Live Amoebae Research Articles

Presence of Non-Tuberculous Mycobacteria Including Mycobacterium avium subsp. paratuberculosis Associated with Environmental Amoebae.

One of the obstacles to eradicating paratuberculosis or Johne's Disease (JD) seems to be the persistence of Mycobacterium avium subsp. paratuberculosis (Map) in the environment due to its ability to survive alone or vectorized. It has been shown that Map is widely distributed in soils and water. Previously, we isolated amoebae associated with Map strains in the environment of bovines from an infected herd. This work aims to verify our working hypothesis, which suggests that amoebae may play a role in the transmission of JD. In this study, we sampled water in the vicinity of herds infected with Map or Mycobacterium bovis (M. bovis) and searched for amoebae and mycobacteria. Live amoebae were recovered from all samples. Among these amoebae, four isolates associated with the presence of mycobacteria were identified and characterized. Map and other mycobacterial species were detected by qPCR and, in some cases, by culture. This study suggests that amoebae and Map may be found in the same environment and might represent a risk of exposure of animals to pathogenic mycobacteria. These data open up new perspectives on the control measures to be put in place to prevent contamination by Map.

Gongolarones as antiamoeboid chemical scaffold

Free Living Amoeba (FLA) infections caused by Acanthamoeba genus include chronic nervous system diseases such as Granulomatous Amoebic Encephalitis (GAE), or a severe eye infection known as Acanthamoeba keratitis (AK). Current studies focused on therapy against these diseases are aiming to find novel compounds with amoebicidal activity and low toxicity to human tissues. Brown algae, such as Gongolaria abies-marina (previously known as Cystoseira abies-marina, S.G. Gmelin), presents bioactive molecules of interest, including some with antiprotozoal activity. In this study, six meroterpenoids were isolated and purified from the species Gongolaria abies-marina. Gongolarones A (1), B (2) and C (3) were identified as new compounds. Additionally, cystomexicone B (4), 1′-methoxyamentadione (5) and 6Z-1′-methoxyamentadione (6) were isolated. All compounds exhibited amoebicidal activity against Acanthamoeba castellanii Neff, A. polyphaga and A. griffini strains. Gongolarones A (1) and C (3) showed the lowest IC50 values against the two stages of these amoebae (trophozoite and cyst). Structure-activity relationship revealed that the cyclization by ether formation from C-12 to C-15 of 1, and the isomerization Δ2 t to Δ3 t of 3, increased the antiamoeboid activity of both compounds. Furthermore, gongolarones A (1) and C (3) triggered chromatin condensation, mitochondrial malfunction, oxidative stress, and disorganization of the tubulin-actin cytoskeleton in treated trophozoites. Moreover, transmission electron microscopy (TEM) images analysis revealed that compounds 1 and 3 induced autophagy process and inhibited the encystation process. All those results suggest that both compounds could induce programmed cell death (PCD) in Acanthamoeba.

Free-living amoebae in an oil refinery wastewater treatment facility

Free Living Amoebae (FLA) are ubiquitous microorganisms reported from harsh environmental conditions. Oil refinery facilities consume vast volumes of water during their processes, generating a large amount of wastewater. The present study aimed to evaluate the wastewater treatment process in an oil refinery wastewater treatment facility (ORWWTF) for the presence of FLA. Water samples were collected from an oil refinery wastewater (ORWW) for nine months. After recording physical-chemical features, samples were cultivated onto non-nutrient agar (NNA). The discriminative fragments of the ribosomal RNA (rRNA) gene were amplified and sequenced to characterize the isolated FLA. Phylogenetic tree, and network analysis were employed to evaluate genetic relationships. The thermo- and osmotolerant tests were performed on the isolated FLA. Twenty-five (32.9%) samples were positive for FLA cultivation. Acanthamoeba spp., Vahlkampfiids, and Vermamoeba spp. were detected, of which Acanthamoeba species were predominant. There was no statistical correlation between pH, NH3, PO4, H2S, and TDS with the presence of FLA. A statistical correlation between the presence of FLA and the type of wastewater treatment plants (WWTPs) was significant (P-value = 0.011). All Acanthamoeba spp. isolates belonged to the genotypes T4 (17/21; 80.95%) and T11 (4/21; 19.05%). Vahlkampfiids were Naegleria spp., (7/10; 70%), Tetramitus aberdonicus (1/10; 10%), Learamoeba spp., (1/10; 10%), and Vahlkampfia spp., (1/10; 10%). All three Vermamoeba spp. were V. vermiformis. The ORWW contains toxic materials, and a few microorganisms can stay active in these environments. This is the first study which isolates FLA from such super harsh conditions. For the first time, T. aberdonicus, and Learamoeba spp., were isolated from oily wastewater. Our findings signify the concern due to the distribution of potentially pathogenic FLA to downstream lands via treated wastewater that may be released after treatment processing.

