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Endocytobiosis & Cell Res. 10, 167-183 (1993/94) 167<strong>FREE</strong>-<strong>LIVING</strong> <strong>AMOEBA</strong>: <strong>INTERACTIONS</strong> <strong>WITH</strong><strong>ENVIRONMENTAL</strong> PATHOGENIC BACTERIA.C. HARFLaboratoire d'Hydrobiologie de l'Environnement, Faculté de Pharmacie, Université LouisPasteur Strasbourg, BP. 24 - 67401 - ILLKIRCH-Cedex FRANCEKey Words : Free-living amoeba; Acanthamoeba sp.; Legionella pneumophila;Listeria monocytogenes; Vibrio cholerae; Xanthomonas maltophilia; Flavobacteriumsp.; interactions; environment.Summary: Free-living amoeba live in close association with other microorganisms inthe same ecological niches, feeding on bacteria and fungi. The efficiency ofconsumption is different, depending on protozoan type and growth state, temperature,bacterial abundance, and size and nature of bacterial prey. Many of the bacteria aredigested, especially enterobacteria which are used as nutriment. Gram positivebacteria seem to be digested slower, owing to their thick cell wall. Some bacteria mayavoid digestion by liberation out of the phagosome before lysosomal fusion or resistthe cellular mechanisms of digestion in phagosomes and escape into the host cellcytoplasm where they multiply.A number of bacteria have been reported to survive as parasites orendosymbionts of free-living amoeba, and some of these bacteria are also potentialpathogens to vertebrates. Free-living amoeba are incriminated in the ecology andepidemiology of legionellosis. They are also likely to be natural hosts for well-knownpathogens like Vibrio cholerae and Listeria monocytogenes, or nosocomial newpathogens, like Xanthomonas maltophilia and Flavobacterium sp.Their interacting with protozoa in biofilms associated to intracellularreplication and inclusion of pathogen bacteria in cysts give these pathogens apossibility of survival and transmission in unfavourable conditions.INTRODUCTIONFree-living amoeba are largely distributed in environmental waters and soils;they occur world-wide. The vegetative forms or trophozoites move with theirpseudopodia and multiply by binary division. Under hostile environmental conditions,the trophozoite is transformed into the cystic resistance form. They offer a greatvariety of species, some of them are described as amphizoic protozoa, capable ofliving as parasites or as free-living.


168 HarfRecognition of human disease caused by free-living amoeba is relatively recentand gave a new interest for the study of these amoeba. Culbertson (1961) describedthe neuropathogenicity in experimental animals of Acanthamoeba isolated fromkidney cell cultures, and predicted the possible role of these amoeba as causal agentsof undiagnosed granulomatous disease. Soon thereafter, Fowler and Carter (1965)identified the first human cases of amoebic meningoencephalitis. Virulent free-livingamoeba implicated in acute infections of the central nervous system areamoeboflagellates of the genus Naegleria. They penetrate through the nasal mucosain children and young adults who have a history of swimming in or contact withheated fresh water, and cause acute primary amoebic meningoencephalitis.Acanthamoeba spp. and the recently described Balamuthia mandrillaris (Visvesvaraand Stehr-Green, 1990; Visvesvara et al., 1990; 1993) cause a chronic disease,granulomatous amoebic encephalitis, predominantly in immunosuppressed patients. Inaddition, Acanthamoeba spp. also cause a painful, vision-threatening disease of thehuman cornea, Acanthamoeba keratitis, by penetrating through a lesion of the corneadue to an accident or to contact lenses, thus invading and destructing the cornealstroma. In recent years, a rapid growth in the number of cases of Acanthamoebakeratitis occurred, due to the increase of contact lens wearers and to the resistance ofAcanthamoeba cysts to water chlorination and contact lens disinfection systems (DeJonckheere, 1991; Harf, 1991).Whereas pathogenic Naegleria are thermophilic and are found throughout theworld mostly in heated aquatic environments, whether man-made or natural,Acanthamoeba species are present in all types of environments all over the world :fresh water, chlorinated swimming pools, medicinal pools, hot tubs, seawater, oceansediment, tap water, bottled mineral water, industrial cooling water, air conditioners,soil, sewage, compost, dust in air, dental treatment units, dialysis units,gastrointestinal washings, dialysis units, contact lens-care solutions and cases, inaddition to bacterial, fungal and mammalian cell cultures (Derr-Harf and DeJonckheere, 1984; Harf and Monteil, 1989; Visvesvara and Stehr-Green, 1990).The notion of pure culture discovered by Pasteur and Koch was necessary tostudy free-living amoeba, as they coexist with other microorganisms in naturalenvironment. They are mainly cultured on Agar plates spread with a thin suspensionof E. coli or E. aerogenes for nutritional purpose. Their taxonomy was at firstestablished on a morphological and pathogenic basis, with the observation of cyststructure and trophozoites on thin-plate cultures (Pussard and Pons, 1977) or hangingdrops (Page, 1976), and experimental infections. Axenic culture techniques oftrophozoites in liquid medium allowed to progress in the knowledge of the cellularand molecular biology of Acanthamoeba and Naegleria. These two genera are knownto contain pathogenic species, moreover they are both relatively easy to axenize.


