthamoeba keratitis.

Although rare, amoebic keratitis (AK) is a disease caused by Acanthamoeba spp. that can lead to blindness. The drugs currently available for its treatment are very toxic, which has motivated the investigation for more effective and safe therapeutic options. In this study, the in vitro activity of ß-caryophyllene (BCP) was exploited taking into account its action against other protozoans as well as its well-known healing and anti-inflammatory properties (aspects relevant for the AK pathogenesis). On the other hand, high volatilization and oxidation phenomena are found for this compound, which led to its incorporation into nanoemulsions (NEs). Two emulsifying agents were tested, resulting in monodisperse systems with reduced droplet size (<265 nm) and high surface charge (positive and negative for NEs prepared with cetrimonium bromide -CTAB and Phosal® 50+, respectively). NEs prepared with CTAB were shown to be more stable after long-term storage at 4 and 25 °C than those prepared with Phosal®. Pure BCP, at the highest concentration (500 µM), resulted in a level of inhibition of Acanthamoeba trophozoites equivalent to that of reference drug (chlorhexidine). This activity was even greater after oil nanoencapsulation. The reduced droplet size could improve the interaction of the oil with the microorganism, justifying this finding. Changes in surface charge did not impact the activity. Positively charged NEs improved the interaction and retention of BCP in the cornea and thus should be prioritized for further studies.

Nitric oxide signaling through three receptors regulates virulence, biofilm formation, and phenotypic heterogeneity of Legionella pneumophila.

The causative agent of Legionnaires' disease, Legionella pneumophila, is an environmental bacterium, that replicates in macrophages, parasitizes amoeba, and forms biofilms. L. pneumophila employs the Legionella quorum sensing (Lqs) system and the transcription factor LvbR to control various bacterial traits, including virulence and biofilm architecture. LvbR negatively regulates the nitric oxide (NO) receptor Hnox1, linking quorum sensing to NO signaling. Here, we assessed the response of L. pneumophila to NO and investigated bacterial receptors underlying this process. Chemical NO donors, such as dipropylenetriamine (DPTA) NONOate and sodium nitroprusside (SNP), delayed and reduced the expression of the promoters for flagellin (PflaA) and the 6S small regulatory RNA (P6SRNA). Marker-less L. pneumophila mutant strains lacking individual (Hnox1, Hnox2, or NosP) or all three NO receptors (triple knockout, TKO) grew like the parental strain in media. However, in the TKO strain, the reduction of PflaA expression by DPTA NONOate was less pronounced, suggesting that the NO receptors are implicated in NO signaling. In the ΔnosP mutant, the lvbR promoter was upregulated, indicating that NosP negatively regulates LvbR. The single and triple NO receptor mutant strains were impaired for growth in phagocytes, and phenotypic heterogeneity of non-growing/growing bacteria in amoebae was regulated by the NO receptors. The single NO receptor and TKO mutant strains showed altered biofilm architecture and lack of response of biofilms to NO. In summary, we provide evidence that L. pneumophila regulates virulence, intracellular phenotypic heterogeneity, and biofilm formation through NO and three functionally non-redundant NO receptors, Hnox1, Hnox2, and NosP. IMPORTANCE: The highly reactive diatomic gas molecule nitric oxide (NO) is produced by eukaryotes and bacteria to promote short-range and transient signaling within and between neighboring cells. Despite its importance as an inter-kingdom and intra-bacterial signaling molecule, the bacterial response and the underlying components of the signaling pathways are poorly characterized. The environmental bacterium Legionella pneumophila forms biofilms and replicates in protozoan and mammalian phagocytes. L. pneumophila harbors three putative NO receptors, one of which crosstalks with the Legionella quorum sensing (Lqs)-LvbR network to regulate various bacterial traits, including virulence and biofilm architecture. In this study, we used pharmacological, genetic, and cell biological approaches to assess the response of L. pneumophila to NO and to demonstrate that the putative NO receptors are implicated in NO detection, bacterial replication in phagocytes, intracellular phenotypic heterogeneity, and biofilm formation.

The ecto-3'-nucleotidase activity of Acanthamoeba castellanii trophozoites increases their adhesion to host cells through the generation of extracellular adenosine.

