Comparative genomics and antimicrobial activity of recombinant phage endolysins against multi-drug resistant bacteria

The treatment of infections caused by multidrug-resistant Gram-negative bacteria is a challenge. Bacteriophage-derived lytic enzymes have been studied as a potential solution for the treatment of these infections. Several studies have evaluated the use of phage endolysins against Gram-positive bacteria. In contrast, the use of endolysins against Gram-negative bacteria has been little explored. The exogenous action of endolysins against Gram-negative bacteria is hampered by the outer membrane. Studies evaluating the exogenous action of endolysins against Gram-negative bacteria are limited to the: (I) use of native endolysins; (II) use of native endolysins combined with outer membrane permeabilizers; and (III) use of engineered endolysins. Recent research has shown that endolysins with signal-anchor-release (SAR)-like domains can permeate the outer membrane of Gram-negative bacteria. The objective of this dissertation was to evaluate the arsenal of endolysins encoded by bacteriophages that infect Gram-negative bacteria, focusing on endolysins containing SAR signal sequences, and to evaluate the antimicrobial activity of SAR-endolysins against Gram-negative bacteria. This dissertation is divided into four chapters. The first chapter summarized the state of the art on the treatment of infections caused by Gram-negative bacteria with endolysins. Chapter two analyzed the arsenal of endolysins encoded by bacteriophages that infect Gram-negative bacteria of clinical interest. The third chapter analyzed the arsenal of SAR-endolysins from metagenomic data. Finally, the fourth chapter evaluated the antimicrobial activity of a representative SAR-endolysin against Gram-negative bacteria of clinical interest, including multidrug-resistant isolates. SAR-endolysin LysKpV475 showed bacteriostatic activity ranging from 8.125 µg.ml-1 for P. aeruginosa ATCC 27853 and 32.50 µg.ml-1 for K. pneumoniae ATCC BAA-2146 and E. cloacae P2224. Bactericidal activity was observed for P. aeruginosa ATCC 27853 (32.50 µg.ml-1) and P. aeruginosa P2307 (65.00 µg.ml-1). However, further studies are needed to determine the role of the SAR domain and the polyhistidine tag in the antimicrobial activity of SAR-endolysins. This study showed the potential of SAR-endolysins against Gram-negative bacteria (AU)