High-throughput fabrication of antimicrobial phage microgels and example applications in food decontamination.

Engineered by nature, biological entities are exceptional building blocks for biomaterials. These entities can impart enhanced functionalities on the final material that are otherwise unattainable. However, preserving the bioactive functionalities of these building blocks during the material fabrication process remains a challenge. We describe a high-throughput protocol for the bottom-up self-assembly of highly concentrated phages into microgels while preserving and amplifying their inherent antimicrobial activity and biofunctionality. Each microgel is comprised of half a million cross-linked phages as the sole structural component, self-organized in aligned bundles. We discuss common pitfalls in the preparation procedure and describe optimization processes to ensure the preservation of the biofunctionality of the phage building blocks. This protocol enables the production of an antimicrobial spray containing the manufactured phage microgels, loaded with potent virulent phages that effectively reduced high loads of multidrug-resistant Escherichia coli O157:H7 on red meat and fresh produce. Compared with other microgel preparation methods, our protocol is particularly well suited to biological materials because it is free of organic solvents and heat. Bench-scale preparation of base materials, namely microporous films (the template for casting microgels) and pure concentrated phage suspension, requires 3.5 h and 5 d, respectively. A single production run, that yields over 1,750,000 microgels, ranges from 2 h to 2 d depending on the rate of cross-linking chemistry. We expect that this platform will address bottlenecks associated with shelf-stability, preservation and delivery of phage for antimicrobial applications, expanding the use of phage for prevention and control of bacterial infections and contaminants.

Low Molecular Weight Shuttle Molecules Enhance Polychloramide Antimicrobial Activity.

Investigated were the influences of succinimide (SI), 5,5-dimethylhydantoin (DMH), and 3-(hydroxymethyl)-5,5-dimethylhydantoin (HDMH) on the biocidal activity of chlorinated, water-soluble polyamide prepared by the reaction of isopropylamine with poly(styrene-alt-maleic anhydride). The resulting polymer was a negatively charged, water-soluble polymer bearing a carboxylic acid and an isopropylamide moiety on nearly every repeat unit. Subsequent treatment with NaOCl chlorinated the polymers to up to 4.4% Cl while inflicting some polymer chain scission. SI, DMH, or HDMH increased the biocidal activity of polychloramides toward planktonic Escherichia coli and Staphylococcus aureus. Independent solution studies confirmed that oxidative chlorine spontaneously transferred from aqueous polychloramides to small molecules. We concluded that SI, DMH, and HDMH acted as shuttles that extracted oxidative Cl from the polymer chloramides and transported oxidative Cl more efficiently to microbial surfaces. Succinimide was the most effective shuttle. These results warn that some low molecular weight soluble molecules in antimicrobial testing solutions may exaggerate the effectiveness of the polymer or immobilized antimicrobial agents.

Purification of phage for therapeutic applications using high throughput anion exchange membrane chromatography.

As cases of multidrug resistant bacterial infections increase, scientists and clinicians around the world are increasingly turning to bacteriophages as alternatives to antibiotics. Even though our understanding of phage has increased significantly since the early days of its discovery, over a century ago, the currently used tools and technologies for phage purification for therapeutic applications are severely limited. Bacteriophages are produced by bacterial cultures, and impurities such as endotoxins must therefore be removed before clinical use. We present an anion exchange bind-and-elute membrane chromatographic method for purifying T7 bacteriophage from Escherichia coli culture supernatant that removes undesirable impurities, while ensuring a high viable phage count in the purified product. Our method does not involve the use of chemicals such as organic solvents and caesium chloride that could typically leave residual toxicity in the final product. It also does not require expensive equipment, such as an ultracentrifuge. Using our method, that is based on an in-house designed membrane module, 65% of viable T7 phage was recovered, and up to 94% endotoxins could be removed. The method, which took approximately 15 min, is rapid and scalable, and produces quite pure bacteriophage samples in a single step. It therefore potentially represents a major improvement over the status quo, and shows the way ahead for streamlining phage manufacturing for therapeutic use.

Impacts of non-microbial soils on polychloramide disinfectants.

