Composition of morphotypes of naked amebae in soils of the forest-steppe zone of Ukraine

In the soils of the forest-steppe zone of Ukraine we found 12 morphotypes of naked amoebae: eruptive (Vahlkampfa sp. (1), Vahlkampfa sp. (2), Willaertia sp.), politactic (Polychaos dubium Schaeffer, 1917, Deuteramoeba mycophaga Page, 1988), monotactic (Saccamoeba stagnicola Page, 1974, Hartmannella vermiformis Page, 1967, Cashia limacoides Page, 1974), lens-like (Cochliopodium sp. (1)), striate (Thecamoeba striata Penard, 1890, Thecamoeba similis Lepsi, 1960), rugose (Thecamoeba terricola (Greef, 1866) Lepsi, 1960), fan-shaped (Vannella sp., Ripella platypodia Smirnov, Nassonova, Chao et Cavalier-Smith, 2007), mayorellian (Mayorella viridis Leidy, 1874, Mayorella cantabrigiensis Page, 1983, Mayorella sp.), dactylopodial (Korotnevella sp. (1), Vexillifera sp.), acanthopodial (Acanthamoeba sp. (1)), branched (Rhizamoeba sp. (1)), lingualate (Stenamoeba stenopodia Page, 1969). In terms of frequency of occurrence, the most common were amoebae of eruptive (91 %), fan-shaped (82), striate (69), lens-like (62), acanthopodial (62), mayorellian (55.5 ), monotactic (53 %) morphotypes, the least common are amoeba of the politactic (24 %) morphotype. In the soils of meadows, there are no amoeba of polytactic and rugose morphotypes, in soils under shrubs – rugose and branched morphotypes. The composition of morphotypes of naked amoebas in the soils of the forest-steppe zone of Ukraine is divided into two complexes: the first complex includes amoebae from the soils of forests and shrubs, the second – from the soils of meadows. The formation of the first complex of morphotypes of amoebae is influenced by the increased humidity and higher temperature of the soils in comparison with the other complex of morphotypes of amoebas.

Голі амеби в ґрунтах Харківської області (Україна)

У ґрунтах Харківської області (Україна) нами ідентифіковано 17 видів голих амеб. Це такі види: Rhizamoeba sp., Saccamoeba stagnicola Page, 1974, Hartmannella vermiformis Page, 1967, Deuteramoeba mycophaga (Pussard et al., 1980) Page, 1988, Thecamoeba striata (Penard, 1890) Schaeffer, 1926, Stenamoeba stenopodia (Page, 1969) Smirnov et al., 2007, Mayorella cantabrigiensis Page, 1983, Mayorella sp., Korotnevella sp., Vexillifera sp., Vannella sp., Ripella platypodia Smirnov, Nassonova, Chao et Cavalier-Smith, 2007, Acanthamoeba sp. (1), Cochliopodium sp., Vahlkampfia sp. (1), Vahlkampfia sp. (2), Naegleria gruberi Schardinger, 1899. Серед ідентифікованих видів найбільш поширеними виявились M. cantabrigiensis, Acanthamoeba sp. (1), Cochliopodium sp., N. gruberi, Vexillifera sp., Vahlkampfia sp. (1), Vahlkampfia sp. (2), S. stenopodia, найменш поширеними – D. mycophaga, Korotnevella sp., Rhizamoeba sp., Mayorella sp., середнє положення за частотою трапляння займають види H. vermiformis, Vannella sp., S. stagnicola, T. striata, R. platypodia. Найбільша кількість видів характерна для ґрунтів лісів (16 видів), найменша – для ґрунтів галявин (9 видів), у ґрунтах чагарників траплялось 11 видів голих амеб. Ідентифіковані нами види належать до 11 морфотипів: розгалуженого (Rhizamoeba sp.), моноподіального (S. stagnicola, H. vermiformis), політактичного (D. mycophaga), стріатного (T. striata), язикоподібного (S. stenopodia), майорельного (M. cantabrigiensis, Mayorella sp.), дактилоподіального (Korotnevella sp., Vexillifera sp.), віялоподібного (Vannella sp., R. platypodia), акантоподіального (Acanthamoeba sp. (1)), лінзоподібного (Cochliopodium sp.), еруптивного (Vahlkampfia sp. (1), Vahlkampfia sp. (2), N. gruberi). За видовим складом населення голих амеб поділяється на два комплекси: з ґрунтів лісів і ґрунтів галявин та чагарників. Таку ж подібність демонструють і морфотипи голих амеб. На формування видових комплексів амеб та їх морфотипів за результатами непараметричного багатовимiрного шкалювання (MDS) впливають в більшій мірі вологість та кислотність ґрунтів, в меншій мірі – температурний фактор.

