Enolase inhibitors as therapeutic leads for Naegleria fowleri infection.

Infections with the pathogenic free-living amoebae Naegleria fowleri can lead to life-threatening illnesses including catastrophic primary amoebic meningoencephalitis (PAM). Efficacious treatment options for these infections are lacking and the mortality rate remains >95% in the US. Glycolysis is very important for the infectious trophozoite lifecycle stage and inhibitors of glucose metabolism have been found to be toxic to the pathogen. Recently, human enolase 2 (ENO2) phosphonate inhibitors have been developed as lead agents to treat glioblastoma multiforme (GBM). These compounds, which cure GBM in a rodent model, are well-tolerated in mammals because enolase 1 (ENO1) is the predominant isoform used systemically. Here, we describe findings that demonstrate these agents are potent inhibitors of N. fowleri ENO (NfENO) and are lethal to amoebae. In particular, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) was a potent enzyme inhibitor (IC50 = 0.14 ± 0.04 µM) that was toxic to trophozoites (EC50 = 0.21 ± 0.02 µM) while the reported CC50 was >300 µM. Molecular docking simulation revealed that HEX binds strongly to the active site of NfENO with a binding affinity of -8.6 kcal/mol. Metabolomic studies of parasites treated with HEX revealed a 4.5 to 78-fold accumulation of glycolytic intermediates upstream of NfENO. Last, nasal instillation of HEX increased longevity of amoebae-infected rodents. Two days after infection, animals were treated for 10 days with 3 mg/kg HEX, followed by one week of observation. At the end of the one-week observation, eight of 12 HEX-treated animals remained alive (resulting in an indeterminable median survival time) while one of 12 vehicle-treated rodents remained, yielding a median survival time of 10.9 days. However, intranasal HEX delivery was not curative as brains of six of the eight survivors were positive for amoebae. These findings suggest that HEX requires further evaluation to develop as a lead for treatment of PAM.

Amoebicidal effect of chlorine dioxide gas against pathogenic Naegleria fowleri and Acanthamoeba polyphaga.

The pathogenic free-living amoebae, Naegleria fowleri and Acanthamoeba polyphaga, are found in freshwater, soil, and unchlorinated or minimally chlorinated swimming pools. N. fowleri and A. polyphaga are becoming problematic as water leisure activities and drinking water are sources of infection. Chlorine dioxide (ClO2) gas is a potent disinfectant that is relatively harmless to humans at the concentration used for disinfection. In this study, we examined the amoebicidal effects of ClO2 gas on N. fowleri and A. polyphaga. These amoebae were exposed to ClO2 gas from a ready-to-use product (0.36 ppmv/h) for 12, 24, 36, and 48 h. Microscopic examination showed that the viability of N. fowleri and A. polyphaga was effectively inhibited by treatment with ClO2 gas in a time-dependent manner. The growth of N. fowleri and A. polyphaga exposed to ClO2 gas for 36 h was completely inhibited. In both cases, the mRNA levels of their respective actin genes were significantly reduced following treatment with ClO2 gas. ClO2 gas has an amoebicidal effect on N. fowleri and A. polyphaga. Therefore, ClO2 gas has been proposed as an effective agent for the prevention and control of pathogenic free-living amoeba contamination.

Next generation imidazothiazole and imidazooxazole derivatives as potential drugs against brain-eating amoebae.

