J Med Microbiol 57 (2008), 428-431; DOI: 10.1099/jmm.0.47694-0
© 2008 Society for General Microbiology
ISSN 1473-5644
Balamuthia mandrillaris resistance to hostile conditions
Ruqaiyyah Siddiqui1,
Antonio Ortega-Rivas2 and
Naveed Ahmed Khan1
1 School of Biological and Chemical Sciences, Birkbeck College, University of London, London WC1E 7HX, UK
2 Department of Parasitology, La Laguna University, La Laguna, Canary Islands, Spain
Correspondence
Naveed Ahmed Khan
n.khan{at}sbc.bbk.ac.uk
Received 15 October 2007
Accepted 7 January 2008
The resistance of Balamuthia mandrillaris to physical, chemical and radiological conditions was tested. Following treatments, viability was determined by culturing amoebae on human brain microvascular endothelial cells for up to 12 days. B. mandrillaris cysts were resistant to repeated freeze–thawing (five times), temperatures of up to 70 °C, 0.5 % SDS, 25 p.p.m. chlorine, 10 µg pentamidine isethionate ml–1 and 200 mJ UV irradiation cm–2.
Abbreviations: HBMEC, human brain microvascular endothelial cell.
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INTRODUCTION
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Balamuthia mandrillaris is an emerging protozoan pathogen that can cause fatal encephalitis in humans and animals (Visvesvara et al., 1990). The life cycle of B. mandrillaris consists of two stages: an infective trophozoite stage and a dormant cyst stage (reviewed by Visvesvara et al., 2007; Siddiqui & Khan, 2008). Trophozoites range in size from 12 to 60 µm with a mean size of 30 µm. They actively feed on other eukaryotic cells, and divide mitotically. However, under unfavourable conditions, such as lack of food, trophozoites differentiate into a cyst form (12 to 60 µm). Under the light microscope, the cyst appears double-walled with a thin irregular outer wall and a thick, round, inner wall. Both walls are separated by a middle amorphous fibrillar layer (Visvesvara et al., 2007). Although B. mandrillaris is a soil amoeba and is closely related to Acanthamoeba, unlike Acanthamoeba, it is unable to prey on prokaryotes as a food source (Schuster & Visvesvara, 1996). In vitro studies have shown that B. mandrillaris feed on small amoebae suggesting that they may play a role in the regulation of amoebae and perhaps other eukaryote populations in the environment (reviewed by Schuster & Visvesvara, 2004). Furthermore, the free-living nature of this organism, and its ability to encyst in human tissues establishing latent infections, suggests that B. mandrillaris cysts have adapted to occupy diverse environments and may be resistant to a variety of hostile conditions. Here, we determined whether B. mandrillaris cysts are resistant to various physical, chemical and radiological conditions.
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METHODS
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B. mandrillaris cultures.
Primary human brain microvascular endothelial cells (HBMECs) were used as food source for B. mandrillaris. The HBMECs were routinely grown on rat-tail collagen-coated dishes in RPMI 1640 containing 10 % heat inactivated fetal bovine serum, 10 % Nu-Serum, 2 mM glutamine, 1 mM pyruvate, 100 U penicillin ml–1, 100 µg streptomycin ml–1, non-essential amino acids and vitamins as described by Alsam et al. (2003) and Stins et al. (1997). B. mandrillaris (isolated from baboon brain tissue) were obtained from the American Type Culture Collection (ATCC50209; www.atcc.org) and grown on HBMEC monolayers (Matin et al., 2006, 2007). Briefly, B. mandrillaris were inoculated in 10 ml RPMI 1640 (105 amoebae ml–1) on HBMEC monolayers grown in T-75 tissue culture flasks. The amoebae consumed HBMECs within 48 h, and produced approximately 5x106–8x106 amoebae (>95 % in trophozoite form). To obtain cysts, B. mandrillaris trophozoites were inoculated in RPMI 1640 (2x105 ml–1) and plates incubated at 37 °C for up to 7 days or until no trophozoites were observed, as previously described (Siddiqui et al., 2007). Cysts were collected by centrifugation at 1000 g for 10 min, counted using a haemocytometer and used for subsequent experiments.
