Search Results (229)

Soft X-ray microscopy (SXM) is a powerful technique for high-resolution biomedical imaging, enabling the observation of bio–nano interactions in near-native conditions without the need for heavy metal staining and fluorescence labeling. A laboratory soft X-ray microscope (LSXM) was developed to bridge the resolution gap between light microscopy and electron microscopy in cellular imaging. However, LSXMs employ a lower-brightness X-ray source in comparison to those operated in synchrotron facilities, which can negatively affect the contrast of X-ray micrographs. Therefore, proper sample preparation is essential to achieve optimal imaging results. This paper details an LSXM sample preparation protocol for investigating cellular nanoparticle uptake. Samples are prepared using optimized parameters for both manual plunge-freezing and automated vitrification, ensuring the rapid transition of biological material into a solid state with controllable thickness in the 5–10 $\mathsf{\mu }$m range, preserving cellular structures and enabling optimal X-ray transmission for cellular imaging. We demonstrate the effectiveness of this protocol in facilitating the observation of nanoparticle uptake in two different biological samples: murine macrophages and acanthamoeba. Controlling ice thickness improves X-ray transmission through the specimen, enhancing the contrast and image quality of SXM. Full article

It is little known that Acanthamoeba trophozoites and Toxocara canis eggs can reduce tumors in vitro and animal models. Although this has been known for many years, the mechanism that induces the antitumor effect in these parasites is still not known. We employed Western blot (WB) and immunofluorescence (IFC) by confocal microscopy to explore the potential protein binding between neuroblastoma (NB) SH-SY5Y cells and anti-Acanthamoeba and anti-Toxocara canis antibodies. Using WB, we detected two fragments of 70 kDa and 60 kDa recognized by the anti-Acanthamoeba antibodies, and two fragments of 115 kDa and 70 kDa recognized by the anti-Toxocara canis antibodies. In both cases, the IFC results were positive in the cell membrane of the SH-SY5Y cells. Our findings suggest a potential overlap of similar molecules between these parasites and tumor cells, which may contribute to tumor elimination. Investigating the relationship between anti-Acanthamoeba and anti-Toxocara canis antibodies in neoplastic cells could provide evidence for the future use of these anti-parasitic antibodies in targeting NB or other cancers. Full article

Background: Diseases in humans caused by amphizoic amoebae that can result in visual impairment and even blindness, have recently been identified more frequently worldwide. Etiologically complex incidents of keratitis, including those connected with Acanthamoeba strains detected in Poland, were evaluated in this study. Methods: Corneal samples from cases resistant to antimicrobial therapy assessed for epidemiological, microbiological and parasitological aspects were investigated by phase-contrast microscope, slit lamp and by confocal microscopy. In vitro techniques were applied for detection of bacteria and fungi, and corneal isolates cultured under axenic condition using BSC medium—for detection of Acanthamoeba spp.; molecular techniques were applied for amoeba species identification. Results: Most etiologically complicated keratitis cases, detected in ~84% of incidents, was due to exposure of contact lenses to tap water or pool water; trophozoites and cysts of Acanthamoeba, concomitant bacteriae, e.g., Pseudomonas aeruginosa, fungi and microfilariae were identified in contact lens users. Conclusions: In samples from contact lens wearers where microbial keratitis is identified along with some connection with the patient’s exposure to contaminated water environments, a risk of Acanthamoeba spp. infections should be considered. Understanding the complicated relationship between Acanthamoeba spp., co-occurring pathogens including associated endosymbionts is needed. In vivo confocal microscopy and in vitro cultivation were necessary to identify potentially contagious concomitant factors affecting the complex course of the keratitis. Full article

This study aims to investigate three endemic ethanolic leaf extracts from Cyprus for anti-Acanthamoeba activities: Odontites linkii subsp. cyprius (Boiss.) Bolliger, Ptilostemon chamaepeuce subsp. cyprius (Greuter) Chrtek & B. Slavík, and Quercus alnifolia Poech. Screening for radical scavenging activity, total phenolic content (TPC), and total flavonoid content (TFC) were performed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABST) methods, Folin–Ciocalteu method, and aluminum chloride method, respectively. An antibacterial-susceptibility test (AST) was performed using a broth microdilution assay to estimate the minimum inhibitory concentration (MIC) using iodonitrotetrazolium chloride (INT). Trypan blue (0.5%) was used to assess in vitro anti-Acanthamoeba cell viability of the ethanolic leaf extracts after 24-, 48-, and 72-h exposure—screening of ethanolic leaf extracts with liquid chromatography–mass spectrometry (LC-MS) for known compounds with biological activity. The ethanolic leaf extract of Odontites linkii subsp. cyprius demonstrated the highest anti-Acanthamoeba activity, with an inhibitory concentration (IC₅₀) of 7.02 mg/mL after 72 h. This extract also showed an in vitro minimum inhibitory concentration (MIC) of 0.625 mg/mL against Enterococcus faecalis, a common nosocomial pathogen. The LC-MS analysis revealed the presence of bioactive iridoid compounds in O. linkii subsp. cyprius, further highlighting its potential as a source for new drug compounds. The ethanolic extract of O. linkii subsp. cyprius demonstrated a dose-dependent and time-dependent anti-Acanthamoeba effect in vitro. This study is the first to report the presence of iridoid compounds and anti-Acanthamoeba activities in the ethanolic extract of O. linkii subsp. cyprius. These promising findings highlight the potential of plant extracts, particularly O. linkii subsp. cyprius, as a source for new drug compounds for Acanthamoeba infections. Full article

