Antimicrobial Actions and Applications of Chitosan

Chitosan is a naturally originating product that can be applied in many areas due to its biocompatibility, biodegradability, and nontoxic properties. The broad-spectrum antimicrobial activity of chitosan offers great commercial potential for this product. Nevertheless, the antimicrobial activity of chitosan varies, because this activity is associated with its physicochemical characteristics and depends on the type of microorganism. In this review article, the fundamental properties, modes of antimicrobial action, and antimicrobial effects-related factors of chitosan are discussed. We further summarize how microorganisms genetically respond to chitosan. Finally, applications of chitosan-based biomaterials, such as nanoparticles and films, in combination with current clinical antibiotics or antifungal drugs, are also addressed.


Antimicrobial Actions of Chitosan
The mechanisms of action of chitosan against bacteria and fungi have been investigated and reported in many articles [72,. Although the antimicrobial properties of chitosan are highly associated with its structure, physicochemical characteristics, and environmental conditions, in addition to the reactive hydroxyl groups at the C-3 and C-6 positions [4,37,39,43,90,103,105,[108][109][110][111][112][113][114][115][116][117][118][119][120], the mode of action of chitosan against microbes can be classified as extracellular effects, intracellular effects, or both based on the targeting site of the antimicrobial effects [36, 68,90,103,107,121]. Because high-MW chitosan is generally unable to penetrate the cell wall and cell membrane, its potential antimicrobial effects involved acting as a chelator of essential metals, preventing nutrients from being taken up from cells extracellularly, and altering cell permeability [68,84,90]. However, low-MW chitosan not only has extracellular antimicrobial activity but also intracellular antimicrobial activity, thereby affecting RNA, protein synthesis, and mitochondrial function [68,84,90,122,123]. Furthermore, the mode of antimicrobial action of chitosan is highly dependent on the type of targeted microorganism.

Antimicrobial Activity against Bacteria
Gram-positive and gram-negative bacteria exhibit remarkable differences in their cell wall structure, in which gram-positive bacteria have thicker peptidoglycans and gramnegative bacteria are enriched in lipopolysaccharide (LPS) [124][125][126][127]. Differences in the cell surface structure of these types of bacteria also lead to distinct susceptibilities to chitosan. For example, gram-negative bacteria present a more negative charge than gram-positive bacteria because LPS is often attached to phosphorylated groups [128,129]. More negatively charged cell surfaces allow the binding of cationic chitosan to phospholipids when the environmental pH is below 6.5 [2,68,90,103,107,121,130,131]. The potential antibacterial action of chitosan is shown in Figure 1A,B. It has been suggested that gram-negative bacteria could be more susceptible to chitosan than gram-positive bacteria [114,[132][133][134], but some studies have shown that gram-positive bacteria are more sensitive to chitosan [88].
Teichoic acids in gram-positive bacteria are also negatively charged due to the presence of phosphate groups in their structure [124,125]. However, deletion of the teichoic acid biosynthesis pathway in Staphylococcus aureus resulted in increased resistance to chitosan [107], indicating that the mode of action of chitosan is more complex than simple electrostatic interactions. In addition, unlike gram-negative bacteria, gram-positive bacteria have a thick cell wall, which might prevent chitosan from binding directly to the cell membrane. However, some chitosan oligomers (<5 kDa) penetrate the cell wall and influence DNA/RNA or protein synthesis [68,84,90,122,123]. Interestingly, reports have demonstrated that chitosan (≤50 kDa) can pass through the cell wall and inhibit DNA transcription [68]. Thus, although the molecular size of chitosan plays an important role in targeting, the structure rather than the MW of chitosan also determines its extracellular, intracellular, or both extracellular and intracellular antibacterial activity.

