# 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 E. coli  
Initial adhesion is mediated by genes such as _fli_, _flg_, and _flt_. At this stage, the bacteria are in a planktonic state. The _flhDC_ gene encodes proteins for the structural assembly of flagella, representing the class 1 gene set [90; 91; 92]. The class 2 gene set includes the _fli_ gene set, which is related to the formation of the basal body and hook, comprising _flgAMN_, _fliFGHIJK_, _fliMNOPQR_, _fliE_, _flgBCDEFGHJ_, _fliAZY_, and _flhBAE_[90]. The class 3 gene set includes _notAB-cheAW_, _fliDST_, _flgKL_, _fliC_, _tar-tap-cheRBYZ_, and _flgMN_. The expression of these gene sets is regulated by class 2 gene sets and is important to produce chemotactic signals and flagellar filaments [90].  
Irreversible attachment is facilitated by fimbiae-encoding genes, specifically the _fim_ genes, which include the _fimAICDFGH_ gene sets. Here, FimA serves as the major subunit forming the rod of type 1 fimbiae, FimC acts as a chaperone that binds to the SecYEG channel, FimD is the translocon subunit, and FimF, FimG, and FimH are located at the tips of the fimbiae [93]. FimH binds to the lectin domain of eukaryotic cells, allowing _E. coli_ to adhere to eukaryotic cell surfaces and other abiotic components [94]. FimI acts as the terminator component of this type of fimbiae [95]. Additionally, biofilm formation is mediated through the production of curli fimbiae by the _csgBAC_ operon, which generates fibrous components of the fimbiae, and _csgDEFG_, where _CsgD_ is the regulatory protein and produces cellulose, while _CsgEG_ functions as the transport protein [96; 97]. Antigen 43, encoded by the flu genes, also contributes to cellular aggregation alongside _AidA_ and _TibA_ proteins, promoting biofilm formation [98] while autoaggregation requires motility and is strongly enhanced by chemotaxis in _E. coli_ at physiological cell densities [99].  
Maturation occurs with the production of poly-\(\beta\)-1,\(\ell\)-N-acetyl-D-glucosamine (PGA) polymer and cellulose. PGA is synthesized by the PgaC glycosyltransferase encoded by the _pgaABCD_ operon [100; 101]. Cellulose is synthesized by the _bcsABZC_ operon, which encodes _BcsA_ cellulose synthase, contributing to the rigidity of the biofilm [102]. Cyclic di-GMP inhibits flagellar movement by increasing the production of the YgcR protein. Conversely, PdeH proteins inactivate YgcR, reducing the concentration of cyclic di-GMP and increasing flagellar movement [103]. The two-component system also facilitates the production of poly-\(\beta\)-1,\(\ell\)-N-acetyl-D-glucosamine through the CpxAR complex, which downregulates curli fimbiae production by activating OmpC, inhibiting flagellar motion and thereby promoting biofilm formation [104; 105]. The EnvZ/OmpR system primarily functions to inhibit flagellar motion [106]. The RcsCDB proteins regulate the synthesis of colonic acid and inhibit the _flhDC_ operon [107].  
Biofilm formation is also significantly influenced by the production of autoinducer 2 (AI-2), a furanosyl borate diester, as _E. coli_ cannot produce autoinducer 1. _E. coli_ encodes an AI-1 sensor through the _sdiA_ gene, which is a _luxR_ homolog. AI-2 is produced by the _luxS_ gene, pumped out of the cell, and then taken up by the _LsrABCD_ proteins and ABC transporters. _LsrK_ kinase subsequently phosphorylates AI-2, with _LsrK_ being repressed by _LsrR_, which enhances the uptake of the AI-2 molecule [108].