key: cord-0005518-71o3epej authors: Iyigundogdu, Zeynep Ustaoglu; Demir, Okan; Asutay, Ayla Burcin; Sahin, Fikrettin title: Developing Novel Antimicrobial and Antiviral Textile Products date: 2016-10-12 journal: Appl Biochem Biotechnol DOI: 10.1007/s12010-016-2275-5 sha: ebc8574f9847c7dfbcdc8dccea247be5db8e259b doc_id: 5518 cord_uid: 71o3epej In conjunction with an increasing public awareness of infectious diseases, the textile industry and scientists are developing hygienic fabrics by the addition of various antimicrobial and antiviral compounds. In the current study, sodium pentaborate pentahydrate and triclosan are applied to cotton fabrics in order to gain antimicrobial and antiviral properties for the first time. The antimicrobial activity of textiles treated with 3 % sodium pentaborate pentahydrate, 0.03 % triclosan, and 7 % Glucapon has been investigated against a broad range of microorganisms including bacteria, yeast, and fungi. Moreover, modified cotton fabrics were tested against adenovirus type 5 and poliovirus type 1. According to the test results, the modified textile goods attained very good antimicrobial and antiviral properties. Thus, the results of the present study clearly suggest that sodium pentaborate pentahydrate and triclosan solution-treated textiles can be considered in the development of antimicrobial and antiviral textile finishes. Textiles are woven or non-woven products produced by natural and/or synthetic fibers. Textile materials have a wide array of usage in areas such as clothing, food industry, building material, automotive industry, military, medical industry, sports equipment, agriculture, and home coli and inactivates influenza viruses on its surface [32] . Also, several sulfated polysaccharides and copolymers of acrylic acid with vinyl alcohol sulfate have been demonstrated to have antiviral activity against human cytomegalovirus (CMV) [31] . However, there is lack of knowledge and study about antiviral textile products so far. Occurrence of polio and vaccinia virus on wool and cotton fabrics has been investigated previously. These viruses were recovered up to 20 and 14 weeks, respectively, from wool fabrics that were exposed to the virus, but they persisted for shorter periods of time on the cotton fabrics [10] . Imai et al. investigated the effect of cotton textiles treated with copper ion-exchanged zeolites on the inactivation of avian influenza virus (AIVs), and they showed that highly pathogenic H5N1 and low pathogenic H5N3 viruses were inactivated on the copper ion-exchanged zeolite textile materials even after short incubations [16] . With increasing health awareness, people have started to pay attention about the removal or minimization of harmful pathogenic organisms from the environment or surfaces; therefore, various kinds of materials are being produced as antimicrobial. Recently, many scientists have investigated antibacterial, antifungal, or antiyeast materials, including textile goods. So far, there have been a number of studies conducted to develop antimicrobial and antiviral textile products, but success is yet to be achieved. This is the first study demonstrating that a novel formulation including triclosan and boron compounds can be used in broad spectrum antimicrobial and antiviral textile products. In this study, the antibacterial activities of textile products were tested against six bacterial species. In addition, one yeast and two fungi species were tested. The list of microbial species is given in Table 1 . The bacteria were obtained from ATCC, and the yeast and fungal species were provided by the culture collection unit of Yeditepe University, Department of Genetics and Bioengineering. Adenoid 75 strain of human adenovirus type 5 (ATCC VR-5) and Chat strain of human poliovirus type 1 (ATCC VR-1562) viruses were used for determining textile products' antiviral activities. HEp-2 (ATCC CCL-23) cells were used for antiviral activity tests. The standard NCCLS disk diffusion assay [23] was modified and used to assess antimicrobial activity against each microorganism tested. Briefly, 100 μL of suspensions containing 10 8 colony-forming unit (CFU)/mL bacteria, 10 6 CFU/mL yeast, and 10 4 spore/mL fungi were prepared from freshly grown cultures and spread on tryptic soy agar (TSA), Sabouraud dextrose agar (SDA), and potato dextrose agar (PDA), respectively. The blank disks (6 mm in diameter) were impregnated with 19 μL of 0.03 % triclosan (T) and 3 % sodium pentaborate pentahydrate (SPP) solutions and placed on the inoculated agar. Ofloxacin (5 μg/disk) and nystatin (100 μg/disk) were used as positive controls for bacteria and fungi, respectively. The inoculated plates were incubated for 24 h for bacterial strains and 48 h for yeast strains at 36 ± 1°C and 72 h at 27 ± 1°C for fungal species. Antimicrobial activity in the modified disk diffusion assay was evaluated by measuring the zone of inhibition against test microorganisms [18] . Each test was repeated at least twice. Antimicrobial solution that contained 7 % Glucapon 215 CS UP, 0.03 % T, and 3 % SPP