Antimicrobial Activity Against Fungi
Chitosan has been shown to have fungicidal effects on several fungal pathogens in plants and humans [82,83,108,[135][136][137][138][139][140][141][142][143][144][145][146][147]. Its antifungal properties are mainly related to the interaction of chitosan with the cell wall or cell membrane. Nevertheless, the minimum inhibitory concentrations (MICs) of chitosan against fungi vary and are highly associated with the MW and degree of deacetylation (DDA) of chitosan, solvent pH, and the type of fungus being targeted [2,90,107,136,140,144,148,149]. Furthermore, the unsaturated fatty acid contents on the cell membrane might be positively correlated with chitosan susceptibility [150] because a higher content of unsaturated fatty acids exhibits better membrane fluidity, leading to a more negative charge on the cell membrane [151]. For example, the opposite characteristics of chitosan-sensitive and chitosan-resistant Neurospora crassa strains are related to the content of unsaturated fatty acids on the cell membranes [150]. These data may account for, at least in part, why Candida albicans, Candida tropicalis, and other Candida species have remarkable differences in susceptibility to the same chitosan [83,152]. Indeed, C. tropicalis exhibited an increase in susceptibility of more than 1,000-fold to certain chitosans compared with C. albicans [83,152]. Similarly, in addition to its extracellular antifungal effects, low-MW chitosan is able to penetrate the cell wall and cell surface, leading to the inhibition of DNA/RNA and protein synthesis [39,68,82,91,122,125,132]. Interestingly, a previous report has further suggested that chitosan affects mitochondrial activity [123]. The mode of action of chitosan against fungi is shown in Figure 1C.

pH
A major antimicrobial effect of chitosan is electrostatic interactions between this cationic molecule and negatively charged cell walls [72,. The pKa values of the amino groups of chitosan are 6.3-6.5, indicating that it is insoluble in alkaline solutions, organic solvents, and water when the pH is higher than 6.5 [36, 68,90,103,107,121]. Addi-tionally, the solubility increases with decreasing solution pH, which leads to an increase in the positive charge on the -NH 3 groups of chitosan and stronger antimicrobial activity [36, 68,90,103,107,121]. In fact, a large number of articles have demonstrated that chitosan exhibits excellent antimicrobial activity under acidic conditions, as summarized in several review articles [36, 68,90,103,107,121].

Molecular Weight
The molecular weight of chitosan determines whether it penetrates the cell surface to exert intracellular antimicrobial activity. Furthermore, the abundance of polysaccharides and a few proteins that compose the complex layers of the cell wall in both bacteria and fungi not only play important roles in pathogenesis, biotic surface adhesion, and abiotic surface adhesion, and induction of the immune response, but also offer mechanical strength and a barrier from the environment [124,[153][154][155]. In fact, the rigid cell wall transports molecules across the outer layer barrier via several delicate mechanisms or by simple diffusion [156][157][158], and the cell wall porosity and pore size determine whether a compound or molecule passes through the bacterial or fungal cell wall [156][157][158]. The pore sizes vary between different bacteria and fungi, with a range of 2-4 nm up to 8 nm [154,[159][160][161][162][163]. For example, the pore sizes determined by fluorescein-labeled dextran are 2.06 and 2.12 nm in Escherichia coli and Bacillus subtilis, respectively, whereas Pseudomonas aeruginosa exhibits larger pores of 13 ± 5 nm [159][160][161][162]. Additionally, it has been proposed that the cell wall pore sizes in Saccharomyces cerevisiae, C. albicans and Cryptococcus neoformans are approximately 5.8 nm [160,163]. Based on pore size, only~5 kDa (minimum radius: 1.1 nm) globular molecules or proteins can penetrate most bacterial cell walls, and 50 kDa (minimum radius: 2.4 nm) spherical molecules or proteins should be able to pass through fungal cell walls [164]. However, the hydration state influences sphere size, and the hydrodynamic radii of proteins are usually larger. For example, the radii of beef pancreas ribonuclease A (14 kDa), beef pancreas chymotrypsinogen A (25 kDa), and hen egg ovalbumin (43 kDa) are 1.05, 1.21 and 1.27 nm, respectively, in a nonhydrated state, but these radii increase to 1.64, 2.09 and 3.05 nm, respectively, in their hydrated state [164]. These data suggest that globular proteins with a molecular weight of 30 kDa or less can cross the microbial cell wall under physiological conditions. Similarly, chitosan has a diameter of~1.1 nm in its linear extended form [165]; however, the hydrodynamic radius of hydrated chitosan  is 25.59 nm [107].
Reports have shown that oligo-chitosan (<5 kDa) can penetrate the cell wall, leading to intracellular antimicrobial activity [122]. Therefore, the question is how a~50 kDa molecular weight chitosan might be able to penetrate the bacterial cell wall to inhibit DNA transcription [68,166]. Several possibilities might explain how larger chitosan molecules could enter cells: (1) Cell walls are dynamic structures that vary during replication, hyphal development, and age [124,[153][154][155], and this flexibility may allow various molecules to pass through the cell wall. Indeed, a recent article has provided solid evidence of this phenomenon. Amphotericin B liposomes (AmBisomes) are liposomal delivery systems containing the antifungal drug amphotericin B [163]. Interestingly, AmBisome, which is 60-80 nm, is able to penetrate the cell walls of C. albicans and C. neoformans (pore size of 5.8 nm) [163]. These data suggest that the fungal cell wall is capable of remodeling and that the viscoelastic properties of the cell wall help larger molecules or compounds migrate through the outer layer. (2) Chitosan might affect cell wall porosity. Many reports have shown that environmental conditions and stresses profoundly influence cell wall porosity. For example, in S. cerevisiae, cell wall porosity increases after treatment with polyethylene glycol (PEG), dithiothreitol (DTT), or ethylenediaminetetraacetic acid (EDTA), whereas glucanase-soluble mannoproteins decrease the cell wall porosity of yeasts [160,[167][168][169]. In addition to cell wall penetration, AmBisomes also transiently affect the cell wall porosity of C. albicans [163]. Therefore, chitosan may influence the cell wall pore sizes, but there is no evidence to support this hypothesis.

