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Science against microbial pathogens: communicating current research and technological advances
______________________________________________________________________________
A. Méndez-Vilas (Ed.)

Antimicrobial efficiency of functionalized cellulose fibres as potential

medical textiles
Tijana Ristić1,2, Lidija Fras Zemljič2, Monika Novak1,3, Marjetka Kralj Kunčič1, Silva Sonjak3, Nina
Gunde Cimerman3 and Simona Strnad2
1
Health care department, Tosama d.o.o., Vir, Domžale, Slovenia
2
Institute for engineering materials and design, Faculty of mechanical engineering, University of Maribor, Slovenia
3
Department of biology, Biotechnical faculty, University of Ljubljana, Slovenia

This chapter presents an overview of methods for cellulose fibres functionalization in order to introduce antimicrobial
activity. In view the need for ecologically friendly textiles antimicrobial finishing is introduced, together with some
strategies for the functionalization of fibres using biodegradable polysaccharides such as the use of chitosan. Additionally,
the methods used for the microbiological testing of these fibres are discussed and the current disadvantages of these
methods indicated. Moreover, a new strategy for a reliable methodology regarding the antimicrobial testing of oriented
fibre-based polymers such as cellulose is discussed, which could also be useful within several other polymer industrial
fields.

Keywords antimicrobial agents; medical textiles; cellulose fibres; microbiological tests; antimicrobial efficiency

1. Introduction
Textile materials (woven, nonwoven, knitted, and composites) have found different end-uses in medical and healthcare
applications. Depending on the specific end-use, different products have to meet the demands for the specific end-use
performances [1]. Irrespective of their applications, internal (surgical threads and various implants) or external (gauzes,
bandages, surgical masks, gowns and apparel, nappies, tampons, and so on), medical textiles have to be comprised of
basic bioactive properties, especially antimicrobial [2].
Common problem in hospitals and healthcare institutions is microbial contamination of surfaces, including textile
fabrics, which can lead to infections and consequently to cross-infections. Therefore, it is essential to reduce the
transmission of harmful microorganisms and spreading of the secondary infections within a curative environment. Not
only that hospital acquired infections are prolonging the healing of patients, and causing potential risks for serious
illness, but are also representing the extra costs to the health service.
Accordingly, it is extremely important that protective clothing (surgical masks, caps, gowns, etc.) and hospital linen
meet the demands for antimicrobial protection. Thus, materials for use in surgery have to ensure adequate protection
against microorganisms, biological fluids and aerosols, i.e. impermeability for microorganisms in wet and dry
atmospheres, and also for air-borne microorganisms. Disease transmission prevention is a very important consideration
for intracorporeal or implantable devices within the human body (e.g. vascular grafts and sutures) and for
extracorporeal devices such as catheters and hollow fibres for dialyzers [3]. Furthermore, wound dressings also need to
prevent infection and promote faster wound healing. Therefore, controlling the undesirable effects of microorganisms
on textiles is becoming an important issue, especially within the medical textile industry.
The abuses of antibiotics, new virus occurrence and prevalence of multi-resistant bacteria, such as meticillin-resistant
Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), along with ageing of the population
have generated high consumptions of medical textiles. The fact that textile materials are one of the main factors for
disease transmission and also the need to enhance the quality of peoples' life (medical staff, patients, and visitors) have
stimulated intensive research and development of antimicrobial textiles. Thus, it is crucial to impart antimicrobial
activity to textile materials in order to protect the user from microorganisms' contamination. Another aspect of
antimicrobial functionalization of textiles is to add a therapeutic value to the material, intended for example, for wound
healing.
A number of chemicals have been employed to impart antimicrobial activity to textile materials. These chemicals
include inorganic salts, organometallics, iodophors (substances that slowly release iodine), phenols and thiophenols,
antibiotics, heterocyclics with anionic groups, nitro compounds, ureas, formaldehyde derivatives, and amines [4]. Many
of these chemicals, however, are toxic to humans and are difficult to degrade within the environment. The major
concerns regarding commercial antimicrobial textiles is the inducement of bacterial resistance to the biocides used. The
textile industry continues to search for eco-friendly processes as substitutes for toxic textile chemicals. An increasing
interest has been noticed in the functionalization based on environmentally-friendly and biodegradable reagents such as,
for example, until now rarely used polysaccharides and their derivatives, which possess the necessary bioactive
properties. From this perspective, amino polysaccharides, including chitosan, are excellent candidates for eco-friendly
textile finishing.

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Cellulose fibres have found a broad application in medical textile field owing to the unique characteristic, such as
high moisture and liquids' adsorption, low impurity content, antistatic behaviour, and good mechanical properties.
However, cellulose fibres provide an excellent surface for microorganisms' growth. Due to their molecular structure and
a large active surface area, cellulose fibres, may be an ideal matrix for the design of bioactive, biocompatible, and
intelligent materials [3, 5, 6]. According to the literature cellulose fibres are one of the most interesting basic materials
for antimicrobial functionalization. The surface modification of the cellulose fibres is currently considered to be the best
route for obtaining modern functionality on textiles for the use in medical applications [4].
However, in spite of various techniques used for fibre functionalization in order to impart antimicrobial properties
and to develop biomedical products, there is still a large gap within the research field of interactions between bacterial
and fungal systems and bioactive surfaces of medical textile materials. Standard test methods are commonly applied to
determine the efficiency of antimicrobial agents. These methods do not usually reflect in-use circumstances, because the
majority of tests have only been performed in liquid media and not on dry, complex heterogeneous systems such as
functionalized fibrous materials. Testing and evaluating antimicrobial efficiency in laboratory conditions with respect to
the real-life environment is rather challenging. Thus, the test selected and interpretations made may vary on the basis of
the different capability of antimicrobial action.
The evaluation of any antimicrobial test results requires a thoughtful and basic understanding of microbiology,
understanding the strengths and limitations of each test, and understanding the mode of action of the antimicrobial agent
in question [1].

