Fast bacterial growth reduces antibiotic accumulation and efficacy

Abstract

Phenotypic variations between individual microbial cells play a key role in the resistance of microbial pathogens to pharmacotherapies. Nevertheless, little is known about cell individuality in antibiotic accumulation. Here, we hypothesise that phenotypic diversification can be driven by fundamental cell-to-cell differences in drug transport rates. To test this hypothesis, we employed microfluidics-based single-cell microscopy, libraries of fluorescent antibiotic probes and mathematical modelling. This approach allowed us to rapidly identify phenotypic variants that avoid antibiotic accumulation within populations of Escherichia coli, Pseudomonas aeruginosa, Burkholderia cenocepacia, and Staphylococcus aureus. Crucially, we found that fast growing phenotypic variants avoid macrolide accumulation and survive treatment without genetic mutations. These findings are in contrast with the current consensus that cellular dormancy and slow metabolism underlie bacterial survival to antibiotics. Our results also show that fast growing variants display significantly higher expression of ribosomal promoters before drug treatment compared to slow growing variants. Drug-free active ribosomes facilitate essential cellular processes in these fast-growing variants, including efflux that can reduce macrolide accumulation. We used this new knowledge to eradicate variants that displayed low antibiotic accumulation through the chemical manipulation of their outer membrane inspiring new avenues to overcome current antibiotic treatment failures.

Microfluidics for antibiotic susceptibility testing

“…One of the most prevalent microfluidic single-phase designs for microbiology is the mother machine, 102 which has been used on numerous occasions to study bacteria in an antibiotic context or close to it: e.g., mother machine was used for the analysis of mutation frequency in E. coli, 103 for studying viable but non-culturable bacteria during antibiotic treatment, 104 for describing the accumulation of polar multidrug efflux pumps that grant antibiotic resistance in older cells and less so in daughter cells, 105 for studying bacterial persistance, 106,107 uptake and efflux of antibiotics by bacteria 108 with fluorescently labelled antibiotics, 109 and measuring the efficacy of bacteria clearance with phages. 110,111 The basic mother machine consists of a large main channel through which growth medium flows and of multiple shallow and narrow dead-end channels that are perpendicular to the main channel, forming a comb-like structure.…”

Environmental feedback drives oxidative stress response heterogeneity in bacterial populations

“…This indicates that while cells can reliably sense the onset of H2O2 stress and respond rapidly, the response magnitude depends on an unidentified factor that varies substantially between cells. This heterogeneity could be caused by a variety of mechanisms, including molecular stochasticity in the specific regulatory circuits or general gene expression machinery (Golding et al, 2005;Uphoff et al, 2016), cell-to-cell variation in growth or morphology (Łapińska et al, 2022;Ojkic et al, 2022;Sampaio et al, 2022) , variable cellto-cell interactions (Dal Co et al, 2020;Snoussi et al, 2018;van Gestel et al, 2021), or differences in the local environment of cells (Snoussi et al, 2018;van Vliet et al, 2018). To pinpoint the mechanisms, we designed a machine learning model using random forest regression [Figure 2A].…”

“…Variation in cell growth and morphology can also affect the influx, dilution, and reaction-diffusion dynamics of stressor molecules such as ROS (Łapińska et al, 2022; Ojkic et al, 2022; Sampaio et al, 2022). Furthermore, cells in a population can interact and modulate their environment in response to stress (Dal Co et al, 2020; Snoussi et al, 2018; van Gestel et al, 2021).…”

A fluorescently labelled quaternary ammonium compound (NBD-DDA) to study mode-of-action and resistance mechanisms in bacteria

“…The potential of fluorescent probes derived from antimicrobial substances to study drug susceptibility in single bacterial cells has recently been demonstrated for a range of antibiotics 22,23 . Here we demonstrate that this approach is also promising for BACs and related QACs, which are used as disinfectants, antiseptics, and preservatives 1 .…”