DDA
Given that the amino group (−NH 2 ) of chitosan is the most important functional group, the DDA of chitosan influences the performance of chitosan in many applications [2,68,90,103,107,121,122,131]. The DDA of chitosan is highly associated with the preparation method, particularly the processing time and temperature used during chemical treatment [4,5]. Longer processing times and higher temperatures usually result in a high DDA [3,5,8,11,12]. Furthermore, chitosan with a high DDA has been shown to exhibit a more positive charge than chitosan with a low DDA in the same acidic environment [68,90,103,107,121,122,170,171]. Thus, chitosan with a high DDA has stronger electrostatic interactions with the microbial cell surface, which often results in better antimicrobial activity. Indeed, studies have shown that high DDAs of chitosan exhibit stronger antimicrobial activity against bacteria [170,171].

Bacterial Responses
There have been few reports regarding the transcriptional responses of bacteria to chitosan. The microarray profile of chitosan-treated S. aureus SG511 showed that 84 genes and 82 genes were significantly upregulated and downregulated, respectively (Table 1) [107]. Chitosan treatment remarkably inhibited bacterial growth through downregulation of the genes involved in growth and metabolism, such as genes for RNA, protein, carbohydrate, amino acid, nucleotide, and lipid biosynthesis [107]. Furthermore, genetic profiles have suggested that chitosan impaired oxygen consumption and preferred anaerobic respiration [107]. A similar finding was found in Bacillus cereus after treatment with either type of polysaccharide or chitosans A and B, in which both chitosans significantly inhibited nitrogen, amino acid, and pyruvate metabolism, and gluconeogenesis (Table 1) [185]. Moreover, several genes involved in ion transport, particularly potassium transport, were upregulated [185]. However, B. cereus deficient in the genes required for potassium transport (the Kdp system) exhibited similar susceptibility to chitosan A and chitosan B compared to the wild-type strain [185], which may have been due to the Kdp system loss in B. cereus not being sufficient to block potassium uptake and enhance chitosan susceptibility.

