Prevalence of <Emphasis Type="Italic">Acanthamoeba</Emphasis> 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.     

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°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.     

Isolation and Genotyping of Acanthamoeba Strains from Soil Sources from Jamaica,West Indies

Acanthamoeba spp. are opportunistic pathogens that are ubiquitous in nature. Many species of this genus are responsible for a fatal encephalitis and keratitis in humans and other animals. Seventy‐two soil samples were collected from the parishes across Jamaica and assessed for the presence of Acanthamoeba spp. Cultivation was carried out on non‐nutrient agar plates seeded with heat killed Escherichia coli. PCR and sequencing of the DF3 region were carried out in order to genotype the isolated strains of Acanthamoeba. Thermotolerance and osmotolerance assays were utilized to investigate the pathogenic potential of the Acanthamoeba isolates. Acanthamoeba spp. was isolated from 63.9% of soil samples. Sequencing of the DF3 region of the 18S rDNA resulted in the identification of genotypes T4, T5, and T11. T4 genotype was most frequently isolated. Most isolates were thermotolerant or both thermotolerant and osmotolerant, indicating that they may present the potential to cause disease in humans and other animals.     

Characterization of Isolates of Acanthamoeba from the Nasal Mucosa and Cutaneous Lesions of Dogs

Acanthamoeba spp. are free-living amoebae that are ubiquitously distributed in the environment and can cause encephalomyelitis in animals and humans. The factors that contribute to Acanthamoeba infections include parasite biology, genetic diversity, environmental spread, and host susceptibility. The aim of the present study was to characterize isolates of Acanthamoeba from the nasal mucosa and cutaneous lesions of dogs in order to access the occurence and pathogenicity of these organisms in this animal group. We studied 13 isolates of Acanthamoeba confirmed by polymerase chain reaction. They were sequenced, the genotype was determined, and their potential of pathogenicity was evaluated.     

Is Acanthamoeba pathogenicity associated with intracellular bacteria?

In addition to the possible role of Acanthamoeba as an evolutionary precursor of pathogenicity in microbial pathogens, it has been suggested that intracellular bacteria or other microbial endosymbionts may also enhance the pathogenicity of Acanthamoeba. Using transmission electron microscopy, polymerase chain reaction and simple culturing, our findings did not reveal any apparent evidence of microbial presence intracellularly of a recently recovered clinical isolate of Acanthamoeba. Based on these findings, it is tempting to speculate that the virulence of Acanthamoeba may not be attributed to the pathogenicity of the endosymbiont alone.     

Acanthamoeba Can Be Differentiated by the Polymerase Chain Reaction and Simple Plating Assays

Acanthamoeba are opportunistic pathogens with invasive and noninvasive species. For clinical purposes it is important to differentiate potentially pathogenic from nonpathogenic isolates. For the rapid and sensitive identification of Acanthamoeba at the genus level, we used a polymerase chain reaction (PCR)-based method which detected as few as five cells. Further, we tested nine isolates of Acanthamoeba for their ability to produce cytopathic effects (CPE) on corneal epithelial cells. On the basis of the results, Acanthamoeba were divided into pathogenic or nonpathogenic groups. However, because CPE assays are not available to every diagnostic laboratory, we developed a simple plating assay based on osmotolerance which correlated well with the CPE assays. Pathogenic Acanthamoeba showed growth on higher osmolarity (agar plates containing one molar mannitol), while growth of nonpathogens was inhibited on these plates. In conclusion, we have developed methods for the rapid identification and differentiation of Acanthamoeba. Received: 5 January 2001/Accepted: 6 February 2001     

Molecular characterization of <Emphasis Type="Italic">Acanthamoeba</Emphasis> strains isolated from domestic dogs in Tenerife,Canary Islands,Spain

The present study describes two cases of Acanthamoeba infections (keratitis and ascites/peritonitis) in small breed domestic dogs in Tenerife, Canary Islands, Spain. In both cases, amoebic trophozoites were observed under the inverted microscope and isolated from the infected tissues and/or fluids, without detecting the presence of other viral, fungal or bacterial pathogens. Amoebae were isolated using 2 % non-nutrient agar plates and axenified for further biochemical and molecular analyses. Osmotolerance and thermotolerance assays revealed that both isolates were able to grow up to 37 °C and 1 M of mannitol and were thus considered as potentially pathogenic. Moreover, the strains were classified as highly cytotoxic as they cause more than 75 % of toxicity when incubated with two eukaryotic cell lines. In order to classify the strains at the molecular level, the diagnostic fragment 3 (DF3) region of the 18S rDNA of Acanthamoeba was amplified and sequenced, revealing that both isolates belonged to genotype T4. In both cases, owners of the animals did not allow any further studies or follow-up and therefore the current status of these animals is unknown. Furthermore, the isolation of these pathogenic amoebae should raise awareness with the veterinary community locally and worldwide.     

