Keywords

5.1 Introduction

Control and prevention of microbial contamination represent major challenges for researchers, doctors, and population. Over time, pathogenic microorganisms have developed some features that have helped them to survive even to the most aggressive antimicrobials of time. Usually, after a long period of efficient usage of new antimicrobials, resistance to those substances develops. Antimicrobial agents with low molecular weight generally have a series of disadvantages like environmental toxicity and short term antimicrobial capacity (Kenawy et al. 2007). In order to minimize these problems, specialists have focused in last years on design and study of polymeric materials with enhanced antimicrobial efficacy, reduced toxicity toward organisms or environment, and prolonged action time, compared with their small molecular weight counterparts (Jain et al. 2014). In this respect, various methods of introducing atoms or functional groups on polymer chain in order to obtain materials able to destroy structural integrity of the bacteria, fungi or algae have been tested. Antimicrobial macromolecular substances have been classified as: quaternary ammonium polymers, guanidine polymers, polymers designed to mimic natural peptides, halogenated macromolecules, polymers containing phosphonic or sulfonic derivatives and silver ion bearing polymers (Munoz-Bonilla and Fernandes-Garcia 2012; Siedenbiedel and Tiller 2012).

Attachment of substances with potential antimicrobial activity to the surface of microorganisms is possible due to the specific interactions between extracellular polymeric components like exopolysaccharides, proteins, lipids or humic substances, and reactive functional groups of antimicrobials. After cell surface adhesion, antimicrobial agents can act by various mechanisms: breakdown of cell membrane structure, inhibition of protein synthesis or metabolic pathways, interference with synthesis of nucleic acids, etc. Due to the surface negative charge of microbial entities, the most probable and efficient interactions will be with cationic contact surfaces. This category also includes quaternary ammonium compounds. Cationic quaternary ammonium antimicrobial agents are part of some formulations used as disinfectants, which act through electrostatic attraction, infiltration and diffusion (Rajkowska et al. 2016).

Importance of this type of materials is evidenced by a large number of reviews that generally discussed various aspects of mechanism of action and medical applications of antimicrobial polymers, as well as of quaternary ammonium based materials (Kenawy et al. 2007; Timofeeva and Kleshcheva 2011; Kenawy et al. 2014; Xue et al. 2015; Santos et al. 2016; Francolini et al. 2017; Jiao et al. 2017).

Present chapter covers most recent studies, predominantly over past 5 years, focused on natural or synthetic quaternary ammonium polymeric compounds and factors that influence their antimicrobial activity. It is generally assumed that molecular weight variation of polymers, hydrophilic-hydrophobic balance, conformation of polymer chain in solution, strength and type of intramolecular and intermolecular interactions will influence antimicrobial efficacy of polymers containing quaternary ammonium moieties. Also, this chapter highlighted the diversity of polymers, especially those biocompatible that can be modified with quaternary ammonium groups to create new antimicrobial materials.

5.2 Polymeric Antimicrobials with Quaternary Ammonium Moieties

Specialized literature reports that various modifications of polymers in solution or in bulk, enhance their antimicrobial activity. From this point of view, antimicrobial applications of polymers with quaternary ammonium moieties are represented by coatings, modified surfaces and solutions. If, in some cases, coating implies application of electrospinning, dip-coating and polyelectrolyte complex formation onto polymeric support; in other ones, modified surfaces resulted from covalent linkage of quaternary ammonium salts directly onto polymeric support. Polymers with quaternary ammonium moieties and potential antimicrobial properties were synthesized from solution, purified by dialysis, concentrated and precipitated in non-solvent, filtered, dried for solvent removal, stored and then dispersed or dissolved in suitable solvent for antimicrobial tests.

In order to prevent biofouling phenomenon, researchers use different strategies to improve antibacterial properties of surfaces via creation of superhydrophobic surfaces; incorporation and controlled diffusion over time of biocide compounds from surfaces; and conjugation of antibiotic functional groups on surfaces. By such modifications, bacterial adhesion is suppressed, biocide leaching is initiated, and contact killing is activated (Lichter et al. 2009). Cationic quaternary ammonium groups could be immobilized or anchored onto polymeric chains via different approaches: chemical coupling reactions, surface initiated polymerization of monomers bearing antibacterial moieties and post-polymerization modifications to synthesize surface-tethered antimicrobial polymer brush (Gettings and White 1987; Lee et al. 2004; Lenoir et al. 2006; Wiarachai et al. 2012; Yao et al. 2010). In next sections, polymeric antimicrobials with quaternary ammonium moieties are classified and analyzed according to the nature of polymer matrix: natural or synthetic.

5.3 Antimicrobials Based on Natural Polymers

Natural polymers like cellulose, chitosan, dextran, starch, pullulan, gum, poly(lactic acid) are suitable for chemical modifications with quaternary ammonium moieties. The new polymers are characterized by improved biological, chemical and physical properties compared with their precursors viz better water solubility, moisture absorption, flocculation, nontoxicity, biocompatibility and antimicrobial activity.

5.3.1 Cellulose Type Antimicrobials

Cellulose is a polysaccharide with linear chain structure consisting of β(1→4) linked D-glucose units, and is a suitable macromolecule for chemical changes for applications in pharmaceutical industry in form of membrane, fibers or hydrogels. Meng et al. (2015) described a one-step modification method for direct covalent linking of quaternary ammonium salts onto membrane surface of regenerated cellulose, based on alkoxy silane polycondensation reaction and silane coupling agents like trimethoxysilylpropyl trimethyl ammonium chloride and trimethoxysilylpropyl octadecyldimethyl ammonium chloride. Reaction mechanism ensured chemical coupling of cationic antibacterial alkyl groups responsible for enhanced hydrophobicity of regenerated cellulose membrane and infiltration of ammonium groups through hydrophobic membranes of tested microorganisms. In same culture conditions, it was reported that Staphylococcus aureus ATCC 6538 more easily formed colonies on modified cellulose membrane compared with Escherichia coli DH5α. In addition, length of hydrophobic alkyl chains from regenerated cellulose membranes modified with trimethoxysilylpropyl trimethyl ammonium chloride or trimethoxysilylpropyl octadecyldimethyl ammonium chloride induced different behavior against the viability of bacteria. Thus, viability of S. aureus or E. coli was about 99% in presence of membrane modified with trimethoxysilylpropyl trimethyl ammonium chloride, while membrane modified with trimethoxysilylpropyl octadecyldimethyl ammonium chloride reduced the cell viabilities up to about 0.5%.

In order to avoid growth of pathogenic microorganisms on skin, researchers designed a hydrogel as a component of diapers, based on blending of native cellulose solution with quaternized cellulose solution (Peng et al. 2016). In this way, novel hydrogel formed by chemical cross-linking in aqueous solution of NaOH-urea mixture and in presence of epichlorohydrin, represented a potential antimicrobial material with improved mechanical properties, too. Varying molecular weight and substitution degree of quaternized cellulose (Mw = 94000 g/mol or 256000 g/mol; DS = 0.23–0.69), different hydrogels encoded as Gel9–2, Gel9–4, Gel9–6, Gel25–2, Gel25–4, and Gel25–6 were prepared and microbiologically tested. Quaternary ammonium groups introduced in hydrogel network by quaternization reaction of cellulose with 3-chloro-2-hydroxypropyl-trimethylammoniumchloride were responsible for inhibition of Saccharomyces cerevisiae cultures. The best antibacterial activity of Gel25–6 sample compared with other hydrogel samples suggested that a higher substitution degree of quaternized cellulose has intensified electrostatic type interactions between polycationic quaternary ammonium groups of cellulosic hydrogel and anionic components of microbial membranes leading to disruption and death of cells.