The presence of some nonmicrobial chemicals and surfaces, herein called "soils", are known to degrade the performance of biocides, and biocidal assays often include mixtures of materials to mimic the effects of soils. We hypothesized that water-soluble anionic polychloramide biocides were less sensitive to soil interference than cationic polymeric biocides. The relationships between soil composition and antimicrobial polymer biocidal activity were compared for an anionic polychloramide, a cationic polychloramide, and a cationic poly(quaternary ammonium) biocide. The nanoscale soil models individually investigated were polyacrylic acid (PAA), cellulose nanocrystals (CNCs), and bovine serum albumin. The low molecular weight model soils were ammonium chloride, glycine, and succinimide. Three types of soil impacts were identified: 1) sequestration, whereby the soil physically inhibited transport of the biocide to microbes; 2) extraction, whereby the soil reduced or extracted oxidative chlorine, decreasing or eliminating the oxidative chlorine strength; and 3) extraction whereby the biocidal activity increases in the presence of a low molecular weight chemical that carries oxidative Cl from the polymer to the microbes. PAA and CNCs inhibit cationic biocides by sequestration but have little impact on anionic polychloramide. Glycine and BSA extract oxidative chlorine, lowering the biocidal activity of the anionic and cationic polychloramides while not impacting the poly(quaternary ammonium) biocide. Finally, the presence of succinimide increased bacteria deactivation of both anionic and cationic polychloramides. We propose that succinimide extracts oxidative chlorine from the polychloramides and transports it to the bacteria.

Functionalization of 3D Printed Scaffolds Using Polydopamine and Silver Nanoparticles for Bone-Interfacing Applications.

The prevention of bacterial colonization and the stimulation of osseointegration are two major requirements for bone-interfacing materials to reduce the incidence of complications and promote the restoration of the patient's health. The present investigation developed an effective, two-step functionalization of 3D printed scaffolds intended for bone-interfacing applications using a simple polydopamine (PDA) dip-coating method followed by the formation of silver nanoparticles (AgNPs) after a second coating step in silver nitrate. 3D printed polymeric substrates coated with a ∼20 nm PDA layer and 70 nm diameter AgNPs proved effective in hindering Staphylococcus aureus biofilm formation, with a 3000-8000-fold reduction in the number of bacterial colonies formed. The implementation of porous geometries significantly accelerated osteoblast-like cell growth. Microscopy characterization further elucidated homogeneity, features, and penetration of the coating inside the scaffold. A proof-of-concept coating on titanium substrates attests to the transferability of the method to other materials, broadening the range of applications both in and outside the medical sector. The antibacterial efficiency of the coating is likely to lead to a decrease in the number of bacterial infections developed after surgery in the presence of these coatings on prosthetics, thus translating to a reduction in revision surgeries and improved health outcomes.

A Bifunctional Spray Coating Reduces Contamination on Surfaces by Repelling and Killing Pathogens.

Surface-mediated transmission of pathogens is a major concern with regard to the spread of infectious diseases. Current pathogen prevention methods on surfaces rely on the use of biocides, which aggravate the emergence of antimicrobial resistance and pose harmful health effects. In response, a bifunctional and substrate-independent spray coating is presented herein. The bifunctional coating relies on wrinkled polydimethylsiloxane microparticles, decorated with biocidal gold nanoparticles to induce a "repel and kill" effect against pathogens. Pathogen repellency is provided by the structural hierarchy of the microparticles and their surface chemistry, whereas the kill mechanism is achieved using functionalized gold nanoparticles embedded on the microparticles. Bacterial tests with methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa reveal a 99.9% reduction in bacterial load on spray-coated surfaces, while antiviral tests with Phi6─a bacterial virus often used as a surrogate to SARS-CoV-2─demonstrate a 98% reduction in virus load on coated surfaces. The newly developed spray coating is versatile, easily applicable to various surfaces, and effective against various pathogens, making it suitable for reducing surface contamination in frequently touched, heavy traffic, and high-risk surfaces.

Self-assembling nanofibrous bacteriophage microgels as sprayable antimicrobials targeting multidrug-resistant bacteria.

