Symposium SM02—Next-Generation Antimicrobial Materials—Combating Multidrug Resistance and Biofilm Formation

The availability of surface-charged polymer-based materials opens up new perspectives for many industrial applications. In recent years, we developed a new technique for achieving such kind of surfaces through a chemical-free procedure that what we call pyro-electrification (PE). It exploits the electrostatic fields spontaneously generated on the surface of pyroelectric lithium niobate (LN) crystals. We present here how this technique can be used in various applications ranging from live cell patterning to biofilm formation for bacteria. In fact, the bacteria intrinsically become more resistant to environmental stresses under biofilm configuration, and may be a serious problem in different industrial sectors. Therefore, the possibility of monitoring the adhesion and the biofilm formation of bacteria is a very important issue, and has numerous advantages, since a fast detection of the presence of bacteria can significantly reduce the risk of contamination. In particular, we show the characterization of biofilm formation through qualitative and quantitative procedures in order to demonstrate the reliability of technique even for different bacterial strains.

References
1) Rega, R., Gennari, O., Mecozzi, L., Grilli, S., Pagliarulo, V., & Ferraro, P. (2016). Bipolar patterning of polymer membranes by pyroelectrification. Advanced Materials, 28(3), 454-459.
2) Mandracchia, B., Palpacuer, J., Nazzaro, F., Bianco, V., Rega, R., Ferraro, P., & Grilli, S. (2018). Biospeckle decorrelation quantifies the performance of alginate-encapsulated probiotic bacteria. IEEE Journal of Selected Topics in Quantum Electronics, 25(1), 1-6.
3) Gennari, O., Marchesano, V., Rega, R., Mecozzi, L., Nazzaro, F., Fratianni, F., ... & Ferraro, P. (2018). Pyroelectric effect enables simple and rapid evaluation of biofilm formation. ACS applied materials & interfaces, 10(18), 15467-15476.
4) Rega, R., Gennari, O., Mecozzi, L., Pagliarulo, V., Mugnano, M., Oleandro, E., ... & Grilli, S. (2019). Pyro-electrification of freestanding polymer sheets: a new tool for cation-free manipulation of cell adhesion in vitro. Frontiers in chemistry, 7, 429.

We synthesized "cationic molecular umbrellas", which are dendrons of multivalent cationic charge attached to a hydrophobic alkyl chain. The generation number and alkyl chain length are key determinants of activity against bacteria and red blood cells. In the best case, we identified a very promising composition that exerts potent, broad-spectrum antibacterial activity (MIC = 4–8 μg/mL) while remaining non-hemolytic (HC₅₀ ~ 5000 μg/mL) even at concentrations 1000x higher than the effective antibacterial dose. Although these compounds do self-assemble into stable micelles in aqueous solutions, the antibacterial activity is observed at concentrations far lower than the CMC, suggestioning that the active species is the individually solvated dendron. Mechanistic studies strongly point to a mechanism involving membrane disruption.

Antibacterial nanomaterials, or nanoantibiotics, are emerging contenders to fend off drug-resistant bacteria when conventional antibiotics fail. Nanostructure itself is widely and sometimes blithely speculated to instigate added benefits in killing bacteria, but whether it plays any role on defining the encounter between nanoantibiotics with bacteria that seals the dour fate of the microbes is not clear. In order for nanoantibiotics to stay relevant in the clinical battlegrounds against bacterial infections, it is imperative to dissect the antibacterial role of nanostructures from the inherent and external chemical moieties associated with the nanoantibiotics that are detrimental to both bacteria and mammalian cells. In this talk, I will discuss our effort to elucidate the antibiotic role of nanostructures using model spherical and rod-like polymer molecular brushes (PMBs) that mimic the two basic structural motifs of bacteriophages. While the individual linear-chain polymer branch that makes up the PMBs is hydrophilic and a weak antimicrobial, amphiphilicity is not a required antibiotic trait once nanostructures come into play. The phage-mimicking PMBs induce an unusual topological transition of bacterial but not mammalian membranes to form pores. The sizes and shapes of the nanostructured PMBs further help define their antibiotic activity and selectivity against different families of bacteria. This nanostructure-induced transformation of antibacterial activities further suggests that nanoantibiotics have the potential to serve as a generic platform for the design of “smart” antibiotics with in-demand activity and selectivity in response to external stimuli by assembly or disassembly of their nanostructures. To exploit this concept for the design of environmentally benign antibiotics that remain fully active in clinical services but become deactivated rapidly once released into the environment, I will discuss an example of antibiotic PMB design that epitomizes the concept of carrying a built-in “OFF” switch responsive to natural stimuli. In their nanostructured forms in services, these PMBs are potent killers for both Gram-positive and Gram-negative bacteria, including clinical multidrug-resistant strains; after services and being discharged into the environment, they are shredded into antimicrobially inactive pieces by bioorthogonal chemistry that does not exist in human body but occurs abundantly in natural habitats.

