Elastase in the pathogenic free-living amoebae Naegleria and Acanthamoeba spp.

The data showed that pathogenic free-living amoebae contain the proteolytic enzyme elastase. The levels of enzyme were similar in Naegleria fowleri, N. australianis italica, and Acanthamoeba culbertsoni A-1. No difference was found between elastase levels in a highly pathogenic N. fowleri and those in the same organism which had lost pathogenicity as a result of long-term axenic maintenance.     

Biosynthesis of prostaglandins in pathogenic and nonpathogenic strains of Acanthamoeba spp.

  The aim of the present study was to examine the biosynthesis of prostaglandins and to investigate factors conditioning their biosynthesis in pathogenic and nonpathogenic strains of Acanthamoeba spp. We established that the activity of the synthase of prostaglandins was almost identical in pathogenic and non-pathogenic strains and that the synthesis of endoperoxide prostaglandins was similar to that of other organisms up to the point at which prostaglandin H₂ was produced. The course of biosynthesis in vitro can be activated by various compounds such as glutathione, albumin, and p-chloromercuribenzoic acid (p-CMB), which are either activators or inhibitors of the enzymes. We suggest that the course of biosynthesis of prostaglandins in vivo is most probably activated by tissues or constitutional liquids surrounding the parasites. Received: 16 July 1996 / Accepted: 1 October 1996     

Acanthamoeba spp. as agents of disease in humans

Acanthamoeba spp. are free-living amebae that inhabit a variety of air, soil, and water environments. However, these amebae can also act as opportunistic as well as nonopportunistic pathogens. They are the causative agents of granulomatous amebic encephalitis and amebic keratitis and have been associated with cutaneous lesions and sinusitis. Immuno compromised individuals, including AIDS patients, are particularly susceptible to infections with Acanthamoeba. The immune defense mechanisms that operate against Acanthamoeba have not been well characterized, but it has been proposed that both innate and acquired immunity play a role. The ameba's life cycle includes an active feeding trophozoite stage and a dormant cyst stage. Trophozoites feed on bacteria, yeast, and algae. However, both trophozoites and cysts can retain viable bacteria and may serve as reservoirs for bacteria with human pathogenic potential. Diagnosis of infection includes direct microscopy of wet mounts of cerebrospinal fluid or stained smears of cerebrospinal fluid sediment, light or electron microscopy of tissues, in vitro cultivation of Acanthamoeba, and histological assessment of frozen or paraffin-embedded sections of brain or cutaneous lesion biopsy material. Immunocytochemistry, chemifluorescent dye staining, PCR, and analysis of DNA sequence variation also have been employed for laboratory diagnosis. Treatment of Acanthamoeba infections has met with mixed results. However, chlorhexidine gluconate, alone or in combination with propamidene isethionate, is effective in some patients. Furthermore, effective treatment is complicated since patients may present with underlying disease and Acanthamoeba infection may not be recognized. Since an increase in the number of cases of Acanthamoeba infections has occurred worldwide, these protozoa have become increasingly important as agents of human disease.     

Apoptosis of primary-culture rat microglial cells induced by pathogenic Acanthamoeba spp

To determine whether trophozoites and lysates of pathogenic Acanthamoeba spp. induce apoptosis in primary-culture microglial cells, transmission electron microscopic (TEM) examinations, assessment of DNA fragmentation by agarose gel electrophoresis, and the TdT-mediated dUTP nick-end labeling assay were performed. When a trophozoite of pathogenic Acanthamoeba culbertsoni came in contact with a microglial cell, the digipodium was observed by TEM. Nuclear chromatin condensation was observed in 10% of microglial cells, while it was not revealed when they were cocultured with weakly pathogenic Acanthamoeba royreba trophozoites. DNA fragmentation in microglial cells cocultured with the A. culbertsoni lysate was detected by electrophoresis, showing DNA ladder formation, whereas it was hardly observed in microglial cells cocultured with A. royreba. DNA fragmentation of microglial cells was also confirmed by flow cytometry analysis. The fluorescence of TdT-stained apoptotic bodies became intensely visible with microglial cells cocultured with the A. culbertsoni lysate. In contrast, with microglial cells cocultured with the A. royreba lysate, only a background level of fluorescence of TdT-stained apoptotic bodies was detected. These results suggest that some rat microglial cells cocultured with pathogenic A. culbertsoni undergo cytopathic changes which show the characteristics of the apoptotic process, such as nuclear condensation and DNA fragmentation.     

