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Self-Decontaminating Surfaces and Antimicrobial Materials – Exploiting Cell Lytic and Reagent-Requiring Enzymes

Antimicrobial enzymes, either specific cell lytic enzymes or wide-spectrum bacterial destroying enzymes such as proteases and oxidative enzymes, can be immobilized onto or incorporated into a variety of materials for the development of self-decontaminating surfaces (Figure SD-1). These surfaces have potential applications in antifouling, environmental decontamination, and food packaging.

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Figure SD-1. Antimicrobial activity of enzyme-based nanoconjugates and coatings. Antimicrobial enzymes are immobilized onto nanomaterials to form enzyme nanoconjugates, which can be further incorporated into paints and films to generate antimicrobial coatings. Bacterial cells can be killed efficiently upon contact with the conjugates or coatings.

 

Anti-MRSA Paints

Infection with antibiotic-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) is one of the primary causes of hospitalizations and deaths. To address this issue, we have designed antimicrobial coatings incorporating carbon nanotube-enzyme conjugates that are highly effective against antibiotic-resistant pathogens. Specifically, we incorporated conjugates of carbon nanotubes with lysostaphin, a cell wall degrading enzyme, into films to impart bactericidal properties against Staphylococcus aureus and Staphylococcus epidermidis [Pangule et al. ACS Nano 4, 3993-4000 (2010)]. We fabricated and characterized nanocomposites containing different conjugate formulations and enzyme loadings. These enzyme-based composites were highly efficient in killing MRSA (>99% within 2 h) without release of the enzyme into solution (Figure SD-2). Additionally, these films were reusable and stable under dry storage conditions for a month. Such enzyme-based film formulations may be used to prevent growth of pathogenic and antibiotic-resistant microorganisms on various common surfaces in hospital settings. Polymer and paint films containing such antimicrobial conjugates, in particular, could be advantageous to prevent risk of staphylococcal-specific infection and biofouling. Finally, Lst-based paints were used in the space shuttle [Kim et al. PLoS One 8, e62437 (2013)] to evaluate the effect of microgravity on the ability of Lst to kill S. aureus as an example to prevent biofilms for long-duration space flights.

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Figure SD-2. Antistaphylococcal activity lysostaphin-CNT-paint films against four different strains of methicillin-resistant Staphylococcus aureus (MRSA). The plot shows percent viability in case of lysostaphin-containing films normalized to that obtained for control paint samples [Pangule et al. ACS Nano 4, 3993-4000 (2010)].

 

Cell Lytic Enzymes for Wound Healing

With the emergence of "super bacteria" that are resistant to antibiotics, e.g., methicillin-resistant Staphylococcus aureus, novel antimicrobial therapies are needed to prevent associated hospitalizations and deaths. Bacteriophages and bacteria use cell lytic enzymes to kill host or competing bacteria, respectively, in natural environments. Taking inspiration from nature, we have employed a cell lytic enzyme, lysostaphin (Lst), with specific bactericidal activity against S. aureus, to generate anti-infective bandages (Figure SD-3) [Miao et al. Biomaterials 32, 9557-9567 (2011)]. Lst was immobilized onto biocompatible fibers generated by electrospinning homogeneous solutions of cellulose, cellulose-chitosan, and cellulose-poly(methylmethacrylate) (PMMA) from 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]), room temperature ionic liquid. Electron microscopic analysis shows that these fibers have submicron-scale diameter. The fibers were chemically treated to generate aldehyde groups for the covalent immobilization of Lst. The resulting Lst-functionalized cellulose fibers were processed to obtain bandage preparations that showed activity against S. aureus in an in vitro skin model with low toxicity toward keratinocytes, suggesting good biocompatibility for these materials as antimicrobial matrices in wound healing applications.

  

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Figure SD-3. Anti-staphylococcal activity on electrospun cotton fibers. The fibers were prepared using cellulose dissolved in an ionic liquid followed by electrospinning. Lysostaphin was chemically attached using periodate treatment into cotton balls. We then placed the enzyme-incorporated cotton onto a 2D layer of human keratinocytes that were seeded with S. aureus, and evaluated killing of the bacteria with the Lst-containing cotton overlay. The non-enzymatic and BSA-sham controls showed no bacterial killing, while the Lst-containing cotton showed complete killing of S. aureus [Miao et al. Biomaterials 32, 9557-9567 (2011)].

 

Anti-Listeria Materials

Ply500 is an endolysin against Listeria monocytogenes, one of the major pathogens contaminating the food supply chain and causing severe infections and death. We covalently attached Ply500 to the FDA-approved SNPs and achieved ~5-log killing of Listeria innocua (a reduced pathogenic surrogate) in 24 h (Figure SD-4) [Solanki et al. Sci. Rep. 3, 1584 (2013)]. The conjugate, while stabilizing the enzyme, completely decontaminated lettuce leaves inoculated with L. innocua. In addition, affinity binding of Ply500 to edible crosslinked starch nanoparticles killed L. innocua by 3-log units in 24 h.

 

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Figure SD-4. (Left) Schematic representation of oriented affinity immobilization of MBP-linker10Ply500 fusion protein on crosslinked starch particles via MBP. (Right) Activity of Ply500 against isolated L. innocua using a viable plate counting assay after 3 h (grey), 6 h (white) and 24 h (black). L. innocua was grown in BHI media for 7 h ) [Solanki et al. Sci. Rep. 3, 1584 (2013)].

