A long-term prospecting study on giant viruses in terrestrial and marine Brazilian biomes.

The discovery of mimivirus in 2003 prompted the search for novel giant viruses worldwide. Despite increasing interest, the diversity and distribution of giant viruses is barely known. Here, we present data from a 2012-2022 study aimed at prospecting for amoebal viruses in water, soil, mud, and sewage samples across Brazilian biomes, using Acanthamoeba castellanii for isolation. A total of 881 aliquots from 187 samples covering terrestrial and marine Brazilian biomes were processed. Electron microscopy and PCR were used to identify the obtained isolates. Sixty-seven amoebal viruses were isolated, including mimiviruses, marseilleviruses, pandoraviruses, cedratviruses, and yaraviruses. Viruses were isolated from all tested sample types and almost all biomes. In comparison to other similar studies, our work isolated a substantial number of Marseillevirus and cedratvirus representatives. Taken together, our results used a combination of isolation techniques with microscopy, PCR, and sequencing and put highlight on richness of giant virus present in different terrestrial and marine Brazilian biomes.

Production of a polyclonal antibody against inosine-uridine preferring nucleoside hydrolase of Acanthamoeba castellanii and its access to diagnosis of Acanthamoeba keratitis.

Acanthamoeba keratitis (AK) is a rare disease but its prevalence throughout the globe continues to grow, primarily due to increased contact lens usage. Since early-stage symptoms associated with AK closely resemble those from other corneal infections, accurate diagnosis is difficult and this often results in delayed treatment and exacerbation of the disease, which can lead to permanent visual impairment. Accordingly, developing a rapid Acanthamoeba-specific diagnostic method is highly desired. In the present study, a rapid and differential method for AK diagnosis was developed using the secretory proteins derived from the pathogenic Acanthamoeba. Among the vast quantities of proteins secreted by the pathogenic Acanthamoeba, an open reading frame of the inosine-uridine preferring nucleoside hydrolase (IPNH) gene was obtained. After expressing and purifying the IPNH protein using the pGEX 4T-3 vector system, mice were immunized with the purified proteins for polyclonal antibody generation. Western blot was performed using protein lysates of the human corneal cell, non-pathogenic amoeba, pathogenic amoeba, and clinical amoeba isolate along with lysates from other causes of keratitis such as Staphylococcus aureus, Pseudomonas aeruginosa, and Fusarium solani to confirm Acanthamoeba-specificity. Western blot using the polyclonal IPNH antibody revealed that IPNH was Acanthamoeba-specific since these proteins were only observed in lysates of Acanthamoeba origin or its culture media. Our findings indicate that the IPNH antibody of Acanthamoeba may serve as a potential agent for rapid and differential AK diagnosis.

Antiamoebic activity of synthetic tetrazoles against Acanthamoeba castellanii belonging to T4 genotype and effects of conjugation with silver nanoparticles.

