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).