In vitro bactericidal effects of photodynamic therapy combined with four tetracyclines against Clostridioides difficile KCTC5009 in planktonic cultures

Surface disinfection in health-care facilities is critical to prevent dissemination of Clostridioides difficile (C. difficile). Tetracyclines (TCs) are broad-spectrum antibiotics that are associated with a low risk of development of C. difficile infection (CDI) and are used as photosensitizers (PS) in photodynamic therapy (PDT). We evaluated whether TCs may be useful environmental cleansing agents. We compared the in vitro ability to kill C. difficile of four TCs (TC, doxycycline, minocycline, and tigecycline) combined with PDT using ultraviolet A (UVA). We included chitosan, a cationic material, as a booster to increase the photodynamic bactericidal efficacy of TCs. PDT-induced bactericidal effects were assessed by the number of viable cells and the degree of DNA damage and membrane integrity. To avoid the intrinsic antibacterial activity of TCs at high concentrations, we used low concentrations of TCs (0.05 and 0.1 mg/mL). The bactericidal effect of treatment with chitosan plus PDT was over 100 times higher than that with PDT alone for each of the four TCs. DNA damage measured by ethidium bromide monoazide and real-time quantitative polymerase chain reaction was also greater for PDT plus chitosan treatment than for PDT alone or under control conditions: the threshold cycle (Ct) values for the control, PDT, and PDT plus chitosan were 14.67 ± 0.22, 20.46 ± 0.12, and 25.54 ± 0.17, respectively. All four TCs caused similar levels of severe cell membrane damage during PDT compared with control conditions. These data suggest that PDT combined with any of the four TCs plus chitosan might be an available tool to kill efficiently planktonic form of C. difficile.