5.3.2 Chitosan Type Antimicrobials

As a natural material derived from chitin, chitosan is a non-toxic, biodegradable and linear macromolecule with cationic behaviour and comprises of β-(1 → 4) linked copolymer of 2-acetamido-2-deoxy-D-glucopyranose units and 2-amino-2-deoxy-D-glucopyranose, often used to design biocompatible materials. Even if it has antimicrobial properties, opportunity to enhance them was exploited by insertion of quaternary ammonium groups. Usually, chitosan is functionalized by quaternization, carboxymethylation, acylation or sulfhydrylation of amino groups in C2 position of glucopyranose unit. Chitosan based materials with quaternary ammonium moieties are diverse in shape, state or functionality: hydrogels (Mohamed et al. 2015; Fan et al. 2015), fibers based on polyelectrolyte complexes (Ignatova et al. 2016), chitosan ammonium salts (Tang et al. 2015; Li et al. 2015; Tan et al. 2016; Li et al. 2016; Wang et al. 2016; Tang et al. 2016; Chen et al. 2016; Oyervides-Munoz et al. 2017) or copolymers (Song et al. 2016).

Hydrogels represent 3-D networks able to retain large amounts of biological fluids or water, being used in different fields such as biomedical devices, food packing or water purification. Also, quaternized chitosan based hydrogels serve as antimicrobial or wound healing materials. When Mohamed et al. (2015) designed hydrogels based on N-trimethyl ammmonium chitosan chloride and poly(vinyl alcohol) in weight ratios of 1:3, 1:1 and 3:1, they used glutaraldehyde in weight ratio of 1–5% as chemical cross-linking agent to ensure formation of chemical bonds between glutaraldehyde and hydroxyl groups of poly(vinyl alcohol) or residual amino groups from N-trimethyl ammmonium chitosan chloride. Beside different physico-mechanical properties, resulted materials exhibited specific antibacterial or antifungal activities. Thus, antimicrobial activity of N-trimethyl ammmonium chitosan chloride/poly(vinyl alcohol) hydrogels was higher than N-trimethyl ammmonium chitosan chloride itself, while antimicrobial effect increased with glutaraldehyde concentration.

Fan et al. (2015) performed gamma radiation-crosslinking to prepare hydrogels based on solution of hyaluronic acid and quaternary ammonium chitosan, poly(ethylene oxide) and poly(vinyl alcohol). Preliminary, quaternary ammonium chitosan was synthesized from chitosan and N-(3-chloro-2-hydroxypropyl)trimethyl ammonium chloride in NaOH solution. Then, solution of quaternary ammonium chitosan mixed with poly(vinyl alcohol)/poly(ethylene oxide) solution was subjected to irradiation with a 60Co source. The sample irradiated with a dose of 40 kilograys was tested against E. coli RCMB 000107 and S. aureus RCMB 000106. In both cases, polymeric hydrogels containing quaternary amino N+(CH3)3 groups showed a clear inhibition zone compared with poly(vinyl alcohol)/poly(ethylene oxide) hydrogel. Due to excellent moisture properties, these hydrogels were proposed as valuable materials for wound dressing applications.

By applying electrospinning, dip-coating and polyelectrolyte complex formation, Ignatova et al. (2016) designed various fibrous materials with antimicrobial and antioxidant properties, based on quaternized chitosan derivative named N-trimethyl ammonium chitosan iodide, poly(3-hydroxybutyrate), caffeic acid and k-carrageenan. Thus, system represented by caffeic acid /poly(3-hydroxybutyrate) fibers was coated with N-trimethyl ammonium chitosan iodide/k-carrageenan polyelectrolyte complex, while system based on poly(3-hydroxybutyrate) fibers was coated with N-trimethyl ammonium chitosan iodide/k-carrageenan complex containing caffeic acid. Ability of fibrous materials containing N-trimethyl ammonium chitosan iodide and/or caffeic acid to inhibit the growth of E. coli and S. aureus was due to the fact that caffeic acid and N-trimethyl ammonium chitosan iodide exercised synergic antibacterial activity (bacteriostatic or bactericidal). Both materials containing caffeic acid and quaternized chitosan suppressed bacteria growth after 3–4 h of exposure. In addition, by studying S. aureus cells adhesion to the surface of fibrous materials, it was observed from scanning electron microscopy images that a large number of viable S. aureus cells with unaltered morphology adhered on surface of hydrophobic neat poly(3-hydroxybutyrate) fibers. In return, on contact with surface of treated poly(3-hydroxybutyrate) fibers, adhesion of pathogenic S. aureus bacteria was suppressed such that no bacteria or only a limited number of adhered bacteria were observed.

When antimicrobial properties of molecular iodine were combined with those of N-(2-hydroxy) propyl-3-trimethylammonium chitosan chloride, a new material with stronger antimicrobial activity was obtained (Tang et al. 2015). Thus, a stable charge transfer complex of N-(2-hydroxy) propyl-3-trimethylammonium chitosan chloride with iodine resulted by combination of those two components in a molar ratio of 1:1.33, due to the attraction between large electron cloud density of iodine and positive charge of quaternary ammonium salt. By testing antibacterial efficacies of samples against S. aureus and E. coli, it was observed that the antibacterial inhibitory effect increased in order: chitosan > N-(2-hydroxy) propyl-3-trimethylammonium chitosan chloride > N-(2-hydroxy) propyl-3-trimethylammonium chitosan chloride-iodide. Other approach to improve antimicrobial activity of chitosan consisted of synthesis of chitosan quaternary ammonium salts with halogens: chitosan halo-acetates and chitosan halo-1,2,3-triazoles (Tan et al. 2016; Li et al. 2016). In this way, electronegativity of halogenic substituent groups in chitosan ammonium salts enriched positive charge density of cationic amino groups, and antimicrobial activity of chitosan derivatives, consequently.

Antifungal activity of a series of water soluble chitosan ammonium salts with halogens namely, chitosan-bromoacetate, chitosan-chloroacetate, chitosan-dichloroacetate, chitosan-trichloroacetate and chitosan-trifluoroacetate was evaluated in vitro by hypha measurement of three phytopathogens viz. Fusarium oxysporum, Phomopsis asparagi and Colletotrichum lagenarium. For example, in case of 1.0 mg/mL of potential antimicrobial agent, and in presence of F. oxysporum, inhibitory index values of 55.8%, 69.6%, 71.0%, 73.9% and 78.5% were reported for chitosan-bromoacetate, chitosan-chloroacetate, chitosan-dichloroacetate, chitosan-trichloroacetate and chitosan-trifluoroacetate, respectively. These increased values compared with respect to chitosan (11.8%) were in relation with substitution degree of polysaccharide and protonation degree of amino groups. As electronegativity of halogenic substituent groups in chitosan ammonium salts decreased in order trifluoromethyl > trichloromethyl > dichloromethyl > chloromethyl > − bromomethyl, the antifungal activity followed same tendency: chitosan-trifluoroacetate > chitosan-trichloroacetate > chitosan-dichloroacetate > chitosan-chloroacetate > chitosan-bromoacetate > chitosan); such that a stronger adherence of antimicrobials to outer membranes of fungi produced more damage to fungal integrity and transport of nutrients was affected (Tan et al. 2016).

Starting from 6-bromo-6-deoxy-N-phthaloyl-chitosan, Li et al. (2016) synthesized triazole chitosan quaternary ammonium iodide, chloro-1,2,3-triazole chitosan quaternary ammonium iodide and bromo-1,2,3-triazole chitosan quaternary ammonium iodide. In case of chloro-1,2,3-triazole chitosan quaternary ammonium iodide and bromo-1,2,3-triazole chitosan quaternary ammonium iodide, the growth inhibition of phytopathogens like F. oxysporum f. sp. niveum ATCC36116, F. oxysporum. f. sp. cucumebrium Owen ATCC42357 and C. lagenarium (Pass) Ell.et halst ATCC30016 was due to an interactive effect of halogen and triazole moieties. Chitosan quaternization by chemical grafting reactions with different low molecular weight quaternary ammonium salts represents a usual practice to obtain chitosan derivatives. In this way, limited antibacterial and antifungal activities of chitosan which are related to its cationic character below pH 6.5, were improved. In addition, even solubility of new chitosan derivatives was better compared with pure chitosan.