B. mandrillaris treatment assays.
To determine the effect of repeated freezing–thawing on the cell numbers and the viability of amoebae, B. mandrillaris cysts (4x105 amoebae ml–1) and trophozoites (4x105 amoebae ml–1) were frozen at –80 °C for 20 min in separate 1.5 ml microfuge tubes and then thawed by placing the tubes in a polystyrene rack and incubated at 37 °C in a water bath, through ambient temperature. This process was repeated five times. Subsequently, amoebae were counted using a haemocytometer. To determine their viability, treated and untreated amoebae were transferred onto HBMEC monolayers grown in 24-well plates. Plates were incubated at 37 °C in a 5 % CO2 incubator, and B. mandrillaris growth was observed for up to 12 days. The presence of actively feeding trophozoites was considered as the presence of viable amoebae. The viability was confirmed further by inoculating amoebae on fresh HBMEC monolayers.
For high temperatures, B. mandrillaris trophozoites or cysts were prepared as described for the freeze–thaw method, and tubes were incubated at various temperatures including 42, 50, 60, 70, 80 and 90 °C for 60 min, and at 100 °C for 5 min in a heated block. Following this, the viability of B. mandrillaris trophozoites or cysts was determined as described for the freeze–thaw method.
For radiological conditions, B. mandrillaris cysts (4x105 amoebae ml–1) and trophozoites (4x105 amoebae ml–1) were placed in 24-well plates, with the lids removed, and exposed to UV light for up to 60 min (total irradiation 200 mJ UV cm–2) at room temperature. The viability of treated and untreated B. mandrillaris was determined as described above.
For chemical conditions, amoebae were incubated with chlorine, pentamidine isethionate and SDS. For chlorine, B. mandrillaris were incubated with various concentrations of chlorine, including 0.05, 0.5, 1.25, 2.5, 5, 10 and 25 p.p.m., at room temperature for 1 h. Following this incubation, amoebae were collected by centrifugation and resuspended in fresh RPMI 1640 and inoculated onto HBMEC monolayers for viability testing as described above. Similar methods were employed using the anti-amoebic drug pentamidine isethionate. To determine the effects of the detergent SDS on the viability of B. mandrillaris, amoebae were incubated with various concentrations of SDS, including 0.01, 0.05, 0.1 and 0.5 %, and their viability was determined as described above.
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RESULTS AND DISCUSSION
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Neither repeated freeze–thawing nor high temperature affected the viability of B. mandrillaris cysts
To determine the effect of harsh conditions on cell numbers and the viability of amoebae, B. mandrillaris cysts and trophozoites were freeze–thawed five times. The repeated freeze–thawing halved trophozoite numbers but had no significant effect on cyst numbers (data not shown). Next, to determine their viability, treated and untreated amoebae were transferred onto HBMEC monolayers. As shown in Table 1
, repeated freeze–thawing did not affect the survival of either trophozoites or cysts, both of which emerged as actively feeding trophozoites within 5–6 days. In contrast, untreated amoebae emerged within 48 h. Although freeze–thawing delayed emergence of amoebae, they were considered as viable as long as actively feeding trophozoites were observed within 12 days. The results are representative of at least three independent experiments.
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Table 1. Effect of repeated freeze–thawing and high temperatures on the viability of B. mandrillaris trophozoites and cysts
For the freeze–thaw method, both cysts (4x105 amoebae ml–1) and trophozoites (4x105 amoebae ml–1) were frozen at –80 °C for 20 min and then thawed by incubating at 37 °C. This process was repeated five times. For testing the effect of high temperatures, amoebae were incubated at 42, 50, 60, 70, 80 or 90 °C for 60 min, or at 100 °C for 5 min in a heated block. For UV irradiation, amoebae were placed in 24-well plates, with the lids removed and exposed to UV. Results are representative of at least three independent experiments. +, Amoebae remained viable following treatment; –, amoebae non-viable following treatment.
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To determine the effects of high temperatures, amoebae were incubated at various temperatures. Cysts withstood heating up to 70 °C for 60 min and produced viable, proliferating amoebae (Table 1
). Temperatures higher than 70 °C had a limited effect on cyst lysis (data not shown), but were detrimental to viability as no excystation was observed (Table 1). In contrast, trophozoites were killed effectively at 60 °C for 60 min (Table 1). Of note, previous studies have shown that Acanthamoeba cysts can be effectively killed by heating at 60 °C for more than 5 min (Aksozek et al., 2002). The results are representative of at least three independent experiments.
