Chitosan and their derivatives: Antibiofilm drugs against pathogenic bacteria

Journal Pre-proof Chitosan and their derivatives: Antibiofilm drugs against pathogenic bacteria Fazlurrahman Khan, Dung Thuy Nguyen Pham, Sandra Folarin Oloketuyi, Panchanathan Manivasagan, Junghwan Oh, Young-Mog Kim

PII:

S0927-7765(19)30771-4

DOI:

https://doi.org/10.1016/j.colsurfb.2019.110627

Reference:

COLSUB 110627

To appear in:

Colloids and Surfaces B: Biointerfaces

Received Date:

30 July 2019

Revised Date:

29 October 2019

Accepted Date:

30 October 2019

Please cite this article as: Khan F, Pham DTN, Oloketuyi SF, Manivasagan P, Oh J, Kim Y-Mog, Chitosan and their derivatives: Antibiofilm drugs against pathogenic bacteria, Colloids and Surfaces B: Biointerfaces (2019), doi: https://doi.org/10.1016/j.colsurfb.2019.110627

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Chitosan and their derivatives: Antibiofilm drugs against pathogenic bacteria

Running title: Antibiofilm properties of chitosan and their derivatives

Fazlurrahman Khan1, Dung Thuy Nguyen Pham2, Sandra Folarin Oloketuyi3, Panchanathan Manivasagan1, Junghwan Oh4 and Young-Mog Kim1, 2* Marine-Integrated Bionics Research Center, Pukyong National University, Busan 48513, Korea

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Department of Food Science and Technology, Pukyong National University, Busan 48513, Korea

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Laboratory of Environmental and Life Sciences, University of Nova Gorica, Vipavska 13, SI-5000,

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Department of Biomedical Engineering, Pukyong National University, Busan 48513, Korea

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Corresponding author: Young-Mog Kim

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Nova Gorica, Slovenia

Phone: +82-51-629-5832; Fax: +82-51-629-5824

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E-mail: [email protected]

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Graphical Abstract

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Highlights

Biofilm formation by pathogenic bacteria is one of the major resistance mechanisms.

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Chitosan is a natural product exploited for combating bacterial infections.

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Several alternative strategies have been developed for the application of chitosan.

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Strategies include chemical modifications or combination with another active agent.

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Abstracts Biofilm formed by several pathogenic bacteria results in the development of resistance against antimicrobial compounds. The polymeric materials present in the biofilm architecture hinder the entry of antimicrobial compounds through the surface of bacterial cells which are embedded as well as enclosed beneath the biofilm matrix. Recent and past studies explored the alternative approaches to inhibit the formation of biofilm by different agents isolated from plants, animals,

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and microbes. Among these agents, chitosan and its derivatives have got more attention due to

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their properties such as biodegradability, biocompatibility, non-allergenic and non-toxicity. Recent researches have focused on employing chitosan and its derivatives as effective agents to inhibit

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biofilm formation and attenuate of virulence properties by various pathogenic bacteria. Such

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antibiofilm activity of chitosan and its derivatives can be further enhanced by conjugation with a wide range of bioactive compounds. The present review describes the antibiofilm properties of

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chitosan and its derivatives against the pathogenic bacteria. This review also summarizes the mechanisms of biofilm inhibition exhibited by these molecules. The knowledge of the antibiofilm

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activities of chitosan and its derivatives as well as their underlying mechanisms provides essential

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insights for widening their applications in the future.

Keywords: Antibiofilm; biofilm inhibition; chitosan; chitosan derivatives; pathogenic bacteria

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Introduction The biofilm formed by pathogenic bacteria is considered as one of the major resistance mechanisms against the rationally used antibiotics [1-3]. Biofilm is a structural polymeric architecture composed of extracellular polysaccharides, extracellular DNA (e-DNA) and proteins, which act as a barrier for the antimicrobial compounds used during the treatment [4-6]. Biofilm formation involves different complex stages from the initial attachment to biotic or abiotic surfaces,

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maturation to dispersal [7]. In the current scenario, research is focused on development of

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alternative strategies to combat the pathogenesis by inhibiting biofilm formation at the initial stage and also eradicating the pre-formed mature biofilm by several active agents [8-15]. These agents

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are either chemically synthesized or isolated from organisms such as plants, animals and

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microorganisms [16, 17]. However, reports showed that the applications of these antibiofilm drugs pose some cytotoxicity effects on the cells [18-21]. Thus, further research is extended to search

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for a biocompatible, biodegradable, non-toxic, non-allergenic, cost-effective and ecologically safe antibiofilm molecule from biological origin. Chitosan holds the above-mentioned properties and

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is identified as a potent antimicrobial and antibiofilm compound [22-27]. Chitosan is a linear polycationic amino-polysaccharide composed of D-glucosamine (GlcN) and N-acetyl glucosamine (GlcNAc) linked by β-1, 4-glycosidic bond. Chitosan is derived from the partial alkaline deacetylation of chitin, which is the second largest polymer after cellulose, present in the body of

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insects, crustacean, molluscs, etc.) by the process of partial N-deacetylation using chemical methods or by the action of microbial enzymes [28, 29]. It has been applied in several fields such as agriculture, pharmaceuticals, foods, and biomedical [23, 24, 27, 30, 31]. Water insolubility, high viscosity, and tendencies to coagulate proteins at high pH are the limitations associated with its application [27, 32]. Thus, the modified form of chitosan (by chemical means) [32] as well as low

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molecular weight form of chitosan named chitooligosaccharides (COS) were exploited [27, 29, 31]. Chitosan is either prepared from chitin through homogenous and/or heterogeneous deacetylation [33] while COS and other derivatives of chitosan are produced from chitosan either by enzymatic or acid hydrolysis [34] (Figure-1). Reports found that the molecular weight and degree of deacetylation of chitosan and COS are important factors for their biological activities [35-37]. Moreover, chitosan and COS are also employed as a carrier molecule for the antibiofilm

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drugs that are chemically synthesized or isolated from other organisms in order to minimize the

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limitations of traditional delivery systems, which involves poor stability, uncontrollable and unsustainable drug release and development of acquired resistances [38-44]. The present review

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article describes different size ranges of chitosan and COS used as the biofilm inhibitors against

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various pathogenic bacteria, namely, Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumonia [12, 45-48]. This

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review also discusses the application of chitosan and COS conjugates with other active agents as

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composites for biofilm treatment in synergistic ways.

Biofilm formation by pathogenic bacteria as a challenge A majority of bacteria possess the ability to form biofilm in the environment where they express a different phenotypic characteristic from other planktonic bacteria [49-51].