Endocytobiosis & Cell Res. 10, 167-183 (1993/94) 169Refined biochemical techniques such as isoenzyme analysis (De Jonckheere, 1982; DeJonckheere, 1983), restriction fragment length polymorphism (RFLP) analysis andpolymerase chain reaction (PCR) of whole cell or ribosomal DNA (De Jonckheere1988; Kilvington et al., 1991; Vodkin et al., 1992) have shown that some speciesdescribed on minor morphological differences were synonyms, while other strains aredifficult to group or seem to belong to as yet undefined species. Thus, while thegenus is easily recognized, the species identification has grown rather difficult.Currently the morphology of the cyst can be used to separate 3 major groups withinthe genus Acanthamoeba (Pussard and Pons, 1977) and freshly isolated strains areoften referred to as Acanthamoeba sp..Free-living amoeba live in close association with other microorganisms in thesame ecological niches, feeding on bacteria and fungi. An amoeba much used as alaboratory model for biochemical research, Acanthamoeba castellanii, wasdiscovered in a yeast culture (Castellani, 1930). Free-living amoeba can live in anymoist environment as far as there are bacteria (Alexeieff, 1972).ASSOCIATION <strong>WITH</strong> BACTERIABacteriophagous amoeba regulate the microbial population density, asprotozoan grazing is reported to be the major factor responsible for bacteriadecreases in natural waters and in sludge, although they seem unable to eradicate thepopulations of prey on which they feed, escaping the threat of self-elimination(Alexander, 1981). Amoeba in fact also stimulate the activity of their prey byrejecting wastes reutilized by bacteria, thus recycling the bacterial biomass,(Couteaux et al., 1988) and by the production of stimulatory factors, (Levrat et al.,1992).Free-living amoeba interact with a variety of bacteria. This interaction rangesfrom bacteria that serve as food-sources or are sequestered as endosymbionts withinamoeba to bacterial species that are amplified by amoeba.EndocytosisThe mechanisms of endocytosis and of digestion are similar to those observed inmacrophages. In Acanthamoeba bacteria are internalized individually, then grouped inone vacuole; after fusion with lysosomes, creating a phagolysosome, acid pH andlysosomal enzymes lyse the phagocyted bacteria (Drozanski and Chmielewski,1979). This similarity suggests that amoeba might be useful as a model to elucidate theinteraction of bacteria with human phagocytic cells. However, virulence determinantsfor infection of, and mutiplication in amoeba and phagocytes do not appear to be the


