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Parasites, Hosts and Diseases May 2024Free-living amoebae (FLA) are found in diverse environments, such as soils, rivers, and seas. Hence, they can be used as bioindicators to assess the water quality based...
Free-living amoebae (FLA) are found in diverse environments, such as soils, rivers, and seas. Hence, they can be used as bioindicators to assess the water quality based solely on their presence. In this study, we determined the presence of FLA in river water by filtering water samples collected from various sites and culturing the resulting filtrates. FLA were detected in all the water samples with varying quality grades (Grades Ι-V). The significant increase in the size of the amoebae population with the deterioration in the water quality. Monoxenic cultures of the amoebae were performed, and genomic DNAs were isolated, among which 18S rDNAs were sequenced to identify the amoeba species. Of the 12 species identified, 10 belonged to the Acanthamoeba genus; of the remaining 2 species, one was identified as Vannella croatica and the other as a species of Vermamoeba. Acanthamoeba was detected in samples with Grades Ι to VI quality, whereas the Vermamoeba species was present only in Grade Ι water. V. croatica was found exclusively in water with Grade ΙΙ quality. Following morphological observations, genomic DNA was sequenced using 16S rDNA to determine whether the species of Acanthamoeba harbored endosymbionts. Most of the isolated Acanthamoeba contained endosymbionts, among which 4 species of endogenous bacteria were identified and examined using transmission electron microscopy. This study provides evidence that the distribution of amoebae other than Acanthamoeba may be associated with water quality. However, further confirmation will be required based on accurate water quality ratings and assessments using a more diverse range of FLA.
Topics: Amoeba; Water Quality; Phylogeny; Rivers; DNA, Protozoan; Acanthamoeba; RNA, Ribosomal, 18S; DNA, Ribosomal; Biodiversity; Sequence Analysis, DNA; RNA, Ribosomal, 16S
PubMed: 38835259
DOI: 10.3347/PHD.24020 -
Antimicrobial Agents and Chemotherapy 2015Acanthamoeba is a protist pathogen that can cause serious human infections, including blinding keratitis and a granulomatous amoebic encephalitis that almost always...
Acanthamoeba is a protist pathogen that can cause serious human infections, including blinding keratitis and a granulomatous amoebic encephalitis that almost always results in death. The current treatment for these infections includes a mixture of drugs, and even then, a recurrence can occur. Photochemotherapy has shown promise in the treatment of Acanthamoeba infections; however, the selective targeting of pathogenic Acanthamoeba has remained a major concern. The mannose-binding protein is an important adhesin expressed on the surface membranes of pathogenic Acanthamoeba organisms. To specifically target Acanthamoeba, the overall aim of this study was to synthesize a photosensitizing compound (porphyrin) conjugated with mannose and test its efficacy in vitro. The synthesis of mannose-conjugated porphyrin was achieved by mixing benzaldehyde and pyrrole, yielding tetraphenylporphyrin. Tetraphenylporphyrin was then converted into mono-nitrophenylporphyrin by selectively nitrating the para position of the phenyl rings, as confirmed by nuclear magnetic resonance (NMR) spectroscopy. The mono-nitrophenylporphyrin was reduced to mono-aminophenylporphyrin in the presence of tin dichloride and confirmed by a peak at m/z 629. Finally, mono-aminoporphyrin was conjugated with mannose, resulting in the formation of an imine bond. Mannose-conjugated porphyrin was confirmed through spectroscopic analysis and showed that it absorbed light of wavelengths ranging from 425 to 475 nm. To determine the antiacanthamoebic effects of the derived product, amoebae were incubated with mannose-conjugated porphyrin for 1 h and washed 3 times to remove extracellular compound. Next, the amoebae were exposed to light of the appropriate wavelength for 1 h. The results revealed that mannose-conjugated porphyrin produced potent trophicidal effects and blocked excystation. In contrast, Acanthamoeba castellanii incubated with mannose alone and porphyrin alone did not exhibit an antiamoebic effect. Consistently, pretreatment with mannose-conjugated porphyrin reduced the A. castellanii-mediated host cell cytotoxicity from 97% to 4.9%. In contrast, treatment with porphyrin, mannose, or solvent alone had no protective effects on the host cells. These data suggest that mannose-conjugated porphyrin has application for the targeted photodynamic therapy of Acanthamoeba infections and may serve as a model in the development of therapeutic interventions against other eukaryotic infections.
