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.

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.

Diagnosis and treatment of Acanthamoeba Keratitis: A scoping review demonstrating unfavorable outcomes.

Acanthamoeba spp. are pathogens that cause Acanthamoeba keratitis (AK), a serious cornea inflammation that can lead to gradual loss of vision, permanent blindness, and keratoplasty. The efficacy of AK treatment depends on the drug's ability to reach the target tissue by escaping the protective eye barrier. No single drug can eradicate the living forms of the amoeba and be non-toxic to the cornea tissue. The treatment aims to eradicate both forms of protozoan life but is hampered by the resistance of the cysts to the most available drugs, leading to prolonged infection and relapses. Drug therapy is currently performed mainly using diamidines and biguanides, as they are more effective against cysts. However, they are cytotoxic to corneal cells. Drugs are applied topically, and hourly. Over time, the frequency of administration decreases, but the treatment time varies from month to years. This study aims to obtain an up-to-date summary of the literature since 2010, allowing us to identify the trends and gaps and address future research involving new alternatives for treating AK. The results were divided into three phases, pre-treatment, empirical treatment, and the treatment after diagnosis confirmation. The drugs prescribed were stratified into antiamoebic, antibiotic, antifungal, antivirals, and steroids. It was possible to observe the transition in drug prescription during three different stages until the diagnosis was confirmed. There were more indications for antibiotic, antifungal, and antiviral drugs in the early stages of the disease. The antiamoebic drugs were only prescribed after exhausting other treatments. This can be directly involved in developing complications and no responsiveness to medical treatment.

Super aggregated amphotericin B with a thermoreversible in situ gelling ophthalmic system for amoebic keratitis treatment.

Acanthamoeba spp. are the causative agents of a sight-threatening infection of the cornea known as Acanthamoeba keratitis (AK). Amphotericin B - deoxycholate (AB) is used in the treatment of infectious keratitis, however, its topical administration has side effects as blepharitis, iritis, and painful instillation. In this context, the preheating of AB can decrease its toxicity by the formation of super aggregates (hAB). hAB associated with a thermoreversible in situ gelling ophthalmic system is a promising option due to the latter biocompatibility, low toxicity, and high residence time on the ocular surface. Our objective was to develop a topical ocular formulation of hAB for the treatment of AK. After heating at 70°C for 20 min, hAB was incorporated into a thermoreversible gelling system. The amebicidal activity of AB and hAB was evaluated against trophozoites and cysts of A. castellanii (ATCC 50492) and a regional clinical isolate (IC01). The results showed that the preheating of AB did not change the pharmacological action of the drug, with the amebicidal effect of AB and hAB under trophozoites and cysts of Acanthamoeba spp. The thermoreversible system remained stable, allowing the increase of drug retention time. For assessment of cytotoxicity, HUVEC (ATCC® CRL-1730) cells were challenged with AB and hAB for 48h. Cell viability was assessed, and hAB did not show cytotoxicity for HUVEC cells. As far as we know this was the first study that showed the preheated AB associated with a thermoreversible in situ gelling ophthalmic system as a promising system for topical ocular topical administration of hAB for AK therapy.

Free-living amoebae and their relationship to air quality in hospital environments: characterization of Acanthamoeba spp. obtained from air-conditioning systems.

Free-living amoebae (FLA) are widely dispersed in the environment, can cause opportunistic and non-opportunistic infections in humans and other animals. The aim of the present study was characterize FLA obtained from air-conditioners of a public hospital in the city of Florianópolis, SC, Brazil. Fifty-four dust samples were collected of air conditioners, and were inoculated on 1.5% non-nutrient agar, overlaid with layers of Escherichia coli. Subsequently the isolates were axenised in PYG growth medium. The morphological and molecular characterization of the isolates was performed, as well as the tolerance (physiological) assays were used to evaluate the pathogenic potential. The results revealed the presence of FLA in 42 (77.8%) of the collected samples. Of these, 39 (92.9%) axenic isolates of FLA were obtained for morphological and genotypic studies. All the isolates characterized belong to the genus Acanthamoeba. Nineteen (48.7%) isolates belong to the genotype T4, 16 (41.0%) to the T5 genotype and 4 (10.3%) to genotype T11. Seven (18.0%) isolates were considered potentially pathogenic in tolerance assays. These findings require attention, considering the isolation environment and immunocompromised characteristics of many hospitalized patients.