170 Harfsame (Dowling et al., 1992). Endocytosis and intracellular multiplication of L.pneumophila in amoeba and in a monocyte-like cell line (U937) was investigated byusing cytocholasin D, and methylamine (King et al., 1991). Cytocholasin D is aninhibitor of actin filament polymerization involved in plasma membrane invaginationduring phagocytosis. Methylamine is an inhibitor of transglutaminase, a plasmamembrane enzyme involved in aggregation of ligand-receptor complexes required forreceptor-mediated pinocytosis in fibroblasts and macrophages. Methylamine inhibitedreplication of L. pneumophila in amoeba. Intra amoebal multiplication of L.pneumophila was not inhibited by cytocholasin D. Thus it is microfilamentindependent,in contrast to macrophages, and according to Fields (1993) a newterminology is needed to describe bacterial uptake by a microfilament-independentprocess.The efficiency of consumption is different, depending on protozoan type andgrowth state, temperature, bacterial abundance, and size and nature of bacterial prey(Gonzalez et al., 1990). Many of the bacteria are digested, especially enterobacteriawhich are used as nutriment. Gram positive bacteria seem to be digested slower,owing to their thick cell wall.Escaping digestionSome bacteria, as shown for Gram negatives by Cavalier-Smith and Lee (1985),may avoid digestion by liberation out of the phagosome before lysosomal fusion orresist the cellular mechanisms of digestion in phagosomes and escape into the hostcell cytoplasm where they multiply. (Hall and Voelz, 1985; Reisser et al., 1985).According to Choi et al., 1992; and Jeon, 1993, non-fusible vesicles such assymbiosomes include symbiont derived lipopolysaccharides which prevent lysosomesfrom fusing with them. Fields (1993), investigating a model of L. pneumophilaendocytosis by Hartmannella vermiformis, described a fusion of the endosómecontaining the internalized Legionella with the host cell's endoplasmic reticulum. Theendoplasmic reticulum becomes the site of bacterial multiplication as it is rich innutrients and would prevent exposure to lysosomes.Escaping digestion can evolve in different types of interactions between amoebaand bacteria :Endosymbionts are permanent harboured bacteria and cannot be cultured so far.Some are described as toxic killer symbionts (Görtz, 1993). An obligateintracellular bacterial parasite, decribed as Sarcobium lyticum, causing fatalinfection in Acanthamoeba castellana, shows a close relationship to anintracellular Legionella (Drozanski 1991). According to Fritsche et al, (1993),


Endocytobiosis & Cell Res. 10, 167-183 (1993/94) 171Fig. 1: Acanthamoeba harbouring Aeromonas: growth curvescontrol culture without Hg,culture with HgCl 2 at 0.33mg.ml -1 ,culture with P.M.A. at 0.33 mg.ml -1 ,Growth at 30° was monitored by counting trophozoites in a haemocytometerA) Acanthamoeba harbouring narrow spectrum Hg-resistant A. caviae with a reductase gene.B) Acanthamoeba harbouring broad spectrum Hg-resistant A. sobria with lyase and reductase genes


172 Harfendosymbiosis occurred in 14 of 57 axenically grown Acanthamoeba isolates; theendosymbionts comprised Gram negative rods and cocci. Michel et al (1994) gaveanother example for bacterial obligate parasites in Acanthamoeba. Jeon (1983)described a symbiosis with a dependency of the amoeba on the presence ofharboured bacteria; prokaryotic xenosomes, from initially virulent for the hostcell, become vital for the latter.Amoeba can use bacterial metabolism to protect themselves against xenobioticpollutants. Our group showed a nonobligate or transitory endocytobiosis, whereamoeba acquired Hg-resistance by using harboured resistant bacteria (Hagneréand Harf, 1993) (Fig. 1).On the other hand bacteria use amoeba as host; bacterial multiplication in amoebais dependent on an intracellular environment and not on soluble nutrients secretedby amoeba (King et al., 1991). Bacteria also use amoeba as dispersal vector andprotection system against fluctuating environmental parameters (Harf andMonteil, 1988). Several authors have shown that bacteria grown in free-livingamoeba are less susceptible to chlorination and biocides (Barker et al., 1992;Kilvington and Price, 1990; King et al., 1988). According to King et al. (1988),resistance to digestion by predatory protozoa might be an evolutionary precursorof bacterial pathogenicity and a survival mechanism for bacteria in aquaticenvironment.PATHOGENIC BACTERIAAs the cell-cycle of Acanthamoeba consists of growth and replication by binaryfission under optimal growth conditions and encystment under environmental stress,harboured bacteria are thus disseminated and are protected from chemical inactivationin cysts. Jadin (1973), isolating Mycobacteria from free-living amoeba, foresaw theirpart as hosts for virus and bacteria. Free-living amoeba were incriminated in theecology and epidemiology of legionellosis (Fields, 1991; Rowbotham, 1984; Tyndalland Domingue, 1982). They are also likely to be natural hosts for well-knownpathogens like Vibrio cholerae and Listeria monocytogenes, and nosocomial newpathogens, i.e. Pseudomonas sp., Xanthomonas maltophilia, Flavobacterium sp..LegionellapneumophilaLegionella appear to have the capacity to survive as intracellular parasites offree-living protozoa as well as intracellular pathogens in humans. Legionellosisconsists of two distinct clinical syndromes, Legionnaire's disease and Pontiac fever.Legionnaire's disease is characterized by pneumonia while Pontiac fever is a self-


