ANTIBACTERIAL ACTIVITY OF CETYLPYRIDINIUM CHLORIDE TREATED COTTON WOVEN FABRICS
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1 ANTIBACTERIAL ACTIVITY OF CETYLPYRIDINIUM CHLORIDE TREATED COTTON WOVEN FABRICS İkilem Göcek 1, Burak Çömlekçi 1, Ziya Çevik 1, Sedat Yalcinkaya 2, İpek Ergal 2, Halil Kurt 2, Burçak Karagüzel Kayaoğlu 1, Alper T. Akarsubaşı 2, Argun Talat Gökçeören 3, A. Sezai Saraç 3 1 Istanbul Technical University, Textile Engineering Department, Istanbul, Turkey 2 Istanbul Technical University, Department of Molecular Biology and Genetics, Istanbul, Turkey 3 Istanbul Technical University, Chemistry Department, Istanbul, Turkey goceki@itu.edu.tr ABSTRACT In the current study, 100% cotton woven fabrics were treated with 2%, 4% and 6% wt/vol cetylpyridinium chloride (CPC) with respect to liquor volume to impart antibacterial property. Antibacterial activity of test samples was determined according to ISO 20743:2007 standard entitled as Textiles-Determination of antibacterial activity of antibacterial finished products. Two bacteria, one from gram negative (Klebsiella pneumonia ATCC 4352) and the other from gram positive (Staphylococcus aureus ATCC 6538) were used to test the antibacterial activity. An adequate antibacterial efficacy could be achieved even at the lowest salt concentration of 2%. Key Words: Woven fabric, cotton, antibacterial activity, quaternary ammonium salts, cetylpyridinium chloride 1. INTRODUCTION There has been an increasing interest in textiles with antimicrobial properties especially for products made from natural fibers such as cotton and wool. Such fibers provide a suitable media for the proliferation of microorganisms such as pathogenic or odor-generating bacteria and fungi [1, 2]. Antimicrobial functions were imparted to nylon fabrics via chemical finishing with quaternary ammonium salts. Processing parameters such as ph, finishing temperature and time were found to affect the exhaustion of the salts on the fabrics [3]. Quaternary ammonium salt containing monomers were grafted on cotton fabrics through high energy gamma radiation and antibacterial efficacy against various bacteria were obtained on the treated samples [4]. It is reported that polycationic antimicrobial agents comprising quaternary ammonium groups form an electrostatic bond with negatively charged sites on the cell wall of the bacteria and penetration of apolar alkyl chains of QAS into the cell wall leads to the disruption of the cell wall, resulting in cell lysis and death [4-6]. In the present work, the antimicrobial activity of quaternary ammonium salt, namely cetylpyridinium chloride (CPC), treated cotton fabrics were tested against two different bacteria namely, one from gram negative (Klebsiella pneumonia ATCC 4352) and the other from gram positive (Staphylococcus aureus ATCC 6538) according to ISO 20743:2007 standard [7]. 2. EXPERIMENTAL 2.1 Materials A pretreated, bleached and scoured, 100% cotton woven fabric supplied by Zorlu Tekstil, Turkey was selected as material. Properties of woven fabric are given in Table
2 Yarn Count Table 1. Properties of woven fabric Warp yarn Ne30 2 nd INTERNATIONAL CONGRESS ON Weft yarn Ne30 Density 26 ends/cm 24picks/cm Yarn Twist 600 turns/m (z) 800 turns/m (z) Weight 57 g/m 2 57 g/m 2 Weave Plain Cetylpyridinium Chloride, an organic salt, was purchased from Sigma-Aldrich was used to impart antibacterial property to the woven fabrics. It is known that this salt is used in various personal hygienic products such as toothpaste, nasal sprays, and antiseptic liquids, etc. The chemical structure of CPC is given in Fig Methods Figure 1. Chemical structure of cetylpyridinum chloride (CPC) Fabric samples were agitated for 60 min in a finishing bath with a liquor ratio of 30:1 using a James H. Heal gyrowash machine. The finishing temperature was 90 C. The treated samples were rinsed thoroughly in tap water, and allowed to dry in the open air. Woven fabrics were treated with 2%, 4% and 6% wt/vol cetylpyridinium chloride (CPC) with respect to liquor volume to impart antibacterial property. Antibacterial activity of test samples were determined according to ISO 20743:2007 ( Textiles determination of antibacterial