Antimicrobial lab coats prevent the cross-contamination of pathogen accumulation and accidental bio-spills. Here, we describe the protocol for developing a skin-friendly antimicrobial fabric using nano-herbal encapsulation and modified standard tests to precisely evaluate the efficacy and suitability for typical usage of the lab coat.
Lab coats are widely used in biohazard laboratories and healthcare facilities as protective garments to prevent direct exposure to pathogens, spills, and burns. These cotton-based protective coats provide ideal conditions for microbial growth and attachment sites due to their porous nature, moisture-holding capacity, and retention of warmth from the user's body. Several studies have demonstrated the survival of pathogenic bacteria on hospital garments and lab coats, acting as vectors of microbial transmission.
A common approach to fix these problems is the application of antimicrobial agents in textile finishing, but concerns have been raised due to the toxicity and environmental effects of many synthetic chemicals. The ongoing pandemic has also opened a window for the investigation of effective antimicrobials and eco-friendly and toxic-free formulations. This study uses two natural bioactive compounds, carvacrol and thymol, encapsulated in chitosan nanoparticles, which guarantee effective protection against four human pathogens with up to a 4-log reduction (99.99%). These pathogens are frequently detected in lab coats used in biohazard laboratories.
The treated fabrics also resisted up to 10 wash cycles with 90% microbial reduction, which is sufficient for the intended use. We made modifications to the existing standard fabric tests to better represent the typical scenarios of lab coat usage. These refinements allow for a more accurate evaluation of the effectiveness of antimicrobial lab coats and for the simulation of the fate of any accidental microbial spills that must be neutralized within a short time. Further studies are recommended to investigate the accumulation of pathogens over time on antimicrobial lab coats compared to regular protective coats.
The protective white coat is a mandatory personal protective equipment (PPE) item in microbiology laboratories and healthcare facilities, and it protects from direct exposure to pathogens, spills, and burns. These cotton coats promote microbial growth due to many factors-the woven fabric provides attachment sites and aeration, cotton and starch used in the manufacturing process along with exfoliated epithelial cells from the user supply nutrients, and the proximity to the user gives warmth and moisture. The accumulation of microbes on textiles can also cause health problems such as allergies and nosocomial infection, unpleasant odors, and fabric deterioration1.
Unlike regular clothing, protective coats are infrequently washed or disinfected, as found in many surveys2,3. Many studies show evidence of lab coats acting as a vector of microbial transmission and the risk of nosocomial infections in the healthcare setting2,4, particularly resistant strains3 such as methicillin-resistant Staphylococcus aureus (MRSA); thus, they raise health concerns of PPE, which is meant to protect from microbial contamination. There are not enough cross-sectional studies on lab coat-associated infections in the context of Biosafety Level 2 (BSL-2) facilities or microbiology teaching labs, but many regulatory authorities restrict the use of lab coats within the containment level. However, many academic institutions in North America struggle to meet the requirements due to practical constraints, such as laundering and storing inside the facility, the incidents of wearing lab coats in public areas such as cafeterias and libraries are common. One practical solution to these issues is the application of antimicrobial agents in textile finishing.
Antimicrobial fabrics are gaining increasing popularity in sportswear, activewear, and socks, mainly intended to reduce body odor. However, the use of these fabrics is not common in PPE development, except for some silver-coated cotton masks and healthcare garments5. We report the development of an antimicrobial fabric for lab coats, which inhibits common pathogens found in BSL-2 labs and renders effective protection from the cross-contamination of common pathogens.
Currently, a variety of antimicrobial fabrics and finishings are available in the market, but most of these use heavy metal colloidal particles (e.g., silver, copper, zinc), organometallics, or synthetic chemicals such as triclosan and quaternary ammonium compounds, which are not environmentally friendly1 and may lead to health issues such as skin irritation and allergies6. Some synthetic formulations pose concerns due to non-target microbes, such as normal flora or inducing antimicrobial resistance (AMR). The US Food and Drug Administration (FDA) regulates commercial antimicrobial fabrics, which must be non-toxic to the user and free from eco-toxicity. Therefore, antimicrobial fabrics based on natural biocides that inhibit a broad spectrum of microbes are preferable. Essential oils (EOs) are used widely as antimicrobial and therapeutic agents, but their use in antimicrobial finishing is limited due to their durability6,7,8. Based on our knowledge and market research on nano-herbal finishing8, no herbal-based antimicrobial fabric is commercially available. This is because synthetic coatings are easy to manufacture and have long durability. A few nano-herbal-coated textiles reported only for research purposes include neem7, moringa9, and curry leaves9.
