Antibiotic resistance is one of the most serious global threats for public health. When we are thinking of antibiotic resistance, we often link it to the intensive application of antibiotics for medical, veterinary or agricultural purposes. Although non-antibiotic pharmaceuticals occupy 95% of the global pharmaceutical market 1,2, little is known about their contribution to the spread of antibiotic resistance. We are wondering if non-antibiotic pharmaceuticals are playing a role, invisibly.
To address this question, we started from horizontal gene transfer, which is the principal process for the spread of antibiotic resistance. Horizontal gene transfer includes three pathways, conjugation, transformation, and transduction. Among them, transformation is the direct uptake and incorporation of exogenous genetic elements (i.e., cell-free DNA) from the surroundings of the bacterium. In this study, we established a bacterial transformation model by applying naturally competent wild-type strain Acinetobacter baylyi ADP1 and cell-free pWH1266 plasmid which encodes resistance to ampicillin (blaTEM-1) and tetracycline (tetA). If A. baylyi strains swallow this plasmid via transformation, then they will become antibiotic resistant against ampicillin and tetracycline. We selected six commonly consumed non-antibiotic pharmaceuticals, including ibuprofen, naproxen, diclofenac (three nonsteroidal anti-inflammatories), gemfibrozil (a lipid-lowering drug), propranolol (a β-blocker), and iopromide (a contrast media).
Surprisingly, we found five non-antibiotic pharmaceuticals (except iopromide) promoted the transformation significantly. That means under exposure of these drugs, more A. baylyi have become antibiotic resistant bacteria after uptaking the plasmid. These results inspired us to explore the underlying mechanisms. We then measured reactive oxygen species (ROS) generation and cell membrane permeability by a flow cytometry, and conducted whole-genome RNA sequencing and proteomic analysis. We revealed that the enhanced transformation was affiliated with promoted bacterial competence, enhanced stress levels, over-produced ROS and increased cell membrane permeability. In addition, we proposed a mathematical model to simulate the dynamics of transformation during the long-term exposure to non-antibiotic pharmaceuticals. Mathematically speaking, these non-antibiotic pharmaceuticals indeed accelerate the transformation process in the long-term.
Given the high consumption of non-antibiotic pharmaceuticals, our findings highlight a new concern that non-antibiotic pharmaceuticals can speed up the dissemination of antibiotic resistance. They are invisible or neglected factors for antibiotic resistance, like all icebergs lying underwater.