Contact lens-related polymicrobial keratitis: Acanthamoeba spp. genotype T4 and Candida albicans.

A 31-year-old female daily user of contact lenses sought medical attention, reporting blurred vision and irritation of the left eye. Slit-lamp examination revealed hyperemia and an irregular corneal epithelium surface, and empirical treatment was started. A corneal scrape was obtained and examined for the presence of fungi, bacteria, and Acanthamoeba spp. The results of the microbial culture revealed growth of Acanthamoeba spp. and Candida albicans. The Acanthamoeba isolate was characterized by cyst morphology as belonging to group II according to Pussard and Pons. Sequencing of the diagnostic fragment 3 (DF3) region located on the 18S ribosomal DNA identified the isolate as genotype T4. The patient was treated with chlorhexidine 0.02% and polyhexamethylene biguanide (PHMB) 0.02% drops for 5 months until the infection resolved. Lately, rare cases of polymicrobial keratitis associated with Acanthamoeba and Candida albicans have been reported. Cases of co-infection are more difficult to treat, since the specific treatment depends on precise identification of the agents involved.

Genotypic characterization and assessment of infectivity of human waterborne pathogens recovered from oysters and estuarine waters in Brazil.

Waterborne, food-borne and sewage-borne pathogens are a major global concern, with the annual recurrence, most notably during the summer, of outbreaks of gastroenteritis of unconfirmed etiology associated with recreational activities in marine environments. The consumption of contaminated water-based foodstuffs is also related to outbreaks of human illness. The main goals of the present study were: i) to identify the genetic assemblages of Giardia duodenalis cysts in growing and depurated oysters destined for human consumption on the southern coast of São Paulo, Brazil; ii) to verify the main circulating G. duodenalis assemblages and their subtypes in different brackish waters used for the production of mollusks and for recreational purposes; iii) to track the contamination of growing and depurated oysters by the human adenovirus and identify the infectivity of adenoviral particles recovered from oysters before and after depuration; iv) to evaluate the occurrence and genotype of the free-living amoebae of the genus Acanthamoeba in brackish water and oysters from all the sites described above. Four sampling sites in the Cananeia estuary were selected to search for pathogenic and amphizoic protozoa (Giardia and Acanthamoeba respectively): site 1: oyster growth, site 2: catchment water (before UV depuration procedure), site 3: filter backwash (filtration stage of water treatment) and site 4: oyster depuration tank. Oysters at sites 1 and 4 were evaluated for the presence of adenovirus (HAdV). Analysis consisted of conventional microbiological as well as molecular methods. Giardia duodenalis were detected in all the water sites analyzed and the molecular analysis revealed that sub-assemblage AII was the most frequently distributed throughout the estuarine environment, although one sample was identified as belonging to the assemblage C. Acanthamoeba were also isolated from different locations of the estuarine area, and were detected at all the analyzed sites. The majority of isolates belonged to the T3 genotype, while the T4 genotype was identified once. The sequencing reaction of Giardia duodenalis revealed the contamination of three batches of depurated oysters by the sub-assemblage AII. With respect to viruses, seven batches of oysters (four growing and three depurated) were found to be harboring infectious HAdV particles when submitted to plaque assay. Overall, the results of the sequencing reactions combined with the plaque assay revealed that the isolates of Giardia duodenalis and the infectious HAdV particles identified in oyster tissues have the potential to infect humans and pose a threat if consumed raw or lightly cooked. This is the first report on the sub-assemblage AII identified in oysters which are submitted to a cleaning and disinfection procedure prior to human consumption in Brazil. Acanthamoeba specific genotypes were also identified for the first time in a recreational estuarine area in Brazil, contributing to knowledge of their molecular and environmental epidemiology, which is considered scarce even in marine and estuarine areas of the world.

Proteomic analysis of Toxocara canis excretory and secretory (TES) proteins.

Toxocariasis is a neglected disease, and its main etiological agent is the nematode Toxocara canis. Serological diagnosis is performed by an enzyme-linked immunosorbent assay using T. canis excretory and secretory (TES) antigens produced by in vitro cultivation of larvae. Identification of TES proteins can be useful for the development of new diagnostic strategies since few TES components have been described so far. Herein, we report the results obtained by proteomic analysis of TES proteins using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach. TES fractions were separated by one-dimensional SDS-PAGE and analyzed by LC-MS/MS. The MS/MS spectra were compared with a database of protein sequences deduced from the genome sequence of T. canis, and a total of 19 proteins were identified. Classification according to the signal peptide prediction using the SignalP server showed that seven of the identified proteins were extracellular, 10 had cytoplasmic or nuclear localization, while the subcellular localization of two proteins was unknown. Analysis of molecular functions by BLAST2GO showed that the majority of the gene ontology (GO) terms associated with the proteins present in the TES sample were associated with binding functions, including but not limited to protein binding (GO:0005515), inorganic ion binding (GO:0043167), and organic cyclic compound binding (GO:0097159). This study provides additional information about the exoproteome of T. canis, which can lead to the development of new strategies for diagnostics or vaccination.

