Growth characteristics, cytopathic effect in cell culture, and virulence in mice of 36 type strains belonging to 19 different Acanthamoeba spp.

A total of 36 strains belonging to 19 different species of Acanthamoeba were compared for temperature tolerance, ability to grow in an axenic medium, cytopathic effect in Vero cell culture, and virulence in mice. Pathogenic strains appeared to belong to different species, whereas pathogenic and nonpathogenic strains occurred in one species. Although growth at high temperatures and readiness to grow axenically indicated a potential for pathogenicity, each such strain had to be tested in cell cultures or laboratory mice to determine whether or not it was virulent. This study was not intended to differentiate Acanthamoeba spp., but to provide methods to be used for the specific isolation and identification of pathogenic Acanthamoeba strains.     

Growth characteristics, cytopathic effect in cell culture, and virulence in mice of 36 type strains belonging to 19 different Acanthamoeba spp

A total of 36 strains belonging to 19 different species of Acanthamoeba were compared for temperature tolerance, ability to grow in an axenic medium, cytopathic effect in Vero cell culture, and virulence in mice. Pathogenic strains appeared to belong to different species, whereas pathogenic and nonpathogenic strains occurred in one species. Although growth at high temperatures and readiness to grow axenically indicated a potential for pathogenicity, each such strain had to be tested in cell cultures or laboratory mice to determine whether or not it was virulent. This study was not intended to differentiate Acanthamoeba spp., but to provide methods to be used for the specific isolation and identification of pathogenic Acanthamoeba strains.     

Free-living freshwater amoebae differ in their susceptibility to the pathogenic bacterium Legionella pneumophila

Legionella pneumophila is known as a facultative intracellular parasite of free-living soil and freshwater amoebae, of which several species have been shown to support the growth of the pathogenic bacteria. We report for the first time the behaviour of two strains (c2c and Z503) of the amoeba Willaertia magna towards different strains of L. pneumophila serogroup 1 and compared it with Acanthamoeba castellanii and Hartmannella vermiformis , known to be L. pneumophila permissive. In contrast to the results seen with other amoebae, W. magna c2c inhibited the growth of one strain of Legionella ( L. pneumophila , Paris), but not of others belonging to the same serogroup ( L. pneumophila , Philadelphia and L. pneumophila , Lens). Also, the different L. pneumophila inhibited cell growth and induced cell death in A. castellanii, H. vermiformis and W. magna Z503 within 3–4 days while W. magna c2c strain remained unaffected even up to 7 days. Electron microscopy demonstrated that the formation of numerous replicative phagosomes observed within Acanthamoeba and Hartmannella is rarely seen in W. magna c2c cocultured with L. pneumophila . Moreover, the morphological differences were observed between L. pneumophila cultured either with Willaertia or other amoebae. These observations show that amoebae are not all equally permissive to L. pneumophila and highlight W. magna c2c as particularly resistant towards some strains of this bacterium.     

Acanthamoeba sp. promotes the survival and growth of Acinetobacter baumanii

Acinetobacter baumanii, which may be found in water, is an important emerging hospital-acquired pathogen. Free-living amoebae can be recovered from the same water networks, and it has been shown that these protozoa may support the growth of other bacteria. In this paper, we have studied potential relationships between A. baumanii and Acanthamoeba species. Two strains of A. baumanii isolated from hospital water were co-cultivated with the trophozoites or supernatants of two free-living amoebae strains: Acanthamoeba castellanii or Acanthamoeba culbertsoni. Firstly, the presence of the amoebae or their supernatants induced a major increase in A. baumanii growth, compared with controls. Secondly, A. baumanii affected only the viability of A. culbertsonii, with no effect on A. castellanii. Electron microscopy observations of the cultures investigating the bacterial location in the protozoa showed persistence of the bacteria within cyst wall even after 60 days of incubation. In our study, the survival and growth of A. baumanii could be favored by Acanthamoeba strains. Special attention should consequently be paid to the presence of free-living amoebae in hospital water systems, which can promote A. baumanii persistence.     