Identification of two genetic markers that distinguish pathogenic and nonpathogenic strains of Acanthamoeba spp.

Species-level identification of Acanthamoeba isolates is difficult and gives little or no indication of the isolate's pathogenicity. We identified two amplification-based genetic markers that were highly correlated with pathogenicity in Acanthamoeba spp. One marker, designed to amplify a 485-bp fragment of the small-subunit ribosomal RNA gene (ssrDNA), was preferentially amplified from the nonpathogenic strains; amplifications from the pathogenic strains yielded anomalous fragments of 650 and 900?bp. A second marker was developed on the basis of the anomalous 650-bp fragment. Primers to this sequence preferentially amplified a noncoding locus (called Ac6) only from the pathogenic strains. These two genetic markers may be useful for identification of pathogenic Acanthamoeba spp. strains.     

Voriconazole as a first-line treatment against potentially pathogenic Acanthamoeba strains from Peru

Pathogenic strains of Acanthamoeba genus are the causative agents of fatal granulomatous amoebic encephalitis and a serious sight-threatening infection of the eye known as Acanthamoeba keratitis. In a previous study, Acanthamoeba strains were isolated from nasal swabs collected from healthy individuals in Peru. In the present study, the pathogenic potential of the isolated strains was established based on temperature and osmotolerance assays as well as the secretion rate of extracellular proteases. Based on these experiments, four strains that showed the highest pathogenic potential were selected for sensitivity assays against two molecules (voriconazole and chlorhexidine) which are currently used for the treatment of Acanthamoeba infections. After performing sensitivity and activity assays, it was found that both drugs were active against the tested strains. However, voriconazole showed higher activity against the studied strains compared to chlorhexidine. Therefore, voriconazole should be established as a first-line treatment against Acanthamoeba infections at least in the studied region of Peru.     

Amebicidal activity of plant extracts from Southeast Asia on Acanthamoeba spp.

The effect of 100 polar and 100 nonpolar plant extract materials obtained from Southeast Asia were evaluated for amebicidal activity in vitro against three species of Acanthamoeba.A. culbertsoni, A. castellanii, and A. polyphaga, the causative agents of granulomatous amebic encephalitis and amebic keratitis, were studied in vitro to determine whether the plant extracts exhibited amebicidal activity or induced encystment of the amebae. Of the 200 plant extracts tested, extracts obtained from three plants (Ipomoea sp., Kaempferia galanga, and Cananga odorata) were amebicidal for all three species of Acanthamoeba and a fourth extract prepared from Gastrochilus panduratum was lytic for A. polyphaga and growth-inhibitory for A. castellanii and A. culbertsoni. Three plant extracts induced encystment of all three species of Acanthamoeba. Select plant extracts were tested as well for tumoricidal activity against B103 neuroblastoma cells. Some plant extracts that exhibited tumoricidal activity for B103 cells were not amebicidal for Acanthamoeba spp. Additionally, the polar and nonpolar extracts that exhibited amebicidal activity were also tested for activity against primary murine peritoneal macrophage cultures. Plant extracts that demonstrated tumoricidal or amebicidal activity were not lytic for normal macrophage cultures. Received: 23 February 1998 / Accepted: 18 May 1998     

Mitochondrial DNA fingerprinting of Acanthamoeba spp. isolated from clinical and environmental sources.

Restriction fragment length polymorphism analysis of mitochondrial DNA (mtDNA fingerprinting) was evaluated as an epidemiologic tool for identifying potential reservoirs of Acanthamoeba infection. Fingerprints for 15 clinical isolates recovered by our affiliated laboratories were compared with those for 25 environmental isolates from western Washington State and 10 American Type Culture Collection (ATCC) strains. Seven different fingerprint groups emerged from the analysis of clinical isolates with six selected restriction enzymes (BamHI, BglII, EcoRI, HindIII, KpnI, and SalI). Fourteen (56%) environmental and 4 (40%) ATCC isolates displayed fingerprints similar to those of clinical isolates. In all, five of the seven groups contained one or more environmental and/or ATCC isolates. Comparisons with published mtDNA fingerprints for Acanthamoeba isolates showed that two groups have counterparts in Europe and Japan and in Europe and Australia. The inclusion of environmental isolates demonstrated that the most common clinical isolates do have counterparts readily recoverable from the surrounding environment and that some of these counterparts appear to be geographically widespread.     

