Antimicrobial activity of chemically modified dextran derivatives

Carbohydrate Polymers 161 (2017) 181–186

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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Antimicrobial activity of chemically modified dextran derivatives Cristina G. Tuchilus a , Marieta Nichifor b,∗ , Georgeta Mocanu b , Magdalena C. Stanciu b a b

University of Medicine and Pharmacy Grigore T. Popa, Department of Microbiology, Faculty of Medicine, Iasi, Romania Petru Poni Institute of Macromolecular Chemistry, Department of Natural Polymers, Bioactive and Biocompatible Materials, Iasi, Romania

a r t i c l e

i n f o

Article history: Received 27 September 2016 Received in revised form 22 November 2016 Accepted 3 January 2017 Available online 6 January 2017 Keywords: Antimicrobial Dextran Amphiphiles Quaternary ammonium groups

a b s t r a c t Cationic amphiphilic dextran derivatives with a long alkyl group attached to the reductive end of the polysaccharide chain and quaternary ammonium groups attached as pendent groups to the main dextran backbone were synthesized and tested for their antimicrobial properties against several bacteria and fungi strains. Dependence of antimicrobial activity on both polymer chemical composition (dextran molar mass, length of end alkyl group and chemical structure of ammonium groups) and type of microbes was highlighted by disc-diffusion method (diameter of inhibition zone) and broth microdilution method (minimum inhibitory concentrations). Polymers had antimicrobial activity for all strains studied, except for Pseudomonas aeruginosa ATCC 27853. The best activity against Staphylococcus aureus (Minimun Inhibitory Concentration 60 ␮g/mL) was provided by polymers obtained from dextran with lower molecular mass (Mn = 4500), C12 H25 or C18 H37 end groups, and N,N-dimethyl-N-benzylammonium pendent groups. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction Infections caused by bacteria are an increasing threat to human safety, causing a large number of deaths every year (Gabriel, Som, Madkour, Eren, & Tew, 2007). The complex epidemiological situation is worsened by the bacterial resistance developed against traditional antibiotics and the lack of new and more active antibiotics (Davies & Davies, 2010; Wright, 2012). Therefore, it is essential to continuously develop antimicrobial agents with novel modes of action to face the evolving resistance. Antimicrobial polymers are a class of new antimicrobial agents with a rapid expansion in the last decade (Kenawy, Worley, & Broughton, 2007; Munoz-Bonilla & Fernandez-García, 2012; Siedenbiedel & Tiller, 2012; Sobczak, Debek, Oledzka, & Kozłowski, 2013; Timofeeva & Kleshcheva, 2011). In comparison with conventional agents of low molecular weight, polymeric antimicrobials have advantages such as longerterm activity, non-volatilization, inability to permeate the skin, longer circulatory time and reduced residual toxicity to the environment (Wang et al., 2015; Waschinski et al., 2008). Several classes of materials such as biocidal polymers, biocide-releasing polymers and antibiotic-conjugated polymers were developed (Siedenbiedel

∗ Corresponding author at: “Petru Poni” Institute of Macromolecular Chemistry, Department of Natural Polymers, Bioactive and Biocompatible Materials, Aleea Grigore Ghica Voda 41 A, 700457 Iasi, Romania. E-mail address: [email protected] (M. Nichifor). http://dx.doi.org/10.1016/j.carbpol.2017.01.006 0144-8617/© 2017 Elsevier Ltd. All rights reserved.

& Tiller, 2012; Strassburg et al., 2015). Amphiphilic cationic polymers are macromolecular counterparts to quaternary ammonium compounds, a well known class of broad-spectrum antibacterial agents, which are constantly applied as disinfectants in medical, industrial, or household areas. Antimicrobial activity of both types of cationic derivatives is based on a similar mechanism: the positively charged molecules adsorb on negatively charged microbial cell surface, diffuse through the cell wall and interact with the cytoplasmic membrane, leading to an irreversible damage of the cell membrane integrity and eventually to the cell death. This action mechanism is considered less prone to acquired resistance (Jennings, Minbiole, & Wuest, 2015; Munoz-Bonilla & FernandezGarcía, 2012). Chemical composition and relative position of cationic and hydrophobic groups have a decisive influence on cationic amphiphilic polymers antimicrobial activity. Polymers with both groups located on the same polymer side groups (pendent type) or with cationic and hydrophobic groups located on different side chains (random copolymers) (Oda, Kanaoka, Sato, Aoshima, & Kuroda, 2011) were designed and tested for their antimicrobial activity. Some recent studies have shown that antimicrobial activity of a polymer can be tuned and enhanced by separation of a quaternary ammonium group and a hydrophobic group (a long alkyl chain) by placing them at the opposite ends of a polymer chain (poly(methyloxazoline)) (Krumm et al., 2014; Waschinski et al., 2008). This chemical structure and the length of the end alkyl groups determine the degree of cell wall penetration by