This phenotype

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involves the formation of the extracellular polymeric substances (EPS) where cells are embedded and serve as a form of protection against shear forces, antimicrobial agents and other extreme conditions [6, 52]. Over decades, biofilm formation has remained a serious threat to different fields such as food, textile, pharmaceutical, metallurgy and biomedical. Furthermore, during hospitalization, patients are exposed to biofilm-forming pathogens from sources within the

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environment including medical devices, catheters, equipment, other infected patients, and healthcare staff, thus, they are susceptible to the risk of nosocomial infections [53]. With regards to food industry, the contact surfaces serve as a good substrate for bacterial colonization, growth and biofilm development [54-56]. Despite the application of surface-cleaning, disinfections and hygienic conditions, these strategies have failed to completely inhibit and eliminate biofilm [45]. These sessile cells are transferred into the foods, strived and could cause

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food spoilage and foodborne infections to the consumers [57, 58]. Regarding to metal industries,

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biofilm formation is also of great interest to material scientists and engineers as bacteria can also form biofilm on the surfaces of ship hulls, heat exchangers, wastewater pipelines and valves,

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resulting in biofouling and corrosion [59-61]. Basically, some bacteria possess the ability to

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degrade metallic materials (e.g. steel and iron) by forming biofilms [62, 63]. Such metals corrosion causes discoloration of surfaces, health issues and environmental impacts, resulting in serious

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economic burden to the industries [64-66]. It has been shown that Burkholderia cepacia exhibited the biofilm-forming characteristics by resisting physiochemical and metal stress, B. cepacia

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possess appendages, expresses virulence genes such as rpoN sigma factor, rpfR, fatty acid signalbased, cciIR and cepIR quorum-sensing system which contributed to its biofilm formation [67-69]. Depending on the surface and metallurgical characteristics of certain metal (e.g. steel), the biofilm forming bacteria such as Serratia marcescens initially attach to the metal surface and start biofilm

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formation, resulting in the corrosion and deterioration [70]. Overall, the formation of biofilm by pathogenic bacteria on numerous biotic and abiotic surfaces have posed a serious threat to public health. To combat this issue, different strategies have been researched on using chemical treatments such as disinfectants, physical or mechanical methods by

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cleaning biofouling surfaces as well as combinatory methods. Despite all efforts to develop effective antibiofilm substance, problems associated with biofilm formation still persist.

Antibiofilm properties of chitosan and chitooligosaccharides Sourced from partial alkaline deacetylation of chitin, chitosan is consisted of two monosaccharides: glucosamine (GlcN) and N-acetyl glucosamine (GlcNAc). There are three reactive functional

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groups located on each monosaccharide unit, one amino group at C-2 position and two hydroxyl

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groups at C-3 and C-6 positions. The varied ratios of two monosaccharides GlcN and GlcNAc give rise to different primary physiochemical properties of chitosan such as degree of deacetylation

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(DD), molecular weight (MW) and viscosity [71]. DD and MW greatly determine antimicrobial

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and anti-biofilm activities of chitosan, and DD further determine chitosan solubility and viscosity [72]. Previous studies have agreed that the unique antibiofilm property of chitosan is mainly

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attributed to its polycationic nature given by the functional amino groups (NH2) of Nacetylglucosamine units [73-76]. The positive charge of chitosan is expected to react

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electrostatically with the negatively-charged biofilm components such as EPS, proteins and DNA, resulting in an inhibitory effect on bacterial biofilm [44, 77]. Likewise, the antimicrobial property of chitosan and COS was also derived from the interaction of positively-charged chitosan and negatively-charged residues such as carbohydrate, proteins and lipids present on the microbial

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cytoplasmic or cell membrane [78]. These types of interactions change the cell membrane permeability and result in the leakage of cytoplasmic content, which ultimately leads to cell death [37, 78-81]. This ability to electrostatically interact with cell membrane components can be further enhanced by conjugation of chitosan with cationic antimicrobial peptides (AMPs), which has been evidenced to actively targeted the growth of a broad range of Gram-positive and Gram-negative

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bacteria [82, 83]. The polymeric nature of chitosan and COS also allows the chelation with several important metals such as calcium, zinc, magnesium which are required in the transcription and translation of the bacterial genes, thus this process get stopped and cell dies [37, 77, 84]. Chitosan has various applications in biomedical industry, pharmaceuticals, food preservation, wastewater treatment, and ophthalmology due to its antimicrobial and antibiofilm activities as well as environment-friendly biological properties such as biodegradability, biocompatibility, and safety

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[85-88]. Regarding the biomedical field, chitosan has been used to protect the medical devices

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such as catheters and orthopedic implants from infectious biofilm-forming methicillin-resistant Staphylococcus aureus (MRSA), P. aeruginosa and beta-hemolytic Streptococci [89]. Chitosan

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coating also eradicates a large amount of pre-existing Staphylococcus epidermidis and S. aureus

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viable biofilm formed on implant surfaces made of pure titanium, titanium alloy and stainless steel and cause significant bacterial cell shrinkage [90]. Such active protection of medical device surface

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by chitosan coating can be considered significant in reducing the high risk of device-related infections [91, 92]. Regarding to food preservation, chitosan has also been used as packaging

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material for various foods ranging from fruits, vegetables, seafood and processed meat in form of a gel, fiber, edible film, and nanoparticle [93, 94]. Chitosan and chitosan-based edible films being a biodegradable material with high antimicrobial property can effectively protect food nutritional qualities from moisture, gaseous and microbial spoilage and extend food shelf-life without causing

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health and environmental impacts [95, 96]. Overall, chitosan owing to its highly reactive chemical structure has been tremendously applied in numerous life aspects in order to prevent microbial attack and minimize health and environmental concerns. The biofilm inhibition property of chitosan and COS is exploited in different structural forms such as unmodified [47, 97], hydrogel [97], high molecular weight, low molecular weight, chemically-modified chitosan, chitosan

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nanoparticles [98, 99], and conjugates (conjugated with antibiofilm drugs, antibiotics and several antimicrobial agents). Furthermore, chitosan concentrations as well as treatment timing can also affect its antibiofilm activity [100].

Chemical modifications of chitosan and their antibiofilm properties The poor water solubility and high viscosity of purified chitosan partially challenge its activity and

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remain disadvantageous for their applications [101, 102]. Being a polycationic polymer in nature

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the chitosan dissolves only in organic acids such as acetic acid, formic acid and inorganic acids such as sulfuric acid and hydrochloric acid [103]. For this reason, modifications either by physical

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(blending) or chemical means (N-substitution, O-substitution, copolymerization, and grafting)

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have majorly been performed on the reactive functional amino group and hydroxyl groups of chitosan backbone under mild conditions. The modifications of chitosan and COS which results

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in improved water solubility can be performed by alkylation, acylation, quaternization and saccharization [104], resulting in products such as N, N, N-trimethyl chitosan (TMC),

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carboxymethyl chitosan (CMCS), hydroxypropyl trimethyl ammonium chloride chitosan (HACC) and COS, respectively (Figure-2). Despite physiochemical changes, the resulted chitosan derivatives with modified structure also exhibit anti-biofilm activity competitively to chitosan either in single form or in combination [42, 105-110]. Chemically modified chitosan with