(1) Background: Microbial keratitis is a serious eye infection that carries a significant risk of vision loss. Acanthamoeba spp. are known to cause keratitis and their bacterial endosymbionts can increase virulence and/or treatment resistance and thus significantly worsen the course of the disease. (2) Methods and Results: In a suspected case of Acanthamoeba keratitis, in addition to Acanthamoeba spp., an endosymbiont of acanthamoebae belonging to the taxonomic order of Holosporales was detected by chance in a bacterial 16S rDNA-based pan-PCR and subsequently classified as Candidatus Paracaedibacter symbiosus through an analysis of an enlarged 16S rDNA region. We used Oxford Nanopore Technology to evaluate the usefulness of whole-genome sequencing (WGS) as a one-step diagnostics method. Here, Acanthamoeba castellanii and the endosymbiont Candidatus Paracaedibacter symbiosus could be directly detected at the species level. No other microbes were identified in the specimen. (3) Conclusions: We recommend the introduction of WGS as a diagnostic approach for keratitis to replace the need for multiple species-specific qPCRs in future routine diagnostics and to enable an all-encompassing characterisation of the polymicrobial community in one step. Full article

Introduction: Metagenomic research has allowed the identification of numerous viruses present in the human body. Viruses may significantly increase the likelihood of developing intrauterine fetal growth restriction (FGR). The goal of this study was to examine and compare the virome of normal and FGR placentas using proteomic techniques. Methods: The study group of 18 women with late FGR was compared with 18 control patients with physiological pregnancy and eutrophic fetus. Proteins from the collected afterbirth placentas were isolated and examined using liquid chromatography linked to a mass spectrometer. Results: In this study, a group of 107 viral proteins were detected compared to 346 in the controls. In total, 41 proteins were common in both groups. In total, 64 proteins occurred only in the study group and indicated the presence of bacterial phages: E. coli, Bacillus, Mediterranenean, Edwardsiella, Propionibacterium, Salmonella, Paenibaciilus and amoebae Mimiviridae, Acanthamoeba polyphaga, Mimivivirus, Pandoravirdae, Miroviridae, Pepper plant virus golden mosaic virus, pol proteins of HIV-1 virus, and proteins of Pandoravirdae, Microviridae, and heat shock proteins of the virus Faustoviridae. Out of 297 proteins found only in the control group, only 2 viral proteins occurred statistically significantly more frequently: 1/hypothetical protein [uncultured Mediterranean phage uvMED] and VP4 [Gokushovirus WZ-2015a]. Discussion: The detection of certain viral proteins exclusively in the control group suggests that they may play a protective role. Likewise, the proteins identified only in the study group could indicate a potentially pathogenic function. A virome study may be used to identify an early infection, evaluate its progress, and possible association with fetal growth restriction. Utilizing this technology, an individualized patient therapy is forthcoming, e.g., vaccines. Full article

The molecular classification of Acanthamoeba is currently based on the analysis of 18S rDNA sequences, delimiting around twenty genotypes (T1–T23). In some cases, however, the resolution of 18S is limited, and other genetic markers could be useful for unravelling poorly resolved lineages. In this study, the partial large subunit (LSU) of rDNA and ITS were used to re-examine the Acanthamoeba palestinensis group (T2/T6 lineage), which consists of various poorly defined lineages, including the T2 and T6 genotypes. New sequences overlapping 18S, ITS, and LSU were recovered. The analysis placed previously identified partial ITS-LSU sequences as T2/T6 and further confirmed the separation of the OX1 lineage from T2. In addition, analysis of the second internal transcribed spacer (ITS-2) suggests that multiple species may be present within the T6 and OX1 lineages. The results obtained from the T2/T6 lineage analysis confirm the utility of partial LSU and ITS for the study of Acanthamoeba, suggesting their advantage for disentangling complex lineages. Full article