Managing primary amoebic meningoencephalitis, induced by Naegleria fowleri poses a complex medical challenge. There is currently no specific anti-amoebic drug that has proven effectiveness against N. fowleri infection. Ongoing research endeavours are dedicated to uncovering innovative treatment strategies, including the utilization of drugs and immune modulators targeting Naegleria infection. In this study, we explored the potential of imidazo[2,1-b]thiazole and imidazooxazole derivatives that incorporate sulfonate and sulfamate groups as agents with anti-amoebic properties against N. fowleri. We assessed several synthesized compounds (1f, 1m, 1q, 1s, and 1t) for their efficacy in eliminating amoebae, their impact on cytotoxicity, and their influence on the damage caused to human cerebral microvascular endothelial (HBEC-5i) cells when exposed to the N. fowleri (ATCC 30174) strain. The outcomes revealed that, among the five compounds under examination, 1m, 1q, and 1t demonstrated notable anti-parasitic effects against N. fowleri (P ≤ 0.05). Compound 1t exhibited the highest anti-parasitic activity, reducing N. fowleri population by 80%. Additionally, three compounds, 1m, 1q, and 1t, significantly mitigated the damage inflicted on host cells by N. fowleri. However, the results of cytotoxicity analysis indicated that while 1m and 1q had minimal cytotoxic effects on endothelial cells, compound 1t caused moderate cytotoxicity (34%). Consequently, we conclude that imidazo[2,1-b]thiazole and imidazooxazole derivatives containing sulfonate and sulfamate groups exhibit a marked capacity to eliminate amoebae viability while causing limited toxicity to human cells. In aggregate, these findings hold promise that could potentially evolve into novel therapeutic options for treating N. fowleri infection.

The type 2 statins, cerivastatin, rosuvastatin and pitavastatin eliminate Naegleria fowleri at low concentrations and by induction of programmed cell death (PCD).

Primary Amoebic Encephalitis due to Naegleria fowleri species is a fatal infection of the Central Nervous System mostly affecting children and young adults. Infections often occur after performance of risk activities in aquatic habitats such as swimming and splashing. PAMs therapy remain a key issue to be solved which needs an urgent development. Recently, statins have been highlighted as possible novel compounds to treat PAM. Furthermore, type 2 statins due to improved pharmacological properties and lower toxicity could be use in the future. In the present work, three type 2 statins were checked for their activity against two type strains of N. fowleri. In addition, the effects at the cellular level triggered in treated amoebae were checked in order to evaluate if programmed cell death was induced. The obtained results showed that the tested statins, rosuvastatin, pitavastatin and cerivastatin were able to eliminate N. fowleri trophozoites and also induced PCD. Therefore, type 2 statins could be used in the near future for the treatment of PAM.

Enzymatic chokepoints and synergistic drug targets in the sterol biosynthesis pathway of Naegleria fowleri.

Naegleria fowleri is a free-living amoeba that can also act as an opportunistic pathogen causing severe brain infection, primary amebic meningoencephalitis (PAM), in humans. The high mortality rate of PAM (exceeding 97%) is attributed to (i) delayed diagnosis, (ii) lack of safe and effective anti-N. fowleri drugs, and (iii) difficulty of delivering drugs to the brain. Our work addresses identification of new molecular targets that may link anti-Naegleria drug discovery to the existing pharmacopeia of brain-penetrant drugs. Using inhibitors with known mechanism of action as molecular probes, we mapped the sterol biosynthesis pathway of N. fowleri by GC-MS analysis of metabolites. Based on this analysis, we chemically validated two enzymes downstream to CYP51, sterol C24-methyltransferase (SMT, ERG6) and sterol Δ8-Δ7 -isomerase (ERG2), as potential therapeutic drug targets in N. fowleri. The sterol biosynthetic cascade in N. fowleri displayed a mixture of canonical features peculiar to different domains of life: lower eukaryotes, plants and vertebrates. In addition to the cycloartenol→ergosterol biosynthetic route, a route leading to de novo cholesterol biosynthesis emerged. Isotopic labeling of the de novo-synthesized sterols by feeding N. gruberi trophozoites on the U13C-glucose-containing growth medium identified an exogenous origin of cholesterol, while 7-dehydrocholesterol (7DHC) had enriched 13C-content, suggesting a dual origin of this metabolite both from de novo biosynthesis and metabolism of scavenged cholesterol. Sterol homeostasis in Naegleria may be orchestrated over the course of its life-cycle by a "switch" between ergosterol and cholesterol biosynthesis. By demonstrating the growth inhibition and synergistic effects of the sterol biosynthesis inhibitors, we validated new, potentially druggable, molecular targets in N. fowleri. The similarity of the Naegleria sterol Δ8-Δ7 -isomerase to the human non-opioid σ1 receptor, implicated in human CNS conditions such as addiction, amnesia, pain and depression, provides an incentive to assess structurally diverse small-molecule brain-penetrant drugs targeting the human receptor for anti-Naegleria activity.

