Name: Quantitative Screening of Nanoparticle-Treated Fabric: The Log Reduction Method
Uploaded: 2023-04-07T14:35:00
Description: Watch this Scientific Journal Video about Quantitative Screening of Nanoparticle-Treated Fabric: The Log Reduction Method at JoVE.com

The video describes a protocol for testing the antimicrobial efficacy of nanoparticles-treated fabrics. It outlines preparing microbial cultures, spiking fabric swatches, conducting control experiments, and analyzing colony counts to calculate microbial log reduction. The results highlight the effectiveness of thymol-encapsulated chitosan nanoparticles against specific pathogens and their decreased efficacy after wash cycles.

Quantitative Screening of Nanoparticle-Treated Fabric: The Log Reduction Method

quantitative screening of nanoparticle-treated fabric: the log reduction method

Efficacité antimicrobienne des tissus enduits de nano-herbes

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 &#34;Parallel Streak Method&#34;19 can provide a relatively quick and easy evaluation of diffusible antimicrobial formulation. However, if the bactericidal effects are to be determined, &#34;Assessment of Antibacterial Finishes on Textile Materials&#34;20 can be employed, which provides the log reduction of the spiked pathogen.

Pleins feux sur l’auteur : Un tissu antimicrobien utilisant l’encapsulation d’huiles essentielles à base de nanoplantes  

Un tissu antimicrobien utilisant l’encapsulation nano-végétale d’huiles essentielles

This research outlines the protocol to develop a safe and eco-friendly antimicrobial fabric which can be used to design lab coats. The study answers many key questions in the field of antimicrobial fabric development. For example, how effectively the treated fabric neutralizers pathogens.The key challenge in antimicrobial fabric development is to choose the right ratios of bioactive compounds without compromising antimicrobial efficacy while maintaining skin friendly finishes. Our antimicrobial fabric provides effective protection against four human pathogens with up to four log reduction, or simply 99.99%effectiveness. It also resists up to 10 wash cycles while retaining an efficacy of up to 90%The major advantage of this method over existing standard techniques is that it provides a simplified and realistic approach to accurately evaluate the efficacy of antimicrobial fabrics.It better represents the typical scenarios of lab coat usage, such as any accidental microbial spills that must be neutralized within a short time. The application of the technique in the manufacturing of protective coats will reduce the biohazard risk in biosafety labs and nosocomial infections in healthcare setting. We are in the process of testing full length antimicrobial lab coats in Biosafety Level 2 labs.Our future research questions would focus on the effectiveness of preventing microbial contamination in the long run and continuous usage.

Watch this Scientific Journal Video about Quantitative Screening of Nanoparticle-Treated Fabric: The Log Reduction Method at JoVE.com

Criblage quantitatif de tissus traités aux nanoparticules : la méthode de réduction logarithmique

Prenez un tissu traité aux nanoparticules et coupez-le en échantillons carrés de 50 millimètres par 50 millimètres. Préparer les cultures de nuit de microbes d’intérêt en inoculant les colonies isolées des plaques de pureté au bouillon de soja Trypticase stérile. Incuber à 35 degrés Celsius pendant 18 à 24 heures.Diluer les cultures de nuit à 150 millions d’UFC par millilitre ou correspondant à la turbidité de 0,5 McFarland standard. Ensuite, pour déterminer le volume approprié de cultures pour l’épistage, ajoutez une série de volumes de la culture de bouillon dilué sur les échantillons de tissu et choisissez le volume de telle sorte que l’échantillon absorbe complètement l’eau et ne laisse aucun liquide résiduel ou libre. Augmentez le volume de chaque culture sur les échantillons placés dans des plaques de Petri stériles.De même, effectuez le contrôle négatif avec le même type d’échantillons de tissu non traités pour chaque test correspondant. Pour récupérer les microbes par numération sur plaque viable, sécher à l’air les échantillons inoculés à l’intérieur des plaques de Petri à température ambiante pendant les périodes de contact requises à tester. Transférer les échantillons de manière aseptique pour séparer les tubes centrifugés stériles et visser fermement les bouchons.Ajouter le bouillon Letheen ou tout tampon neutralisant pertinent pour obtenir une dilution de 1:100. Après avoir fermé les tubes à centrifuger, vortex pendant une minute à une vitesse moyenne. Diluer en série la suspension avec le PBS stérile lors des dilutions 1:10 suivantes, de sorte que le nombre de colonies du groupe non traité devienne trop faible pour être compté.Planter les dilutions sur des plaques de milieux appropriées qui favorisent la croissance du micro-organisme ou sur tout milieu qui optimise la croissance et offre un bon contraste pour que le comptage des colonies soit précis. Incuber les plaques bactériennes à 35 degrés Celsius pendant deux jours et les plaques fongiques à température ambiante pendant cinq jours. Comptez les unités viables formant des colonies directement à l’aide d’un compteur de colonies ou d’un logiciel d’imagerie.Calculez la réduction logarithmique microbienne R due au tissu antimicrobien à l’aide de l’équation illustrée à l’écran. Ici, A est la valeur logarithmique du nombre d’unités formant colonies récupérées à partir de tissus non traités, et B est la même pour le tissu traité. Les résultats de la réduction logarithmique du tissu antimicrobien recouvert de nanoparticules de chitosane encapsulées dans du thymol testés contre S. aureus sur gélose au sang, E. coli sur gélose au lactose pourpre, P. aeruginosa sur gélose cétrimide et C. albicans sur gélose au dextrose Sabouraud sont présentés ici.Les agents pathogènes ont été enrichis sur des échantillons non traités et traités pendant 30 minutes et récupérés après neutralisation et dilution. Les taux de dilution sont indiqués entre la série traitée et la série non traitée. La réduction logarithmique de trois bactéries et d’un champignon due au contact de tissus antimicrobiens imprégnés de deux composés bioactifs est illustrée ici.L’efficacité antimicrobienne est affaiblie après les cycles de lavage pour les tissus enduits de carvacrol et de thymol.

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