Detection and quantification of human adenovirus genomes in Acanthamoeba isolated from swimming pools.

Acanthamoeba is the most common free-living environmental amoeba, it may serve as an important vehicle for various microorganisms living in the same environment, such as viruses, being pathogenic to humans. This study aimed to detect and quantify human adenoviruses (HAdV) in Acanthamoebas isolated from water samples collected from swimming pools in the city of Porto Alegre, Southern Brazil. Free-living amoebae of the genus Acanthamoeba were isolated from water samples, and isolates (n=16) were used to investigate the occurrence of HAdVs. HAdV detection was performed by quantitative real-time polymerase chain reaction (qPCR). HAdVs were detected in 62.5% (10/16) of Acanthamoeba isolates, ranging from 3.24x103 to 5.14x105 DNA copies per milliliter of isolate. HAdV viral loads found in this study are not negligible, especially because HAdV infections are associated with several human diseases, including gastroenteritis, respiratory distress, and ocular diseases. These findings reinforce the concept that Acanthamoeba may act as a reservoir and promote HAdV transmission through water.

Supporting data for comparative proteomic analysis of Listeria monocytogenes ATCC 7644 exposed to a sublethal concentration of nisin.

Here we provide the LC-MS/MS data from a comparative analysis of Listeria monocytogenes ATCC 7644 treated and non-treated with a sublethal concentration of nisin (10(-3) mg/mL). Protein samples were analyzed by multidimensional protein identification technology (MudPIT) approach, in an off-line configuration. The raw MS/MS data allowed the detection of 49,591 spectra which resulted in 576 protein identifications. After Scaffold validation, 179 proteins were identified with high confidence. A label-free quantitative analysis based of normalized spectral abundance factor (NSAF) was used and 13 proteins were found differentially expressed between nisin-treated and non-treated cells. Gene ontology analysis of differentially expressed proteins revealed that most of them are correlated to metabolic process, oxidative stress response mechanisms and molecular binding. A detailed analysis and discussion of these data may be found in Miyamoto et al. [1].

Identification of Paenibacillus as a Symbiont in Acanthamoeba.

Amoebae of the genus Acanthamoeba occur worldwide and in addition to being pathogens, are important vehicles for microorganisms with clinical and environmental importance. This study aimed to evaluate the profiling of endosymbionts in 12 isolates of Acanthamoeba using V3 region of 16S rDNA denaturing gradient gel electrophoresis (DGGE) and sequencing. The DGGE enabled us to characterize the endosymbionts diversity in isolates of Acanthamoeba, and to identify Paenibacillus sp., an emerging pathogen, as an amoebic endosymbiont. The results of this study demonstrated that Acanthamoeba is capable of transporting a large number of endosymbionts. This is the first study that reports, the presence of Paenibacillus sp. as amebic symbiont.

Comparative proteomic analysis of Listeria monocytogenes ATCC 7644 exposed to a sublethal concentration of nisin.

Listeria monocytogenes infections have been frequently reported in many food poisoning outbreaks around the world. In this work, the protein repertoires of L. monocytogenes ATCC 7644 cells treated or not with a 10(-3)mg/mL nisin sublethal concentration, established by antimicrobial susceptibility tests, were analyzed by LC-MS/MS. Overall, 179 proteins were identified, 9 of them more abundant in nisin-treated samples, and 4 more abundant in non-treated control samples. In nisin treated cells, proteins associated to oxidative stress response showed higher abundance. Also, the higher abundance of an enzyme related to the production of cell membrane lipids upon nisin exposure is suggestive of both a failure in conventional cell division mechanism and the activation of an alternative L-form mediated division mechanism. Finally, flagellar and motility proteins' overexpression upon nisin exposure is indicative of increased bacterial motility in response to the bacteriocin. Taken together, these results provide new insights on nisin effects on L. monocytogenes cells and on how this bacterium may overcome a bacteriocin-containing environment. BIOLOGICAL SIGNIFICANCE: The antimicrobial mechanism of nisin on target bacterial cells has been extensively studied since discovery of this bacteriocin. The nisin pore-forming mechanism is mediated by its binding to the pyrophosphate portion of membrane lipid II [1], but some evidences point out to alternative mechanisms. Results from assays with mutacin 1140 hybrids [2] showed that the portion of nisin that is not involved with lipid II binding could damage the bacterial cell, independently of pore formation [3,4]. Moreover, there are insufficient data to explain how nisin affects the bacterial survival. In this scenario, proteomics is an interesting approach, as a comparison between treated and untreated cells may provide insights of both antimicrobial mechanisms of action and bacterial response mechanisms [5].