Acanthamoeba causes diseases such as Acanthamoeba keratitis (AK) which leads to permanent blindness and granulomatous Acanthamoeba encephalitis (GAE) where there is formation of granulomas in the brain. Current treatments such as chlorhexidine, diamidines, and azoles either exhibit undesirable side effects or require immediate and prolonged treatment for the drug to be effective or prevent relapse. Previously, antifungal drugs amphotericin B, nystatin, and fluconazole-conjugated silver with nanoparticles have shown significantly increased activity against Acanthamoeba castellanii. In this study, two functionally diverse tetrazoles were synthesized, namely 5-(3-4-dimethoxyphenyl)-1H-tetrazole and 1-(3-methoxyphenyl)-5-phenoxy-1H-tetrazole, denoted by T1 and T2 respectively. These compounds were evaluated for anti-Acanthamoeba effects at different concentrations ranging from 5 to 50 µM. Furthermore, these compounds were conjugated with silver nanoparticles (AgNPs) to enhance their efficacy. Particle size analysis showed that T1-AgNPs and T2-AgNPs had an average size of 52 and 70 nm respectively. After the successful synthesis and characterization of tetrazoles and tetrazole-conjugated AgNPs, they were subjected to anti-Acanthamoeba studies. Amoebicidal assay showed that at concentration 10 µM and above, T2 showed promising antiamoebic activities between the two compounds while encystation and excystation assays reveal that both T1 and T2 have inhibited differentiation activity against Acanthamoeba castellanii. Conjugation of T1 and T2 to AgNP also increased efficacy of tetrazoles as anti-Acanthamoeba agents. This may be due to the increased bioavailability as AgNP allows better delivery of treatment compounds to A. castellanii. Human cell cytotoxicity assay revealed that tetrazoles and AgNPs are significantly less toxic towards human cells compared with chlorhexidine which is known to cause undesirable side effects. Cytopathogenicity assay also revealed that T2 conjugated with AgNPs significantly reduced cytopathogenicity of A. castellanii compared with T2 alone, suggesting that T2-conjugated AgNP is an effective and safe anti-Acanthamoeba agent. The use of a synthetic azole compound conjugated with AgNPs can be an alternative strategy for drug development against A. castellanii. However, mechanistic and in vivo studies are needed to explore further translational values.

Clinical presentations, genotypic diversity and phylogenetic analysis of Acanthamoeba species causing keratitis.

Introduction. Acanthamoeba keratitis is a sight-threatening corneal infection that is commonly reported among contact lens users and those suffering from corneal trauma. The prevalence of Acanthamoeba species or genotypes in causing keratitis infection is not well known.Aim. This study was conducted to identify and genotype Acanthamoeba isolates from keratitis patients, targeting the ribosomal nuclear subunit (Rns) region, and describe the associated clinical presentation and treatment outcome.Methodology. Thirty culture-confirmed patients with Acanthamoeba keratitis, identified in a tertiary eye care centre in South India during the period from December 2016 to December 2018, were included in this study. The data collected from patient records include demographic details, history of illness, mode of trauma, treatment history and follow-up status. The genotype and species were identified based on the Rns sequence and phylogenetic tree analysis.Results. Acanthamoeba culbertsoni was the most predominant keratitis-causing species, followed by Acanthamoeba quina, Acanthamoeba castellanii, Acanthamoeba healyi, Acanthamoeba hatchetti, Acanthamoeba polyphaga and Acanthamoeba stevensoni. Three major genotypes were identified (T4, T11 and T12), with the T4 genotype being the most predominant, with four subclusters, i.e. T4A, T4B, T4D and T4E. This is the first report on corneal infection by the A. stevensoni T11 genotype and the A. healyi T12 genotype. No significant correlation was observed between the clinical outcomes of corneal disease and the genotypes or species.Conclusion. Rns genotyping is very effective in identifying the Acanthamoeba species and genotype in keratitis. Genotyping of Acanthamoeba spp. will help to advance our understanding of genotype-specific pathogenesis and geographical distribution.

N-chlorotaurine Inactivates Acanthamoeba and Candida albicans in the Porcine Ex Vivo Corneal Infection Model.

PURPOSE: N-chlorotaurine (NCT) is an anti-infective belonging to the class of chloramines and an investigative drug for the topical treatment of keratoconjunctivitis. The aim of the present study was to demonstrate its efficacy against Acanthamoeba and Candida in corneas infected ex vivo. METHODS: Corneal buttons from porcine eyes were contaminated with Acanthamoeba castellanii trophozoites or Candida albicans Centraalbureau voor Schimmelcultures 5982 and incubated for 7 and 3 days, respectively. Subsequently, they were treated with 1% NCT for 5 to 120 minutes. After further incubation for 2 days in the absence of NCT in tests with A. castellanii, the buttons were homogenized, and the amoebae grown for a further 5 days before they were counted in a light microscope. For C. albicans, quantitative cultures were performed from corneal homogenates. RESULTS: Incubation of 120 minutes in NCT completely inhibited the regrowth of A. castellanii and reduced the number of C. albicans colony-forming unit counts by 4 log10. In addition, at 60 minutes, significant reductions of both pathogens could be observed. Histology showed penetration of pathogens into the stroma of the corneal buttons. CONCLUSIONS: NCT inactivates A. castellanii and C. albicans in corneal tissue.

