Free-living pathogenic and nonpathogenic amoebae in Maryland soils

Tests for potentially pathogenic amoebae were carried out on soil samples from the following sites: (i) farmlands fertilized with municipal sewage wastes, (ii) a stream receiving sewage effluent from a sludge lagoon, (iii) a ravine receiving storm runoff from a cattle farm, (iv) farmlands not fertilized with sewage wastes, and (v) a vegetated shoreline of a waterfront estate not used for farming or livestock production. Study sites were located on the eastern shore of Maryland, bordered to the north by Delaware and to the south by Virginia. Twenty-four species of soil amoebae, including five potentially pathogenic Acanthamoeba species (members of the family Acanthamoebidae), were identified. All of the sites yielded two or more of the potential pathogens.     

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.     

Viability of pathogenic and nonpathogenic free-living amoebae in long-term storage at a range of temperatures.

The long-term storage of pathogenic and nonpathogenic strains of both Naegleria and Acanthamoeba spp. were tested on Page amoeba saline agar slopes for 24 months at room temperature and for 8 months at -10, 4, 10, and 15 degrees C. Acanthamoeba strains showed better survival potential than Naegleria strains, particularly when they were stored at temperatures equal to or lower than room temperature.     

Viability of pathogenic and nonpathogenic free-living amoebae in long-term storage at a range of temperatures

The long-term storage of pathogenic and nonpathogenic strains of both Naegleria and Acanthamoeba spp. were tested on Page amoeba saline agar slopes for 24 months at room temperature and for 8 months at -10, 4, 10, and 15 degrees C. Acanthamoeba strains showed better survival potential than Naegleria strains, particularly when they were stored at temperatures equal to or lower than room temperature.     

Free-living amoebae promote Candida auris survival and proliferation in water

Candida auris is an emerging species responsible for life-threatening infections. Its ability to be resistant to most systemic antifungal classes and its capacity to persist in a hospital environment have led to health concerns. Currently, data about environmental reservoirs are limited but remain essential in control of C. auris spread. The aim of our study was to explore the interactions between C. auris and two free-living amoeba (FLA) species, Vermamoeba vermiformis and Acanthamoeba castellanii, potentially found in the same water environment. Candida auris was incubated with FLA trophozoites or their culture supernatants. The number of FLA and yeasts was determined at different times and transmission electron microscopy (TEM) was performed. Supernatants of FLAs promoted yeast survival and proliferation. Internalization of viable C. auris within both FLA species was also evidenced by TEM. A water environmental reservoir of C. auris can therefore be considered through FLAs and contamination of the hospital water networks would consequently be possible.     

Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals

Knowledge that free-living amoebae are capable of causing human disease dates back some 50 years, prior to which time they were regarded as harmless soil organisms or, at most, commensals of mammals. First Naegleria fowleri, then Acanthamoeba spp. and Balamuthia mandrillaris, and finally Sappinia diploidea have been recognised as etiologic agents of encephalitis; Acanthamoeba spp. are also responsible for amoebic keratitis. Some of the infections are opportunistic, occurring mainly in immunocompromised hosts (Acanthamoeba and Balamuthia encephalitides), while others are non-opportunistic (Acanthamoeba keratitis, Naegleria meningoencephalitis, and cases of Balamuthia encephalitis occurring in immunocompetent humans). The amoebae have a cosmopolitan distribution in soil and water, providing multiple opportunities for contacts with humans and animals, as evidenced by antibody titers in surveyed human populations. Although, the numbers of infections caused by these amoebae are low in comparison to other protozoal parasitoses (trypanosomiasis, toxoplasmosis, malaria, etc.), the difficulty in diagnosing them, the challenge of finding optimal antimicrobial treatments and the morbidity and relatively high mortality associated with, in particular, the encephalitides have been a cause for concern for clinical and laboratory personnel and parasitologists. This review presents information about the individual amoebae: their morphologies and life-cycles, laboratory cultivation, ecology, epidemiology, nature of the infections and appropriate antimicrobial therapies, the immune response, and molecular diagnostic procedures that have been developed for identification of the amoebae in the environment and in clinical specimens.     

