EPIGENETIC AND GENETIC RESPONSES OF ESCHERICHIA COLI PERSISTER CELLS TO CIPROFLOXACIN STRESS: INSIGHTS INTO THE ROLE OF NUCLEOID-ASSOCIATED PROTEINS

Abstract

Persister cells in Escherichia coli (E. coli) represent a transient, non-mutational form of antibiotic tolerance that contributes to treatment failure and chronic infections. This study investigated the genetic and epigenetic responses of E. coli persister cells exposed to ciprofloxacin-induced stress, with a focus on the role of nucleoid-associated proteins (NAPs) in promoting DNA protection and persistence. Fifty clinical E. coli isolates were screened for persister cells using the Rapid Killing and Replica Plating Tolerance Isolation System (REPTIS). The expression of selected NAP genes (hns, hupA, hupB, and dps) was analyzed using real-time PCR (RT-qPCR), while hns gene methylation was assessed via qMethyl PCR. DNA integrity was evaluated by the neutral comet assay, and Dps protein expression was measured by Western blotting. Persister cells were detected in 4% of isolates, with isolate E3 confirmed by REPTIS. RT-qPCR showed significant upregulation of NAP genes in persisters, with hupB showing the highest fold increase (685-fold). qMethyl PCR revealed elevated hns methylation levels (61.55%), suggesting epigenetic regulation. The comet assay showed reduced DNA damage in persister cells, while Western blotting indicated a 1.2-fold increase in Dps expression under ciprofloxacin stress. These findings suggest that E. coli persistence involves a multifaceted mechanism, including transcriptional activation, epigenetic modifications, and protein-level regulation of NAPs. The enhanced expression of Dps under antibiotic stress highlights its protective role and supports the potential of targeting NAPs in developing novel therapeutic strategies against persistent and recurrent E. coli infections.