Isolation and Identification of Free Living Amoeba from Patients and Contact Lens Users in Iran.

Background:Free-living amoebae (FLA) such as Acanthamoeba spp., are considered as opportunistic and pathogenic protozoans. Acanthamoeba granulomatous encephalitis (AGE) is a serious threat for immunodeficient patients and Acanthamoeba keratitis (AK) for contact lens users. We aimed to identify the presence of free living amoebae in nasal swabs of patients and contact lens users in Qazvin, Iran.Methods:During 2019, 251 nasal and oral swabs (including the pharynx and mouth) were collected from patients with diabetes, AIDS and those under periodic dialysis in Qazvin, Iran. In addition, 27 soft contact lenses were collected from the participants. Following DNA extraction, PCR and sequencing were conducted to identify the genotypes of the amoeba. Phylogenetic analysis of the identified sequences was performed using MEGA 7 software.Results:A strain of Acanthamoeba belonging to the T3 genotype was isolated from hemodialysis patients. Two specimens of Acanthamoeba with T3 genotype were isolated from keratitis patients.Conclusion:The clinicians should pay attention to the possible complication of this organism because this amoeba is potentially pathogenic for immunocompromised patients. Since the amoeba is present in environmental resources, the use of contact lenses should be accompanied by considering proper hygiene.

Molecular Comparison of Free -Living Amoeba Isolated From Iraqi, Iranian and Turkish Waters

Amoebas live freely in different climates and parts of the world. Several species of Free Living Amoeba (FLA) are capable of causing serious as well as fatal infections in human beings. The aim of this study was to identify and compare genotypes of water-polluting FLA in major rivers and lakes of Iraq and compare them with FLA isolates from Iran and Turkey. For this purpose, the study included 20 water samples from the Tigris River, Euphrates River , Najaf Sea and Dukan lake in Iraq, 20 water samples from Marivan, Velasht, and Soleimanshah lakes and Caspian sea in Iran, and 20 water samples from Sabanca, Seyfi , Hazar and Yay lakes in Turkey. The samples were studied by culture methods, invert microscope, and molecular methods.
 After inoculation and microscopic examination, cysts and trophozoites were detected in 18 cultured specimens. Overall, out of 60 water samples, 30 cases (30%) were found to be contiminated in the three countries. The highest pollution was in Turkish waters (40%), while the rate in the Iraqi and Iranian water samples was the same (25%). Because of the various species of FLA , it may be difficult to distinguish pathogenic from non-pathogenic species by culture on non-nutrient agar. Therefore, the molecular technology was applied in this study. Only a specific band of Acanthamoeba Rns genes which ranged from 423 to 551 nucleotides was observed. The isolates belonged to the T3 genotype . In addition, it was a new isolate that differs from what exists in other neighboring countries , registered in the GenBank under accession number MN462973 as the Acanthamoeba genotype T3 isolate T3 Iraq . This is the first study to detect pathogenic FLA in Iraq by PCR and Sequencing techniques. Given the high prevalence of Acanthamoeba potential pathogenic genotypes in various environmental sources and the evidence of T3 genotype in Iraqi specimens , more studies about Acanthamoeba and other pathogenic FLA for various environmental sources in Iraq are required.