The present study uses two bioactive components extracted from oregano EOs, carvacrol and thymol, which are effective against a wide range of bacterial pathogens and viruses but are generally recognized as safe for humans10. However, these bioactive components are volatile, and therefore their antimicrobial potential is short-lived if applied directly to the fabric. Nano-herbal encapsulation is a process in which bioactive components or drugs are loaded inside a polymeric shell that protects the core from environmental degradation, and thus enhances the shelf life. In addition, the small size of the polymeric particles, which generally range from 10 nm to 100 nm, enhances the efficacy of the application and slows the release of the bioactive compounds onto the fabric. These bioactive compounds are used for various purposes, such as food preservation10, but not for textile coating.
Among many polymeric encapsulants, chitosan is an attractive candidate due to many of its attributes, such as nontoxicity, biodegradability, mucoadhesivity, and biocompatibility11. It is a natural polysaccharide, obtained by the deacetylation process from chitin, which is found in seashells and fungal cell walls. It is used in biochemical and food preservation applications such as drug or protein delivery11,12,13, controlled release14, and antimicrobial films10. Chitosan is not readily soluble in water but forms a colloidal suspension in acidic media. Bioactive molecules are loaded into chitosan nanoparticles (NPs) by a simple two-step ionic gelation method14,15,16. In this process, hydrophobic bioactive compounds such as carvacrol and thymol form an oil-in-water emulsion, which is aided by a surfactant, Tween 80. Subsequently, a polyanionic compound, pentasodium tripolyphosphate (TPP), is used to form the cross-linkages between the amino groups along the polycationic polymer molecules and phosphate groups of TPP molecules to stabilize the complex. This complexation process solidifies the bioactive compounds within the matrix of chitosan, which is subsequently purified and coated onto cotton swatches to produce antimicrobial fabric.
The nano-formulations must be tested first for antimicrobial effectiveness in emulsion form before being applied to the fabric. This can be conveniently evaluated by a qualitative method, such as Kirby-Bauer disk diffusion, well diffusion, and the cylinder plate assay. However, the cylinder plate assay17 provides the flexibility to load varying volumes of the formulation and compare the zone of clearance. In this method, the antimicrobial formulations are loaded in stainless steel cylinders and placed on a soft agar layer, which is inoculated with the test microorganism or pathogen. The diameter of the zone of clearance produced against the test organism is proportional to the inhibitory potential of the antimicrobial formulation, and therefore can be used as an alternative to broth dilution methods. However, the size of the clear zones is only a comparative or qualitative measure within a specific plate unless specific standards are maintained. Antimicrobial agents act against the pathogens either by inhibiting their growth (biostatic) or killing the cells (biocidal), which can be quantified by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), respectively. However, the efficacy and behavior of the bioactive chemicals are different in their formulations (liquid state) and when coated on a substrate such as a fabric18. This is because multiple factors play a role in the efficacy, such as the stability of the adherence of the antimicrobial agents to the fabric, moisture content, substrate type, and adherence of the microbes. If the intended purpose is only bacteriostatic activity, a qualitative assay such as the "Parallel Streak Method"19 can provide a relatively quick and easy evaluation of diffusible antimicrobial formulation. However, if the bactericidal effects are to be determined, "Assessment of Antibacterial Finishes on Textile Materials"20 can be employed, which provides the log reduction of the spiked pathogen.
1. Preparation of nanoparticles
2. Cylinder plate assay for screening of nanoparticles
3. Parallel streak method (modified from AATCC 147)
4. Quantitative log reduction method (modified from AATCC 100)
Initial screening of the synthesized NPs
Following the two-step oil-in-water emulsion technique16, the bioactive compounds (carvacrol and thymol) were successfully encapsulated in chitosan. This was confirmed by UV-Vis spectrophotometry for the peak absorption of the respective bioactive compounds compared to controls, which were the chitosan NPs without any bioactive compounds. The constituted NPs were homogeneous and stable over 12 months at 4 °C. The initial screening of the antimicrobial effectiveness was verified by the cylinder plate method (Figure 1). This is a qualitative method as the zone of clearance is influenced by multiple factors, such as agar thickness, the strength of the inoculum, and the concentration of the test samples. Many simpler methods, such as the Kirby-Bauer disk diffusion method and well diffusion method, can be employed for this purpose, but the cylinder plate method provides the opportunity to vary the concentrations (Figure 1A) by diluting the NPs, and each cylinder can hold the test sample volume up to 200 µL. In addition, the agar overlay with cultures forms a smooth inoculum, enabling the determination of the clear zones with better precision17. The results demonstrated that the clear zones are proportionate to the progressively increasing concentrations (Figure 1A). This adds validity to the data and differentiates it from any artifacts or abnormal zones. Based on the size of the clear zones (usually over 20 mm), the correct concentration of NPs can be selected for coating. Any previously characterized or old NPs, which were stored appropriately (4 °C), can also be verified by the large zones (Figure 1B,C) before coating onto the fabric.