2. Requirements for antimicrobial finishing

The term 'antimicrobial' refers to a broad range of technologies that provide varying degrees of protection for textile
materials against microorganisms. Antimicrobials are very different in their chemical nature, mode of action, impact on
people and the environment, handling characteristics, durability, costs, regulatory compliance, and how they interact
with microorganisms [1].
The purpose of imparting antimicrobial activity to textiles is to protect the material from microbial attack, prevent the
transmission and spreading of pathogenic microorganisms, inhibit odour development resulting from microbial
degradation, and creating a material that will act as preventive and/or curative treatment.
Ideal antimicrobial finishing needs to fulfil a number of requirements in order to achieve the maximum benefit from
antimicrobially functionalized textile products. An antimicrobially-treated material is defined as being hygienic and,
therefore, should have the following requirements [4, 7, 8]:
 effective inhibition against a broad spectrum of bacterial and fungal species,
 non-toxicity to the consumer, manufacturer and the environment,
 durability,
 compatibility with resident skin microbiota, and other finishing processes,
 avert from irritations and allergies,
 applicability with no adverse effects on the quality or appearance of the textile.

3. Modes of antimicrobial action

There are several different classifications for antimicrobial agents in the literature [4, 9-12] according to the chemistry,
the mechanism of antimicrobial activity, efficiency, and washing-resistance. Consistent with these studies, antimicrobial
substances can be divided into biocides and biostats, leaching and bound antimicrobials, controlled-release and barrier-
forming agents, synthetic and natural, and agents of poor and of good washing resistance [11]. In general, the
antimicrobial agents can have a biocidal or biostatic effect on microbial growth rates. Whilst the biocides (bactericides
and fungicides) cause the death of microorganisms, the biostats (bacteriostats and fungistats) lead to the inhibition of
the microorganisms’ growth (see Figure 1). The mode of action is strongly dependent upon the concentration of the
active substance in the textile. The minimum inhibitory concentration (MIC) is required for biostatic activity, while the
minimum biocidal concentration (MBC) should be exceeded for biocidal activity [11]. Many commercially-used
antimicrobial products, e.g. silver, triclosan, polyhexamethylen biguanid (PHMB) and quaternary ammonium
compounds, are biocides [4].

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Fig. 1: Biocidal/biostatic effect of antibacterial agents on microbial growth rates (x indicates addition of an agent; CFU colony
forming unit) [13].

The actual mechanisms by which antimicrobial substances control microbial growth are extremely varied and depend
on the type of agent used. Generally, antimicrobial agents prevent cell reproduction, damage cell walls or cell
permeability, denature proteins, block enzymes and make cell survival impossible [3, 4, 10].
It is believed that polycationic antimicrobial compounds target the cytoplasmic membranes of microorganisms and
thus the mechanism usually takes place in six-step process [13]:
(1) adsorption onto the microbial cell surface
(2) diffusion through the cell wall
(3) binding to the cytoplasmic membrane
(4) disruption of the cytoplasmic membrane
(5) release of cytoplasmic constituents such as K+ ion, DNA and RNA
(6) death of the cell.

Antimicrobial agents can act in two distinct ways (Fig. 2):

(i) by contact; the antimicrobial agent inhibits microbes only on the fibre surface (substances are permanently
attached to the fibre surface),
(ii) by diffusion; the antimicrobial agent is slowly released onto the fibre surface and/or from the surface
(substances with controlled-release mechanism).

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Fig. 2: Modes of antimicrobial action

Bound antimicrobials are chemically bound to the textile fibre's surface, where they form a barrier against
microorganisms, and control the spread of those microorganisms that come into contact with the fibre surface [10, 11].
The main advantage of these agents is that they do not leach-off the textile substrates into the surroundings, so the
probability of microorganisms developing resistance to them is small. Because bound antimicrobials are firmly attached
onto the fibre surface they are more durable to laundering than leaching antimicrobials. However, the washing
durability of an agent cannot ensure its durability regarding the antimicrobial function [11]. Even though antimicrobial
agent is present on the surface, it might lose its activity, i.e. become deactivated, or can be abraded away. Chemical
bond formation, such as covalent binding, is achieved under certain conditions and is strongly dependent upon proper
anchoring groups available in the agent and in the fibre structure. The major drawback of this kind of process is that by
attaching active agents to the fibres, chemical bonds are formed between the functional groups, which results in
blocking of those functional groups responsible for antimicrobial effectiveness. Therefore, antimicrobial activity
decreases. A further disadvantage of this process is the use of auxiliary chemicals and cross-linking agents, such as
glutaraldehyde, dimethyloldihydroxyethyleneurea (DMDHEU), or epichlorohydrin (ECH), which may not comply with
the safety regulations and could affect the biocompatibility of those fibres intended for medical applications.
Another type of chemical function is the controlled-release mechanism. These substances are called leaching
antimicrobials, because they 'leach' onto the fibre surface or in the surrounding environment [10]. Since they are not
chemically-bonded on the textile surface and the 'reservoir' of agent to be delivered is limited, the concentration of
active agent decreases eventually, and gradually falls under the limit of effectiveness, which can consequently induce
resistance to these substances in microorganisms [10, 11]. Furthermore, releasing of the agent can provoke serious
health problems, while the antimicrobial may interfere with skin resident microbiota, and cause rashes, skin irritations
and allergies, or even lead to the outgrowing of pathogenic bacteria. Leaching antimicrobials do not usually withstand
repeated washing cycles and their release into water can have a negative effect on desirable microorganisms needed for
successful waste-water treatment.
Despite many negative properties, the leaching of an antimicrobial agent is sometimes desirable, for example when
the agent is incorporated into the fibres and thus ineffective unless it migrates onto the surface, or when it is intended
for wound healing, where the penetration of agent into infected part is required.

