Using the reactive dye method to covalently attach antibacterial compounds to cotton

The Journal of Cotton Science 11:154–158 (2007), The Cotton Foundation 2007 TEXTILE TECHNOLOGY
Using the Reactive Dye Method to Covalently Attach
Antibacterial Compounds to Cotton
More recently, an awareness of general sanitation, contact disease transmission, and personal protection Fabric quality and durability are a concern
have led to the development of antibacterial fibers with fibers that contain natural antibacterial
to protect wearers against the spread of bacteria and properties or are treated to provide antibacterial
diseases rather than to protect the quality and durability properties. The textile industry has developed
of the textile material. Most of these approaches entail antibacterial fabric to address the public’s desire
the attachment of a biocidal or bacteriostatic agent for improved sanitation and personal protection
to the fabric surface. The mechanisms used to attach against disease transmission. The approach has
these agents to the fabric include the layer deposition been to attach biocidal or some bacteriostatic
of silver nanoparticles onto fabric structures (Dubas groups to the fabric surface. In this study, well
et al., 2006), graft polymerization of N-halamide described antibacterial drugs were attached to
monomers onto cellulosic substrates (Lui and Sun, cotton fabric with the goal that if this could be
2006), placement of quaternary ammonium salts onto accomplished easily, treated fabric could act
cot on fabrics using a covalently bound adduct (Son et as barriers against specific diseases or wound
al., 2006), covalent attachment of a chloromelamine infections. Trimethoprim and sulfamethoxazole
derivative (Sun et al., 2005), and the attachment of were modified to act as reactive dyes and were
chitosan to cotton fabric via cross-linking agents (El- covalently bonded to the surface of cotton in
talawy et al., 2005; Ye et al., 2006).
order to impart antibacterial properties. Some
The approach in this study was to modify two of the treated fabric was subjected to multiple
well described antibacterial drugs, previously or washings to determine durability. The treated
currently in use for treating diseases, for direct fabrics were then assayed for antibacterial prop-
attachment to cotton fabric. Assuming this can be erties. The preliminary results suggest that the
performed easily and cost effectively, the poten- antibacterial compound trimethoprim is tightly
tial exists to attach specific antibacterial drugs in bound to the cotton fabric and imparts to the
situations where treated fabric could act as a barrier fabric antibacterial properties, which are durable
against specific diseases or wound infections (Parikh through multiple washes. The results show that
et al., 2005). Such a situation would be advantageous, both trimethoprim and sulfamethoxazole impart
because the history and action of most antibacterial antibacterial properties to cotton fabric. These
drugs have been well researched and established, results indicate that other compounds may be
making its regulatory acceptance less of a burden. used to attach specific antibacterial compounds
Also, many more antibacterial compounds or drugs to fabric to create specific usage, designer, or tai-
have come on the scene in the last 50 years. Some lored fabrics to meet specialized needs.
of these antibacterial drugs have become so cheap and readily available that they are routinely added to Antibacterial finishing of textiles first appeared in animal feed as supplements to promote rapid weight 1941 in response to a need to protect the apparel of gain of the animal. Since these drugs are antibacterial, military personnel from hot and humid environmental by covalently attaching them to fabric the chance conditions in the South Pacific, which were ideal for of imparting desired antibacterial properties to the the growth of organisms on natural fiber substrates. fabric is expected to be high, which would provide the possibility of creating designer or tailored anti- microbial fabric or yarn for specific medical, as well general usage. The approach in this research also has D.T.W. Chun and G.R. Gamble, USDA-ARS Cotton Quality the potential for obtaining bacteriostatic fabric that Research Station, P.O. Box 792, Clemson, SC 29633 *Corresponding author: does not degrade upon washing, as occurs in some ChuN AND GAMbLE: uSiNG REACTivE DYES To ATTACh ANTibACTERiALS of the other described methods of attachment. in maintained at 5 °C during the drop-wise addition of addition, treating for bacteriostatic properties by a reactive dye mechanism will potentially prevent an Synthesis of 4- (2,4-dichloro-6-amino-s-triazino) alteration in the hand and moisture migration proper- -N-(5-methyl-3-isoxazolyl)benzenesulfonamide was ties of the treated fabric, which is one drawback of a accomplished by suspending 7.59 g (0.03 mole) sul- polymer graft mechanism of attachment.