Later, Overvidez-Munoz et al. (2017) functionalized amine groups of chitosan with different quaternary ammonium salts such as 4-bromobutyl-benzalkonium bromide, 4-bromobutyl-triethylammonium bromide or 4-bromobutyl-pyridinium bromide, resulting in following derivatives of chitosan: 4-bromobutyl-benzalkonium bromide chitosan, 4-bromobutyl-triethylammonium bromide chitosan and 4-bromobutyl-pyridinium bromide chitosan. Amongst these modified chitosan samples, 4-bromobutyl-pyridinium bromide chitosan showed highest antibacterial activity, while 4-bromobutyl-triethylammonium bromide chitosan was the least active. Chitosan bearing benzalkonium, triethylammonium and pyridinium moieties, resulting from mixing of 4-bromobutyl-benzalkonium bromide, 4-bromobutyl-triethylammonium bromide and 4-bromobutyl-pyridinium bromide in a weight ratio of 1:1:1, was less soluble, and displayed a reduced antimicrobial activity due to an improper contact of insoluble material with bacterial cells.

Wang et al. (2016) intended to modify the hydroxyl group of chitosan from C2 position, and prepared five water-soluble O-quaternary ammonium salt-chitosans bearing N-methyl-N-R-N, N-bis(2-hydroxyethyl) ammonium bromides where radical R represented benzyl, dodecyl, tetradecyl, hexadecyl or octadecyl. Because the new amphiphilic chitosan derivatives presented lipotropic character due to long carbon chains, and hydrophilic character due to quaternary ammonium salt moiety within O-quaternary ammonium salt-chitosan backbone, its antibacterial abilities and cytotoxicity were consistently improved compared to their low molecular weight quaternary ammonium salt homologues. In other works, a chitosan quaternary ammonium salt, named N-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride, obtained by grafting glycidyl trimethylammonium chloride on chitosan, was the subject of subsequent transformations: combination with commercial-grad Reactive Red x-3b (Tang et al. 2016) or phthalic anhydride (Chen et al. 2016). Using Reactive Red x-3b, a color reactive bearing sulfonic groups, suitable for textile and wood, a biopolymer dye with improved water solubility and antibacterial properties compared with pure low molecular weight dye was achieved. It was observed that in presence of new biopolymer dye, number of bacterial colony was reduced from 79% to 99% in case of E. Coli, and from 73% to 99% in case of S. aureus due to a synergic effect of quaternary ammonium and sulfonic groups from structure of biopolymer dye (Tang et al. 2016).

Following esterification reaction of N-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride with phthalic anhydride, subsequent introduction of NCO-sulfobetaine onto OH position of N-phathaloyl quaternized chitosan derivative were confirmed. Amine groups of resulting N-phathaloyl quaternized betainized chitosan were recovered using an aqueous solution of hydrazine monohydrate (Chen et al. 2016). The final zwitterionic product (O-sulfobetaine-N-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride) fulfilled role of materials with enhanced antibacterial activity and biocompatibility based on synergic action of sulfobetaine and quaternary ammonium groups. Improved water solubility, low cytotoxicity and hemolytic activity of zwitterionic chitosan derivatives are among the necessary parameters for their application under in vitro and in vivo condition. Beside sulfonic and sulfobetaine groups, thiourea may synergistically act, potentiating the antimicrobial properties of the compound carrying quaternary ammonium groups. Usually, benzoyl thiourea and acyl thiourea derivatives of chitosan display antifungal activity (Eweis et al. 2006; Mohamed and El-Ghany 2012). Earlier, Li et al. (2015) introduced double antibacterial groups, namely O-quaternary ammonium and N-acyl thiourea, onto OH and NH2 positions of macromolecular chains of chitosan following few steps: synthesis of N-benzylidene-chitosan; quaternization of N-benzylidene-chitosan with glycidyl trimethylammonium chloride resulting in O-quaternary ammonium N-benzylidenechitosan; synthesis of O-quaternary ammonium chitosan; reaction of acyl thiocyanate with O-quaternary ammonium chitosan resulting in O-quaternary ammonium-N-acyl thiourea chitosan. In case of S. aureus , Bacillus subtilis, E. coli, Pseudomonas aeruginosa and Aspergillus niger, antibacterial activity increased in order: O-quaternary ammonium-N-acyl thiourea chitosan > O-quaternary ammonium chitosan > chitosan. Zeta potential measurements and transmission electron microscopy images confirmed hypothesis related to electrostatic interactions between cationic amino groups and negative charged cell membranes. Thus, higher values of zeta potential in case of quaternized chitosans compared with pure chitosan indicated an increase in positive charge of chitosan derivatives and an increased number of electrostatic interactions. Also, changes in bacterial morphology after treatment with O-quaternary ammonium-N-acyl thiourea chitosan were due to the rupture of cell membranes and modification of cell metabolism as a result of quaternized chitosan infiltration.

Knowing that chitosan quaternary ammonium salt has antimicrobial and good antioxidant properties, Song et al. (2016) attempted to integrate it in a traditional dental material in order to form a resin base which could improve the overall oral health. They used N-trimethyl ammmonium chitosan chloride, denture water (containing methyl methacrylate monomer, cross-linking agent, inhibitor and an UV absorber) and denture powder (containing poly(methyl methacrylate)), and prepared two types of formulations in accordance with order of material mixing. Material 1 was prepared physically by adding varying amount of chitosan quaternary ammonium salt directly to denture powder, and then denture water. Material 2 was prepared chemically by adding variable amount of N-trimethyl ammmonium chitosan chloride to denture water, which determined the graft copolymerization of methyl methacrylate monomer with the chitosan quaternary ammonium salt. Both formulations presented low cytotoxicity and insignificantly modified tensile strength. In contrast, these materials recorded different antimicrobial properties and degrees of corrosion resistance. Antimicrobial tests were carried out with microorganisms specific to oral cavity: Streptococcus mutans and Candida albicans. In case of both materials, antibacterial rate increased with concentration of chitosan quaternary ammonium salt. As material 1 may have higher specific surface area over chemically produced material 2; it recorded a better performance in both antibacterial and antifungal tests as compared to material 2. In addition, material 2 did not recorded any antifungal property.

5.3.3 Dextran Type Antimicrobials

Naturally synthesized by Leuconostoc mesenteroides and S. mutans bacteria, dextrans are complex glucans composed of glucose molecules linked by α-1,6 glycosidic linkages in main chain or by α-1,3 glycosidic linkages in branching points. These polysaccharides with various molecular weights due to branched glucose chains of variable lengths are intensively used in medicine and laboratory as anticoagulants, size-exclusion chromatography matrices, cell osmotic pressure or blood sugar level regulators, and components of biosensors or bioreactors. To diversify its degree of application, dextran could be chemically modified such that to obtain cationic amphiphilic dextran derivatives with potential application as broad spectrum external biocides.