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4. Antimicrobial finishing methods

Various approaches have been used for antimicrobial functionalization of textile materials depending on the particular
active agent and fibre type. In general, two different antimicrobial finishing methods can be distinguished.
Antimicrobial agents can be either applied in an after-treatment process or incorporated into the polymer solution prior
to extrusion or into the spinning bath (Fig. 3a) [4, 13]. Substance embedded within the fibre structure has to migrate to
the surface, and should be slowly released during use in order to be active [9]. Incorporation of antimicrobial substance
within a fibre matrix is suitable only for synthetic fibres.
As after-treatment processes for antimicrobial finishing of natural, as well as synthetic fibres, conventional exhaust
and pad-dry-cure methods have been used (Fig. 3b). In addition, methods like padding, spraying, coating and foam
finishing have been developed [4, 13]. Many other methods have also been reported, such as the use of nanosized
colloidal solutions, nanoparticles, chemical modification of the biocide for covalent bond formation with the fibre (Fig.
3c), crosslinking of the active agent onto the fibre using crosslinker and sol-gel processes [4].

Fig. 3: Antimicrobial agent is: (a) incorporated into the fibres; (b) applied on the fibre surface; (c) chemically bonded onto the fibres
[7].

5. Antimicrobial agents
There are several antimicrobial agents used for antimicrobial functionalization of textiles. The most contemporary are
discussed below.

5.1 Triclosan
The antimicrobial effectiveness of textiles could be achieved by several different ways. One of the most widely used
antimicrobial products today is 2,4,4'-trichloro-2'-hydroxydiphenyl ether, more commonly known as triclosan (Fig. 4)
[2, 14]. Triclosan acts on microbial growth mainly by inhibiting fatty acid biosynthesis trough blocking lipid
biosynthesis, as well as by interacting with amino acid residues of the enzyme-active site within a membrane [14].
As a textile finish, it is mostly used for the protection of industrial and transport filters, the production of antimicrobial
shoe-socks, socks, towels, cleaning wipes and for household textiles [2, 14]. Cotton knitted fabric treated with 6 %
(owf) triclosan solution by the conventional exhaust process has shown an effective reduction of bacteria
Staphylococcus aureus and Escherichia coli. After 50 laundering cycles antimicrobial activity decreased, but the
reduction was still over 70 %. The activity in acidic, basic and synthetic urine conditions was also reduced [14].
In order to achieve more durable finishing, triclosan has been inserted into the hydrophobic cavity of β-cyclodextrins
to form an inclusion complex [15], which was then embedded within a polymer film or a fibre, or encapsulated in
microspheres which were subsequently attached to viscose [16]. Poly(L,L-lactide) microspheres loaded with triclosan
have been synthesised and immobilized onto nonwoven viscose textiles [17].
Bacterial resistance to triclosan has been well-documented [4, 18, 19]. Furthermore, exposure to sunlight causes the
break-down of triclosan and the formation of toxic polychlorinated dioxins. There is an increasing concern about the
harmful consequences of triclosan for humans, animals, and the environment [4, 19, 20].

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Fig. 4: Chemical structure of triclosan [4]

5.2 Quaternary ammonium compounds

Quaternary ammonium compounds (QAC), particularly those containing chains of 12-18 carbon atoms, have been
widely used as antiseptic agents and disinfectants for many years [4, 10, 11]. The antimicrobial activity of QAC
depends on the length of the alkyl chain and the number of quaternary ammonium groups in the molecule that carry a
positive charge at the N atom. The antimicrobial function arises from attractive interactions between the cationic
ammonium group of the QAC and the negatively charged cell membrane of the microbe. This causes the interruption of
all essential functions of the cell membrane and, consequently, the interruption of protein activity. QAC also affects
bacterial DNA multiplication ability [11].
Cotton fabrics have been treated using three different quaternary ammonium salts during finishing process. QAC
have formed ionic interactions with sulfonate groups on the dyed cotton fabrics, which contributed to higher exhaustion
uptakes of the salts and better antimicrobial activities against Escherichia coli of the dyed fabrics compared with non-
dyed. The chemical structures of salts have significantly affected their uptakes on cotton fabrics. However, the washing
durability of the antimicrobial functions on finished cotton fabrics was still low because of the easy dissociation of ionic
interactions between the sulfonate and pyridinum groups in water [21].
Commercially used QAC, 3-trihydroxysilyl propyldimethyloctadecyl ammonium chloride (AEM 5700, AEGIS
Enivironments), forms covalent bonds with the textile (Fig. 5). AEM 5700 has a MIC value of 10 – 100 µg/ml [48]. So
far, it has been used on cotton, polyester and nylon, and can be applied by padding, spraying, and foam-finishing [22 –
24]. Bacterial resistance to some QAC has been observed, lately [4].