In-vitro activity of miltefosine and voriconazole on clinical isolates of free-living amebas: Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri

The anticancer agent miltefosine and the antifungal drug voriconazole were tested in vitro against Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri. All three amebas are etiologic agents of chronic (Balamuthia, Acanthamoeba) or fulminant (Naegleria) encephalitides in humans and animals and, in the case of Acanthamoeba, amebic keratitis. Balamuthia exposed to <40 microm concentrations of miltefosine survived, while concentrations of >or=40 microM were generally amebacidal, with variation in sensitivity between strains. At amebastatic drug concentrations, recovery from drug effects could take as long as 2 weeks. Acanthamoeba spp. recovered from exposure to 40 microM, but not 80 microM miltefosin. Attempts to define more narrowly the minimal inhibitory (MIC) and minimal amebacidal concentrations (MAC) for Balamuthia and Acanthamoeba were difficult due to persistence of non-proliferating trophic amebas in the medium. For N. fowleri, 40 and 55 microM were the MIC and MAC, respectively, with no trophic amebas seen at the MAC. Voriconazole had little or no inhibitory effect on Balamuthia at concentrations up to 40 microg/ml, but had a strong inhibitory effect upon Acanthamoeba spp. and N. fowleri at all drug concentrations through 40 microg/ml. Following transfer to drug-free medium, Acanthamoeba polyphaga recovered within a period of 2 weeks; N. fowleri amebas recovered from exposure to 1 microg/ml, but not from higher concentrations. All testing was done on trophic amebas; drug sensitivities of cysts were not examined. Miltefosine and voriconazole are potentially useful drugs for treatment of free-living amebic infections, though sensitivities differ between genera, species, and strains.     

A molecular approach to the phylogeny of Acanthamoeba

Isoenzyme electrophoresis of three different enzyme systems was used to compare 71 strains assigned to the 15 currently recognized species of Acanthamoeba. A phylogenetic (cladistic) analysis of the zymograms indicated an arrangement of strains in 15 distinguishable lineages, but not all corresponding to current taxonomic assignments. Five of the groups corresponded to the recognized species A. castellanii, A. culbertsoni, A. griffini, A. lenticulata and A. royreba. But none of these groups consisted of only strains which had been previously assigned to each respective species. The type-equivalent strains for two species, A. hatchetti and A. tubiashi, were not closely aligned to any other strain and thus are considered to be monotypic. Strains of A. triangularis, A. astronyxis and A. palestinensis occurred together in a single group suggesting possible synonymy; however, on morphologic criteria, the strains assigned to these species are readily distinguishable. Strains assigned to A. polyphaga and A. rhysodes were interspersed throughout the other species groups. The strains of these two species were either misidentified or the species could not be recognized. Two groups previously not recognized as unique formed distinctive clusters which could be considered as new species. The analysis also made it possible to place strains which had previously been identified only to genus into species complexes. These results therefore suggest that previous criteria which have been used to classify Acanthamoeba are not adequate for fully resolving taxa at the species level.     

Immunological inter-strain crossreactivity correlated to 18S rDNA sequence types in Acanthamoeba spp

Various species of the genus Acanthamoeba have been described as potential pathogens; however, differentiation of acanthamoebae remains problematic. The genus has been divided into 12 18S rDNA sequence types, most keratitis causing strains exhibiting sequence type T4. We recently isolated a keratitis causing Acanthamoeba strain showing sequence type T6, but being morphologically identical to a T4 strain. The aim of our study was to find out, whether the 18S rDNA sequence based identification correlates to immunological differentiation. The protein and antigen profiles of the T6 isolate and three reference Acanthamoeba strains were investigated using two sera from Acanthamoeba keratitis patients and one serum from an asymptomatic individual. It was shown, that the T6 strain produces a distinctly different immunological pattern, while patterns within T4 were identical. Affinity purified antibodies were used to further explore immunological cross-reactivity between sequence types. Altogether, the results of our study support the Acanthamoeba 18S rDNA sequence type classification in the investigated strains.     

