Ultrastructural changes of <i>Acanthamoeba</i> cyst of clinical isolates after treatment with minimal cysticidal concentration of polyhexamethylene biguanide

, Kong, H H; , Chung, D I

doi:1998.36.1.7

Korean J Parasito > Volume 36(1):1998 > Article

Original Article


Korean J Parasitol. 1998 Mar;36(1):7-13. English. Published online Mar 20, 1998. http://dx.doi.org/10.3347/kjp.1998.36.1.7
Copyright © 1998 by The Korean Society for Parasitology


Ultrastructural changes of Acanthamoeba cyst of clinical isolates after treatment with minimal cysticidal concentration of polyhexamethylene biguanide
Hyun-Hee Kong and Dong-Il Chung^*
Department of Parasitology, Kyungpook National University School of Medicine, Taegu, Korea.

Received November 06, 1997; Accepted February 04, 1998.
Abstract
In order to understand the action mechanism of polyhexamethylene biguanide (PHMB) to the cyst of Acanthamoeba on the morphological basis, the cysts of four corneal isolates of Acanthamoeba were treated with minimal cysticidal concentration (MCC) of PHMB and their ultrastructural changes were examined by transmission electron microscopy. The most striking change of cysts treated with PHMB compared with normal cysts was the shrinkage of intracystic amoebae, which resulted in the separation of the plasma membrane of intracystic amoeba from endocystic wall. Subplasmalemmal lipid droplets became irregularly shaped. In severely damaged cysts, cytoplasm was aggregated and organelles were severely deformed. Cytoplasmic materials were leaked out through the damaged plasma membrane. Most cysts showed aggregation of nuclear chromatin material. Number of mitochondrial cristae was also reduced. Ecto- and endo-cystic walls were relatively well tolerated. Findings in the present study revealed that PHMB affected mainly on plasma membrane, but lesser on organellar membrane of intracystic amoeba. It seemed likely that PHMB might kill cystic forms of Acanthamoeba by similar mechanism in which this environmental biocide can damage the cell wall of Escherichia coli by binding with acidic phospholipids.

Figures


	Fig. 1 Agarose gel electrophoretic restriction fragment patterns for mitochondria DNA of 4 Korean corneal isolates of Acanthamaoeba. M: Hind III digested λ phage DNA as DNA molecular size standard.

Figs. 2-10
Fig. 2 & 3. Normal cyst of Acanthamaoeba KA/E2 isolate. The plasma membrance (arrows) is lined along with endocystic wall. The morphology of nucleus. mitochondria (arrow heads) and other intracellular organelles are normal. The mitochondria had 4-10 cristae. Fig. 2, ×5,500; Fig. 3, ×18,500. Bars = 1µm. Fig. 4. The nucleus with typical central karyosome of control cyst of KA/E3 isolate. ×16,500. Bar = 1µm. Fig. 5. Severely damaged cyst of KA/E1 strain treated with MCC8. ×7,600. Bar = 1µm. Fig. 6. Separation of plasma membrane from endocyst of KA/E2 cyst treated with MCC8. ×23,700. Bar = 1µm. Fig. 7. Irregular shaped subplasmalemmal lipid droplet of KA/E4 cyst treated with MCC48. ×75,000. Bar = 0.1µm. Fig. 8. Severely deformed intracellular organelles and aggregation of intracytoplasmic materials of KA/E3 cyst treated with MCC48. ×9,600. Bar = 1µm. Fig. 9. The number of mitochondrial cristae was decreased, and autophagic vacuole was observed in the cyst of KA/E4 treated with MCC8. ×38,300. Bar = 1µm. Fig. 10. Leakage of intracellular materials (arrows) and lipid droplets through plasma membrane (arrow heads) in KA/E3 cyst treated with MCC8. ×14,000. Bar = 1µm.

Figs. 11-13
Fig. 11. Intranuclear aggregation of chromatin materials of KA/E1 cyst treated with MCC48. ×11,300. Bar = 1µm. Figs. 12 & 13. In the cyst treated with MCC8 for 2 hr, invagination of plasma membrance was observed, and mitochondria and intracellular organelles were not deformed. Fig. 12, ×27,300; Fig. 13, ×16,900. Bar = 1µm. APV, autophagic vacuole; CW, cyst wall; EC, ectocyst; EN, endocyst; K, karyosome; L, lipid droplet; M, mitochondria; N, nucleus; O, ostyole; P, peroxisome; PM, plasma membrane.

Tables


	Table 1 Average MCC (µm/ml) of PHMB against four Acanthamaoeba isolates

References


1.	Bowers B, Korn ED. The fine structure of Acanthamoeba castellanii (Neff strain). II. Encystment. J Cell Biol 1969;41(3):786–805.