Detection of Free Living Amoeba Acanthamoeba in the Nasal and Oral Discharges of HIV + / HIV­ Sample in Arak

Detection of Free Living Amoeba Acanthamoeba in the Nasal and Oral Discharges of HIV + / HIV­ Sample in Arak

Detection of Free Living Amoeba infection in patients with suspected central nervous system and keratitis disease in Shiraz, southern Iran

BackgroundFree Living Amoebas, as opportunistic protozoa, can cause more problems such as meningoencephalitis, encephalitis and keratitis in human being. These protozoa have been isolated from many sources in Iran. This study was undertaken to determine the diseases due to these parasites in the south of Iran. Methods In this cross-sectional study, 200 cerebrospinal fluid (CSF) samples and 15 corneal scrapings were collected from patients admitted in clinics of Shiraz city. The samples were examined by light microscopy, cultivation and molecular methods. Phylogenetic relationship was also conducted among the sequences and various Acanthamoeba spp. based on nucleotide sequences in NCBI GenBank. Results No infection in CSF samples was seen, while one patient suspected with keratitis was positive to Acanthamoeba sp. infection only by PCR. Using Sequencing technique and Phylogenetic tree, the genotype of the parasite was demonstrated T4. This sample belonged to a 26 year old woman who used a contact lens. Conclusion Our results indicate that it is necessary to pay attention to the complexity of the free living amoeba infections, especially in soft contact lens wearers. Also, PCR as an appropriate method in diagnosis is recommended for the detection of free living amoebae. However, it is unavoidable to suspect these protozoa as an infectious agent in patients with central nervous system infection due to increased immunodeficiency disorders.

Special issue on Free Living Amoebae (FLA): Recent advances presented at the XVIIth International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM, 2017), Zarzis, Tunisia

Special issue on Free Living Amoebae (FLA): Recent advances presented at the XVIIth International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM, 2017), Zarzis, Tunisia

Isolation of Balamuthia mandrillaris (Free Living Amoeba) from Shatt Al-Arab River in Basrah, South of Iraq

Isolation of Balamuthia mandrillaris (Free Living Amoeba) from Shatt Al-Arab River in Basrah, South of Iraq

Bio-characterization of Some Free Living Amoebae in the Surface Water of the City of Yaounde: Relationship to Physico-chemical Parameters of the Medium

A study was carried in Yaounde to evaluate the level of organic pollution in the surface water and to determine the distribution of pathogenic free living amoeba in the aquatic ecosystem. Six points were selected on the Olezoa and the Abiergue streams of Yaounde for our study. Physico-chemical pollution was carried out by standard methods of assessment. The parameters measured were suspended solids, electric conductivity, pH, TDS, Oxydability, BOD5, COD, humidity, Dissolved Oxygen. The free living amoeba were identified by direct observation on microscope and observation after culture on Non Nutritive Agar (NNA)and Page’s Agar Saline Solution (PAS) which are all enriched with Escherichia coli. The data obtained reveal a high pollution of these urban streams of the Mfoundi River system and presents a high densities of telluric amoebae such as Acanthamoeba (21 Cell./L), Balamuthia (11 Cell./L), Naegleria (6 Cell./L), Vanella (17 Cell./L) and Vermamoeba (5 Cell./L). The sizes vary between 12 to 94 μm for the different forms isolated and identified in our study. The presence of amoeba are significantly correlated with hydrometry, temperature, nitrates suspended solids, COD and BOD5 (p <0.001). These organisms in water show contamination of the groundwater by wastewater during the study period.

Competition between Naegleria fowleri and Free Living Amoeba Colonizing Laboratory Scale and Operational Drinking Water Distribution Systems.

Free living amoebae (FLA), including pathogenic Naegleria fowleri, can colonize and grow within pipe wall biofilms of drinking water distribution systems (DWDSs). Studies on the interactions between various FLA species in biofilms are limited. Understanding the interaction between FLA and the broader biofilm ecology could help better predict DWDS susceptibility to N. fowleri colonization. The aim of this study was to determine if N. fowleri and other FLAs ( Naegleria, Vermamoeba, Willaertia, and Vahlkampfia spp.) cocolonize DWDS biofilm. FLAs commonly isolated from DWDSs ( N. fowleri, V. vermiformis, and N. lovaniensis) were introduced into laboratory-scale biomonitors to determine the impact of these amoebae on N. fowleri's presence and viability. Over 18 months, a single viable amoebae ( N. fowleri, N. lovaniensis, or V. vermiformis) was detected in each biofilm sample, with the exception of N. lovaniensis and N. fowleri, which briefly cocolonized biofilm following their coinoculation. The analysis of biofilm and bulk water samples from operational DWDSs revealed a similar lack of cocolonization with a single FLA detected in 99% ( n = 242) of samples. Interestingly, various Naegleria spp. did colonize the same DWDS locations but at different times. This knowledge furthers the understanding of ecological factors which enable N. fowleri to colonize and survive within operational DWDSs and could aid water utilities to control its occurrence.