Fig. 5: Chemical structure of 3-trihydroxysilyl propyldimethyloctadecyl ammonium chloride (AEM 5700) [4]

5.3 Polyhexamethylen biguanide

Polyhexamethylen biguanide – PHMB (Fig. 6) is widely-used biocide for numerous applications. It has been used as
disinfectant in swimming pools, in the food industry and in hospitals as an antiseptic agent for preventing wound
infections. PHMB is also used as preservative in cosmetics, personal care products, fabric softeners, contact-lens
solutions, and hand washes [25].
It has also attracted attention for antimicrobial finishing for textiles, especially cellulose materials. Polyhexamethylen
biguanide, owing to its cationic nature, forms ionic as well as hydrogen bonding with cellulose materials. At lower
concentrations, electrostatic interactions between the PHMB and carboxylic acid groups within the cellulose dominate,
but in the contrast, as the concentration of PHMB increases, hydrogen bonding with cellulose becomes increasingly
dominant. At high PHMB concentrations, any observations of increasing PHMB sorption are attributed to monolayer
aggregation and the multilayer stacking of PHMB through electrostatic interactions with counterions and the hydrogen
bonding of biguanide groups [26].
PHMB can be directly exhausted onto cellulose material or applied during a pad-dry-cure process [4]. The
antimicrobial effect of positively charged materials is based on the interactions of the cationic molecule with anionic
phospholipids, within the bacterial cell-wall, which causes its damage. Wound dressings containing PHMB as an
antimicrobial agent are already available on the market [5].

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Fig. 6: Chemical structure of polyhexamethylen biguanide – PHMB [4]

5.4 N-halamine compounds

N-halamines are hetreocyclic organic compounds containing one or more nitrogen-halogen (N – X) covalent bonds that
are normally formed by the halogenation of imide, amide, or amine groups [27]. Their antimicrobial properties are
based on the electrophilic substitution of Cl in the N–Cl bond with H. This reaction can be carried out in the presence of
water, and results in the presence of Cl+ ions that bind to the acceptor regions on microorganism and, subsequently,
hinder their enzymatic and metabolic processes, leading to destruction of microorganism [11]. During the deactivation
of microorganisms, the N-halamine bond is reversibly reacted to N–H and has to be exposed to hypochlorite solution
(e.g. during bleaching or laundering) for regeneration of its antimicrobial activity (Fig. 7) [10].
Cellulose treated with methylol-5,5-dimethylhydantoin in combination with hypochlorite forms chloramines on the
fibre surface [10]. Nevertheless, the yellowing of fabrics and strength-loss have proven to be problematic when using
higher concentrations of chloramines, and are thus useless from the practical point of view [3, 4].

Fig. 7: Reaction mechanism of N-halamine compounds [4]

5.5 Silver and silver compounds

Silver is, in general, the most widely-used metal in the textile industry, although some other metals such as zinc, copper,
and cobalt have shown effective inhibition of microbes [4, 6]. It can be used to reduce infections in the treatment of
burned areas, and for preventing bacterial colonisation on medical devices, as well as on other textile fabrics [28].
It is believed that heavy metals react with proteins by combining the thiol (–SH) groups, which leads to the
inactivation of the proteins [29]. In the presence of moisture (e.g. from air), metal ions are formed which inhibit
microbial replication. A brief explanation of its antimicrobial mechanism can be given as follows: normally metal ions
destroy or pass through the cell membrane, and bond to the –SH group of cellular enzymes. The consequent critical
decrease of enzymatic activity causes microorganism's metabolisms to undergo change and their growth to be inhibited,
up to the death of the cell. The metal ions also catalyze the production of oxygen radicals that oxidize the molecular
structure of bacteria. Silver ions can lead to denaturation of proteins, and cell death because of their reaction with
nucleophilic amino acid residues in proteins, and their attachments to sulphydryl, amino, imidazole, phosphate and
carboxyl groups of membrane or enzyme proteins. Silver is also known to inhibit a number of oxidative enzymes such
as yeast alcohol dehydrogenase, the uptake of succinate by membrane vesicles and the respiratory chain of Escherichia
coli, causing metabolite efflux and interference with DNA replication [30].
Different types of silver preparations were explored for cellulose finishing. Woven cotton fabrics were treated using
silver nanoparticles, silver microparticles obtained by the chemical reduction of silver nitrate with L-ascorbic acid, and
with silver chloride obtained by the chemical reaction of silver nitrate with hydrochloric acid in presence of a
crosslinking agent. Fabrics treated with silver compounds have shown good antibacterial efficacy against two bacterial
strains, Bacillus subtilis and Escherichia coli. Fabrics have also demonstrated good washing resistance [31]. In another
study El-Rafie and co-workers [29] investigated the antimicrobial effect of cotton fabrics finished with silver
nanoparticles prepared by fungi-based technique. Produced silver nanoparticles had size distribution within the range of
3 – 8 nm, and were applied in a pad-dry-cure process. Silver nanoparticle-treated cotton fabrics have demonstrated a
bacterial reduction over 90 % against Staphylococcus aureus and Escherichia coli. Nevertheless, almost 50 % of the
imparted antibacterial properties were lost after 20 washing cycles. This problem had been solved with incorporation of
a binder in the finishing bath, which enhanced the antibacterial properties even after 20 washing cycles, respectively.
Li and co-workers [32] developed nanoparticles, a mix of silver ions and titanium dioxide, with the intention of
reducing the number of silver resistant bacteria species. Surgical masks coated with prepared nanoparticles were
effective against S. aureus and E. coli. Silver nanoparticles have extremely large specific surface area, thus increasing
their contact with bacteria or fungi, and vastly improving their bactericidal and fungicidal effectiveness [31].