solution was prepared, and pH of the solution was adjusted between 5 and 7 with citric acid or acetic acid. The solution was homogenized by mixing 1-1.5 h. Cotton fabrics were immersed into the solutions and shaken for 30 min; the fabrics were then dried at between 25 and 90°C. ICP-MS was used to indicate the boron content of the antimicrobial solution-treated textile surfaces. ICP-MS results were obtained by using X Series 2 ICP-MS Thermo Scientific (MA, USA). Treated textile samples (4 × 4 cm (0.28 g)) were mixed with 10 mL nitric acid (65 % HNO 3 ) and poured into microwave sample holders. The microwave was operated at 1600 W, 200°C, and 600 psi. Samples were filtered through 0.2-μm membrane filters and diluted with 40 mL distilled water. One milliliter of the solution was diluted to 50 mL, and the boron content was measured. UHPLC was used to indicate the triclosan content of antimicrobial solution-treated textile surfaces. Results were obtained by using Thermo Scientific UltiMate™ 3000 UHPLC systems (MA, USA). The instrument was equipped with an ACQUITY UPLC BEH reversed-phase column of C18 (2.1 × 50 mm, 1.7 μm). The UV wavelength used in the experiment was 230 nm for triclosan. Stock solution of triclosan (0.2 mg/mL) was prepared, and then, standard working solutions were prepared by dilution with a concentration ranging from 0.002 to 0.04 mg/mL in methanol/water (70:30, v/v). These standard samples were run on the HPLC, and a calibration curve was prepared. Treated textile samples were sonicated for 45 min in mobile phase in ultrasonic bath. Sonicated samples were filtered through 0.2-μm regenerated cellulose filters. Then, samples were prepared in appropriate to standard concentration amounts and analyzed by HPLC. The mobile phase of methanol/water (70:30 (v/v)) was delivered at a constant flow rate of 0.3 mL/min. The total run time for an HPLC analysis was 4 min. The antimicrobial activity of the surface-modified cotton textile products were cut as approximately 1 × 1 cm sizes, and antimicrobial activities of these products were investigated for selected microorganisms (Table 1 ). TSA, SDA or PDA were inoculated with bacteria, yeast, and fungus as described in Microbial tests section, and antimicrobial fabrics were placed onto inoculated medium. Sterile, distilled water-impregnated blank textile products were used as negative controls. HEp-2 cells, derived from an epidermoid carcinoma of the larynx, were maintained in minimum essential medium (MEM) (1×), supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin-streptomycin amphotericin (PSA). After 1 day, passage of HEp-2 cells at 1:4 in plastic flasks was infected by poliovirus type 1 (PV-1) and adenovirus type 5 (AV-5) for preparing viral stocks. Cytopathic effect (CPE) of prepared viral stocks was checked by microscopy. At 50-75 % CPE, the cell line was added to 10 % FBS and frozen at −80°C. After one freeze thawing of infected cells, supernatant was collected by low-speed centrifugation at 4-8°C for 30 min at 3300 rpm. All gross debris was discarded, and the supernatant was used as virus stocks in the experiment. To measure virus titer, HEp-2 cells were seeded into 96-well plates at a density of 2 × 10 4 cells and incubated at 37°C for 24 h in 5 % CO 2 and each individual sample was serially diluted from 10 −1 to 10 −9 in 10-fold increments. Each dilution was inoculated into HEp-2 cells and incubated for 3 days at 5 % CO 2 and 37°C. PV-1 and AV-5 titers in the cell culture were calculated by Spearman-Karber method [12] . HEp-2 cells were seeded at 2 × 10 4 in 96-well plates at cells in MEM and incubated at 37°C in 5 % CO 2 . Textile surfaces treated with chemical mixture were cut and placed on the top of the 0.5-cm diameter vials. Viral stocks (0.1 mL) were passed through the fabrics, and collected virus stocks in vials were sterilized by passing from filter of pore size 0.22 μm. Then, PV-1 and AV-5 were serially diluted from 10 −1 to 10 −9 in 10-fold increments. Each dilution was inoculated into HEp-2 and incubated for 3 days at 37°C in 5 % CO 2 . PV-1 and AV-5 titers in the cell culture were calculated by Spearman-Karber method [12] . Same procedure was repeated for untreated textiles as control group. Antimicrobial activities of the chemicals and chemical-treated textile goods were investigated against six bacteria, one yeast, and two fungi species based on the disk diffusion assay by evaluating the presence of inhibition zones. Zone diameters measured from the disk diffusion assay are given in Table 2 . The disk diffusion assay revealed that both SPP and T solutions displayed variable antimicrobial activities on the microorganisms tested. SPP (3 %) and 0.03 % T solution both showed antimicrobial effect against bacteria, yeast, and fungi. SPP has stronger anticandidal and antifungal activity than T. On the other hand, T had stronger antibacterial activity than SPP and even stronger than the ofloxacin disk that is used as a positive control. Antimicrobial mixture-treated textile goods antimicrobial test results showed that those fabrics have remarkable