Morphological,physiological, molecular and phylogenetic characterization of new environmental isolates of <Emphasis Type="Italic">Acanthamoeba</Emphasis> spp. from the region of Bratislava,Slovakia

Protozoa of the genus Acanthamoeba are organisms that can be generally found in the environment. The focus of this study is the detection of the presence of Acanthamoeba in different water sources and samples taken from airconditioning units. The identification of Acanthamoeba isolates was based on the morphology of cysts and trophozoites as well as PCR amplification with a genus specific primer pair JDP1 and JDP2. Growth characteristics and temperature tolerance were monitored. The pathogenic potential was tested in vitro on Vero cell cultures. Genotype identification was based on the sequencing of the GTSA.B1 PCR amplimer of 18S ribosomal DNA. The data obtained revealed that the isolates belong to T3 and T4 genotypes. One T3 and one T4 isolate contain a group I intron. The 933 base pair intron found in a genotype T4 isolate is considerably larger compared to formerly described introns of Acanthamoeba griffini (genotype T3) and A. Lenticulata (genotype T5). This is the first report detailing the environmental distribution of the Acanthamoeba genotypes in the region of Bratislava, Slovakia.     

Biological characterization of a clinical and an environmental isolate of <Emphasis Type="Italic">Acanthamoeba polyphaga</Emphasis>: analysis of relevant parameters to decode pathogenicity

Acanthamoeba spp. consists of free-living amoebae, widespread in nature, which occasionally can cause human infections including granulomatous amoebic encephalitis and amoebic keratitis. Acanthamoeba pathogenesis is not entirely known and correlations between pathogenic potential and taxonomy are complex issues. In order to decipher the definition of a pathogenic amoeba, the objective of this work was to decipher the definition of pathogenic amoeba by characterizing two isolates of Acanthamoeba polyphaga obtained from different origins (a keratitis patient and freshwater), looking for differences among them. The clinical isolate grew faster in Peptone-yeast extract-glucose (PYG) medium, transformed more rapidly from a trophozoite to cyst and exhibited increased cytopathic effect on cultured cells. Morphological differences were also noted, since freshwater amoebae presented more acanthopodia than the clinical isolate. Moreover, actin labeling demonstrated that microfilament organization varies between isolates, with the presence of locomotory structures as lobopodia and lamellipodia in the keratitis isolate, which were less adherent on plastic. Zymography demonstrated that the keratitis isolates presented higher proteolytic activity and also were more able to invade collagen matrices. Altogether, we conclude that a group of stable physiological characteristics exist in Acanthamoeba that can be related to pathogenicity.     

Vegetative Compatibility,Pathogenicity and Virulence Diversity of Fusarium oxysporum f. sp. melongenae Recovered from Eggplant

Fusarium oxysporum (Schlechtend.: Fr.) f. sp. melongenae (Fomg) recovered from symptomatic eggplants from five eggplant‐growing areas in Turkey, including the south, west, north‐west, north and south‐east regions. The objective of this study was to investigate the genetic diversity of the Fomg isolates from different geographical location by pathogenicity and VCG tests. Three hundred and seventy‐four Fomg isolates were classified as highly virulent, virulent, moderately virulent and low virulent through pathogenicity assays. No correlation was observed between virulence of Fomg isolates and their locations. The nitrate non‐utilizing mutants (nit) were generated as nit1, nit3 and NitM, based on phenotyping of Fomg growth characteristics of the Fomg isolates on diagnostic media with various sources of nitrogen. The majority of nit mutants (39.4%) recovered were nit1 from minimal medium (MM) containing of 2.0% potassium chlorate (MMC). The most of Fomg isolates were identified as heterokaryon self‐compatible (HSC) based on their ability to form a stable heterokaryon, while four isolates were classified as heterokaryon self‐incompatible (HSI). A large amount of Fomg isolates were vegetatively compatible and assigned as members of the same VCG, whereas nit mutants of 10 Fomg isolates that did not complement with tester strains only paired by themselves (HSC), these isolates were termed vegetative incompatible (vic). The complementation of 33 isolates with tester strains was slow and quite weak, but not paired with themselves even though they are HSC. About 96.3% of the Fomg isolates were assigned to VCG 0320, while the remaining 3.7% were classified as vegetative incompatible group.     

