A new strategy to combat biofilm antibiotic tolerance by targeting bacterial dormancy

Introduction: The use of medical devices has become an essential part of today’s healthcare system to diagnose, prevent, treat, and rehabilitate illnesses. Oftentimes, the usage of these devices such as catheters, ventilators, dentures, or prostheses lead to bacterial infections that respond poorly to antibiotic treatments. One major mechanism that contributes to the failure of current antibiotic treatment of biofilm infections is the formation of persister cells, a dormant subpopulation that are tolerant to conventional antibiotics. During antibiotic treatment, persisters remain growth arrested and transiently repopulate once the antibiotic is withdrawn, causing the failure of treatment and relapse of the infection. Despite extensive research, controlling persister cells remains a challenge. In addition, traditional antibiotic discovery screenings focus on target affinity and selectivity for growth inhibition. Thus, most of the discovered agents are unable to eradicate the dormant population. Therefore, a new strategy that target persister cells is essential.

Recently, we reported a new strategy for persister control that leverages the dormancy characteristic of persisters and reduced drug efflux activities to increase the intracellular accumulation of certain antibiotics such as minocycline, eravacycline and rifamycin SV. This led to effective killing of Escherichia coli persisters cells. Based on these results, we proposed that persister control agents should (1) be positively charged under physiological condition to interact with the negatively charged lipopolysaccharides, (2) be able to penetrate persister cells via energy-independent diffusion, (3) be amphiphilic to have membrane activity for penetration, and (4) have strong binding to an intracellular target (Roy S. PLOS Pathogens. 2021;17(12)).

Materials and

Methods: In this study, we were motivated to further test eravacycline (ERV) on other pathogenic species such as Pseudomonas aeruginosa. P. aeruginosa causes major infections that are difficult to eradicate by current antimicrobial therapies. It is a major causative agent of infections associated with surgical wound sites, bloodstream, orthopedic implants, and community-acquired pneumonia. It is also a leading cause of fatal episodes of ventilator-associated pneumonia (VAP), catheter-associated urinary tract infections (CAUTI), and chronic lung infections of cystic fibrosis patients. To determine if ERV is an effective agent against P. aeruginosa biofilms and associated persister cells, we treated mature (48 h) biofilms of PAO1 and PDO300 on silicone (polydimethylsiloxane; PDMS) surfaces with different concentrations of ERV. In addition to in vitro tests, we evaluated ERV for persister killing during wake up in vivo using a murine lung infection of P. aeruginosa. We then optimized a chemoinformatic clustering algorithm based on the criteria mentioned above to screen for new persister drugs. A small chemical compound library (80 compounds from Asinex) was screened using this model to identify leads that have similar chemical properties of our previously reported persister control drugs including minocycline, eravacycline and rifamycin SV. The top 10 potential persister control agents were further evaluated experimentally against persister cells of E. coli HM22. E. coli HM22 was chosen as our strain of model species because it contains the hipA7 allele that leads to high-level persistence, and it was the model strain used in our previous study (Roy S. PLOS Pathogens. 2021;17(12)).

Results, Conclusions, and Discussions: ERV was found effective in killing P. aeruginosa PAO1 (wild-type) and PDO300 (mucoid mutant) persisters cells (2.8 & 2 log reduction, respectively). With100 µg/mL of eravacycline, the number of viable biofilm cells of PAO1 and PDO300 reduced by 3 and 2.8 log, respectively. No significant biofilm-associated persister killing was observed when ERV was added at 10 and 50 µg/mL for PAO1. However, a killing of 2 logs was observed when treated at a higher concentration of 100 µg/mL, indicating a threshold. A dose-dependent killing of biofilm-associated persister cells was observed for PDO300, (1.0, 1.2, & 3.4 logs killing observed after treatment at 10, 50, and 100 µg/mL, respectively). Consistent with in vitro results, persister killing during wakeup by ERV was validated in vivo using a mouse model of lung infection. Next, we optimized a chemoinformatic clustering algorithm based on our recently developed criteria to filter a chemical compound library to a subset that have similar chemical properties of previously proven agents effective against Escherichia coli persisters. Our screening led to the discovery of five new compounds that can effectively kill ~95% of E. coli persisters when treated with 100 µg/mL. These positive hits demonstrate the effectiveness of screening for persister drugs using this predictive chemoinformatic model.

Increasing evidence emphasizes the urgent need for more effective agents to control persistent infections. Persister cells are growth-arrested; thus, most antibiotics that were selected to target active cells fail to eradicate persisters. In this study, we demonstrate that ERV can effectively accumulate in P. aeruginosa persister cells, and exhibit strong killing during wakeup, including both planktonic and biofilm-associated persister cells of the wild-type strain and its mucoid mutant. The effects of eravacycline on persister control were further validated in vivo using a lung infection model in mice. We also identified new leads for persister control based on a set of criteria we developed recently. The results demonstrate an efficient approach to identifying persister control agents using a guided screening that incorporates phenotypic traits with molecular descriptors. Collectively, these results demonstrate the possibility to control persister cells of bacterial pathogens by targeting dormancy.

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