Evolutionary dynamics of resistance development against a biofilm inhibitor in Salmonella Typhimurium
Evolutionaire dynamica van resistentie-ontwikkeling tegen een biofilm inhibitor in Salmonella Typhimurium
Dubey, Akanksha; R0236252
Salmonella is one of the most important pathogens known to infect humans as well as animals. Every year there are millions of cases of salmonellosis which require hospitalization and emergency medical care. The ubiquitous serotype of Salmonella called S. Typhimurium, generally causes gastrointestinal infections which can be fatal if not treated. It is also known to have the capacity to produce typhoid-like infections in mice and humans or asymptomatic intestinal colonization in chickens. Unfortunately, the incidences of this foodborne illness have shown negligible reductions. Due to the worldwide health burden caused by Salmonella, it is ranked amongst the top five foodborne pathogens adversely affecting the world. A major difficulty in combating this pathogen comes from the fact that it can form robust biofilms, both on biotic as well as abiotic surfaces. Biofilms are multicellular assemblages of bacteria enclosed in self-produced matrix which helps them survive against a range of antimicrobial treatments. Since the identification of this biofilm mode of growth in bacteria, scientists have shifted their attention to combat biofilms in order to foil bacterial infections. Aminoimidazoles are a class of compounds which have been shown to prevent biofilm formation of a broad spectrum of microbes. Our research group, S&P, has used different methods to analyse the mode of action of these compounds. In this thesis, we followed the evolutionary dynamics of resistance development against aminoimidazoles using the approach of experimental evolution to unravel the mechanisms of resistance and mode of action of the biofilm inhibitor.In summary, we first optimized a set-up for artificial evolution of Salmonella biofilms under high concentrations of a biofilm inhibitor. After 108 days of parallel evolution in this set-up, 3 independent pools of mutants with high resistance were obtained. The end-pools were heterogeneous in nature and constituted of various morphotypes. At first instance one of the resistant clones- BWSM from the endpoint pool was characterized at the genomic level via whole genome sequencing. Clonal sequencing of BWSM revealed a putative role of a set of six candidate causal mutations in conferring resistance to the end point clone. To investigate the role of each mutation independently, knock-out mutants and overexpression strains were constructed and tested against the biofilm inhibitor. Resistance testing of the mutant derivatives revealed the importance of up-regulation of a specific efflux system in imparting resistance. The clone BWSM was further analysed at the transcriptomic and phenotypic level to analyse the effect of mutations on expression of biofilm related genes. Next, pooled sequencing was performed of the 3 endpoint mutant pools and stored pools originating from earlier time points of the 3 parallel evolution experiments. Analysis of the evolutionary pools revealed that an identical mutation (deletion of 15 nucleotides) occurred in a transcriptional regulator STM14_0676 (ramR) of the efflux system and seems tohave been fixed in the populations of all three replicate evolution lines. Additionally, metagenomic sequencing of the evolved populations was helpful in determining the evolutionary dynamics and population genetics in the course of evolution. A haplotype reconstruction protocol was developed and applied to all sequencing results. Analysis of the pool-sequencing data indicated up-regulation of the efflux system to be a main resistance mechanism for all mutants in the three different endpoint pools. However, since the mutations affecting the efflux pump could not be found in pools at earlier time points, which also already had substantial resistance, other resistance mechanisms should play a role earlier in evolution. In summary, this PhD research was one of the first attempts of experimental evolution on biofilms to study the mode of action of a biofilm inhibitor. This study offered insights into the mechanisms of resistance development against biofilm inhibitors which could be of relevance in medical and industrial establishments. However, additional aspects of the evolution experiment still need to be analysed to gain a complete understanding of the molecular mechanisms and dynamics involved in biofilm resistance.