activity of antibacterial finished products ) standard. Plate count methodology was applied to determine the antibacterial activity of the samples as a quantitative test method. Gram negative K. pneumonia ATCC 4352 and the Gram positive S. aureus ATCC 6538 was used to test antibacterial activity. Total of six fabric samples, each having a weight of 0.40g ± 0.05g, were tested for each species. Tests were performed for untreated control fabric and fabrics treated with three different cetylpyridinium chloride (CPC) concentrations of 2%, 4% and 6% wt/vol. Bacteria were grown on dehydrated yeast extract, casein tryptone and glucose containing enumeration agar, ph 7.2±0.2. Afterwards, one colony was inoculated in 20 ml of nutrient broth, ph 6.9±0.2, composed of beef extract, peptone, and water, for h at 37 C. Inoculums from the previous step (0.4 ml) were placed in 20 ml of nutrition broth, and incubated for an additional 3±1 h in order to obtain bacterial stock concentration of 10 7 Colony Forming Units/ml. Thereafter, stock concentration was diluted in between and CFU/ml. The quantity of bacteria in solution was determined using a SmartSpec Plus spectrophotometer from BIO-RAD, CA,USA. 146
3 1 ml of the inoculum was added into a test tube containing 9.0 ± 0.1 ml of the nutrient broth, and shaken well for a homogenous distribution using a Certomat SII shaker from Sartorius, Germany. Serial dilutions were prepared and 1 ml of each dilution was pipetted into two petri dishes containing 20 ml of LBA for each sample, then all the dishes were incubated at 37± 2 C for h. After incubation, the number of colonies on the petri dishes of dilution series were counted and the bacteria concentration in the solution was calculated according to the following formula: cb = Z x R (1) where cb is the bacteria concentration in Colony Forming Units per milliliter (CFU/ml); Z is the average value in Colony Forming Units (CFU) in the two petri dishes; R is the dilution rate. A Perkin Elmer FTIR spectrophotometer and a UV visible spectrophotometer, UV-1600PC, from VWR, USA were used to make chemical characterization of CPC salt on treated cotton fabrics. The tensile strength and tear strength of treated fabrics were tested using a Titan2 tensile testing machine according to TS EN ISO and TS EN ISO , respectively. Figure 2. Fabric samples during (left) tensile strength (right) tear strength test 3. RESULTS AND DISCUSSION 3.1 Antibacterial Activity Figs. 3 to 5 show the antibacterial activity of control and CPC treated samples with different CPC salt concentrations. The control samples showed no antibacterial activity as seen from the Figures. 147
4 Bacteria type Control sample 2 nd INTERNATIONAL CONGRESS ON Treated sample Klebsiella pneumoniae Staphylococcus aureus Figure 3. Antibacterial activity of control sample and fabric sample treated with 6% CPC salt Control sample Treated sample Klebsiella pneumoniae Staphylococcus aureus Figure 4. Antibacterial activity of control sample and fabric sample treated with 4% CPC salt 148
5 Control sample Treated sample Klebsiella pneumoniae Staphylococcus aureus Figure 5. Antibacterial activity of control sample and fabric sample treated with 2% CPC salt The antibacterial activity of the tested samples against two bacteria are given in Table 2, where the numbers in logarithmic scale show the mean values obtained from duplicate samples for each CPC concentration. Control samples showed no antibacterial activity. For all CPC concentrations, antibacterial activity efficiently observed (>log4). At the same time, CPC was found to be effective on both gram positive and negative bacteria with a slight difference. As the results showed that; 2% CPC treatment was sufficient to achieve an antibacterial efficacy on cotton fabrics. Table 2. Reduction in bacterial activity of control and CPC containing samples in logarithmic scale Samples K. pneumonia - S. aureus+ Control 0 0 2% CPC treated -5,89-5,22 4% CPC treated -5,89-5,52 6% CPC treated Mechanical Properties The tensile test results of the 6% CPC treated samples was around 190 N, whereas the control sample showed a tensile strength of 220 N. The application of CPC salt did not cause a significant reduction in the tensile strength. The tear strength values of control and CPC treated samples were comparable, which was around 8 N. The results showed that the CPC treatment did not have a negative influence on the mechanical properties of the fabric. 149
