Figure 3: Evaluation of the engineered probiotic strain in a C. elegans infection model.

(a) Fluorescence images of C. elegans show the localization of (i) wild-type Nissle expressing mRFP1; (ii) P. aeruginosa (PA) expressing GFP. m=mouth. The scale bars represent 100 μm. (b) C. elegans were given the following for 24 h and viewed by fluorescence microscopy: (i) P. aeruginosa (constitutively expressing GFP) alone; (ii) EcN expressing RFP in the presence of AHL (PA sensor device with RFP); (iii) P. aeruginosa followed by EcN with PA Sensor (for 4 h) before viewing. The scale bars represent 100 μm. (c) At 24 h post-infection, the nematodes were divided into the following treatment groups; untreated (PA); treated with engineered EcN SED and its control that expresses either E7 alone, S5 & E7 only (SE), DspB & E7 (dspB) or Sensor mutant (disrupted lasR with S5, E7 and dspB gene intact). Representative images of nematodes treated with all the variants of EcN are shown to demonstrate the clearance of P. aeruginosa (GFP). The scale bars represent 100 μm. (d) The survival rate of each treatment group was quantified until all the nematodes in the infection group had died (96 h). The significance of the results was determined by the Mantel–Haenszel log-rank test followed by Bonferroni’s correction. Statistically significant differences in survival among the groups were not observed. The data from three independent experiments are shown (n=80–100). *P<0.001.