Nanofilamentous bacteriophages (bacterial viruses) are biofunctional, self-propagating, and monodisperse natural building blocks for virus-built materials. Minifying phage-built materials to microscale offers the promise of expanding the range function for these biomaterials to sprays and colloidal bioassays/biosensors. Here, we crosslink half a million self-organized phages as the sole structural component to construct each soft microgel. Through an in-house developed, biologics-friendly, high-throughput template method, over 35,000 phage-built microgels are produced from every square centimetre of a peelable microporous film template, constituting a 13-billion phage community. The phage-exclusive microgels exhibit a self-organized, highly-aligned nanofibrous texture and tunable auto-fluorescence. Further preservation of antimicrobial activity was achieved by making hybrid protein-phage microgels. When loaded with potent virulent phages, these microgels effectively reduce heavy loads of multidrug-resistant Escherichia coli O157:H7 on food products, leading to up to 6 logs reduction in 9 hours and rendering food contaminant free.

Highly Stable Hierarchically Structured All-Polymeric Lubricant-Infused Films Prevent Thrombosis and Repel Multidrug-Resistant Pathogens.

Thrombus formation and infections caused by bacterial adhesion are the most common causes of failure in blood-contacting medical devices. Reducing the interaction of pathogens using repellent surfaces has proven to be a successful strategy in preventing device failure. However, designing scale-up methodologies to create large-scale repellent surfaces remains challenging. To address this need, we have created an all-polymeric lubricant-infused system using an industrially viable swelling-coagulation solvent (S-C) method. This induces hierarchically structured micro/nano features onto the surface, enabling improved lubricant infusion. Poly(3,3,3-trifluoropropylmethylsiloxane) (PTFS) was used as the lubricant of choice, a previously unexplored omniphobic nonvolatile silicone oil. This resulted in all-polymeric liquid-infused surfaces that are transparent and flexible with long-term stability. Repellent properties have been demonstrated using human whole blood and methicillin-resistant Staphylococcus aureus (MRSA) bacteria matrices, with lubricated surfaces showing 93% reduction in blood stains and 96.7% reduction in bacterial adherence. The developed material has the potential to prevent blood and pathogenic contamination for a range biomedical devices within healthcare settings.

Modeling the Impact of Polychloramide Solution Properties on Bacterial Disinfection Kinetics.

Anionic water-soluble polychloramide biocides are of interest because, compared to conventional cationic antimicrobial polymers, anionic biocides are less likely to be sequestered or deactivated by contact with non-microbial soil. Although electrostatics can prevent anionic polymers from adsorbing on microbes, water-soluble polychloramides appear to transfer oxidative chlorine during transient contacts between polymer chains and microbe surfaces. The Chick-Watson model of disinfection kinetics has been modified to account for the contributions of polychloramide molecular weight (MW) and the polychloramide configuration in solution estimated from the overlap concentration, C*, below which dilute polymer chains exist as discrete objects in solution. The key assumption in the modeling was that the transfer rate of oxidative chlorine from polychloramide chains to microbe surfaces impacts the disinfection kinetics. Because both C* and MW are measurable, the polymer-modified Chick-Watson (PCW) model has one less unknown parameter than the two-parameter Chick-Watson equation. The PCW model predicts that lower MW polymers are more effective biocides compared with high MW counterparts. Additionally, polymers with more compressed configurations in solution are more effective biocides. Experimental evidence supports these conclusions. Based on the estimated time scale of bacteria/polymer collisions compared with disinfection kinetics, arguments are made that bacteria surfaces must be contracted many times by polychloramide chains to achieve sufficient Cl transfer to deactivate bacteria.

Water-soluble anionic polychloramide biocides based on maleic anhydride copolymers.

Our goal was to develop film-forming polymers to extend the antimicrobial lifetimes of cleaned and disinfected surfaces. Antimicrobial polymers were prepared by first reacting poly(ethylene-alt-maleic anhydride) with isopropylamine, partially consuming the anhydride groups, followed by hydrolysis to give water-soluble, highly anionic polyamide PC3. Chlorination with NaOCl gave PC3Cl with oxidative chlorine contents up to 9 wt%. Dried, 5 µm thick, PC3Cl films, gave log 4 reductions in the concentration of Escherichia coli or Staphylococcus aureus exposed to films. A unique feature of the maleic anhydride copolymer platform was the ability to form covalent grafts to surfaces via anhydride reactions. PC3 solution was impregnated into cellulosic filter paper, heated to form ester linkages with cellulose, followed by chlorination with sodium dichloroisocyanurate dihydrate giving grafted PC3Cl. The treated paper (0.3 wt% PC3Cl) gave a log 4 reduction of E. coli concentration in 30 min.