Fungal Responses
Compared with bacteria, there have been relatively more studies showing how budding yeast and fungal pathogens respond to chitosan. In S. cerevisiae, as chitosan treatment time increased (15,30,60,120, and 180 min), the number of up-and downregulated genes increased [186]. Functional analysis showed that genes involved in endoplasmic reticulum (ER), cell wall biogenesis, cell membrane biogenesis, and stress adaptation were significantly differentially expressed ( Table 2). The ER is a key organelle that synthesizes lipids and membrane-associated proteins for the plasma membrane [186]. Furthermore, chitosan-treated S. cerevisiae exhibited less sensitivity to β-1,3-glucanase [186]. These data suggest that the cell wall and cell membrane are the targets of chitosan. A step further is the understanding of the transcription factor (TF) in S. cerevisiae that are involved in chitosan stress responses, which are Cin5p, Crz1, and Rlm1p. Cin5p is a basic leucine zipper (bZIP) that mediates drug resistance and stress tolerance. Crz1p, a calcineurin-responsive zinc finger, is required for calcium hemostasis and is activated in response to calcium. Rlm1p is a protein kinase involved in cell wall integrity [186]. These data suggest that chitosan may also have intracellular activity that influences gene expression.
Haploinsufficiency (HIP), homozygous deletion (Hop), and multicopy suppression (MSP) fitness assays of chitosan oligosaccharide (COS) combined with microarray analyses, showed that the response to COS is associated with the plasma membrane, respiration, and mitochondrial biogenesis, and 21 genes required for chitosan resistance in budding yeast were successfully identified (Table 2) [187]. Among these, overexpression of ARL1, which encodes a GTPase involved in the regulation of membrane organization and trafficking, resulted in reduced chitosan-induced membrane permeabilization [187]. Interestingly, ARL1 overexpression did not confer resistance to salt and sugar stresses, and exhibited increased sensitivity to antifungal drugs, indicating that the chitosan-induced transcriptional response is distinct from those to antifungals and stresses.
Aspergillus ochraceus is one of the most abundant food-contaminating microorganisms due to mycotoxin production [188]. Chitosan treatment caused A. ochraceus to form abnormal hyphal branches and remarkably influenced cell wall and cell membrane architectures [189]. RNA sequencing analysis further demonstrated that chitosan inhibited genes involved in cell surface integrity and protein biosynthesis [189]. Chitosan upregulated phospholipase-related genes involved in membrane degradation and genes involved in steroid metabolism (Table 2) [189].  In N. crassa, chitosan treatment led to higher levels of intracellular reactive oxygen species (ROS), leading to plasma membrane permeabilization [190]. RNA sequencing analysis revealed that genes associated with mitochondrial function (4, 8, and 16 h treatment), peroxisome organization (4 h treatment), oxidative response (4 h treatment), and fatty acid metabolism (4 h treatment) were induced by chitosan ( Table 2). Deletion of either NCU10521, which encodes a glutathione S-transferase involved in ROS detoxification, or NCU07840, which encodes a plasma membrane protein, resulted in increased chitosan susceptibility, which is consistent with the transcriptomic profile [190]. Furthermore, genes associated with the cytoskeleton, cell wall cortex, and vesicle organization were inhibited in response to chitosan (Table 2) [190]. Interestingly, chitosan significantly induced protein synthesis in contrast to the observation in chitosan-treated A. ochraceus [189]. These data suggest that the mode of action of chitosan is greatly dependent on the type of chitosan, the properties of chitosan and the particular fungus.
Interestingly, a recent article showed the potential mechanisms of how a fungus is more resistant to chitosan [191]. Pochonia chlamydosporia is a nematophagous fungus that can be utilized as a biocontrol against the root-knot nematode Meloidogyne javanica [192]. Chitosan not only promotes P. chlamydosporia growth [193] but also improves tomato root colonization by P. chlamydosporia [194]. Furthermore, chitosan in combination with this fungus reduces damage caused by root-knot nematodes [194]. The greater resistance of P. chlamydosporia to chitosan could be due to two mechanisms: (1) The genome of P. chlamydosporia contains more chitosanase genes [195], thereby utilizing chitosan as a nutrient source [196]. (2) Many monosaccharide transport genes of P. chlamydosporia were induced to assimilate chitosan monomers after chitosan was taken up and degraded into monosaccharides. These findings further demonstrate that the antimicrobial activity of chitosan varies among different microorganisms.
C. albicans is the most frequently isolated fungal pathogen in humans [197,198]. Investigation of the mechanisms of chitosan against C. albicans was conducted via mutant library screening [82,123]. These studies identified several genes potentially involved in chitosan resistance (Figure 2). The functions of these genes include adherence, antifungalrelated responses, cell surface integrity, stress adaptation, mitochondrial biogenesis, and virulence-associated functions [82,123]. Furthermore, several signaling pathways, such as the Hog1, Cek1/Cek2, Mkc1, Ras1-cAMP, and calcineurin cascades, were proposed to be associated with chitosan tolerance [123]. In particular, chitosan treatment significantly reduced C. albicans cell wall thickness via inhibition of the expression of the Spt-Ada-Gcn5acetyltransferase (SAGA) complex [82]. Furthermore, chitosan represses mitochondrial function by inhibiting MSS2 [123], which contradicts that observed in N. crassa during the response to chitosan [190]. Finally, several calcineurin components and Crz1 TFs were identified during library screening [82,123]. CRZ1and calcineurin-associated deletion strains exhibited high sensitivity to both chitosan and high CaCl 2 concentrations (unpublished data), suggesting that calcium homeostasis might be associated with chitosan susceptibility. Indeed, in N. crassa, the application of exogenous Ca 2+ could minimize damage caused by chitosan [190].