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CH2

the polymer. The results provide interesting opportunities for the development of new biologically active polymers. Recently, we designed and prepared new cationic amphiphilic polymers which combine the pendent type structure with that placing the hydrophobic group at the end of the polymer chain (Nichifor, Mocanu, & Stanciu, 2014). The new polymers are based on a biocompatible and biodegradable polysaccharide, dextran, which can be selectively modified at its reductive chain end and at its numerous OH groups. These selective chemical modifications provided dextran derivatives carrying a hydrophobic alkyl chain at the reducing end and quaternary ammonium groups with moderate amphiphilicity attached as pendent groups to dextran backbone. The polymers form aggregates in aqueous solutions, and their selfassembling ability depends on the alkyl chain length and cationic group amphiphilicity (Nichifor et al., 2014). In the present work, we tested the activity of some of these new polymers as antimicrobial agents, using several bacterial and yeast strains. The influence of polymer chemical composition (dextran molar mass, end alkyl chain length, pendant quaternary ammonium group structure) on antimicrobial activity was followed. 2. Experimental part

CH2

O

OH

OH

O OH

OH

OH O

OH OH

n

Dextran

OH

(1) Alkylamine NaBH3CN DMSO/MeF 65oC, 48 h

(2) Tertiary amine/ECH 1/1 mol/mol Water 70oC, 6 h

CH2

CH2

O

OH

O O

OH

CH2

O

OH

x

O OH

OH y

OH

OH

R1

OH

CH2 HC CH2 R2 OH

A1- A8 R1 =

NH

CH2

n CH3

or

N

CH2

11

CH3

CH2 CH3 11

2.1. Materials 2.1.1. Chemicals Dextran samples from Leuconostoc mesenteroides with molecular weights Mr (as indicated by the supplier) 6000 (D6) and 9000–11000 (D10) were purchased from Sigma. The numberaverage molar masses (Mn ) determined by size exclusion chromatography were 4500 (D6) and 8000 (D10). All the other reagents were from Aldrich and used as received. DMSO and Nmethylformamide (MeF) were dried on molecular sieves. 2.1.2. Microorganisms Gram positive bacteria (Staphylococcus aureus ATCC 25923, Sarcina lutea ATCC 9341), Gram negative bacteria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853) and pathogenic yeasts (Candida albicans ATCC 90028, Candida glabrata ATCC MYA 2950, Candida parapsilosis ATCC 22019) used as reference strains were obtained from the Culture Collection of the Department of Microbiology, Faculty of Pharmacy, “Gr. T. Popa” University of Medicine and Pharmacy, Iasi, Romania. Staphylococcus aureus species of clinical provenience were isolated from surgical wounds (S. aureus 65, S. aureus 68, S. aureus 100) and blood culture (S. aureus 4828). S. aureus 68 and S. aureus 100 are methicillin resistant (MRSA), the other two species are methicillin sensitive (MSSA). 2.2. Polymer synthesis Polymer synthesis was realized by a procedure described in detail elsewhere (Nichifor et al., 2014). In a first step, dextran (D6 or D10) with an alkyl end group was obtained by reductive amination of dextran reductive end with a large excess of dodecyl, octadecyl or di(dodecyl)amine, using a mixture DMSO-MeF as a solvent and NaCNBH3 as a reducing agent. The product was purified by repeated precipitation from DMSO in methanol. The integrals of the specific peaks found in 1 H NMR spectrum (DMSOd6 , Bruker Avance DRX 400 spectrometer) were used to calculate the modification degree with formula: 100(ACH3 /3)/(Adex /DPdex ), where ACH3 and Adex are the integrals of the peaks assigned to the methyl protons of the alkyl chain (0.85 ppm,) and anomeric protons of dextran (4.7 ppm), respectively, and DPdex is the dextran degree of polymerization. The obtained values were in the range 92–98%. The complete reduction of intermediate unstable Schiff base to amine group was proved by UV analysis (325–330 nm)

