Diabetes-related infections have become challenging and important public health problems in China and around the world. P. aeruginosa plays an important role in diabetic foot infections. As a gram-negative opportunistic pathogen, P. aeruginosa causes recurrent and refractory infections that are characterized by biofilm formation. Previous studies have demonstrated that biofilm-challenged wounds typically take longer to heal than non-biofilm-challenged normal wounds in diabetic mouse models. In the present study, we sought to explore the mechanism via which insulin treatment affects cyclic di-GMP signaling in P. aeruginosa-infected chronic wounds in db/db diabetic mice. We found that the wounds of diabetic mice healed more slowly than those of nondiabetic mice. Moreover, wound healing in diabetic mice treated with insulin exhibited a considerable delay. Peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) was used to detect biofilms on P. aeruginosa-infected wound tissues. Increased intracellular c-di-GMP levels promoted biofilm formation in wound tissues from nondiabetic mice. Greater biofilm formation was observed in the wounds of insulin-treated diabetic mice than in the wounds of untreated diabetic mice or nondiabetic mice, in both the PAO1/p_lac-yhjH- and PAO1-infected groups. Quantitative RT-PCR indicated that upon infection with PAO1/P_lac-yhjH (the low c-di-GMP expression strain), the expression of IL-4 RNA was significantly higher in diabetic mice treated with insulin than in untreated diabetic mice or nondiabetic mice at each observation time point. Peak expression of IFN-γ occurred earlier in diabetic mice treated with insulin than in untreated diabetic mice with each of the experimental strains. Finally, P. aeruginosa harboring the plasmid pCdrA: gfp ^s was used as a reporter strain to monitor c-di-GMP levels. We found that insulin could promote biofilm formation by increasing intracellular c-di-GMP levels in vitro. Taken together, these data demonstrate that insulin treatment increases intracellular c-di-GMP levels, promotes biofilm formation and prolongs the inflammation period during the healing of infected wounds, resulting in delayed wound healing.

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