Naegleria fowleri: Sources of infection, pathophysiology, diagnosis, and management; a review.

Naegleria fowleri, a thermophilic flagellate amoeba known as a "brain-eating" amoeba, is the aetiological agent of a perilous and devastating waterborne disease known as primary amoebic meningoencephalitis (PAM), both in humans as well as in animals. PAM is a rare but fatal disease affecting young adults all around the world, particularly in the developed world but recently reported from developing countries, with 95%-99% mortality rate. Swimmers and divers are at high risk of PAM as the warm water is the most propitious environment adapted by N. fowleri to cause this infection. Infective amoeba in the trophozoite phase enter the victim's body through the nose, crossing the cribriform plate to reach the human brain and cause severe destruction of the central nervous system (CNS). The brain damage leads to brain haemorrhage and death occurs within 3-7 days in undiagnosed cases and maltreated cases. Though the exact pathogenesis of N. fowleri is still not known, it has exhibited two primary mechanisms, contact-independent (brain damage through different proteins) and contact-dependent (brain damage through surface structures food cups), that predominantly contribute to the pathogen invading the host CNS. For the management of this life-threatening infection different treatment regimens have been applied but still the survival rate is only 5% which is ascribed to its misdiagnosis, as the PAM symptoms closely resembled bacterial meningitis. The main objectives of this review article are to compile data to explore the sources and routes of N. fowleri infection, its association in causing PAM along with its pathophysiology; latest techniques used for accurate diagnosis, management options along with challenges for Pakistan to control this drastic disorder.

Synthetic nanoparticle-conjugated bisindoles and hydrazinyl arylthiazole as novel antiamoebic agents against brain-eating amoebae.

Balamuthia mandrillaris and Naegleria fowleri are free-living amoebae that can cause life-threatening infections involving the central nervous system. The high mortality rates of these infections demonstrate an urgent need for novel treatment options against the amoebae. Considering that indole and thiazole compounds possess wide range of antiparasitic properties, novel bisindole and thiazole derivatives were synthesized and evaluated against the amoebae. The antiamoebic properties of four synthetic compounds i.e., two new bisindoles (2-Bromo-4-(di (1H-indol-3-yl)methyl)phenol (denoted as A1) and 2-Bromo-4-(di (1H-indol-3-yl)methyl)-6-methoxyphenol (A2)) and two known thiazole (4-(3-Nitrophenyl)-2-(2-(pyridin-3-ylmethylene)hydrazinyl)thiazole (A3) and 4-(Biphenyl-4-yl)-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazole (A4)) were evaluated against B. mandrillaris and N. fowleri. The ability of silver nanoparticle (AgNPs) conjugation to enrich antiamoebic activities of the compounds was also investigated. The synthetic heterocyclic compounds demonstrated up to 53% and 69% antiamoebic activities against B. mandrillaris and N. fowleri respectively, while resulting in up to 57% and 68% amoebistatic activities, respectively. Antiamoebic activities of the compounds were enhanced by up to 71% and 51% against B. mandrillaris and N. fowleri respectively, after conjugation with AgNPs. These compounds exhibited potential antiamoebic effects against B. mandrillaris and N. fowleri and conjugation of synthetic heterocyclic compounds with AgNPs enhanced their activity against the amoebae.

Phenotypic Screens Reveal Posaconazole as a Rapidly Acting Amebicidal Combination Partner for Treatment of Primary Amoebic Meningoencephalitis.

Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis (PAM), which is fatal in >97% of cases. In this study, we aimed to identify new, rapidly acting drugs to increase survival rates. We conducted phenotypic screens of libraries of Food and Drug Administration-approved compounds and the Medicines for Malaria Venture Pathogen Box and validated 14 hits (defined as a 50% inhibitory concentration of <1 µM). The hits were then prioritized by assessing the rate of action and efficacy in combination with current drugs used to treat PAM. Posaconazole was found to inhibit amoeba growth within the first 12 hours of exposure, which was faster than any currently used drug. In addition, posaconazole cured 33% of N. fowleri-infected mice at a dose of 20 mg/kg and, in combination with azithromycin, increased survival by an additional 20%. Fluconazole, which is currently used for PAM therapy, was ineffective in vitro and vivo. Our results suggest posaconazole could replace fluconazole in the treatment of PAM.

Antiparasitic Activity of Auranofin against Pathogenic Naegleria fowleri.

We report that the gold containing antirheumatoid drug auranofin is amoebicidal against human pathogenic Naegleria fowleri. Treatment of N. fowleri cultures at biologically relevant concentrations of 0.75-3.0 µg/ml auranofin reduced amoeba counts, metabolic activity, and increased cell permeability. These results suggest that the addition of auranofin may benefit the treatment of N. fowleri-infected patients afflicted by the rapidly fatal disease primary amoebic meningoencephalitis.

Identification of cysteine protease inhibitors as new drug leads against Naegleria fowleri.

Primary amebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living ameba Naegleria fowleri. PAM occurs principally in healthy children of less than 13 years old with a history of recent exposure to warm fresh water. While as yet not a reportable disease, the Centers for Disease Control and Prevention (CDC) documents a total of 143 cases in the United States. Only four patients have survived. Infection results from water containing N. fowleri entering the nose, followed by migration of the amebae to the brain. Within the brain, N. fowleri infection results in extensive necrosis, leading to death in 3-7 days. Mortality among patients with PAM is greater than 95%. The drugs of choice in treating PAM are the antifungal amphotericin B, and the antileishmanial, miltefosine. However neither drug is FDA-approved for this indication and the use of amphotericin B is associated with severe adverse effects. Moreover, very few patients treated with amphotericin B have survived PAM. Therefore, development of new, safe and effective drugs is a critical unmet need to avert future deaths of children. The molecular mechanisms underlying the pathogenesis of PAM are poorly understood but it is known that cysteine proteases of N. fowleri play a role in the progression of PAM. We therefore assessed the in vitro activity of the synthetic vinyl sulfone cysteine protease inhibitor, K11777, and 33 analogs with valine, phenylalanine or pyridylalanine at P2 position, against cysteine protease activity in the lysate of N. fowleri. Inhibitors with phenylalanine or pyridylalanine at P2 position were particularly effective in inhibiting the cysteine protease activity of N. fowleri cell lysate with IC50 ranging between 3 nM and 6.6 µM. Three of the 34 inhibitors also showed inhibitory activity against N. fowleri in a cell viability assay and were 1.6- to 2.5-fold more potent than the standard of care drug miltefosine. Our study provides the first evidence of the activity of synthetic, small molecule cysteine protease inhibitors against N. fowleri.

Expression of matrix metalloproteinases in Naegleria fowleri and their role in invasion of the central nervous system.