UV light did not affect B. mandrillaris viability
B. mandrillaris were exposed to UV for up to 60 min (total irradiation 200 mJ UV cm–2) at room temperature. The results revealed that both B. mandrillaris cysts and trophozoites survived this dosage of UV (Table 1). This was unexpected and to confirm that the UV irradiation was effective, Escherichia coli K-12 strain HB101 (4x106 bacteria ml–1) was exposed to UV for 30 and 60 min. Both exposures effectively killed bacteria but not B. mandrillaris. The results are representative of at least three independent experiments. Interestingly, other studies have shown that Acanthamoeba also exhibits resistance to a high dosage of irradiation without any loss of viability (Aksozek et al., 2002). It was suggested that DNA in encysted cells is not engaged in translational activities, which may explain their resistance to a high dosage of irradiation, and/or cells may be able to repair DNA without any loss of viability, in the case of trophozoites. Similar mechanisms may explain the ability of B. mandrillaris to tolerate DNA damage.
B. mandrillaris cysts are resistant to chlorine, pentamidine isethionate and SDS
Chlorine is a commonly used disinfectant for water treatment. The effects of chlorine on the viability of amoebae were determined. The viability of neither B. mandrillaris cysts nor trophozoites was affected by 0.05, 0.5, 1, 1.25, 2.5, 5, 10 and 25 p.p.m. chlorine (Table 2). Again, this was unexpected and to confirm that the chlorine was effective, E. coli K-12 strain HB101 (4x106 bacteria ml–1) was incubated with the aforementioned concentrations of chlorine. The findings clearly demonstrated that concentrations as low as 2.5 p.p.m. effectively killed bacteria but not B. mandrillaris. Similarly, the effects of the anti-amoebic drug, pentamidine isethionate, were determined. The findings revealed that at physiological concentrations, pentamidine isethionate did not exhibit amoebicidal effects (Table 2). In the control experiments, pentamidine isethionate effectively killed Acanthamoeba trophozoites (data not shown). The results are representative of at least three independent experiments.
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Table 2. Effect of chemical treatments on B. mandrillaris viability
For chemical treatment, both cysts and trophozoites were prepared as described for the freeze–thaw method, and then chlorine, SDS or pentamidine isethionate was added. Results are representative of at least three independent experiments. +, Amoebae remained viable following treatment; –, amoebae non-viable following treatment.
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For SDS, the results revealed that all tested concentrations of SDS killed trophozoites, but had no effect on the viability of cysts (Table 2). However, it is noteworthy that cysts treated with 0.5 % SDS showed delayed excystation and it took up to 10–11 days to observe active trophozoites. Our finding that SDS (0.5 %) is ineffective in killing B. mandrillaris cysts is significant. The majority of protozoan pathogens that form cysts possess proteins and/or pores in their outer walls, which allow them to monitor environmental changes and/or to detect stimuli for excystation, and are susceptible to SDS. For example, Acanthamoeba, a close relative of Balamuthia is known to possess pores (ostioles), as well as cyst wall-specific proteins/carbohydrate, which makes it susceptible to even lower concentration of SDS (Hirukawa et al., 1998; Neff & Neff, 1969; Page, 1967; Tomlinson & Jones, 1962). Ultrastructurally, B. mandrillaris cysts possess three walls and the presence of ostioles is yet to be demonstrated (Visvesvara et al., 1993); thus, at present, it is unclear how cysts would resist 0.5 % SDS. Moreover, heat inactivation of up to 100 °C did not lyse cysts but affected their viability. As for other protozoan pathogens, it is likely that the B. mandrillaris cyst wall largely comprises carbohydrates, a subject for future studies.
In conclusion, these findings suggest that B. mandrillaris cysts are highly resistant to physical, chemical and radiological conditions, which may allow them to occupy wide environmental conditions, including hostile surroundings. These results should provide a basis for determining the susceptibility of B. mandrillaris cysts to a range of disinfectants and/or may help develop strategies for their eradication from environments common to humans. Recent studies have shown that B. mandrillaris can harbour pathogenic bacteria such as Legionella pneumophila (Shadrach et al., 2005). L. pneumophila cells are known to be parasitic for a variety of amoeba, and this property allows them to survive in the environment, as well as in the amoebic intracellular environment, which might assist the bacteria in adapting to survive in mammalian phagocytic cells. Thus the ability of B. mandrillaris cysts to survive under harsh conditions may protect and help distribute L. pneumophila to a variety of hostile environmental settings, which may lead to bacterial transmission to susceptible hosts. Taken together, these results have significance for the epidemiology and control of infections due both to B. mandrillaris and L. pneumophila.