Analysis of Cultural and Genetic Diversity in Alternaria helianthi and Determination of Pathogenic Variability Using Wild Helianthus Species

The study aims at assessment of morphological, molecular and pathogenic variability of Alternaria helianthi, incitant of leaf blight of sunflower. Morphological characteristics determined for 26 isolates of A. helianthi from India revealed variations in shape of culture, pigmentation, conidial measurements, number of septa and colony growth. The conidia of isolate Ah‐7 were long, while conidia of isolate Ah‐15 were short. Based on cultural characters, isolates were classified into four groups. Genetic variability of the isolates was assessed by random amplified polymorphic DNA analysis. Good polymorphism was observed and cluster analysis indicated presence of six genetically distinct groups among the isolates. The isolates Ah‐1, Ah‐7 and Ah‐14 were genetically distinct. Resistant sources are not available in cultivated sunflower, while wild Helianthus species possess resistance to multiple stresses. We evaluated reaction of wild Helianthus species to isolates of A. helianthi. Among wild Helianthus species, H. tuberosus followed by H. occidentalis showed moderately to highly resistant reaction to all the isolates and recorded less disease incidence. The species H. argophyllus followed by H. laevigatus showed more disease incidence. The cultivated sunflower recorded susceptible reaction to most of the isolates and recorded high disease incidence. The isolates differed significantly for pathogenic reaction and were grouped into three pathogenicity groups; low, medium and high. Six isolates induced <20% disease incidence and were included in the low pathogenicity group. Majority of isolates were in the medium pathogenicity group. Six isolates i.e. Ah‐9, Ah‐10, Ah‐18, Ah‐20, Ah‐24 and Ah‐26 induced more than 50% disease incidence and were considered high pathogenicity group. Our results demonstrate the existence of considerable variation in resistance of Helianthus species to A. helianthi and also in morphological and genetic characters of A. helianthi isolates prevalent in India.     

Acanthamoeba spp. in domestic tap water in houses of contact lens wearers in the metropolitan area of Mexico City

A survey was carried out in the metropolitan area of Mexico City to determine the presence of Acanthamoeba in the tap water of houses of contact lens wearers. Water samples were taken from the mains water entry, bathroom sinks and storage containers (roof tanks, cisterns) of 27 houses; and from the solution contained in the contact lens cases. Samples were filtered and cultured onto NNE medium. The isolates were identified based on their morphological features and pathogenicity. Total and fecal coliforms, water temperature, pH, dissolved oxygen and residual free-chlorine were measured by standard methods. Forty five isolates of Acanthamoeba from 200 water samples were obtained. The highest number of amoebae was isolated from cisterns and roof tanks. Most Acanthamoeba isolates were non-pathogenic, however, their presence in tap water is a potential hazard since some species can cause Acanthamoeba keratitis and granulomatous amoebic encephalitis.     

Evaluation of real‐time PCR methods for quantification of Acanthamoeba in anthropogenic water and biofilms

Aims: To assess two real‐time PCR methods (the Riviere and Qvarnstrom assays) for environmental Acanthamoeba. Methods and Results: DNA extracted from Acanthamoeba castellanii taken from water and biofilms of cooling towers was analysed by the Riviere and Qvarnstrom assays. To quantify environmental Acanthamoeba, the calibration curves (DNA quantity vs cell number) were constructed with samples spiked with A. castellanii. The calibration curves for both quantitative PCR assays showed low variation (coefficient of variation of C_t≤ 5·7%) and high linearity (R² ≥ 0·99) over six orders of magnitudes with detection limit of three cells per water sample. DNA quantity determined by Qvarnstrom assay was equivalent between trophozoites and cysts (P = 0·49), whereas a significant difference was observed with Riviere assay (P < 0·0001). Riviere assay failed to detect Acanthamoeba in 21% (15/71) of the environmental samples which were positively detected by Qvarnstrom assay, while one sample (1·4%) was shown positive by Riviere assay but negative by Qvarnstrom assay. Moreover, Acanthamoeba counts by Qvarnstrom assay were greater than those by Riviere assay (P < 0·0001). Conclusions: Qvarnstrom assay performs better than Riviere assay for detection and quantification of Acanthamoeba in anthropogenic water and biofilms. Significance and Impact of the Study: Qvarnstrom assay may significantly contribute to a better knowledge about the distribution and abundance of Acanthamoeba in environments.     