In Vitro Evaluation of Adhesion of Two Acanthamoeba Strains to Cosmetic Contact Lenses.

PURPOSE: To evaluate the factors affecting the adhesion of Acanthamoeba trophozoites to the surface of cosmetic contact lenses (CCLs). METHODS: Acanthamoeba castellanii and A. hatchetti trophozoites were inoculated onto CCLs (hema copolymer [HM] [38.5% H2O], phemfilcon [PF] [55% H2O], polymacon [PM] [38% H2O], polyhema [PH] [%42 H2O], and hema [HM55] [55% H2O]), and the number of trophozoites adhered to the lens surfaces was assessed over time, that is, at 15 min, 1, and 24 hr. In addition, scanning electron microscopy (SEM) analysis of the lens surfaces was performed to evaluate the effect of lens surface topology on adhesion. RESULTS: The number of amoeba adhered to the contact lens surface was found lower with PF and PH production materials, than lenses with HM, PM, and HM55 production materials (P<0.05). No significant difference was detected in amoebic strains adhered in all the contact lens types (P>0.05). No significant difference was found on average amoeba adhesion between contact lenses with hema production material but with different water contents (45%, 55%), to see the effect of water content on amoebic adhesion (P>0.05). As a result of SEM analysis, surface topology showed no effect on adhesion. CONCLUSION: (1) Chemical composition of lenses seemed to be mostly responsible for the adhesion of Acanthamoeba. (2) Different numbers of trophozoites, obtained after the adhesion experiment, could also indicate that adherence capacity can also differ among Acanthamoeba species.

Characterizing clinical isolates of Acanthamoeba castellanii with high resistance to polyhexamethylene biguanide in Taiwan.

BACKGROUND/PURPOSE: Acanthamoeba keratitis (AK), a painful infectious corneal disease, is caused by the free-living pathogenic species Acanthamoeba. The symptoms include corneal infiltrate, epithelial, and stromal destruction, and loss of vision. Current treatment generally involves an hourly application of polyhexamethylene biguanide (PHMB) over a period of several days; however, even this is not entirely effective against all strains/isolates. The aims of this study were to confirm the existence of pathogenic strains in Taiwan which are highly resistant to drugs and to characterize the behavior of these strains. METHODS: An in vitro Acanthamoeba species culture platform was established to observe the effectiveness of treatment and chart the morphological changes that occur under the effects of drugs using a light microscope and time-lapse recording. Changes in gene expression were examined using reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. RESULTS: Over 90% of the standard strain cells (ATCC 30010) were lysed after being treated with PHMB for 1 hour; however, clinical isolates of Acanthamoeba castellanii that differed in their susceptibility to the treatment drug were only partly lysed. Following treatment with PHMB, National Cheng Kung University Hospital isolation B (NCKH_B) transformed into a pseudocyst under the effects of drug stress; however, National Cheng Kung University Hospital isolation D (NCKH_D), an isolate with higher tolerance for PHMB, did not transform. CONCLUSION: Our results confirm the existence of clinical isolates of A. castellanii with high resistance to PHMB in Taiwan and present the alternative drug tolerance of A. castellanii in addition to the transformation of pseudocyst/cyst.

Continuous positive airway pressure-associated cutaneous amoebiasis in an immunosuppressed patient.