2.	Broxton P, Woodcock PM, Heatley F, Gilbert P. Interaction of some polyhexamethylene biguanides and membrane phospholipids in Escherichia coli. J Appl Bacteriol 1984;57(1):115–124.

3.	Burger RM, Franco RJ, Drlica K. Killing acanthamoebae with polyaminopropyl biguanide: quantitation and kinetics. Antimicrob Agents Chemother 1994;38(4):886–888.

4.	Callicott JH Jr. Amebic meningoencephalitis due to free-living amebas of the Hartmannella (Acanthamoeba)-Naegleria group. Am J Clin Pathol 1968;49(1):84–91.

5.	Centers for Disease Control (CDC). Acanthamoeba keratitis associated with contact lenses--United States. MMWR Morb Mortal Wkly Rep 1986;35(25):405–408.

6.	Davies A, Bentley M, Field BS. Comparison of the action of vantocil, cetrimide and chlorhexidine on Escherichia coli and its spheroplasts and the protoplasts of gram positive bacteria. J Appl Bacteriol 1968;31(4):448–461.

7.	Davies A, Field BS. Action of biguanides, phenols and detergents on Escherichia coli and its sphereoplasts. J Appl Bacteriol 1969;32(2):233–243.

8.	De Jonckheere JF. Ecology of Acanthamoeba. Rev Infect Dis 1991;13 Suppl 5:S385–S387.

9.	Gray TB, Gross KA, Cursons RT, Shewan JF. Acanthamoeba keratitis: a sobering case and a promising new treatment. Aust N Z J Ophthalmol 1994;22(1):73–76.

*10.*	Ikeda T, Ledwith A, Bamford CH, Hann RA. Interaction of a polymeric biguanide biocide with phospholipid membranes. Biochim Biophys Acta 1984;769(1):57–66.

*11.*	Ikeda T, Tazuke S, Watanabe M. Interaction of biologically active molecules with phospholipid membranes. I. Fluorescence depolarization studies on the effect of polymeric biocide bearing biguanide groups in the main chain. Biochim Biophys Acta 1983;735(3):380–386.

*12.*	Jones DB, Visvesvara GS, Robinson NM. Acanthamoeba polyphaga keratitis and Acenthamoeba uveitis associated with fatal meningoencephalitis. Trans Ophthalmol Soc U K 1975;95(2):221–232.

*13.*	Kong HH, Park JH, Chung DI. Interstrain polymorphisms of isoenzyme profiles and mitochondrial DNA fingerprints among seven strains assigned to Acanthamoeba polyphaga. Korean J Parasitol 1995;33(4):331–340.

*14.*	Larkin DF. Acanthamoeba keratitis. Int Ophthalmol Clin 1991;31(2):163–172.

*15.*	Larkin DF, Kilvington S, Dart JK. Treatment of Acanthamoeba keratitis with polyhexamethylene biguanide. Ophthalmology 1992;99(2):185–191.

*16.*	Mills RA, Wilhelmus KR, Osato MS, Pyron M. Polyhexamethylene biguanide in the treatment of Acanthamoeba keratitis. Aust N Z J Ophthalmol 1993;21(4):277–278.

*17.*	Moore MB, McCulley JP, Luckenbach M, Gelender H, Newton C, McDonald MB, Visvesvara GS. Acanthamoeba keratitis associated with soft contact lenses. Am J Ophthalmol 1985;100(3):396–403.

*18.*	Shin JW, et al. Kyungpook Med J 1996;37:297–307.

*19.*	Theodore FH, Jakobiec FA, Juechter KB, Ma P, Troutman RC, Pang PM, Iwamoto T. The diagnostic value of a ring infiltrate in acanthamoebic keratitis. Ophthalmology 1985;92(11):1471–1479.

*20.*	Tirado-Angel J, Gabriel MM, Wilson LA, Ahearn DG. Effects of polyhexamethylene biguanide and chlorhexidine on four species of Acanthamoeba in vitro. Curr Eye Res 1996;15(2):225–228.

*21.*	Wilhelmus KR. Rev Inf Dis 1991;13 S:367–368.

*22.*	Yagita K, Endo T. Restriction enzyme analysis of mitochondrial DNA of Acanthamoeba strains in Japan. J Protozool 1990;37(6):570–575.

*23.*	Yee E, Fiscella R, Winarko TK. Topical polyhexamethylene biguanide (pool cleaner) for treatment of acanthamoeba keratitis. Am J Hosp Pharm 1993;50(12):2522–2523.