antimicrobial (antibacterial, anticandidal, and antifungal) effects against all tested microorganisms. Figure 1 shows the results of the antimicrobial tests concerning microorganisms given in Table 1 . The petri dishes that are presented in Fig. 1 correspond to untreated cotton fabric used as negative control, and modified cotton fabrics give qualitative results. In addition, Table 2 showed the inhibition zone diameters of antimicrobial textiles. Fabrics treated with 3 % SPP and 0.03 % T solution showed remarkable antimicrobial activity against all tested microorganisms where untreated fabrics exhibit no antimicrobial performance. Antimicrobial solution loaded on cotton fabrics revealed highest antimicrobial effect against Staphylococcus epidermidis where lowest antimicrobial activity was observed against S. aureus. The active ingredient amounts attached to the surface of the cotton fabric were determined quantitatively. According to the ICP-MS analysis, the boron ion contents of the treated textile samples were measured as 0.70 ± 0.050 % (w/w) that has an equivalent value of 3.8 ± 0.272 % SPP. Triclosan amount attached to the surface of the fabric was determined as 0.0243 %. Figure 2 shows the HPLC chromotogram for triclosan recovered from treated textile surface. HPLC chromotogram represents the triclosan peak relevant to the active compound for the retentition time at 2.97 min. Known concentrations of triclosan standards were overlayed with textile samples, and triclosan amounts of samples were obtained. Unfortunately, the mechanism of action of boron compounds against microorganisms is not totally understood, but previous reports support the antimicrobial activity. Remarkable antimicrobial activity against a wide range of microorganisms including bacteria (both gram positive and gram negative), fungi, and yeast have been stated [7, 9, 15] . Due to the large surface area and ability to retain moisture, textiles become a favorable medium for the growth of microorganisms on the fabric. Thus, scientists study antimicrobial textiles to control microbial growth, to prevent odors and discolorations caused by microorganisms and moreover to diminish the threat to public health [41] . Despite antimicrobial activity of boron compounds being reported in a few studies, there is no study about antimicrobial textile goods treated with boron compounds up to now. Contrary to boron, the antimicrobial mechanism of action of triclosan is well understood. It has been reported that triclosan blocks lipid biosynthesis and inhibits microbial growth [11] . The antimicrobial activity of triclosan has been known for decades, and it is used with textiles to obtain antimicrobial properties [11, 25, 27, 37, 42] . In the current study, the Triclosan has no or weak antimicrobial effect against Candida albicans and Aspergillus niger. However, due to the strong antimicrobial effect of SPP against these microorganisms, SPP + T combination-treated cotton textiles have shown remarkable inhibition zones around textile goods. As mentioned above, the antiviral tests were performed with AV-5 and PV-1. Initial AV-5 and PV-1 virus titers were both 5 log [10]/ml. Collected viral stocks that passed through the treated and untreated textiles were serially diluted and inoculated into HEp-2 cells. After 72 h, the antiviral effects were determined by the 50 % tissue culture-infected dose (TCID 50 ) and the virus-induced cell death was recorded by analyzing cell Table 3 ). Rate of decline in virus titers between wells was calculated by observing cell deaths. Figures 3 and 4 showed the view of death and viable cells at 72 h of AV-5 and PV-1 virus titers that were passed through the fabrics and serially diluted in 10-fold increments and the control groups. Even though antiviral activity of triclosan against a number of viruses has been studied previously [8, 17, 22] , there has not been any study about the antiviral activity of triclosantreated textile products so far. Just like triclosan, boron-containing compounds' antiviral activity has been indicated in a few studies. The boron-containing antibiotic, boromycin, was found as an antiviral agent against human immunodeficiency virus type 1 (HIV-1) strain in in vitro conditions [20] . Also, boronic acid antiviral activity against hepatitis C virus (HCV) has been indicated previously [40] . However, there is no study that reveals the antiviral activity of textile materials treated with boron compounds, up to now. In the current study, T and SPP were applied to cotton textile materials for the first time, and these modified textile materials showed antiviral effects against PV-1 and AV-5. The modified textile materials decreased both virus titers 60 %. In this study, antiviral tests performed against both DNA and RNA virus models which are AV-5 and PV-1, respectively. From this aspect, combination of 0.03 % T and 3 % SPP applied on textile materials should be effective against enveloped and non-enveloped DNA viruses such as hepatitis B virus (HBV), and they are assumed to be effective against enveloped and