Repeated Sublethal Photodynamic Inactivation Does Not Increase Biofilm Formation or Induce Resistance in Acinetobacter baumannii

• After 15 cycles of sublethal photodynamic therapy (sPDT) there was no increase in biofilm-forming capacity or upregulation of biofilm-related gene expression • After 15 cycles of sPDT, the biofilms formed showed no decreased susceptibility to the same aPDT regimens. • The addition of potassium iodide enhanced the antibacterial efficacy of photodynamic therapy with methylene blue towards biofilm after 15 cycles of sPDT. • sPDT was defined as 0.5-2 log10 reduction (68%-99% eradication) per cycle. . Antimicrobial photodynamic inactivation (aPDI) could be an effective and novel approach to address the increasingly severe issue of antibiotic resistance. However, no standardized dosing protocol exists for aPDI administration, and clinical applications have frequently used sublethal doses. This study evaluated whether repeated sublethal antimicrobial photodynamic therapy (sPDT) could not only trigger resistance in Acinetobacter baumannii , but also increase biofilm biomass accumulation and upregulate key biofilm-related genes. In this study, we evaluated the response of five clinical antimicrobial-resistant A. baumannii strains to sPDT. A “cycle” was defined as one round of sPDT followed by an overnight regrowth and subsequent resuspension in fresh medium (3-3.5 h) to reach the logarithmic phase. To compare the effects of biofilm properties and antimicrobial responses, bacteria were exposed to 15 cycles of sPDT under three conditions: (1) no treatment (control group); (2) methylene blue (MB) mediated sPDT alone (MB-sPDT group); (3) a combination of MB and potassium iodide (KI) to mediate sPDT (MB/KI-sPDT group). Our findings showed that the biofilms formed by A. baumannii after consecutive sPDT exhibited no resistance to subsequent aPDT, and that MB/KI-aPDT demonstrated superior efficacy in biofilm eradication. Moreover, some strains exhibited reduced biofilm-forming capacity after 15 cycles of sPDT. Compared to untreated controls (0th cycle), the expression levels of biofilm-associated genes (bap, csuE, ompA, and abaI genes) in most strains decreased after the 5th, 10th, and 15th cycles of MB-sPDT and MB/KI-sPDT, except for the upregulation of ompA observed in one multidrug-resistant strain after 5th cycles of MB-sPDT. These results indicate that after 15 cycles of sPDT there was no increase in biofilm-forming capacity or upregulation of biofilm-related gene expression, and the biofilms formed showed no decreased susceptibility to the same aPDT regimens.