Characteristics of pathogenic Neisseria spp. isolated from homosexual men.

Oropharyngeal, urethral, and rectal cultures for pathogenic Neisseria spp. were collected from 815 homosexual men attending a community clinic in Chicago. Meningococci were characterized by serogrouping and antimicrobial susceptibility testing. Gonococci were auxotyped, and susceptibilities to penicillin and tetracycline were determined. Of the 815 men tested, 42.5% carried meningococci in the oropharynx. Gonococci were recovered from the urethra, rectum, and oropharynx of 18.5, 16.3, and 5.6%, respectively. Meningococci were also recovered from the urethra (6 patients) and the rectum (15 patients). Some of these isolates were identical to the isolates from the oropharynges of the same patients, whereas others were distinct from the oropharyngeal isolates by serogroup or antimicrobial susceptibilities. Serogroups B, W135, and C comprised over 90% of the meningococci. Almost 80% of the gonococcal strains required minimal inhibitory concentrations greater than 0.06 micrograms of penicillin per ml, whereas greater than 90% of the meningococci were inhibited at this concentration. Auxotyping demonstrated three major auxotypes: Zero (required none of the nutrients tested), 60%; arginine requiring, 19.4%; and proline requiring, 12.3%. Only four strains (1.2%) required arginine, hypoxanthine, and uracil.     

Proteases as markers for differentiation of pathogenic and nonpathogenic species of Acanthamoeba

Acanthamoeba keratitis is a vision-threatening infection caused by pathogenic species of the genus Acanthamoeba. Although not all Acanthamoeba spp. can cause keratitis, it is important to differentiate pathogenic species and isolates from nonpathogens. Since extracellular proteases may play a role in ocular pathology, we used colorimetric, cytopathic, and zymographic assays to assess extracellular protease activity in pathogenic and nonpathogenic Acanthamoeba. Colorimetric assays, using azo-linked protein as a substrate, showed extracellular protease activity in Acanthamoeba-conditioned medium and differentiated pathogenic and nonpathogenic Acanthamoeba. Monolayers of immortalized corneal epithelial cells in four-well plates were used for cytopathic effect (CPE) assays. Pathogenic Acanthamoeba isolates exhibited marked CPE on immortalized corneal epithelial cells, while nonpathogenic isolates did not exhibit CPE. Protease zymography was performed with Acanthamoeba-conditioned medium as well as with Acanthamoeba- plus epithelial-cell-conditioned medium. The zymographic protease assays showed various banding patterns for different strains of Acanthamoeba. In pathogenic Acanthamoeba isolates, all protease bands were inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting serine type proteases, while in nonpathogenic strains only partial inhibition was observed by using PMSF. The pathogenic Acanthamoeba strains grown under typical laboratory conditions without epithelial cells exhibited one overexpressed protease band of 107 kDa in common; this protease was not observed in nonpathogenic Acanthamoeba strains. The 107-kDa protease exhibited activity over a pH range of 5 to 9.5.     

Survival of pathogenic Mycobacterium abscessus subsp. massiliense in Acanthamoeba castellanii

We used an amoeba model to study the intracellular growth and cytotoxicity of clinical strains of Mycobacterium abscessus subsp. massiliense (Mabsm) isolated from 2 patients (one with cystic fibrosis, the other one with idiopathic bronchiectasis) during the early (smooth colonies) and late stage (rough colonies) of chronic pulmonary infection. Acanthamoeba castellanii were infected with Mabsm (MOI 100) and samples collected every 24 h for 72 h. Results showed Mabsm is able to survive in trophozoites and persist in cysts for at least 7 days. Late Mabsm demonstrated higher cytotoxicity toward A. castellanii when compared to early strains. A. castellanii is a useful in vitro host model to study infection of Mabsm clinical isolates.     