Organisms of the genus Acanthamoeba are environmentally ubiquitous and colonizers of the oral mucosa in humans. While largely asymptomatic in healthy persons, Acanthamoeba infection can cause disseminated disease with poor prognosis in immunosuppressed populations. Here we report a unique case of cutaneous amoebiasis associated with continuous positive airway pressure use in an immunosuppressed patient.

Cytotoxic activity and degradation patterns of structural proteins by corneal isolates of Acanthamoeba spp.

BACKGROUND: Proteolytic enzymes secreted by trophozoites (amoebic secretome) are suggested as the main virulence factor involved in the severity of Acanthamoeba keratitis. The degradation profile of the main glycoprotein components of anterior and posterior portions of the cornea and the cytopathic effect of secretomes on endothelial cells by contact-independent mechanism were evaluated. METHODS: Trophozoites were isolated primarily from corneal tissue samples (n = 11) and extracellular proteins were collected from axenic cell culture supernatants. The molecular weights of proteolytic enzymes were estimated by zymography. Enzymatic cleavage of laminin and fibronectin substrates by amoebic secretome was investigated and cluster analysis was applied to the proteolysis profiles. Primary cultures of endothelial cells were used in both qualitative and quantitative assays of cytophatogenicity. RESULTS: Differential patterns of proteolysis were observed among the Acanthamoeba secretomes that were analysed. The uniformity of laminin degradation contrasted with the diversity of the proteolysis profiles observed in the fibronectin substrate. Acanthamoeba secretome extracted from four clinical isolates was shown to be toxic when in contact with the endothelial cell monolayer (p < 0.01). Induction of apoptosis and membrane permeability, at different percentual values, were suggested as the main mechanisms that could induce endothelial cell death when in contact with amoebic secretome. CONCLUSIONS: Our results provide evidence that virulence factors secreted by Acanthamoeba trophozoites can be related to an increased pathogenicity pattern by an independent contact-trophozoite mechanism, through induction of endothelial cell death by apoptosis at a higher percentage than providing the lack of cell viability by the membrane-associated pore-forming toxin activity.

Lethal effects of Helianthemum lippii (L.) on Acanthamoeba castellanii cysts in vitro.

Acanthamoeba spp. commonly cause Acanthamoeba keratitis which is typically associated with the wear of contact lenses. Therefore, finding an economic, efficient, and safe therapy of natural origin is of outmost importance. This study examined the in vitro lethal potential of ethyl acetate and methanol extracts of Helianthemum lippii (L.) (sun roses) against Acanthamoeba castellanii cysts isolated from patients with amoebic keratitis. Both extracts proved to be potent as regard to their lethal effects on A. castellanii cysts with comparable results to chlorhexidine. The ethyl acetate was more promising with cumulative lethality. It showed a highly significant lethal percentage along the duration of treatment. The analysis of the more potent ethyl acetate extract revealed the presence of 2.96 mg/100 g of total phenolics, 0.289 mg/100 ml of total flavonoids and 37 mg/100 mg of total tannins which highlighted their phytomedicinal role.

Characterization of cyst and trophozoite proteins of environmental isolates of Acanthamoeba castellanii by two-dimensional gel electrophoresis.

Acanthamoeba castellanii has been known to possess pathogenic properties, such as acanthamoebic keratitis and granulomatous amoebic encephalitis. The role of proteases and proteins in the pathogenesis of these infections is still poorly understood. As Acanthamoeba sp is a ubiquitous protozoon found in the natural environment they can potentially cause human infections. This study characterized cyst and trophozoite proteins of 3 environmental A. castellanii isolates in comparison with a clinical isolate, ATCC 50492. The latter and environmental IMU1 isolate had 100% genotype identity with A. castellanii and demonstrated protein spots with higher molecular weights (> 95 kDa) at relatively higher isoelectric values (> pI 7.00) compared to the two other environmental isolates (IMU4 and IMU5) that had 99% genotype identity to A. castellanii based on 16 S rDNA sequence. Thus such trophozoite proteins may be involved with the parasite's ability to cause acanthamoebic keratitis.