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Despite the efficacy of antimicrobials based on silver, concerns about bacterial resistance have been reported in
several papers [32 – 34]. However, the use of medical devices containing silver must be undertaken with caution, since
a concentration-dependent toxicity has been demonstrated. Braydich-Stolle et al. [35] assessed the suitability of a mouse
spermatogonial stem cell line, as an in vitro model for assessing the nanotoxicity of silver. Concentrations of silver
nanoparticles between 5 g/mL and 10 g/mL have induced necrosis or apoptosis of mouse spermatogonial stem cells
[35].

5.6 Natural bioactive compounds

Natural bioactive compounds with antimicrobial properties are gaining considerable attention as attractive ecofriendly
alternative to synthetic antimicrobial agents for textile applications, especially in medical and health care textiles, as
they are safe, non-toxic and skin-friendly [36]. These naturally active compounds such as, for example, the until now
less employed polysaccharides and their derivatives, natural dyes, as well as some other extracts from roots, stem,
leaves, flowers fruits and seed of diverse species of plants exhibiting antibacterial properties, have already been
explored as textile finishing agents in their crude form or as microcapsules [36-38].
Polysaccharides that play an important part in surface coatings for materials used for medical applications are
dextran, hyaluronic acid, carboxymethyl cellulose, heparin, alginate and others [6, 39]. Alginate and alginate
copolymers are mainly used in wound dressings due to their high absorption capacity. Hyaluronic acid is used for
healing wounds in wet conditions [5]. Artificial veins from polyethylene terephthalate are usually coated with heparin
for improved biocompatibility. Vascular grafts, made from a mixture of polyurethane and heparin, have shown
excellent antithrombogenicity and biocompatibility [40]. Special attention is being paid to amino polysaccharides,
including chitosan, a chitin derivative, and apart from cellulose the most abundant biopolymer on Earth. Chitosan, as an
amino polysaccharide representative, is described in more details in the following section 5.7.
Plant-based products (Aloe vera, tea-tree and eucalyptus oil, neem, grapefruit seed and tulsi leaf extracts, etc.)
represent the major group of antimicrobial agents, which consist of substances such as phenols (simple phenols,
phenolic acids, quinines, flavonoids, flavones, tannins and cumarines), terpenoids, essential oils, alkaloids, lectines,
polypeptides and polyacetylenes [36]. These components show, not only antimicrobial, but also antioxidant properties.
This is extremely important when developing innovative biomaterials for medical devices, such as bioactive dressings
and wound-healing isolation materials. For these kinds of applications it is essential to provide, besides antimicrobial
inhibition, a reduction in those reactive oxygen species that are strongly implicated in the pathogenesis of e.g. wounds,
causing injury with bio-molecules such as lipids, proteins and nucleic acids, as well as the depletion of mitochondrial
DNA from human skin. Flavonoids are referred to be powerful antioxidants, i.e. to counteract free-radicals and prevent
any damage caused by them [41], and are, therefore, used as anti-inflammatory, antimicrobial, and anti-cancer agents.
Cotton fabrics treated with Aloe vera extract by pad-dry-cure process exhibited antimicrobial activity against
Staphylococcus aureus and have retained durability even after 50 washing cycles [42].
However, one of the major limitations in antimicrobial finishing based on natural agents is the non-durability of the
finish since most of them cannot form any bonds with textile materials [43]. Microencapsulation methods are often used
for increasing the durability of antimicrobial finish on textiles [36, 37, 43, 44], the grafting of active compound by using
crosslinker [36], and immobilization of bioactive liquids in sol-gel matrices are also described in the literature [45].
Honey has been used for its medicinal properties since ancient times and has recently regained its importance as
natural remedy for the treatment of infected wounds [46-49]. It has been recognized to possess a broad spectrum of
antimicrobial activity, which originates from hydrogen peroxide production, but is also assigned to non-peroxide
mechanisms of action such as methyl-glyoxal, high sugar content, acidity, and content of phenolics and oligopeptides
[50-53]. Comparative studies, which included different types of honeys, have revealed that manuka honey has the
highest antimicrobial activity [47, 54-56]. In-vitro tests of antimicrobial activity of different Slovenian honeys showed
that chestnut honey has the best inhibitory effect against common pathogenic microorganisms present in the wound
[57]. Alginate nonwoven wound dressing coated with medical chestnut honey has demonstrated faster wound healing in
clinical studies of infected and chronic wounds [58]. Wound dressings based on alginate and chestnut honey are already
available on the market and are suitable for wide range of wound types (burns, venous leg ulcers, diabetic foot ulcers,
pressure ulcers, wounds infected with antibiotic resistant bacteria, such as MRSA and VRE, etc.).
Natural dyes and pigments, derived from plants, insects, animals, and minerals, also exhibit antimicrobial activities
[36, 59-62] and have been used in food, cosmetic, pharmaceutical and textile industries [59]. In this way, by choosing
specific dyes, antimicrobial finishing and dyeing of textile materials can be achieved simultaneously [4, 63].