Naegleria fowleri is a free-living amoeba found in freshwater lakes and ponds and is the causative agent of primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system (CNS). PAM occurs when amoebae attach to the nasal epithelium and invade the CNS, a process that involves binding to, and degradation of, extracellular matrix (ECM) components. This degradation is mediated by matrix metalloproteinases (MMPs), enzymes that have been described in other pathogenic protozoa, and that have been linked to their increased motility and invasive capability. These enzymes also are upregulated in tumorigenic cells and have been implicated in metastasis of certain tumours. In the present study, in vitro experiments linked MMPs functionally to the degradation of the ECM. Gelatin zymography demonstrated enzyme activity in N. fowleri whole cell lysates, conditioned media and media collected from invasion assays. Western immunoblotting indicated the presence of the metalloproteinases MMP-2 (gelatinase A), MMP-9 (gelatinase B) and MMP-14 [membrane type-1 matrix metalloproteinase (MT1-MMP)]. Highly virulent mouse-passaged amoebae expressed higher levels of MMPs than weakly virulent axenically grown amoebae. The functional relevance of MMPs in media was indicated through the use of the MMP inhibitor, 1,10-phenanthroline. The collective in vitro results suggest that MMPs play a critical role in vivo in invasion of the CNS and that these enzymes may be amenable targets for limiting PAM.

Amphotericin B induces apoptosis-like programmed cell death in Naegleria fowleri and Naegleria gruberi.

Naegleria fowleri and Naegleria gruberi belong to the free-living amoebae group. It is widely known that the non-pathogenic species N. gruberi is usually employed as a model to describe molecular pathways in this genus, mainly because its genome has been recently described. However, N. fowleri is an aetiological agent of primary amoebic meningoencephalitis, an acute and fatal disease. Currently, the most widely used drug for its treatment is amphotericin B (AmB). It was previously reported that AmB has an amoebicidal effect in both N. fowleri and N. gruberi trophozoites by inducing morphological changes that resemble programmed cell death (PCD). PCD is a mechanism that activates morphological, biochemical and genetic changes. However, PCD has not yet been characterized in the genus Naegleria. The aim of the present work was to evaluate the typical markers to describe PCD in both amoebae. These results showed that treated trophozoites displayed several parameters of apoptosis-like PCD in both species. We observed ultrastructural changes, an increase in reactive oxygen species, phosphatidylserine externalization and a decrease in intracellular potassium, while DNA degradation was evaluated using the TUNEL assay and agarose gels, and all of these parameters are related to PCD. Finally, we analysed the expression of apoptosis-related genes, such as sir2 and atg8, in N. gruberi. Taken together, our results showed that AmB induces the morphological, biochemical and genetic changes of apoptosis-like PCD in the genus Naegleria.

NLRP3 Inflammasome Activation in THP-1 Target Cells Triggered by Pathogenic Naegleria fowleri.

Naegleria fowleri, known as the brain-eating amoeba, causes acute primary amoebic meningoencephalitis. During swimming and other recreational water activities, N. fowleri trophozoites penetrate the nasal mucosa and invade the olfactory bulbs, resulting in intense inflammatory reactions in the forebrain tissue. To investigate what kinds of inflammasome molecules are expressed in target cells due to N. fowleri infection, human macrophage cells (THP-1 cells) were cocultured with N. fowleri trophozoites in a noncontact system, and consequently, interleukin-1ß (IL-1ß) production was estimated. Caspase-1 activation and IL-1ß production from THP-1 cells by Western blotting and the culture supernatant by enzyme-linked immunosorbent assay analysis were observed at 3 h after cocultivation. In addition, the increased expression of ASC and NLRP3, which make up an inflammasome complex, was also observed at 3 h after cocultivation. To confirm the caspase-1 activation and IL-1ß production via the NLRP3 inflammasome in THP-1 cells triggered by N. fowleri trophozoites, THP-1 cells were pretreated with several inhibitors. The inhibition assay showed that CA-074 (a cathepsin B inhibitor), glybenclamide (an NLRP3 molecule inhibitor), and N-benzyloxycarbony-Val-Ala-Asp(O-methyl)-fluoromethylketone (Z-VAD-FMK; a caspase-1 inhibitor) reduced the levels of caspase-1 activation and IL-1ß production from THP-1 cells. This study suggests that N. fowleri infection induces the NLRP3 inflammasome, which activates caspase-1 and subsequently produces IL-1ß, thus resulting in inflammation.

Use of the Novel Therapeutic Agent Miltefosine for the Treatment of Primary Amebic Meningoencephalitis: Report of 1 Fatal and 1 Surviving Case.

