Deciphering Genetic Contributions to Antibiotic Persistence: Insights from Multi-Gene Knockout Studies

Antibiotic resistance is a critical global health challenge, complicating efforts to treat bacterial infections effectively. Antibiotic persistence plays a significant role in driving resistance. Persisters are a subpopulation of non-dividing bacterial cells that often evade lethal antibiotic treatment. Once antibiotics are removed, these cells "resurrect," resuming growth and may lead to relapsing infections. Notably, the progeny of persister cells exhibit a heightened tendency to acquire mutations or incorporate antibiotic resistance genes, further compounding the challenge of antibiotic resistance. In our recent study, we knocked out seven hypothetical genes and observed a significant reduction in persister levels. Compared to the wild type, mutants exhibited 4-6-fold fewer persisters after 3 hours and 10-15-fold fewer persisters after 6 hours of lethal ampicillin treatment. However, at 24 hours and beyond (48 and 72 hours), no significant difference in survival was detected between the mutants and the wild type. These findings align with our previous hypothesis that individual cells within a clonal population use distinct survival strategies, resulting in multiple persister genotypes. The seven genes may function in a single pathway or in multiple pathways, contributing to persister survival during the medium-term phase of antibiotic exposure. To investigate further, we constructed double and triple gene knockouts. While most of the mutants showed no growth defects, one double mutant did. Triple mutants exhibited significantly reduced persister levels at 3 hours compared to the wild type. Utilizing the Minimum Inhibitory Concentration (MIC) Agar Dilution Test, we quantitatively demonstrated that several mutants exhibit increased antibiotic sensitivity compared to the wild type. This assay precisely measures the effects of even minimal antibiotic concentrations on bacterial growth, yielding critical insights that other methods we tried could not provide. We show that in some cases, conventional antibiotic strip tests lacked the sensitivity needed to detect subtle reductions in MIC for some mutants, a limitation overcome by the Agar Dilution Test. Researchers often compare MIC values between wild-type and mutant strains to assess the impact of specific genetic alterations, and our findings highlight that the Agar Dilution Test can offer a more precise and sensitive alternative to strip tests in certain contexts.