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beneficial biological properties include water soluble chitosan and its derivatives-chitosan salts, zwitterionic chitosan and chitosan oligomers by enzymatic or chemical hydrolysis; conjugation of chitosan and its derivatives with anion substrates such as sodium alginate, hyaluronic acid, sodium cellulose sulfate, poly L-aspartic acid, pectin, carboxymethylcellulose, hydroxypropyl trimethyl ammonium chloride to form polyelectrolyte complexes and nanoparticles [111-115]. The intrinsic

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mechanism of biofilm inhibition by TMC, which is the product of chitosan reaction with methyl iodide, has remained unclear. The chitosan derivative was found to bind rapidly to pre-existing S. epidermidis and E. coli and subsequently eradicate a significant number of sessile bacterial cells [77]. Unlike TMC, CMCS was produced by applying O-substitution method to chitosan. CMCS was found to highly dissolve in aqueous solution, thus it is used to produce biomaterials, nanomaterials and drug delivery system [116]. In terms of bacterial biofilm inhibition, previous

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studies have employed CMCS grafted onto titanium and silicone orthopedic implanted surfaces

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together with dopamine and vascular endothelial growth factor in the former study and polydopamine in the latter study. Both studies showed that the inhibitory activity exhibited majorly

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towards adhesion stage of S. aureus, E. coli and Proteus mirabilis, respectively [117, 118]. HACC,

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on the other hand, was synthesized from the reaction of chitosan with glycidyl trimethyl ammonium chloride [105]. The N-substituted quaternized chitosan HACC was reported to inhibit

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Staphylococcus epidermidis biofilm formation at the initial adhesion stage by suppressing the expression of a biofilm-forming determinant known as polysaccharide intercellular adhesin-

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encoded gene locus ica [105].

Antibiofilm properties of low molecular weight chitosan With similar attempt as chitosan chemical derivatives and low molecular weight chitosan (LMWC)

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to overcome the naturally limited solubility and viscosity of chitosan, COS was produced either by chemical, physical, enzymatic or electrochemical degradation methods [29]. The chemical method employs acid (phosphoric acid, hydrochloric acid and nitrous acid) and oxidative reagents (hydrogen peroxide, ozone, potassium persulfate, and sodium perborate). The physical method employs ultrasonic, microwave and gamma rays. The electrochemical method uses Ti/Sb-SnO2

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and Ti/TiO2-RuO2 electrode. While physical method is economic and easy for modifications accordingly to specific purposes, the compatibility between chitosan and the blended polymers remains a challenge. The chemical methods, however, offer high specificity and lower price [119]. Recently, the enzymatic method using specific (chitosanases) or non-specific enzymes such as proteases, lipases, and amylase has become more preferable due to high production yield, minimized environmental risks as well as chemical conversion despite its high cost [29, 120, 121].

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The resulted COS with the altered glycosidic backbone has shorter chain length, along with free

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amino groups. LMW and degree of deacetylation (DD) are thus genuinely lowered (maximum 3.9 KDa and 20%, respectively) and aqueous solubility is increased, in comparison with pure chitosan

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[122]. Such advantages have benefited for COS biological functions, making them active fungal,

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bacterial, viral and tumor inhibitors, antioxidant agents and immuno-regulators as shown by several studies [123-127].

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A recent report showed that different molecular weight of COS exhibited antibiofilm, antivirulence and antihemolytic properties against S. aureus [109]. Another report also showed the biofilm

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inhibition properties of COS with molecular weight of 2000 Da against Cronobacter spp. [128] . Similarly, Quintero-Villegas et al. [129] also reported that anti-biofilm activity of COS against enteropathogenic Escherichia coli (EPEC) was varied accordingly to its DD, in which the lower was the DD value, the more effective was the COS biofilm inhibition. COS exhibited its activity

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at the initial adhesion of biofilm-forming cells onto human epithelial cells (HEp-2) and removed more than 90% of adherent cells. Although the study was unable to identify the molecular mechanism of such inhibition, a conclusion was drawn referring COS with low DD as a potential anti-adherence agent against E. coli. Similar observation was recorded by Rhoades et al. [130] and

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Altamimi et al. [131], in which COS treatment was reported to prevent up to 30% and 85% of E. coli and clostridia adhesion on the human epithelial cell (HT29), respectively.

Antibiofilm properties of chitosan in different nanomaterial forms With the advances in nanotechnology, several modified nanomaterial forms of chitosan has been developed such as nanoparticle, microsphere, hydrogel, beads, and nanofibers [78, 93, 99]. Since

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the structural barrier formed by the biofilm matrix in pathogenic bacteria is the major hindrance

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for most of the antimicrobial drugs, formulating chitosan in the form of nanomaterial enables the polymer to easily cross the biofilm matrix and kill the sessile cells. There are several methods such

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as spray drying, ionic-gelation, emulsion cross-linking and complex coacervation available for the

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preparation of chitosan nanoparticles [41, 78, 115, 132-134]. However, chitosan nanoparticles are now commonly prepared by ionic gelation, in which positively-charged chitosan is dissolved in

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acetic acid buffer at pH~5-5.5 with stirring and then electrostatically cross-linked with negativelycharged ions of tri-polyphosphate pentasodium (TPP) at room temperature, since this method is

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mild, time-saving and allows controlling the surface charge and size of resulted chitosan nanoparticles [99, 135].

Chitosan nanoparticles derive its anti-biofilm activity in a similar way as unmodified chitosan, which is due to the electrostatic interaction between positively charged chitosan molecules and

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negatively charged biofilm constituents such as extracellular polysaccharides, proteins and DNA [44, 77]. In most cases, chitosan with low MW is known to exhibit the highest anti-biofilm activity compared to medium to high MW, as having low MW allows high diffusion into biofilm membrane and reduces aggregation in the biofilm [99, 136]. The most important advantage in the application of nanoparticle forms of chitosan is that even at neutral pH, chitosan present inside the

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nanoparticle retains the positively charged amino groups [99]. Additional advantages of nanometric size, flexible structure and predictable kinetics have aided the nanoparticle penetration and stability against high temperature, enzymatic or microbial degradations [135, 137], recognizing it as an excellent drug-carrier as well as the encapsulation material. The antibiofilm activity of chitosan cross-linked to alginate microspheres (Figure 2) against S. aureus, E. faecalis, P. aeruginosa and P. vulgaris was reported to derive from their actively entrance through the

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bacterial biofilm [138]. The nitric oxide (NO)-releasing COS resulted from N-diazeniumdiolate

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modification of 2-Methylaziridine COS has also been investigated [107]. Results have shown that the biofilm-disrupting effect of NO-releasing chitosan oligosaccharide against P. aeruginosa could

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be attributed to increasing NO storage or release and its rapid association with the biofilm-forming

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bacteria [108]. This mechanism has also been further improved by the presence of polyethylene glycol (PEG) chains (Figure 2), resulting in rapid penetration of nitric oxide through the biofilm

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matrix and killing of biofilm cells [107]. Apart from the antibiofilm properties of chitosan nanoparticles, it can be also used for the delivery of several other drugs that will be helpful for

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treating the complex biofilm forming pathogenic bacteria [98, 139-142]. In this way, several natural antibiofilm substances and metals can maximize their biofilm inhibition properties in conjugation with chitosan nanoparticle. For instance, Antimicrobial Photodynamic Inactivation (APDI) employed photosynthesizer methylene blue against methicillin-resistant S. aureus (MRSA)

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and multidrug-resistant (MDR) P. aeruginosa in combination with chitosan nanoparticles [143].