Free-living amoebae (FLA) co-occurring with legionellae in industrial waters

Legionella pneumophila is known as the causative agent of Legionnaires’ disease and free-living amoebae (FLA) can serve as vehicles for legionellae. The aim of this study was to screen industrial waters for the occurrence of FLA and their co-occurrence with legionellae. A total of 201 water samples, including 129 cooling waters and 72 process waters, and 30 cooling lubricants were included in the study. Treated waters were screened periodically, pre and post treatment. Altogether, 72.6% of the water samples were positive for FLA, acanthamoebae being most prevalent (in 23.9% of the samples) followed by Vermamoeba vermiformis (19.4%). Only one cooling lubricant was positive (Acanthamoeba genotype T4). Legionella spp. were detected in 34.8% of the water samples and in 15% in high concentrations (>1000 CFU/100 ml). Altogether, 81.4% of the Legionella-positive samples were positive for FLA by standard methods. By applying a highly sensitive nested PCR to a representative set of random samples it was revealed that Legionella spp. always co-occurred with Acanthamoeba spp. Although the addition of disinfectants did influence amoebal density and diversity, treated waters showed no difference concerning FLA in the interphases of disinfection. It appears that FLA can re-colonize treated waters within a short period of time.     

Pore-forming toxins from pathogenic amoebae

Some amoeboid protozoans are facultative or obligate parasites in humans and bear an enormous cytotoxic potential that can result in severe destruction of host tissues and fatal diseases. Pathogenic amoebae produce soluble pore-forming polypeptides that bind to prokaryotic and eukaryotic target cell membranes and generate pores upon insertion and oligomerization. This review summerizes the current knowledge of such small protein toxins from amoebae, compares them with related proteins from other species, focuses on their three-dimensional structures, and gives insights into divergent activation mechanisms. The potential use of pore-forming toxins in biotechnology will be briefly outlined.     

Free-living amoebae isolated from water-hyacinth root (Eichhornia crassipes)

Free-living amoebae are widely distributed in aquatic environments and their hygienic, medical and ecological relationships to man are increasingly important. The purpose of this study was to isolate free-living amoebae from water-hyacinth root (Eichhornia crassipes) and the water of an urban lake in Mexico City. Five grams of wet root were seeded on non-nutritive agar with Enterobacter aerogenes (NNE). Water samples were concentrated by centrifugation at 1200g for 15 min and the pellet was seeded on NNE. Of the 16 isolated genera, 10 were detected in both habitats. The most frequent were Vannella in root and Acanthamoeba and Naegleria in water. The total number of isolates and genera isolated from root was higher than that isolated from water. The differences between root and water are probably due to the morphological characteristics of water-hyacinth root, which provides a large habitat and refuge area for many organisms.     

Molecular and physiological differentiation between pathogenic and nonpathogenic Acanthamoeba

In this study, 14 isolates of Acanthamoeba from both clinical and environmental sources belonging to seven different species were assayed for tolerance of high osmotic pressure, temperature tolerance, extracellular proteases, and cytopathic effects (CPE) on immortalized rabbit corneal epithelial cells. On the basis of the results, amoeba isolates were divided into pathogenic and nonpathogenic groups. Ribosomal DNA sequencing was performed on these isolates. Phylogenetic relationships revealed that all the pathogenic strains tested clustered together as one group, while nonpathogenic strains clustered into other groups. Sequence comparisons with previously published sequences determined that among the six new pathogenic isolates used in this study, five belong to T4 genotype and one to T11. This is the first report of a T11 genotype being found in Acanthamoeba keratitis. Received: 1 November 2001 / Accepted: 31 December 2001     