famethoxazole (4-amino-N-(5-methyl-3-isoxazolyl) This study looked at the feasibility of utilizing benzenesulfonamide (Sigma Chemical Co.; St. Louis, two common antibacterial drugs and chemically Mo) in 20 ml deionized water in an ice bath at 5 converting them in order to obtain a reactive dye- °C. To this suspension, 5.52 g (0.03 mole) cyanuric type molecule, which could be applied to cotton chloride was added. The suspension was maintained fabric with the goal of imparting the antibacterial at 5 °C during the drop-wise addition of 30 ml 1 N properties of the antibiotic compounds to the fabric. Naoh (0.03 mole).
The two compounds used were trimethoprim and Bonding the reactive antibiotic to cotton fabric.
sulfamethoxazole, which both possess bacteriostatic An exhaust dyeing method was used to bind the reac- properties effective against a wide range of bacteria. tive antibiotic to the cotton fabric. The dye bath was The drugs are often used together as part of a syner- prepared by adding 0.5 ml of Triton-X-100 (octylphe- gistic combination for the treatment and prevention nol ethoxylates; Dow Chemical Co.; Midland, Mi), 75 of urinary tract infections, diarrhea, respiratory infec- g of sodium sulfate, and 6.5 g of the reactive antibiotic, tions, and prevention and treatment of infections by or 3.25 g of each of the two reactive antibiotics to Pneumocystis (beers and berkow, 1999). The drugs 1.2 L of deionized water. Three, 20-g squares of the interfere with folic acid synthesis in bacteria and cotton fabric were submerged in the dyebath heated eventually with bacterial DNA synthesis. These two to 60 °C. After 30 min of incubation, 12 g Naoh that compounds were reacted with cyanuric chloride, and had been dissolved in 100 ml of deionized water was the resultant product was then cross-linked to cotton added. The temperature was then raised to 80 °C, and fabric using an exhaust methodology. Treated fabrics the fabrics heated for another 30 min. The fabric was were then assayed for antibacterial properties.
then rinsed in deionized water and heated for 10 min at 80 °C in deionized water, then rinsed and kept in a MATERIALS AND METHODS
convection oven at 105 °C until dried.
Assay for antibacterial properties. The assay
Test fabric. The fabric from a commercial
used for measuring antibacterial properties was producer was supplied by Cotton inc. (Cary, NC). based on the ‘AATCC Test Method 100-1999, An- The fabric was a white, 100% cotton, tight-weave tibacterial Finishes on Textile Materials: Assessment denim-like fabric, weighing approximately 271.3 of” (Anonymous, 1999a), which has been described g/m², which had been commercially scoured and previously (Chun et al., 2006). Prior use of the assay bleached. The fabric was cut into large squares, ap- found that the population densities after incubation proximately 12.8 cm x 12.8 cm, before being treated. often remained the same or increased even in the After treatment, the large squares were ironed to controls, so the population densities of the zero remove wrinkles, cut into 18.1 cm2 squares, 4.25 time of incubation for the controls and treatments cm on a side, and sterilized in an autoclave using a were not determined. Test and control swatches dry cycle prior to the antibacterial assay.
were inoculated with challenge bacteria and after a Synthesis of a reactive trimethoprim and sul-
period of incubation, the bacteria were eluted from famethoxazole to covalently bond with the cotton
the swatches with known volumes of extraction so- fabric. Synthesis of 2,4-bis (2,4-dichloro-6-amino-
lution. Then the numbers of viable bacteria present s-triazino)-5-(3,4,5-trimethoxybenzyl)pyrimidine in the extraction solutions were determined and the was accomplished by suspending 5.80 g (0.02 mole) trimethoprim (2,4-diamino-5-3,4,5-trimethoxybenzyl Two bacterial species, Gram-positive Staphylo- pyrimidine) (Sigma Chemical Co.; St. Louis, Mo) coccus aureus (ATCC No. 6538), and Gram-negative in 20 ml deionized water in an ice bath at 5 °C. To Klebsiella (ATCC No. 4352) were used throughout this suspension, 7.36 g (0.04 mole) cyanuric chloride the experiment. Stock cultures were maintained on (2,4,6-trichloro-1,3,5-triazine) (Aldrich Chemical slants of Difco brain heart infusion Agar (Difco Co.; St. Louis, Mo) was added. The suspension was Laboratories; Detroit, Mi). The stock cultures were JouRNAL oF CoTToN SCiENCE, volume 11, issue 3, 2007 transferred once every 3 to 4 wk by incubating a and plates were incubated at 37 ± 2°C for at least 24 freshly inoculated slant at 37 ± 2 °C for 2 d before Experimental design and statistical analysis.