Similarly, Shrestha and Kishen [144] reported that the photo-activated rose bengal against Enterococcus faecalis (Figure 2) was significantly effective as a result of conjugation with chitosan nanoparticles. The action was found to derive from nanoparticle ability to disrupt the biofilm structure, thus enabling the contained antibiofilm agent to expose to cells within the structure.

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Chitosan nanoparticles loaded with antibiotic ciprofloxacin, coated by fucoidan polysaccharide was possibly able to electrostatistically interact with the negatively charged biofilm components, thus sustainably releasing the antibiotic and finally eradicating the Salmonella biofilm [145]. For stabilization property, natural antimicrobial agents such as essential oil and natural substances encapsulated within chitosan nanoparticles are effectively protected from environmental degradation. Clove oil carried by chitosan nanoparticles embedded on gelatin nanofibers

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drastically interrupted and removed Escherichia coli O157:H7 biofilm population formed in vitro

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[146]. Propolis naturally produced by honey bee encapsulated by chitosan nanoparticles effectively inhibited the growth of both planktonic or sessile multi-drug resistant Enterococcus

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faecalis cells by significantly altering the expression of most of the bacterial invasion genes and

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virulence genes [147].

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Chitosan as a carrier molecule for antibiofilm drugs

Recent and past studies showed that antibiofilm drugs isolated from different sources exhibited

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cytotoxicity effects, poor stability and water insolubility during the time of treatments [41, 148150]. In order to combat this limitation, the controlled release technology emerged where the antibiofilm drugs are loaded to different formulation of chitosan as carrier molecules (e.g. microspheres, nanofibers, hydrogels, beads, nanocomposites, nanoparticles) [78, 87, 98, 102, 151-

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154]. These carrier molecules allow the controlled release of antibiofilm drugs such as quercetin [155], cinnamaldehyde [156], ferulic acid [157], caffeic acid [158], lysostaphin [159] and kaempferol [139] which resulted in prolonged efficiency and the least cytotoxicity effects. Due to the biodegradability, biocompatibility and cost-effectiveness of chitosan and COS, it has been also exploited for the dosage forms preparation and delivery of several antibiofilm drugs. Delivery of

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antibiofilm drugs to the site of biofilm matrix can also be considered as the indirect role of chitosan in biofilm inhibition [108]. Different approaches are adapted for the immobilization/conjugation of active agents to different structural forms of chitosan. For example, cross-linking multilayer of chitosan and alginate onto titanium surfaces increased loading capacity of the desired antibioticminocycline, which in turn removed S. aureus initial adhesion and colonization [160]. Immobilization of chitosan-lauric acid conjugate onto titanium surfaces triggered similar biofilm

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inhibitory action, which was to disrupt S. aureus and P. aeruginosa adhesion [161].

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Photosensitizers such as Rose Bengal being cross-linked to chitosan in Photodynamic therapy exhibited bacteriostatic activity towards sessile cells (Figure-2) [143, 144]. Chitosan stabilized

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onto metal nanoparticles such as silver and gold nanoparticles was shown to disrupt a significant

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amount of pre-existing bacterial biofilms [162, 163]. Diffusion of chitosan-embedded metal nanoparticles was proposed to limit the production of exopolysaccharide and suppress intra-

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biofilm activities [164]. Xu and colleagues [165] have formulated sodium tri-polyphosphatemodified N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride nanoparticle based on

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ion gelatin method as a protein carrier using bovine serum albumin as a model drug. The in-vitro drug release studies showed a slow and continuous release rate of about 45%, though sodium tripolyphosphate decreased the release rate, but the encapsulation efficiency (the percentage of drug that is successfully entrapped into the nanoparticle) was improved. Similarly, Tan et al. [133]

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showed the functionality of positively charged chitosan nanoparticles prepared by ion gelatin method, loaded with oxacillin and Deoxyribonuclease I (DNase I) as a potential carrier for drug carrier against S. aureus biofilm on silicone platelets with an in-vitro drug (oxacillin) release rate of 88.4% and 100% antibiofilm activity at the concentration ≥0.5 µg/ml. The chitosan nanoparticles displayed the ability to facilitate oxacillin diffusion and penetrate through the biofilm

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matrix, disrupting mature biofilm formed on the surface by DNase, thereby causing about 97% biofilm reduction. The released NO from chitosan-loaded NO is involved in the disruption of the structural integrity of biofilm architecture and leads to the eradication of biofilm. The mechanism of biofilm inhibition is similar to antimicrobial activity [166, 167] by exerting nitrosative and oxidative stresses to the component of biofilm matrix such as extracellular-protein, exopolysaccharide and e-DNA [168-170]. Apart from the antibiofilm inhibition properties [109,

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128], COS has also been exploited for the synthesis of metallic nanoparticles which acted as a

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potent antibiofilm agent [171]. Similarly, the application of COS has also been used for the treatment of biofilm-forming pathogenic bacteria either in conjugate form or in combination with

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another active agent [42, 107, 108].

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Synergistic action of chitosan in combination with other active agents Inhibition of biofilm using the combination of two different drugs is considered as a promising approach to combat bacterial pathogenesis and biofilm formation [10, 42, 172]. Although the

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combinatorial effects of several antibiotics are commonly reported, the structural part of the biofilm matrix remains a barrier for the entry of the antibiotics [173-175]. The recent strategy is adopted by combining two drugs in which one disrupts the biofilm matrix and interferes the

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quorum sensing signaling circuit while the others enters inside the matrix and directly acts on the cellular levels. Furthermore, nanotechnology-based delivery system has made it much easier where they can easily cross the biofilm matrix in a controllable manner and perform sustainable drug release. Some reports are available on LMW chitosan nanoparticles which can easily cross the biofilm matrix and kill the bacterial cell [99, 147, 176]. Immobilization of either antibiotics or

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antibiofilm drugs to the chitosan nanoparticles is very helpful in biofilm inhibition, followed by complete killing of the cells in a synergistic way. He et al. [42] reported that by conjugating onto a low molecular weight alginate-derived oligosaccharide, COS and azithromycin were found to effectively inhibit the growth of planktonic and sessile P. aeruginosa by quorum sensing modulation, suppression of virulence factors such as elastase B and pyocyanin, thus inhibiting biofilm formation and reducing antibiotic resistance. Chitosan nanoparticles encapsulated with