Quaternary ammonium groups were attached to dextran main chain bearing alkyl or dialkyl end groups by reaction of modified polysaccharide with an equimolar mixture of a tertiary amine (N,N-dimethyl-N-benzylamine, N,N-dimethyl-N-octylamine or 1-methylimidazol) and epichlorohydrin. Obtained cationic amphiphilic dextran derivatives were tested from microbiological point of view (Tuchilus et al. 2017). It was observed that all samples were ineffective against P. aeruginosa ATCC 27853, but displayed antimicrobial activity against E. coli ATCC 25922, S. aureus ATCC 25923, Sarcina lutea ATCC 9341, Candida glabrata ATCC MYA2950, C. albicans ATCC 90028 and C. parapsilosis ATCC 22019. Besides type of microbes, antimicrobial activity of samples was correlated with polymer chemical composition. Thus, self-assembling properties, and consequently interactions with microbial cellular components, were tuned by dextran with relative molar mass of 6000 or 10,000, chemical structure of ammonium groups e.g. R3 = benzyl, octyl; R2 = dimetyl-R3-quaternary ammonium, imidazol; R1 = dodecyl, octadecyl, and length of end alkyl group, meaning by hydrophilic/hydrophobic balance. Even that length of alkyl or dialkyl chains like C12, C18 or (C12)2 did not significantly influenced antimicrobial activity of samples originating from dextran with the same molecular mass, modified polymers based on dextran 10,000 recorded lower activity compared with those coming from dextran 6000. In addition, polymers with N,N-dimetyl-N-benzyl-quaternary ammonium groups showed the best results in case of all tested microorganisms, while samples with N,N-dimetyl-N-octyl-quaternary ammonium groups have a preference for E. coli, and imidazolium based polymers induced only a weak antifungal activity.

5.3.4 Pullulan Type Antimicrobials

Pullulan is a neutral homopolysaccharide with linear chain consisting of almost regularly repeating α-(1 → 4)–maltotriosyl units (3-D-glucopyranosyl) joined through α-(1 → 6) linkages. It looks like an amorphous slime formed by aerobic fermentation of Aureobasidium pullulans polymorphic fungus (Singh et al. 2008; Cheng et al. 2011). Molecular weight of this exopolysaccharide is from 4.5 × 104 to 6 × 105 Da and varies with yeast cultivation parameters. Hydroxyl functional groups as a part of flexible polysaccharide chain are the subject for chemical modifications and can result into a series of derivatives: pullulan succinylate, pullulan acetate, pullulan amine, carboxymethyl pullulan, cholesterol bearing pullulan (Singh et al. 2015). In this way, chemical and physical properties of pullulan such as hygroscopicity, biodegradability, viscosity, non-reducing agent, water solubility, formation of oxygen impermeable films are improved due to solubility in organic solvents or enrichment with new reactive functional groups.

Since its introduction as food additive by the Japanese, pullulan and its derivatives recorded numerous applications in foods, pharmaceutical and medical industries. Exploiting antimicrobial potential of quaternary ammonium groups, Grigoras et al. (2013) designed three pullulan derivatives. Length and frequency of grafts influenced all solutions properties, and consequently antimicrobial activity. Diameter of inhibition zones, recorded in presence of S. aureus ATCC 25923 strains and salted aqueous polymer solutions, varied from 7 mm to 10 mm in accordance with solution concentration and poly(3-acrylamidopropyl) trimethylammonium chloride content in grafted polymer. Antibacterial activity of grafted pullulans was due to electrostatic interactions between quaternary ammonium cation of poly(3-acrylamidopropyl)trimethylammonium chloride and phosphatidylethanolamine, a negatively charged component from bacterial cell wall.

5.3.5 Starch Type Antimicrobials

Starch (amylum) is a polysaccharide synthesized by plants and structurally formed from amylase and amylopectin molecules in variable proportions. Because of their widespread availability, biodegradability and environmental friendliness, this macromolecule and its derivatives frequently are used as flocculants and disinfectants in water treatment industry. Cationic starch based flocculants possessing quaternary ammonium functional groups on backbone have been developed by grafting reaction or esterification, a more convenient, cost saving, and reliable reaction. Huang et al. (2017) synthesized four different starch-graft-poly(2-methacryloyloxyethyl) trimethyl ammonium chlorides with various grafting ratios, while Liu et al. (2017) reported synthesis of four starch-3-chloro-2-hydroxypropyl triethylammonium chlorides having various quaternary ammonium salt groups on starch backbone. Tests on synthetic turbid and microbiological contaminated waters revealed that starch based derivatives have dual functionality: flocculation effect and antimicrobial performance. Three dimensional excitation emission matrix fluorescence spectra of supernatants of bacterial cultures and scanning electron microscopy images of E. coli CMCC 44102 and S. aureus CMCC 26003 before and after flocculation using cationic starch based flocculants are used to represent antibacterial properties of these starch derivatives. Fluorescence signals from wavelength intervals of 200–240 nm and 280–380 nm, attributed to protein like extracellular organic matters, were more intense for E. coli than for S. aureus suggesting the higher breakdown of E. coli cells. Also, after flocculation, treatment with cationic starch based derivatives, changed shape of bacteria from uniform elliptical to anamorphic one in case of E. coli, while most of S. aureus cells preserved their spherical shape or appeared “pinched”. The study suggested that due to synergistic effect from inorganic particles of kaolin and cationic starch based flocculants, bacterial cell walls were effectively destroyed through strong electrostatic interactions.

5.3.6 Cashew Gum Type Antimicrobials

Cashew gum is a chemical constituent of Anacardium occidentale L. tree stem, extracted in order to prepare viscous and emulsifying suspensions for food and pharmaceutical industries. This anionic heteropolysaccharide is composed of β-D-galactose, α-D-glucose, arabinose, rhamnose and glucuronic acid in proportion of 72%, 14%, 4.6%, 3.2% and 4.6%, respectively. Galactose units are joined by β (1 → 3) links in main chain of macromolecule or by β (1 → 6) links in side chain (de Paula et al. 1998). To improve its weak antimicrobial activity (Campos et al. 2012), Quelemes et al. (2017) chemically modified cashew gum by quaternization reaction. Firstly, cashew gum alkoxide was reacted with epoxide- or N-(3-chloro-2-hydrohypropyl) trimethylammonium chloride. In this way, alkoxide groups of cashew gum were nucleophilic substituted by cationic quaternary ammonium moieties. All three resulted quaternized cashew gum derivatives with degree of substitution of 42%, 68% and 73% were tested against eleven strains of Staphylococcus genus. Generally, it was observed that minimum inhibitory concentration and minimum bactericidal concentration values decreased with the increase in degree of substitution; anti-staphylococcal activity was enhanced due to more incorporated positive charges in quaternized cashew gum. Influence of the most promising derivative on bacterial morphology was evaluated using atomic force microscopy. After a 24 h exposure of methicilin sensitive S. aureus ATCC 29213 culture to solution of quaternized cashew gum having 73% degree of substitution, mean height of cells changed from 0.9 μm (in case of untreated cultures) to 1.4 μm or 1.3 μm at minimum inhibitory concentration or minimum bactericidal concentration. In addition, integrity of cell wall was gradually affected due to partial damage of bacterial cell. Also, all quaternized cashew gum derivatives showed good biocompatibility on erythrocytes, fibroblasts and keratinocytes which recommended them as promising agents against skin pathogens.

5.3.7 Poly(lactide) Type Antimicrobials

Being a non-toxic, biodegradable and bioactive bioplastic intensively used globally, poly(lactic acid) or poly(lactide) represents a thermoplastic aliphatic polyesther extracted from natural sources like sugarcane, corn starch or cassava roots, and used as fibers or films. Thus, poly(lactide) fibers could be more sustainable compared with other polyesther fibers derived from petroleum.

Usually, finishing of poly(lactide) fibers is carried out by their treatment with an antimicrobial film of silver or zinc oxide nanoparticles, essential oils, antibiotics or chitosan. Some of these antimicrobial coatings operate by mechanism of controlled release or by formation of a biological barrier. To increase the effectiveness of antimicrobial protection in case of poly(lactide) fibers, Logar et al. (2016) tailored antimicrobial coatings with dual and synergistic activities using silver and quaternary ammonium functional groups. They developed a three-stage finishing procedure which involved creation of a silica matrix (I), in situ synthesis of AgCl from silver nitrate and sodium chloride (II) and in third stage, application of silver chloride and 3-(trimethoxysilyl)-propyldimethyltetradecyl ammonium chloride. Even though application of this coating generated some esthetic disadvantages for poly(lactide) fibers such as reduced lightness and increased yellowing; from microbiological point of view it displayed excellent bactericidal activity with a 99.99% reduction in S. aureus and E. coli colonies.