Acanthamoeba castellanii, a free-living amoeba, can be pathogenic to humans causing a corneal infection named Acanthamoeba keratitis (AK). The mannose-binding protein (MBP) is well established as the major factor related to Acanthamoeba pathogenesis. However, additional factors that participate in the adhesion process and protect trophozoites from cytolytic effects caused by host immune responses remain unknown. Ectonucleotidases, including 3'-nucleotidase/nuclease (3'-NT/NU), a bifunctional enzyme that was recently reported in A. castellanii, are frequently related to the establishment of parasitic infections. We verified that trophozoites can hydrolyze 3'-AMP, and this activity is similar to that observed in other protists. The addition of 3'-AMP increases the adhesion of trophozoites to LLC-MK2 epithelial cells, and this stimulation is completely reversed by DTT, an inhibitor of ecto-3'-nucleotidase activity. Lesions in corneal cells caused by AK infection may elevate the extracellular level of 3'-AMP. We believe that ecto-3'-nucleotidase activity can modulate the host immune response, thus facilitating the establishment of parasitic infection. This activity results from the generation of extracellular adenosine, which can bind to purinergic receptors present in host immune cells. Positive feedback may occur in this cascade of events once the ecto-3'-nucleotidase activity of trophozoites is increased by the adhesion of trophozoites to LLC-MK2 cells.

Understanding Acanthamoeba Keratitis: An In-Depth Review of a Sight-Threatening Eye Infection.

Acanthamoeba keratitis (AK) is a rare but potentially sight-threatening corneal infection caused by the Acanthamoeba parasite. This microorganism is found ubiquitously in the environment, often in freshwater, soil, and other sources of moisture. Despite its low incidence, AK presents significant challenges due to delayed diagnosis and the complex nature of therapeutic management. Early recognition is crucial to prevent severe ocular complications, including corneal ulceration and vision loss. Diagnostic modalities and treatment strategies may vary greatly depending on the clinical manifestation and the available tools. With the growing reported cases of Acanthamoeba keratitis, it is essential for the ophthalmic community to thoroughly understand this condition for its effective management and improved outcomes. This review provides a comprehensive overview of AK, encompassing its epidemiology, risk factors, pathophysiology, clinical manifestations, diagnosis, and treatment.

Amoebicidal effect of chlorine dioxide gas against pathogenic Naegleria fowleri and Acanthamoeba polyphaga.

The pathogenic free-living amoebae, Naegleria fowleri and Acanthamoeba polyphaga, are found in freshwater, soil, and unchlorinated or minimally chlorinated swimming pools. N. fowleri and A. polyphaga are becoming problematic as water leisure activities and drinking water are sources of infection. Chlorine dioxide (ClO2) gas is a potent disinfectant that is relatively harmless to humans at the concentration used for disinfection. In this study, we examined the amoebicidal effects of ClO2 gas on N. fowleri and A. polyphaga. These amoebae were exposed to ClO2 gas from a ready-to-use product (0.36 ppmv/h) for 12, 24, 36, and 48 h. Microscopic examination showed that the viability of N. fowleri and A. polyphaga was effectively inhibited by treatment with ClO2 gas in a time-dependent manner. The growth of N. fowleri and A. polyphaga exposed to ClO2 gas for 36 h was completely inhibited. In both cases, the mRNA levels of their respective actin genes were significantly reduced following treatment with ClO2 gas. ClO2 gas has an amoebicidal effect on N. fowleri and A. polyphaga. Therefore, ClO2 gas has been proposed as an effective agent for the prevention and control of pathogenic free-living amoeba contamination.

[First isolation of Acanthamoeba spp. in seawater from the southeast of Buenos Aires (SE, Argentina)]. / Primer aislamiento de Acanthamoeba spp. en agua de mar del sudeste bonaerense, Argentina.

Free-living amoebae (FLA) of the genus Acanthamoeba are ubiquitous and amphizoic protozoa that colonize aquatic and terrestrial habitats and can serve as reservoirs for other microorganisms. They are considered econoses that can cause severe and rare pathologies. Due to limited epidemiological data available, the objective of this study was to investigate the presence of Acanthamoeba in coastal wetlands of the southeast of Buenos Aires province and evaluate their association with bacteriological and environmental variables. From February 2021 to July 2022, 22 seawater samples were collected at different points along the coast of the city of Mar del Plata (Buenos Aires, Argentina). Environmental parameters were determined and physicochemical and bacteriological studies, morphological identification, cultures and molecular typification were conducted. Regardless of the environmental and bacteriological variables, the presence of Acanthamoeba spp. was molecularly confirmed in 54.54% of the samples, being the first report of these protozoa in seawater in Argentina.

Anti-Acanthamoeba metallopharmaceuticals: Amoebicidal activity and synergistic effect of copper(II) coordination compound.