Infection in a rat model reactivates attenuated virulence after long-term axenic culture of Acanthamoeba spp

Prolonged culturing of many microorganisms leads to the loss of virulence and a reduction of their infective capacity. However, little is known about the changes in the pathogenic strains of Acanthamoeba after long culture periods. Our study evaluated the effect of prolonged culturing on the invasiveness of different isolates of Acanthamoeba in an in vivo rat model. ATCC strains of Acanthamoeba, isolates from the environment and clinical cases were evaluated. The in vivo model was effective in establishing the infection and differentiating the pathogenicity of the isolates and re-isolates. The amoebae cultured in the laboratory for long periods were less virulent than those that were recently isolated, confirming the importance of passing Acanthamoeba strains in animal models.     

Entomopathogenicity of beauveria bassiana and metarhizium anisopliae to the cowpea aphid,aphis craccivora koch (homoptera: Aphididae)

The pathogenicity of four isolates each of the entomopathogenic fungi, Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) Sorok. to apterous adult Aphis craccivora Koch was evaluated in the laboratory at 4 concentrations of conidia. All fungi isolates tested were found to be pathogenic to the insect but their virulence varied among species and isolates within species. Three isolates, B. bassiana CPD 11 and M. anisopliae CPD 4 and 5 caused significantly higher mortality than the other isolates at the various concentrations tested causing mortality of between 58–91%, 64 to 93% and 66–100%, respectively, at 7 days post treatment. At the highest concentration of 1 × 10⁸conidiaml^‐1, these isolates produced the shortest LT₅₀s of 3.5, 3.6 and 3.4 days, respectively. Their LC₅₀s were 6.8 × 10⁵, 3.1 × 10⁵ and 2.7 × 10⁵ conidia ml^‐1, respectively. The results indicate that these isolates are promising candidates for the control of the cowpea aphid but their pathogenicity to various aphid non‐target beneficial organisms within the cowpea agroecosystem warrant further investigation before initiating field control.     

Phenotypic traits,virulence‐associated gene profile and genetic relatedness of Edwardsiella tarda isolates from Japanese eel Anguilla japonica in Korea

Edwardsiella tarda is the predominant bacterium in farm‐cultured eel in Korea. Here, we evaluated the heterogeneity of 37 E. tarda isolates derived from Japanese eel with various origins (olive flounder, common carp and ornamental fish) between 2003 and 2010. Regardless of origins, the biochemical characteristics of E. tarda isolates were homogenous except hydrogen sulfide production, citrate utilization and mannitol fermentation. Based on the phylogenetic analysis of 16S rRNA, E. tarda isolates could be classified into two subgroups and displayed a close relation with Edwardsiella ictaluri and Edwardsiella hosinae lineages, suggesting that the subgroup I has been a predominant type in the Jeonnam and Jeonbuk provinces. I‐CeuI‐based pulsed‐field gel electrophoresis (PFGE) typing showed that the isolates from Japanese eels belonged to 11 pulsotypes, indicating that the presence of highly genomic diversity. Additionally, two isolates, ET‐060 and ET‐191, showed a high frequency of virulence genes (100%) and caused 90% and 60% mortality in Japanese eel, respectively. This finding suggests a substantial congruence of virulence gene profiles and pathogenicity. Our results demonstrate that the intraspecific diversity within E. tarda strains from Japanese eel has been in prior existence.

Significance and Impact of the Study

Based on the biochemical characteristics, the phylogenetic property of the 16S rRNA gene and PFGE types of Edwardsiella tarda, we could identify the intraspecific diversity of isolates from Japanese eel, Anguilla japonica in Korea. In addition, this study describes the strong congruence of virulence‐related genes and pathogenicity, suggesting that the virulence profile may be useful tool for prediction of pathogenicity.     

Correlations between Morphological, Molecular Biological, and Physiological Characteristics in Clinical and Nonclinical Isolates of Acanthamoeba spp.

Eleven Acanthamoeba isolates, obtained from Acanthamoeba keratitis patients, from contact lens cases of non-Acanthamoeba keratitis patients, from asymptomatic individuals, from necrotic tissue, and from tap water and two reference strains were investigated by morphological, molecular biological, and physiological means in order to discriminate clinically relevant and nonrelevant isolates. All clinically relevant isolates showed Acanthamoeba sp. group II morphology. 18S ribosomal DNA sequencing revealed sequence type T4 to be the most prevalent group among the isolates and also the group recruiting most of the pathogenic strains. Interestingly, within T4 the strains of no clinical relevance clustered together. Moreover, physiological properties appeared to be highly consistent with initial pathogenicity and with sequence clustering. Altogether, the results of our study indicate a correlation between the phylogenetic relationship and pathogenicity.     