In vitro inhibition of protease-activated receptors 1, 2 and 4 demonstrates that these receptors are not involved in an Acanthamoeba castellanii keratitis isolate-mediated disruption of the human brain microvascular endothelial cells.

Granulomatous amoebic encephalitis is a rare but serious human disease leading almost always to death. The pathophysiology of amoebic encephalitis is better understood, while events leading to the constitution of brain infection are largely unknown. Traversal of the blood-brain barrier is a key step in amoebae invasion of the central nervous system and facilitated by amoebic extracellular proteases. By using specific inhibitors of protease-activated receptors 1, 2 and 4, here we studied the role of these host receptors in Acanthamoeba castellanii-mediated damage to human brain microvasculature endothelial cells (HBMEC), which constitute the blood-brain barrier. The primary HBMEC were incubated with A. castellanii-conditioned medium in the presence or absence of FR-171113 (selective inhibitor of protease-activated receptor 1), FSLLRY-NH2 (inhibitor of protease-activated receptor 2), and tcY-NH2 (inhibitor of protease-activated receptor 4). The HBMEC monolayer disruptions were assessed by microscopy using Eosin staining, while host cell cytotoxicity was determined by measuring the release of cytoplasmic lactate dehydrogenase. Zymographic assays were performed to determine the effects of inhibitors of protease-activated receptors on the extracellular proteolytic activities of A. castellanii. A. castellanii-conditioned medium produced severe HBMEC monolayer disruptions within 60 min. The selective inhibitors of protease-activated receptors tested did not affect HBMEC monolayer disruptions. On the contrary, pre-treatment of A. castellanii-conditioned medium with phenylmethylsulfonyl fluoride, a serine protease inhibitor, or heating for 10 min at 95°C abolished HBMEC monolayer disruptions. Additionally, inhibitors of protease-activated receptors tested, failed to block A. castellanii-mediated HBMEC cytotoxicity and did not affect extracellular proteolytic activities of A. castellanii. Protease-activated receptors 1, 2 and 4 do not appear to play a role in A. castellanii-mediated dysfunction of HBMEC, which constitute the blood-brain barrier. The role of additional protease-activated receptors in amoebic invasion of the central nervous system is discussed further.

Silicone hydrogel contact lenses surface promote Acanthamoeba castellanii trophozoites adherence: qualitative and quantitative analysis.

PURPOSE: To describe the adhesion properties of Acanthamoeba castellanii trophozoites to silicone hydrogel contact lenses of first generation (lotrafilcon A), second generation (galyfilcon A), and third generation (comfilcon A) and correlate the results with their specific surface characteristics, time of interaction, and suspension media. METHODS: Qualitative and quantitative assessments of the adhesion of 200 trophozoites of A. castellanii on contact lenses in culture medium (Bacto Casitone) and isotonic saline (IS) at different time points (15 minutes and 6 hours) were determined. RESULTS: By scanning electron microscopy, A. castellanii trophozoites were observed firmly adhered to the surface of hydrogel lenses after 15 minutes of interaction. The surface of lotrafilcon A lenses on which amoebae adhere better (16.4±10.2 amoebae/lens section) is rough and folded, which increases the contact surface with trophozoites, allowing acanthopodia to attach firmly. Contrarily, galyfilcon A lenses have a smoother surface, and lower numbers of amoebae were observed adhered to these lenses (4.7±2.9 amoebae/lens section). Even fewer amoebae adhered to the smoother surface of the comfilcon A lens (2.2±1.7 amoebae/lens section). Trophozoites showed similar behavior in both Bacto Casitone medium and IS. CONCLUSION: A rough surface may contribute to better adhesion of amoebae to silicone hydrogel lenses. Although a reduced numbers of trophozoites adhered to smooth lenses, trophozoites are a risk factor for amoebic keratitis. Isotonic saline facilitated trophozoite survival, suggesting that homemade saline solutions may contribute to the persistence of trophozoites, especially when there is no proper hygiene regimen used with the contact lens cases.