6 3.3 FTIR Analysis Fig. 6 shows the FTIR spectra of control and 2% CPC treated fabric samples. The orange line belongs to 2% CPC treated fabric and blue line corresponds to the control sample. Slight differences in the absorbance peaks at wavelengths of 1000, 3000 and 3500 cm 1 were observed which may be due to the addition of CPC salt to the fabric structure. The band with peak positions at 2900 and 2850 cm -1 corresponding to the C-H stretching vibration of the CH 2 and CH groups, while the strong sharp peak at 3433 cm 1 and the band at 1042 cm 1 are due to in-plane deformation of C H bond and N H bond seen for the pyridine and quaternized ammonium functionalities. 3.4 UV Visible Spectroscopy Figure 6. FTIR spectra of control and 2%CPC treated cotton fabric Fig. 7 shows the UV visible spectra of 0.05 M CPC in distilled water (blue) and 2%CPC treated cotton fabric (red). The graph shows the absorbance behavior of CPC salt in distilled water. Based on this, the release of CPC salt over time can be observed. The observed peak maxima at 405 nm for CPC could be used to determine the in-take of the cotton fabric. As it can be seen a peak was also be observed for the 2%CPC treated cotton fabric sample. But as seen from figure 7 the presence of CPC is lower for the treated sample. Figure 7. UV visible spectra of 0.05 M CPC in distilled water (blue) and 2%CPC treated cotton fabric (red) 150
7 4. CONCLUSION In this study, cotton woven fabrics were treated with CPC salt at different concentrations in a gyrowash machine. The presence of CPC salt on cotton fabric was verified by FTIR analysis. The treatment did not adversely affect the mechanical performance of the fabric. Antibacterial efficacy could be achieved even at the lowest salt concentration of 2%. The potential application of the proposed product would be a disposable wound covering material. The extension of this work will focus on the use of lower salt concentrations to minimize the use of QAS without sacrificing antibacterial activity. Currently, the durability of quaternary ammonium salt treatment on the fabric against washing has been being studied. This would expand the applicability of this treatment on different products such as hospital beddings and multiple-use medical gowns. 5. REFERENCES 1. Kim H.W., Kim B.R., Rhee Y.H., Imparting durable antimicrobial properties to cotton fabrics using alginate quaternary ammonium complex nanoparticles, Carbohydrate Polymers, 2010, Vol.79, Goel N.K., Rao M.S., Kumar V., Bhardwaj Y.K., Chaudhari C.V., Dubey K.A., Sabharwal, S., Synthesis of antibacterial cotton fabric by radiation-induced grafting of [2- (methacryloyloxy)ethyl] trimethylammonium chloride (MAETC) onto cotton, Radiation Physics and Chemistry, 2009, Vol.78, Son Y.-A., Sun G., Durable Antimicrobial nylon 66 fabrics: ionic interactions with quaternary ammonium salts, Journal of Applied Polymer Science, 2003, Vol.90, Goel N.K., Kumar V., Rao M.S., Bhardwaj Y.K., Sabharwal S., Functionalization of cotton fabrics by radiation induced grafting of quaternary salt to impart antibacterial property, Carbohydrate Polymers, 2010, Vol.79, Chang H, Yang MS, Liang M. The Synthesis, Characterization and antibacterial activity of quaternized poly(2,6-dimethyl-1,4-phenylene oxide)s modified with ammonium and phosphonium salts, Reactive & Functional Polymers, 2010, Vol.70, Gazzarri M, Bartoli C, Mota C, Puppi D, Dinucci D, Volpi S, and Chiellini F., Fibrous star poly(ε-caprolactone) melt-electrospun scaffolds for wound healing applications, Journal of Bioactive and Compatible Polymers, 2013, Vol.28, ISO 20743:2007, Textiles-Determination of antibacterial activity of anti-bacterial finished products, First edition , Externe elekronische Auslegestelle-Beuth-SNV shop Schweizer. Normen-Vereinigung ein Joint Venture mit TFV-KdNr ID, DF B0A2F10E7012C EB , 16:20:30, Available from: htm?csnumber=34261, Accessed: February 10,
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