Pathogen–pathogen interactions: a comparative study of Escherichia coli interactions with the clinical and environmental isolates of Acanthamoeba

In this study, we compared the interactions of invasive and non-invasive strains of E. coli with clinical and environmental isolates of Acanthamoeba. The environmental isolate of Acanthamoeba exhibited significantly higher association with E. coli compared with the clinical isolates of Acanthamoeba. The ratio of E. coli per amoebae was more than 8-fold higher in the environmental isolate compared with the clinical isolates of Acanthamoeba. Interestingly, non-pathogenic environmental Acanthamoeba showed uptake and/or survival of the non-invasive E. coli. In contrast, clinical isolates of Acanthamoeba did not support uptake and/or survival of non-invasive E. coli. Using several mutants derived from K1, we demonstrated that outer membrane protein A (OmpA) and lipopolysaccharide (LPS) are crucial bacterial determinants responsible for E. coli K1 interactions and in the intracellular survival of E. coli in Acanthamoeba. The use of Acanthamoeba as a model to study E. coli K1 pathogenesis and to understand bacterial immune evasion strategies is discussed further.     

Caspofungin‐induced in‐vitro post‐antifungal effect and its impact on adhesion related traits of oral Candida dubliniensis and Candida albicans isolates

Adhesion to buccal epithelial cells (BEC) and denture acrylic surfaces (DAS), germ tube (GT) formation and cell surface hydrophobicity (CSH) are all virulence traits involved in the pathogenicity of Candida. Post‐antifungal effect (PAFE) also have a bearing on pathogenicity and virulence of Candida. Candida dubliniensis is associated with oral and systemic candidosis, which can be managed with caspofungin. There is no published information on caspofungin‐induced PAFE and its impact on adhesion traits of C. dubliniensis isolates. Thus, the purpose of this investigation was to determine the in vitro duration of PAFE on 20 C. dubliniensis isolates following transient exposure to caspofungin. Furthermore the impacts of caspofungin‐induced PAFE on adhesion to BEC and DAS, GT formation and CSH of these isolates were also determined. After establishing the minimum inhibitory concentration (MIC) of caspofungin, C. dubliniensis isolates were exposed to sub‐lethal concentrations (×3 MIC) of caspofungin for 1 hr. Thereafter the duration of PAFE, adhesion to BEC and DAS, GT formation and CSH were determined by previously described in‐vitro assays. MIC (μg/mL) of C. dubliniensis isolates to caspofungin ranged from 0.004 to 0.19. Caspofungin‐induced mean PAFE on C. dubliniensis isolates was 2.17 hr. Exposure to caspofungin suppressed the ability of C. dubliniensis isolates to adhere to BEC and DAS, form GT and CSH by 69.97%, 71.95%, 90.06% and 32.29% (P < 0.001 for all), respectively. Thus, transient exposure of C. dubliniensis isolates to caspofungin produces an antifungal effect not only by suppressing its growth but also by altering its adhesion traits.     

The Characterization of an Adrenergic Signalling System Involved in the Encystment of the Ocular Pathogen Acanthamoeba spp.

The aim of this study was to identify and characterize the receptor system involved in controlling encystment in Acanthamoeba using specific agonists and antagonists and to examine whether endogenous stores of catecholamines are produced by the organism. Acanthamoeba trophozoites suspended in axenic growth medium were exposed to adrenoceptor agonists and antagonists to determine which compounds promoted or prevented encystment. Second, trophozoites were cultured in medium containing a catecholamine synthesis inhibitor to investigate the effect this had on natural encystment. Nonspecific adrenoceptor agonists including epinephrine, isoprotenerol, and the selective β1 adrenoceptor agonist dobutamine were found to cause > 90% encystment of Acanthamoeba trophozoites compared to < 30% with the controls. The selective β1 antagonist metoprolol was able to inhibit epinephrine mediated encystment by > 55%. Cultures of Acanthamoeba with the catecholamine synthesis inhibitor α‐methyl‐p‐tyrosine significantly reduced the level of amoebic encystment compared to controls. In conclusion, Acanthamoeba appear to contain a functional adrenergic receptor system of unknown structure which is involved in initiating the encystment process that can be activated and blocked by β1 agonists and antagonists respectively. Furthermore, the presence of this receptor system in Acanthamoeba indicates that topical β adrenoceptor blockers may be effective adjunct therapy by reducing the transformation of trophozoites into the highly resistant cyst stage.