ParasitologyNovel culture medium for the axenic growth of Balamuthia mandrillaris
Introduction
To isolate and cultivate Gymnamoebae from the environment including pathogenic free-living amoebae Naegleria and Acanthamoeba, several culture media have been developed, and some of recipes can be find in Page (1988). According to Dykova and Kostka (2013), there are some “difficult” strains, e.g., Mayorella gemmifera, Saccamoeba, and several Naegleria strains, which need well-grown monolayers of cell cultures for subculturing them, as Balamuthia mandrillaris needs them for its isolation from the environment; thus, it has been scanty (Lares-Jiménez et al., 2014).
B. mandrillaris is an opportunistic protist pathogen causative of necrotizing hemorrhagic granulomatous encephalitis among other infections. More than 200 cases have been reported worldwide with its majority within the Americas (Lorenzo-Morales et al., 2013). Infections due to B. mandrillaris have been estimated with a fatality rate approaching 98%, and no specific successful treatment was reported (Siddiqui and Khan, 2015). For advances in the study of free-living amoebae infections, it is primordial to grow those organisms in the laboratory. B. mandrillaris, unlike most of other free-living amoebae, does not feed on Gram-negative bacteria; thus, the use of nonnutrient agar coated with bacterial cultures has resulted to be ineffective for its growth. Until now, complex media such as the BM-3 culture medium and the modified Chang's special medium are the only ones that have been recommended for axenic cultivation of B. mandrillaris (Kiderlen et al., 2006, Schuster and Visvesvara, 1996). BM-3 medium is excellent for massive growth of B. mandrillaris due to its richness in nutrients, but it has some disadvantages, as it requires 11 components plus newborn calf serum and its preparation is laborious for it has to be prepared and sterilized separately, by different methods, and then put them together. The modified Chang's special medium was created by Kiderlen et al. (2006) to sustain the axenic culture of B. mandrillaris based on another liquid medium developed to cultivate and differentiate pathogenic and nonpathogenic Naegleria fowleri strains (De Jonckheere, 1977); it is a good medium to cultivate B. mandrillaris as the BM-3 medium, but it is also complex in composition and preparation. Cerva's medium is a simpler medium based on Bacto™ Casitone and designed originally for Acanthamoeba (Hartmannella) castellanii (Cerva, 1966), modified later to grow N. fowleri by adding sterile fresh horse serum and antibiotics, and now is used for axenic cultivation of most of free-living amoebae (Cerva, 1969). The objective of this study was to find an easy-to-prepare culture medium for axenic growth of B. mandrillaris using Cerva's medium as basal component.
Section snippets
Materials and methods
Ten strains of B. mandrillaris, including the original isolate (CDC:V039), upon which the description of the amoeba as a new genus and species was based (Visvesvara et al., 1990, Visvesvara et al., 1993), and 9 environmental isolates from water and soil (Lares-Jiménez et al., 2014), were used during trials. Stock cultures of the amoebae were maintained in axenic conditions at 37 °C in BM-3 medium by serial subcultivation (Schuster and Visvesvara, 1996). For assays, Cerva's medium, consisted in
Results and discussion
After the first week of daily observations of the tested combinations of BM-3 components and Cerva's medium, amoebae numbers diminished in every flask. At the end of the second week of incubation, no trophozoites nor cysts were seen in any modification, except for the one consisting of the basal medium complemented with 10× Hank's balanced salt solution (34.0 mL/500 mL) (Table 1) where growth was recovered. Although Hank's balanced salt solution is a very common component of several culture
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Facial Balamuthia mandrillaris infection with neurological involvement in an immunocompetent child
2022, The Lancet Infectious DiseasesCitation Excerpt :For the axenic culture of B mandrillaris, BM-3 culture medium and modified Chang's special medium have been recommended in previous studies, but they have some disadvantages, insofar as both require many components and their preparation is laborious.23,24 Lares-Jimenez and colleagues developed a modified medium for B mandrillaris axenic culture that contains 10·0 g Bacto Casitone (Difco, Detroit, MI, USA), 34·0 mL of 10 × Hank's balanced salt solution (Gibco, Grand Island, NY, USA), 456·0 mL of ddH2O, 10% fetal calf serum, and penicillin–streptomycin (200 UI/mL–200 μg/mL), which is as useful as BM-3 for axenisation.25 B mandrillaris infection has two different clinical presentations.
Identification of plicamycin, TG02, panobinostat, lestaurtinib, and GDC-0084 as promising compounds for the treatment of central nervous system infections caused by the free-living amebae Naegleria, Acanthamoeba and Balamuthia
2019, International Journal for Parasitology: Drugs and Drug ResistanceCitation Excerpt :Since T4 is the predominant genotype in brain infections caused by Acanthamoeba (Booton et al., 2005), we selected a strain representing T4 genotype. Balamuthia mandrillaris: Trophozoites (ATCC strain PRA-291, gift from Joseph L. DeRisi lab) were grown in axenic Cerva's medium (Lares-Jimenez et al., 2015) at 37 °C and 5% CO2 and sub-cultured every 3–4 days. All the experiments were performed using trophozoites harvested during the logarithmic phase of growth.
Potentially pathogenic genera of free-living amoebae coexisting in a thermal spring
2018, Experimental ParasitologyCitation Excerpt :Amoebae that grew massively when incubated at 45 °C, which is characteristic of thermophilic Naegleria, or grew below the surface of the agar, which is characteristic of B. mandrillaris and some species of Acanthamoeba, when incubated at 37 °C, were reseeded until pure cultures were obtained. Specifically, when B. mandrillaris was targeted, fungal-free samples with a suspicious morphology similar to this amoeba were transferred to cell monolayers for massive growth and, finally, axenic cultures were attempted using BMI culture medium (Lares-Jiménez et al., 2015). DNA was extracted from amoebae growing on monoxenic cultures, using the DNeasy extraction kit (Qiagen, Inc., Valencia, CA, USA), followed by measurement of the DNA concentration with a NanoDrop 2000c spectrophotometer (Thermo Scientific, USA).
Detection of serum antibodies in children and adolescents against Balamuthia mandrillaris, Naegleria fowleri and Acanthamoeba T4
2018, Experimental ParasitologyCitation Excerpt :Axenic cultures of Balamuthia mandrillaris, Naegleria fowleri and pathogenic Acanthamoeba sp. from our strain collection were grown in 75 cm2 disposable tissue culture flasks. B. mandrillaris was cultured using BMI axenic medium (Lares-Jiménez et al., 2015). Naegleria and Acanthamoeba were cultured using Červa's axenic medium (Cerva, 1980).
Molecular identification of waterborne free living amoebae (Acanthamoeba, Naegleria and Vermamoeba) isolated from municipal drinking water and environmental sources, Semnan province, north half of Iran
2017, Experimental ParasitologyCitation Excerpt :Previously, studies conducted in Iran reported the occurrence of B. mandrillaris in the dust of a hospital, soils of recreational areas and recreational hot springs (Niyyati et al., 2009, 2016; Latifi et al., 2016). B. mandrillaris has often been detected from soil and water worldwide (Lares-Jiménez et al., 2015). More studies are needed to evaluate the exact niches of B. mandrillaris, in Iran and worldwide.