Cytotoxic effect of organic solvents and surfactant agents on Acanthamoeba castellanii cysts.

Acanthamoeba castellanii is a protozoan parasite that may cause sight-threatening keratitis in some individuals. Its eradication is difficult because the trophozoites encyst making organisms highly resistant to anti-amoebic drugs. To test new anti-Acanthamoeba agents, usually having low water solubility, organic solvents and surfactant agents should be used. Therefore, the lethal effect of different concentrations of the solvents acetone, methanol, ethanol, and DMSO and surfactant agents Tween 20, Tween 80, and Triton X-100 was tested. The minimal inhibitory concentrations (MIC) were determined against Acanthamoeba cysts. Results of the present study showed that the MIC for ethanol, methanol, acetone and DMSO was 25, 12.5, 12.5, and 10%, respectively and for Tween 20, Tween 80, and Triton X-100 was 0.25, 0.06, and 0.03%, respectively. There was no significant inhibitory effect on the multiplication of Acanthamoeba cysts as compared to parasite control when using the concentrations 3.12% for ethanol, 1.6% for methanol and acetone, 1.25% for DMSO, and 0.016% for Tween 20. On the other hand, both Tween 80 and Triton X-100 showed highly significant difference in comparison to parasite control almost among all the range of concentrations used in this study, and both showed lethal effect of 19 and 27.2%, respectively at their least concentration.

Failure of chemotherapy in the first reported cases of Acanthamoeba keratitis in Pakistan.

Acanthamoeba keratitis is a painful and progressive infection of the cornea that can result in loss of vision. Here, for the first time in Pakistan, we report two cases of Acanthamoeba keratitis. The first patient was a 37-year-old female who presented with severe itching, redness, pain, along with loss of vision. The patient was a regular soft contact lens wearer. The second patient was a 25-year-old female who had been using soft contact lenses for the past two years. She presented with a burning sensation and extreme pain, along with loss of vision. Both patients were treated for a possible microbial keratitis with topical moxifloxacin hydrochloride drops, vancomycin drops, propamidine isethionate ointment, amphotericin B drops, and amikacin drops. However, the response was inadequate and both patients were referred for corneal transplant. Acanthamoeba castellanii was isolated by placing contact lenses and contact lens cases on non-nutrient agar plates containing a lawn of non-invasive Escherichia coli K-12 HB101 bacteria. The polymerase chain reaction (PCR) using genus-specific probes confirmed the identity of Acanthamoeba spp., whereas the morphological characteristics of trophozoites and cysts were suggestive of A. castellanii in both cases. With growing use of contact lenses for vision correction/cosmetic use coupled with sub-standard lens care in this region and the possibility of non-contact lens-associated Acanthamoeba keratitis, a need for increased awareness of this sight-threatening infection is discussed further.

Identification and quantification of the Acanthamoeba species and genotypes from reservoirs in Taiwan by molecular techniques.

The occurrence of Acanthamoeba was investigated from 21 main reservoirs of Taiwan with 12 (57.1%) testing positive. Analysis of the 18S rRNA gene PCR product was performed in order to identify the Acanthamoeba isolates. Acanthamoeba spp. concentrations were determined according to TaqMan real-time qPCR. Acanthamoeba genotypes of all isolates were identified T4. The species were categorized to Acanthamoeba culbertsoni, Acanthamoeba polyphaga, Acanthamoeba castellanii and Acanthamoeba hatchetti. The concentration of Acanthamoeba spp. in detected positive reservoir water samples was in the range of 3.0-1.8 × 10(3) cells/L. These results highlight the importance of Acanthamoeba in reservoirs of potential pathogens and its possible role in the spread of bacterial genera with interest in public and environmental health.