Antimicrobial Surfaces Using Reagent-Requiring Enzymes

While cell-lytic enzymes require just water as a reactant, there are numerous enzymes that can serve as strong antimicrobials in the presence of a co-reactant. We have employed several of these enzymes in decontaminating environments, including chloroperoxidase, laccase and perhydrolase (Figure SD-5). In several cases, we employed conjugates of these enzymes with carbon nanotubes and then embedded these conjugates into a composite paint material.

  

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Figure SD-5. Mechanisms of reagent-dependent antimicrobial enzymes. (a) Antimicrobial system combining glucose oxidase (GOx) with (halo)peroxidase or perhydrolase for producing potent antimicrobial agents (red colored compounds). (b) Laccase-catalyzed reaction for generating I2 [Wu et al. Annu. Rev. Chem. Biomol. Eng. 8, 87-113 (2017)]. 

CPO-MWNT conjugates, mixed with a commercial ecofriendly paint to fabricate, generated ~1.4 mM HOCl within 30 min, resulting in killing >99.99% of S. aureus and E. coli (Figure SD-6). In this system, the production of HOCl by haloperoxidases makes them useful candidates for disinfection, sanitization, and decontaminating formulations [Grover et al. Enzyme Microb. Technol. 50, 271-279 (2012)]. Similarly, perhydrolase (AcT) was covalently immobilized onto oxidized MWNTs in the presence of an amphiphilic PEG spacer. The AcT–PEG–MWNT conjugates were then incorporated into a latex-based paint [Grover et al. Appl. Microbiol. Biotechnol. 97, 8813-8821 (2013); Dinu et al. Adv. Funct. Mater. 20, 392-398 (2010)]. The paint showed a 6-log reduction in the viability of spores of B. cereus and B. anthracis Sterne within 1 h (Figure SD-7). AcT-containing paint composites also exhibited antiviral activity; >4-log reduction in the titer of influenza virus (X-31) (initially challenged with 107 PFU/mL) was observed within 10 min (Figure SD-8) [Grover et al. Appl. Microbiol. Biotechnol. 97, 8813-8821 (2013)]. The crosslinked AcT-based conjugates were highly stable and had no loss of activity over 6-months. Laccase was also immobilized onto MWNTs and subsequently mixed with a commercial latex paint to generate an antimicrobial biocatalytic coating. The generation of I2 from laccase-based paint coating led to 6-log reduction within 30 min for S. aureus and 60 min for E. coli. Furthermore, an optimized laccase-based paint formulation was able to kill >90% of B. cereus and B. anthracis spores within 1 h. Thus, the immobilization of laccase on nanomaterials offers environmentally benign approaches to generate surface decontaminating coatings.

 

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Figure SD-6. Rate of generation of HOCl by CPO; and Bactericidal activity of CPO catalyzed generation of hypochlorous acid (HOCl) against (Left) S. aureus and (Middle) E. coli. (♦ PBS control, ■ H2O2, ▲ CPO + H O + NaCl). Experimental Conditions: Bacterial cells, 106 CFU/mL; CPO, 5 μg/mL; H2O2, 2 mM; NaCl, 100 mM. (Right) Bactericidal activity of CPO-MWNT conjugates containing paint composites against S. aureus (□) and E. coli (■). Experimental Conditions: Bacterial cells, 106 CFU/mL; CPO, 0.14% (w/w); H2O2,2 mM; NaCl, 100 mM (* more than 99.99% killing) [Grover et al. Enzyme Microb. Technol. 50, 271-279 (2012)].

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Figure SD-7. Sporicidal activity of AcT-PEG-MWNT containing paint composites (0.30%, w/w, AcT) with urea hydrogen peroxide (UHP) and propylene glicol diacetate (PGD) against B. cereus spores (♦ control, ■ 200 mM UHP, ▲ 400 mM UHP, □ 0.30% (w/w) AcT with 200 mM UHP and Δ 0.30 % (w/w) ACT with 400 mM UHP). Experimental conditions: Spores, 106 CFU/mL; PGD, 100 mM; UHP, 200-400 mM, catalase, 100 U/mL and sodium thiosulfate, 0.05% (w/w) [Grover et al. Appl. Microbiol. Biotechnol. 97, 8813-8821 (2013); Dinu et al. Adv. Funct. Mater. 20, 392-398 (2010)].

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Figure SD-8. Kinetics of antiviral activity of AcT-PEG-MWNT containing paint composites against Influenza A virus strain X-31 (inset displays antiviral activity as a function of H2O2 concentration; control and ■ 0.30%, w/w, AcT). Experimental conditions: virus, 4x107 PFU/mL; PGD, 10 mM; H2O2, 5 mM; catalase, 100 U/mL and sodium thiosulfate, 0.05% (w/w) [Grover et al. Appl. Microbiol. Biotechnol. 97, 8813-8821 (2013)].

 

Collaborators :

Ravi Kane – Georgia Tech

Jungbae Kim – Korea University

Cynthia Collins - Rensselaer Polytechnic Institute