Bacteria can cause an array of diseases ranging from mildly inconvenient to deadly. In fact, every year around the world, five million people succumb to a bacterial infection. Antibiotics can kill bacteria or stop their growth, but many bacterial species are now able to evade these drugs.
To be efficient, most antibiotics first need to get inside a bacterium; there, they accumulate until they reach the concentration they need to act. Often, the drugs make their way through channel-like structures (‘pores’) studded through the external membranes of bacteria and which control the passage of molecules in and out of cells.
Resistance usually emerges when genetic changes provide the microorganism with an advantage against antibiotics, or when the microorganism performs the biochemical reactions necessary for life at a slower pace.
In contrast, Łapińska, Pagliara et al. decided to examine how genetically similar Escherichia coli bacteria which differed in their growth rate would fare against antibiotics. The drug targeted ribosomes, the machinery that produces proteins in a cell. A combination of techniques was used to follow individual cells, revealing that fast-growing variants better managed to survive. A closer look showed that bacteria which were growing quickly had a surplus of ribosomes, which then produced more pores that could pump the antibiotic out the cell. Next, Łapińska, Pagliara et al. exposed the bacteria to both the antibiotic and a compound that weakens bacterial membrane; this erased the advantage shown by the fast-growing variants. Overall, this work gives a finer understanding of the mechanisms that underlie antibiotic resistance, which could help pave the way to new strategies to combat harmful bacteria.
Experimental assessment of single-cell real-time drug accumulation dynamics
We combined our recently developed single-cell microfluidics-microscopy platform with a library of fluorescent derivatives representing most major classes of antibiotics, including macrolides (roxithromycin) (, oxazolidinones (linezolid) , glycopeptides (vancomycin) , fluoroquinolones (ciprofloxacin) , antifolates (trimethoprim) , and membrane-targeting lipopeptides/peptides (polymyxin B, octapeptin, and tachyplesin)
Each antibiotic was functionalised at a site that minimises any changes in biological activity, adding a substituent that allows for facile coupling with a small fluorophore, nitrobenzoxadiazole as previously reported . Using minimum inhibitory concentration (MIC) assays we found that the fluorescent derivatives of polymyxin B, octapeptin, tachyplesin, vancomycin, and linezolid maintained the antibiotic activity of the parent drug against E. coli, whereas the fluorescent derivatives of roxithromycin, trimethoprim, and ciprofloxacin displayed a 3-fold, 64-fold, and 256-fold increase compared to the parent drug, respectively . Next we used each probe in our microfluidics-microscopy platform to quantify the dynamics of the accumulation of each antibiotic in individual bacteria in real-time . Briefly, we loaded an aliquot of a stationary phase clonal bacterial culture in a microfluidic device equipped with small parallel channels, each hosting between one and six bacteria . Then we continuously flowed lysogeny broth (LB) medium into xthe device for 2 hr to stimulate cell growth and reproduction. During this period bacteria displayed an average elongation rate of (5.3±1.2) µm hr–1. We also performed separate experiments flowing LB for a longer period of time and found that the elongation rate (averaged across the bacterial population) did not further increase after the first 2 hr exposure to LB, corroborating previous bulk data showing that E. coli is in exponential phase between 2 and 5 hr after passage in fresh medium. Therefore after 2 hr incubation in LB, we injected one of the antibiotic probes and imaged the real-time intracellular probe accumulation in hundreds of individual live bacteria . Typically, upon onset drug accumulation increased until reaching steady-state saturation levels due to probe efflux, compound transformation , or target saturation , although several bacteria displayed divergent accumulation dynamics
You must be logged in to post a comment.