Molecular identification of Vermamoeba vermiformis from freshwater fish in lake Taal, Philippines

Free Living Amoebae (FLA) are considered ubiquitous. FLAs may infect various biological organisms which act as reservoir hosts. Infected freshwater fishes can pose a public health concern due to possible human consumption. This study aims to identify possible pathogenic FLAs present in freshwater fishes. Seventy five (75) Oreochromis niloticus were studied for the presence of FLAs. Fish organs were suspended in physiologic saline pelleted and cultured in non-nutrient agar (NNA) lawned with Escherichia coli and were incubated in 33 °C for 14 days. Eighteen (18) fish gills and nineteen (19) fish intestine samples presented with positive growth. Trophozoites and cystic stages of FLAs were subcultured until homogenous growth was achieved. Cells were harvested from cultured plates and DNA was extracted using Chelex resin. DNA was subjected to polymerase chain reaction using universal forward primer EukA and reverse primer EukB targeting the 18s RNA. Of the 37 plates that presented with positive amoebic growth, 9 samples showed the presence of DNAs and were sent for further purification and sequencing. Basic Local Alignment Search Tool (BLAST) results showed that protists isolated from fish organs in Lake Taal include: Eocercomonas (HM536152), Colpoda steinii (KJ607915) and Vermamoeba vermiformis (KC161965). The results showed that fresh-water fishes can harbour FLAs in the gut. It is proposed that freshwater reservoirs utilized for aquaculture be monitored for the presence of FLAs and extensive study be conducted on the pathogenicity of bacterial endosymbionts and infecting viruses to its mammalian and non-mammalian host.

Entamoeba histolytica Trophozoites and Lipopeptidophosphoglycan Trigger Human Neutrophil Extracellular Traps.

Neutrophil defense mechanisms include phagocytosis, degranulation and the formation of extracellular traps (NET). These networks of DNA are triggered by several immune and microbial factors, representing a defense strategy to prevent microbial spread by trapping/killing pathogens. This may be important against Entamoeba histolytica, since its large size hinders its phagocytosis. The aim of this study was to determine whether E. histolytica and their lipopeptidophosphoglycan (EhLPPG) induce the formation of NETs and the outcome of their interaction with the parasite. Our data show that live amoebae and EhLPPG, but not fixed trophozoites, induced NET formation in a time and dose dependent manner, starting at 5 min of co-incubation. Although immunofluorescence studies showed that the NETs contain cathelicidin LL-37 in close proximity to amoebae, the trophozoite growth was only affected when ethylene glycol tetra-acetic acid (EGTA) was present during contact with NETs, suggesting that the activity of enzymes requiring calcium, such as DNases, may be important for amoeba survival. In conclusion, E. histolytica trophozoites and EhLPPG induce in vitro formation of human NETs, which did not affect the parasite growth unless a chelating agent was present. These results suggest that NETs may be an important factor of the innate immune response during infection with E. histolytica.

Acanthamoeba and Fusarium interactions: A possible problem in keratitis

The incidence of Acanthamoeba and Fusarium species has increased in contact lens-related infectious keratitis. They share several environments and cases of co-infection have been reported. The interaction between the amoebae and other microorganisms may result in significant changes for both, like increased virulence in mammalian hosts. In this study, we evaluated the interaction of three Acanthamoeba castellanii strains with Fusarium conidia and the possible implications on keratitis. F. conidia were internalized by A. castellanii strains and were able to germinate inside the amoebae. The co-culture with the live amoebae, as well as the amoebal culture supernatant and lysate, increased the fungal growth significantly. Moreover, live F. solani and its culture supernatant enhanced the survival of amoebae, but in a different way in each amoebal strain. The encystment of the A. castellanii strain re-isolated from rat lung was increased by the fungus. These results show that A. castellanii and F. solani interaction may have an important influence on survival of both, and specially indicate a possible effect on virulence characteristics of these microorganisms. These data suggest that the A. castellanii–F. solani interaction may cause severe impacts on keratitis.

