Synergistic In Vitro Effects Of Indocyanine Green And Ethylenediamine Tetraacetate-Mediated Photodynamic Antimicrobial Chemotherapy Combined With Antibiotics For Resistant Bacteria In Diabetic Foot Infection

Objective: Antibiotic resistance has emerged as one of the most important determinants in diabetic foot infections outcomes.Photodynamic antimicrobial chemotherapy(PACT)has been proposed as an alternative approach for inactivating bacteria,especially resistant bacterial biofilms.This research investigated the synergistic effects of PACT mediated by the photosensitizer indocyanine green(ICG)and ethylenediamine tetraacetate(EDTA)combined with antibiotics against common pathogens and resistant bacterial biofilm of diabetic foot ulcer infection,including Staphylococcus aureus and Pseudomonas aeruginosa,in vitro.Methods: Staphylococcus aureus and Pseudomonas aeruginosa standard strains,Methicillin-resistant Staphylococcus aureus(MRSA)and Multidrug-resistant Pseudomonas aeruginosa(MRPA)were selected as the research subjects,and incubated with ICG and(or)EDTA,respectively,and then exposed to laser treatment.Quantitative viable counting estimates the bactericidal effect on S.aureus and P.aeruginosa.The susceptibility of methicillin-resistant S.aureus(MRSA)and multidrug-resistant P.aeruginosa(MRPA)to PACT treatment mediated by ICG and EDTA wasdetected by disk diffusion and micro-broth dilution methods.The micro-broth dilution method was used to detect the minimum biofilm inhibitory concentration(MBIC)of MRSA and MRPA.Confocal microscopy was used to detect the morphology of biofilms treated with PACT and antibiotics and the resazurin assay was used to quantify the metabolic activity of bacteria in biofilms.Results: PACT mediated by ICG and EDTA can significantly reduce the number of CFU against S.aureus and P.aeruginosa,and has a more pronounced bactericidal effect compared with ICG alone-mediated PACT.P.aeruginosa was more sensitive to ICG and EDTA-mediated PACT than S.aureus.After PACT,MRSA and MRPA significantly reduced the MIC of susceptibility test drugs,and the MBIC reduced to a quarter of the control.Furthermore,PACT combined with antibiotic treatment significantly contributed to killing bacteria in the biofilm,disrupting biofilm structure and reduce the metabolic activity of bacteria in biofilms.Conclusions: The addition of EDTA to the photosensitizer ICG increased the antibacterial effect of ICG-mediated PACT.The photosensitizer ICG combined with EDTA-mediated PACT can significantly inactivate the common pathogens of diabetic foot infection,Staphylococcus aureus and Pseudomonas aeruginosa,while Gramnegative bacteria are more sensitive than Gram-positive bacteria.ICG and EDTA-mediated PACT combined with antibiotics synergistically enhanced the effects of sterilization,the sensitivity of commonly used antibiotics and biofilm destruction.The results may provide an interesting treatment strategy and approach for the treatment of patients with diabetic foot ulcer resistant bacteria infection.