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cinnamaldehyde also inhibited P. aeruginosa biofilm formation via anti quorum-sensing activity

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and motility alteration [156]. Apart from QS and QS-related virulence factors, the antibiofilm activity of chitosan and its derivatives was widely proposed to derive from the electrostatic

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interaction between their positively charged amino group and the negatively charged constituents

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of biofilm matrix such as DNA, techoic acid and surface proteins. For instance, this effect of chitosan has been synergistically combined with chlorhexidine by Decker and colleagues [177] in

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their attempts to inhibit the initial adherence of Streptococcus sanguinis to human enamel which ultimately prevents caries and periodontal diseases. Antibiotics such as streptomycin, gentamycin

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and cefotaxime upon conjugating with chitosan also effectively penetrated through biofilm matrix and exhibited significant antibiofilm action against a wide range of Gram-positive and Gramnegative bacteria [43, 48, 73, 178]. Gentamycin sulphate encapsulated to Polyvinyl AlcoholPolyethylene Glycol coating with chitosan showed significant antibiofilm activity in comparison

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to coating without chitosan [179]. Not only compounds, enzymes which was immobilized with modified chitosan also showed inhibition of biofilm in a synergistic mode of the action. For example, the enzyme DspB (β-N-acetylglucosaminidase) isolated from Aggregatibacter actinomycetemcomitans CU 100 synergistically exhibited antibiofilm activity against S. epidermidis and S. aureus with carboxymethyl chitosan [41]. Besides, the metabolic state of

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bacterial biofilm-forming population has been reported as another target of chitosan combination by Zhang et al. [180]. They demonstrated that inulin conjugated chitosan increased readily eradicated the preformed mature biofilm of S. aureus, Streptococcus hyovaginalis and P. aeruginosa as compared to conventional antibiotics by interfering with the bacterial metabolism. Overall, chitosan as well as its derivatives can synergistically cooperate with other antibiofilm agents to inhibit bacterial biofilm formation and eradicate preformed biofilm via multiple targets

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which are (1) biofilm matrix constituents, (2) QS system and (3) metabolism.

Conclusion and future perspectives

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Chitosan is a nontoxic, biodegradable, biocompatible and commonly found marine product which

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is exploited in different fields such as chemistry, nanotechnology, biomedical, environmental, and agriculture for different purposes. Chitosan and its derivatives are known as excellent agents for

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inhibiting biofilm formation by a wide range of pathogenic bacteria. One major limitation for application of chitosan is the insolubility in water at neutral pH, leading to different physical and

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chemical modifications, alteration in sizes, and formulation in various forms in order to improve their applications. Different forms of chitosan and COS (Figure 3) can be used in (1) inhibition of initial adhesion stage, (2) disruption of matured biofilms and (3) inhibition of QS system as well as QS-regulated virulence factors production (Figure 4). The primary mechanism of antimicrobial

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and antibiofilm properties of chitosan and COS is widely accepted as the electrostatic interaction between the positively charged amino group of chitosan and negatively charged constituents of biofilm matrix such as exopolysaccharides, DNA, surface proteins and lipids. This interaction leads to the change of membrane permeability and dispersal of biofilm matrix. In recent trends, chitosan and COS are also used in conjugation with several antimicrobial and antibiofilm

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compounds isolated from different sources for synergistic inhibition of bacterial biofilms. Furthermore, chitosan and COS act as a suitable carrier molecule for several antibiofilm drugs to the infection site and help in the controllable and sustainable release of drugs. Although significant achievements in the application of chitosan and their derivatives for the treatment of bacterial biofilms have been reported, still there are several aspects/issues that need to be addressed as future perspectives;

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1. The antimicrobial and antibiofilm properties of chitosan and its derivatives are usually

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functional under the acidic condition. Therefore, its clinical application is limited as the body pH is close to the neutral. Thus, modification and formulation of chitosan and their

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derivatives should be done in such a way so that it can function in neutral pH.

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2. Future study is required to check the antivirulence properties of chitosan and its derivatives at molecular and biochemical levels of pathogenic bacteria.

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3. Targeting the QS signaling circuit in biofilm-forming pathogenic bacteria is another approach to minimize the pathogenesis [181]. Hence, quenching of QS signaling circuits

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by chitosan and its derivatives must be also investigated. 4. Furthermore, the in-vivo trial/test of these antibiofilm chitosan needs to be conducted. 5. Preparation of nanoparticles especially the low molecular weight chitosan would be promising to eradicate the biofilm and show antimicrobial actions since nanoparticles can

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diffuse rapidly and penetrate through the biofilm matrix [99, 182]. Also, chitosan in the

form of nanoparticles retains their positive charge, which helps in the interaction with bacterial cell membrane as well as the components of the biofilm matrix.

6. Combination of chitosan nanoparticle with antibiotics and antibiofilm drugs would be very promising for treatment of bacterial infections.

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Funding This work was supported by Marine Biotechnology Program (Grant number 20150220) funded by Ministry of Oceans and Fisheries, Republic of Korea. This research was also supported by Basic Science Research Program through the National Research Foundation of Korea funded by the

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Ministry of Education (NRF-2019R1A2C1087156).

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Compliance with ethical standards

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Conflict of interest

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The authors declare that they have no conflict of interest.

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Research involving human participants and or animals

This article does not contain any studies associated with human participants or animals

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performed by any of the authors.

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Figure legends Figure-1. Different sources of chitin and possible methods for the preparation of chitosan and

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chitooligosaccharides (Information obtained from literature) [34, 140].

Figure-2. Chemical structure of different types of chemically modified and conjugated forms of

chitosan. The information obtained from the literature such as N,N,N-trimethyl-chitosan [77], HACC [105], chitosan-rose bengal [134], chitosan-streptomycin [48] carboxymethyl chitosan

34

[106, 193], chitosan-caffeic acid [158], PEGylated 2-methyl aziridine-chitosan oligosaccharide

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[107] and chitosan-alginate [138].

35

of ro -p re lP ur na Jo Figure-3. Flow chart of different forms of chitosan and their derivatives.

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Figure-4. Antibiofilm and antivirulence properties of different forms of chitosan.

