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     

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.     

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.     

Distinct immunomodulatory properties of extracellular vesicles released by different strains of Acanthamoeba

Free living amoeba of the genus Acanthamoeba are opportunist protozoan involved in corneal, systemic, and encephalic infections in humans. Most of the mechanisms underlying intraspecies variations and pathogenicity are still unknown. Recently, the release of extracellular vesicles (EVs) by Acanthamoeba was reported. However, comparative characterization of EVs from distinct strains is not available. The aim of this study was to evaluate EVs produced by Acanthamoeba from different genotypes, comparing their proteases profile and immunomodulatory properties. EVs from four environmental or clinical strains (genotypes T1, T2, T4, and T11) were obtained by ultracentrifugation, quantitated by nanoparticle tracking analysis and analyzed by scanning and transmission electron microscopy. Proteases profile was determined by zymography and functional properties of EVs (measure of nitrite and cytokine production) were determined after peritoneal macrophage stimulation. Despite their genotype, all strains released EVs and no differences in size and/or concentration were detected. EVs exhibited a predominant activity of serine proteases (pH 7.4 and 3.5), with higher intensity in T4 and T1 strains. EVs from the environmental, nonpathogenic T11 strain exhibited a more proinflammatory profile, inducing higher levels of Nitrite, tumor necrosis factor alpha and interleukin-6 via TLR4/TLR2 than those strains with pathogenic traits (T4, T1, and T2). Preincubation with EVs treated with protease inhibitors or heating drastically decreased nitrite concentration production in macrophages. Those data suggest that immunomodulatory effects of EVs may reflect their pathogenic potential depending on the Acanthamoeba strains and are dependent on protease integrity.     

Genotyping of Acanthamoeba isolates from clinical and environmental specimens in Iran

In this study, 15 Acanthamoeba isolates from AK patients and 10 environmental samples (water, soil and animal-origin samples) were classified at the genotype level based on the sequence analysis of the Diagnostic Fragment 3 (DF3) of Acanthamoeba small subunit rRNA gene. The obtained results revealed that most of these Acanthamoeba strains belonged to genotype T4 both in clinical and environmental samples. The presence T11 genotype in clinical samples was also revealed after the genotyping analysis and to our knowledge this is the first report of T11 genotype in Iran. Moreover, the isolation of T4 genotype from cow faeces in this study highlights a possible transmission of Acanthamoeba through animal faeces in Iran.Overall, the widespread distribution of pathogenic Acanthamoeba T4 across the environmental sources and the increasing number of contact lens wearers in Iran, demands more awareness within the public and health professionals as this pathogen is emerging as a risk for human health in Iran and worldwide.     

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.     

Pathogenicity, Morphology, and Differentiation of Acanthamoeba

Acanthamoeba keratitis is sight threatening corneal infection caused by pathogenic Acanthamoeba. Previous studies have shown the genotypic differences between pathogenic and non-pathogenic species/strains of Acanthamoeba. In this study, we examined the morphological differences between pathogenic and non-pathogenic species/strains using scanning electron microscopy. Pathogenic Acanthamoeba exhibited higher number of acanthopodia (structures associated with the binding of amoeba to the target cells) as compared to non-pathogens. In addition, interactions of amoeba with the corneal epithelial cells were studied. Only pathogenic amoeba exhibited adhesion to epithelial cells. Further results indicated that phagocytosis occurs in the pathogenic amoeba by the formation of amoebastome (characteristic of amoeba phagocyte). This study showed that Acanthamoeba phagocytosis may be both an efficient means of obtaining nutrients for the amoeba and a significant factor in the pathogenesis of Acanthamoeba infections. Received: 2 April 2001 / Accepted: 12 April 2001     

Molecular Characterization of Acanthamoeba spp. Occurring in Water Bodies and Patients in Poland and Redefinition of Polish T16 Genotype

