SummaryNaegleria gruberi is a free-living non-pathogenic amoeboflagellate and relative of Naegleria fowleri, a deadly pathogen causing primary amoebic meningoencephalitis (PAM). A genomic analysis of N. gruberi exists, but physiological evidence for its core energy metabolism or in vivo growth substrates is lacking. Here, we show that N. gruberi trophozoites need oxygen for normal functioning and growth and that they shun both glucose and amino acids as growth substrates. Trophozoite growth depends mainly upon lipid oxidation via a mitochondrial branched respiratory chain, both ends of which require oxygen as final electron acceptor. Growing N. gruberi trophozoites thus have a strictly aerobic energy metabolism with a marked substrate preference for the oxidation of fatty acids. Analyses of N. fowleri genome data and comparison with those of N. gruberi indicate that N. fowleri has the same type of metabolism. Specialization to oxygen-dependent lipid breakdown represents an additional metabolic strategy in protists.

Naegleria gruberi is a free-living non-pathogenic amoeboflagellate and relative of Naegleria fowleri, a deadly pathogen causing primary amoebic meningoencephalitis (PAM). A genomic analysis of N. gruberi exists, but physiological evidence for its core energy metabolism or in vivo growth substrates is lacking. Here we show that N. gruberi trophozoites are, in contrast to earlier reports, strict aerobes and that they furthermore shun both glucose and amino acids as growth substrates.Trophozoite growth depends mainly upon lipid oxidation via a mitochondrial branched respiratory chain, both ends of which require oxygen as final electron acceptor. N. gruberi thus has a strictly aerobic energy metabolism with a marked substrate preference for the oxidation of fatty acids. Analyses of N. fowleri genome data and comparison with those of N. gruberi indicate that N. fowleri has the same type of metabolism. Specialization to oxygen-dependent lipid breakdown represents a hitherto unprecedented metabolic strategy in protists.

Verona-Integron-encoded-Metallo-b-lactamase-positive Pseudomonas aeruginosa (VIM-PA) is a cause of hard-to-treat nosocomial infections, and can colonize hospital water networks alongside Acanthamoeba. We developed an in-vitro disinfection model to examine whether Acanthamoeba castellanii can harbour VIM-PA intracellularly, allowing VIM-PA to evade being killed by currently used hospital disinfectants. We observed that A. castellanii presence resulted in significantly increased survival of VIM-PA after exposure to chlorine for 30 s or for 2 min. This undesirable effect was not observed after disinfection by 70% alcohol or 24% acetic acid. Confocal microscopy confirmed the presence of VIM-PA within A. castellanii pseudocysts. Our data indicate that A. castellanii contributes to persistent VIM-PA colonization of water systems after chlorine treatment.

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One of the reasons for the lengthy tuberculosis (TB) treatment is the difficulty to treat the nonmultiplying mycobacterial subpopulation. In order to assess the ability of (new) TB drugs to target this subpopulation, we need to incorporate dormancy models in our preclinical drug development pipeline. In most available dormancy models, it takes a long time to create a dormant state, and it is difficult to identify and quantify this nonmultiplying condition. The Mycobacterium tuberculosis 18b strain might overcome some of these problems, because it is dependent on streptomycin for growth and becomes nonmultiplying after 10 days of streptomycin starvation but still can be cultured on streptomycin-supplemented culture plates. We developed our 18b dormancy time-kill kinetics model to assess the difference in the activity of isoniazid, rifampin, moxifloxacin, and bedaquiline against log-phase growth compared to the nonmultiplying M. tuberculosis subpopulation by CFU counting, including a novel area under the curve (AUC)-based approach as well as time-to-positivity (TTP) measurements. We observed that isoniazid and moxifloxacin were relatively more potent against replicating bacteria, while rifampin and high-dose bedaquiline were equally effective against both subpopulations. Moreover, the TTP data suggest that including a liquid culture-based method could be of additional value, as it identifies a specific mycobacterial subpopulation that is nonculturable on solid media. In conclusion, the results of our study underline that the time-kill kinetics 18b dormancy model in its current form is a useful tool to assess TB drug potency and thus has its place in the TB drug development pipeline.