Proteomic profiling of the infective trophozoite stage of Acanthamoeba polyphaga.

Acanthamoeba polyphaga is a free-living protozoan pathogen, whose infective trophozoite form is capable of causing a blinding keratitis and fatal granulomatous encephalitis in humans. The damage caused by A. polyphaga trophozoites in human corneal or brain infections is the result of several different pathogenic mechanisms that have not yet been elucidated at the molecular level. We performed a comprehensive analysis of the proteins expressed by A. polyphaga trophozoites, based on complementary 2-DE MS/MS and gel-free LC-MS/MS approaches. Overall, 202 non-redundant proteins were identified. An A. polyphaga proteomic map in the pH range 3-10 was produced, with protein identification for 184 of 370 resolved spots, corresponding to 142 proteins. Additionally, 94 proteins were identified by gel-free LC-MS/MS. Functional classification revealed several proteins with potential importance for pathogen survival and infection of mammalian hosts, including surface proteins and proteins related to defense mechanisms. Our study provided the first comprehensive proteomic survey of the trophozoite infective stage of an Acanthamoeba species, and established foundations for prospective, comparative and functional studies of proteins involved in mechanisms of survival, development, and pathogenicity in A. polyphaga and other pathogenic amoebae.

Isolation and genotyping of free-living environmental isolates of Acanthamoeba spp. from bromeliads in Southern Brazil.

Species of Acanthamoeba are frequently isolated from distinct environmental sources such as water, soil, dust and air. They are responsible to cause infections and disease in humans and animals. In addition, Acanthamoeba sp. are considered an important reservoir of bacteria, virus and fungi, which act as "Trojan horses" to protect these microorganisms of harsh environmental conditions. In this study, nine Acanthamoeba isolates from bromeliads phylloplane were identified based on the morphology of cyst and trophozoite forms. The genotype level was accessed by the sequence analysis of Acanthamoeba small-subunit rRNA gene. Genotypic characterization grouped five isolates in the genotype T2/T6, three in the T4 genotype and one in the genotype T16. The results obtained indicate that the genotype T2/T6 is common on phylloplane. To predict the pathogenic potential of the Acanthamoeba isolates, thermo and osmotolerance assays were employed, although all isolates were capable of surviving at temperatures of 37°C, other tests will be conducted in the future to determine the potential pathogenic of the isolates. Altogether, our results revealed the importance of the presence of Acanthamoeba associated with bromeliads in Rio Grande do Sul, Brazil, and the necessity for further studies to determine the environmental distribution and the role of these species.

Prevalence of acanthamoeba from tap water in rio grande do Sul, Brazil.

A total of 136 samples of tap water were collected from state and municipal schools between March and November 2009. The samples were filtered through cellulose nitrate membranes that were seeded at non-nutrient agar 1.5% containing an overlayer of Escherichia coli suspension. Thirty-one (22.79%) tap water samples investigated were found positive for free-living amoebae (FLA). From these, 13 presented as FLA that seems to belong to the genus Acanthamoeba. All samples of FLA were cloned and identified as belonging to the genus Acanthamoeba by the morphology of cysts and trophozoites and by PCR using genus-specific primers that amplify the ASA.S1 region of 18S rDNA gene. Physiological tests of thermotolerance and osmotolerance were used to evaluate the pathogenicity of the isolates. The sequencing analysis by comparing the sequences submitted to GenBank, showed genotype distribution into groups T2, T2/T6, T6, and T4. In tests of thermotolerance and osmotolerance, 50% of the isolates had a low pathogenic potential. The results indicated the presence of Acanthamoeba in tap water in Rio Grande do Sul, Brazil, revealing its importance and the need for more epidemiological studies to determine their distribution in the environment and its pathogenic potential.