Alkyl

Dialkyl

CH3 2

R =

N

R3 Cl

or

N

N CH3

Cl

CH3

DMR3

Imidazol

Scheme 1. Synthesis pathways and general chemical structure of cationic amphiphilic dextran samples A1–A8. (1) Reductive amination of dextran end aldehyde group; (2) Chemical modification of dextran OH groups with formation of pendant quaternary ammonium groups. Detailed chemical composition of each sample is given in Table 1.

performed on 0.1 wt% aqueous solutions. In the second step, the end modified polymers were reacted with an equimolar mixture of a tertiary amine (N,N-dimethyl-N-octylamine, N,N-dimethyl-Nbenzylamine or 1-methylimidazol) and epichlorohydrin, in order to attach quaternary ammonium groups to the dextran main chain. The final polymers A1–A8 were obtained by precipitation from water-methanol mixture 1/1 v/v in acetone. The general chemical structure depicted in Scheme 1 was confirmed by the presence of several peaks (1 H NMR, D2 O) assigned to pendent groups (-CH-OH at 4.2 ppm and -N-CH3 at 3.1 ppm) besides the peaks characteristics to dextran and end group. The amino group content (expressed as mol%) was determined by elemental analysis (content in chloride ions measured by potentiometric titration with AgNO3 ), using formula DS = x = 100 (162 · Cl) / (3550 − Cl · Mp) where Cl and Mp are cloride content, in wt%, and molecular weight of attached pendent group, respectively. 2.3. Methods 2.3.1. Polymer characterization The onset of polymer aggregation, critical aggregation concentration (CAC), was determined by fluorescence measurements in the presence of pyrene as a fluorescent probe (Nichifor et al., 2014). Steady-state fluorescence emission spectra were obtained with a LS 55 Perkin Elmer fluorescence spectrometer, using an excitation wavelength of 337 nm. Zeta potential and size of aggregates formed in 1 wt% aqueous solutions of cationic polymers was measured with a Zetasizer Nano-ZS, ZEN-3500 model (Malvern Instruments) with

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183

Table 1 Chemical composition and some characteristics of cationic polymers with chemical structure presented in Scheme 1. DS = 29 ± 2 mol%. Polymer sample

Mn,dex

R1

R2

R3

CAC (mg/ml)

Dh a (nm)

Zeta potentiala (mV)

A1 A2 A3 A4 A5 A6 A7 A8

8000 8000 8000 8000 8000 4500 4500 4500

Alkyl, n = 18 – Alkyl, n = 12 Alkyl, n = 12 Dialkyl Alkyl, n = 18 Alkyl, n = 12 Alkyl, n = 12

DMR3 DMR3 DMR3 DMR3 DMR3 DMR3 DMR3 Imidazol

Benzyl Octyl Octyl Benzyl Benzyl Benzyl Benzyl –

1.06 3.00 1.02 2.02 1.81 0.83 1.02 1.25

230 380 254 240 250 133 155 235

26 40 28 26 27 30 29 30

a

Values measured for 1 g/dL polymer aqueous solutions.

suspension (106 CFU/mL). MIC was the lowest concentration of extract where complete inhibition of visible growth was observed after 24 h incubation at 37 ◦ C (for antibacterial test) and 24 ◦ C (for antifungal test). MBC/MFC values were determined by transferring 0.1 mL of samples showing complete inhibition of visible growth on the surface of an agar plate. The subcultures were incubated 24 h at 37 ◦ C (for antibacterial test) and 24 ◦ C (for antifungal test). The MBC/MFC was the lowest concentration of extracts required to kill more than 99.9% of microorganisms being tested. MIC and MBC/MFC of ciprofloxacin/fluconazol towards bacterial/yeast strains were also evaluated. 3. Results and discussion 3.1. Design of cationic polymer samples and their characteristics

Fig. 1. TEM image of polymer A6. Bar represents 50 nm.