Acanthamoeba genus is divided into 20 genotypes (T1–T20) on the basis of the gene encoding 18S rRNA sequence. Using of at least 2 kbp gene fragments is strongly recommended to identify new genotypes and 5% difference is commonly used as a criterion of new genotypes, however, this value is questionable. In this paper, Polish Acanthamoeba strains described earlier on the basis of ~850 bp Ami fragment of 18S rRNA gene as T4, T11 and a new T16 genotype, have been analyzed using near‐complete sequence of the gene. This analysis was needed because the Ami fragment does not reveal full variability within 18S rRNA gene. Phylogenetic analysis based on Ami fragment is biased by artifacts in the construction of the tree, so the fragment should not be used for identification of new putative Acanthamoeba genotypes. The analysis confirmed that the Polish sequences represent T4 and T11 genotypes and that the strains described earlier as T16 genotype are in fact a new subgroup of the T20 genotype and that this genotype should be divided into two subgroups: T20a (two strains described by [J. Eukaryot. Microbiol. 62 (2015) 69]) and T20b (11 Polish strains described in this study). The T20b subgroup was isolated from both clinical samples and water bodies used by people as bathing places and there is a risk of infection for humans during contact with water.     

A systematic analysis of Acanthamoeba genotype frequency correlated with source and pathogenicity: T4 is confirmed as a pathogen-rich genotype

Acanthamoeba is a genus of facultative human parasites that is currently classified into 17 genotypes (T1–T17) each of which arguably represents a species. These amoebae cause Acanthamoeba Keratitis (AK) a disease of the eye, and a rare but usually fatal Granulatomous Acanthamoeba Encephalitis (GAE). A database of strains derived from the literature and a number of fresh isolates has been constructed to detect trends of pathogenic and other associations with these genotypes. One genotype in particular, T4, was found to be over represented in human disease. The prevalence of this genotype has been commented upon previously, however T4 is also the most common type isolated from the environment. Our statistical analysis of the database allows us to claim that T4 is in fact the genotype most often associated with human disease, even after its abundance in the general environment is taken into account. T3 and T11 are closest relatives to T4 and they are the second and third most often associated with AK. A number of other more subtle correlations also emerge from this analysis.     

Isolation and identification of Acanthamoeba species related to amoebic encephalitis and nonpathogenic free‐living amoeba species from the rice field

Aims: Isolation and characterization of the clinically relevant amphizoic amoebas in vegetated farmlands, which may present a risk to farmers’ health. Methods and Results: Acanthamoeba species was isolated and characterized via morphological and molecular means in the rice field where the patient was exposed to rice paddy water which most probably was the point of infection. An Acanthamoeba sp. abundant in the rice field was identified. Genotyping showed the strain to be genotype T4, which was identical to the amoebic parasite found in patient’s cerebrospinal fluid. During the course of the study, three nonpathogenic free‐living amoeba species were also isolated and characterized for the first time in Taiwan. Conclusions: This study successfully located a possible source of granulomatous amoebic encephalitis in a patient and provided the first evidence that Acanthamoeba genotype T4 may be a potential pathogen in Taiwan. Significance and Impact of the Study: The integration of field survey, clinical data and morphological and genetic examination represents a sound strategy for investigation of the possible role of free‐living amoebae in causing human diseases. Future work should include investigating the potential contributory role of other nonpathogenic free‐living protozoa in disease of livestock or even human.     

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.     

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.     

Detection of type III secretion gene as an indicator for pathogenic Edwardsiella tarda

Aims: To differentiate pathogenic and nonpathogenic Edwardsiella tarda strains based on the detection of type III secretion system (T3SS) gene using polymerase chain reaction (PCR). Methods and Results: Primers were designed to amplify Edw. tarda T3SS component gene esaV, catalase gene katB, haemolysin gene hlyA and 16S rRNA gene as an internal positive control. Genomic DNAs were extracted using a commercial isolation kit from 36 Edw. tarda strains consisting of 18 pathogenic and 18 nonpathogenic strains, and 50 ng of each DNA was used as the template for PCR amplification. PCR was performed with a thermocycler (TaKaRa TP600) in a 25‐μl volume. Products of esaV were detected in all pathogenic strains, but not in nonpathogenic strains; katB was detected in all pathogenic strains and one of nonpathogenic strains; hlyA was not detected in any strains. Conclusions: The detection of esaV gene can be used for the assessment of pathogenic Edw. tarda strains. Significance and Impact of the Study: The strategy using T3SS gene as the virulence indicator provides a useful tool for the clinical assessment of pathogenic Edw. tarda strains and prediction of edwardsiellosis risk in fish culture environments.     