Primary amebic meningoencephalitis (PAM) is a fulminant central nervous system infection caused by the thermophilic free-living ameba Naegleria fowleri. Few survivals have been documented and adequate treatment is lacking. We report 2 PAM cases, 1 fatal and 1 surviving, treated with the novel antiparasitic agent miltefosine.

Naegleria fowleri: pathogenesis, diagnosis, and treatment options.

Naegleria fowleri has generated tremendous media attention over the last 5 years due to several high-profile cases. Several of these cases were followed very closely by the general public. N. fowleri is a eukaryotic, free-living amoeba belonging to the phylum Percolozoa. Naegleria amoebae are ubiquitous in the environment, being found in soil and bodies of freshwater, and feed on bacteria found in those locations. While N. fowleri infection appears to be quite rare compared to other diseases, the clinical manifestations of primary amoebic meningoencephalitis are devastating and nearly always fatal. Due to the rarity of N. fowleri infections in humans, there are no clinical trials to date that assess the efficacy of one treatment regimen over another. Most of the information regarding medication efficacy is based on either case reports or in vitro studies. This review will discuss the pathogenesis, diagnosis, pharmacotherapy, and prevention of N. fowleri infections in humans, including a brief review of all survivor cases in North America.

Bis-benzimidazole hits against Naegleria fowleri discovered with new high-throughput screens.

Naegleria fowleri is a pathogenic free-living amoeba (FLA) that causes an acute fatal disease known as primary amoebic meningoencephalitis (PAM). The major problem for infections with any pathogenic FLA is a lack of effective therapeutics, since PAM has a case mortality rate approaching 99%. Clearly, new drugs that are potent and have rapid onset of action are needed to enhance the treatment regimens for PAM. Diamidines have demonstrated potency against multiple pathogens, including FLA, and are known to cross the blood-brain barrier to cure other protozoan diseases of the central nervous system. Therefore, amidino derivatives serve as an important chemotype for discovery of new drugs. In this study, we validated two new in vitro assays suitable for medium- or high-throughput drug discovery and used these for N. fowleri. We next screened over 150 amidino derivatives of multiple structural classes and identified two hit series with nM potency that are suitable for further lead optimization as new drugs for this neglected disease. These include both mono- and diamidino derivatives, with the most potent compound (DB173) having a 50% inhibitory concentration (IC50) of 177 nM. Similarly, we identified 10 additional analogues with IC50s of <1 µM, with many of these having reasonable selectivity indices. The most potent hits were >500 times more potent than pentamidine. In summary, the mono- and diamidino derivatives offer potential for lead optimization to develop new drugs to treat central nervous system infections with N. fowleri.

Identification of peptidases in highly pathogenic vs. weakly pathogenic Naegleria fowleri amebae.

Naegleria fowleri, a free-living ameba, is the causative agent of Primary Amebic Meningoencephalitis. Highly pathogenic mouse-passaged amebae (Mp) and weakly pathogenic axenically grown (Ax) N. fowleri were examined for peptidase activity. Zymography and azocasein peptidase activity assays demonstrated that Mp and Ax N. fowleri exhibited a similar peptidase pattern. Prominent for whole cell lysates, membranes and conditioned medium (CM) from Mp and Ax amebae was the presence of an activity band of approximately 58 kDa that was sensitive to E64, a cysteine peptidase inhibitor. However, axenically grown N. fowleri demonstrated a high level of this peptidase activity in membrane preparations. The inhibitor E64 also reduced peptidase activity in ameba-CM consistent with the presence of secreted cysteine peptidases. Exposure of Mp amebae to E64 reduced their migration through matrigel that was used as an extracellular matrix, suggesting a role for cysteine peptidases in invasion of the central nervous system (CNS). The collective results suggest that the profile of peptidases is not a discriminative marker for distinguishing Mp from Ax N. fowleri. However, the presence of a prominent level of activity for cysteine peptidases in N. fowleri membranes and CM, suggests that these enzymes may serve to facilitate passage of the amebae into the CNS.