a He-Ne laser ( 633 nm). All the reported values are averages of three separate measurements, with a standard deviation of ±4%. The TEM images were taken with a HITACHI T7700 microscope (Tokyo, Japan), operated at 120 kV in high resolution mode using samples placed on a carbon-coated copper grid and negatively stained with an aqueous solution of phosphotungstic acid. 2.3.2. Antimicrobial activity 2.3.2.1. Antimicrobial susceptibility tests. Antimicrobial tests of selected microorganisms were carried out using a disc-diffusion method (CLSI, 2009, 2016). A small amount of each microbial culture was diluted in sterile 0.9% NaCl until the turbidity was equivalent to McFarland standard no. 0.5 (106 CFU/mL). The suspensions were further diluted 1:10 in Mueller Hinton agar for bacteria (Oxoid) and Mueller-Hinton agar for yeasts (HiMedia) and then spread on sterile Petri plates (25 mL/Petri plate). Sterile stainless steel cylinders (5 mm internal diameter; 10 mm height) were applied on the agar surface in Petri plates. Than, 0.1 mL of each compound (A1–A8, as 1 wt% aqueous solutions) were added into cylinders. Commercial available discs containing Ciprofloxacin (5 ␮g/disc), Fluconazol (25 ␮g/disc) and Nystatin (100 ␮g/disc) were used as positive controls. The plates were incubated at 37 ◦ C for 24 h (bacteria) and at 24 ◦ C for 48 h (yeasts). After incubation the diameters of inhibition zones were measured in mm, including disc size. 2.3.2.2. Broth microdilution method. The compunds were tested for the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)/Minimum Fungicidal Concentration (MFC) against S. aureus ATCC 25923 and Candida albicans ATCC 90028. Serial double dilutions of each extract in Mueller Hinton broth (Oxoid) were inoculated with equal volumes of bacterial/yeast

Chitosan and its derivatives were polymers of choice for the design of antimicrobial agents based on natural biocompatible polymers, due to chitosan weak intrinsic antimicrobial properties (Hosseinnejada & Jafari, 2016; Kong, Chen, Xing, & Park, 2010). Few other polysaccharides with attached cationic groups were studied for their antimicrobial activity, for example Konjac glucomannan (Yu, Huang, Ying, & Xiao, 2007) and cashew gum (Quelemes et al., 2017). We used here another polysaccharide, dextran, and chemically modified it in order to impart it new biological properties. General chemical structure of polymers selected for antimicrobial tests is shown in Scheme 1, and chemical composition of each polymer sample is detailed in Table 1. The choice of chemical compositions was based on some previously established relationships between structure and self-assembling properties. It is well known that the polymer charge density is one of the key factors in achieving a good attachment to the microbial cell surface. Therefore, the content in quaternary ammonium groups of all used polymers was kept at values of about 30 mol%, the highest charge density for which cationic polymers still preserve their amphiphilic character, that means they have a proper hydrophilic/lipophilic balance to allow self-assembling in aqueous solution (Nichifor et al., 2014). A chemical modification of dextran up to 30 mol% can also preserve a significant level of dextran backbone biodegradability, as revealed by earlier studies about dextran derivative with biomedical application (Verkauteren, Bruneel, Schacht, & Duncan, 1990). The chemical structure of cationic groups was limited to groups with moderate hydrophobicity of R3 substituents (octyl and benzyl), because some cationic polymers having shorter (ethyl, butyl) and longer (dodecyl, cetyl) alkyl substituents at the nitrogen of pendent quaternary ammonium groups did not display antibacterial activity (Nichifor, Stanciu, & Simionescu, 2010; Wang et al., 2015). A sample with imidazolium pendent groups (A8) was also used since imidazolium salts have proved to be very active as microbicidal agents (Colonna et al., 2012). Some polymer aggregate properties (CAC, hydrodynamic radius Dh , zeta potential values) were also included in Table 1 and the aggregate shape was assessed by

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Table 2 Antibacterial and antifungal activities of the tested polymers. Polymer samples

A1 A2 A3 A4 A5 A6 A7 A8 Ciprofloxacin(5 ␮g/disc) Fluconazol (25 ␮g/disc) Nystatin (100 ␮g/disc) a

Diameter of inhibition zones (mm) S. aureus ATCC 25923

S. lutea ATCC 9341

E. coli ATCC 25922

Pseudom. aeruginosa ATCC 27853

C. albicans ATCC 90028

C. glabrata ATCC MYA 2950

C. parapsilosis ATCC 22019

13.6 ± 0.57 13.6 ± 0.57 13.0 ± 0.00 13.3 ± 0.57 10.0 ± 0.00 15.3 ± 0.57 15.0 ± 0.00 0 24.7 ± 0.06 NTa NTa

14.0 ± 0.00 15.0 ± 0.57 13.7 ± 0.06 13.0 ± 0.00 14.0 ± 0.00 17.8 ± 0.28 17.8 ± 0.28 0 25.0 ± 0.00 NTa NTa

