Fluorescence photography of patterns and waves of bacterial adaptation at high antibiotic doses

Abstract

Fisher suggested advantageous genes would spread through populations as a wave so we sought genetic waves in evolving populations, as follows. By fusing a fluorescent marker to a drug efflux protein (AcrB) whose expression provides Escherichia coli with resistance to some antibiotics, we quantified the evolution and spread of drug-resistant E. coli through spacetime using image analysis and quantitative PCR. As is done in hospitals routinely, we exposed the bacterium to a gradient of antibiotic in a 'disk diffusion' drug susceptibility test that we videoed. The videos show complex spatio-genomic patterns redolent of, yet more complex than, Fisher's predictions whereby a decelerating wave front of advantageous genes colonises towards the antibiotic source, forming bullseye patterns en route and leaving a wave back of bacterial sub-populations expressing AcrB at decreasing levels away from the drug source. qPCR data show that E. coli sited at rapidly-adapting spatial hotspots gain additional copies of acr, the operon that encodes AcrB, within h and imaging data show resistant sub-populations thrive most near the antibiotic source due to non-monotone relationships between inhibition due to antibiotic and distance from the source. In the spirit of Fisher, we provide an explicitly spatial nonlinear diffusion equation that exhibits these properties too. Finally, linear diffusion theory quantifies how the spatial extent of bacterial killing scales with increases in antibiotic dosage, predicting that microbes can survive chemotherapies that have been escalated to × the clinical dosage if the antibiotic is diffusion-limited.

Antibiotic heteroresistance generated by multi-copy plasmids

“…13 Heterogeneous drug susceptibility within a population can also arise from the stochastic expression of genes encoding intrinsic antibiotic-resistance mechanisms, notably efflux pumps. 14,15 Rapid adaptation to antibiotics can also be achieved through genomic duplications that increase the dosage of known drug-resistance genes, 8,16,17 for instance through amplification of efflux pump operons 18,19 or genes encoding drug-modifying enzymes. 20,21 Laboratory studies have shown that genomic amplifications scale up with the strength of the selective pressure, 16 and are unstable in the absence of selection due to the fitness burden associated with the duplication of large chromosome regions.…”

Inexpensive Apparatus for High-Quality Imaging of Microbial Growth on Agar Plates

“…Lab scanners are widely used for high throughput colony counting and growth analysis ( Hartman and Tippery, 2004 ; Michel et al, 2008 ; Clarke et al, 2010 ; Levin-Reisman et al, 2014 ), although the mode of image capture through the bottom of the agar plate limits the resolution of morphological features. Other apparatuses have been custom-made for automated colony counting ( Clarke et al, 2010 ; Brugger et al, 2012 ), imaging colonies in soft agar ( Parkinson, 2007 ), or for imaging plates over time ( Parkinson, 2007 ; Cobo et al, 2018 ; Reding et al, 2019 ). The specialization of these apparatuses increases the cost of construction as they include illuminators that provide transmitted light through the bottom of the plate ( Parkinson, 2007 ; Clarke et al, 2010 ; Brugger et al, 2012 ), climate control equipment ( Cobo et al, 2018 ; Reding et al, 2019 ), or fluorescence imaging capability ( Reding et al, 2019 ).…”

“…Other apparatuses have been custom-made for automated colony counting ( Clarke et al, 2010 ; Brugger et al, 2012 ), imaging colonies in soft agar ( Parkinson, 2007 ), or for imaging plates over time ( Parkinson, 2007 ; Cobo et al, 2018 ; Reding et al, 2019 ). The specialization of these apparatuses increases the cost of construction as they include illuminators that provide transmitted light through the bottom of the plate ( Parkinson, 2007 ; Clarke et al, 2010 ; Brugger et al, 2012 ), climate control equipment ( Cobo et al, 2018 ; Reding et al, 2019 ), or fluorescence imaging capability ( Reding et al, 2019 ).…”