Acanthamoeba spp. emerged as a clinically important pathogen related to amoebic keratitis. It is among the main causes of corneal transplantation and vision loss in ophthalmology. The treatment protocols have a low cure rate, high toxicity, and need for drug combination. Transition metal compounds have shown promising antiprotozoal effects. This study evaluates the amoebicidal activity of copper(II) coordination compounds in combination with chlorhexidine and the cytotoxicity to topical ocular application. These copper(II) coordination compounds were screened against Acanthamoeba castellanii trophozoites (ATCC 50492). The cytotoxicity on rabbit corneal cell line (ATCC-CCL 60) was performed. The compounds showed high amoebicidal potential, with inhibition of trophozoite viability above 80%. The Cp12 and Cp13 compounds showed Minimal Inhibitory Amoebicidal Concentration (MIAC) at 200 µM and mean inhibitory concentration (IC50) values lower than 10 µM. Against the cysts, Cp12 showed a reduction in viability (48%) in the longest incubation period. A synergistic effect for Cp12 with chlorhexidine was observed. The compounds have a dose-dependent effect against rabbit corneal cells. Compound Cp12 has potential for future application in developing ophthalmic formulations against Acanthamoeba keratitis and its use in multipurpose solutions is highlighted.

Influence of salt and temperature in the growth of pathogenic free-living amoebae.

Introduction: Free-living amoebae are an extensive group of protistans that can be found in a wide variety of environments. Among them, the Acanthamoeba genus and Naegleria fowleri stand out as two of the most pathogenic amoebae and with a higher number of reported cases. N. fowleri is mainly found in warm freshwater water bodies whereas amoebae of the Acanthamoeba genus are broadly distributed through natural and anthropogenic environments. In this regard, the management and the control of the amoebic populations in swimming pools has become a major public health challenge for institutions. Methods: The aim of this work was to evaluate the growth pattern of trophozoites of A. griffini and N. fowleri at different temperatures and salt concentrations. Results and discussion: Our results showed that A. griffini resisted a higher concentration of salt than N. fowleri. Moreover, no trophozoites could withstand the salt levels of the sea in in vitro conditions. This work supports the contention that salinity could represent an important and useful tool for the control of the most pathogenic amoebic populations in recreational water bodies.

Comparison of therapeutic effects between big-bubble deep anterior lamellar keratoplasty and penetrating keratoplasty for medically unresponsive Acanthamoeba keratitis.

PURPOSE: To compare the outcomes of big-bubble deep anterior lamellar keratoplasty (BB-DALK) and penetrating keratoplasty (PKP) in the management of medically unresponsive Acanthamoeba keratitis (AK). METHODS: This retrospective study included 27 eyes of BB-DALK and 24 eyes of PKP from a tertiary ophthalmology care centre. Glucocorticoid eye drops were subsequently added to the treatment plan 2 months postoperatively based on the evaluation using confocal laser scanning microscopy. The clinical presentations, best-corrected visual acuity (BCVA), postoperative refractive outcomes, graft survival, and Acanthamoeba recurrence were analyzed. RESULTS: The AK patients included in the study were in stage 2 or stage 3, and the percentage of patients in stage 3 was higher in the PKP group (P = 0.003). Clinical presentations were mainly corneal ulcers and ring infiltrates, and endothelial plaques, hypopyon, uveitis and glaucoma were more common in the PKP group (P = 0.007). The BCVA and the graft survival rate showed no statistically significant differences between the two groups at 1 year after surgery. However, 3 years postoperatively, the BCVA of 0.71 ± 0.64 logMAR, the graft survival rate of 89.5%, and the endothelial cell density of 1899 ± 125 cells per square millimeter in the BB-DALK group were significantly better than those of the PKP group (P = 0.010, 0.046, and 0.032, respectively). 3 eyes (11.1%) in the BB-DALK group and 2 eyes (8.3%) in the PKP group experienced Acanthamoeba recurrence, but the rates showed no statistically significant difference between the two groups (P = 1.000). In the PKP group, immune rejection and elevated intraocular pressure were observed in 5 and 6 eyes, respectively. CONCLUSION: Corneal transplantation is recommended for AK patients unresponsive to antiamoebic agents. The visual acuity and graft survival can be maintained after BB-DALK surgery. Acanthamoeba recurrence is not related to the surgical approach performed, whereas complete dissection of the infected corneal stroma and delayed prescribing of glucocorticoid eye drops were important to prevent recurrence.

Molecular detection and characterization of Acanthamoeba infection in dogs and its association with keratitis in Korea.

Acanthamoeba infection is associated with keratitis in humans; however, its association with keratitis in dogs remains unclear. To investigate this possibility, we collected 171 conjunctival swab samples from dogs with eye-related diseases (65 with keratitis and 106 without keratitis) at Chungbuk National University Veterinary Teaching Hospital, Korea, from August 2021 to September 2022. Polymerase chain reaction identified 9 samples (5.3%) as Acanthamoeba positive; of these, 3 were from dogs with keratitis (4.6%) and 6 were from dogs without keratitis (5.7%). Our results indicated no significant association between Acanthamoeba infection and keratitis, season, sex, or age. All Acanthamoeba organisms found in this study had the genotype T4, according to 18S ribosomal RNA analysis. Acanthamoeba infection in dogs might have only a limited association with keratitis.