5.7 Chitosan
Chitosan is a modified natural carbohydrate derived from chitin – a base component of the skeletons of crustaceans,
mussels, fungi, insects, and some algae. Chitin is a linear polysaccharide composed of 2-acetamino-2-deoxy-β-D-
glucosic units connected with β-1,4- glycosidic linkages (Fig. 8). Chitosan has the ability to inhibit bacteria, fungi and
viruses [64]. The antimicrobial activity of chitosan is assigned to the amino groups, which in acidic media form

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ammonium salts [65]. There are two proposed mechanisms for the antibacterial activity of chitosan, which both
emphasise the importance of the amount of active amino groups. In one mechanism, the polycationic nature of chitosan
interferes with bacterial metabolism by stacking the cell's surface. The other mechanism is the binding of chitosan with
DNA to inhibit mRNA synthesis. The important structural parameters influencing chitosan's antimicrobial capacity are
the degree of deacetylation (DD), the molecular weight (MW), and the ratio between protonated and un-protonated
amino groups [66].

CH3

O C
OH
NH
O
HO
O
HO O
O
NH2
OH
n m

Fig. 8: Chemical structure of chitin and chitosan – chitin mainly consists of monomers in the N-acetyl form (form 'm'), while
chitosan, depending on the degree of deacetylation, consists mainly in the amine form (form 'n') [66].

The use of chitosan in the textile area can be divided into two groups, namely the use of chitosan and its derivates for
[67]: i) the production of chitin and chitosan fibres, and ii) the treatment of fibrous materials in classical or alternative
finishing procedures. Chitosan fibres are used as nonwovens for dressings and as drug carriers in the form of hollow
fibres [68]. Chitosan fibres blended with collagen were produced by viscose spinning route. Results have shown
improved blood compatibility for use in sutures [69].
Niekraszewicz [68] evaluated a chitosan-polypropylene nonwoven prepared according to the wet-paper method
production. Microbiological tests showed antimicrobial activity, no cytotoxicity was detected. The use of chitosan as an
antimicrobial agent for the treatment of polypropylen nonwovens for surgical covers has been researched on different
types of hospital bacteria. Textiles treated in such a way were effective against Gram-negative and Gram-positive
bacteria [67]. Sakai et al. [70] dissolved chitosan in water using carbonic acid gas (CO2) via preparation of the gel.
Cotton gauze treated with chitosan-CO2 solution effectively inhibited Staphylococcus aureus. A very important field of
chitosan application is wound-care management. When proposed for skin tissue repair, chitosan was found to produce a
less dense scar and a slightly quicker healing than other standard materials [65, 69-72].
Cotton fabric treated with water-soluable carboxymethyl chitosan derivative showed good antibacterial activity
against E. coli and S. aureus at 0.1 % concentration, as well as improved wrinkle recovery [73]. Lim and Hudson [74]
synthesized a fiber-reactive chitosan derivative, O-acrylamidomethyl-N-[(2-hydroxy-3-trimethylammonium)propyl]
chitosan chloride, which can be covalently bonded to cellulose fibers. The derivative showed good reduction of bacteria
S. aureus and E. coli.
Chitosan based core-shell particles of poly(n-butyl acrylate) (PBA) core and chitosan shell has been designed as a
novel antibacterial coating for textiles [75]. Cotton fabrics treated with PBA-chitosan particles during a pad-dry-cure
process demonstrate an excellent antibacterial activity against S. aureus with bacterial reduction over 90 %.

6. Evaluation of antimicrobial efficiency

To date, testing the antimicrobial activity of functionalized textiles is consisted of two categories of standardized test
methods, qualitative (AATCC TM147 and AATCC TM30 (antifungal) (American Association of Textile Chemists and
Colorists Test Method), ISO/DIS 20645, EN ISO 20645 and ISO 11721 (International Standards Organization), and SN
195 920 (921 - antifungal) (Swiss Norm)) and quantitative (AATCC TM100, ISO 20743, SN 195924, JIS L 1902
(Japanese Industrial Standard) and ASTM E 2149 (American Society for Testing and Materials)). Qualitative methods
are mostly based on the agar diffusion test. They are relatively quick, cheap, simple and well-defined but subjective (use
of ratings) and not appropriate for all kind of textiles and for analyses of efficacy of different antimicrobial agents as
they diffuse through agar at different rates or not at all. Quantitative methods are on the other hand more broadly
applicable but more time-intense and expensive as they involve actual microbe enumeration, indicating level of
bactericidal/ fungicidal activity. They can be used for all types of textiles and antimicrobials and comparisons can be
made between different antimicrobial treatments as well as various treatment levels on the same textile [9, 10, 76, 77].
The more commonly used tests for evaluation of the antimicrobial efficiency are Parallel Streak Method AATCC
TM147 and ISO 20645, from among qualitative tests and AATCC TM100, JIS L 1902 and ISO 20743, from among
quantitative tests (Table 1).

