Discussion
In this study, we optimized the extraction of chitosan from the white shrimp species (Metapenaeus affinis ) by manipulating concentration, temperature, reaction time, and v/w ratio. Our results showed an impressive DD% of 93.98%, surpassing previous studies conducted with different material sources. Unlike most previous research that focused on only three variables (Hwang et al., 2002, Younes et al., 2014), our study considered five significant variables for optimization. The results of FTIR analysis confirmed the authenticity of the extracted chitosan. The peak at 2860 cm-1 indicates stretching vibrations related to the branching of C-H bonds, specifically attributed to CH2 and CH3 groups (Kumirska et al., 2010). The 1652 cm-1band corresponds to the amide I absorption resulting from interactions between hydrogen and hydroxyl groups, indicating the removal of the acetyl group. At 1587 cm-1, the band corresponds to amide II (-NH2 bending). The peak observed at 1429 cm-1 represents C-H stretching, while the band at 1373 cm-1 corresponds to amide III and signifies C-N stretching of N-acetyl-glucosamine. The absorption peak at 1148 cm-1 indicates the presence of a symmetric glycosidic linkage (C-O-C), and at 1027 cm-1, an absorption band is observed, indicating stretching vibrations of the C-O ring. The absorption peak around 880 cm-1 is attributed to the β-anomer (1-4) glycosidic linkage (C-O-C) (Abdel-Rahman et al., 2015).
The present study demonstrated the synergistic effect of combined chitosan and antibiotic treatment, resulting in a decreased minimum inhibitory concentration (MIC) in P. aeruginosa. This finding highlights the antimicrobial efficacy of chitosan. Ceftazidime, a member of the β-Lactam antibiotics group, is widely recognized as one of the most effective and safe antibiotics. This group encompasses numerous antibiotics that share the common β-Lactam ring structure and can be further classified into five main subgroups based on various criteria (Brooks et al., 2014). These bactericidal antibiotics exert their mode of action by inhibiting cell wall synthesis, specifically by interfering with the transpeptidation reaction through covalent binding to the target site of Penicillin-binding proteins (PBPs). This binding ultimately halts the synthesis of peptidoglycan, leading to bacterial cell death (Guilfoile and Alcamo, 2007). It is important to note that β-Lactam antibiotics are effective against actively growing bacteria that are in the process of synthesizing their cell walls (Drlica and Fong, 2008). The antimicrobial properties of chitin, chitosan, and their derivatives have gained significant attention in recent years, particularly in their effectiveness against various microorganisms. Several mechanisms have been proposed to explain the inhibitory effects of chitosan on microbial cells. These multiple mechanisms contribute to the antimicrobial efficacy of chitosan and its potential as an effective agent in combating microbial infections. Chitosan’s polycationic nature enables it to interact with anionic groups on the cell surface. This interaction forms an impermeable layer surrounding the cell, which hinders the transport of essential solutes through the outer membrane of gram-negative bacteria. Consequently, it induces structural changes in the cell membrane, increases permeability, and causes the leakage of proteins and other intracellular components (Kamala et al., 2013). Chitosan acts as a chelating agent, selectively binding trace metals. This subsequently inhibits toxin production and microbial growth (Muslim et al., 2018b). Chitosan triggers various defense processes in the host tissue, acts as a water-binding agent, and inhibits specific enzymes. Low molecular weight (LMW) chitosan is capable of entering the cytosol of microorganisms (Kašparová et al., 2022). Upon binding with DNA, it interferes with mRNA and protein synthesis. Chitosan forms an impermeable polymeric layer on the cell surface, altering cell permeability and preventing nutrient uptake. Furthermore, chitosan has the ability to adsorb electronegative substances within the cell, leading to their flocculation. This disturbance of the microorganism’s physiological activities ultimately results in cell death (Badawy and Rabea, 2011). Chitosan possesses a broad spectrum of antimicrobial properties, but its practical application is hindered by its low solubility at neutral pH. Nonetheless, research has demonstrated that enhancing the positive charge of chitosan enables stronger binding to bacterial cell walls (Kamala et al., 2013). The antimicrobial efficacy of chitosan correlates directly with its degree of deacetylation, which determines the number of amino groups present (Kim et al., 2003). Consequently, a higher degree of deacetylation leads to greater quantities of protonated amino groups in acidic conditions. This increased solubility allows for enhanced interaction between chitosan and negatively charged cell walls of microorganisms, thereby augmenting its antimicrobial activity (Silva et al., 2021).
QS genes, which frequently encode virulence factors, play a crucial role in the interaction between bacteria and their hosts (Warrier et al., 2021). The formation of QS-mediated biofilms confers antibiotic resistance to bacteria and impedes the effectiveness of the host immune (Warrier et al., 2021). In P.aeruginosa , the lasR and rhlR systems govern QS and are responsible for the synthesis of various virulence enzymes, including LasA protease, elastase, pyocyanin, and rhamnolipids (Qin et al., 2022). In this study, chitosan extract effectively reduced the production of pyocyanin, rhamnolipids, and protease in P. aeruginosa . It also decreased the expression of lasR and rhlR, as well as swimming motility and biofilm formation in a dose-dependent manner. Similar findings were observed by Rubini et al ( 2019)., where pyocyanin production in clinical isolates of P. aeruginosa was reduced by 40%-80% upon treatment with chitosan extract from crab shell (Pseudomonas sanguinolentus ) (Rubini et al., 2019). This confirms chitosan’s capability to disrupt the rhl quorum sensing system, which regulates pyocyanin pigment production (Le Berre et al., 2008). Additionally, a previous report by Overhage et al. (2007) demonstrated that a swarming-associated gene in P. aeruginosa is responsible for biofilm formation and the production of virulence enzymes (Overhage et al., 2007). The results clearly demonstrate that extracted chitosan has a significant inhibitory effect on the swarming motility of Pseudomonas aeruginosa strains, leading to the prevention of biofilm formation.
In this investigation, the extracted chitosan demonstrated a significant decrease in the ability of P. aeruginosa to form biofilms at sub-MIC levels. The presence of cationic charge in chitosan facilitates its penetration into the biofilm, disrupting the ionic charge of the cell membrane and preventing adherence to both living and non-living surfaces (Zhang et al., 2013). A study by Orgaz et al. (2011) showed that mature biofilms of Pseudomonas spp. are highly susceptible to chitosan treatment. Consistent with this finding, extracted chitosan exhibited a powerful effect against preformed biofilms of P. aeruginosa , reinforcing its potential as an antibiofilm agent (Orgaz et al., 2011). The biofilm matrix consists of various macromolecules, with the extracellular polysaccharide substance (EPS) playing a crucial role in the three-dimensional structure of biofilms (Limoli et al., 2015). EPS has been implicated in limiting the entry of antibiotics into the biofilm matrix, contributing to antibiotic resistance (Pinto et al., 2020). Extracted chitosan disrupts the structural integrity of the biofilm architecture by reducing the EPS layer (Rubini et al., 2019). Mu et al. (2014) reported the antibiofilm efficacy of chitosan, which resulted in a reduction in biomass of Listeria species through biofilm dispersal (Mu et al., 2014).