Ciprofloxacin pre-exposure influences individual cell MIC and heteroresistance of bacteria inside microfluidic droplets

Bacterial cells in general present different phenotypes even within the monoclonal populations. Some phenotypes may offer selective advantages under antibiotic stress. In clinical practice such advantage may lead to selection of resistant phenotypes and to treatment failure. Improving treatment regimens thus requires thorough understanding of the underlying cause of developing phenotypic variations within bacterial populations exposed to antibiotics. Here, we use droplet microfluidics to study the response of individual bacterial cells. We investigate how a short-term exposure of ciprofloxacin or streptomycin at the sub-minimum inhibitory concentration levels changes the single-cell distribution of susceptibility on subsequent exposure to either of the antibiotics. We used three different concentrations of pre-exposure for each antibiotic on Escherichia coli MG1655 and followed with re-exposure of the same bacterial cells individually in microfluidic droplets. Experiments with various concentrations of re-exposure on single cells showed that pre-exposures to sub-lethal levels of ciprofloxacin can elevate the individual cell MIC and the level of heteroresistance to ciprofloxacin. Exposure to quarter and half MIC of ciprofloxacin in separate experiments increased the heteroresistance of pre-exposed cells by more than 20-fold on subsequent treatment with ciprofloxacin. Interestingly, the same ciprofloxacin pre-exposure pattern made the cells more fragile and, in some cases, decreased the individual MIC values to streptomycin. The results in our study can be very important in developing new better treatment regimen, addressing the wider problem of antibiotic resistance.