non-enveloped RNA viruses such as HIV, HCV, Ebola, MERS, and SARS. Further studies should be conducted for other types of woven and non-woven textile products. The examinations and tests performed show that the modification of cotton fabrics with 3 % SPP + 0.03 % T solution makes it possible to obtain antibacterial, anticandidal, antifungal, and antiviral effects as expected. The inhibition zones of microbial growth ranging from 16 to 46 mm obtained in modified disk diffusion assay indicates notable antimicrobial activity of modified cotton fabrics. Modified cotton textile fabrics showed antiviral activity against adenovirus type 5 and poliovirus type 1 and reduced both virus titers 60 %, while untreated fabrics cause no decline in viral titers. This formulation may be used not only in medical applications but also for manufacturing textile products of daily use and technical textiles. In the future, newly developed antimicrobial textiles are recommended for use in the military, health care, work/uniforms, home fashions and domestic products, and sports apparel. With these new technologies, the growing needs of the consumer in antimicrobial textile related to safety, human health, and environment are fulfilled. Efficacy Assessment of Treated Articles: A guidance Contamination of protective clothing and nurses' uniforms in an isolation ward Structural characterizations of metal ion binding transcriptional regulator CueR from opportunistic pathogen Pseudomonas aeruginosa to identify its possible involvements in virulence Evaluation of comfort properties of polyester knitted spacer fabrics finished with water repellent and antimicrobial agents A review on the application of inorganic nano-structured materials in the modification of textiles: focus on anti-microbial properties A new method to stabilize nanoparticles on textile surfaces Antifungal mechanisms supporting boric acid therapy of Candida vaginitis The antiviral action of common household disinfectants and antiseptics against murine hepatitis virus, a potential surrogate for SARS coronavirus Antibacterial and cytotoxic properties of boron-containing dental composite Quantitative studies on fabrics as disseminators of viruses II. Persistence of poliomyelitis virus on cotton and wool fabrics Recent advances in antimicrobial treatments of textiles Trimmed Spearman-Karber method for estimating median lethal concentrations in toxicity bioassays Control of methicillin-resistant Staphylococcus aureus in a hospital and an intensive care unit Physical properties of textile fibres Discovery of a novel class of boron-based antibacterials with activity against gramnegative bacteria Inactivation of high and low pathogenic avian influenza virus H5 subtypes by copper ions incorporated in zeolite-textile materials Triclosan: a review of effectiveness and safety in health care settings Determination of antimicrobial properties of Picaridin and DEET against a broad range of microorganisms Deodorant properties of wool fabrics dyed with acid mordant dyes and a copper salt Boromycin, an anti-HIV antibiotic Exogenous or endogenous reservoirs of nosocomial Pseudomonas aeruginosa and Staphylococcus aureus infections in a surgical intensive care unit In-vivo efficacy of hand sanitisers against feline calicivirus: a surrogate for norovirus Manual on antimicrobial susceptibility testing. Performance standards for antimicrobial testing: Twelfth Informational Supplement Antibacterial effect of nanosized silver colloidal solution on textile fabrics Molecular basis of triclosan activity Application of a fiber-reactive chitosan derivative to cotton fabric as an antimicrobial textile finish Antibiotic Resistance Antimicrobial finishing of cotton with zinc pyrithione Antibacterial activity of cellulose fabrics modified with metallic salts Survival of enterococci and staphylococci on hospital fabrics and plastic Sulfated polymers inhibit the interaction of human cytomegalovirus with cell surface heparan sulfate Antiviral and antibacterial polyurethanes of various modalities Enzymatic hydrolyzed feather peptide, a welcoming drug for multiple-antibiotic-resistant Staphylococcus aureus: structural analysis and characterization Simultaneous sonochemicalenzymatic coating of medical textiles with antibacterial ZnO nanoparticles Durable and regenerable antimicrobial textiles: synthesis and applications of 3-methylol-2, 2, 5, 5-tetramethyl-imidazolidin-4-one (MTMIO) Investigation of antibacterial activity on cotton fabrics with cold plasma in the presence of a magnetic field Structures of novel antimicrobial agents for textiles-a review Antibacterial properties of antimicrobial-finished textile products Epidemiology of infections with Pseudomonas aeruginosa in burn patients: the role of hydrotherapy Boronic acids in medicinal chemistry: anticancer, antibacterial and antiviral applications Antimicrobial finishing of cotton textile based on water glass by sol-gel method Triclosan and antimicrobial resistance in bacteria: an overview Antibacterial activity of cotton coated with ZnO and ZnO-CNT composites