Ciprofloxacin and sulfadiazine at environmentally relevant concentrations induce a viable but non-culturable state in Escherichia coli.

Large numbers of bacteria can enter a viable but nonculturable (VBNC) state to survive adverse conditions, and antibiotic-resistant Escherichia coli cells in a VBNC state pose public health risks. This study aimed to investigate whether prolonged exposure to environmentally relevant concentrations (10 µg/L) of two typical antibiotics, ciprofloxacin and sulfadiazine, could induce Escherichia coli to enter the VBNC state and whether cells regrown from this state would exhibit multidrug resistance. Experimental results showed that after exposing to 10 µg/L ciprofloxacin and sulfadiazine for consecutive 10 days, cultivability of E. coli decreased significantly (p < 0.05) and entered VBNC state. E. coli in VBNC state started to recover in nutrient-rich Luria-Bertani (LB) broth after 16 h at 37 °C. Minimum inhibition concentration (MIC) measurements demonstrated that ciprofloxacin- and sulfadiazine- induced regrown E. coli were resistant to nine commonly used antibiotics, including amoxicillin, ampicillin, chloramphenicol, ciprofloxacin, colistin, enrofloxacin, kanamycin, sulfadiazine, and tetracycline. Quantitative reverse transcription-polymerase chain reaction assays showed that efflux pumps were activated when bacteria challenged with antibiotics. In addition, genes related to oxidative stress and outer membrane porins up-regulated significantly (p < 0.05) in VBNC E. coli cells. Consequently, morphological alterations in cell membrane were observed after antibiotics’ treatment. This study reveals that antibiotic at environmentally relevant concentrations can induce E. coli to enter VBNC state with multidrug resistance, enhancing our grasp of health risks from antimicrobial resistance, antibiotics, and VBNC bacteria in environmental settings.