For each assay, the challenge bacteria were Four main fabric treatments were (A) a control fabric incubated in either a trypticase soy broth (TSb; that was not chemically altered; (b) fabric that had becton Dickinson and Co.; Cockeysville, MD) or on trimethoprim covalently bonded to it; (C) fabric that trypticase soy agar slants (TSA; becton Dickinson had sulfamethoxazole covalently bonded to it; and (D) and Co.) at 37 ± 2 °C for 1 to 3 d before being used fabric that had both trimethoprim and sulfamethox- to inoculate broth (TSb) cultures for testing. The azole covalently bonded to it. before assaying the 4 inoculated broth cultures were incubated in a shake treatments, a trial run using just the control and fabric incubator (37 ± 2 °C and 300 rpm) for 24 h. At the treated with trimethoprim was conducted to see if the end of incubation, the broth cultures were placed in antibiotic would covalently bond to the fabric, and if the attachment of the antibiotic would persist through To get a standardized density of bacteria for multiple washings. The fabric was washed by Cotton inoculation, the chilled cultures were diluted with inc. (Cary, NC), based on the AATCC Test Method TSB to a pre-determined turbidity that provided 124-1996 (Anonymous, 1999b) laundering procedure, approximately 2 x 109 CFU/ml or 2 x 108 CFU/ml. which used a normal/cot on sturdy cycle, 1.81 kg (4 lb) The turbidity was measured at 500 nm on a beckman load, warm water temperature, and AATCC detergent Du-7 Spectrophotometer (beckman instruments, without optical brightener. The treated and control inc.; irvine CA). Chilled TSb from the same batch samples were washed 3 and 10 times, respectively. For as the cultures were grown in was used to zero the the trial run, only results with Klebsiella pneumoniae instrument. Then the diluted broth cultures were serially diluted with chilled diluent without Tween- in the antibacterial assays, the bacterial in- 80 or gelatin (Chun and Perkins, 1996) for a final oculum was dispersed over 3 swatches per replicate approximate bacterial density of 1 to 2 x 105 CFU/ sample as droplets. Replicate tests were done and the ml. based on prior experience, 0.5 ml of the initial observations were combined and used for statistical broth culture was added early in the dilution series to analysis. A log10(CFu +1), where CFu = microbial compensate for the observed loss from the expected population as colony forming units, transformation population starting density to the actual starting was used for the analysis dealing with the microbial density used in the assays. The bacterial suspensions data. Data were analyzed with SAS (release 8.00; were kept in an ice bath. A magnetic stir bar and stir- SAS institute inc., Cary, NC) for Duncan mean ring plate was used to keep the bacteria suspended comparisons when the anlysis of variance analysis yielded significant F-values to indicate a high degree For each replicate sample, 1.0 ± 0.1 ml of inocu- of difference of the variable. Microsoft office Excel lum was dispersed as droplets over the 3 swatches us- 2003 (Microsoft Corporation, Redmond, WA) was ing a Rainin EDP-Plus Electronic Pipette (RAiNiN used to randomize treatment assignments, to enter instrument Co., inc.; Woburn, MA). The swatches and store data, to sort data and prepare for SAS anal- were inoculated in pre-sterilized 237 ml (half pint) ysis, to transform data, to summarize and tabulate canning jars. The band and lid of the canning jar were results, to obtain simple treatment statistics (means, screwed on the jar to prevent evaporation. After all standard deviations, regressions, t-test comparison, the samples were inoculated, the jars were incubated etc.), and to perform other spreadsheet functions.
at 37 ± 2 °C for 24 h before being assayed for bacte-rial population density.