Subgenus Systematics of Acanthamoeba: Four Nuclear 18S rDNA Sequence Types

ABSTRACT Classification of Acanthamoeba at the subgenus level has been problematic, but increasing reports of Acanthamoeba as an opportunistic human pathogen have generated an interest in finding a more consistent basis for classification. Thus, we are developing a classification scheme based on RNA gene sequences. This first report is based on analysis of complete sequences of nuclear small ribosomal subunit RNA genes ( Rns ) from 18 strains. Sequence variation was localized in 12 highly variable regions. Four distinct sequence types were identified based on parsimony and distance analyses. Three were obtained from single strains: Type T1 from Acanthamoeba castellanii V006, T2 from Acanthamoeba palestinensis Reich, and T3 from Acanthamoeba griffini S-7. T4, the fourth sequence type, included 15 isolates classified as A. castellanii, Acanthamoeba polyphaga, Acanthamoeba rhysodes , or Acanthamoeba sp., and included all 10 Acanthamoeba keratitis isolates. Interstrain sequence differences within T4 were 0%–4.3%, whereas differences among sequence types were 6%–12%. Branching orders obtained by parsimony and distance analyses were inconsistent with the current classification of T4 strains and provided further evidence of a need to reevaluate criteria for classification in this genus. Based on this report and others in preparation, we propose that Rns sequence types provide the consistent quantititive basis for classification that is needed.     

Development of a real-time PCR assay for quantification of Acanthamoeba trophozoites and cysts

Free-living amoebae have been found to be a reservoir for various pathogenic bacteria in aquatic environments. For example, the Acanthamoeba genus renders possible the intracellular multiplication of Legionella pneumophila, which is responsible for legionellosis. It consequently matters to quantify Acanthamoeba cells and thereby enhance our assessment of the risk of contamination. The classical microbiological method of quantification relies on amoebae growth and most probable number calculation. We have developed a real-time PCR assay based on a TaqMan probe that hybridizes onto 18S rDNA. This probe is specific to the Acanthamoeba genus. The assay was successful with both the trophozoite and the cyst forms of Acanthamoeba. Highly sensitive, it proved to permit detection of fewer than 10 cells, even those that are not easily cultivable, such as the cyst forms.     

Novel Chlamydiales strains isolated from a water treatment plant

Chlamydiae are obligate intracellular bacteria infecting free-living amoebae, vertebrates and some invertebrates. Novel members are regularly discovered, and there is accumulating evidence supporting a very important diversity of chlamydiae in the environment. In this study, we investigated the presence of chlamydiae in a drinking water treatment plant. Samples were used to inoculate Acanthamoeba monolayers ( Acanthamoeba co-culture), and to recover autochthonous amoebae onto non-nutritive agar. Chlamydiae were searched for by a pan-chlamydia 16S rRNA gene PCR from both Acanthamoeba co-cultures and autochthonous amoebae, and phylotypes determined by 16S rRNA gene sequencing. Autochthonous amoebae also were identified by 18S rRNA gene amplification and sequencing. From a total of 79 samples, we recovered eight chlamydial strains by Acanthamoeba co-culture, but only one of 28 amoebae harboured a chlamydia. Sequencing results and phylogenetic analysis showed our strains belonging to four distinct chlamydial lineages. Four strains, including the strain recovered within its natural host, belonged to the Parachlamydiaceae ; two closely related strains belonged to the Criblamydiaceae ; two distinct strains clustered with Rhabdochlamydia spp.; one strain clustered only with uncultured environmental clones. Our results confirmed the usefulness of amoeba co-culture to recover novel chlamydial strains from complex samples and demonstrated the huge diversity of chlamydiae in the environment, by identifying several new species including one representing the first strain of a new family.     

Area 51: How do Acanthamoeba invade the central nervous system?

Acanthamoeba granulomatous encephalitis generally develops as a result of haematogenous spread, but it is unclear how circulating amoebae enter the central nervous system (CNS) and cause inflammation. At present, the mechanisms which Acanthamoeba use to invade this incredibly well-protected area of the CNS and produce infection are not well understood. In this paper, we propose two key virulence factors: mannose-binding protein and extracellular serine proteases as key players in Acanthamoeba traversal of the blood-brain barrier leading to neuronal injury. Both molecules should provide excellent opportunities as potential targets in the rational development of therapeutic interventions against Acanthamoeba encephalitis.     