0 11.0 ± 0.00 10.6 ± 0.57 0 0 0 10.6 ± 0.57 0 30.5 ± 0.50 NTa NTa

0 0 0 0 0 0 0 0 30.0 ± 0.00 NTa NTa

11.0 ± 0.00 12.0 ± 0.00 12.0 ± 0.00 12.3 ± 0.57 13.0 ± 0.00 14.7 ± 0.06 14.3 ± 0.57 10.3 ± 0.57 a NT 30.5 ± 0.50 23.5 ± 0.50

12.5 ± 0.50 13.0 ± 0.00 12.0 ± 0.00 12.3 ± 0.57 12.3 ± 0.57 13.0 ± 0.00 15.0 ± 0.00 10.7 ± 0.06 a NT 23.5 ± 0.50 22.0 ± 0.00

13.0 ± 0.00 20.3 ± 0.57 20.3 ± 0.57 20.0 ± 0.00 20.3 ± 0.57 22.3 ± 0.57 23.3 ± 0.63 15.0 ± 0.00 a NT 22.0 ± 0.00 23.0 ± 0.00

NT-not tested.

Fig. 2. Disc diffusion assays of A2–A7 on S. aureus ATCC 25923 (left) and Candida parapsilosis ATCC 22019 (right).

Table 3 MIC and MBC/MFC values of tested polymers. Polymer sampleS. aureus ATCC

A1 A2 A3 A4 A5 A6 A7 Ciprofloxacin Fluconazol a b

25923

3.2. Antimicrobial activity C. albicans

ATCC 90028

MIC (mg/mL)

MBC (mg/mL)

MIC (mg/mL)

MFC (mg/mL)

1.25 1.25 1.25 1.25 1.25 0.06 0.06 1a n.d.b

2.5 2.5 2.5 2.5 2.5 1.25 1.25 2a n.d.b

2.5 2.5 2.5 2.5 2.5 1.25 1.25 n.d.b 8a

5 5 5 5 5 2.5 2.5 n.d.b 16a

Values are expressed in ␮g/mL. Not determined.

the TEM image presented in Fig. 1. Existence of a detectable CAC is a proof for a polymer amphiphilic character and its value is a measure of self-assembling ability. According to the TEM image (Fig. 1), and Dh values (Table 1) polymers form spherical micellelike aggregates, which are large in aqueous solution (150–350 nm, DLS), and small in dry state (10–20 nm, TEM). The rather high size of these aggregates in solution is due to extension of each charged dextran chains by intramolecular electrostatic repulsions. Zeta potential of polymer particles formed in aqueous solutions can play a role in the adherence of positively charged polymer to negatively charged microbe surface, consequently it could also influence the antimicrobial activity. As expected, all samples have similar zeta potentials due to similar charge density (Table 1), therefore this parameter might not be responsible for differences in polymer activity.

Antimicrobial activity was first evaluated by measuring the diameter of the inhibition zone (Fig. 2), and the results are summarized in Table 2. According to these data, the tested polymer samples have antimicrobial activity against all strains used, except for Pseudomonas aeruginosa ATCC 27853. Their activity is lowest for E. coli ATCC 25922 (only three sample, A2, A3 and A7, had some activity against this Gram-negative strain) and highest against C. parapsilosis. This behavior is similar to many other quaternary ammonium compounds. Gram-positive organisms, such as S. aureus, are known to be more susceptible than Gram-negative organisms (Russell, 2004), fungi of the Candida type have an intermediate susceptibility (McDonnel & Russell, 1999) and P. aeruginosa is the most resistant species (Lambert, 2002). All tested samples are less active than specific agents used as positive controls, except for C. parapsilosis, for which most of the polymer samples gave similar results with Nystatin. A lower activity than that of standard antibiotics was reported for other cationic polymers (Quelemes et al., 2017), but it is balanced by broader activity. Correlation of antimicrobial activity with polymer chemical structure highlighted the decisive role of the ratio between hydrophilic and hydrophobic part, tuned by dextran molecular mass. All samples based on D10 (2–3 wt% content in end hydrophobic block) have lower activity than those obtained from D6 (3.6–5.3 wt% hydrophobic block). The length of the alkyl chains –C12 , C18 or (C12 )2 (dialkyl) - did not have a significant influence on the activity of polymers with the same dextran sample (see samples A1–A5 and A6–A7). Presence of quaternary ammonium groups with R3 = benzyl gave the best results, in all cases, but samples with R3 = octyl seem to have a better affinity for E. coli (A2 and A3). Polymer with imidazolium groups had only a weak antifungal activity,

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Table 4 Antibacterial activity against clinical pathogenic S. aureus species. Bacterial strain