Isolation and Molecular Characterization of Acanthamoeba Strains from Dental Units in Costa Rica

Free‐living amoebae are protozoa widely distributed in nature, which can be found in a variety of environments. Four genera are recognized as causal agents of infections in humans and animals: Acanthamoeba, Naegleria, Balamuthia, and Sappinia. In this study, the presence of Acanthamoeba in dental units was determined and the isolates obtained were molecularly characterized; osmotolerance and thermotolerance assays were also performed to evaluate multiplication under these conditions, frequently associated with pathogenicity. The morphological analysis and partial sequencing of the 18S rDNA gene revealed the presence of Acanthamoeba genotype T4 in 14% of the units sampled. Osmotolerance and thermotolerance tests were positive for more than 80% of the isolates. Up to date, this is the first study that reports the detection, identification, and genotyping of Acanthamoeba isolated from dental units in Costa Rica and even in Latin‐America. Further assays to determine the potential pathogenicity of these Acanthamoeba isolates are underway.     

Identification and characterization of Francisella species from natural warm springs in Utah, USA

Aims: To characterize Francisella isolated from two natural warm springs in Utah and compare them to a strain isolated from a patient with probable exposure to one of the springs in 2001. Methods and Results: A total of 39 presumptive Francisella isolates were obtained from two springs, Wasatch Hot Spring and Hobo Warm Spring, just north of Salt Lake City, Utah. All isolates were characterized by a combination of biochemical and molecular analyses, including novel PCR/electrospray ionization‐mass spectrometry (ESI‐MS) typing assays. Thirty‐one were identified as F. philomiragia, while the remaining eight were identified as F. tularensis ssp. novicida. Phylogenetic analysis of the 16S rRNA sequences revealed 27 isolates, which clustered with F. philomiragia, albeit into two distinct clades. The remaining isolates clustered along with other F. tularensis strains including the Utah clinical isolate. Testing with the PCR/ESI‐MS assays confirmed the identities of the isolates, but both yielded DNA signatures distinct from that of the clinical isolate. Conclusion: We were successful in isolating several Francisella strains from natural warm springs; however, none appeared to genetically match the original 2001 clinical isolate. Significance and Impact of the Study: This work highlights the presence of viable, potentially pathogenic Franscisella species living in the unique environmental niche of natural warm springs.     

Genetic relationships of Edwardsiella strains isolated in China aquaculture revealed by rep-PCR genomic fingerprinting and investigation of Edwardsiella virulence genes

Aims: The aim of this study was to classify Edwardsiella strains isolated from China aquaculture based on biochemical and molecular methods. Methods and Results: In this study, biochemical characterization of 19 Edwardsiella tarda isolates and two Edwardsiella ictaluri isolates was performed with API 20E system. Other pathogenicity‐related phenotypes such as haemagglutination, haemolytic activities and lethality to fish were also examined in these strains. As it was difficult to categorize the subgroups of Edw. tarda according to their origins or phenotypic properties, three PCR‐based methods, i.e. PCR amplification of virulence genes, Enterobacterial repetitive intergenic consensus‐PCR and BOX‐PCR, were carried out to further resolve the relatedness of the Edw. tarda isolates. As a result, all Edw. tarda isolates could be generally grouped into pathogenic and nonpathogenic branches before being classified into strain‐specific or origin‐specific clades. Conclusions: Biochemical characterization was sensitive for interspecific typing, while PCR‐based approaches permitted a more accurate discrimination for intraspecific typing resulting in pathogenic and nonpathogenic clusters and further more delicate clades for Edwardsiella. Significance and Impact of the Study: PCR‐based genomic fingerprinting to study the relatedness and trace the pathogenicity of the Edwardsiella strains will be helpful in investigating the virulence factors of Edwardsiella and in the development of vaccines and diagnostics for edwardsiellosis.     

Starch Gel Electrophoresis: An Effective Method for Separation of Pathogenic and Nonpathogenic Naegleria Strains*

SYNOPSIS. Isoenzyme electrophoresis of 7 different enzyme systems was used to compare 24 strains of Naegleria fowleri and 6 strains of N. gruberi. The 30 strains could be grouped into 4 distinct categories based upon zymogram patterns. No interstrain band variation in all enzyme systems was demonstrated in pathogenic strains of N. fowleri. Three nonpathogenic high temperature-tolerant strains of Naegleria had similar zymograms. Four of the 5 remaining nonpathogenic Naegleria strains had no interstrain band variation. Based upon zymograms, the 22 pathogenic strains constitute a homogenous species. Similarly the high temperature-tolerant nonpathogenic strains formed a cohesive group. The remaining nonpathogenic strains could be separated into 2 groups.     

