Mitigation of Bacterial Virulence Using Compounds Derived from the Human Microbiome

Abstract

Although antibiotics play an important role in eliminating pathogens by targeting their central growth pathways, these pathways are shared among diverse bacteria and therefore antibiotic resistance (AMR) has emerged and increased rapidly. AMR necessitates research into new control strategies that may exert less evolutionary pressure on bacteria to develop resistance. Anti-virulence agents represent a potential strategy to target microbial pathogenicity without inhibiting microbial growth. The general aim of this doctoral project is to screen bacteria isolated from the human commensal and associated bacteria for the production of compounds that could potentially mitigate the virulence and biofilm formation of selected pathogenic bacteria. Screening bacterial lysates or supernatants instead of live cells is a route by which the bioprospecting process can be streamlined and where cell-free agents have useful therapeutic potential, the route to human use is considerably more direct than for live biotherapeutics. Initially, cell-free extracts (CFEs) from the human commensal and associated bacteria (25 species) were tested for antibacterial and antibiofilm activity against selected wound-associated pathogens (S. aureus, P. aeruginosa, S. epidermidis, E. coli and MRSA). Inhibitory activities on growth dynamics and biofilm formation were determined using a microtiter plate-based method. Planktonic growth dynamics and biofilm formation of the pathogens were not significantly affected by CFEs of the isolates except for cell-free supernatants (SFS) derived from E. coli Nissle 1917. These significantly inhibited biofilm formation and dispersed extant pseudomonas biofilms without inhibiting planktonic bacterial growth. No inhibitory effects against P. aeruginosa were observed for other tested E. coli strains. eDNA was reduced in biofilms following exposure to Nissle CFS, as visualized by confocal microscopy. Nissle CFS exhibiting biofilm inhibitory activity against P. aeruginosa were exposed to enzymatic and heat treatments or size fractionation prior to screening for anti-biofilm activity to determine the nature of the active compound(s) involved. Moreover, P. aeruginosa biofilms were analysed by proteome analysis after treatment with Nissle CFS to gain an understanding of the potential mechanisms involved in the anti-biofilm effect. Data indicate that Nissle CFS downregulated the expression of several P. aeruginosa proteins involved in motility (Flagellar secretion chaperone FliSB, B-type flagellin fliC, Type IV pilus assembly ATPase PilB), and quorum sensing (acyl-homoserine lactone synthase lasI and HTH-type quorum-sensing regulator rhlR), which are associated with biofilm formation. Physicochemical characterization of the putative antibiofilm compound(s) indicates the involvement of heat-labile proteinaceous factors of greater than 30 kDa molecular size. Nissle CFS were further assessed for their potential protective activity against P. aeruginosa virulence using an invertebrate infection model utilising Galleria melonella, and the effects on swimming, swarming, and twitching motility were also assessed. The data indicate that pretreatment with Nissle CFS significantly protected the larvae from the lethal effect of P. aeruginosa however simultaneous injection of Nissle CFS did not show any protective activities against P. aeruginosa. Moreover, swarming motility was significantly reduced in cultures to which Nissle CFS were added. In conclusion, the work presented in this doctoral thesis demonstrates some promising effects of E. coli Nissle 1917 cell-free supernatants against biofilm and other virulence factors of P. aeruginosa.