# Electrospinning of Chitosan for Antibacterial Applications--Current Trends
## 6 Antibacterial Effect of Chitosan Nanofibers
### Susceptibility of Different Bacterial Strains to Chitosan  
Conflicting opinions regarding the greater or lower sensitivity of chitosan towards Gram-positive and Gram-negative bacteria are found in the literature. The different strain behaviors are attributed to the variance in the membrane structures of Gram-positive and Gram-negative bacteria and the electrostatic charges involved. Most bacterial cell surfaces have a net negative charge due to proton dissociation of carboxyl, phosphate, and amino groups on bacterial cell surfaces [138; 139]. The cell wall of Gram-positive bacteria consists of two layers. The first layer consists of a thick peptidoglycan mesh layer (Murein), which is located over the plasma membrane. The other layer is a lipid bilayer made of interconnected sugars and peptides that are functionalized with teichoic acids and are mainly responsible for the negative charge on the bacterial membrane. Gram-negative bacteria exhibit a sandwich membrane structure with an outer wall composed of phospholipids, lipopolysaccharidesaccharides (endotoxins), and lipoproteins, as well as a thin peptidoglycan layer and an inner cytoplasmic membrane. The phosphate groups of LPS on the outer cell wall make the cell membrane more hydrophilic and highly polar, with a negative surface charge density [140]. The cell wall compositions of Gram-positive (see Figure 6a) and Gram-negative (see Figure 6b) bacteria are shown in Figure 6.  
Some researchers discovered that chitosan showed bactericidal effect against the Gram-positive bacteria L. monocytogenes, two Bacillus strains, S. aureus, and three Lactobacillus strains. However, it was only able to stop further growth of Gram-negative bacteria such as _Escherichia coli_ (_E. coli_), _P. fluorescens_, and _V. parahaemolyticus_, without killing them. Meanwhile, _S. typhimurium_ was totally resistant to chitosan [142]. One research group theorized that chitosan acts on Gram-positive bacteria due to a polymer barrier on the cell surface that prevents food penetration [143]. Another research group suggested that low-molecular-weight chitosan penetrates the cell wall, interacts with  
Figure 6: Cell wall compositions of (**a**) Gram-negative and (**b**) Gram-positive bacteria. Preprinted from [141], originally published under a CC-BY 4.0 license. the electronegative lipopolysaccharideaccharides, and upsets cell metabolism, which consequently inhibits the growth of _E. coli_[144]. An investigation was conducted to analyze the bacterial susceptibility or resistance to chitosan nanofibers with regards to the membrane hydrophilicity, surface charge density, and pathogenicity of each bacteria strain. In typical conditions, bacterial growth phases were clearly noticed. Per contra, the presence of CNFs in a bacterial culture entirely suppressed _E. coli_ growth, while _S. typhimurium_ development was substantially affected. Moreover, there was no growth recovery after deprotonation of chitosan when the pH of the suspension was neutralized with NaOH, indicating that CNFs possess a bactericidal effect that is irreversible and permanent. In another attempt to find out the main factor behind this effect, CNFs were treated with the anionic surfactant sodium dodecyl sulfate (SDS) in order to conceal their positive charges. The slight decrease in bacteria growth can be either attributed to the cell lysis typically caused by SDS solution at high concentrations or to chitosan chains forming a barrier layer that prevents cell exchange, as well as to the fact that certain amino groups of CNFs tend to be protonated after the addition of salt. To rule out other possibilities, NaCl was also applied to screen the charges of NH3\({}^{+}\) at concentrations lower than that which can normally cause the cell lysis of _E. coli_, although the same growth with a decreasing effect was reached. This contradicts the fact that SDS caused the lysis. Furthermore, the slight decrease in OD was insignificant compared to the antibacterial effect that could be achieved by the positively charged groups.