Free-living amoebae (FLA) are prevalent in diverse environments, representing various genera and species with different pathogenicity. FLA-induced infections, such as the highly fatal amoebic encephalitis, with a mortality rate of 99%, primarily affect immunocompromised individuals while others such as Acanthamoeba keratitis (AK) and cutaneous amebiasis may affect immunocompetent individuals. Despite the prevalence of FLA, there is a lack of standardized guidelines for their detection near human habitats. To date, no studies on the isolation and identification of FLA in environmental soil samples in Warsaw have been published. The aim of this study was to determine the presence of amoebae in soil samples collected from Warsaw parks and squares frequented by humans. The isolated protozoa were genotyped. Additionally, their pathogenic potential was determined through thermophilicity tests. A total of 23 soil samples were seeded on non-nutrient agar plates (NNA) at 26 °C and monitored daily for FLA presence. From the total of 23 samples, 18 were positive for FLA growth in NNA and PCR (78.2%). Acanthamoeba spp. was the most frequently isolated genus, with a total of 13 positive samples (13/18; 72.2%), and the T4 genotype being the most common. Moreover, Platyamoeba placida (3/18; 16.7%), Stenamoeba berchidia (1/18; 5.6%) and Allovahlkampfia sp. (1/18; 5.6%), also potentially pathogenic amoebae, were isolated. To our knowledge, this is the first report of FLA presence and characterization in the Warsaw area. Full article

Francisella is a highly infectious gram-negative bacterium that causes tularemia in humans and animals. It can survive and multiply in a variety of cells, including macrophages, dendritic cells, amoebae, and arthropod-derived cells. However, the intracellular life cycle of a bacterium varies depending on the cell type. Shortly after the infection of mammalian cells, the bacterium escapes the phagosome into the cytosol, where it replicates. In contrast, in the amoebae Acanthamoeba castellanii and Hartmannella vermiformis, the bacterium replicates within the membrane-bound vacuole. In recent years, the amoeba Dictyostelium discoideum has emerged as a powerful model to study the intracellular cycle and virulence of many pathogenic bacteria. In this study, we used D. discoideum as a model for the infection and isolation of Francisella novicida-containing vacuoles (FCVs) formed after bacteria invade the amoeba. Our results showed that F. novicida localized in a vacuole after invading D. discoideum. Here, we developed a method to isolate FCV and determined its composition by proteomic analyses. Proteomic analyses revealed 689 proteins, including 13 small GTPases of the Rab family. This is the first evidence of F. novicida-containing vacuoles within amoeba, and this approach will contribute to our understanding of host–pathogen interactions and the process of pathogen vacuole formation, as vacuoles containing bacteria represent direct contact between pathogens and their hosts. Furthermore, this method can be translocated on other amoeba models. Full article

Access to safe water and sanitation is a critical global challenge, posing significant health risks worldwide due to waterborne diseases. This study investigates the efficacy of ultraviolet type C radiation as a disinfection method for improving water quality. The research elucidates UV-C’s mechanism of action, highlighting its ability to disrupt DNA and RNA replication, thereby inactivating pathogens. Furthermore, the study analyses the influence of key factors on UV-C disinfection effectiveness, including water turbidity and the presence of dissolved organic matter, which can attenuate UV-C penetration and reduce treatment efficiency. The experimental results demonstrate a substantial reduction in microbial content following UV-C treatment. River water samples exhibited a 57.143% reduction in microbial load, while well water samples showed a 50% reduction. Notably, Escherichia coli (E. coli) concentrations decreased significantly, with an 83.33% reduction in well water and a 62.5% reduction in borehole water. This study makes a novel contribution to the understanding of UV-C disinfection by identifying the presence of resistant organisms, including Adenoviruses, Bacterial spores, and the Protozoan Acanthamoeba, in water samples. This finding expands the scope of UV-C research beyond easily culturable bacteria. To address this challenge, future investigations should explore synergistic disinfection strategies, such as combining UV-C treatment with advanced oxidation processes. Optimising UV-C system designs and developing robust, real-time monitoring systems capable of detecting and quantifying known and emerging UV-resistant pathogens are crucial for ensuring comprehensive water decontamination. Full article