Efficacy of dental unit waterlines disinfectants on a polymicrobial biofilm

Due to their high surface-volume ratio, their laminar flow and frequent stagnation periods, dental unit waterlines (DUWL) foster the attachment of microorganisms and the development of biofilm, resulting in the continuous contamination of the outlet water from dental units; this contamination may be responsible for a potential risk of infection due to the exposure of patients and medical staff to droplet inhalation or splashed water. In this study, the anti-biofilm activity of three disinfectants recommended by dental unit manufacturers –Calbenium©, Oxygenal 6© and Sterispray© – was evaluated. A dynamic model simulating DUWL conditions was developed and polymicrobial biofilms containing bacteria (Pseudomonas aeruginosa), fungi (Candida albicans) and Free Living Amoeba (FLA: Vermamoeba vermiformis) were allowed to form. The ability of disinfectants to reduce biofilm formation or to eradicate an already formed biofilm was evaluated. Results showed the various effects of the tested disinfectants according to their composition, concentration and the targeted species. V. vermiformis was resistant to disinfectants, regardless of the tested concentrations and the concentrations recommended by manufacturers were not the most appropriate. Results also showed that Calbenium© was the most effective disinfectant to reduce already formed biofilms; its maximum efficiency was observed from 0.5% on both P. aeruginosa and C. albicans compared to 2 and 3% respectively for Sterispray©. The maximum efficiency of Oxygenal© was observed from 3% on P. aeruginosa but Oxygenal© was unable to totally eliminate C. albicans in the tested conditions, contrary to other disinfectants. Calbenium© was able to prevent biofilm formation efficiently even if it displayed no prophylactic activity against V. vermiformis. Overall, the FLA survival may contribute to maintaining other species. Finally the tested disinfectants were partially active against sessile microorganisms and more suitable concentrations could be used to increase their efficacy. Their use in a prophylactic rather than curative way should be recommended.

Risks & Threats Comes with Global Warming: Pathogenic Free Living Amoeba

Free living amoebae like Naegleria , Acanthamoeba , Balamuthia and Sappinia are known appearing opportunistic and also fatal protozoa in humans and other animals. They are widely distributed in soil and water in the world. They cause “Primer Amoebic Meningoencephalitis” the host immune response to these protist pathogens differs from each other to evidence by the postmortem laboratory findings from the affected patients. This review was performed with a search in Medline, PubMed, Science Direct, Ovid, and Scopus literatures by the search terms of “pathogenic free-living amoeba infections”. Analysis of a detailed review and literature shown that Naegleria fowleri , Acanthamoeba and Balamuthia and also Sappinia sp . infections are causing extensive brain damage to the host immune response. In human infection due to related to brain, skin, lung and eyes have increased significantly during the last years. They have different effects on epidemiology, immunology, pathology, and clinical features of the infections produced. This particular review planned to raise awareness about free-living amoeba, which found in a patient who applied to ESOGU Hospital Neurology Clinic because of suddenly unconsciousness and coma and diagnosed with Naegleria fowleri . Clinicians should be aware of PAM infections and include in differential diagnosis of meningoencephalitis. PAM should be suspected in young adults and children with acute neurological symptoms as described below and recent exposure to fresh water. Preventive and control measures include public health education and awareness among medical practitioners and adequate control of public water supplies. Key words : Pathogenic free-living amoebae, global warning, Naegleria, Acanthamoeba , Balamuthia, Sappinia

High Occurrence of Potentially Pathogenic Free Living Amoebae in Water Bodies of Kaleybar and Khodaafarin, East Azerbaijan Province

Free living amoebas are widespread protozoans that can be found in different environmental resources such as water bodies, soil and clays. Free living amoebae particularly Acanthamoeba could lead to diseases with poor prognosis including ameobic keratitis (AK) and granulomatous amebic encephalitis (GAE) especially in contact lens wears and immunocompromised individuals, respectively. In the present study 50 samples were collected from water resources (hot-springs, cold springs, rivers, wells, refined drinking water and subterranean canals) of Kaleybar and Khodaafarin, East Azarbayjan Province, Iran. The samples were transferred to laboratory and cultured on 1.5% non-nutrient agar medium containing Escherichia coli bacteria at room temperature and finally examined by microscopic methods. The results showed that out of 50 water samples, 40% were positive samples for free-living amoebae (FLA) including 60% Acanthamoebae genus, %35 Haetmannellidae, %20 Valkamphiids, %20 Vannella, %10 Thechamoeba (45% of plates had mix contamination). The present research was the first study to address the presence of potentially pathogenic free living amoebae in Kaleybar and Khodaafarin, East-Azerbaijan Province.