Problems Associated with Chitosan
Despite the potential uses of chitosan against microbial infections, there are several concerning issues regarding its properties that may hinder its application: (1) Molecular weight: Chitosan does not have a defined molecular weight, and the molecular weight distribution of each chitosan increases the application difficulty of passing regulatory rules, particularly in the medical field. (2) Purity: Chitosan is made from deacetylated chitin. In general, chitosan with a higher DDA exhibits stronger antimicrobial activity. However, even after treatment with NaOH for a long time and incubation at a high temperature, chitosan with a high DDA (>90%) is produced, indicating that there is less than 10% N-acetylglucosamine in the sample [4,5]. The purity of chitosan might be an issue for application, given that the small amount of N-acetylglucosamine product might affect bioactivity against microbes. (3) Solubility: Chitosan has extremely low solubility under neutral or alkaline pH conditions, and it is dissolved only in acidic environmental conditions [36, 68,90,103,107,121], which limits its applications in many areas. Furthermore, a low pH results in more positive charges on chitosan, leading to stronger antimicrobial properties. However, low pH conditions may harm cells, tissues, or organs of the human body.

Applications of Chitosan-Based Nanoparticles and Films in Combination with Clinical Drugs against Microbes
Chitosan has been widely applied in many areas. However, the antimicrobial effects of pure chitosan and most of its derivatives are still remarkably lower than those of clinical antimicrobial drugs. Several articles have also shown that pure chitosan in combination with clinical drugs exhibits great antimicrobial activity [83,152,[199][200][201][202][203]. Thus, this section focuses on the antimicrobial effects of developed chitosan-based biomaterials with current antibacterial and antifungal drugs because chitosan not only has intrinsic antimicrobial properties but is also able to deliver extrinsic antimicrobial drugs (Table 3).

Films
PEG-chitosan hydrogels containing ciprofloxacin improved the growth inhibition of E. coli compared with drug-free hydrogels and sustainably released the antibiotic for 24 hr [70]. Similarly, the high DDA of chitosan films loaded with different antibiotics exhibited better activity against different pathogenic bacteria [55,[215][216][217][218]. Fibrin-chitosan loaded with two antibiotics (metroidazole and ciprofloxacin) enhanced anti-Enterococcus faecalis activity [78]. A chitosan hydrogel containing ciprofloxacin and fluconazole nanoparticles exhibited significant antimicrobial activities against C. albicans, E. coli, and S. aureus [219]. Finally, a chitosan gel with metronidazole showed great anti-Candida activity to treat vaginal infection [220].

Implants
Chitosan-coated titanium containing tetracycline or chlorhexidine digluconate effectively inhibited Actinobacillus actinomycetemcomitans and Staphylococcus epidermidis [221]. Interestingly, a chitosan bar containing gentamicin prepared using crosslinking, solvent evaporation, and a cylinder model cutting technique, which was implanted into rabbit tibias, exhibited significant antibacterial activity, suggesting that this chitosan bar would be effective against chronic osteomyelitis [222].

Conclusions
Approximately 30,000 original research and review articles related to chitosan have been reported [223], indicating that this naturally occurring product has great potential applications. This review suggests that chitosan as a natural antimicrobial agent can be applied in agriculture, food, and biomedical areas. Transcriptomic analyses in chitosantreated microbes have further concluded that the mode of action of chitosan against bacteria or fungi may have multiple intracellular and extracellular effects. Although chitosan shows great promising antimicrobial potential, most of these studies are still at the laboratory level. Furthermore, the low water solubility and the lack of defined molecular weight and purity are the major issues for future application of chitosan. The development of better strategies and optimized conditions against pathogenic bacteria and fungi is necessary.