Experimental evolution in morbidostat reveals converging genomic trajectories on the path to triclosan resistance

Abstract

Understanding the dynamics and mechanisms of acquired drug resistance across major classes of antibiotics and bacterial pathogens is of critical importance for the optimization of current anti-infective therapies and the development of novel ones. To systematically address this challenge, we developed a workflow combining experimental evolution in a morbidostat continuous culturing device with deep genomic sequencing of population samples collected in time series. This approach was applied to the experimental evolution of six populations of Escherichia coli BW25113 towards acquiring resistance to triclosan (TCS), an antibacterial agent in various consumer products. This study revealed the rapid emergence and expansion (up to 100% in each culture within 4 days) of missense mutations in the fabI gene, encoding enoyl-acyl carrier protein reductase, the known TCS molecular target. A follow-up analysis of isolated clones showed that distinct amino acid substitutions increased the drug IC90 in a 3–16-fold range, reflecting their proximity to the TCS-binding site. In contrast to other antibiotics, efflux-upregulating mutations occurred only rarely and with low abundance. Mutations in several other genes were detected at an earlier stage of evolution. Most notably, three distinct amino acid substitutions were mapped in the C-terminal periplasmic domain of CadC protein, an acid stress-responsive transcriptional regulator. While these mutations do not confer robust TCS resistance, they appear to play a certain, yet unknown, role in adaptation to relatively low drug pressure. Overall, the observed evolutionary trajectories suggest that the FabI enzyme is the sole target of TCS (at least up to the ~50 µm level), and amino acid substitutions in the TCS-binding site represent the main mechanism of robust TCS resistance in E. coli . This model study illustrates the potential utility of the established morbidostat-based approach for uncovering resistance mechanisms and target identification for novel drug candidates with yet unknown mechanisms of action.

A low-cost, open-source evolutionary bioreactor and its educational use

“…The morbidostat therefore adjusts the selective pressure to maintain near-constant growth inhibition as the population evolves resistance. Although this device has advanced our understanding of the repeatability of resistance evolution in bacteria ( Toprak et al, 2012 ; Dößelmann et al, 2017 ; Yoshida et al, 2017 ; Leyn et al, 2021) and temperature stress response in yeast ( Wong et al, 2018 ), the lack of open-source software and high commercial cost may limit its widespread use, particularly in educational settings.…”

A broad-spectrum synthetic antibiotic that does not evoke bacterial resistance

“…The morbidostat measures the growth rates of evolving microbial populations and automatically adjusts drug concentrations to maintain a constant drug-induced inhibition. 34 , 35 The hardware, software components and experimental methods and work-flow were as described. 34 Briefly, bacterial populations were placed under increasing selective drug pressure in parallel reactors.…”

Grand challenge of antibiotics resistance: A global, multidisciplinary effort is needed

“…Repeated exposure of microbes to chemicals other than antibiotics, such as triclosan (Leyn et al, 2021), a biocide commonly used in household and personal care cleaning agents to prevent microbial growth and an emerging pollutant, can increase the selection pressure for antibiotic resistance and cross-resistance. The triclosan can select and enrich mutations in genes that encode bacterial efflux pumps and fatty acid biosynthesis, which can expel antibiotics and confer broadspectrum tolerance to antibiotics.…”