A water soluble cationic benzanthrone derivative namely 1-[(7-oxo-7H-benzo[de]anthracen-3-ylcarbamoyl)-methyl]-triethylammonium chloride was synthesized and incorporated into a poly(lactide) thin film in order to test its antimicrobial activity (Staneva et al. 2015). The compound was observed to be very photostable and exhibited a prolonged release of benzanthrone derivative into aqueous solution. The system composed of poly(lactide) matrix and benzanthrone derivative was tested against a wide spectra of microorganisms (Bacillus cereus, B. subtilis, S. lutea, Micrococcus luteus, E. coli, Pseudomonas aeruginosa, Acinetobacter johnsonii, S. cerevisiae, Xanthomonas oryzae, Candida lipolytica), and minimum inhibitory concentration was in range of 48–125 μg/mL and zones of inhibition were in range of 12–24 mm. Also, optical densities of growth media for E. coli and P. aeruginosa strains decreased in the presence of composite material compared with untreated poly(lactide) matrix. Authors attributed the antimicrobial activity of new system to action of cationic quaternary ammonium groups onto plasma membrane of yeasts and cytoplasmic (inner) membrane of bacteria.

5.4 Antimicrobials Based on Synthetic Polymers

Because natural sources are limited, sometimes researchers attempt to design synthetic polymers with antimicrobial activities. Biocompatible synthetic polymers like poly(amidoamine), poly(urethane)s, poly(siloxane)s, poly(methacrylate)s and poly(ethylene) have found applications in various medical subdomains. In addition, these have efficient antimicrobial properties and are non-toxic for rest of cells, too.

5.4.1 Poly(amidoamine) Type Antimicrobials

A new trend to enhance the contact surface between pathogenic cells and antimicrobial substances involves the use of tree like branched polymers, with a sphere like shape, named dendrimers. Modulating synthesis conditions of these molecules, the number of branches could exponentially increase with each new generation of branches. Poly(amidoamine) is a representative of dendrimer class, composed of repetitively branched subunits of amide and amine functionality. Because it is biocompatible, easy to synthesize and has similar properties with globular proteins, some researchers had functionalized this macromolecule in order to fabricate novel antibacterial agent for water treatment. Thus, Maleki et al. (2017) modified poly(amidoamine) dendrimers (generations G2 and G4) into quaternary ammonium salts using Cl, Br, I halogens groups. Then, antimicrobial activity was determined against E. coli, Klebsiella oxytoca, B. subtilis, and S. aureus isolated from contaminated water samples. Compared with unmodified polymers, quaternary ammonium salts exhibited an enhanced antimicrobial efficacy against bacteria, the most effective being G4 generation of poly(amidoamine) modified with iodine.

5.4.2 Poly(urethane) Type Antimicrobials

Versatile materials like poly(urethane)s, intensively applied in medical and automotive fields as biomaterials or coatings, are synthesized in forms of foams, starting from di- or triisocyanates and polyols. To avoid potential leaching of physically bonded biocides from polyurethane matrix, some authors chosed chemically bonded cationic groups like imidazolium, pyrrolidonium or ammonium to design antimicrobials with prolonged usage. Thus, Udabe et al. (2017) prepared porous materials with antibacterial properties using glycerol propoxilate, tri- or tetra-hydroxyl functionalized quaternary ammonium compounds, and poly(hexamethylene diisocyanate) or toluene diisocyanate in different compositions. They observed that poly(urethane) ability to grow was enhanced in accordance with amount of quaternary ammonium compound in foam formulation. Also, 20% of quaternary ammonium component in poly(urethane) foams ensured a high antimicrobial activity. Chemically incorporated ammonium groups into poly(urethane) backbone were responsible for killing of S. aureus ATCC 29737 and E. coli ATCC 25922. In case of E. coli, an efficiency of 89% was observed for poly(urethane) formulation with 16% quaternary ammonium content. However, in case of S. aureus , maximum killing efficiency was observed with 7% of quaternary ammonium content. Besides, different chemical composition of microorganism structure and different content of reactive antimicrobial groups, type of isocyanate was another influencing factor for antibacterial behavior. In comparison with poly(hexamethylene diisocyanate), the more hydrophobic molecule toluene diisocyanate ensured a higher antimicrobial rate.

In another study, dressings based on poly(urethane) foams containing poly(diallyl-dimethylammonium chloride), known as polyquaternium-6, were applied on wounds infected with different microorganisms: S. aureus AH133; P. aeruginosa strain PAO1 Lux; a clinical isolate of Acinetobacter baumannii; S. aureus Lux; MRSA strains of S. aureus (Tran et al. 2017). Wound dressing material was placed on back of laboratory animals and bacterial biofilm formation in tissue under bandage was evaluated. It was observed that poly(diallyl-dimethylammonium chloride) containing poly(urethane) dressings totally inhibited biofilm formation, for all test organisms, reducing wound overinfection.

To endow them with antimicrobial properties, the biodegradable and biocompatible poly(urethane) coatings were successfully prepared by Liu et al. (2015) starting from a mixture of terpene-based polyol, various quaternary ammonium salts and hydrophilically modified hexamethylene diisocyanate tripolymer. In this case, quaternary ammonium salts were synthesized by quaternization reaction of terpene-based carbamate with alkyl bromides. Beside excellent mechanical properties and water resistance, resulted products recorded considerable antimicrobial activities against both E. coli and S. aureus even at reduced concentrations (5 wt%). As expected, antimicrobial activity of poly(urethane) coatings was improved by introduction of quaternary ammonium groups.

In order to prevent damage of medical devices covered by antimicrobials, but subsequently overpopulated with dead microorganisms, a research team designed a complex reverse surface structure, composed of an contact-active antibacterial upper-layer and a long-lasting antifouling sub-layer (He et al. 2016). Thus, gemini quaternary ammonium salt waterborne poly(urethane) films casted from solutions have been prepared using: isophorone diisocyanate, poly(tetramethylene glycol), poly(ethylene glycol), N,N,N′,N′-tetramethyl-N,N′-bisdodecyl-2,6-bis(ammonium bromide)–L–lysine-(1′,3′-propylene diamide)-L-lysine, and L-lysine. Poly(tetramethylene glycol) soft segment of modified poly(urethane) films and isophorone diisocyanate were hydrophobic, while soft segment poly(ethylene glycol) and L-lysine formed anti-fouling sub-layer, and N,N,N′,N′-tetramethyl-N,N′-bisdodecyl-2,6-bis(ammonium bromide)–L–lysine-(1′,3′-propylene diamide)-L-lysine antibacterial brushes formed upper layer. By varying molar fraction of components, different gemini quaternary ammonium salt waterborne poly(urethane) samples were obtained. Killing efficiency of this novel surface structure was tested against both types of Gram bacteria. It was observed that after a critical concentration of N,N,N′,N′-tetramethyl-N,N′-bisdodecyl-2,6-bis(ammonium bromide)–L–lysine-(1′,3′-propylene diamide)-L-lysine (4.96 wt%), all gemini quaternary ammonium salt waterborne poly(urethane) samples displayed strong antibacterial activity. Simultaneously, antifouling properties were related with surface hydrophilicity determined by contact angle measurements. Decreasing of time related water contact angle and enhancing of hydrophilicity at the same time with increase in N,N,N′,N′-tetramethyl-N,N′-bisdodecyl-2,6-bis(ammonium bromide)–L–lysine-(1′,3′-propylene diamide)-L-lysine, were attributed to the cationic nature of gemini quaternary ammonium fragments from modified poly(urethane)s.