Enhancing osseointegration and mitigating bacterial biofilms on medical-grade titanium with chitosan-conjugated liquid-infused coatings.

Titanium alloys, in particular, medical-grade Ti-6Al-4 V, are heavily used in orthopaedic applications due to their high moduli, strength, and biocompatibility. Implant infection can result in biofilm formation and failure of prosthesis. The formation of a biofilm on implants protects bacteria from antibiotics and the immune response, resulting in the propagation of the infection and ultimately resulting in device failure. Recently, slippery liquid-infused surfaces (LIS) have been investigated for their stable liquid interface, which provides excellent repellent properties to suppress biofilm formation. One of the current limitations of LIS coatings lies in the indistinctive repellency of bone cells in orthopaedic applications, resulting in poor tissue integration and bone ingrowth with the implant. Here, we report a chitosan impregnated LIS coating that facilitates cell adhesion while preventing biofilm formation. The fabricated coating displayed high contact angles (108.2 ± 5.2°) and low sliding angles (3.56 ± 4.3°). Elemental analysis obtained using X-ray photoelectron spectroscopy (XPS) confirmed the availability of fluorine and nitrogen, indicating the presence of fluorosilane and chitosan in the final coating. Furthermore, our results suggest that chitosan-conjugated LIS increased cell adhesion of osteoblast-like SaOS-2 cells and significantly promoted proliferation (a fourfold increase at 7-day incubation) compared to conventional titanium liquid-infused surfaces. Furthermore, the chitosan conjugated LIS significantly reduced biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA) by up to 50% and 75% when compared to untreated titanium and chitosan-coated titanium, respectively. The engineered coating can be easily modified with other biopolymers or capture molecules to be applied to other biomaterials where tissue integration and biofilm prevention are needed.

Inducing Microscale Structural Order in Phage Nanofilament Hydrogels with Globular Proteins.

Biological hydrogels play important physiological roles in the body. These hydrogels often contain ordered subdomains that provide mechanical toughness and other tissue-specific functionality. Filamentous bacteriophages are nanofilaments with a high aspect ratio that can self-assemble into liquid crystalline domains that could be designed to mimic ordered biological hydrogels and can thus find applications in biomedical engineering. We have previously reported hydrogels of pure cross-linked liquid crystalline filamentous phage formed at very high concentrations exhibiting a tightly packed microstructure and high stiffness. In this work, we report a method for inducing self-assembly of filamentous phage into liquid crystalline hydrogels at concentrations that are several orders of magnitude below that of lyotropic liquid crystal formation, thus creating structural order but a less densely packed microstructure. Hybrid hydrogels of M13 phage and bovine serum albumin (0.25 w/v%) were formed and shown to adsorb up to 16× their weight in water. Neither component gelled on its own at the low concentrations used, suggesting synergistic action between the two components in the formation of the hydrogel. The hybrid hydrogels exhibited repetitive self-healing under physiological conditions and at room temperature, autofluorescence in three channels, and antibacterial activity toward Escherichia coli host cells. Furthermore, the hybrid hydrogels exhibited a more than 2× higher ability to pack water compared to BSA-only hydrogels and 2× lower compression modulus compared to tightly packed M13-only hydrogels, suggesting that our method could be used to create hydrogels with tunable mechanical properties and pore structure through the addition of globular proteins, while maintaining bioactivity and microscale structural order.

Stress Exposure of Evolved Bacteriophages under Laboratory versus Food Processing Conditions Highlights Challenges in Translatability.