Topics: Acanthamoeba castellanii; Antiprotozoal Agents; Benzaldehydes; Cell Line; Cells, Cultured; Humans; Magnetic Resonance Spectroscopy; Mannose; Porphyrins; Pyrroles
PubMed: 25753633
DOI: 10.1128/AAC.05126-14 -
Nature Reviews. Microbiology Apr 2017The accidental discovery of the giant virus of amoeba - Acanthamoeba polyphaga mimivirus (APMV; more commonly known as mimivirus) - in 2003 changed the field of... (Review)
Review
The accidental discovery of the giant virus of amoeba - Acanthamoeba polyphaga mimivirus (APMV; more commonly known as mimivirus) - in 2003 changed the field of virology. Viruses were previously defined by their submicroscopic size, which probably prevented the search for giant viruses, which are visible by light microscopy. Extended studies of giant viruses of amoebae revealed that they have genetic, proteomic and structural complexities that were not thought to exist among viruses and that are comparable to those of bacteria, archaea and small eukaryotes. The giant virus particles contain mRNA and more than 100 proteins, they have gene repertoires that are broader than those of other viruses and, notably, some encode translation components. The infection cycles of giant viruses of amoebae involve virus entry by amoebal phagocytosis and replication in viral factories. In addition, mimiviruses are infected by virophages, defend against them through the mimivirus virophage resistance element (MIMIVIRE) system and have a unique mobilome. Overall, giant viruses of amoebae, including mimiviruses, marseilleviruses, pandoraviruses, pithoviruses, faustoviruses and molliviruses, challenge the definition and classification of viruses, and have increasingly been detected in humans.
Topics: Acanthamoeba; Amoeba; Genome, Viral; Giant Viruses; Mimiviridae; Virophages; Virus Internalization
PubMed: 28239153
DOI: 10.1038/nrmicro.2016.197 -
International Journal of Molecular... Nov 2022keratitis is an aggressive and rapidly progressing ocular pathology whose main risk factor is the use of contact lenses. An early and differential diagnosis is...
keratitis is an aggressive and rapidly progressing ocular pathology whose main risk factor is the use of contact lenses. An early and differential diagnosis is considered the main factor to prevent the progression and improve the prognosis of the pathology. However, current diagnosis techniques require time, complex and costly materials making an early diagnosis challenging. Thus, there is a need for fast, accessible, and accurate methods for detection by practitioners for timely and suitable treatment and even for contact lens user as preventive diagnosis. Here, we developed a dual-mode colorimetric-based method for fast, visual, and accurate detection of using gold nanoparticles (AuNPs). For this strategy, AuNPs were functionalized with thiolated probes and the presence of target genomic sequences, produce a colorimetric change from red to purple. This approach allows the detection of 0.02 and 0.009 μM of the unamplified genome by the naked eye in less than 20 min and by color analysis using a smartphone. Additionally, real samples were successfully analyzed showing the potential of the technology considering the lack of point-of-care tools that are mostly needed.
Topics: Humans; Acanthamoeba; Gold; Metal Nanoparticles; Acanthamoeba Keratitis; Early Diagnosis
PubMed: 36499204
DOI: 10.3390/ijms232314877 -
Microbiology Spectrum Jun 2022Traditional cysticidal assays for species revolve around treating cysts with compounds and manually observing the culture for evidence of excystation. This method is...
Traditional cysticidal assays for species revolve around treating cysts with compounds and manually observing the culture for evidence of excystation. This method is time-consuming, labor-intensive, and low throughput. We adapted and trained a YOLOv3 machine learning, object detection neural network to recognize Acanthamoeba castellanii trophozoites and cysts in microscopy images to develop an automated cysticidal assay. This trained neural network was used to count trophozoites in wells treated with compounds of interest to determine if a compound treatment was cysticidal. We validated this new assay with known cysticidal and noncysticidal compounds. In addition, we undertook a large-scale bioluminescence-based screen of 9,286 structurally unique marine microbial metabolite fractions against the trophozoites of A. castellanii and identified 29 trophocidal hits. These hits were then subjected to this machine learning-based automated cysticidal assay. One marine microbial metabolite fraction was identified as both trophocidal and cysticidal. The free-living can exist as a trophozoite or cyst and both stages can cause painful blinding keratitis. Infection recurrence occurs in approximately 10% of cases due to the lack of efficient drugs that can kill both trophozoites and cysts. Therefore, the discovery of therapeutics that are effective against both stages is a critical unmet need to avert blindness. Current efforts to identify new anti- compounds rely primarily upon assays that target the trophozoite stage of the parasite. We adapted and trained a machine learning, object detection neural network to recognize trophozoites and cysts in microscopy images. Our machine learning-based cysticidal assay improved throughput, demonstrated high specificity, and had an exquisite ability to identify noncysticidal compounds. We combined this cysticidal assay with our bioluminescence-based trophocidal assay to screen about 9,000 structurally unique marine microbial metabolites against A. castellanii. Our screen identified a marine metabolite that was both trophocidal and cysticidal.
