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

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 and viruses can be found in water. This thesis analysed and identified protozoal species and Vibrio cholerae in water samples from Sudan and investigated the interactions of Swedish clinical isolates of V. cholerae with the free-living amoeba Acanthamoeba species. The effect of outer membrane protein A (OmpA) and vesicles on the survival of V. cholerae was also investigated, using molecular methods, cell culture, viable cell count, gentamicin assay, vital staining, light and electron microscopy and statistical analysis.

Analysis of natural water samples collected from different cholera endemic areas in Sudan showed that most V. cholerae bacteria occurred together with Acanthamoeba species in the same samples. The percentage of samples containing V. cholerae only was 11% and that of V. cholerae together with Acanthamoeba was 89%. Moreover, sequencing identified 66 protozoa 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 suppressed survival of wild-type V. cholerae cultivated alone. Co-cultivation with A. castellanii enhanced survival of wild-type and OmpA mutant strains. However, in co-cultivation, OmpA had no effect on attachment, engulfment or intracellular growth of V. cholerae interacting with the amoeba, although the OmpA mutant released more OMVs and inhibited viability of the amoeba more than the wild type. Surprisingly, treatment of amoebal cells with 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. cholerae O11 and V. cholerae O160 V. cholerae were able to grow and survive outside and inside the 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 be concluded 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 survived inside 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 to survive better in association with eukaryotes and Acanthamoeba might be a biological factor enhancing survival of V. cholerae in nature.

List of scientific papers

I. Shanan S, Abd H, Hedenstrom I, Saeed A, Sandstrom G. Detection of Vibrio cholerae and Acanthamoeba species from same natural water samples collected from different cholera endemic areas in Sudan. BMC Res Notes. 2011; 4:109. Epub 2011/04/08
https://doi.org/10.1186/1756-0500-4-109

II. Shanan S, Abd H, Bayoumi M, Saeed A, Sandstrom G. Prevalence of protozoa species in drinking and environmental water sources in Sudan. BioMed research international. 2015;2015:345619. Epub 2015/03/20
https://doi.org/10.1155/2015/345619

III. Valeru SP, Shanan S, Alossimi H, Saeed A, Sandstrom G, Abd H. Lack of outer membrane protein A enhances the release of outer membrane vesicles and survival of Vibrio cholerae and suppresses viability of Acanthamoeba castellanii. International Journal of Microbiology. 2014; 2014:610190. Epub 2014/05/07
https://doi.org/10.1155/2014/610190

IV. Shanan S, Magdi Bayoumi, Amir Saeed, Gunnar Sandström and Hadi Abd. Swedish isolates of Vibrio cholerae enhance their survival when interacted intracellularly with Acanthamoeba castellanii. [Manuscript]