Bacterial viruses, or bacteriophages, are highly potent, target-specific antimicrobials. Bacteriophages can be safely applied along the food production chain to aid control of foodborne pathogens. However, bacteriophages are often sensitive to the environments encountered in food matrices and under processing conditions, thus limiting their applicability. We sought to address this challenge by exposing commercially available Listeria monocytogenes bacteriophage, P100, to three stress conditions: desiccation, elevated temperature, and low pH, to select for stress-resistant bacteriophages. The stressed bacteriophage populations lost up to 5.1 log10 in infectivity; however, the surviving subpopulation retained their stress-resistant phenotype through five passages with a maximum of 2.0 log10 loss in infectivity when exposed to the same stressor. Sequencing identified key mutation regions but did not reveal a clear mechanism of resistance. The stress-selected bacteriophage populations effectively suppressed L. monocytogenes growth at a modest multiplicity of infection of 0.35-0.43, indicating no trade-off in lytic ability in return for improved survivability. The stressed subpopulations were tested for survival on food grade stainless steel, during milk pasteurization, and within acidic beverages. Interestingly, air drying on stainless steel and pasteurization in milk led to significantly less stress and titer loss in bacteriophage compared to similar stress under model lab conditions. This led to a diminished benefit for stress-selection, thus highlighting a major challenge in real-life translatability of bacteriophage adaptational evolution.

Bacteria repellent protein hydrogel decorated with tunable, isotropic, nano-on-micro hierarchical microbump array.

We report the development of ordered shape-controllable microbump structures on protein hydrogels using polystyrene honeycomb templates. Addition of protein nanogels results in the formation of hierarchical nano-on-micro structures and increases surface hydrophilicity by over 55%, exhibiting bacteria repellency 100 times stronger than a flat hydrogel surface composed of the same protein.

Antibiotic-Impregnated Liquid-Infused Coatings Suppress the Formation of Methicillin-Resistant Staphylococcus aureus Biofilms.

Medical device-associated infections are an ongoing problem. Once an implant is infected, bacteria create a complex community on the surface known as a biofilm, protecting the bacterial cells against antibiotics and the immune system. To prevent biofilm formation, several coatings have been engineered to hinder bacterial adhesion or viability. In recent years, liquid-infused surfaces (LISs) have been shown to be effective in repelling bacteria due to the presence of a tethered liquid interface. However, local lubricant loss or temporary local displacement can lead to bacteria penetrating the lubrication layer, which can then attach to the surface, proliferate, and form a biofilm. Biofilm formation on biomedical devices can subsequently disrupt the chemistry tethering the slippery liquid interface, causing the LIS coating to fail completely. To address this concern, we developed a "fail-proof" multifunctional coating through the combination of a LIS with tethered antibiotics. The coatings were tested on a medical-grade stainless steel using contact angle, sliding angle, and Fourier transform infrared spectroscopy. The results confirm the presence of antibiotics while maintaining a stable and slippery liquid interface. The antibiotic liquid-infused surface significantly reduced biofilm formation (97% reduction compared to the control) and was tested against two strains of Staphylococcus aureus, including a methicillin-resistant strain. We also demonstrated that antibiotics remain active and reduce bacteria proliferation after subsequent coating modifications. This multifunctional approach can be applied to other biomaterials and provide not only a fail-safe but a fail-proof strategy for preventing bacteria-associated infections.

Filamentous Phages as Building Blocks for Bioactive Hydrogels.

Filamentous bacteriophages (bacterial viruses) are semiflexible proteinous nanofilaments with high aspect ratios for which the surface chemistry can be controlled with atomic precision via genetic engineering. That, in addition to their ability to self-propagate and replicate a nearly monodisperse batch of biologically and chemically identical nanofilaments, makes these bionanofilaments superior to most synthetic nanoparticles and thus a powerful tool in the bioengineers' toolbox. Furthermore, filamentous phages form liquid crystalline structures at high concentrations; these ordered assemblies create hierarchically ordered macro-, micro-, and nanostructures that, once cross-linked, can form hierarchically ordered hydrogels, hydrated soft material with a variety of physical and chemical properties suitable for biomedical applications (e.g., wound dressings and tissue engineering scaffolds) as well as biosensing, diagnostic assays. We provide a critical review of these hydrogels of filamentous phage, and their physical, mechanical, chemical, and biological properties and current applications, as well as an overview of limitations and challenges and outlook for future applications. In addition, we present a list of design parameters for filamentous phage hydrogels to serve as a guide for the (bio)engineer and (bio)chemist interested in utilizing these powerful bionanofilaments for designing smart, bioactive materials and devices.