Topics: Acanthamoeba Keratitis; Acanthamoeba castellanii; Amebicides; Animals; Machine Learning; Trophozoites
PubMed: 35467370
DOI: 10.1128/spectrum.00077-22 -
Journal of Clinical Microbiology Jan 2021is a free-living amoeba of extensive genetic diversity. It may cause infectious keratitis (IK), which can also be caused by bacteria, fungi, and viruses. High...
Detection and Identification of and Other Nonviral Causes of Infectious Keratitis in Corneal Scrapings by Real-Time PCR and Next-Generation Sequencing-Based 16S-18S Gene Analysis.
is a free-living amoeba of extensive genetic diversity. It may cause infectious keratitis (IK), which can also be caused by bacteria, fungi, and viruses. High diagnostic sensitivity is essential to establish an early diagnosis of -associated keratitis. Here, we investigated the applicability of next-generation sequencing (NGS)-based ribosomal gene detection and differentiation (16S-18S) compared with specific real-time PCR for the detection of Two hundred DNAs extracted from corneal scrapings and screened by -specific real-time PCR were analyzed using an in-house 16S-18S NGS assay. Of these, 24 were positive by specific real-time PCR, of which 21 were positive by the NGS assay. Compared with real-time PCR; the specificity and sensitivity of the NGS assay were 100% and 88%, respectively. Genotypes identified by the NGS assay included T4 ( = 19) and T6 ( = 2). Fungal and bacterial species of potential clinical relevance were identified in 31 of the samples negative for , exemplified by ( = 11), spp. ( = 6), ( = 2), spp. ( = 4), and ( = 1). In conclusion, the 16S-18S assay was slightly less sensitive than real-time PCR in detecting -specific DNA in corneal scrapings. Robust information on genotypes was provided by the NGS assay, and other pathogens of potential clinical relevance were identified in 16% of the samples negative for NGS-based detection of ribosomal genes in corneal scrapings could be an efficient screening method for detecting nonviral causes of IK, including .
Topics: Acanthamoeba; Acanthamoeba Keratitis; High-Throughput Nucleotide Sequencing; Humans; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 33239372
DOI: 10.1128/JCM.02224-20 -
Parasites, Hosts and Diseases Nov 2023Acanthamoeba species are free-living amoebae those are widely distributed in the environment. They feed on various microorganisms, including bacteria, fungi, and algae....
Acanthamoeba species are free-living amoebae those are widely distributed in the environment. They feed on various microorganisms, including bacteria, fungi, and algae. Although majority of the microbes phagocytosed by Acanthamoeba spp. are digested, some pathogenic bacteria thrive within them. Here, we identified the roles of 3 phagocytosis-associated genes (ACA1_077100, ACA1_175060, and AFD36229.1) in A. castellanii. These 3 genes were upregulated after the ingestion of Escherichia coli. However, after the ingestion of Legionella pneumophila, the expression of these 3 genes was not altered after the consumption of L. pneumophila. Furthermore, A. castellanii transfected with small interfering RNS (siRNA) targeting the 3 phagocytosis-associated genes failed to digest phagocytized E. coli. Silencing of ACA1_077100 disabled phagosome formation in the E. coli-ingesting A. castellanii. Alternatively, silencing of ACA1_175060 enabled phagosome formation; however, phagolysosome formation was inhibited. Moreover, suppression of AFD36229.1 expression prevented E. coli digestion and consequently led to the rupturing of A. castellanii. Our results demonstrated that the ACA1_077100, ACA1_175060, and AFD36229.1 genes of Acanthamoeba played crucial roles not only in the formation of phagosome and phagolysosome but also in the digestion of E. coli.
Topics: Acanthamoeba castellanii; Escherichia coli; Phagocytosis; Legionella pneumophila; Phagosomes
PubMed: 38043535
DOI: 10.3347/PHD.23088 -
PLoS Neglected Tropical Diseases Aug 2023Acanthamoeba and Klebsiella pneumoniae are both environmental commensals. Recently, clinical harm caused by hypermucoviscous K. pneumoniae has been observed. However,...
