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.

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.

Polyphenolic extract from maple syrup potentiates antibiotic susceptibility and reduces biofilm formation of pathogenic bacteria.

Phenolic compounds are believed to be promising candidates as complementary therapeutics. Maple syrup, prepared by concentrating the sap from the North American maple tree, is a rich source of natural and process-derived phenolic compounds. In this work, we report the antimicrobial activity of a phenolic-rich maple syrup extract (PRMSE). PRMSE exhibited antimicrobial activity as well as strong synergistic interaction with selected antibiotics against Gram-negative clinical strains of Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa. Among the phenolic constituents of PRMSE, catechol exhibited strong synergy with antibiotics as well as with other phenolic components of PRMSE against bacterial growth. At sublethal concentrations, PRMSE and catechol efficiently reduced biofilm formation and increased the susceptibility of bacterial biofilms to antibiotics. In an effort to elucidate the mechanism for the observed synergy with antibiotics, PRMSE was found to increase outer membrane permeability of all bacterial strains and effectively inhibit efflux pump activity. Furthermore, transcriptome analysis revealed that PRMSE significantly repressed multiple-drug resistance genes as well as genes associated with motility, adhesion, biofilm formation, and virulence. Overall, this study provides a proof of concept and starting point for investigating the molecular mechanism of the reported increase in bacterial antibiotic susceptibility in the presence of PRMSE.

Effects of environmental and clinical interferents on the host capture efficiency of immobilized bacteriophages.

Bacteriophage-functionalized surfaces are a new class of advanced functional material and have been demonstrated to be applicable for use as antimicrobial surfaces in medical applications (e.g., indwelling medical devices or wound dressings) or as biosensors for bacterial capture and detection. However, the complex composition of many real life samples (e.g., blood, natural waters, etc.) can potentially interfere with the interaction of phage and its bacterial host, leading to a decline in the efficiency of the phage-functionalized surface. In this study, the bacterial capture efficiency of two model phage-functionalized surfaces was assessed in the presence of potential environmental and biomedical interferents. The two phage-bacteria systems used in this study are PRD1 with Salmonella Typhimurium and T4 with Escherichia coli. The potential interferents tested included humic and fulvic acids, natural groundwater, colloidal latex microspheres, host extracellular polymeric substances (EPS), albumin, fibrinogen, and human serum. EPS and human serum decreased the host capture efficiency for immobilized PRD1 and T4, and also impaired the infectivity of the nonimmobilized (planktonic) phage. Interestingly, humic and fulvic acids reduced the capture efficiency of T4-functionalized surfaces, even though they did not lead to inactivation of the suspended virions. Neither humic nor fulvic acids affected the capture efficiency of PRD1. These findings demonstrate the inadequacy of traditional phage selection methods (i.e., infectivity of suspended phage toward its host in clean buffer) for designing advanced functional materials and further highlight the importance of taking into account the environmental conditions in which the immobilized phage is expected to function.

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.

Predation in homogeneous and heterogeneous phage environments affects virulence determinants of Pseudomonas aeruginosa.

The rise of bacterial variants in the presence of lytic phages has been one of the basic grounds for evolution studies. However, there are incongruent results among different studies investigating the effect of phage resistance acquisition on bacterial fitness and virulence. We used experimental evolution to generate three classes of Pseudomonas aeruginosa variants under selective pressure from two different homogeneous phage environments and one heterogeneous phage environment. The fitness and virulence determinants of the variants, such as growth, motility, biofilm formation, resistance to oxidative stress, and the production of siderophores and chromophores, changed significantly compared to the control. Variants with similar colony morphology that were developed through different phage treatments have different phenotypic traits. Also, mRNA transcription for genes associated with certain phenotypic traits changed significantly; however, sequencing did not reveal any point mutations in selected gene loci. Furthermore, the appearance of small colony variants and melanogenic variants and the increase in pyocyanin and pyoverdin production for some variants are believed to affect the virulence of the population. The knowledge gained from this study will fundamentally contribute to our understanding of the evolutionary dynamics of bacteria under phage selective pressure which is crucial to the efficient utilization of bacteriophages in medical contexts.

Evolution of Pseudomonas aeruginosa virulence as a result of phage predation.

The rapid increase in the emergence of antibiotic-resistant bacteria has attracted attention to bacteriophages for treating and preventing bacterial infections. Bacteriophages can drive the diversification of Pseudomonas aeruginosa, giving rise to phage-resistant variants with different phenotypes from their ancestral hosts. In this study, we sought to investigate the effect of phage resistance on cytotoxicity of host populations toward cultured mammalian cells. The library of phage-resistant P. aeruginosa PAO1 variants used was developed previously via experimental evolution of an isogenic host population using phages PP7 and E79. Our results presented herein indicate that the phage-resistant variants developed in a heterogeneous phage environment exhibit a greater ability to impede metabolic action of cultured human keratinocytes and have a greater tendency to cause membrane damage even though they cannot invade the cells in large numbers. They also show a heightened resistance to phagocytosis by model murine macrophages. Furthermore, all isolates produced higher levels of at least one of the secreted virulence factors, namely, total proteases, elastase, phospholipase C, and hemolysins. Reverse transcription-quantitative PCR (RT-qPCR) revealed upregulation in the transcription of a number of genes associated with virulence of P. aeruginosa for the phage-resistant variants. The results of this study indicate a significant change in the in vitro virulence of P. aeruginosa following phage predation and highlight the need for caution in the selection and design of phages and phage cocktails for therapeutic use.

Formation of biofilms under phage predation: considerations concerning a biofilm increase.

Bacteriophages are emerging as strong candidates for combating bacterial biofilms. However, reports indicating that host populations can, in some cases, respond to phage predation by an increase in biofilm formation are of concern. This study investigates whether phage predation can enhance the formation of biofilm and if so, if this phenomenon is governed by the emergence of phage-resistance or by non-evolutionary mechanisms (eg spatial refuge). Single-species biofilms of three bacterial pathogens (Pseudomonas aeruginosa, Salmonella enterica serotype Typhimurium, and Staphylococcus aureus) were pretreated and post-treated with species-specific phages. Some of the phage treatments resulted in an increase in the levels of biofilm of their host. It is proposed that the phenotypic change brought about by acquiring phage resistance is the main reason for the increase in the level of biofilm of P. aeruginosa. For biofilms of S. aureus and S. enterica Typhimurium, although resistance was detected, increased formation of biofilm appeared to be a result of non-evolutionary mechanisms.

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.

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.

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.

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.

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.

Bacterial capture efficiency and antimicrobial activity of phage-functionalized model surfaces.

The rise of antibiotic-resistant bacteria has directed substantial attention toward the use of bacteriophages as a means to control bacterial populations. It has been proposed that bacteriophages can be applied as a coating on surfaces in healthcare settings or on indwelling medical devices to create an antimicrobial surface. In this study, antimicrobial model surfaces functionalized with five different types of bacteriophage were prepared and characterized with X-ray photoelectron spectroscopy and atomic force microscopy. The bacterial capture efficiency of these functionalized surfaces was studied for two common bacteria, Escherichia coli and Salmonella typhimurium. Binding of the phages to a solid surface affected their biofunctionality as expressed by the capture efficiency and rate of host membrane disruption. Moreover, the size and shape of the bacteriophage and positioning of its specific binding proteins significantly affected its bacterial capture capability in the immobilized state. Symmetric bacteriophages were found to be a better choice for antibacterial surfaces compared to more asymmetric tailed bacteriophages. Immobilized phages were found to disrupt the membranes of attached bacteria and are thus proposed as a candidate for antimicrobial surfaces.