Qualitative screening of the treated fabric samples
Despite the antimicrobial effectiveness of the encapsulated NPs confirmed by large clear zones, the NP-coated fabrics must be tested. This is because the antimicrobial agents may work differently when applied to the fabric compared to their original formulations. Many factors, such as the properties of the fabric (thickness, hydrophobicity), coating efficacy, and degradation of bioactive compounds while coating, influence the effectiveness20. As such, the parallel streak method was used to evaluate the antimicrobial effects of the treated fabrics qualitatively. The negative controls (untreated fabrics) demonstrated no antimicrobial effects by the uninterrupted microbial growth along all five streak lines (Figure 3 A,B). The treated fabric showed interrupted microbial growth along the streak lines (Figure 3 C,D), which was due to the diffused bioactive compounds from the fabric. However, the average width of the clear zones was low (<5 mm) as the test samples were subjected to 10 wash cycles before testing. As the inoculum concentrations decrease along the parallel steaks from the first to the fifth streak (Figure 2), the clear zones are prominent in the subsequent streaks. If the first streak (high inoculum) shows a clear zone, the antimicrobial potential of the fabric is usually high. Some irregularities, such as the fifth line in Figure 3D, may occur due to the qualitative nature of the test. The clear zone (interrupted growth) due to the bacteriostatic activity provides an indication of the antimicrobial potential, but does not give an adequately sensitive guideline18, which is calculated by the "log reduction test". However, the parallel streak method is useful for a relatively quick and easily executed procedure to screen a large number of swatches6,7,20, particularly when testing for a wash durability test over many cycles.
Quantitative analysis of the treated fabric samples
The log reduction test (also known as the percentage reduction test) demonstrated a significant (<0.001) reduction of microbial cultures upon contact with the treated fabric for 30 min (Figure 4 and Figure 5). The antimicrobial fabric swatches were significantly effective (>99%) against a Gram-positive bacterium (S. aureus), Gram-negative bacteria (E. coli and P. aeruginosa), and a skin fungal species (C. albicans). Since the untreated fabric (negative control) had no antimicrobial activity, the recovered CFUs were too numerous to count until diluted to extinction, up to 106 dilutions (Figure 4). The number of CFUs recovered from the treated fabrics at "0" contact time (plated immediately upon inoculation and neutralization) was very similar to that of the untreated fabric, and data were omitted for simplicity. The neutralizer used (Letheen broth) is effective in neutralizing the effects of phenolic derivatives such as carvacrol and thymol. Compared to carvacrol NPs, thymol NPs showed slightly higher antimicrobial effects against all four microbes (Figure 5). Both the thymol and carvacrol-coated fabric worked equally effectively against three microbes (S. aureus, E. coli, and C. albicans) with over a 4-log reduction (99.99%), except P. aeruginosa, which ranged from a 2.8- to a 3.2-log reduction (99.9). This was expected, as P. aeruginosa is intrinsically resistant to a range of antimicrobials22. The wash durability test demonstrated that the treated fabric was able to exhibit effective antimicrobial resistance (>99%) against three species (S. aureus, E. coli, and C. albicans) and moderate resistance against P. aeruginosa after 10 wash cycles.
Figure 1: Cylinder plate assay of synthesized nanoparticles with a range of concentrations tested against bacteria. (A) Serially diluted thymol NPs for an initial screening against E. coli showing the placement of cylinders and clear zones after 18 h of incubation. Progressively increasing concentrations "o" to "r" resulted in proportionately higher zones. The clear zone produced by the highest concentration is indicated by a red circle. The negative control (chitosan NPs without the bioactive compounds) is represented by "t" and the supernatant extracted during purification is represented by "s". (B) Two out of three concentrations of carvacrol NPs (12 months old, stored at 4 °C) screened for the fabric treatment showing effective zones (>20 mm) against S. aureus. (C) Three concentrations of thymol NPs (12 months old, stored at 4 °C) screened for the fabric treatment showing effective zones against S. aureus. Abbreviation: NP = nanoparticle. Please click here to view a larger version of this figure.