Reduced Efficiency of Chlorine Disinfection of Naegleria fowleri in a Drinking Water Distribution Biofilm.

Naegleria fowleri associated with biofilm and biological demand water (organic matter suspended in water that consumes disinfectants) sourced from operational drinking water distribution systems (DWDSs) had significantly increased resistance to chlorine disinfection. N. fowleri survived intermittent chlorine dosing of 0.6 mg/L for 7 days in a mixed biofilm from field and laboratory-cultured Escherichia coli strains. However, N. fowleri associated with an attached drinking water distribution biofilm survived more than 30 times (20 mg/L for 3 h) the recommended concentration of chlorine for drinking water. N. fowleri showed considerably more resistance to chlorine when associated with a real field biofilm compared to the mixed laboratory biofilm. This increased resistance is likely due to not only the consumption of disinfectants by the biofilm and the reduced disinfectant penetration into the biofilm but also the composition and microbial community of the biofilm itself. The increased diversity of the field biofilm community likely increased N. fowleri's resistance to chlorine disinfection compared to that of the laboratory-cultured biofilm. Previous research has been conducted in only laboratory scale models of DWDSs and laboratory-cultured biofilms. To the best of our knowledge, this is the first study demonstrating how N. fowleri can persist in a field drinking water distribution biofilm despite chlorination.

ANTI-AMEBIC ACTIVITY OF DIOSGENIN ON NAEGLERIA FOWLERI TROPHOZOITES.

The aim of this study was to investigate the activity of diosgenin against Naegleria fowleri trophozoites at the cellular and molecular levels. Diosgenin (100 µg/ml; 241.2 µM) had a 100% inhibitory effect on N. fowleri trophozoites (5 x 10(5) cell/ml). Scanning electron micrograph revealed diosgenin decreased the number of sucker-like apparatuses and food cup formation among N. fowleri trophozoites at 3 and 6 hours post-exposure, respectively. Diosgenin down-regulated the nf cysteine protease gene expression of N. fowleri trophozoites at 6 and 12 hours post-exposure. The toxicity to mammalian cells caused by diosgenin at therapeutic dose was less than amphotericin B, the current drug used to treat N. fowleri infections. Our findings suggest diosgenin has activity against the surface membrane and the nf cysteine pro tease of N. fowleri trophozoites. However, the other mechanisms of action of diosgenin against N. fowleri trophozoites require further exploration.

Biocidal efficacy of monochloramine against planktonic and biofilm-associated Naegleria fowleri cells.

AIMS: Free-living amoebae (FLA) in aqueous systems are a problem for water network managers and health authorities because some are pathogenic, such as Naegleria fowleri, and they have also been reported to operate as reservoirs and vectors of several pathogenic bacteria. Therefore, to better control the occurrence of such amoebae, we evaluate the efficacy of monochloramine against planktonic forms (trophozoites and cysts) and also biofilm-associated cells of N. fowleri as FLA are often associated with biofilms. METHODS AND RESULTS: From a freshwater biofilm growing in a pilot reactor and inoculated with N. fowleri, we obtained Ct values ranging from 4 to 17 mg Cl2 min l(-1) at 25°C and pH 8·2 on both planktonic and biofilm associated cells. In addition, the inactivation pattern of biofilm associated was intermediate between those of trophozoïtes and cysts. CONCLUSIONS: The monochloramine efficiency varies with the life stage of N. fowleri (trophozoïte, cyst, and biofilm-associated). The sensitivity to disinfectant of amoeba, that is, trophozoïtes and cysts, in the biofilm life stage is as high as that of their planktonic cyst form. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives Ct values for cysts and biofilm-associated N. fowleri. This may impact on water treatment strategies against amoebae and should be considered when controlling N. fowleri in man-made water systems such as cooling towers or hot water systems.