Effect of Monovalent Thallium Ions on Differentiation and Multiplication of Acanthamoeba castellanii

The vegetatively multiplying Acanthamoeba castellanii cells are transformed into cysts under unfavourable feeding conditions. The cyst formation may also be induced by treatment of the cells with DNA-synthesis inhibitors or by placing the cells into special ionic medium containing magnesium and calcium at pH 9, with aeration. During Acanthamoeba encystment the morphology of the cells changes significantly, namely a cellulose-protein cyst wall appears which is easily seen under the light and electron microscope. The process of encystment in Acanthamoeba castellanii is considered as a useful simple model of cytodifferentiation of eukaryotic cells.
This communication describes the effects of monovalent thallium ions on the differentiation and multiplication of Acanthamoeba cells growing in optimal feeding conditions. Thallium ions being potassium analogues are readily accumulated by cells. On the other hand, thallium ions, unlike potassium ions, are able to form complexes with some anions, which results in disturbances of some cellular functions.
Thallium ions, added to the growth medium of 2–3-days old Acanthamoeba culture at a concentration of 0.05–1.0 mM inhibit the population growth inducing the differentiation of cells into cysts. The increase of the thallium ion concentration up to 5 or 10 mM in the growth medium causes the very fast multiplication of Acanthamoeba cells. However, at these thallium ion concentrations no cysts can be observed.
Thus, on the basis of the experimental data it seems likely that thallium ions play some role in increasing the rate of multiplication and in switching on the differentiation process (encystment) in Acanthamoeba cells.     

Specimen housing unit for cinemicrographic studies in the vertical plane.

The destructive action of chlorine on the pathogenic Naegleria fowleri and Acanthamoeba culbertsoni, the nonpathogenic N. gruberi, and an avirulent Acanthamoeba isolate was investigated. N fowleri is somewhat more sensitive to chlorine than N. gruberi, whereas the two Acanthamoeba strains are very resistant. This study yields information needed for the destruction of amoebic cysts in drinking water and swimming pools. It also gives some explanation for the occurence of Acanthamoeba strains in these waters.     

Pathogenic free-living amoebae in Korea

Acanthamoeba and Naegleria are widely distributed in fresh water, soil and dust throughout the world, and cause meningoencephalitis or keratoconjunctivitis in humans and other mammals. Korean isolates, namely, Naegleria sp. YM-1 and Acanthamoeba sp. YM-2, YM-3, YM-4, YM-5, YM-6 and YM-7, were collected from sewage, water puddles, a storage reservoir, the gills of a fresh water fish, and by corneal washing. These isolates were categorized into three groups based on the mortalities of infected mice namely, highly virulent (YM-4), moderately virulent (YM-2, YM-5 and YM-7) and nonpathogenic (YM-3). In addition, a new species of Acanthamoeba was isolated from a freshwater fish in Korea and tentatively named Korean isolate YM-4. The morphologic characters of its cysts were similar to those of A. culbertsoni and A. royreba, which were previously designated as Acanthamoeba group III. Based on experimentally infected mouse mortality, Acanthamoeba YM-4 was highly virulent. The isoenzymes profile of Acanthamoeba YM-4 was similar to that of A. royreba. Moreover, an anti-Acanthamoeba YM-4 monoclonal antibody reacted only with Acanthamoeba YM-4, and not with A. culbertsoni. Random amplified polymorphic DNA marker analysis and RFLP analysis of mitochondrial DNA and of a 18S small subunit ribosomal RNA, placed Acanthamoeba YM-4 in a separate cluster based on phylogenic distances. Thus Acanthamoeba YM-4 was identified as a new species, and assigned Acanthamoeba sohi. Up to the year 2002 in Korea, two clinical cases were found to be infected with Acanthamoeba spp. These patients died of meningoencephalitis. In addition, one case of Acanthamoeba pneumonia with an immunodeficient status was reported and Acanthamoeba was detected in several cases of chronic relapsing corneal ulcer, chronic conjunctivitis, and keratitis.     