Antimicrobial resistance (AMR) has become a global severe problem and is aggravated by the slow pace of antibiotic development. Antimicrobial peptides and its mimics have emerged as alternative therapeutic agents but most failed in clinical trials due to varied reasons like limited antimicrobial spectrum, reduced activity in physiological condition, toxicity issue etc. Here we applied one-pot multicomponent polymerization and prepared a series of main-chain cationic polyimidazoliums (PIMs). The lead compound, PIM1, demonstrated good biocompatibility and potent broad-spectrum antimicrobial efficacy against both multi-drug resistant Gram-positive and Gram-negative bacteria even including colistin-resistant burkholderia, and it’s an anti-mycobacterial agent. Unlike classic antimicrobial peptides, PIM1 does not permeabilize bacterial cell membrane but accumulates into cells with assistance of membrane potential, and ultimately leads to cell death. PIM1 does not develop resistance in Gram-negative P. aeruginosa cells while has menaquinone mutation in Gram-positive S. aureus. In murine wound model, PIM1 demonstrated good potency in preventing topical skin infection, but systemic toxicity in mice was found via intraperitoneal injection of PIM1. We further developed PIM1 derivative, PIM1D, which has amide bond and become less hydrophobic. Experiments showed that PIM1D does not cause systemic toxicity to mice even with 7-days repetitive dosing but maintained good antimicrobial potency and demonstrated the ability to rescue mice suffering sepsis infection. Overall, we successfully showed a potential drug candidate in fighting against pan-resistant bacterial infections.

There are about 1.1 million burn injuries that receive medical attention every year in the United States, of which majority are related to first- and second-degree burns. Burns are among the most painful and debilitating wounds and often turn deadly when infection sets in. Approximately 50,000 of these require hospitalization; around 20,000 suffer from major third degree burns, and an estimated 4,500 die from burn wounds. Patients who are admitted to the hospital after sustaining a large burn injury are at high risk for developing hospital-associated infections. If patients survive the initial three days after a burn injury, infections are the most common cause of death. The risk of infections caused by multidrug-resistant bacteria increases as the patients stays longer time in the hospitals. While susceptible gram-positive organisms predominate in the initial days, the more resistant gram-negative organisms are found later. These findings affect the choice of empiric antibiotics in critically ill burn patients.

To combat such infections only a handful of FDA-approved products are available in the market to treat second and third degree burns, and hardly a handful treat scars associated with such burns. Topical agents in treating wounds such as chlorhexidine, proflavine, iodine, hydrogen peroxide, silver etc. have been used to combat wound infections. However, the relentless emergence of antibiotic resistant strains of pathogens, often with multiple antibiotic resistances, together with the discovery of novel antibiotics has led to the need to find alternative treatments.

We have developed a series of hybrid membranes engineered using material modification through intercalation and exfoliation of silicate-based materials and metal ions to prevent infections from ESKAPE pathogens sans the expensive or environmentally toxic ions or nanoparticles while promoting rapid wound and scar healing. Further, we will also display a metal-less hybrid organic-inorganic system that shows huge promise in antimicrobial efficacy against gram-negative and gram-positive bacteria. Complete characterization including physico-chemical, spectroscopic and mechanical analyses corroborates our hypothesis for the structures, properties, mechanisms, mechanical durability and microbial activity of such membranes. The membranes also exhibit optimal water vapor transport through the pores and skin - an important feature that helps with the healing and quick recovery. Cost-effective, near-zero toxicity, biodegradable and durability features, together with easy application on the wound areas, make these hybrid membranes unique for burn treatments.