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Table 1: Comparisons between the more commonly used methods for evaluation of antimicrobial textiles activities [10, 77, 92]

Method Title Procedure Advantages Disadvantages

AATCC 147 Antibacterial Activity
Assessment of Textile
Materials: Parallel
or Streak Method
The agar surface is covered by thin Quick, easily Non realistic,
strips of test fabrics, previously executed,
non-quantitative,
inoculated with test culture. After inexpensive,
in-sensitive,
incubation the inhibition zones are well-defined
qualitatively defined non-reproducible

ISO 20645 Textile fabrics -

Determination of
antibacterial activity -
Agar diffusion plate
test

AATCC 100 Antibacterial Finishes
on Textile Materials:
Assessment of
Test and control fabrics are Quantitative, Time-consuming
inoculated with suspension of sensitive,
microorganisms. Concentration is realistic
enumerated at »time zero« and after
a defined contact incubation period.
Microbial reduction or growth
inhibition is quantitatively analyzed
on the basis of differences between
test and control fabrics.

ASTM E2149 Standard Test Method Test and control fabrics are placed Quantitative, Non realistic,
for Determining the individually in 50 ml of a liquid easily
no correlation
Antimicrobial media, where test microorganisms consistent
between other
Activity or are also inoculated. Flasks are then
quantitative tests
Immobilized vigorously shaken. At »time zero«
Antimicrobial Agents and after a defined contact period
Under Dynamic (usually 1 h) microbial
Contact Conditions concentration in solution is
determined and microbial reduction
is calculated

JIS I 1902 Testing for Test and control fabrics (in three Quantitative, Time-consuming,
Antibacterial Activity replicates) are inoculated in liquid realistic, expensive
and Efficacy on media with specified concentration flexible,
or Textile Products of microbial suspension. At »time sensitive,
zero« and after the contact period reproducible
the microorganisms are enumerated. results
ISO 20743 Textiles - Results are presented in log
Determination of reduction.
antibacterial activity
of antibacterial
finished product

Qualitative methods (Parallel Streak Method AATCC TM147 and ISO 20645) are both based on agar diffusion test.
The Parallel Streak Method is a modification of traditionally used method for antibiotic susceptibility (Kirby-Bauer test)
and ISO 20645 is standardized variant by International Standards Organization. In qualitative tests, textile samples (e.g.
a strip of fabric) are laid on nutrient agar plates, inoculated with the test bacteria (Fig. 9). After incubation (24 – 48 h,
depending on microorganism used), the plates are examined for bacterial growth directly underneath the fabric and
around its edges (zone of inhibition). On the surfaces, where no bacterial growth is observed, the antimicrobial agent

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was acting inhibitory. A zone of inhibition should not be expected if the antimicrobial agent is covalently attached to
the textile, which prevents its diffusion into the agar. If the agent diffuses into the agar, the zone of inhibition becomes
apparent and its size provides some indication of the antimicrobial activity potency or the release-rate of the active
agent [4]. Although, zone of inhibition does not necessarily imply that microorganisms have been killed, they might
have only been prevented from growing. These methods ensure simple and quick performance, but can lead to
subjective determination of contact inhibition. It could also happen that antimicrobial agents diffuse through agar at
different rates or not at all (depend also on the weight and texture of the fabric); therefore the efficacy of different
agents cannot be compared.

Fig. 9: Inhibition zone of treated fabric sample (agar diffusion test)

A frequently-used quantitative method is the Shake flask method ASTM E 2149 (or its modification, ASTM
standard), which presents a standard test method for determining the antimicrobial activity of immobilized antimicrobial
agents under dynamic contact conditions [78]. It was developed for routine quality control and screening tests, in order
to overcome the difficulties of using classical static antimicrobial test methods during the evaluation of substrate-bound
antimicrobials. These difficulties are about ensuring contact of inoculum with treated surface, use of inappropriately
applied static conditions, sensitivity, and reproducibility. The dynamic shake flask method is particularly appropriate for
non-leaching antimicrobials whilst the dynamic contact conditions are applied to the samples [77]. This method has
been used for testing antimicrobial activity of cotton fabrics (or cellulose fibres) treated with the nanoparticles [79, 80],
as well as functionalized wool [81], cotton and viscose fibres coated with chitosan [82, 83] and some other fabrics.

Fig. 10: Shake flask method - ASTM E 2149

The fabric sample is immersed in an inoculated buffer solution in a flask, which is then agitated using a wrist action
shaker. The bacterial concentration is usually ~105 mL-1, but with no nutrient beyond that transferred with the