Atomic force microscopic imaging of Acanthamoeba castellanii and Balamuthia mandrillaris trophozoites and cysts.

Light microscopy and electron microscopy have been successfully used in the study of microbes, as well as free-living protists. Unlike light microscopy, which enables us to observe living organisms or the electron microscope which provides a two-dimensional image, atomic force microscopy provides a three-dimensional surface profile. Here, we observed two free-living amoebae, Acanthamoeba castellanii and Balamuthia mandrillaris under the phase contrast inverted microscope, transmission electron microscope and atomic force microscope. Although light microscopy was of lower magnification, it revealed functional biology of live amoebae such as motility and osmoregulation using contractile vacuoles of the trophozoite stage, but it is of limited value in defining the cyst stage. In contrast, transmission electron microscopy showed significantly greater magnification and resolution to reveal the ultra-structural features of trophozoites and cysts including intracellular organelles and cyst wall characteristics but it only produced a snapshot in time of a dead amoeba cell. Atomic force microscopy produced three-dimensional images providing detailed topographic description of shape and surface, phase imaging measuring boundary stiffness, and amplitude measurements including width, height and length of A. castellanii and B. mandrillaris trophozoites and cysts. These results demonstrate the importance of the application of various microscopic methods in the biological and structural characterization of the whole cell, ultra-structural features, as well as surface components and cytoskeleton of protist pathogens.

Genotyping of potentially pathogenic Acanthamoeba strains isolated from nasal swabs of healthy individuals in Peru

Free Living Amoebae (FLA) of Acanthamoeba genus are widely distributed in the environment and can be found in the air, soil and water; and have also been isolated from air-conditioning units. In humans, they are causative agents of a sight-threating infection of the cornea, Acanthamoeba keratitis (AK) and a fatal infection of the central nervous system known as Granulomatous Amoebic Encephalitis (GAE). In this study, a survey was conducted in order to determine the presence and pathogenic potential of free-living amoebae of Acanthamoeba genus in nasal swabs from individuals in two regions of Peru. Identification of isolates was based on cyst morphology and PCR-sequencing of the Diagnostic Fragment 3 to identify strains at the genotype level. The pathogenic potential of the isolates was also assayed using temperature and osmotolerance assays and extracellular proteases zymograms. The obtained results revealed that all isolated strains exhibited pathogenic potential. After sequencing the highly variable DF3 (Diagnostic Fragment 3) region in the 18S rRNA gene as previously described, genotype T4 was found to be the most common one in the samples included in this study but also genotype T15 was identified. To the best of our knowledge, this is the first study on the characterization of Acanthamoeba strains at the genotype level and the first report of genotype T4 and T15 in Peru.

Survival of Campylobacter jejuni in co‐culture with Acanthamoeba castellanii: role of amoeba‐mediated depletion of dissolved oxygen

Campylobacter jejuni is a major cause of infectious diarrhoea worldwide but relatively little is known about its ecology. In this study, we examined its interactions with Acanthamoeba castellanii, a protozoan suspected to serve as a reservoir for bacterial pathogens. We observed rapid degradation of intracellular C.jejuni in A.castellanii 5 h post gentamicin treatment at 25°C. Conversely, we found that A.castellanii promoted the extracellular growth of C.jejuni in co-cultures at 37°C in aerobic conditions. This growth-promoting effect did not require amoebae - bacteria contact. The growth rates observed with or without contact with amoeba were similar to those observed when C.jejuni was grown in microaerophilic conditions. Preconditioned media prepared with live or dead amoebae cultivated with or without C.jejuni did not promote the growth of C.jejuni in aerobic conditions. Interestingly, the dissolved oxygen levels of co-cultures with or without amoebae - bacteria contact were much lower than those observed with culture media or with C.jejuni alone incubated in aerobic conditions, and were comparable with levels obtained after 24 h of growth of C.jejuni under microaerophilic conditions. Our studies identified the depletion of dissolved oxygen by A.castellanii as the major contributor for the observed amoeba-mediated growth enhancement.