The bacterial population density was determined by extracting the bacteria from the fabric by adding initial testing determined whether reactive 100 mL of diluent to each jar and shaking the jars antibiotics would covalently bond to the cotton on a tabletop shaker for 1 min. Then aliquots were fabric and impart antibiotic properties to the fabric. removed and plated directly into Petri dishes or A pilot test was done with trimethoprim using both further diluted before being plated. The pour plate the Staphylococcus aureus and Klebsiella pneumo- method was used to determine the bacterial density niae as challenge bacteria. With S. aureus, bacterial (Chun and Perkins, 1996). No antibiotics were used, populations of the control swatches averaged 6.291 ChuN AND GAMbLE: uSiNG REACTivE DYES To ATTACh ANTibACTERiALS [log10(CFu + 1)], and the swatches treated with S. aureus significantly after 24-hr incubation. Sul- trimethoprim averaged 5.101 [log10(CFu + 1)]. famethoxazole was less effective than trimethoprim With K. pneumoniae, the control swatches averaged alone or when both trimethoprim and sulfamethox- 7.743 [log10(CFu + 1)], and the swatches treated azole were attached to the fabric. but the fabric with trimethoprim averaged 4.333 [log10(CFu + treated with trimethopriman and trimethoprim and 1)]. The difference between the means of the control sulfamethoxazole were not significantly different. and the trimethoprim-treated swatches were highly Although fabric treated with sulfamethoxazole was significant using t-test analysis for S. aureus and K. bacteriostatic compared with the untreated control pneumoniae, P = 0.011 and P < 0.001, respectively. for K. pneumoniae, sulfamethoxazole was not sig- This indicated that the trimethoprim was bound to nificantly different than trimethoprim alone. The the cotton fabric, and the antibacterial activity of the total amount of trimethoprim and sulfamethoxazole in the 1:1 mixture treatment, however, was half the The large swatches of treated and untreated cot- amount used to evaluate the compounds individually. ton fabric were washed 3 and 10 times at Cotton inc. There is a possibility that the two compounds may to determine if the antibiotic binding to the fabric have had a synergistic effect to account for the low would be durable through normal washing. After bacterial density compared with trimethoprim alone washing, these large swatches were cut into smaller or the amount of the trimethoprim applied alone was swatches, sterilized, and then assayed for antibacte- in excess to what is needed to effectively lower the rial properties. Both S. aureus and K. pneumoniae bacterial density to this level. of course, this also were used, but only the results for K. pneumoniae suggests that the reactive trimethoprim may have will be reported. Populations of S. aureus between been preferentially attached to the cotton fabric and the unwashed control and the trimethoprim treated that even at half the dose could account for the lower swatches were 5.758 and 4.202 [log10(CFu + 1)], bacterial density when combined sulfamethoxazole. respectively, and were significantly different. The re- To resolve this, further research must be carried out sults from the washed portions of the assay, even after comparing different concentrations of the antibiot- being repeated, were unexplainable and anomalous. ics, both alone and together, in influencing bacterial The observations from two tests using K. pneumoniae densities, and the potential influence of competition were combined for analysis. The bacterial density for between dyes for the same hydroxyl groups on cot- the untreated fabric was 7.2, 6.8, and 6.7 log10 (CFu ton fabric. Since trimethoprim and sulfamethoxazole + 1) for the unwashed, washed 3 times, and washed were both easily prepared to act as reactive dyes and 10 times, respectively. These averages were not sig- that many, if not most, of other commonly known nificantly different from one another, which indicated antibiotic compounds have the same or similar that washing alone did not affect the bacterial density. reactive sites, many other antibiotics may be used The bacterial density of the treated fabric was 3.4, 3.3, in a similar manner, and future research should be and 3.6 log10 (CFu + 1) for the unwashed, washed 3 times, and washed 10 times, respectively. both un- Table 1. Bacterial density after 24-hr incubation on cotton
washed and washed treated fabrics had significantly fabric treated with trimethoprim, sulfamethoxazole, or
lower bacterial density than the untreated fabric, and both compounds
the averages were not significantly different among Density [log10(CFU + 1)]z
the three treated fabrics. These results indicate Treatment
that the antibacterial property imparted by binding pneumoniae
trimethoprim to cotton was durable and retained at Treated with
in a second experiment, fabric was treated with trimethoprim
trimethoprim, sulfamethoxazole, and a 1:1 mixture Treated with
of trimethoprim and sulfamethoxazole each at half sulfamethoxazole
the strength. The observations from three separate Treated with
antibacterial assays were combined for analysis trimethoprim and
(Table 1). The results indicate that both trimethop- z Means within a column followed by the same letter are
rim and sulfamethoxazole individually or together not significantly different according to Duncan’s multiple
depressed the bacterial density of K. pneumonia and range test (P = 0.05)
JouRNAL oF CoTToN SCiENCE, volume 11, issue 3, 2007 expanded to include testing a wide spectrum of Chun, D.T.W., Foulk, J.A., and McAllister iii, D.D. 2006. antibiotic compounds. This creates an opportunity Modification, use of, and description of AATCC test to design or tailor antimicrobial fabric or yarn us method 100-1999 to test for antibacterial properties of denim made from flax fabric. p. 2050. In Proc. beltwide ing this reactive dye method to attach antibacterial compounds to cotton. For example, one area where Cotton Conf., San Antonio, TX. 3 – 6 Jan. 2006. Natl. Cotton Counc. Am., Memphis, TN. Available online this approach may prove to be of value would be to at attach scarce antibacterial drugs to dressings to act as barriers to specific drug resistant bacteria to help prevent or reduce infection and its spread.
