Molecular detection of Vibrio cholerae and protozoa from water and interaction of V. cholerae clinical isolates with Acanthamoeba castellanii

摘要

Microbial contamination of water is a major public concern due to outbreaks of cholera,giardiasis and cryptosporidiosis. Despite this, a large number of bacteria, protozoa andviruses can be found in water. This thesis analysed and identified protozoal species andVibrio cholerae in water samples from Sudan and investigated the interactions of Swedishclinical isolates of V. cholerae with the free-living amoeba Acanthamoeba species. Theeffect of outer membrane protein A (OmpA) and vesicles on the survival of V. choleraewas also investigated, using molecular methods, cell culture, viable cell count, gentamicinassay, vital staining, light and electron microscopy and statistical analysis.Analysis of natural water samples collected from different cholera endemic areas in Sudanshowed that most V. cholerae bacteria occurred together with Acanthamoeba species in thesame samples. The percentage of samples containing V. cholerae only was 11% and that ofV. cholerae together with Acanthamoeba was 89%. Moreover, sequencing identified 66protozoa species, of which 19 (28.8%) were amoebae, 17 (25.7%) apicomplexa, 25 (37.9%)ciliates and 5 (7:6%) flagellates.Examination of the role of OmpA and outer membrane vesicles (OMVs) in survival of V.cholerae alone and during its interaction with A. castellanii showed that OmpA suppressedsurvival of wild-type V. cholerae cultivated alone. Co-cultivation with A. castellaniienhanced survival of wild-type and OmpA mutant strains. However, in co-cultivation,OmpA had no effect on attachment, engulfment or intracellular growth of V. choleraeinteracting with the amoeba, although the OmpA mutant released more OMVs and inhibitedviability of the amoeba more than the wild type. Surprisingly, treatment of amoebal cellswith OMVs isolated from the OmpA mutant significantly decreased their viable counts.The Swedish clinical isolates V. cholerae O3, V. cholerae O4, V. cholerae O5, V. choleraeO11 and V. cholerae O160 V. cholerae were able to grow and survive outside and insidethe amoeba and the presence of the amoeba enhanced the survival of all bacteria strains,indicating that all strains can be considered facultative intracellular bacteria. It can beconcluded that the presence of Acanthamoeba species supports the presence of V. cholerae.In co-cultivation, Swedish clinical isolates V. cholerae and other strains grew and survivedinside A. castellanii. The OmpA gene of V. cholerae suppressed production of OMVs,which decreased the viability of A. castellanii. Thus V. cholerae might be adapted tosurvive better in association with eukaryotes and Acanthamoeba might be a biologicalfactor enhancing survival of V. cholerae in nature.