Figure 2: Parallel streak method layout. Placement of a fabric swatch on Mueller-Hinton agar inoculated with five subsequent parallel streaks. Please click here to view a larger version of this figure.
Figure 3: Parallel streak method results showing clear zones for the treated fabric swatches. The swatches were placed on top of the inoculum (bottom row) compared to untreated (top row). (A) Untreated fabric against S. aureus, (B) untreated fabric against C. albicans, (C) thymol NP-coated fabric (after 10 wash cycles) against S. aureus, and (D) thymol NP-coated fabric (after 10 wash cycles) against C. albicans. Please click here to view a larger version of this figure.
Figure 4: Log reduction results of antimicrobial fabric coated with thymol encapsulated chitosan nanoparticles tested agaisnt four pathogens. The lactose agar plates in panel B were donated by the Canadian Food Inspection Agency, which include their labels at the back side and look like swatches. Other agar plates were prepared in the lab, without labels. Fabric swatches were not placed on top of the agar plates, as in the experiment shown in Figure 3. Rather, the microbes were recovered from the swatches (after vortexing in PBS) and plated. (A) S. aureus on blood agar, (B) E. coli on purple lactose agar, (C) P. aeruginosa on cetrimide agar, (D) C. albicans on SDA. The pathogens were spiked on "untreated" (top row) and "treated" (bottom row) swatches for 30 min and recovered upon neutralization and dilution plating. The dilution ratios are shown between the treated and untreated series. Please click here to view a larger version of this figure.
Figure 5: Log reduction of three bacteria (S. aureus, E. coli, and P. aeruginosa) and one fungus (C. albicans) due to the contact of antimicrobial fabrics impregnated with two bioactive compounds (carvacrol and thymol separately). The antimicrobial efficacy is weakened after washed cycles (five times and 10 times, respectively) for both carvacrol- and thymol-coated fabrics. Please click here to view a larger version of this figure.
The antimicrobial efficacy of biocides is conventionally tested by quantitative assays, such as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), in which the bacteria are immersed in an antimicrobial liquid for 24 h. However, these assays are not suitable for coated fabrics, where the liquid interface is lacking and the biocides are diffused slowly along the fabric fibers. Therefore, many standard fabric tests have been established, such as AATCC 147, ISO 20645, AATCC 100, and JIS L 1902. A comparison study of these standards by Pinho et al.23 acknowledges that there is no consensus on the most suitable method to be used. This study modified the protocol slightly to better represent the use of antimicrobial lab coats in biosafety laboratories. The test microorganisms used were the species detected frequently from the lab coats in BSL-2 labs, based on a previous study (unpublished). They also represent a wide variety of pathogenic microbes that are commonly used in pharmaceutical tests, for example, a Gram-positive human pathogen (S. aureus), a Gram-negative indicator species (E. coli), a highly resistant species (P. aeruginosa), and a dermal pathogenic fungus (C. albicans).
The overall antimicrobial efficacy observed in this study (99.99%) was higher than the efficacy reported in similar studies7,9,23,24, which ranged from 80% to 99%. However, the way the experiments were carried out in the other studies (according to AATCC 100)19 provides a restriction to refine the efficacy, particularly when the efficacy is 100%. The modified protocol with a series of five plates (Figure 5) used in this study allows for calculating the log reduction with more accuracy. Upon inoculation of the fabric swatches, the incubation was carried out at R/T for 30 min, compared to the standard O/N incubation at 37 °C. The modification better represents the typical usage of the lab coat at R/T and any accidental microbial spills that must be neutralized within a short time (20-30 min) to consider the antimicrobial lab coat effective. The wash durability tests show that the antimicrobial efficacy (>90%) remained after 10 wash cycles. The antimicrobial retention of the fabric is a bit lower compared to some studies7,9, which produced efficacies up to 20-30 cycles. However, protective garments such as lab coats, unlike regular clothing, are infrequently washed2,3, and therefore 10 wash cycles would be adequate.