The genitalia and associated glands of five British species belonging to the family Chthoniidae (Pseudoscorpiones: Arachnida)

The male and female genitalia of five species representing the genus Chthonius are fundamentally similar in form. However minor differences do occur which are of possible taxonomic significance at both the sub-generic and specific levels. Thus, the two species C. kewi and C. tetrachelatus of the sub-genus Ephippiochthonius have a number of characters in common which differ from those of the remaining three species, C. ischnocheles, C. tenuis and C. orthodactylus of the subgenus Chthonius (s.st). Other genital characters can be used to separate the five species.
Such small differences in the genitalia suggests that spermatophore formation and deposition in the four species C. tenuis, C. tetrachelatus, C. kewi and C. orthodactylus is not different from that of C. ischnocheles (Legg, 1973). What differences that do occur, for example, the thickening of the roof of the medial diverticulcum, size of the males and the pheromone produced by the lateral glands, probably are aids to species isolation.     

Acanthamoeba: could it be an environmental host of Shigella?

Shigellosis is a serious public health problem in Korea, because large outbreaks of Shigella sonnei infections were recorded in many parts of the country during the period 1998-2000. However, the epidemiological features of shigellosis are not well known. In this study, we devised conditions suitable for the growth and replication of Shigella in an amoebic intracellular environment, and investigate whether medium conditions affect the survival and replication of Shigella within Acanthamoeba. We evaluated the uptake rates of invasive and non invasive S. sonnei strains by three Acanthamoeba species, namely, A. castellanii Neff, A. astronyxis Ray & Hayes, and A. healyi OC-3A. When A. castellanii Neff was infected with S. sonnei 99OBS1 or 80DH248, shigellae was maintained for a longer time in cytoplasms than in other Acanthamoeba species. S. sonnei 99OBS1 strain (a virulent strain) was recovered in higher numbers than the non-virulent S. sonnei 80DH248 strain in all experiments. Moreover, S. sonnei was more easily engulfed by Acanthamoeba at 18 degrees C. The shigellae uptake rates of Neff strain, which was cultured in free-media (less nutrition), were higher (>10-fold) than those observed in original amoeba culture media (PYG medium) in all time points. S. sonnei 99OBS1 was localized, with an intact membrane, to the vacuoles of Acanthamoeba. We conclude that free-living amoebae more likely act as environmental hosts for shigellae, and thus, may have contributed to outbreaks of shigellosis in Korea.     

Detection of a serine proteinase gene in Acanthamoeba genotype T6 (Amoebozoa: Lobosea)

Cytopathic proteins are assumed to contribute to the pathogenicity of Acanthamoeba spp. due to their degrading capacity that is required for tissue invasion. In this study, a serine proteinase gene was demonstrated in a highly virulent Acanthamoeba keratitis causing strain with genotype T6. This gene was detected in both, the genomic DNA and the cDNA by PCR and subsequent sequencing. The gene fragment comprises about 500 bp and exhibits high sequence similarity to the serine proteinases of Acanthamoeba strains with genotype T4 and T12. The detection of a serine proteinase in this Acanthamoeba T6 strain is significant, because while T4 is the most common genotype among pathogenic Acanthamoeba strains and also T12 is known to be associated with disease, this is the only virulent Acanthamoeba T6 strain known to date. Obviously, this serine proteinase represents a common tool in pathogenic processes during Acanthamoeba infection.     

Phenotypic Characterization and Deoxyribonucleic Acid Homologies of Pseudomonas solanacearum

Twenty-six strains and colony variants of Pseudomonas solanacearum belonging to four described biotypes were characterized, by using 169 phenotypic characters previously found useful in distinguishing among strains of other Pseudomonas species. Deoxyribonucleic acid (DNA) hybridization (intra- and interspecific DNA-DNA hybridizations) was performed by using the in vitro "DNA competition" technique. P. solanacearum appears to be a moderately homogeneous species, which is, at most, only remotely related to all other species of the genus studied to date. The four biotypes are not clearly distinct from one another with respect to nutritional characters or DNA homologies. Discrepancies between acid production and growth with some carbohydrates were noted. Difficulties were encountered in certain DNA competition experiments and some problems of the methodology are discussed.