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Table 1 List of chitosan and their derivatives as antibiofilm drugs against the different pathogenic bacteria Molecu lar weight (KDa)

Chitosan (HMW) Chitosan (LMW)

624 107

Degree of deacetyl ation (%) >75 75-85

Chitosan (HMW)

624 107

>75 75-85

Vancomycin-resistant Staphylococcus aureus, Vancomycin-resistant Enterococcus faecalis

Chitosan beads

125

13

Acidithiobacillus ferrooxidans

Chitosan (shrimp) Chitosan (crab)

NA

75 70.79 ± 0.04

Methicillin-resistant Staphylococcus aureus

lP

na

Pathogenic bacteria

re

Chitosan/chitooligosac charides

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ur

Chitosan (LMW)

Streptococcus mutans

39

Inhibitory Activity

References

HMW chitosan was recommended for effectively inhibiting adhesion and eradicating mature biofilm of Streptococcus mutans. In both vancomycin-resistant bacteria tested, chitosan actively inhibited (1) the growth of planktonic cells and (2) adhesion and biofilm formation of sessile cells. These activities were claimed to derive from bacterial cell wall permeability in the presence of chitosan. Acidithiobacillus ferrooxidans was stabilized and treated by chitosan flow released chitosan beads Chitosan inhibited the bacterial biofilm formation, reduced the production of staphyloxanthin and disrupted the EPS matrix of mature

[183]

[184]

[154]

[185]

of ro

NA

NA

Vibrio parahaemolyticus

Carboxymethyl chitosan

30

90

Chitosan (LMW)

107

75-85

Lactobacillus gasseri, Streptococcus salivarius, Rothia dentocariosa, and Staphylococcus epidermidis Methicillin-resistant Staphylococcus aureus (MRSA), Methicillinsusceptible Staphylococcus aureus (MSSA) Methicillin-resistant Staphylococcus epidermidis (MRSE) Streptococcus mutans

Jo Chitosan

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lP

na

ur

Chitosan

-p

Chitosan

30005000

≥75

30005000

70

Hydroxypropyl20.091.83 trimethyl ammonium 3.0 x chloride chitosan 104 (HACC)

biofilm without causing cytotoxicity. Chitosan showed antibacterial [186] activities, inhibited biofilm formation and eradicated the preexisting mature biofilm. Carboxymethyl chitosan inhibited [106] multi-species biofilm as well as metabolic activities within the biofilm. Chitosan actively inhibited MSSA [78] and MRSA growth and biofilm formation while prevented MRSE adhesion.

Chitosan at all tested [100] concentrations inhibited bacterial adhesion; whereas chitosan at the highest concentration inhibited biofilm accumulation through prolonged exposure time. Chitosan reduced both planktonic [187] cell growth and biofilm viability of Streptococcus mutans.

Streptococcus mutans ACTT 25175 and Streptococcus sanguinis ACTT 10556 Staphylococcus aureus and Increasing HACC concentration Staphylococcus inhibited biofilm formation by epidermidis disrupting the expression of an

40

[105]

of Chitosan (MMW) Crab shell Chitosan Chitosan low viscous Chitosan middle viscous

Medium

75–85 >85

Chitosan (HMW) Chitosan (LMW)

535.4 97.8

lP

na

≥ 90 ≥ 90

150 50

> 75 75-85

Chitosan (HMW) Chitosan (LMW)

624 107

>75 75-85

Chitosan (LMW)

107

75–85

Jo

ur

Chitosan (HMW) Chitosan (LMW)

ro

0.82

Actinomyces naeslundii, Streptococcus oralis, Streptococcus mutans, Staphylococcus mutans, Streptococcus sobrinus Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae

-p

182

re

Chitosan

Klebsiella pneumoniae and Escherichia coli

extracellular polysaccharidesencoded gene named as icaA. Chitosan treatment minimized the [188] initial adhesion, biofilm formation and growth of most tested bacteria.

Chitosan coating actively disrupted bacterial biofilmforming cells on device’s surface over a long period of time.

Inhibition and eradication of Escherichia coli and Klebsiella pneumoniae biofilm were rational to exposure time to chitosan Klebsiella pneumoniae and The antibiofilm and antimicrobial Escherichia coli activities of chitosan, which acted on separating and destroying bacterial cell membrane, were dependent on MW and environmental pH. Methicillin-resistant Chitosan of both low and high MW Staphylococcus aureus and at low concentration actively methicillin-sensitive inhibited the bacterial growth and Staphylococcus aureus biofilm formation by MRSA and MSSA. Staphylococcus aureus, Chitosan inhibited the growth of Staphylococcus all bacteria and Staphylococcus epidermidis, Acinetobacter epidermidis biofilm formation and baumannii metabolism.

41

[189]

[47]

[92]

[190]

[191]

of

Staphylococcus aureus, Staphylococcus epidermidis, Acinetobacter baumannii

Carboxymethyl chitosan NA

90

Low viscosity chitosan

~80

Lactobacillus gasseri, Streptococcus salivarius, Rothia dentocariosa and Staphylococcus epidermidis Staphylococcus epidermidis

-p

ro

75–85

re

107

150

lP

Chitosan (LMW)

75–85

na

Chitosan (MMW) 190Pronase-treated chitosan 300

Jo

ur

Chitosan sponge

251 17

± 82.5 1.7

Chitosan nanoparticulates

NA

NA

Chitosan nanoparticles

NA

NA

Listeria monocytogene, Salmonella enterica, Staphylococcus aureus, Pseudomonas fluorescens and Bacillus cereus ± Staphylococcus aureus, Pseudomonas aeruginosa and co-culture of Staphylococcus aureus and Pseudomonas aeruginosa Enterococcus faecalis

Enterococcus faecalis, Streptococcus mutans

42

Biofilm formation of [192] Staphylococcus epidermidis which cause catheter-related bloodstream infections was actively reduced by chitosan. Carboxymethyl chitosan reduced [193] biofilm production on silicone surface in a long term.

Low viscosity chitosan exhibited bacteriostatic and bactericidal activities in concentration and pHdependent manners. Chitosan exhibited various levels of biofilm inhibition. Pronasetreated chitosan further increased the antimicrobial effectiveness as compared to MMW chitosan. Chitosan sponge was shown as a potential delivery system for 2 antibiotics (vancomycin and amikacin) against the bacterial biofilm formation. Similar to ZnO nanoparticle, chitosan nanoparticles dispersed the pre-existing biofilm structure. The antibacterial activity of both nanoparticles was maintained over a long time period. Chitosan nanoparticles reduced individual- and mixed-species biofilm of Enterococcus faecalis

[194]

[45]

[195]

[182]

[196]

of N,N,Ntrimethylchitosans

4.09±0. 83 x 102, 1.96±1. 37 x 102 and 2.86±0. 47 x 102

NA

Pseudomonas aeruginosa

lP

na

Staphylococcus epidermidis and Escherichia coli

Jo

ur

NA-not available

ro

NA

-p

NA

re

2-methylaziridinemodified chitooligosaccharide (MMW)

43

and Streptococcus mutans. Synergism between chitosan nanoparticles with ozonated olive oil effectively eradicated the mature mixed-species biofilm. 2-methylaziridine-modified [108] chitooligosaccharides actively altered Pseudomonas aeruginosa biofilm viscoelasticity as well as the bacterial cell membrane by releasing nitric oxide. N,N,N-trimethyl chitosan actively [77] bound and eradicated the preformed biofilms by both Grampositive and Gram-negative bacteria.

