Sensitivity of Vermamoeba (Hartmannella) vermiformis cysts to conventional disinfectants and protease.

Vermamoeba vermiformis is a free-living amoeba (FLA) widely distributed in the environment, known to colonize hot water networks and to be the reservoir of pathogenic bacteria such as Legionella pneumophila. FLA are partly resistant to biocides, especially in their cyst form. The control of V. vermiformis in hot water networks represents an important health issue, but there are very few data on their resistance to disinfection treatments. The sensitivity of cysts of two strains of V. vermiformis to three disinfectants frequently used in hot water networks (chlorine, heat shock, peracetic acid (PAA) mixed with hydrogen peroxide (H2O2)) was investigated. In vitro, several concentrations of biocides, temperatures and exposure times according to the French regulation were tested. Cysts were fully inactivated by the following conditions: 15 mg/L of chlorine for 10 min; 60 °C for 30 min; and 0.5 g/L equivalent H2O2 of PAA mixed with H2O2 for 30 min. For the first time, the strong efficacy of subtilisin (0.625 U/mL for 24 h), a protease, to inactivate the V. vermiformis cysts has been demonstrated. It suggests that novel approaches may be efficient for disinfection processes. Finally, V. vermifomis cysts were sensitive to all the tested treatments and appeared to be more sensitive than Acanthamoeba cysts.

Efficacy of dental unit disinfectants against Candida spp. and Hartmannella vermiformis.

Human oral commensal Candida yeasts, as well as environmental free-living amoebae (FLA) such as Hartmannella, are known to be direct or indirect human pathogens. These microorganisms may be isolated from dental unit waterlines (DUWL), because of contamination coming from the tap water and/or a patient's mouth. This study compared the efficacy of commonly used DUWL disinfectants (chlorine, H2 O2 , and Oxygenal 6©) against three species of Candida (C. albicans, C. glabrata, and C. parapsilosis) and one FLA species (H. vermiformis), growing either as single or as mixed biofilms in tap water. Results showed variable efficacies: H2 O2 had no significant activity, while chlorine was effective but only at the highest doses tested, probably not compatible with DUWL uses. Oxygenal 6© was the most efficacious in preventing the growth of yeasts in tap water. However, in the presence of FLA, Oxygenal 6© displayed a reduced antimicrobial activity against sessile C. albicans. In conclusion, none of the tested disinfectants could eradicate yeasts or FLA. Moreover, the antiyeast activity of Oxygenal 6© was reduced in the presence of FLA. Both sessile or planktonic and mixed or single-species conditions should be considered when evaluating the activity of disinfectants for DUWL maintenance. This study also highlighted that FLA should be included in the testing protocols.

Impact of silver and copper on the survival of amoebae and ciliated protozoa in vitro.

The efficacy of 1:10 silver/copper combinations for inactivation of Hartmannella vermiformis amoebas and the ciliated protozoan Tetrahymena pyriformis in vitro was studied. Tetrahymena and Hartmannella/isolate 19 were inactivated for 2 log steps by 100 + 1000 micrograms/l Ag + Cu. Hartmannella/isolate 21 was more resistant. 500 + 5000 micrograms/l produced only a 0.6 log reduction. The investigations clearly showed that levels within the limit of the German drinking water regulation (10 + 100 micrograms/l Ag + Cu) could not inactivate these protozoas in vitro.

Acanthamoeba keratitis with symbiosis of Hartmannella ameba.

PURPOSE: To report a case of severe amebic keratitis in which both Hartmannella and Acanthamoeba were isolated simultaneously from the same lesion. METHOD: Case report. The deep corneal lesion was scraped for cytopathology and isolation of the pathogens. We tested the in vitro sensitivities of the pathogens to several drugs. RESULTS: Cultures of the corneal scrapings and of the solution in the patient's contact lens storage case were positive for Acanthamoeba E9 cysts and trophozoites. Hartmannella ameba coexisted with Acanthamoeba in the cornea. When tested in vitro, Acanthamoeba trophozoites were sensitive to both miconazole nitrate and natamycin, while cysts were sensitive only to natamycin. However, the patient did not respond to these antiamebic drugs. CONCLUSIONS: This case suggests that Acanthamoeba is not the only origin of amebic keratitis. Hartmannella may also cause severe drug-resistant keratitis.

Effects of cytochalasin D and methylamine on intracellular growth of Legionella pneumophila in amoebae and human monocyte-like cells.

A cloned and axenically cultured strain of Hartmannella vermiformis was used as a model to study intracellular multiplication of Legionella pneumophila in amoebae. The growth of L. pneumophilia in both H. vermiformis and a human monocyte-like cell line (U937) was investigated with cytoskeletal and metabolic inhibitors. L. pneumophila replicated only intracellularly in these cellular models, and electron microscopy showed ultrastructural similarities in the initial phase of multiplication. Treatment of amoebae with an inhibitor of microfilament-dependent phagocytosis (cytochalasin D, 0.5 or 1.0 micrograms/ml) did not inhibit intracellular growth of L. pneumophila; however, intracellular multiplication was inhibited by treatment of U937 monocytes with the same concentrations of cytochalasin D. Methylamine (10 to 100 mM), an inhibitor of adsorptive pinocytosis, inhibited the replication of L. pneumophila in amoebae in a dose-dependent manner. All doses of methylamine tested (10 to 50 mM) inhibited growth of L. pneumophila in U937 monocytes. Cytochalasin D and methylamine had no effect on the multiplication of L. pneumophila in culture medium or on the viability of amoebae or U937 monocytes. Intracellular replication of L. pneumophila in H. vermiformis may be accomplished by a cytochalasin D-independent mechanism, such as adsorptive pinocytosis. In contrast, both cytochalasin D- and methylamine-sensitive mechanisms may be essential for the intracellular multiplication of L. pneumophila in U937 monocytes.

Excystment of axenically prepared cysts of Hartmanella culbertsoni.

Axenically prepared cysts of Hartmannella culbertsoni readily excysted in the presence of heat stable factors prepared from Escherichia coli, Klebsiella aerogenes, Staphylococcus aureus, Sarcina lutea, Bacillus subtilis, Bacillus megaterium and several fungi. Peptone, proteose peptone, tryptone or amino acids also promoted excystment. Crowding of the cysts and dilution of bacterial extracts adversely affected the excystment. Continual presence of the factors in the medium was essential for excystment.

The inhibitory effect of glucose on the differentiation of trophic Hartmannella culbertsoni into viable cysts.

During encystation of Hartmannella culbertsoni induced by taurine or epinephrine, 60-70% of the reserve glycogen is degraded. Glycogen phosphorylase is activated and glycogen synthetase is inhibited after 6-8 hr of exposure to the encystation medium. The carbon skeleton of glycogen but not that of protein is utilised in the synthesis of cyst wall cellulose. Exogenously added glucose (225 and 550 mM) blocks encystation, degradation of glycogen and synthesis of cellulose. Cyclic AMP synthesis is also very much reduced in cells exposed to glucose.