5.4.3 Poly(siloxane) Type Antimicrobials

Poly(siloxane)s, generically called silicones, are biocompatible and biodurable materials widely applied in healthcare industry. State of aggregation of final products could be tuned by varying –Si–O– chain length, side organic groups attached to silicon atoms and crosslinking degree. Thus, silicones with properties characteristic to liquids or hard plastics such as thermal and chemical stability, hydrophobicity or low surface tension, represented matrix for various medical equipment and instruments. When the latter are to be used for therapeutic purposes, conditions of sterility and asepsis should also be considered. In this regard, antimicrobial materials based on silicones and quaternary ammonium moieties were designed.

Starting from poly(siloxane)s containing tertiary amino group and epichlorohydrin, Cui et al. (2015) obtained poly(siloxane) quaternary ammonium salts containing epoxy group with different molecular weights (Mw = 3150–13,000 g/mol) by a quaternization reaction in ethanol. It was observed that the new products spontaneously formed micelles in aqueous solutions and exhibited surfactant properties, as well as thermal and chemical stabilies. From microbiological point of view, poly(siloxane) quaternary ammonium salts containing epoxy group were active against S. aureus , B. subtilis, and E. coli such that increased molecular weight of polymers led to a high antibacterial activity. In addition, sensitivity of microorganisms in relation to poly(siloxane) quaternary ammonium salts containing epoxy group decreased in order: B. subtilis (most sensitive) > S. aureus (less sensitive) > E. coli (most insensitive).

Beside better wetting, emulsifying and solubilization properties, and lower critical micelle concentration, compared with conventional surfactants, gemini surfactants (molecules composed of two hydrophilic headgroups and two hydrophobic tails connected through a spacer unit) could have antimicrobial properties, especially when they have cationic or zwitterionic character. Taking into account all these advantages, Bao et al. (2017) synthesized some cationic gemini surfactants with poly(ether siloxane) linked group (Cm-PSi-Cm, m = 8, 10, 12, 14, 16, 18). Qualitative and quantitative antimicrobial tests revealed that C10-PSi-C10 polymeric surfactants have strongest antimicrobial ability against E. coli and S. aureus , while C14-PSi-C14 possessed the strongest antimicrobial activity against A. flavus. As expected, antimicrobial activities of C8-PSi-C8 and C18-PSi-C18 compounds were insignificant, because the inadequate length of hydrophobic chain (too long or too short) from structure of polymeric gemini surfactants was unfavorable to interactions with tested pathogenic microorganisms.

Another approach to improve antimicrobial properties of biocompatible synthetic polymers involves synthesis of copolymers, especially block copolymers. Because pure poly(dimethylsiloxane) has tendency to form a brittle silica like layer, it was chosen as candidate for blending with antimicrobial agents and subsequently chemical surface modifications. Thus, Qin et al. (2015) designed an antibacterial coating from block copolymers of poly(dimethylsiloxane) with quaternized poly(N,N-dimethylaminoethyl methacrylate). In this study, hydrophilic poly(N,N-dimethylaminoethyl methacrylate) was converted into cationic form by quaternization of tertiary amino groups with n-octyliodide. Spin coated copolymer films were obtained from acetonitrile solution of block copolymers mixed with hexamethylene diisocyanate. A different chemical composition of quaternized block copolymers was related with variable Si/N ratio: 1.5/1, 0.6/1, 0.7/1 and 1.1/1. In case of some films, mobility of poly(dimethylsiloxane) chains and intermolecular ionic interactions between alkyl side chains and positively charged groups in quaternary ammonium salts have favored appearance of microphase separations. So, surface roughness determined interaction with bacteria such that heterogeneous surfaces ensured an increased contact area with microorganisms and improved antimicrobial activity. From entire series of quaternized block copolymers, the exponent with the higher content of N+ showed antimicrobial activity towards both B. subtilis and E. coli. In contrast, the other three were insensitive towards Gram negative bacteria.

Self-assembling capacity of block copolymers was exploited by Zhou et al. (2015) in order to design new fluorosilicone copolymers containing bloks of dithiocarbonate terminated poly(dimethylsiloxane), poly(2-(dimethylamino)ethyl methacrylate) quaternized with n-octyliodide, poly(hexafluorobutyl methacrylate) and hydroxyethyl methacrylate, respectively. The new quaternized multi block copolymers contained different mass percentages of poly(2-(dimethylamino)ethyl methacrylate) and poly(hexafluorobutyl methacrylate). Visualization of inhibition zones around multi block copolymer films, in presence of representative Gram positive bacteria (B. subtilis ATCC 63501) and Gram negative bacteria (E. coli ATCC 44752), helped to correlate antimicrobial activities of these quaternized multi block copolymers with the stronger tendency of poly(dimethylsiloxane) and quaternary ammonium salts related blocks to migrate to surface of films, compared with poly(hexafluorobutyl methacrylate) blocks. Thus, fluorosilicone multi block copolymer containing appropiate amount of poly(hexafluorobutyl methacrylate), usually lower than 26.1 wt%, recorded antibacterial activity due to a higher C-N+ content and ability to form films with relatively smooth morphology.

5.4.4 Poly(methacrylate) Type Antimicrobials

Monomethacrylates and di-methacrylates are monomers intesivelly used in polymerization reactions resulting in thermoplastic materials with excellent weatherability, good mechanical properties and chemical resistance. Makvandi et al. (2018) reviewed antibacterial properties of dental composite materials composed of a quaternary ammonium modified methacrylate resin matrix, beside inorganic microfillers or nanofillers, coupling agents, and an initiator–accelerator system. Even that dental restoration materials incorporated with quaternary ammonium compounds exert a long time antibacterial effect compared with other antimicrobials, e.g. silver or zinc oxide nanoparticles, a balance between antimicrobial properties and mammalian cell cytotoxicity must always be considered.

Lv and team (2018) designed an electrochemical biosensor for immobilization of redox protein, which possesed antimicrobial properties, too. They used positively charged polymeric quaternary ammonium salts of dimethylaminoethyl methacrylate and negatively charged hemoglobin for constructing a film onto a glassy carbon electrode. Catalytic activity of immobilized hemoglobin was evaluated toward peroxide of hydrogen, and antimicrobial properties were tested against E. coli (ATCC 25922), S. aureus (ATCC 6538), Candida albicans (ATCC 14053) and Aspergillus fumigatus (Af 293). Both properties were influenced by the length of alkyl chain or type of methacrylate molecule (macromolecule or monomer).

5.4.5 Poly(ethylene) Type Antimicrobials

Beside well-known plastics like poly(urethane), poly(styrene), poly(propylene) or poly(vinyl chloride), being intensively used in daily life, poly(ethylene) is another thermoplastic semicrystalline polymer usually applied in packaging industry or medical instruments and devices. Because most of poly(ethylene) type materials come into contact with living beings, it is necessary to ensure their sterility. Thus, Rossetti et al. (2017) mixed poly(ethylene) from melt with mono- and bicationic quaternary ammonium salts. They used nine different salts: benzyldimethylstearylammonium chloride; trimethylstearylammonium chloride; dimethyldistearylammonium chloride; methyltristearylammonium bromide; tetrastearylammonium bromide; 1,12-bis(dimethyloctylammonium)dodecane dibromide; 1,20-bis(dimethyloctylammonium)eicosane dibromide; 1,30-bis(dimethyloctylammonium)triacontane dibromide; bis(dimethyloctylammonium)PEG(400) ditosylate. Resulted antimicrobial poly(ethylene)s were self-organized in such a way that quaternary ammonium salts bounded by Vander-Waals forces were displayed to the surface of polymer, forming nonleaching, permanent biocidal surfaces.