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organisms from the original culture. The time period for the exposure is defined (usually 1 h). After the specified time,
an aliquot of the buffer from the flask is plated onto nutrient agar without neutralization, incubated overnight, and the
number of colonies counted. Any reduction is calculated using the known initial microbial concentration and the final
count after exposure to the test sample or is calculated versus an untreated control. It is recommended that an untreated
control is tested in parallel to ensure a valid test and to run the test in triplicate. The “inoculum only” flask, as specified
in the method, should be tracked alongside all treated and untreated samples with each test, to ensure there is no
antimicrobial effect resulting from the non-nutritive test buffer or due to surfactants added for wetting. This test is
technically easily consistent, and ensures good contact between microorganisms and the treated fibre by constant
agitation of the test specimen in a microbial suspension; however, there is little or no correlation between the Shake
flask method and other quantitative tests. This is due to placing a small piece of fabric within a large amount of fluid
and taking a microbial sample from the fluid and not from the fabric surface, the conditions of this test do not replicate
real-life use conditions. Consecutively, the test is not widely accepted by professionals as an indicator of efficacy.
Other quantitative tests, which are also widely accepted for determining the efficacy of the antimicrobially treated
textiles, include AATCC TM100, JIS L 1902 and ISO 20743.
All these methods are based on similar principle: specified amount (weight, size, and surface area) of sample
swatches are inoculated with a specified number of microorganisms. The inoculum must be completely absorbed into
the fabric and in intimate contact with the treated surface. Surviving organisms are extracted from swatches by shaking
in a known amount of neutralizing broth. Microorganisms are recovered from the broth by re-plating and the number of
surviving organisms counted as colony forming units (CFU; Fig. 10). Results are presented as a percent or log10
reduction in contamination versus either the initial inoculum of microorganisms or the untreated control. Quantitative
testing can be used for all antimicrobials. Comparisons can be made between different antimicrobial treatments, as well
as various treatment levels on the same textile. Methods are long and expensive, because they require a number of
manipulations to the sample and organisms.
AATCC TM100 uses a rich nutrient broth allowing aggressive bacterial growth and reproduction. This method is
cited in military specifications for evaluation of appare,l since such apparel may become heavily soiled.
JIS L 1902 was developed in Japan for testing silver-based antimicrobials. The nutrient broth used in this method is
low in nutrients as to reduce neutralization of antimicrobials with proteins and, as a result, simulates real world usage
and behaviour. It is applicable to all textile products and allows comparisons between different antimicrobial treatments,
as well as various treatment-levels on the same textile [84]. This method is explicit about calculating results for treated
products versus those for untreated controls, and calls for testing in triplicate. The standard for a valid test is that there
should be at least a 1.5 log increase in the untreated control [76].
The standardized method ISO 20743 is modelled largely on the JIS L 1902, but allows more flexibility for setting the
test conditions.

Fig. 10: Assessment of antibacterial textiles - AATCC TM100; Untreated (left) and treated (right) textile sample.

In general, antimicrobial test methods should be performed under controlled, standardized conditions, in order to
guarantee reproducibility of the results. However, as most of the methods do not specify an absolute standard for
efficacy, they are frequently modified and are thus inconsistent amongst laboratories nationally and internationally. A
drawback of the standardized tests is also that they are performed in conditions that are rarely found during the normal
usage of a textile product. Moreover, the accuracies of all the mentioned methods largely depend on the efficiencies of
the microbial extractions from the sample fabrics. In regarding to all this, many laboratories apply, in addition to
standardized methods, also other tests for screening the antimicrobial activities of functionalized surfaces, such as
different colorimetric analytical methods [85] and viability test [86], viability staining and microscopy [77], and
fluorescent staining coupled with flow cytometry [87].
With an increasing need for more reliable, rapid, and inexpensive microbiological test methods, interest in the
applications of new techniques is increasing as well. The reliability of the test depends on precision, sensitivity, and

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accuracy; measures that could be met with a method that detects the total viable count in real-time. Currently two major
rapid technologies for microbiological testing, molecular and immunological are most promising. Both are considered
highly accurate and fast. Of these, PCR and immunoassays are the key methods available. Despite the higher cost per
test, these and other newer methods are being used more frequently, replacing traditional culture-dependent methods
and are, in some specific industries (food, pharmaceutical) covering approximately 40 % of the microbiology tests [88-
91]. Thus in future we can expect their extensive use also within the textile industry as the demand for reliable
antimicrobial tests is increasing especially for sanitary textiles or textiles used in medical applications.
Special methods can be used for testing antifungal activity. When testing activity against yeast, all methods used for
bacteria can be adopted, but for activity against filamentous fungi, qualitative AATCC TM30 test is usually applied.
Two parts of AATCC TM30 cover the activity against moulds. When performing the Part III of mentioned standard, the
textile samples are placed on potato dextrose agar inoculated with spores of Aspergillus niger. After seven-days of
incubation fungal growth is subjectively rated (0 – no growth, 1 – growth seen with a microscope, and 2 – visible
growth). The zone of inhibition, if present, is also measured and reported. In Part IV different procedure allow for better
discrimination between treated and untreated samples: a dry, treated and untreated strip of nutrient saturated fabric,
sprayed with a mixed-spore suspension of moulds is incubated in a humidity jar. The percentage of coverage by fungal
growth is determined after the incubation period. The organism must germinate and establish itself on the treated fabric.

7. Conclusion
The most significant antimicrobial agents used for textile functionalization have been presented in this chapter. Many of
these chemicals, however, are toxic to humans and hard to degrade within the environment. Even more, most of them
promote bacterial resistance. Taping new potential antimicrobial substances, such as antimicrobial polysaccharides, can
considerably minimize the undesirable activities of the antimicrobial products. Only a few of these latest possibilities
have been explored and commercialized, to date. The antimicrobial functionalization of fibres using different
polysaccharides and other natural agents for medical applications is thus the creation of new prosperity, especially due
to the fact that biomedical and biodegradable products will be in the near future largest application of antimicrobial
textiles.
However, in order to reliably ensure the antimicrobial efficiencies of functionalized materials it is extremely
important to choose an appropriate method for a heterogeneous system such as functionalized fibres or fabrics. Many of
the commonly used antimicrobial tests for the evaluation of textiles have failed to produce any successful criteria,
consequently leading to wrong interpretation of results. The methods listed for the usage of fibre-materials, as presented
in this chapter, can thus be understood as representative cases, for indicating the different options for their use regarding
a certain textile applications. The major disadvantages of those methods used until now, have been pointed out.
Considering this, an appropriate microbiological test for functionalized fibre materials can be chosen. Understanding
the antimicrobial methods described in details through this chapter help to establish material's potentials and limitations
within the field of medical usage.

Acknowledgements Operation part financed by the European Union, European Social Fund.

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