In vitro adhesion of Acanthamoeba castellanii to soft contact lenses depends on water content and disinfection procedure.

PURPOSE: To compare the potential of different soft contact lenses to be contaminated with Acanthamoeba castellanii as a function of material parameters and cleaning procedures. METHODS: Different unworn soft hydrogel and silicone hydrogel contact lenses were incubated with human pathogenic A. castellanii. The adhesion of the acanthamoebae was investigated on the contact lenses and put into relation to their material parameters. The efficacy of a recommended contact lens cleaning procedure in reducing A. castellanii adhesion was investigated. RESULTS: We found that material parameters such as elastic modulus, silicone content, ionic properties and swelling do not influence the adhesion of acanthamoebae to soft contact lenses. A material parameter that influenced adhesion significantly was the water content of the lens. With increasing water content, the adhesion of acanthamoebae increased. By following the cleaning instructions of the manufacturer the contamination of the lenses with A. castellanii could be reduced to a minimum, as shown both on contact lenses and in control experiments. CONCLUSION: With this study we show that for the tested lenses, the adhesion of A. castellanii to contact lenses is independent of the silicone content of the lens, but depends nonlinearly on the water content of the lens. Furthermore, we demonstrate that applying proper lens cleaning procedures minimizes the risk of acanthamoebae adhesion to contact lenses.

Density of environmental Acanthamoeba and their responses to superheating disinfection.

Exposure to viable Acanthamoeba may cause fatal encephalitis and blinding keratitis in humans. Quantification of environmental Acanthamoeba by a reliable analytical assay is essential to assess the risk of human exposure and efficacy of control measures (e.g., superheating). Two DNA binding dyes (ethidium monoazide (EMA) and propidium monoazide) coupled with real-time quantitative PCR (qPCR) were tested for the ability in selectively quantifying viable Acanthamoeba castellanii. This newly developed qPCR assay was applied to determine the density of environmental Acanthamoeba and disinfection efficacy of superheating. Results showed qPCR with 2.3 µg/mL EMA performed optimal with a great linearity (R (2) = 0.98) and a wide range of detection (5-1.5 × 10(5) cells). EMA-qPCR analyses on water samples collected from cooling towers, eyewash stations, irrigated farmlands, and various wastewater treatment stages further showed viable Acanthamoeba density from nondetectable level to 6.3 × 10(5) cells/L. Superheating A. castellanii at 75-95 °C for 20 min revealed significant reductions in both EMA-qPCR and qPCR detectable Acanthamoeba target sequences with an adverse association between heating temperature and qPCR-determined DNA quantity (r = -0.76 to -0.93, p < 0.0001). Moreover, A. castellanii trophozoites were more sensitive to superheat stress than the cells being encysted for 6 and 13 d (p < 0.05). This is the first study to quantify environmental Acanthamoeba and characterize their responses to superheating by EMA-qPCR. The quantitative data provided in this study facilitate to understand better the relative risk for human exposed to viable Acanthamoeba and the efficacy of superheating against Acanthamoeba.

Antimicrobial action of biguanides on the viability of Acanthamoeba cysts and assessment of cell toxicity.