Населення голих амеб на межі зони «мох – ґрунт» у лісових екосистемах України

У досліджених мікробіотопах (мох, межа «мох – ґрунт», ґрунт) України нами знайдено 17 видів голих амеб. Це такі види: Vahlkampfia sp. (1), Vahlkampfia sp. (2), Willaertia sp., Deuteramoeba mycophaga (Pussard, Alabouvette & Pons, 1980) Page, 1988, Saccamoeba stagnicola Page, 1974, Hartmannella vermiformis Page, 1967, Korotnevella sp., Vexillifera sp., Vannella sp., Ripella platypodia Glaeser, 1912, Cochliopodium sp., Mayorella cantabrigiensis Page, 1983, Mayorella sp., Thecamoeba striata (Penard, 1890) Schaeffer, 1926, Stenamoeba stenopodia (Page, 1969) Smirnov et al., 2007, Acanthamoeba sp. (1), Filamoeba nolandi Page, 1967. На межі мікробіотопів «мох – ґрунт» не формується специфічний варіант населення голих амеб і не спостерігається збільшення його видового багатства, а залишаються види, характерні для одного з сусідніх мікробіотопів. Для ґрунтової фауни характерні амеби Willaertia sp., D. mycophaga, Korotnevella sp., M. cantabrigiensis, F. nolandi. Не формується специфічний склад морофтипів голих амеб на межі «мох – ґрунт» і сусідні мікробіотопи за складом морфотипів подібні між собою. Амеба політактичного морфотипу надає перевагу ґрунту, язикоподібного та майорельного – ґрунту та межі «мох – ґрунт», що має пристосувальний характер до умов середовища.

Impact of inter-amoebic phagocytosis on the L. pneumophila growth

ABSTRACT Free-living amoebae are known to act as replication niches for the pathogenic bacterium Legionella pneumophila in freshwater environments. However, we previously reported that some strains of the Willaertia magna species are more resistant to L. pneumophila infection and differ in their ability to support its growth. From this observation, we hypothesize that L. pneumophila growth in environment could be partly dependent on the composition of amoebic populations and on the possible interactions between different amoebic species. We tested this hypothesis by studying the growth of L. pneumophila and of a permissive free-living amoeba, Vermamoeba vermiformis (formerly named Hartmannella vermiformis), in co-culture with or without other free-living amoebae (Acanthamoeba castellanii and W. magna). We demonstrate the occurrence of inter-amoebic phagocytosis with A. castellanii and W. magna being able to ingest V. vermiformis infected or not infected with L. pneumophila. We also found that L. pneumophila growth is strongly impacted by the permissiveness of each interactive amoeba demonstrating that L. pneumophila proliferation and spread are controlled, at least in part, by inter-amoebic interactions.

The novel Legionella pneumophila type II secretion substrate NttC contributes to infection of amoebae Hartmannella vermiformis and Willaertia magna

The type II protein secretion (T2S) system of Legionella pneumophila secretes over 25 proteins, including novel proteins that have no similarity to proteins of known function. T2S is also critical for the ability of L. pneumophila to grow within its natural amoebal hosts, including Acanthamoeba castellanii, Hartmannella vermiformis and Naegleria lovaniensis. Thus, T2S has an important role in the natural history of legionnaires’ disease. Our previous work demonstrated that the novel T2S substrate NttA promotes intracellular infection of A. castellanii, whereas the secreted RNase SrnA, acyltransferase PlaC, and metalloprotease ProA all promote infection of H. vermiformis and N. lovaniensis. In this study, we determined that another novel T2S substrate that is specific to Legionella, designated NttC, is unique in being required for intracellular infection of H. vermiformis but not for infection of N. lovaniensis or A. castellanii. Expanding our repertoire of amoebal hosts, we determined that Willaertia magna is susceptible to infection by L. pneumophila strains 130b, Philadelphia-1 and Paris. Furthermore, T2S and, more specifically, NttA, NttC and PlaC were required for infection of W. magna. Taken together, these data demonstrate that the T2S system of L. pneumophila is critical for infection of at least four types of aquatic amoebae and that the importance of the individual T2S substrates varies in a host cell-specific fashion. Finally, it is now clear that novel T2S-dependent proteins that are specific to the genus Legionella are particularly important for L. pneumophila infection of key, environmental hosts.

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.

POTENTIALLY PATHOGENIC FREE-LIVING AMOEBAE IN SOME FLOOD-AFFECTED AREAS DURING 2011 CHIANG MAI FLOOD

SUMMARY The survey was carried out to investigate the presence of potentially pathogenic free-living amoebae (FLA) during flood in Chiang Mai, Thailand in 2011. From different crisis flood areas, seven water samples were collected and tested for the presence of amoebae using culture and molecular methods. By monoxenic culture, FLA were detected from all samples at 37 °C incubation. The FLA growing at 37 °C were morphologically identified as Acanthamoeba spp., Naegleria spp. and some unidentified amoebae. Only three samples (42.8%), defined as thermotolerant FLA, continued to grow at 42 °C. By molecular methods, two non-thermotolerant FlA were shown to have 99% identity to Acanthamoeba sp. and 98% identity to Hartmannella vermiformis while the two thermotolerant FLA were identified as Echinamoeba exundans (100% identity) and Hartmannella sp. (99% identity). This first report of the occurrence of FLA in water during the flood disaster will provide information to the public to be aware of potentially pathogenic FLA.