5.5 Target Microorganisms Affected by Polymeric Antimicrobials Containing Quaternary Ammonium Moieties

Eradication of various pathogens such as viruses, bacteria, fungi, algae from surrounding world, or of opportunistic germs from living organisms, involves the use of more or less aggressive physical or chemical methods. Each method is chosen according to the main structural features of target microorganism. In this regard, it takes in consideration hydrophilic character of Gram positive bacteria or hydrophobic character of Gram negative bacteria. This difference is due to synergic action of main components of cells. Thus, murein layer in Gram positive bacteria cell wall is thick, cross-linked and reinforced with teichoic and lipoteichoic acids, and displayed to external part of cell wall. In return, in case of Gram negative bacteria, thin layer of murein is located in periplasmic space between outer and inner membranes. If membrane of Gram positive bacteria is composed of cardiolipin, predominant phospholipids in inner membrane of Gram-negative bacteria are phospahtidylethanolamine and phosphatidylglycerol. In addition, outer membrane of Gram negative bacteria is covered with a lypopolysaccharides layer, while in case of Gram positive bacteria, outer membrane is missing. Even chemical structure of fungi is variable. For example, mycelium hyphae consisting of branched filament tubes are surrounded by a rigid cell wall with variable chemical composition, most of fungi containing chitin and glucan. Some fungus like organisms have a cell wall made up of considerable amounts of cellulose, while cell surface of Candida albicans is negatively charged due to terminal sialic acid, which is found on the surface of the membrane. There is a wide spectrum of pathogens studied by researchers, but representative microorganisms tested in the presence of polymers containing quaternary ammonium moieties are limited and grouped in Table 5.1 or schematically represented in Fig. 5.1 and Table 5.2, respectively.

Table 5.1 Representative microorganisms tested in presence of polymers with quaternary ammonium moieties
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Fig. 5.1
figure 1

Schematic representation of microorganisms and polymer chain bearing quaternary ammonium moieties. Under favorable conditions, macromolecular chains bearing positive charged groups of quaternary ammonium can interact with components of microbial entities

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Table 5.2 Chemical formulas of polymers with quaternary ammonium moieties and antimicrobial activity (selected from literature)
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A possible lower antimicrobial activity of quaternary ammonium polymers compared with standard antibiotics is balanced by their broader activity. Bacteriostatic/fungistatic and bactericidal/fungicidal effects of quaternary ammonium polymers are related to passive or active mechanism of action of substances in the presence of microorganisms. If a passive action of antimicrobials is supposed to repel pathogenic entity due to hydrophilic/hydrophobic/electrostatic repulsion or low surface energy, an active action involves microorganism killing by electrostatic or biocidal interaction (Huang et al. 2016). In depth, molecular mechanism of antimicrobial action is carried out along several stages: adsorption of antimicrobial agent onto cell surface/to outer membrane; formation of interface complexes between microorganism membrane and antimicrobial substance by diffusion through cell wall and binding of substances to cytoplasmic membrane; cytoplasmic membrane disruption; translocation of negatively charged lipids of membrane from inside part to outside part of membrane and lateral segregation of membrane lipids; destabilization of outer membrane by releasing cytoplasmic constituents like potassium ions, DNA and RNA; interaction of genetic material with biocidal agent; cell death (Munoz-Bonilla and Fernandes-Garcia 2012; Timofeeva and Kleshcheva 2011).

Preliminary qualitative microbiological tests and extensive quantitative microbiological methods are used in order to evaluate antimicrobial activity of substances. Some of parameters directly refer to microorganism number affected by potential antimicrobials; others are indirectly related with the effect of antimicrobials. Usually, colony forming units are counted, while cell viability, inhibitory index, bacterial reduction, and antimicrobial efficiency are assessed in percentage. Other parameters are also estimated from microbiological tests: bactericidal activity in time (h), bacteriostatic efficiency (log reduction), minimum inhibitory concentration, minimum bactericidal concentration and minimum fungicidal concentration. There are different approaches to evaluate inhibition zone; some authors include in diameter of zone of inhibition, even the size of cylinder or disk containing or immersed with antimicrobial solution; while others calculate zone of inhibition (in mm) by separating support “imprinted” with antimicrobial solution from diameter of transparent zone. Table 5.3 presents the antimicrobial activity of polymers containing quaternary ammonium moieties. It could be observed that diameter of inhibition had values of 6–25 mm for quaternized compounds compared with their precursors characterized by lower or nonexistent antimicrobial activity. Also, for a constant concentration of potential biocidal polymer, diameter of inhibition zone increased in accordance with quaternization degree of polymers. In some cases, values of diameter of inhibition zone were lower than those recorded in presence of standard antibiotics (22–32 nm).

Table 3 Table 5.3 Antimicrobial activity of polymers with quaternary ammonium moieties
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After a variable time of microorganisms incubation in presence of polymeric antimicrobials containing quaternary ammonium moieties, e.g. 4 h for hydrogels containing quaternized cellulose and native cellulose (Peng et al. 2016); 12 h for regenerated cellulose membranes including trimethoxysilylpropyl trimethyl ammonium chloride or trimethoxysilylpropyl octadecyldimethyl ammonium chloride (Meng et al. 2015) or 24 h for chitosan biopolymer dye (Tang et al. 2016), cell viability (%) was considerably reduced. This decrease in cell viability was inversely proportional to the alkyl chain dimension, substitution degree of ionizable groups in polymer chain and incubation time period. A similar behavior was observed in case of bacterial reduction. For example, for poly(urethane) coatings containing quaternary ammonium salts, this parameter had the highest value in case of compound with the longest hydrophobic alkyl chains of the whole series (Liu et al. 2015). In other case, if poly(lactic acid) fabrics coated with crosslinked Si-matrix contained 3-(trimethoxysilyl)-propyldimethyltetradecyl ammonium chloride and silver nanoparticles, the bacterial reduction represented a synergistic effect of quaternary ammonium groups and silver (Logar et al. 2016).

Because the quality and antiseptic properties of a substance are not assured only by a bacteriostatic effect, usually bactericidal activity is also examined (https://emerypharma.com/biology/minimum-inhibitory-concentration/). Literature reports that bacteriostatic activity is associated with a ratio of minimum bactericidal concentration to minimum inhibitory concentration greater than 4, although determination of this ratio is affected by numerous factors and technical problems. Regarding in vitro microbiological activity, it has been found that, at high concentrations, some antibacterial agents considered bacteriostatic are often bactericidal towards some susceptible organisms, while at low concentrations, other bactericidal drugs simply exhibit bacteriostatic effect. In vitro bacteriostatic or bactericidal data provide information about potential action of antimicrobial agents because only clinical results are relevant in treatment of bacterial infections. Thus, presumption that bactericidal agents are superior compared with bacteriostatic agents was invalidated by clinical results: meningitis, usually treated with bactericidal drugs, was effectively cured with bacteriostatic agents (Pankey and Sabath 2004).

In case of cationic amphiphilic dextran derivatives with a long alkyl group and quaternary ammonium moieties, minimum bactericidal concentration was two times lower than minimum inhibitory concentration; same results was recorded between minimum fungicidal concentration and minimum inhibitory concentration (Tuchilus et al. 2017). In the same time, values of minimum inhibitory concentration were insignificantly different from minimum bactericidal concentration values for the most quaternized cashew gums with different substitution degree of (3-chloro-2-hydroxypropyl) trimethylammonium chloride (Quelemes et al. 2017), while for poly(amidoamine) dendrimers modified with halogenated quaternary ammonium salts, values of minimum bactericidal concentration were slightly higher than minimum inhibitory concentration values (Maleki et al. 2017). Study about antimicrobial activity of poly(siloxane) quaternary ammonium salts containing epoxy group revealed that microbiological parameters indirectly have been influenced by concentration of quaternary ammonium groups such that minimum bactericidal concentration values generally decreased with increase of molecular weight of polymers (Cui et al. 2015).