PURPOSE: To assess dose- and concentration-dependent rates of biguanides on the viability of Acanthamoeba cysts isolated from severe ulcerative keratitis, and to correlate cysticidal activites with cytotoxic profiles in corneal and endothelial cells. METHODS: Cysticidal activities of polyhexamethylene biguanide and chlorhexidine digluconate were evaluated in the Acanthamoeba castellanii strain and clinical isolates of Acanthamoeba spp obtained from two severe and recurrent cases of ulcerative keratitis. The molecular characterization of protozoa used in the experimental assays was performed by sequencing reactions of the 18S rDNA gene. Acanthamoeba cysts were exposed at different dosages and concentrations of both biguanides; the application of double-biguanides was also evaluated. Automated cell viability assessment of cysts was performed using the trypan blue dye exclusion method. Cytotoxicity assays of biguanides were conducted using primary cultures of endothelial cells alone or in coculture with Acanthamoeba cysts. Human corneal epithelial cells were used as a comparative pattern to assess the toxicity of biguanide compounds. Cell viability was measured using both quantitative and qualitative methods. Statistical analyses were applied to the data. RESULTS: The in vitro study showed that all dosages, concentrations, and combinations of biguanides tested had a cysticidal effect on Acanthamoeba spp strains tested compared with control cultures not exposed to any antimicrobials; the difference in response was statistically significant. The use of both biguanides in combination demonstrated the best cysticidal effect. The use of isolated biguanides was associated with greater cytotoxic effects than with biguanides used in combination. Chlorhexidine digluconate used alone tended to have greater cytotoxicity than polyhexamethylene biguanide. Furthermore, the double-biguanide application had a statistically significant decrease in the deleterious effect on endothelial cells at higher dosage and concentration. Quantitative and qualitative analyses demonstrated the toxic effect of biguanide compounds on the viability of corneal epithelial cells, under single or in combination usage. CONCLUSIONS: We demonstrated that the combined use of biguanides had greater cysticidal activity than individual drug application as well as a possible protective effect on endothelial cells. The biguanide compounds tested were able to induce corneal epithelial cell death in time and concentration-independent fashions. Findings support the hypothesis concerning the cysticidal effect and the differential patterns of toxicity expressed by polyhexamethylene biguanide and chlorhexidine digluconate on the endothelial and corneal cells.

Acanthamoeba castellanii of the T4 genotype is a potential environmental host for Enterobacter aerogenes and Aeromonas hydrophila.

BACKGROUND: Acanthamoeba can interact with a wide range of microorganisms such as viruses, algae, yeasts, protists and bacteria including Legionella pneumophila, Pseudomonas aeruginosa, Vibrio cholerae, Helicobacter pylori, Listeria monocytogenes, Mycobacterium spp., and Escherichia coli. In this capacity, Acanthamoeba has been suggested as a vector in the transmission of bacterial pathogens to the susceptible hosts. METHODS: Here, we used a keratitis isolate of A. castellanii of the T4 genotype and studied its interactions with two bacterial genera which have not been tested before, Enterobacter aerogenes, and Aeromonas hydrophila, as well as E. coli. Assays were performed to determine bacterial association with and invasion of A. castellanii. Additionally, bacterial survival intracellular of A. castellanii trophozoites as well as cysts was determined. RESULTS: All three bacterial isolates tested, associated, invaded, and survived inside A. castellanii trophozoites as well as A. castellanii cysts. However, E. aerogenes and E. coli exhibited significantly reduced association with and invasion of A. castellanii as compared with A. hydrophila (P < 0.01 using paired T-test, one tail distribution). In the long term survival assays, all three bacterial isolates tested remained viable inside A. castellanii trophozoites, while amoeba remained intact; however A. hydrophila exhibited higher survival inside amoebae (14.54 ± 3.3 bacteria:amoeba ratio) compared with E. aerogenes (3.96 ± 0.7 bacteria:amoeba ratio) and E. coli (5.85 ± 1.1 bacteria:amoeba ratio). A. hydrophila, E. coli, and E. aerogenes remained viable during the encystment process and exhibited higher levels of recovery from mature cysts (14.13 ± 0.89 A. hydrophila:amoeba ratio, 10.13 ± 1.17 E. aerogenes:amoeba ratio, and 11.95 ± 0.7 E. coli:amoeba ratio). CONCLUSIONS: A. hydrophila and E. aerogenes also joined the ranks of other bacteria that could benefit from A. castellanii. Because cysts can be airborne, these findings suggest that Acanthamoeba is a potential vector in the transmission of A. hydrophila and E. aerogenes to susceptible hosts.