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

ナノハーブコーティング生地の抗菌効果

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.

著者スポットライト:エッセンシャルオイルのナノハーブカプセル化を使用した抗菌生地  

エッセンシャルオイルのナノハーブカプセル化を使用した抗菌ファブリック

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

ナノ粒子処理布の定量的スクリーニング:対数低減法

ナノ粒子処理された布を取り、それを50ミリメートル×50ミリメートル正方形の見本にカットします。単離されたコロニーを純度プレートから滅菌トリプチカーゼ大豆ブロスに接種することにより、目的の微生物の一晩培養を準備します。摂氏35度で18〜24時間インキュベートします。一晩培養物をミリリットルあたり1億5000万CFU、または0.5マクファーランド標準の濁度に対応するように希釈します。次に、スパイクに適した培養量を決定するために、一連の量の希釈ブロス培養物を布見本に追加し、布見本が水を完全に吸収し、残留または遊離液体を残さないように体積を選択します。滅菌ペトリプレートに入れた見本に各培養物の量をスパイクします。同様に、対応するテストごとに同じタイプの未処理の生地見本を使用してネガティブコントロールを実行します。生存可能なプレート数によって微生物を回収するには、ペトリプレート内の接種スウォッチを室温で風乾し、必要な接触期間をテストします。スウォッチを無菌的に移して滅菌遠心チューブを分離し、キャップをしっかりとねじ込みます。Letheen Brothまたは関連する中和バッファーを加えて、1:100に希釈します。遠沈管を閉じた後、中速で1分間ボルテックスします。その後の1:10希釈で滅菌PBSで懸濁液を連続希釈し、未処理群のコロニー数が低すぎてカウントできなくなるようにします。微生物の成長をサポートする適切な培地プレート、または成長を最適化し、良好なコントラストを提供してコロニーカウントを正確にする培地に希釈液を植えます。細菌プレートを摂氏35度で2日間インキュベートし、真菌プレートを室温で5日間インキュベートします。コロニーカウンターまたはイメージングソフトウェアを使用して、生存可能なコロニー形成単位を直接カウントします。抗菌布による微生物対数減少Rを、画面上の式を用いて算出する。ここで、Aは未処理布から回収されたコロニー形成単位数の対数値であり、Bは処理布についても同様である。チモール封入キトサンナノ粒子でコーティングされた抗菌布を、血液寒天培地上の黄色ブドウ球菌、紫色ラクトース寒天培地上の大腸菌、セトリミド寒天培地上の緑膿菌、およびサブローデキストロース寒天培地上のC.アルビカンスに対して試験した結果をここに示します。病原体を未処理および処理済みの見本に30分間スパイクし、中和および希釈メッキで回収しました。希釈比は、処理された系列と未処理の系列の間で示されます。ここでは、2つの生理活性化合物を含浸させた抗菌布の接触による3つの細菌と1つの真菌の対数減少を示します。抗菌効果は、カルバクロールとチモールコーティングされた布地の両方の洗浄サイクル後に弱まります。

Research

JoVE Journal

Biology

An Antimicrobial Fabric Using Nano-Herbal Encapsulation of Essential Oils

Lab coats are widely used in biohazard laboratories and healthcare facilities as protective garments to prevent direct exposure to pathogens, spills, and ...