Exploring the potential of N-acetyl-cysteine to mitigate the impact of the ciprofloxacin-induced SOS response in communities of Escherichia coli and Klebsiella pneumoniae

Abstract

Introduction: Exposure to fluoroquinolone antibiotics such as ciprofloxacin (CIP) induces the bacterial SOS response involved in repairing DNA damage. This repair can be mutagenic and lead to antibiotic resistance (ABR). Antibiotic-induced stress responses and their role in the development of ABR are not commonly assessed in bacterial communities. We aimed to investigate the CIP-induced SOS response in Escherichia coli and Klebsiella pneumoniae co-cultures, and to determine how the SOS response is affected by treatment with an SOS suppressor, N-acetyl-cysteine (NAC). Methods: Three susceptible strains each of E. coli (EC) and K. pneumoniae (KP) in five co-culture combinations were investigated. Combinations included laboratory reference strains (ECA-KPA), and clinical strains from bloodstream infections (EC1-KP1, EC1-KP2, EC2-KP1 and EC2-KP2). Co-cultures were grown for 2h before adding CIP at 0.5x the minimum inhibitory concentration (MIC) of the E. coli strains with and without 1 mg/ml of NAC. Gene expression was analysed after 4h of antibiotic exposure using RNA-sequencing. Mutation rates were calculated after 24h using mutant counts on chromogenic media containing 8x the nalidixic acid MIC of each species. Results: SOS response genes were upregulated in all isolates in co-culture, with log fold changes (logFC) ranging from 1.847 to 4.527. This response was greater in E. coli in the ECA-KPA combination and in both clinical K. pneumoniae isolates. Genes involved in phage induction were also upregulated in these isolates (logFC = 1.949 – 6.189). Addition of NAC partially or completely abrogated the upregulation. Although resistant mutants were obtained from KP1 and KP2, no significant increase in mutation rates was observed upon CIP exposure. However, NAC reduced the mutation rate of KP2 compared to CIP-exposed cultures (6.72E-07 vs. 2.65E-06 in co-culture with EC1) and unexposed cultures (8.83E-07 vs. 2.40E-06 in co-culture with EC2). Discussion: The SOS response is induced in K. pneumoniae in co-culture even when exposed to very low CIP concentrations. SOS-mediated phage induction upon fluoroquinolone treatment may disrupt the balance of the gut microbiota. The selection of mutants from KP1 and KP2 even without CIP exposure emphasizes the role of bacterial competition in development of ABR. The potential of NAC to reduce SOS and phage induction, as well as ABR development, in bacterial communities is promising and warrants further investigation as a strategy to reduce ABR. Conclusion: The findings of this study highlight the effects of sub-inhibitory fluoroquinolone treatment on the development of ABR and phage induction in bacterial communities, and the potential of NAC to mitigate this.