Activation of complement by pathogenic and nonpathogenic Entamoeba histolytica

Previous studies had demonstrated that strains of Entamoeba histolytica isolated from patients with colitis or amebic liver abscess were resistant to complement-mediated killing, whereas strains from asymptomatic patients were readily lysed by non-immune serum. Both serum-sensitive and serum-resistant strains of E. histolytica depleted complement rapidly as assessed by CH50, C3, and C7, and C5-9 hemolytic activities. Activation of the alternative pathway was important in lysis of nonpathogenic strains, as demonstrated by lysis by NHS (60.9 +/- 15.6%) and NHS + 5 mM EGTA (59.3 +/- 4.5%) as well as by C4-deficient guinea pig serum (72.8 +/- 7.1%) and C2-deficient human serum (64.4 +/- 11.1%), but not by NHS + 5 mM EDTA. Classical pathway activation also occurs as both pathogenic and nonpathogenic strains deplete greater than 98% of C4 activity, although it is not necessary for lysis. Pathogenic strains are not lysed by either the classical or the alternative pathway. These results suggest that pathogenic strains of E. histolytica activate complement but are able to evade an important host defense, complement-mediated lysis.     

Effect of lactose monolaurate on pathogenic and nonpathogenic bacteria

The antimicrobial activities of sucrose monolaurate and a novel ester, lactose monolaurate (LML), were tested. Gram-positive bacteria were more susceptible than Gram-negative bacteria to both esters. The minimal bactericidal concentrations of LML were 5 to 9.5 mM for Listeria monocytogenes isolates and 0.2 to 2 mM for Mycobacterium isolates.     

The biosynthesis and role of phosphorylcholine in pathogenic and nonpathogenic bacteria

Phosphorylcholine (ChoP) can be found in all life forms. Although this molecule was first thought to be uncommon in bacteria, it is now appreciated that many bacteria express ChoP on their surface. ChoP is usually attached to a glycan structure, but in some cases, it is added as a post-translational modification to proteins. Recent findings have demonstrated the role of ChoP modification and phase variation (ON/OFF switching) in bacterial pathogenesis. However, the mechanisms of ChoP synthesis are still unclear in some bacteria. Here, we review the literature and examine the recent developments in ChoP-modified proteins and glycolipids and of ChoP biosynthetic pathways. We discuss how the well-studied Lic1 pathway exclusively mediates ChoP attachment to glycans but not to proteins. Finally, we provide a review of the role of ChoP in bacterial pathobiology and the role of ChoP in modulating the immune response.     

Homologous cysteine proteinases of pathogenic and nonpathogenic Entamoeba histolytica. Differences in structure and expression.

A cDNA clone derived from the gene encoding a cysteine proteinase of pathogenic Entamoeba histolytica was isolated using an antiserum to the purified enzyme. This clone was used to identify the homologous clone in a cDNA library from nonpathogenic E. histolytica. Sequence analysis and comparison of the predicted amino acid sequences revealed a sequence divergence of 16%. Southern blot analyses indicated that (i) pathogenic isolates may contain more genes coding for these or related enzymes than nonpathogenic isolates, (ii) the structure and organization of these genes are conserved within each group of amoebae, and (iii) none of the genes is found in both pathogenic and nonpathogenic E. histolytica, underlining the notion that the two groups are genetically distinct. Northern blot analyses suggested that the cysteine proteinase is expressed by pathogenic isolates in substantially higher amounts than by nonpathogenic isolates. Overexpression of this enzyme may be an important factor in the pathogenicity of E. histolytica.     

Effect of disinfectants on pathogenic free-living amoebae: in axenic conditions.

The amoebicidal properties of chlorine, chlorine dioxide, ozone, and deciquam 222 were examined in axenic conditions. Naegleria spp. were found to be more sensitive to chlorine and chlorine dioxide than Acanthamoeba spp. No marked difference in sensitivity to ozone or deciquam 222 could be detected between the pathogenic (A-1) and nonpathogenic (1501) strains of Acanthamoeba and the pathogenic (MsT) and nonpathogenic (P1200f) strains of Naegleria. Methods of disinfection are discussed with reference to suitability of the disinfectants to real conditions.