The Symbiotic Relationship Of V. Cholerae With Free-living Amoebae

V. cholerae which caused seven pandemic of cholera is still one of important public health issues worldwide. The seasonal outbreak of cholera together with the bacteria's survival story has been highlighted in its epidemiological researches recently. Free-living amoebae (FLA) have been found living in aquatic environments widely and play an important mutual role with other different pathogens in their ecological communities. It has been demonstrated that FLA could be served as reservoirs for Legionella pneumophila, Mycobacterium, Chlamydozoan, etc. But the role of FLA in the epidemiology of cholera is till need to be more fully addressed to explore the mechanisms underlying seasonal outbreaks of cholera. In this study, we investigated the symbiotic relationship between V. cholerae and FLA with different temperatures and disinfectant treatments. The contribution of FLA in the prevention and control of cholera pandemic will be highlighted in the project.It was observed that the free-living amoebae, A. polyphaga, could uptake V. cholerae with phagocytosis when they were co-cultured at 28℃for 8 hr using Gimenez staining and electronic microscope. The more bacteria were internalized and most of which located within vacuoles when they were co-cultured with the amoebae further. Interestingly, a mitotic V. cholerae was observed in the infected trophozoites. Later, not only vacuoles but also cyst wall and its cytoplasm were found containing the symbiotic bacteria after amoebae's encystation. Furthermore, the internalized V. cholerae were survived after the excystmen of A.polyphaga. In addition, the V. cholerae co-cultured with amoebae could maintained in a relative higher concentration when compared with that of control at 28℃. Besides, it was demonstrated that the medium from A.polyphaga could promote the proliferation of V. cholerae in a traswell co-cultural experiment. It suggested that Acanthamoeba might be a reservoir for co-cultured V. cholerae at 28℃.The symbiotic relationship of V. cholerae and amoebae was also been investigated further under low temperature. The viable El Tor biotype V. cholerae decreased from (3.32±0.43)×109cfu/ml to less than 1 cfu/ml in the presence of A.polyphaga at 4℃after Day 30. But the control viable El Tor decreased to non-detectable levels until Day 45. However, it was co-cultured V. cholerae but not control bacteria could be re-isolated from the cysts after excystment at 28℃after Day 50. It indicated that the presence of A.polyphaga improved the survival of V. cholerae at low temperature, but no evidence showed that the bacteria could survival forever with the symbiosis of A.polyphaga in the analogical low temperature environment.Since the symbiosis of standard stain, V. cholerae and A.polyphaga, was observed in analogical conditions, we focused our study on the real living amoebae and cholerae in Huangpu River, Shanghai. Two free-living amoebas (W-HPR and E-HPR) were isolated from the river and both of them belonged to genotype T4, Acanthamoeba strains. A V. cholerae non-01/0139 strain was also found in the same area. We demonstrated that the isolated cholerae strain could live within the trophozoite and vacuoles of amoebae when be co-cultured with W-HPR for 8 hr. Next, the protective role of amoebae against disinfection of disinfectants for V. cholerae was investigated on the co-culture. Although 50 mg/L chlorine was effective as a disinfectant against acanthamoebal trophozoites after exposure for 30 min, it could not kill the cysts unless using 125mg/L chlorine for 30 min or 50 mg/L for 2 hr. Four in a total number of 8 domestic water samples were proved to be positive for amoeba although ozone and chlorine were used in daily drinking water. We demonstrated that A.polyphaga could protect co-cultured El Tor biotype V. cholerae from the treatment of chlorine at 2 mg/L.In conclusion, the data showed that A. polyphaga could be an environmental reservoir of V. cholerae to protect the bacteria from low temperature and disinfections. The detail symbiotic mechanism between A. polyphaga and V. cholerae together with their epidemiological studies should be more addressed in their future research.