Rational Drug Design and Inhibitory Potential of Class II Bacteriocins: Novel Strategies for Mitigating Antibiotic Resistance

Context: Since their commercial usage in the early 20th century, antibiotics have been a transformative force in the management of infectious diseases, substantially lowering mortality rates globally. However, misuse and a concomitant decrease in the production of novel antibiotics have led to an escalating crisis of antimicrobial resistance (AMR). Given this looming crisis, the development of innovative antimicrobial strategies is a necessity for the preservation of global health. 

Objective: This thesis leverages a dual approach to combat the rising tide of AMR: first, by examining the evolutionary trajectory and collateral sensitivity patterns of the widely distributed antibiotic resistance gene: blaCTX-M-15 gene to inform the creation of effective drug combinations; second, by comprehensively delineating the role of class II bacteriocins within the context of gut microbiota, with a view towards unearthing their potential as novel antimicrobial agents and gut modulators.

Methodology: Escherichia coli was employed as a vector to express mutational variants of the blaCTX-M-15 gene. These variants were subsequently subjected to drug testing, followed by amplicon sequencing of selected clones. Further, 75 distinct class II bacteriocins were heterologously expressed in E. coli and tested against a diverse collection of 49 representative human gut microorganisms, which included 21 pathogenic species. Selected bacteriocins were additionally evaluated in a complex gut microbiome environment in an ex vivo study using murine feces. Finally, the possibility to utilize bacteriocin delivery in a heterologous host to modulate the intestinal microbiota was assessed in vivo.

Findings: Our studies discerned that co-administration of the antibiotics mecillinam and cefotaxime, currently utilized to treat urinary tract infections, could inhibit the resistance development of the blaCTX-M-15 gene, a finding corroborated in a mouse model. Moreover,  heterologously expressed bacteriocins displayed selective inhibitory activity against clinically significant species such as Enterococcus faecalis, Bacteroides vulgatus, Eubacterium rectale, and Collinsella aerofaciens, in vitro. Furthermore, the bacteriocins actifencin and Bacteroidetocin A demonstrated the capability to suppress the growth of the bacterial genera Lactobacillus and Bacteroides respectively, in an ex vivo study. These significant
findings were not reproducible in vivo. 

Conclusion: The results delineated in this dissertation validate the efficacy of rational drug design in mitigating the evolution of antimicrobial resistance. They further underscore the promising potential of bacteriocins as innovative antimicrobial agents with the capacity for targeted modulation of the gut microbiota.