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ACKNOWLEDGEMENTS
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This work was partially supported by grants from the Life Sciences Research Fund, Birkbeck, University of London, the Royal Society and the Society for General Microbiology.
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REFERENCES
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Aksozek, A., McClellan, K., Howard, K., Niederkorn, J. Y. & Alizadeh, H. (2002). Resistance of Acanthamoeba castellanii cysts to physical, chemical, and radiological conditions. J Parasitol 88, 621–623.[CrossRef][Medline]
Alsam, S., Kim, K. S., Stins, M., Rivas, A. O., Sissons, J. & Khan, N. A. (2003). Acanthamoeba interactions with human brain microvascular endothelial cells. Microb Pathog 35, 235–241.[CrossRef][Medline]
Hirukawa, Y., Nakato, H., Izumi, S., Tsuruhara, T. & Tomino, S. (1998). Structure and expression of a cyst specific protein of Acanthamoeba castellanii. Biochim Biophys Acta 1398, 47–56.[Medline]
Matin, A., Stins, M., Kim, K. S. & Khan, N. A. (2006). Balamuthia mandrillaris exhibits metalloprotease activities. FEMS Immunol Med Microbiol 47, 83–91.[CrossRef][Medline]
Matin, A., Jeong, S. R., Stins, M. & Khan, N. A. (2007). Effects of human serum on Balamuthia mandrillaris interactions with human brain microvascular endothelial cells. J Med Microbiol 56, 30–35.[Abstract/Free Full Text]
Neff, R. J. & Neff, R. H. (1969). The biochemistry of amoebic encystment. Symp Soc Exp Biol 23, 51–81.[Medline]
Page, F. C. (1967). Re-definition of the genus Acanthamoeba with descriptions of three species. J Protozool 14, 709–724.[Medline]
Schuster, F. L. & Visvesvara, G. S. (1996). Axenic growth and drug sensitivity studies of Balamuthia mandrillaris, an agent of amebic meningoencephalitis in humans and other animals. J Clin Microbiol 34, 385–388.[Abstract]
Schuster, F. L. & Visvesvara, G. S. (2004). Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals. Int J Parasitol 34, 1001–1027.[CrossRef][Medline]
Shadrach, W. S., Rydzewski, K., Laube, U., Holland, G., Ozel, M., Kiderlen, A. F. & Flieger, A. (2005). Balamuthia mandrillaris, free-living ameba and opportunistic agent of encephalitis, is a potential host for Legionella pneumophila bacteria. Appl Environ Microbiol 71, 2244–2249.[Abstract/Free Full Text]
Siddiqui, R. & Khan, N. A. (2008). Balamuthia amoebic encephalitis: an emerging disease with fatal consequences. Microb Pathog 44, 89–97.[CrossRef][Medline]
Siddiqui, R., Matin, A., Warhurst, D., Stins, M. F. & Khan, N. A. (2007). Effect of antimicrobial compounds on Balamuthia mandrillaris encystment and human brain microvascular endothelial cell cytopathogenicity. Antimicrob Agents Chemother 51, 4471–4473.[Abstract/Free Full Text]
Stins, M. F., Gilles, F. & Kim, K. S. (1997). Selective expression of adhesion molecules on human brain microvascular endothelial cells. J Neuroimmunol 76, 81–90.[CrossRef][Medline]
Tomlinson, G. & Jones, E. A. (1962). Isolation of cellulose from the cyst wall of a soil amoeba. Biochim Biophys Acta 63, 194–200.[Medline]
Visvesvara, G. S., Martinez, A. J., Schuster, F. L., Leitch, G. J., Wallace, S. V., Sawyer, T. K. & Anderson, M. (1990). Leptomyxid ameba, a new agent of amebic meningoencephalitis in humans and animals. J Clin Microbiol 28, 2750–2756.[Abstract/Free Full Text]
Visvesvara, G. S., Schuster, F. L. & Martinez, A. J. (1993). Balamuthia mandrillaris, n. g., n. sp., agent of amebic meningoencephalitis in humans and other animals. J Eukaryot Microbiol 40, 504–514.[Medline]
Visvesvara, G. S., Moura, H. & Schuster, F. L. (2007). Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea. FEMS Immunol Med Microbiol 50, 1–26.[CrossRef][Medline]
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INTRODUCTION
METHODS
