Chromobacterium Csp_P Reduces Malaria and Dengue Infection in Vector Mosquitoes and Has Entomopathogenic and In Vitro Anti-pathogen Activities
Csp_P was grown under planktonic and/or biofilm conditions and tested for anti-pathogen activity independent of the mosquito. Five different preparations of Csp_P were tested: (a) planktonic-state liquid culture, (b) biofilm supernatant, (c) fresh biofilm, (d) dessicated biofilm, and (e) heat-inactivated biofilm. A fresh Comamonas sp biofilm was also tested as control. (A) Csp_P 36-h biofilm has anti-parasite activity against asexual-stage P. falciparum. Csp_P cultures were filtered using a 0.2-µm filter and mixed with ring-stage P. falciparum parasite cultures. SYBR green I was then added to each sample, and inhibition of asexual-stage P. falciparum by Csp_P was measured by assaying fluorescence relative to the negative control (parasite medium, standardized to 0% inhibition). Chloroquine was used as a positive control and standardized to 100% inhibition. We performed a Tukey's test on the raw data to determine whether each bacterial treatment differed significantly from the PBS+LB control (*** p<0.001). (B) Csp_P has anti-parasite activity against ookinete-stage P. falciparum. Csp_P bacterial preparations were filtered using a 0.2-µm filter and mixed with blood taken from female Swiss Webster mice infected with Renilla luciferase-expressing transgenic P. berghei. Ookinete-stage P. berghei parasite counts were determined using the Renilla luciferase assay system, and percent inhibition by Csp_P was calculated relative to the negative control (PBS+LB control, standardized to 0% inhibition). We performed a Tukey's test to determine whether each bacterial treatment differed significantly from the control (*p<0.05, ***, p<0.001). (C) Csp_P 42-h biofilm has anti-parasite activity against gametocyte-stage P. falciparum. Csp_P cultures were filtered using a 0.2-µm filter and mixed with gametocyte-stage P. falciparum cultures. Erythrocytes were examined for gametocytes using Giemsa-stained blood films collected 3 days after Csp_P exposure. The red X indicates that the supernatant caused hemolysis and was therefore unusable. We determined gametocyte density per 1000 RBCs for each sample and performed a Tukey's test to determine whether each bacterial treatment significantly differed from the PBS+LB control (*p<0.05, *** p<0.001). (D) Csp_P has anti-dengue activity. Each Csp_P bacterial preparation (75 µl, unfiltered) was mixed with 75 µl MEM containing dengue virus serotype 2 and incubated at room temperature for 45 min. Samples were then filtered through a 0.2-µm filter and used to infect BHK21-15 cells. Percent inhibition was calculated as the percent decrease in PFU/ml relative to the negative control (PBS+LB, standardized to 0% inhibition). We analyzed the significance of pairwise comparisons between each treatment and the control using a Tukey's test (***, p<0.001). (E) Csp_P has anti-dengue activity when virus is suspended in human blood. Biofilms from multiple bacteria were tested for anti-dengue activity. All bacteria tested were isolated from field-caught Ae. aegypti mosquitoes. Ps.sp = Pseudomonas sp., Pr.sp = Proteus sp., Pn.sp = Paenobacillus sp., Co.sp = Comamonas sp., Pa.sp = Pantoea sp., Ae.sp = Aeromonas sp. . The biofilm from each species was grown for 48 h at room temperature, and dengue virus mixed 1∶1 with human blood was added directly to the biofilm. After a 45-min incubation, the virus+blood/bilofilm solution was filtered and used to infect C6/36 cells. Biofilm sup = biofilm supernatant, H. I. biofilm = heat inactivated biofilm, dess. biofilm = dessicated biofilm re-suspended in 1× PBS.