Assmilation of iron by pathogenic Neisseria spp.

Iron assimilation by Neisseria meningitidis and Neisseria gonorrhoeae has been shown to be important to their growth and virulence. Iron acquisition in vitro was studied in an agar diffusion assay employing the iron-binding protein conalbumin. The ability of various iron compounds to alter the growth-inhibitory effect of conalbumin was investigated. On an equimolar iron basis, citrate-containing iron compounds were most effective; hemin was slightly less effective; ferrous sulfate and ferrous ammonium sulfate were even less effective; and the ferric compounds, ferric nitrate, ferric chloride, and ferric dextran (Imferon), were least effective. The results suggested that, as with Escherichia coli and certain other bacteria, the Neisseria spp. may utilize a citrate-mediated iron transport system. Microbial siderophores were also tested for their ability to relieve the growth-inhibitory effect of conalbumin. Two phenolate siderophores and Desferal enhanced growth inhibition in the deferrated form, but were inactive in the ferrated form. Several trihydroxamates of the ferrichrome family and coprogen were inactive in either the deferrated or ferrated forms. Of the 12 different siderophores tested, only the dihydroxamates (schizokinen, arthrobactin, and aerobactin) were stimulatory, but then only in the ferrated forms. Apparently, even though those siderophores could be utilized as specific iron transport agents by the Neisseria spp., they could not compete with conalbumin for iron under these assay conditions.     

Occurrence of bacterial endosymbionts in Acanthamoeba spp. isolated from corneal and environmental specimens and contact lenses.

Free-living and parasitic protozoa are known to harbor a variety of endosymbiotic bacteria, although the roles such endosymbionts play in host survival, infectivity, and invasiveness are unclear. We have identified the presence of intracellular bacteria in 14 of 57 (24%) axenically grown Acanthamoeba isolates examined. These organisms are gram negative and non-acid fast, and they cannot be cultured by routine methodologies, although electron microscopy reveals evidence for multiplication within the amoebic cytoplasm. Examination for Legionella spp. with culture and nucleic acid probes has proven unsuccessful. We conclude that these bacteria are endosymbionts which have an obligate need to multiply within their amoebic hosts. Rod-shaped bacteria were identified in 5 of 23 clinical Acanthamoeba isolates (3 of 19 corneal isolates and 2 of 4 contact lens isolates), 4 of 25 environmental Acanthamoeba isolates, and 2 of 9 American Type Culture Collection Acanthamoeba isolates (ATCC 30868 and ATCC 30871) previously unrecognized as having endosymbionts. Coccus-shaped bacteria were present in one clinical (corneal) isolate and two environmental isolates. There was no statistical difference (P > 0.8) between the numbers of endosymbiont strains originating from clinical (26% positive) and environmental (24% positive) amoebic isolates, suggesting that the presence alone of these bacteria does not enhance amoebic infectivity. Rods and cocci were found in both clinical and environmental isolates from different geographical areas (Seattle, Wash., and Portland, Oreg.), demonstrating their widespread occurrence in nature. Our findings suggest that endosymbiosis occurs commonly among members of the family Acanthamoebidae and that the endosymbionts comprise a diverse taxonomic assemblage. The role such endosymbionts may play in pathogenesis remains unknown, although a variety of exogenous bacteria have been implicated in the development of amoebic keratitis, warranting further evaluation.     

Apoptosis of Primary-Culture Rat Microglial Cells Induced by Pathogenic Acanthamoeba spp.

To determine whether trophozoites and lysates of pathogenic Acanthamoeba spp. induce apoptosis in primary-culture microglial cells, transmission electron microscopic (TEM) examinations, assessment of DNA fragmentation by agarose gel electrophoresis, and the TdT-mediated dUTP nick-end labeling assay were performed. When a trophozoite of pathogenic Acanthamoeba culbertsoni came in contact with a microglial cell, the digipodium was observed by TEM. Nuclear chromatin condensation was observed in 10% of microglial cells, while it was not revealed when they were cocultured with weakly pathogenic Acanthamoeba royreba trophozoites. DNA fragmentation in microglial cells cocultured with the A. culbertsoni lysate was detected by electrophoresis, showing DNA ladder formation, whereas it was hardly observed in microglial cells cocultured with A. royreba. DNA fragmentation of microglial cells was also confirmed by flow cytometry analysis. The fluorescence of TdT-stained apoptotic bodies became intensely visible with microglial cells cocultured with the A. culbertsoni lysate. In contrast, with microglial cells cocultured with the A. royreba lysate, only a background level of fluorescence of TdT-stained apoptotic bodies was detected. These results suggest that some rat microglial cells cocultured with pathogenic A. culbertsoni undergo cytopathic changes which show the characteristics of the apoptotic process, such as nuclear condensation and DNA fragmentation.     