5.6 Factors That Influence Antimicrobial Activity

Beside already presented heterogeneous chemical composition of microorganisms responsible for hydrophilic, hydrophobic, lipophilic or amphiphilic character of microbial entities, there are other factors that influence antimicrobial activity of substances (Table 5.4). Higher is the degree of substitution of ionizable groups, more potent is the antimicrobial activity. Because of a large number of incorporated positive charges in modified polymers, charge density increases with increase in zeta potential values for macromolecules in solution. Molecular weight of polymer is another characteristic that could indirectly influence charge density. By increasing degree of substitution, molecular weight of polymer increase and more electrostatic interactions are established between biomembranes and antimicrobial substances (Wang et al. 2016; Quelemes et al. 2017). A higher antimicrobial activity was recorded with an increase in molecular weight, in case of poly(siloxane) quaternary ammonium salts containing epoxy group compared with their precursor (Cui et al. 2015).

Table 5.4 Physical, chemical and physicochemical factors that influence antimicrobial activity of compounds bearing quaternary ammonium moieties
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Depending on position of the biological active quaternary ammonium groups in polymer structure (usually pendant, and rarely in side chain) and their steric hindrance, the flexibility and conformation of macromolecular chains may vary, influencing thus probability of interactions between cationic quaternary ammonium groups and anionic components of microorganisms (Bao et al. 2017). Flexible chains bearing quaternary ammonium groups have a facile access to components of microorganisms, while an extended conformation enhances antimicrobial effect (Grigoras et al. 2013). Also, probability of electrostatic interactions depends on density/concentration of quaternary ammonium bearing grafts on polymer backbone which is indirectly related to degree of substitution of ionizable groups (Grigoras et al. 2013). For example, in case of wastewater treatment industry, the number of grafts on polymeric backbone is important. Thus, flocculation properties of some quaternary ammonium type materials were enhanced with increase of grafting ratio, because cell wall of pathological E. coli was destroyed by electrostatic interactions established between modified starches and biomembranes (Huang et al. 2017).

Taking into account hydrophilic–hydrophobic balance, some researchers designed antimicrobials with polyampholyte or polyzwitterion character. Presence of hydrophobic alkyl chains with variable lengths between hydrophilic quaternary ammonium groups and polymer backbone could be tuned from synthesis conditions; the length of alkyl chain being thus a critical parameter. In this way, chemical structure of polymer, namely ratio between hydrophobic moiety and hydrophilic part, has a decisive role on antimicrobial activity. If hydrophobicity is enhanced by a long alkyl chain, penetration of quaternary ammonium groups through a hydrophobic biological membrane, mainly composed from proteins and phospholipids, is assured, and efficiency of antimicrobials with quaternary ammonium groups is improved (Meng et al. 2015; Bao et al. 2017). It seems that, usually, an octyl chain in structure of polymers is the best choice to design such polymeric antimicrobials containing quaternary ammonium moieties (Tuchilus et al. 2017; Qin et al. 2015). Since longer alkyl chains are more flexible, they can patch and mask the quaternary ammonium chains, so that electrostatic interactions will be limited and the antimicrobial potential of the substances will decrease (Lv et al. 2018).

Another issue concerns the correlation between self-aggregation state of quaternized macromolecules and their antimicrobial activity. Experiments revealed that some chemical agents were microbiologically active only in self-assembled form when minimum inhibitory concentration value was almost the same with that of critical micelle concentration; others were efficient at concentrations higher or lower than their critical micelle concentration. In addition, bacteriostatic or bactericidal character of a substance is dictated by relationship between minimum bactericidal concentration and minimum inhibitory concentration; for example, in case of quaternized dextrans, minumum inhibitory concentration recorded a value of 20 times greater than minimum bacterial concentration (Singh et al. 2008).

Electronegativity of different substituted groups with halogens in polymeric quaternary ammonium salts is a sensitive factor that influenced antifungal activities of halogenated derivatives of chitosan acetate (Tan et al. 2016) or of chitosan derivatives with halogeno-1,2,3-triazole (Li et al. 2016). It was observed that as electronegativity increased in the order trifluoromethyl > trichloromethyl > dichloromethyl > chloromethyl > bromomethyl, antifungal activity recorded same trend. Halogenated groups with stronger electronegativity have ability to draw more electrons from surrounding cationic amino groups of polymeric ammonium salts, and positive charge densities of cationic amino groups was consequently enhanced. Cationic amino groups possessing higher positive charge densities are more likely to interact with anionic components of fungal entities, such as mannan, glucan, proteins, and lipids, thereby blocking the proper transport of essential nutrients into cell.

pH of medium and type of solvent influence the polymer solubility, conformation of macromolecular chains and accessibility of cationic quaternary ammonium groups to anionic components of microorganisms. For example, chitosan, a biopolymer with intrinsec antimicrobial properties, become soluble in water if a small quantity of acid is added in solution such that glucosamine units are transformed into positively charged moieties. In addition, in order to increase solubility and antibacterial activity of this polysaccharide, introduction of substituting groups represented a feasible choice, especially in case of quaternary ammonium groups.

Compared with macromolecules in solutions, where antimicrobial activity depends especially on flexibility of macromolecular chains, in case of polymeric films containing quaternary ammonium moieties, surface morphology is related with position of biological active quaternary ammonium groups confined on film surface. On the other hand, morphology, roughness and topography of surface are in close relationship with antimicrobial activity. A surface with relatively smooth morphology and lower surface roughness value presents an increased contact area favoring microorganism adhesion (Rossetti et al. 2017).

In order to increase antimicrobial performance of materials, researchers chemically modify macromolecules such that equipped them not just with quaternary ammonium moieties, but also with other types of reactive antimicrobial groups: acyl thiourea, triazole, halogen, benzalchonium or pyridinium (Li et al. 2015; Tang et al. 2015; Tan et al. 2016; Li et al. 2016; Oyervides-Munoz et al. 2017). When they made physical mixture, beside quaternized polymer bearing quaternary ammonium cations, they used molecules with antimicrobial properties like silver, k-carrageenan or caffeic acid (Ignatova et al. 2016; Logar et al. 2016). In some cases, the intrinsic antimicrobial properties of polymers like chitosan were combined with the properties of quaternary ammonium cations (Mohamed et al. 2015). In this way, dual antimicrobial materials have a synergistic antimicrobial effect against a broad spectrum of microorganisms.

5.7 Conclusions

Quaternary ammonium groups are versatile chemical entities with antimicrobial properties that could be introduced in various type of polymers in order to design more efficient biocides. Incorporation of quaternary ammonium groups or moieties can be accomplished by two pathways or approaches: chemical and physical. Each of them has advantages and disadvantages. Antimicrobial agents bonded on matrix by physical forces are released in a controlled manner into environment until their concentration decreases by leaching of antimicrobial system. In return, chemical bonded antimicrobial agents, accomplished by binding biocide moieties to polymer chains, form a permanent bio-barrier and act as a biological obstacle for microorganisms that come in contact with polymeric matrix. Although, from economical point of view, a permanent biocidal material is preferred over physically bonded material, the latter possess a higher specific surface area available for more contacts with pathogenic entities. Even if polymeric quaternary ammonium salts have various advantages like chemical stability, no volatility, and generally do not penetrate mucosa, biocompatibility and cytotoxicity tests are required in order to assess potential toxic effects before introducing them into clinical use.

Currently, there are numerous roughly qualitative studies in which antimicrobial effects are generally observed. For the progress of pharmaceutical industry and responsible use of resources, the need for simulation studies of molecular dynamics regarding interactions between quaternary ammonium compounds and model molecules, which have chemical structures similar to each component of the microorganism, should be considered. Also, it is desirable and should insist on design of materials with dual-antimicrobial functional groups to prevent resistance of the most aggressive microorganisms to antimicrobial substances.