Acanthamoeba T3, T4 and T5 in swimming-pool waters from Southern Brazil.

Species of Acanthamoeba, known to cause keratitis (AK) and granulomatous encephalitis in humans are frequently isolated from a variety of water sources. In this study, 13 Acanthamoeba isolates from swimming pools were classified at the genotype level based on the sequence analysis of the Acanthamoeba small-subunit rRNA gene. Nine of the 13 isolates were genotype T5, three were genotype T4, and one was T3. Several genotypes have been reported worldwide as causative agents of AK, including genotypes T3, T4, and T5. The present study indicates that genotype T5 is a common contaminant in swimming-pool water.

Bacteriocin-like substance from Bacillus amyloliquefaciens shows remarkable inhibition of Acanthamoeba polyphaga.

The effectiveness of a bacteriocin-like substance (BLS) produced by Bacillus amyloliquefaciens was tested against Acanthamoeba polyphaga strains, and its cytotoxic potential on Vero cells was investigated. Amebicidal activity of the purified BLS was tested by plate bioassays with concentrations ranging from 12.5 to 6,400 AU mL(-1). Damage to A. pholyphaga cells was monitored using an inverted microscope and counted in a Fuchs-Rosenthal chamber after 24, 48, and 72 h. According to the results obtained, the BLS showed remarkable amebicidal and amebostatic effect on A. polyphaga and showed no cytotoxicity on the Vero cells. These results may have great relevance in the development of new acanthamoebicidal compounds.

Susceptibility of Aedes aegypti (Diptera: Culicidae) to Acanthamoeba polyphaga (Sarcomastigophora: Acanthamoebidae).

To date there is no report on mosquitoes infected with free-living amoebae. For this reason, the aim of this study was to verify if Aedes aegypti could be susceptible to Acanthamoeba polyphaga under laboratory conditions, so trophozoites were offered as a unique food resource for larvae of first instar. The results show that those amoebae are able to infect and colonize the mosquito gut and could be re-isolated of all stages of the mosquito (larvae, pupae, and adults).

Potentially pathogenic acanthamoeba isolated from a hospital in Brazil.

Studies on free-living amoebae (FLA), has been increased in recent years, especially related to the genus Acanthamoeba, because these organisms are widely found in the environment. The present work isolated and characterized this organism from biofilms and dust in hospital environment. 135 samples were collected in 15 different environments in a hospital at the south of Brazil. Thirty-one (23%) isolates were identified as morphologically belonging to the Acanthamoeba genus and 10 of these were submitted to temperature and osmotolerance tests as criterion for evaluation of the viability and pathogenicity. The tests indicate that four (40%) of these isolates could be potentially pathogenic because grew at high temperature (40 degrees C) and osmolarity (mannitol 1 M). Some isolates genotypes were determined after ribosomal DNA sequencing. These data revealed that three dust isolates belong to T4, two biofilm isolates to T5 and one dust isolate to T3 genotype. Therefore, Acanthamoeba found in the hospital environment represents a risk for people that circulate there.

Acanthamoeba spp. and bacterial contamination in contact lens storage cases and the relationship to user profiles.

Storage cases for contact lenses receive microbiota from the environment, body, and eye, which can form biofilms. These biofilms, in addition to causing discomfort and cloudy vision, can cause local irritation, facilitate the adherence of microorganisms, and lead to infection. The objective of this study was to evaluate the presence of bacteria and Acanthamoeba spp. in the biofilm and solutions in contact lens storage cases, and to assess their relationships to the habits of contact lens wearers. Eighty-one volunteers assembled from the ophthalmology section of a public hospital and from the Central Campus of the federal university, both in the state of Rio Grande do Sul, Brazil, provided the contact lens storage cases. The samples collected were inoculated into sheep blood agar, to isolate bacteria; and into 1.5% non-nutrient agar with an overlayer of Escherichia coli, to isolate free-living amoebas. Of the 81 samples analyzed, 58 (71%) showed bacterial growth and seven (8.6%) were positive for Acanthamoeba spp. The amoebas were identified according to the morphological criteria of Page (A new key to fresh water and soil gymnamoebae, Freshwater Biology Association, Ambleside, UK, 1988) and confirmed by PCR. The storage cases that were positive for Acanthamoeba spp. had a mean of 10(7) UFC/mL and belonged to individuals who had not taken sufficient care with hand washing.