Persistence Depends on Events Before, During, and After Antibiotic Treatment

Abstract

Bacterial persisters are phenotypic variants that constitute sub-populations of bacterial cultures that exhibit increased tolerance to antibiotics when compared to their surrounding kin. Persisters are implicated in the tolerances of biofilm infections, thought to underlie recurring, chronic infections, and facilitate the development of antibiotic resistant mutants. Despite their clinical relevance, knowledge of their physiology has been difficult to gain due largely to difficulties in isolating them from other cell types. To overcome this technical challenge, model systems have been used for persister studies and one type is based on the accumulation of toxins from toxin-antitoxin (TA) modules. TA modules are widespread in prokaryotes and it has been well documented that the accumulation of toxins can increase the tolerances of bacteria to antibiotics. This tolerance-enabling capacity of toxins allowed us to construct model persister systems where the expression of different toxins and antitoxins could be modulated and used to understand extreme antibiotic tolerance and relate that understanding to wild-type persister physiology. Specifically, we used the LdrD and MazF toxins to generate model persisters and examine their capacity to engender tolerance when expressed before, during, and after antibiotic treatment had concluded. In our work, we discovered that controlling LdrD or MazF accumulation to occur only after fluoroquinolone (FQ) treatment in stationary-phase cultures resulted in significant increases in survival that were comparable to those achieved when toxin expression was initiated before or during treatment. Further, we found that in wild-type E. coli, inhibition of transcription and translation after antibiotic FQ exposure produced significant increases in persistence. Further genetic investigation revealed important roles for homologous recombination and nucleotide excision repair machinery in the post-FQ rescue we had observed with model and wild-type persisters. Directing attention toward the nutritional environment of cultures before and during antibiotic treatment revealed that the type of starvation and FQ modulate the level of persistence, although DNA repair machinery was found to be important for all conditions tested. Collectively, this work highlights the importance of bacterial physiology before, during, and after antibiotic treatment on persister survival and underlines the integral role DNA repair enzymes in FQ persistence.