Low Ciprofloxacin Concentrations Select Multidrug-Resistant Mutants Overproducing Efflux Pumps in Clinical Isolates of Pseudomonas aeruginosa

Low antibiotic concentrations present in natural environments are a severe and often neglected threat to public health. Even if they are present below their MICs, they may select for antibiotic-resistant pathogens. Notably, the minimal subinhibitory concentrations that select resistant bacteria, and define the respective sub-MIC selective windows, differ between antibiotics. The establishment of these selective concentrations is needed for risk-assessment studies regarding the presence of antibiotics in different habitats. Using short-term evolution experiments in a set of 12 Pseudomonas aeruginosa clinical isolates (including high-risk clones with ubiquitous distribution), we have determined that ciprofloxacin sub-MIC selective windows are strain specific and resistome dependent. Nonetheless, in all cases, clinically relevant multidrug-resistant (MDR) mutants emerged upon exposure to low ciprofloxacin concentrations, with these concentrations being below the levels reported in ciprofloxacin-polluted natural habitats where P. aeruginosa can be present. This feature expands the conditions and habitats where clinically relevant quinolone-resistant mutants can emerge. In addition, we established the lowest concentration threshold beyond which P. aeruginosa, regardless of the strain, becomes resistant to ciprofloxacin. Three days of exposure under this sub-MIC "risk concentration" led to the selection of MDR mutants that displayed resistance mechanisms usually ascribed to high selective pressures, i.e., the overproduction of the efflux pumps MexCD-OprJ and MexEF-OprN. From a One-Health viewpoint, these data stress the transcendent role of low drug concentrations, which can be encountered in natural ecosystems, in aggravating the antibiotic resistance problem, especially when it comes to pathogens of environmental origin. IMPORTANCE It has been established that antibiotic concentrations below MICs can select antibiotic-resistant pathogens, a feature of relevance for analyzing the role of nonclinical ecosystems in antibiotic resistance evolution. The range of concentrations where this selection occurs defines the sub-MIC selective window, whose width depends on the antibiotic. Herein, we have determined the ciprofloxacin sub-MIC selective windows of a set of Pseudomonas aeruginosa clinical isolates (including high-risk clones with worldwide distribution) and established the lowest concentration threshold, notably an amount reported to be present in natural ecosystems, beyond which this pathogen acquires resistance. Importantly, our results show that this ciprofloxacin sub-MIC selects for multidrug-resistant mutants overproducing clinically relevant efflux pumps. From a One-Health angle, this information supports that low antimicrobial concentrations, present in natural environments, may have a relevant role in worsening the antibiotic resistance crisis, particularly regarding pathogens with environmental niches, such as P. aeruginosa. It has been established that antibiotic concentrations below MICs can select antibiotic-resistant pathogens, a feature of relevance for analyzing the role of nonclinical ecosystems in antibiotic resistance evolution. The range of concentrations where this selection occurs defines the sub-MIC selective window, whose width depends on the antibiotic.