Regenerating heavily biofouled dissolved oxygen sensors using bacterial viruses.

Bacterial biofilms are aggregates of bacterial cells embedded in a self-produced extracellular polymeric matrix. Biofilm formation has always been considered a major challenge for sensors used in underwater measurements, and is a primary source of measurement error, especially when it comes to long-term in situ monitoring. We demonstrate the utility of lytic bacteriophages (bacterial viruses) as a non-invasive strategy for removing bacterial biofilms formed on the gas permeable membrane of electrochemical dissolved oxygen sensors. Our results show that a 4 day Pseudomonas aeruginosa biofilm with a fully developed matrix significantly affected the sensor signal and response time, decreasing the signal by 32% and increasing the response time by 94%. In addition, measurements with the biofouled membrane had a very low signal to nose ratio compared to a clean sensor membrane. A single dose of overnight phage treatment effectively removed the biofilm (as indicated by scanning electron micrographs and fluorescence images of the membrane), without the need for repeated treatments. Furthermore, the sensor signal that had plummeted by 32% for a fully biofouled membrane, was returned to the original value (7.96 ± 0.27 mg L-1) after phage treatment and the signal to noise ratio (calculated as the ratio of mean to standard deviation) increased 8 folds for a phage-treated membrane compared to a biofouled membrane. Our data indicate near complete regeneration and signal recovery for the dissolved oxygen sensor, making the biofouled sensor reusable without the use of harsh chemicals that could destroy the fragile sensor membrane.

Liposomes for Antibiotic Encapsulation and Delivery.

When antibiotics are administered, orally or intravenously, they pass through different organs and layers of tissue on their way to the site of infection; this can cause dilution and/or intoxication. To overcome these problems, drug delivery vehicles have been used to encapsulate and deliver antibiotics, improving their therapeutic index while minimizing their adverse effects. Liposomes are self-assembled lipid vesicles made from at least one bilayer of phospholipids with an inner aqueous compartment. Liposomes are attractive vehicles to deliver antibiotics because they can encapsulate both hydrophobic and hydrophilic antibiotics, they have low toxicity, and they can change the biodistribution of the drug. Furthermore, liposomes have been approved by regulatory agencies. However, most developmental and mechanistic research in the field has been focused on encapsulation and delivery of anticancer drugs, a class of molecules that differ significantly in chemistry from antibiotics. In this critical Review, we discuss the state of knowledge regarding the design of liposomes for encapsulation and delivery of antibiotics and offer insight into the challenges and promises of using liposomes for antibiotic delivery.

Liposomal Nanovesicles for Efficient Encapsulation of Staphylococcal Antibiotics.

Liposomes are attractive vehicles for localized delivery of antibiotics. There exists, however, a gap in knowledge when it comes to achieving high liposomal loading efficiencies for antibiotics. To address this issue, we investigated three antibiotics of clinical relevance against staphylococcal infections with different hydrophilicity and chemical structure, namely, vancomycin hydrochloride, teicoplanin, and rifampin. We categorized the suitability of different encapsulation techniques on the basis of encapsulation efficiency, lipid requirement (important for avoiding lipid toxicity), and mass yield (percentage of mass retained during the preparation process). The moderately hydrophobic (teicoplanin) and highly hydrophobic (rifampin) antibiotics varied significantly in their encapsulation load (max 23.4 and 15.5%, respectively) and mass yield (max 74.1 and 71.8%, respectively), favoring techniques that maximized partition between the aqueous core and the lipid bilayer or those that produce oligolamellar vesicles, whereas vancomycin hydrochloride, a highly hydrophilic molecule, showed little preference to any of the protocols. In addition, we report significant bias introduced by the choice of analytical method adopted to quantify the encapsulation efficiency (underestimation of up to 24% or overestimation by up to 57.9% for vancomycin and underestimation of up to 61.1% for rifampin) and further propose ultrafiltration and bursting by methanol as the method with minimal bias for quantification of encapsulation efficiency in liposomes. The knowledge generated in this work provides critical insight into the more practical, albeit less investigated, aspects of designing vesicles for localized antibiotic delivery and can be extended to other nanovehicles that may suffer from the same biases in analytical protocols.