Endocytobiosis & Cell Res. 10, 167-183 (1993/94) 173limited, non-pneumonic, respiratory illness. In the pneumonic form of legionellosis,the bacteria multiply intracellularly in the alveolar macrophages of an infected oftenimmunodepressed host. This ability to multiply intracellularly is considered thebacterium's primary virulence factor.Inhalation of aerosols containing Legionella is presumed to be the primarymeans of acquiring legionellosis (Fields, 1991). Known sources for transmission ofLegionella via aerosols have been identified: showers, respiratory care equipment,air-conditioning cooling-towers, whirlpool baths, ultrasonic mist machines, lakes andrivers (Breiman, 1993). Legionella are ubiquitous in freshwater environment andwere isolated from a number of aquatic habitats. The contrast between the ubiquity ofthese bacteria and the fastidiousness of their recovery from environmental samples,added to the fact that their growth in the environment in the absence of protozoa hasnot been documented, led to the assumption that protozoa are their primary means ofproliferation in nature (Fields, 1993; Rowbotham, 1980). Rowbotham (1980) was thefirst to report phagocytosis of L. pneumophila by amoeba and suggested that avacuole or an amoeba, full of legionellae might be the infective particle for man ratherthan free legionellae themselves.Several studies confirmed the intracellular growth of Legionella in variousprotozoa (Anand et al., 1984; Fields et al., 1984; Tyndall and Domingue, 1982). FiveCATEGORYAmoebaORGANISMAcanthamoeba castellaniiA. polyphagaA. palestinensisA. royrebaA. culbertsoniNaegleria gruberiN. fowleriN. lovaniensisN. jadiniHartmannella vermiformisH. cantabrigiensisVahlkampfia jugosaEchinamoeba exundansCiliated protozoa(Fields, 1993)TetrahymenaT. voraxpyriformisTable 1: Protozoa supporting the growth of Legionella


174 Harfgenera of amoeba (Acanthamoeba, Naegleria, Hartmannella, Vahlkampfia,Echinamoeba), and one genus of ciliated protozoa (Tetrahymena), have shown tosupport intracellular growth of L. pneumophila (Fields, 1993), (Table I).The occurrence of several cases of legionellosis after immersion in the Rhineriver (Hasselmann et al., 1987), in addition to frequent isolation of free-living amoebain the same natural sites (Harf and Monteil, 1989), led us to investigate theinteractions between Legionella and free-living amoeba. We demonstrated byimmunofluorescence assay (IFA) and inoculation to guinea pigs that washed and lysedamoeba from river water and sediment contained L. pneumophila (Harf and Monteil,1988). Direct culturing of Legionella from environmental samples proved to be verydifficult, due to the abundance of the associated bacterial flora overgrowing thecultures. In order to improve the detection of Legionella spp., we developed apolymerase chain reaction assay (PCR), which was useful in river waters, and inAcanthamoeba cysts lysed by shaking with glass beads (Jaulhac et al., 1993) (Fig. 2).ListeriamonocytogenesL. monocytogenes, a facultative intracellular bacteria is a common pathogenthroughout the world. Its epidemiology is mostly food-borne. Particularly susceptiblehosts are pregnant mothers, their foetuses, and immunocompromised individuals. Themajor symptomatic manifestations of listeriosis are septicemia and meningitis. Thevirulence mechanism enabling L. monocytogenes to cause these invasive diseases isthought to be its ability to survive and replicate in cells of the infected host.In macrophages, after phagocytosis, Listeria secretes hemolysin andphospholipase C. The enzymes break down the phagosomal membrane so that thebacteria can enter the macrophage cytoplasm, where it grows and divides using hostcell nutrients.Within and between host cells Listeria has a spectacular mode of transport(Tilney and Tilney, 1993). By inducing host cell actin to assemble from its surface,the bacteria forms a tail composed of many short, crossbridged actin filaments. Withthis tail Listeria is propelled across the cytoplasm like a comet streaking across thesky at speeds up to 1 μm s -1 , and generate a protuberance when they make contactwith the plasma membrane. This long pseudopod is phagocytosed by the neighbouringmacrophage. The doubly encapsulated Listeria escapes into the cytoplasm bydissolving both membranes by phospholipases and hemolysin, and the cycle isrepeated.Ly and Muller (1990) suggested that protozoa might be the missing link in theecology and pathology of Listeria, and reported that different species of Listeriawere ingested and multiplied intracellularly in protozoa. The authors reported that