The selection of bioactive compounds is paramount, as the compound must be safe for the user. As such, many toxic chemicals and irritants are not compatible. EOs from a range of herbal products were extracted and screened for their suitability as an ideal candidate for nano-herbal encapsulation, considering the efficacy against the four pathogens, stains produced on the fabric, and an acceptable level of odor. For example, pomegranate rind extract showed significant effects against the pathogens, but the stain was not acceptable for a white coat. However, carvacrol and thymol were found to be effective with an acceptable level odor in textile finishing. The encapsulation efficacy was optimal when the weight ratio of chitosan to carvacrol (or thymol) was 1:1.25, which was consistent with previous research findings10,16. The encapsulation of the EOs is facilitated by the cross-linking of poly-cationic groups (NH3+) of chitosan molecules and poly-anionic groups (P3O105-) of TPP molecules. The cross-linker TPP stabilizes the NPs, but too much cross-linker may result in clumped particles. The encapsulation efficacy (EE) is dependent on many factors, such as the weight ratio of polymer to EOs, the rate of dispensing the EOs, and the temperature. Some volatile bioactive compounds can be encapsulated effectively under a cold-water bath with ice10. One easy way to test the EE is to test the supernatant, as shown in Figure 1A. If the supernatant is highly antimicrobial due to the unbound EOs, the EE will be low. All the ingredients used in the process are food-grade materials, and safe for the user and environment. Only a few studies exist on herbal-based NP-coated fabrics that use EOs and chitosan or natural polymers. This study has particularly focused on the development and testing of antimicrobial fabric for lab coats and suggests an effective solution to reduce microbial contamination in biosafety labs. Further studies are recommended to verify the efficacy of the antimicrobial fabric in real life by sampling regular versus antimicrobial lab coats after prolonged use in biohazard labs or healthcare facilities.
The authors have nothing to disclose.
This study was funded by "Applied Research, Innovation and Entrepreneurship Services" (ARIES), Centennial College, Canada.
Acetic acid | Millipore Sigma | 64-19-7 | |
Antibiotic base agar | BD Difco | DF0270-17-4 | Also known as Antibiotic Medium 2 |
Antibiotic seed agar | BD Difco | DF0263-17-3 | Also known as Antibiotic Medium 1 |
Blood Agar (Nutrient Agar with 5% Sheep Blood) | Donated by CFIA | ||
Bromcresol Purple Lactose Agar | Donated by CFIA | ||
Candida albicans | ATCC The Global Bioresource Center | ATTC 10231 | |
Carvacrol | Millipore Sigma | 282197 (CAS# 499-75-2) | |
Centrifuge Allergra X-22R Centrifuge | Beckman Coulter | Model # X-22R | Refrigerated. Wait at least 20 min or until the temperature reach the set low value (e.g., 4 °C) as the refrigeration takes time. |
Chitosan Medium Molecular Weight (CS) | Millipore Sigma | 448877 (CAS # 9012-76-4) | |
Clamshell Heat Press | Intiva | IM1200 | |
Escherichia coli (E. coli) | ATCC The Global Bioresource Center | ATTC 23725 | |
Incubator | Thermo Scientific | 1205M34 | |
Letheen Broth | BD Difco | DF0681-17-7 | Used to neutralize antimicrobial effects. Product from different manufacturers may require to add Polysorbate 80, which is already added in Difco product. |
Milli Q water | Millipore Sigma | ZR0Q16WW | Deionized water |
Mueller-Hinton Agar | BD Difco | DF0252-17-6 | |
Pentasodium tripolyphosphate (TPP) | Millipore Sigma | 238503 (CAS# 7758-29-4) | |
Phospahte Buffered Saline (PBS) | Thermo Scientific | AM9624 | |
Pseudomonas aeruginosa | ATCC The Global Bioresource Center | ATTC 9027 | |
Sabouraud Dextrose Agar | BD Difco | DF0109-17-1 | |
Shaking incubator/ Thermo shaker | VWR | Model# SHKA2000 | |
Staphylococcus aureus | ATCC The Global Bioresource Center | ATTC 6538 | |
Thymol | Millipore Sigma | T0501 (CAS# 89-83-8) | |
Trypticase Soy Agar | BD Difco | 236950 | |
Trypticase Soy Broth | BD Difco | 215235 | |
Tween 80 | Millipore Sigma | STS0204 (CAS # 9005-65-6) | |
UV-Vis Spectrophometer | Thermo Scientific | GENESYS 30 (840-277000) |