Phylogenetic and Variable-Number Tandem-Repeat Analyses Identify Nonpathogenic Xanthomonas arboricola Lineages Lacking the Canonical Type III Secretion System

Xanthomonas arboricola is conventionally known as a taxon of plant-pathogenic bacteria that includes seven pathovars. This study showed that X. arboricola also encompasses nonpathogenic bacteria that cause no apparent disease symptoms on their hosts. The aim of this study was to assess the X. arboricola population structure associated with walnut, including nonpathogenic strains, in order to gain a better understanding of the role of nonpathogenic xanthomonads in walnut microbiota. A multilocus sequence analysis (MLSA) was performed on a collection of 100 X. arboricola strains, including 27 nonpathogenic strains isolated from walnut. Nonpathogenic strains grouped outside clusters defined by pathovars and formed separate genetic lineages. A multilocus variable-number tandem-repeat analysis (MLVA) conducted on a collection of X. arboricola strains isolated from walnut showed that nonpathogenic strains clustered separately from clonal complexes containing Xanthomonas arboricola pv. juglandis strains. Some nonpathogenic strains of X. arboricola did not contain the canonical type III secretion system (T3SS) and harbored only one to three type III effector (T3E) genes. In the nonpathogenic strains CFBP 7640 and CFBP 7653, neither T3SS genes nor any of the analyzed T3E genes were detected. This finding raises a question about the origin of nonpathogenic strains and the evolution of plant pathogenicity in X. arboricola. T3E genes that were not detected in any nonpathogenic isolates studied represent excellent candidates to be those responsible for pathogenicity in X. arboricola.     

Genetic Characterization of Clinical Acanthamoeba Isolates from Japan using Nuclear and Mitochondrial Small Subunit Ribosomal RNA

Because of an increased number of Acanthamoeba keratitis (AK) along with associated disease burdens, medical professionals have become more aware of this pathogen in recent years. In this study, by analyzing both the nuclear 18S small subunit ribosomal RNA (18S rRNA) and mitochondrial 16S rRNA gene loci, 27 clinical Acanthamoeba strains that caused AK in Japan were classified into 3 genotypes, T3 (3 strains), T4 (23 strains), and T5 (one strain). Most haplotypes were identical to the reference haplotypes reported from all over the world, and thus no specificity of the haplotype distribution in Japan was found. The T4 sub-genotype analysis using the 16S rRNA gene locus also revealed a clear sub-conformation within the T4 cluster, and lead to the recognition of a new sub-genotype T4i, in addition to the previously reported sub-genotypes T4a-T4h. Furthermore, 9 out of 23 strains in the T4 genotype were identified to a specific haplotype ({"type":"entrez-nucleotide","attrs":{"text":"AF479533","term_id":"28194589","term_text":"AF479533"}}AF479533), which seems to be a causal haplotype of AK. While heterozygous nuclear haplotypes were observed from 2 strains, the mitochondrial haplotypes were homozygous as T4 genotype in the both strains, and suggested a possibility of nuclear hybridization (mating reproduction) between different strains in Acanthamoeba. The nuclear 18S rRNA gene and mitochondrial 16S rRNA gene loci of Acanthamoeba spp. possess different unique characteristics usable for the genotyping analyses, and those specific features could contribute to the establishment of molecular taxonomy for the species complex of Acanthamoeba.     

Pathogenic and Nonpathogenic Acanthamoeba spp. in Thermally Polluted Discharges and Surface Waters1

During spring and autumn, the total number of amoebae and the number of Acanthamoeba species able to grow at 37°C were determined in six thermally polluted factory discharges and the surrounding surface waters. The isolated Acanthamoeba strains were studied for growth in axenic medium, cytopathic effect in Vero cell cultures, and virulence in mice. Although more amoebae were isolated in autumn, the number of Acanthamoeba species was lower than in spring, when the percent of pathogenic strains among the isolates was highest. Higher concentrations of amoebae were found in warm discharges, and more virulent strains occurred in thermal discharges than in surface waters.