# Development and Control of Biofilms in Diabetic Foot Infections: A Narrative Review
## 10 Biofilm Formation of Gram-Negative Organisms in Diabetic Foot Ulcer  
### Mechanism of Biofilm Formation in Pseudomonas aeruginosa  
Recent studies indicate that the presence of glucose significantly enhances the biofilm formation of _Pseudomonas aeruginosa_. This upregulation is primarily dependent on the extracellular polymeric substance PsIA, which is produced during glucose metabolism. PsIA is a branched pentasaccharide composed of D-glucose, D-mannose, and D-Hamming  
Figure 4: _ica_-independent biofilm formation in diabetic foot infection.  
in a ratio of 3:1:1 [81]. Notably, glucose selectively increases the concentration of PsIA without affecting the synthesis of the pel and _alg_ genes. Furthermore, exogenous glucose has been shown to stimulate Ofloxacin resistance in _Pseudomonas aeruginosa_[1].  
Biofilm formation occurs through three primary pathways: _las_, _rhl_, and _pqs_ systems. The _las_ system requires 3-oxo-C12 homoserine lactone, while the _rhl_ system depends on C4 homoserine lactone. The _pqs_ system involves the interaction of acyl homoserine lactones and quinolone molecules [82]. C12 and C4 HSL are synthesized via a lactonization process, which is directly linked to the increase in cell population. As the cell count rises, the threshold for C12 HSL is reached, allowing the LasR receptor protein to bind to C12 HSL and form the LasR-C12 HSL complex. This complex initiates the transcription of las genes and expresses _psla-L_ genes, leading to the production of more extracellular polymeric substances in the form of PsIA [83].  
The LasR-C12 HSL complex also stimulates the rhlR gene, resulting in the production of the RhlR receptor protein. When the C4 HSL threshold is met, the RhlR-C4 HSL complex forms and further transcribes the _rhl_ genes. This complex additionally transcribes the pelA-G genes, which produce extracellular polymeric substances. The _rhl_ genes encode rhamnolipids, pyocyanin, hydrogen cyanide, Lectins A and B, exoenzymes A and B, and proteins involved in twitching and swarming motility [84]. Moreover, the LasR-C12 HSL complex promotes the _pqs_ system and releases extracellular DNA through the autolysis of older cells [85].  
Biofilm regulation is mediated by the GacS/A system. GacS functions as a sensor kinase that, upon autophosphorylation, transfers a phosphate group to GacA, which upregulates small regulatory RNAs such as RsmZ and RsmY [86]. These RNAs bind to unbound RsmA, thereby reducing its repression of autoinducer formation [87]. The RetS/LadS system also plays a role in determining the phosphorylating state of GacS, influencing AHL production, biofilm formation, and the production of virulence factors [88]. This regulatory system can lead to both chronic infections and acute infections in patients.  
In patients with diabetic foot ulcers, elevated blood glucose levels contribute to the production of PsIA through increased transcription of the _psla_ gene [83]. High glucose concentrations promote PsIA formation, which subsequently regulates the production of cyclic di-GMP by combining two molecules of guanosine triphosphate via diguanylate cyclase. Therefore, PsIA acts as a signaling molecule for cyclic di-GMP production [89]. Cyclic di-GMP further stimulates the production of _alg_ and _pel_ genes, which generate Alg4 and PelD, both of which are extracellular polymeric substances. Conversely, when cyclic di-GMP levels are low, bacterial dispersion occurs, disrupting the biofilm, a scenario that may arise with low glucose concentrations.