In vitro augmented photodynamic bactericidal activity of tetracycline and chitosan against Clostridium difficile KCTC5009 in the planktonic cultures

• The PDT with tetracycline (TC) and chitosan against Clostridium difficile was tested.
• Bactericidal effect was greater in the TC plus chitosan group than TC group.
• DNA damage was greater in the TC plus chitosan group than TC group.
• Bacterial membrane damage was greater in the TC plus chitosan group than TC group.
• Combination of TC and chitosan is more effective than conventional PDT with TC.

Infection with Clostridium difficile (C. difficile) causes a severe colitis with high recurrence. Treatment of C. difficile infection (CDI) is based on antibiotics in spite of the increase of resistance. To interrupt the vicious cycles such as new antibiotics treatment and appearance of resistance strains, photodynamic therapy (PDT) might be a possible alternative therapy for CDI. Tetracycline (TC) has been used as a broad spectrum antibiotic with low risk of CDI and a photosensitizer (PS) in PDT. In vitro PDT against C. difficile was conducted using UVA and TC as a PS before in vivo study. To enhance the photodynamic antibacterial activity of TC, we applied chitosan as a boostering agent. Bactericidal effects after PDT, were measured by counting viable cells, DNA damage and membrane integrity. At 1 mg/mL of TC, chitosan treatment combined with PDT, increased the bactericidal effect by > 10,000-fold of the effect of PDT alone. Membrane damage and cellular DNA damage demonstrated by EMA-qPCR were also greater in the group treated with PDT + chitosan than in that treated PDT alone. The present study showed that PDT using a combination of TC and chitosan is an effective method for killing C. difficile.