of

NA

Jo

ur

na

ChitosanNA Polyvinyl alcohol (PVA)Polyethylene glycol (PEG) Chitosan NA

Chitosan

NA

NA

NA

Pathogenic bacteria Inhibitory Activity

-p

Active agents

re

Chlorhexidine

lP

Chitosan/chitooligosaccharides Name Molecular Degree weight of (KDa) deacetyl ation (%) Chitosan NA NA

ro

Table 2 Conjugates form of chitosan and their derivatives with active agents involved in biofilm inhibition in a synergistic way

Gentamycin sulphate

Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa

Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli

Copper oxide Bacillus subtilis, nanocomposite Pseudomonas aeruginosa, Escherichia coli and Penicillium notatum Protease (from- Staphylococcus Bacillus aureus, Listeria licheniformis) and monocytogenes and neutrase (B. Pseudomonas amyloliqueaeruginosa faciens)

44

References

By loading to [197] monotmorillonite/chitosan nanocomposite, the released of chlorhexidine was prolonged and controlled, thus their antibiofilm activity was improved and cytotoxicity was lowered. Coating of chitosan-PVA-PEG [179] prevented bacterial adhesion and biofilm formation on silicone catheter surface with relatively good biocompatibility. The chitosan–copper oxide [198] nanocomposite dispersed preexisting biofilm of Bacillus subtilis and Pseudomonas aeruginosa The protease- and neutrase- [28] immobilized chitosan showed significant inhibition against bacterial biofilm.

NA

NA

Chitosan

NA

NA

of

Carboxymethyl chitosan

Kaempferol

Chromobacterium violaceum CV026

Encapsulation of kaempferol onto [139] chitosan nanoparticles inhibited biofilm formation of Chromobacterium violaceum by altering quorum sensing system. Metallic salts Staphylococcus Carboxymethyl chitosan [32] combining with metallic salts aureus, Staphylococcus exhibited antibiofilm activity epidermidis, Kocuria against all tested bacteria. rhizophila, Pseudomonas aeruginosa and Burkholderia cepacia Carboxymethyl Streptococcus The nanocomposite formulations [199] starch and mutans in terms of size, morphology and montmorillonite concentrations of chitosan and nanoclay Carboxymethyl starch was optimized for optimal curcumin release against Streptococcus mutans Soybean milk and Streptococcus Addition of chitosan and IgY [200] immunoglobulin mutans significantly reduced dental IgY biofilm formation. Lysostaphin Staphylococcus Lysostaphin-containing chitosan [159] aureus SA113 hydrogel reduced planktonic MRSA growth. Increasing concentration of lysostaphin inhibited MRSA biofilm formation. Clove oil Escherichia coli Clove oil-loaded chitosan [146] nanoparticles exhibited high antibacterial and antibiofilm

ro

75–85%

-p

NA

NA

Jo

Chitosan

ur

na

lP

re

Chitosan (LMW) nanoparticle

NA

Chitosan

100–150

85%

Chitosan nanoparticle

NA

85%

45

of

Chitosan nanoparticle

~100–150

~85

Chitosan

~115

ro

NA

-p

NA

[138]

na

lP

re

Chitosan (LMW) Chitosan (MMW)

against Escherichia coli population. Incorporation of this conjugate into gelatin nanofibers helped maintaining colour and flavours. Alginate Staphylococcus Chitosan-alginate microspheres aureus, showed antibacterial and Enterococcus antibiofilm activity against faecalis, common human health-related Pseudomonas bacteria. The mechanism was aeruginosa, Proteus proposed to be the cell membrane vulgaris permeability and acidic pH. Ciprofloxacin and Salmonella paratyphi Both non-coated ciprofloxacin fucoidan A loaded chitosan nanoparticles (cCNPs) and fucoidan coated cCNPs was effectively entered through cellular compartments and induced biofilm dispersal. Quercetin and Bioengineered E. Combination with chitosan has baicalein coli Top10 biosensor improved the flavonoids inhibitory activity towards QS system and biofilm formation and prevented flavonoids accumulation. Silver nanoparticles Methicillin-resistant Loading of high concentration of Staphylococcus chitosan gel onto silver aureus and nanoparticles prevents bacterial Pseudomonas growth and biofilm production aeruginosa Azithromycin Pseudomonas Chitooligosaccharides and aeruginosa azithromycin combination inhibited bacterial growth and

Jo

ur

~42

Chitosan gel

190-375

80.7

Chitooligosacchar ides

250

∼65

46

[145]

[155]

[162]

[42]

of 50–90

≥85%

Amikacin, clindamycin, vancomycin erythromycin Cloxacillin

Chitosan

150

NA

Hyaluronic acid

Chitosan

NA

NA

na ur 369 ± 4 1278 ± 8 2520 ± 9

86 ± 3 89 ± 2 85 ± 3

∼310 KDa

≥90%

Jo

Chitosan (LMW) Chitosan (MMW) Chitosan (HMW)

Chitosan

Listeria monocytogenes

and

re

lP

~88

ro

3 and 13 180

-p

Chitosan (LMW) Chitosan (HMW) Chitosan (LMW)

Staphylococcus spp.

Staphylococcus aureus and Escherichia coli

Iron oxide coated Methicillin-resistant graphene oxide Staphylococcus nanocomposite aureus, Staphylococcus aureus, Escherichia coli

Gold nanoparticle

Staphylococcus aureus and Pseudomonas aeruginosa

Polypyrrole

Pseudomonas aeruginosa PAO1

47

biofilm formation by altering quorum sensing system. Combination with chitosan [178] enhanced the anti-biofilm efficacy of tested antibiotics.

Chitosan-cloxacillin combination promoted cloxacillin bactericidal activity, biofilm inhibition and eradication of mature biofilm. Chitosan/hyaluronic acid coating helped inhibiting bacterial growth and biofilm formation on the silicone surface. The chitosan−iron oxide coated graphene oxide nanocomposite hydrogel can be considered as a nontoxic nanomaterial that exhibited antimicrobial and antibiofilm activities by disrupting the bacterial cell wall and cell membrane. Chitosan with medium MW and the highest DD grafted to gold nanoparticles shows antibiofilm activity against Staphylococcus aureus and Pseudomonas aeruginosa by disrupting bacterial cell wall with no cytotoxicity to mammalian cells. Chitosan-polypyrrole nanocomposites inhibited the bacterial biofilm formation,

[201]

[202]

[203]

[163]

[102]

of ro

NA

95.6%

ZnO

Chitosan oligosaccharides

>10 kDa

NA

Gold nanoparticles

Chitosan

13

88

Chitosan

re

Pseudomonas aeruginosa PAO1

lP

na ur NA

Jo

Chitosan

Pseudomonas nigrifaciens

-p

Chitosan

NA

NA

NA

Streptomycin and Listeria gold nanoparticles monocytogenes, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium Liposome coating Listeria antibacterial monocytogenes peptide Apep10

Silver nanoparticles Staphylococcus aureus

48

eradicated the mature biofilm and reduced the production of key virulence factors. The antibiofilm and antibacterial [204] activities of chitosan-ZnO nanocomposites was derived from chitosan and reactive oxygen species released by ZnO. Capping gold nanoparticles with [171] chitooligosaccharide helped enhancing the antibiofilm and anti-hemolytic activities of the nanoparticles. Chitosan and streptomycin was [205] effectively delivered through the bacterial biofilm by gold nanoparticles, thereby inhibiting biofilm formation by Grampositive and Gram-negative bacteria.