Migration patterns of pathogenic and nonpathogenic Naegleria spp.

Four species of Naegleria were tested for their ability to migrate under agarose. Pathogenic N. fowleri strains exhibited rapid locomotion at 37 degrees C. Environmental isolates of N. fowleri moved faster than clinical isolates which had been kept in axenic culture for longer periods, and this result was confirmed by using the 84-2205-7 strain kept in axenic culture for 1 or 5 months. Nonpathogenic N. gruberi strains migrated actively at 28 degrees C but not at 37 degrees C; moreover, even at 28 degrees C, active amoebae constituted only a small proportion of the whole. The temperature-tolerant, nonpathogenic species N. lovaniensis moved more slowly than N. fowleri at 37 degrees C. In contrast, N. australiensis, which is temperature tolerant as well as pathogenic for mice, migrated at a rate comparable to that of N. fowleri. There appears to be a direct correlation between the locomotive ability of free-living amoebae and their pathogenic potential.     

Topology of outer membrane porins in pathogenic Neisseria spp.

In Escherichia coli, membrane-spanning amphipathic beta-sheet structures are characteristic of many outer membrane proteins. By applying the principles that have been recognized for them to the four classes of neisserial porins, we have constructed a model for the topology of the porins within the outer membrane. This model predicts eight surface-exposed loops, both in the meningococcal class 1 and 2 proteins and in the gonococcal PIA and PIB proteins. The transmembrane sequences are highly conserved among these porins and are able to form an amphipathic beta-sheet structure. The surface-exposed hydrophilic loops show extensive variation in both length and sequence. Experimental evidence in support of this model has been obtained by using antisera against synthetic peptides which correspond to surface-exposed loops in class 1 and 2 proteins. Thus, binding to the cell surface was observed with antibodies against loops 1, 4, and 5 of class 1 and loops 1 and 5 of class 2. In class 1, these loops are the longest ones and show the highest sequence diversity among strains of different subtypes. Mapping of epitopes recognized by monoclonal antibodies with bactericidal activity has also provided strong support for the model. The epitopes are located in loops 1 and 4 of class 1 protein, loop 5 of PIB, and loop 6 of PIA. A nonbactericidal antibody that binds only weakly to whole cells was shown to recognize loop 3 of PIB. These results suggest that the longest loops are immunodominant, provide the binding sites for bactericidal antibodies, and display the greatest variation among different strains.     

Comparative analyses of different genetic markers for the detection of Acanthamoeba spp. isolates

Acanthamoeba are widespread free-living amoebae which may cause granulomatous amoebic encephalitis (GAE), keratitis, skin ulcerations and disseminated tissue infection. An important diagnostic and prognostic factor for the treatment of infection is a quick and correct diagnosis of amoebae strains. The aim of our study was to develop a rapid method for detection and identification of pathogenic Acanthamoeba spp. strains from diagnostic material collected from water. In this study we analysed five amplification-based genetic markers (Aca 16S, Ac6/210, GP, JDP, Nelson) used for identification of pathogenic Acanthamoeba spp. strains isolated in water sources in Poland, Iceland and Sweden. Our results demonstrated the presence of pathogenic Acanthamoeba strains in tap water. PCR assay appeared to be a more rapid and sensitive method to detect the presence of amoebae than the limited conventional techniques. Based on our observations, we can confirm that the use of four out of five genetic markers (Aca 16S, Ac 6/210, JDP, GP, Nelson) may be helpful in identification of Acanthamoeba spp. strains, but only one Aca 16S primer pair is a highly specific marker that distinguishes between pathogenic strains of Acanthamoeba and other free-living amoeba families.