Chun, D.T.W., and h.h. Perkins, Jr. 1996. Effects of conven- in summary, trimethoprim and sulfamethoxazole tional cotton storage on dust generation potential, bacte-rial survival and endotoxin content of lint and dust. Ann. could be prepared as reactive dyes that can covalently bind to cotton fabric. The treated cotton fabric dis- Dubas, S.T., P. Kumlangdudsana, and P. Potiyaraj. 2006. played antibacterial properties that persisted through Layer-by-layer deposition of antimicrobial silver 10 launderings. Antibiotic concentration should be nanoparticles on textile fibers. Colloids and Surfaces A: investigated to determine efficacious rates. The ease Physicochem. Eng. Aspects 289:105-109.
of application may extend to the use of other anti- biotic drugs to provide value to cotton fabric where El-talawy, K.F., M.A., El-bendary, A.G., Elhendawy, and tailored or designer antibacterial fabric is desired.
S.M. hudson. 2005. The antimicrobial activity of cotton fabrics treated with different crosslinking agents and chitosan. Carbohydrate Polymers 60(4): 421-430.
Liu, S. and G. Sun. 2006. Durable and regenerable biocidal Mention of a trademark, warranty, proprietary polymers: acyclic N-halamine cotton cellulose. ind. Eng. Chem. Res. 45:6477-6482 product or vendor does not constitute a guarantee by the u.S. Department of Agriculture and does not Parikh, D.v., T. Fink, K. Rajasekharan, N.D. Sachinvala, imply approval or recommendations of the product A.P.S. Sawhney, T.A. Calaniari, and A.D. Parikh. 2005. to the exclusion of others that may also be suitable.
Antimicrobial silver/sodium carboxymethyl cotton dress- ings for burn wounds. Textile Res. J. 75:134-138.
Son, Y.A., b.S. Kim, K. Ravikumara, and S.G. Lee. 2006. imparting durable antimicrobial properties to cot- We gratefully acknowledge Cotton inc. for ton fabrics using quaternary ammonium salts through providing the cotton fabric used in the study and 4-aminobenzenesulfonic acid–chloro–triazine adduct. especially Norma M. Keyes and vickki b. Martin for their help in washing the test fabric.
Sun, Y., Z. Chen, and M. braun. 2005. Preparation and physi- cal and antimicrobial properties of a cellulose-supported chloromelamine derivative. ind. Eng. Chem. Res. REFERENCES
Anonymous. 1999a. AATCC test method 100-1999, Anti- Ye, W., J.h. Xin, P.L. K.L. D. Lee, and T.L. Kwong. 2006. bacterial finishes on textile materials: assessment of. p Durable antibacterial finish on cotton fabric by using 147-149. In American Association of Textile Chemists chitosan-based polymeric core-shell particles. J. Appl. and Colorists Technical Manual. vol. 75, 2000. AATCC, Anonymous. 1999b. AATCC test method 124-1996, Ap- pearance of fabrics after repeated home laundering. p. 205-208. In American Association of Textile Chemists and Colorists Technical Manual. vol. 75, 2000. AATCC, beers, M.h. and R. berkow (ed.). 1999. The Merck manual of diagnosis and therapy. 17th ed. Merck Publishing Group,


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