Endocytobiosis & Cell Res. 10, 167-183 (1993/94) 175Fig. 2: Isolation of environmental Legionella


176 Harfhemolytic species, L. monocytogenes and L. seeligeri ruptured their host. However,these reports are preliminary and lack confirmation; as the authors used non-axenicamoeba, there might be other environmental bacteria interacting.Our group investigated the possibility of free-living amoeba and Listeriainteractions by trying cocultures with different species of Listeria and anenvironmental strain of axenic Acanthamoeba sp.. Listeria strains were kindlyprovided by Prof. P. Berche (Paris). By observing in vivo thin-plate cultures, we sawListeria moving fastly intracellularly. Fig. 3 shows intracellular L. monocytogenes inAcanthamoeba sp. trophozoites observed a) in vivo with phase contrast microscopy,and b) after Giemsa staining. Amoeba thus seem to engulf Listeria. Subsequently wetried to prove intracellular multiplication by establishing growth-curves with axenicAcanthamoeba sp. and various Listeria strains, but although Listeria penetrated, theycould not be recovered by culture (Fouchecourt, 1993).Vibrio choleraeA recent investigation (Thorn et al., 1992) was undertaken to determine whetherAcanthamoeba polyphaga and Naegleria gruberi could be hosts for various strains ofV. cholerae. While the existence of an aquatic reservoir of V. cholerae and its longsurvival under nutrient stress were proved by laboratory and field studies, the preciseidentity of the ecological niche favouring its survival in an aquatic environmentremains obscure. The results of this investigation have demonstrated that V. choleraeis capable of survival within amoeba. Furthermore, it was shown that V. choleraecould survive encystment within N. gruberi cysts. V. cholerae had not previously beenassociated with an intracellular mode of existence., and its pathogenesis does notinvolve cellular invasion. The survival of V. cholerae within cysts would provide aprotected niche under unfavourable conditions and a means of dispersal.Multiresistant nosocomial bacteriaIn addition to their function of vectors for highly pathogenic bacteria, weobserved that environmental free-living amoeba harbour various bacteria susceptibleto be pathogens for immunocompromised patients in hospitals (Table II). Harbouredbacteria were isolated in amoeba lysates.Bacteria of the Pseudomonas group (Xanthomonas maltophilia, Pseudomonasspp, Comamonas acidovorans) and of the Flavobacterium group (Flavobacterium sp.,Sphingobacterium multivorum) are the most frequent in environmental waters,associated to Acanthamoeba and Naegleria in natural sites (Harf and Monteil, 1989).Nosocomial infections caused by multiresistant Xanthomonas maltophilia andFlavobacterium sp. occurred in hospitals using non-chlorinated groundwater (Monteil


Endocytobiosis & Cell Res. 10, 167-183 (1993/94) 177a) Thin plate culture, phase contrast (x 1000)b) Giemsa staining (x 1000)Fig. 3: Intracellular Listeria monocytogenes in Acanthamoeba sp.