S. aureus 65 S. aureus 68 S. aureus 100 S. aureus 4828 a

A6

A7

Diameter of inhibition zone, mm

MIC (mg/mL)

MBC (mg/mL)

Diameter of inhibition zone, mm

MIC (mg/mL)

MBC (mg/mL)

13.3 ± 0.57 0.00 12.0 ± 0.00 0.00

1.25 n.d.a 2.5 n.d.a

2.5 n.d.a 5 n.d.a

14.3 ± 0.57 0.00 14.0 ± 0.00 12.7 ± 0.06

1.25 n.d.a 1.25 2.5

1.25 n.d.a 2.5 5

Not determined.

perhaps due to the lack of any hydrophobic moiety in the structure of pendent quaternary ammonium salts. A quantitative evaluation of antimicrobial activity is provided by values of MIC and MBC/MFC presented in Table 3. Two aspects have to be highlighted related to these values. First aspect concerns the correlation between MIC and CAC values. Previous attempts to establish a direct relationship between CAC and MIC did not provide reliable data. Some antibacterial agents were active only in selfassembled form, as they became efficient at concentrations higher than CMC (MIC > CMC) (Wang et al., 2015), but other agents had MIC < CMC (Denny, Novotny, West, Blesova, & Zamocka, 2005). In our case, MICs and CMCs are more or less similar for polymers based on D10 (A1–A5), but MI < CAC for polymers A6–A7. However, in the case of fungal strain C. albicans, MICs are always higher than CMCs. These findings show the absence of a correlation between self-aggregation state and antimicrobial activity and support the role of a fine balance between hydrophobic and hydrophilic groups of amphiphilic polymers which enables pronounced interaction of the polymers with biomembranes, more important than polymer self-assembling state. Secondly, the lowest values found for MIC (60 ␮g/mL) for S. aureus are of the same order of magnitude with values reported for other cationic amphiphilic polymers: 40 ␮g/mL for end modified polyoxazoline (Strassburg et al., 2015), 20–80 ␮g/mL for an acrylic polymers with cationic Gemini surfactant pendant groups (Wang et al., 2015) and for end modified ionene (Strassburg et al., 2015), 35–60 ␮g/mL for cationic cashew gum (Quelemes et al., 2017). It is also worth mentioning the relation between MIC and MBC/MFC. An agent is usually regarded as bactericidal if the MBC is no more than four times its MIC (French, 2006). Most of the samples listed in Table 3 could be considered bactericidal, as they have MBC/MFC ≈ 2MIC. In case of A6 and A7, the relationship between the critical concentrations determined for S. aureus is MBC ≈ 20MIC, therefore these samples could display mainly a bacteriostatic activity. Nevertheless, MBC values for A6 and A7 are lower than for the other polymers. Polymer samples with the best results (A6 and A7) against reference strains were also tested for their activity against clinical pathogenic S. aureus species. Data included in Table 4. show a moderate activity against MRSA 68 and the two MSSA strains (65 and 4828). The only strain resistant to tested cationic polymers is MR S. aureus 68, which is also resistant to Ciprofloxacin (data not shown). A comparison between the activities of the selected polymer samples against reference and clinical S. aureus strains highlighted some differences. MIC and MBC values found for A6 were twice those for A7, and A6 was not active against S.aureus 4828, indicating a higher activity of A7, which was not observed when reference strains were used. Besides, both polymer samples displayed bactericidal activity against clinical strains and bacteriostatic against reference ones.

4. Conclusions Cationic amphiphilic polymers with novel chemical structure, which combines the pendent type structure with that placing the hydrophobic group at the end of the polymer chain, were prepared by selective chemical modification of dextran with low molar mass, and proved to have moderate to good antimicrobial activity against several reference Gram positive or Gram-negative bacteria and fungi strains, as well as against clinical pathogenic strains. The best antibacterial activity was found against Gram positive Staphylococcus aureus ATCC 25923 and the best antifungal activity was displayed against Candida parapsilosis ATCC 22019. The antibacterial activity against S. aureus was significantly improved by decreasing dextran number-average molar masses (Mn ) from 8000 to 4500, due to increase of hydrophobic group relative content. Comparison between CAC and MIC values of tested cationic amphiphilic dextran derivatives showed that an appropriate balance between hydrophobic and hydrophilic groups of polymers is more important for antimicrobial activity than their self-assembling ability. All the results showed that these cationic polymers have potential for application as broad-spectrum external biocides.

Acknowledgement This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS – UEFISCDI, project number PN-II-ID-PCE-2011-3-0622.

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