Background Malaria is one of the most lethal infectious diseases in tropical and subtropical areas of the world. Paratransgenesis using symbiotic bacteria offers a sustainable and environmentally friendly strategy to combat this disease. In the study reported here, we evaluated the disruption of malaria transmission in the Anopheles stephensi-Plasmodium berghei assemblage using the wild-type (WT) and three modified strains of the insect gut bacterium, Enterobacter cloacae. Methods The assay was carried out using the E. cloacae dissolvens WT and three engineered strains (expressing green fluorescent protein-defensin (GFP-D), scorpine-HasA (S-HasA) and HasA only, respectively). Cotton wool soaked in a solution of 5% (wt/vol) fructose + red dye (1/50 ml) laced with one of the bacterial strains (1 × 109cells/ml) was placed overnight in cages containing female An. stephensi mosquitoes (age: 3–5 days). Each group of sugar-fed mosquitoes was then starved for 4–6 h, following which time they were allowed to blood-feed on P. berghei–infected mice for 20 min in the dark at 17–20 °C. The blood-fed mosquitoes were kept at 19 ± 1 °C and 80 ± 5% relative humidity, and parasite infection was measured by midgut dissection and oocyst counting 10 days post-infection (dpi). Results Exposure to both WT and genetically modified E. cloacae dissolvens strains significantly (P < 0.0001) disrupted P. berghei development in the midgut of An. stephensi, in comparison with the control group. The mean parasite inhibition of E. cloacaeWT, E. cloacaeHasA, E. cloacaeS−HasA and E. cloacaeGFP−D was measured as 72, 86, 92.5 and 92.8 respectively. Conclusions The WT and modified strains of E. cloacae have the potential to abolish oocyst development by providing a physical barrier or through the excretion of intrinsic effector molecules. These findings reinforce the case for the use of either WT or genetically modified strains of E. cloacae bacteria as a powerful tool to combat malaria. Graphical Abstract

Background: Malaria is one of the most lethal infectious diseases in tropical and subtropical areas of the world. To fight the disease, paratransgenesis using symbiotic bacteria offers a sustainable and environmentally-friendly strategy. Here we evaluated the disruption of malaria transmission in the Anopheles stephensi-Plasmodium berghei assemblage, using wild and modified insect gut bacterium, Enterobacter cloacae.Methods: The assay was carried out using E. cloacae dissolvens wild-type (WT) and its three engineered strains expressing GFP-defensin (GFP-D), scorpine-HasA (S-HasA), and HasA, The 3-5 day-old female mosquitoes were supplemented overnight with the studied bacteria [1×109cells/mL of 5% (wt/vol), fructose and red dye (1/50 ml)] soaked on cotton-wool. Each group of sugar-fed mosquitoes was then starved for 4-6 hours and fed on a P. berghei–infected mouse for 20 min in the dark at 17-20°C. The blood-fed mosquitoes were kept at 19±1°C and RH 80±5, and parasite infection was measured by midgut dissection and oocyst counting 10 days post-infection (dpi). Results: Both wild-type and genetically modified bacterial strains significantly (P< 0.0001) disrupted the P. berghei development in the An. stephensi midgut, in comparison with the control group. The mean parasite inhibition of E. cloacaeWT, E. cloacaeHasA, E. cloacaeS-HasA, and E. cloacaeGFP-D was measured as 72, 86, 92.5 and 92.8 respectively. Conclusions: The wild and modified E. cloacae might abolish oocyst development by providing a physical barrier or by excretion of intrinsic effector molecules. These findings reinforce the case for the use of either wild or genetically modified E. cloacae bacteria as a powerful tool to combat malaria.