Are Thermotolerant and Osmotolerant Characteristics of Acanthamoeba Species an Indicator of Pathogenicity?

Objective: The aim of this study was to evaluate the pathogenicity of Acanthamoeba strains with T4, T5, T11, and T12 genotypes by comparing the osmotolerance and thermotolerance characteristics of Acanthamoeba strains isolated from genotype groups, within species with the same genotype, and from environmental and keratitis cases. Methods: In this study, after axenic cultures of 22 Acanthamoeba strains with T4 (Neff, A, B, D, E), T5, T11, and T12 genotypes isolated from clinical and environmental samples, thermotolerance (37 °C, 39 °C and 41 °C) and osmotolerance (0.5 M, 1 M) tests were performed. Results: All strains showed growth ability at 37 °C and 0.5 M osmolarity. While all five strains isolated from patients with Acanthamoeba keratitis showed growth ability at 37 °C and 0.5 M osmolarity, no growth was detected at 41 °C and 1 M osmolarity. When the tolerance characteristics of the strains with the same genotype were evaluated, the strains with the T5 and T4E genotypes showed the same characteristics. When Acanthamoeba strains with the T4 genotype were evaluated in general, 31.25% of the strains were found to grow at 39 °C and 6.25% at 41 °C. Of the T4Neff strains, only one strain did not show the ability to reproduce at 39 °C and showed a different feature from the other strains. While the strain with the T11 genotype grew at all temperatures, the strain with the T12 genotype did not grow at 41 °C. Conclusion: According to our research results, we believe that tolerance to 39 °C and 1 M mannitol is not an indicator of pathogenicity. More studies with Acanthamoeba strains are required to clarify this issue.

Zooming in on the intracellular microbiome composition of bacterivorous Acanthamoeba isolates.

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.

Acanthamoeba Sequence Types and Allelic Variations in Isolates from Clinical and Different Environmental Sources in Italy.

The genus Acanthamoeba comprises free-living amoebae distributed in a wide variety of environments. These amoebae are clinically significant, causing opportunistic infections in humans and other animals. Despite this, limited data on Acanthamoeba sequence types and alleles are available in Italy. In the present study, we analyzed all Acanthamoeba sequences deposited from Italy with new positive Acanthamoeba clinical samples from symptomatic AK cases, to provide an overview of the genetic variants' spatial patterns from different sources within the Italian context. A total of 137 Acanthamoeba sequences were obtained. Six sequence types were identified: T2/6, T3, T4, T11, T13, and T15. Only T4 and T15 were found in both sources. The Acanthamoeba T4 sequence type was found to be the most prevalent in all regions, accounting for 73% (100/137) of the Italian samples analyzed. The T4 sequence type demonstrated significant allelic diversity, with 30 distinct alleles from clinical and/or environmental samples. These outcomes enabled a better understanding of the distribution of Acanthamoeba isolates throughout Italy, reaffirming its well-recognized ubiquity. Acanthamoeba isolates analysis from keratitis, together with the environmental strains monitoring, might provide important information on different genotypes spreading. This might be useful to define the transmission pathways of human keratitis across different epidemiological scales.

Adaptation to an amoeba host drives selection of virulence-associated traits and genetic variation in saprotrophic Candida albicans.

Amoebae are micropredators that play an important role in controlling fungal populations in ecosystems. However, the interaction between fungi and their amoebic predators suggests that the pressure from predatory selection can significantly influence the development of fungal virulence and evolutionary processes. Thus, the purpose of this study was to investigate the adaptation of saprotrophic Candida albicans strains during their interactions with Acanthamoeba castellanii. We conducted a comprehensive analysis of survival after co-culture by colony counting of the yeast cells and examining yeast cell phenotypic and genetic characteristics. Our results indicated that exposure to amoebae enhanced the survival capacity of environmental C. albicans and induced visible morphological alterations in C. albicans, particularly by an increase in filamentation. These observed phenotypic changes were closely related to concurrent genetic variations. Notably, mutations in genes encoding transcriptional repressors (TUP1 and SSN6), recognized for their negative regulation of filamentous growth, were exclusively identified in amoeba-passaged isolates, and absent in unexposed isolates. Furthermore, these adaptations increased the exposed isolates' fitness against various stressors, simultaneously enhancing virulence factors and demonstrating an increased ability to invade A549 lung human epithelial cells. These observations indicate that the sustained survival of C. albicans under ongoing amoebic predation involved a key role of mutation events in microevolution to modulate the ability of these isolates to change phenotype and increase their virulence factors, demonstrating an enhanced potential to survive in diverse environmental niches.