BACKGROUND
Acanthamoeba and Klebsiella pneumoniae are both environmental commensals. Recently, clinical harm caused by hypermucoviscous K. pneumoniae has been observed. However, the interaction between these microbes and the origin of hypermucoviscous K. pneumoniae have not been reported.
METHODOLOGY/PRINCIPAL FINDINGS
Here, we report that the bacterial capsule is enlarged when co-cultured with Acanthamoeba using India ink staining, and this effect depends on the number of parasites present. This interaction results in an enhancement of capsular polysaccharide production in the subsequent generations of K. pneumoniae, even without co-culturing with Acanthamoeba. The hypermucoviscosity of the capsule was examined using the sedimentation assay and string test. We also screened other K. pneumoniae serotypes, including K1, K2, K5, and K20, for interaction with Acanthamoeba using India ink staining, and found the same interaction effect.
CONCLUSIONS/SIGNIFICANCE
These findings suggest that the interaction between Acanthamoeba and K. pneumoniae could lead to harmful consequences in public health and nosocomial disease control, particularly hypermucoviscous K. pneumoniae infections.
Topics: Humans; Klebsiella pneumoniae; Acanthamoeba; Klebsiella Infections; Serogroup
PubMed: 37566587
DOI: 10.1371/journal.pntd.0011541 -
Microbiological Research Dec 2016Free-living amoebas (FLA) are ubiquitous environmental protists that have enormously contributed to the microbiological contamination of water sources. FLAs have... (Review)
Review
Free-living amoebas (FLA) are ubiquitous environmental protists that have enormously contributed to the microbiological contamination of water sources. FLAs have displayed resistance to environmental adversities and germicides and have played important roles in the population control of microbial communities due to its predatory behavior and microbicidal activity. However, some organisms have developed resistance to the intracellular milieu of amoebas, as in the case of Acanthamoebas, which in turn, have been functioning as excellent reservoirs for amoeba-resistant microorganisms (ARMs), such as bacteria, viruses and fungi. Little is known about these relationships and interaction mechanisms, but it is speculated that the FLAs need a very broad repertoire or universal class of receptors to bind and recognize these diverse species of microorganisms. By harboring these organisms as a "Trojan Horse", the Achantamoeba has been working as an excellent vector for pathogens. Moreover, studies have demonstrated that the interaction of pathogens with Acanthamoeba results in environmental selective pressure responsible for induction and maintenance of virulence factors and increase in microbial pathogenicity. This phenomenon is correlated to the observation of higher gene number and DNA content of ARMs, when compared to their relatives which are adapted to other hosts, due to allopatric or sympatric gene transfer and acquisition, contradicting the overall genome reduction theory for intracellularly adapted pathogens. Thus, adaptation to FLAs indirectly provided a "learning" environment for pathogens to resist later to macrophages; besides the evolutionary distance, these phagocytes share similar predatory mechanisms, such as phagocytosis and phagolysossomal degradation. In this mini-review, we cover the most important aspects of Acanthamoeba biology and their interactions with endemically important human pathogens.
Topics: Acanthamoeba; Animals; Bacteria; Environmental Microbiology; Fungi; Humans; Microbial Interactions; Virulence; Viruses
PubMed: 27825484
DOI: 10.1016/j.micres.2016.08.001 -
Iranian Journal of Parasitology 2020species are vision-threatening agents by causing cornea infections known as keratitis. A 5 year-old kid with the complaints of erythema, eyelid edema, inflammation,...
species are vision-threatening agents by causing cornea infections known as keratitis. A 5 year-old kid with the complaints of erythema, eyelid edema, inflammation, limitation of eye movements in the right eye, and having no history of wearing contact lenses or trauma, was diagnosed of conjunctivitis through laboratory examinations in the Ophthalmology clinic. The visual sharpness of the patient improved after the treatment. A 44 year-old female patient suffering from pain, stinging, irritation, and inability to see in the left eye with the history of wearing contact lenses or trauma was diagnosed of keratitis through laboratory examinations. The agent was isolated and identified as "" in the Genotype "T2". Examination of the left eye on the 15th day of treatment indicated that all complaints disappeared except for the cataract originated visual loss. However, the first diagnosis of keratitis appeared in the literature on a case with no history of wearing contact lenses and trauma it is found to be attention grabbing. We think that should not be ignored among microbial agents that cause eye infection with or without trauma and contact lens usage history.
PubMed: 32595719
DOI: No ID Found