Primary amoebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living amoeba Naegleria fowleri. The amoeba migrates along the olfactory nerve to the brain, resulting in seizures, coma, and, eventually, death. Previous research has shown that Naegleria gruberi, a close relative of N. fowleri, prefers lipids over glucose as an energy source. Therefore, we tested several already-approved inhibitors of fatty acid oxidation alongside the currently used drugs amphotericin B and miltefosine. Our data demonstrate that etomoxir, orlistat, perhexiline, thioridazine, and valproic acid inhibited growth of N. gruberi. We then tested these compounds on N. fowleri and found etomoxir, perhexiline, and thioridazine to be effective growth inhibitors. Hence, not only are lipids the preferred food source for N. gruberi, but also oxidation of fatty acids seems to be essential for growth of N. fowleri. Inhibition of fatty acid oxidation could result in new treatment options, as thioridazine inhibits N. fowleri growth in concentrations that can be reached at the site of infection. It could also potentiate currently used therapy, as checkerboard assays revealed synergy between miltefosine and etomoxir. Animal testing should be performed to confirm the added value of these inhibitors. Although the development of new drugs and randomized controlled trials for this rare disease are nearly impossible, inhibition of fatty acid oxidation seems a promising strategy as we showed effectivity of several drugs that are or have been in use and that thus could be repurposed to treat PAM in the future.

Acanthamoeba castellanii ( A. castellanii ) can cause Acanthamoeba keratitis, a sight-threatening infection, as well as a fatal brain infection termed granulomatous amoebic encephalitis, mostly in immunocompromised individuals. In contrast, Naegleria fowleri ( N. fowleri ) causes a deadly infection involving the central nervous system, recognized as primary amoebic encephalitis, mainly in individuals partaking in recreational water activities or those with nasal exposure to contaminated water. Worryingly, mortality rates due to these infections are more than 90%, suggesting the need to find alternative therapies. In this study, antiamoebic activity of a peptide based on the structure of the antibiotic tyrocidine was evaluated against A. castellanii and N. fowleri . The tyrocidine-derived peptide displayed significant amoebicidal efficacy against A. castellanii and N. fowleri . At 250 μg/mL, the peptide drastically reduced amoebae viability up to 13% and 21% after 2 h of incubation against N. fowleri and A. castellanii. , whereas, after 24 h of incubation, the peptide showed 86% and 94% amoebicidal activity against A. castellanii and N. fowleri . Furthermore, amoebae pretreated with 100 μg/mL peptide inhibited 35% and 53% A. castellanii and N. fowleri , while, at 250 μg/mL, 84% and 94% A. castellanii and N. fowleri failed to adhere to human cells. Amoeba-mediated cell cytopathogenicity assays revealed 31% and 42% inhibition at 100 μg/mL, while at 250 μg/mL 75% and 86% A. castellanii and N. fowleri were inhibited. Assays revealed inhibition of encystation in both A. castellanii (58% and 93%) and N. fowleri (73% and 97%) at concentrations of 100 and 250 μg/mL respectively. Importantly, tyrocidine-derived peptide depicted minimal cytotoxicity to human cells and, thus, may be a potential candidate in the rational development of a treatment regimen against free-living amoebae infections. Future studies are necessary to elucidate the in vivo effects of tyrocidine-derived peptide against these and other pathogenic amoebae of importance.

Diagnosis of cystic echinococcosis (CE) is at present mainly based on imaging techniques. Serology has a complementary role, partly due to the small number of standardized and commercially available assays. Therefore we examined the clinical performance of the SERION ELISA classic Echinococcus IgG test. Using 10 U/ml as a cut-off point, and serum samples from 50 CE patients and 105 healthy controls, the sensitivity and specificity were 98.0% and 96.2%, respectively. If patients with other infectious diseases were used as negative controls, the specificity decreased to 76.9%, which causes poor positive predictive values. However, if results between 10 and 15 U/ml are classified as indecisive, the specificity of positive results (≥15 U/ml) increased to 92.5% without greatly affecting the sensitivity (92.0%). Using this approach in combination with imaging studies, the SERION ELISA classic Echinococcosis IgG test can be a useful aid in the diagnosis of CE.