Chitosan coating enhanced [206] liposome physical stability, thus supporting the antibacterial and antibiofilm activities of peptidecontaining liposome. Silver nanoparticles coated with [90] chitosan showed antibiofilm activity towards biofilm components (e.g. carbohydrates and proteins) without causing cytotoxicity.

3, 13 and 50, 18 and 88

Chitosan

220

Chitosan

NA

Staphylococcus epidermidis and Staphylococcus aureus

Listeria monocytogenes, Listeria welshimeri and Listeria innocua

Iron oxide Staphylococcus nanoparticles aureus

na

lP

85

of

Chitosan

Titanium (Ti), titanium alloy (Ti6Al-4V) and stainless steel (ASTM F139, 18Cr-14Ni-2.5Mo) 75 Gentamycin

ro

NA

-p

NA

re

Chitosan

Chlorhexidine

Enterococcus faecalis

Jo

ur

NA

Chitosan Oligochitosa n (LMW)

0.4–0.6 98 1.3, 2.6 and 4

Tilmicosin

Staphylococcus aureus

Chitosan (LMW)

50–190

Inulin

Staphylococcus aureus, Pseudomonas

<1

NA

49

Chitosan coating interfered with [207] bacterial cell shape, thus reducing bacterial adhesion and growth on the surface of orthopaedic implants. Chitosan facilitated gentamycin penetration through Listeria biofilm matrix, thus significantly improving antibiofilm activity of gentamycin. Chitosan-loaded iron oxide nanoparticle minimized bacterial growth and biofilm formation, which was possibly due to electrostatic interaction between chitosan and biofilm components (e.g. techoic acid and lipids) Chitosan combined with chlorhexidine was a potential alternative for sodium hypochloride in treating root canal irrigation caused by Enterococcus faecalis biofilm. Chitosan individually exhibited antibacterial and antibiofilm activities while showing synergism when combined with tilmicosin with minimum to no cytotoxicity effects. Conjugation between chitosan oligosaccharides and inulin increased the antibacterial and

[73]

[208]

[209]

[210]

[180]

of

-p

Titania nanotubes

re

2.0 x 105 91.83 (parental (parental chitosan) chitosan)

NA

NA

ur NA

Jo

Chitosan nanoparticle

Chitosan

250

antibiofilm activities and caused low toxicity to mammalian cells.

ro

4–6

Cinnamon oil

na

Chitosan

aeruginosa and Streptococcus hyovaginalis

lP

Chitosan oligosaccharide Chitosan oligosaccharide Hydroxypro pyltrimethyl ammonium chloride chitosan (HACC)

Methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus aureus and Staphylococcus epidermidis Staphylococcus epidermidis

NA

Rose bengal

Enterococcus faecalis

82.46 ± 1.679

Polymer Staphylococcus polyethylene glycol aureus

50

Enlarged-diameter Titania [110] nanotubes (160-200 nm) loaded with 27% HACC showed significant inhibition against staphylococcal adhesion and biofilm formation.

Coating the orthopaedic implant [211] surface with the combination of cinnamon oil and chitosan reduced Staphylococcus epidermidis biofilm formation on the surface. Rose bengal-chitosan [134] nanoparticles exhibited non-toxic antibacterial and antibiofilm activities by permeabilizing the bacterial cell membrane and improved dentin stability. Chitosan/PEG blended sponges [30] can be considered as an effective delivery system for antibiotics and antifungals to prevent polymicrobial musculoskeletal infections.

85

Chitosan hydrogel

NA

75-85

Chitosan

~13

NA

Ferulic acid

of

310

Pseudomonas aeruginosa, Listeria monocytogenes and Staphylococcus aureus

Ferulic acid grafted chitosan [157] conjugate exhibited killing effect to Listeria monocytogenes and Staphylococcus aureus while inhibited Pseudomonas aeruginosa growth and biofilm formation by cell membrane permeability. AmelogeninStreptococcus Chitosan hydrogel carrying QP5 [212] derived peptide mutans actively inhibited Streptococcus QP5 mutans biofilm formation and metabolism in a long term and enhanced remineralisation of enamel lesions. Streptomycin Listeria The presence of chitosan [48] monocytogenes, enhanced the susceptibility of Listeria welshimeri bacterial biofilm to streptomycin, and Listeria innocua, possibly by electrostatically Staphyloccocus interacting with the bacterial cell aureus, membrane components. Enterococcus faecalis, Pseudomonas aeruginosa and Salmonella typhimurium Polydopamine Escherichia coli Coating medical silicone surface [118] and Proteus with carboxymethyl chitosan mirabilis combined with polydopamine significantly reduced Escherichia coli and Proteus mirabilis adhesion and inhibited biofilm formation by these two bacteria.

NA

re

lP

na ur Jo Carboxymet hyl chitosan

-p

ro

Chitosan

≥75

51

of

NA

NA

Polyethylene glycol Staphylococcus aureus and Pseudomonas aeruginosa

Chitosan nanoparticle

NA

>75

Cinnamaldehyde

Chitosan oligosaccharides (MMW)

NA

NA

2-methyl aziridine

Pseudomonas aeruginosa

Chitosan oligosaccharide

~2500, 5000, 10,000 KDa

2-methyl aziridine

Pseudomonas aeruginosa

-p

re

lP

na

Pseudomonas aeruginosa PAO1

Jo

ur

NA

ro

Chitosan

52

Chitosan-polyethylene glycol paste released the antibiotics locally at a constant rate in comparison to unmodified chitosan. Cinnamaldehyde encapsulated chitosan nanoparticles reduced quorum-sensing regulated virulence factors production and motility, thus reducing biofilm formation. The nitric oxide releasing Ndiazeniumdiolates-modified chitosan oligosaccharide can effectively eradicated preexisting Pseudomonas aeruginosa biofilm in low oxygen environment. With improved water solubility, the N-diazeniumdiolatefunctionalized chitosan oligosaccharide synthesized by PEGylation of 2-methyl aziridine-grafted-chitosan oligosaccharide exhibited antibiofilm activity against P. aeruginosa by rapidly entering and eradicating the bacterial biofilm. This action caused low cytotoxicity to the mammalian cells.

[213]

[156]

[214]

[107]

NA

of

NA

Caffeic acid, ferulic Pseudomonas Grafting of chitosan with [158] acid, and sinapic aeruginosa and phenolic compounds increased acid Listeria the bacterial cell permeability, monocytogenes thus biofilm adhesion was inhibited and mature biofilm was eradicated.

-p

ro

Chitosan

Jo

ur

na

lP

re

NA-not available

53