Acanthamoeba is a ubiquitous genus of amoebae that can trigger a severe and progressive ocular disease known as Acanthamoeba Keratitis (AK). Furthermore, current treatment protocols are based on the combination of different compounds that are not fully effective. Therefore, an urgent need to find new compounds to treat Acanthamoeba infections is clear. In the present study, we evaluated staurosporine as a potential treatment for Acanthamoeba keratitis using mouse cornea as an ex vivo model, and a comparative proteomic analysis was conducted to elucidate a mechanism of action. The obtained results indicate that staurosporine altered the conformation of actin and tubulin in treated trophozoites of A. castellanii. In addition, proteomic analysis of treated trophozoites revealed that this molecule induced overexpression and a downregulation of proteins related to key functions for Acanthamoeba infection pathways. Additionally, the ex vivo assay used validated this model for the study of the pathogenesis and therapies of AK. Finally, staurosporine eliminated the entire amoebic population and prevented the adhesion and infection of amoebae to the epithelium of treated mouse corneas. Full article

Acanthamoeba keratitis (AK) is a rare but serious infection of the cornea, typically associated with contact lens wear. Here, we present a case of AK caused by the Acanthamoeba genotype T4D/T4e in a patient without identifiable risk factors: a 34-year-old woman who initially presented with signs and symptoms suggestive of herpetic keratitis, and who did not respond to conventional treatment. Corneal culture and targeted metagenomic analysis (18S rRNA, 16S-like rRNA) revealed the presence of an Acanthamoeba species closely related to the ‘Nagington’ strain. Despite intensive anti-Acanthamoeba therapy, complications arose necessitating penetrating keratoplasty. In conclusion, this case underscores the importance of considering Acanthamoeba as a causal agent of keratitis in non-contact-lens wearers. The identification of Acanthamoeba genotype T4D/T4e challenges the previous understanding of its pathogenic potential. Furthermore, it emphasizes the need for ongoing research into the pathogenicity of different Acanthamoeba subtypes. Early diagnosis and treatment are essential for preventing vision-threatening complications associated with AK. Full article

Background: Early therapeutic penetrating keratoplasty (TKP) for Acanthamoeba keratitis (AK) is thought to have a worse visual prognosis than the delayed optical penetrating keratoplasty (OKP) after successful conservative treatment of AK. This has led to a tendency to prolong conservative therapy and delay penetrating keratoplasty in patients with AK. This retrospective series presents the results of patients with AK that underwent early penetrating keratoplasty after reducing the corneal amoeba load through intensive conservative therapy, so-called “low load keratoplasty” (LLKP). Patients and methods: The medical records of our department were screened for patients with AK, confirmed by histological examination and/or PCR and/or in vivo confocal microscopy, which underwent ab LLKP and had a follow-up time of at least one year between 2009 and 2023. Demographic data, best corrected visual acuity (BCVA) and intraocular pressure at first and last visit, secondary glaucoma (SG), and recurrence and graft survival rates were assessed. Results: 28 eyes of 28 patients were included. The average time from initiation of therapy to penetrating keratoplasty (PKP) was 68 ± 113 days. The mean follow-up time after LLKP was 53 ± 42 months. BCVA (logMAR) improved from 1.9 ± 1 pre-operatively to 0.5 ± 0.6 at last visit (p < 0.001). A total of 14% of patients were under medical therapy for SG at the last visit, and two of them underwent glaucoma surgery. The recurrence rate was 4%. The Kaplan–Meier graft survival rate of the first graft at four years was 70%. The second graft survival rate at four years was 87.5%. Conclusion: LLKP appears to achieve a good visual prognosis with an earlier visual and psychological habilitation, as well as low recurrence and SG rates. These results should encourage us to reconsider the optimal timing of PKP in therapy-resistant AK. Full article

Recently, we published that the monoclonal antibody (D12 mAb) recognizes gp63 of L. mexicana, and it is responsible for COX activity. This D12 mAb exhibited cross-reactivity with Trypanosoma cruzi, Entamoeba histolytica, Acanthamoeba castellanii, and Naegleria fowleri. COX activity assays performed in these parasites suggested the potential presence of such enzymatic activity. In our investigation, we confirmed that wild-type recombinant gp63 exhibits COX-like activity, in contrast to a mutated recombinant gp63 variant. Consequently, our objective was to identify sequences orthologous to gp63 and subsequently analyze the binding of arachidonic acid (AA) to the putative active sites of these proteins. Given the absence of a crystallized structure for this protein in the Protein Data Bank (PDB), it was imperative to first obtain a three-dimensional structure by homology modeling, using leishmanolysin from Leishmania major (PDB ID: LML1) as a template in the Swiss model database. The results obtained through molecular docking simulations revealed the primary interactions of AA close to the Zinc atom present in the catalytic site of gp63-like molecules of several parasites, predominantly mediated by hydrogen bonds with HIS264, HIS268 and HIS334. Furthermore, COX activity was evaluated in commensal species such as E. dispar and during the encystment process of E. invadens. Full article