178 Harfet al., 1992). Environmental amoeba harboured X. maltophilia and Flavobacteriumsp. highly resistant to various antibiotics. Axenisation for further study of amoebastrains was impossible with antibiotics normally used (penicillin, streptomycin);intracellular bacteria persisted after numerous subcultures. Amoeba strains werefinally cured after successive transfers on disks with new b-lactams and 6-fluoroquinolones (Hagneré and Harf, 1993). These new antibiotics were chosen fortheir high activity against multiresistant opportunistic bacteria involved in nosocomialhospital infections. Other nosocomial pathogens, i.e. Acinetobacter sp., Alcaligenessp., Aeromonas spp., and Legionella pneumophila, already mentioned above, werealso harboured by free-living amoeba isolated from environmental waters (Hagneré,1993; Hagneré and Harf, 1993; Harf and Monteil, 1988; Mirgain et al; 1992; Monteilet al, 1992).Amoeba are likely to be natural hosts for resistant nosocomial bacteria. ThisAmoebaAcanthamoebaNaegleria spp.spp.Harboured bacteriaXanthomonas maltophilia (5)Flavobacterium sp. (4)Sphingobacterium multivorum (1)Legionella pneumophila (3)Pseudomonas fluorescens (2)P. aeruginosa (2)P. putida (2)Comamonas acidovorans (1)Alcaligenes denitrificans (2)Aeromonas spp. (2)X. maltophilia (5)Flavobacterium sp. (4)S. multivorum (1)Legionella pneumophila (3)P. aeruginosa (4)P. putida (4)Alcaligenes denitrificans (4)Acinetobacter anitratus (4)(1) Hagneré, 1993(2) Hagneré and Harf, 1993(3) Harf and Monteil, 1992(4) Mirgain et al, 1992(5) Monteil et al, 1992Table 2: Harboured bacteria in environmental free-living amoeba


Endocytobiosis & Cell Res. 10, 167-183 (1993/94) 179potential reservoir of hydric opportunistic bacteria in nonchlorinated or insufficientlytreated water used in multiple ways in hospitals, may represent a high risk forimmunocompromised patients and in intensive care units. Nosocomial infections maybe the result of our man-made environments which favour the growth andtransmission of nosocomial bacteria to susceptible hosts.CONCLUSIONA number of bacteria have been reported to survive as parasites orendosymbionts of free-living amoeba, and some of these bacteria are also potentialpathogens to vertebrates. Free-living amoeba might be dispersal vectors andprotection systems for known human pathogens as Mycobacteria and Vibriocholerae, or nosocomial new pathogens. Legionella pneumophila has the ability tosurvive as intracellular parasite both in protozoan and in mammalian cells. Anothermammalian intracellular parasite, Listeria monocytogenes was also reported tomultiply in Acanthamoeba sp.. This might be a survival mechanism for bacteria inaquatic environment. Their interacting with protozoa in biofilms associated tointracellular replication and inclusion of pathogen bacteria in cysts give thesepathogens a possibility of survival and transmission in unfavourable conditions. Someintracellular pathogens may even have acquired their virulence by first adapting tointracellular life in predatory free-living protozoa.Acknowledgements: This work was supported by PIRE-Eau Alsace, CNRS Franceand Région Alsace. The author thanks Prof. H. Monteil, Institute of Bacteriology,Faculty of Medecine, ULP Strasbourg for helpful criticism of this manuscript..REFERENCESAlexander M., 1981. Why microbial predators and parasites do not eliminate theirprey and hosts. Ann. Rev. Microbiol. 35, 113-133.Alexeieff A., 1972. Sur les caractères cytologiques de la systématique des amibes dugroupe limax. Bull. Soc. Zool. France. 37, 55-74.Anand C, Skinner Α., Malic Α., Kurtz J., 1984. Intracellular replication of Legionellapneumophila in Acanthamoeba palestinensis. In: Legionella, Proc. 2nd Int.Symp. ed. ASM., Washington DC. pp 330-332.Barker J., Brown M.R.W., Collier P.J., Farrell I., Gilbert P., 1992. Relationshipbetween Legionella pneumophila and Acanthamoeba polyphaga: physiologicalstatus and susceptibility to chemical inactivation. Appl. Environ. Microbiol. 58,2420-2425.


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