Nanozymes kill bacteria with reactive oxygen species (ROS) they produce in situ. Because ROS can simultaneously oxidize diverse substances crucial for proper cell functions, nanozymes are recognized as a class of novel antimicrobial agents that are promising for tackling antimicrobial resistance. However, the intrinsic inability of ROS to distinguish bacteria from mammalian cells deprives nanozymes of the selectivity necessary for an ideal antimicrobial. Note that mammalian cells, but not bacteria, actively internalize nanoparticles via endocytosis and that reactive ROS have extremely short lifetimes. We hypothesize that nanozymes that generate surface-bound ROS may selectively kill bacteria over mammalian cells, thereby offering disinfection and biofilm inhibition in a biocompatible manner. To prove this hypothesis, we identified AgPd, a silver-palladium bimetallic alloy nanoparticle, to be an oxidase-like nanozyme that generates surface-bound ROS and, using AgPd as a model for surface-bound ROS-generating nanozymes, carried out antibacterial assays and mammalian cell cytotoxicity tests both in vitro and in animal models. We further excluded the possibility that our hypothesis applies only to AgPd, by examining the performances of another two nanozymes that are distinct in materials from AgPd but generate surface-stabilized ROS in oxidase-like way as well. To examine whether or not the surface-bound nature of ROS on AgPd impacts its potential in eliminating antibiotic-resistant bacteria or delaying the onset of bacterial resistance emergence, we performed serial antibacterial assays in vitro. Moreover, to evaluate the potential of AgPd as antibacterial additive for surface coating, we coated AgPd onto an inert substrate and tested the potency of the resulting surface in thwarting biofilm formation both in vitro and in mouse models. Results from above in vitro assays and animal experiments will be reported.

Emergence of antimicrobial resistance (AMR) caused by superbugs, has threatened the global public health thereby constituting a major share of the annual mortality, worldwide. On top of that, COVID-19 pandemic in the recent times has further worsened the prevailing scenario. In this context, the global community has been realizing the significant role of surfaces in infection transmission and necessity for the development of effective preventive measures and therapeutics. Towards this goal of mitigating the spread of pathogens through fomites, our group has engineered numerous polymeric antimicrobial paints.^1-2 Recently, we have developed a one-step curable, covalent antimicrobial coating, which can be applied to various surfaces such as cotton, plastic, polyurethane, surgical mask, apron, gloves.³ The coating displays excellent activity against drug resistant bacteria, and pathogenic fungi. Remarkably, this coating shows complete killing of human influenza viruses and is also being investigated for ability to inactivate SARS-CoV-2. Our interest into biomaterial researched has also led to antimicrobial hydrogels and injectable antimicrobial sealant with superior adhesive strength and haemostatic ability and potent therapeutic effect against corneal infections.^4-6 Alongside, we have contributed significantly in the development of synthetic peptidomimetic antimicrobial polymers.^7-9 These membrane-active macromolecules demonstrate excellent activity against multidrug resistant pathogenic bacteria and fungi in in-vitro as well as in-vivo, without any detectable resistance development. Overall, the aforementioned inventions hold promise leading to be developed as smart technologies in future to combat microbial infections and antimicrobial resistance.

Reference:
(1) Hoque, J. et al. ACS Biomater. Sci. Eng. 2019, 5, 81. (2) Hoque, J. et al. ACS Appl. Mater. Interfaces, 2019, 11, 39150. (3) Ghosh, S. et al. ACS Appl. Mater. Interfaces 2020, 12, 27853. (4) Hoque, J. et al. ACS Appl. Mater. Interfaces 2017, 9, 15975. (5) Hoque, J. et al. Biomacromolecules 2017, 19, 267. (6) Hoque, J. et al. Mol. Pharmaceutics 2017, 14, 1218. (7) Barman et al, ACS Appl. Bio Mater. 2019, 2, 5404-5414; (8) Barman, S. et al. ACS Appl. Mater. Interfaces 2019, 11, 33559. (9) Mukherjee, S. et al. Front. Bioeng. Biotechnol. 2020, 8, 55.

Mask-wearing has become a new normal in many parts of the world in the COVID-19 pandemic. There has been much interest in enhanced masks that can better protect the wearers. However, a mask or face covering is much more effective in protecting others, because it can block and reroute a large portion of the virus-laden respiratory droplets from symptomatic or asymptomatic infected wearers during coughing and sneezing. Here we report an on-mask chemical modulation strategy, where droplets escaping a masking layer are chemically contaminated with antipathogen molecules (e.g., mineral acids or copper salts) pre-loaded on polyaniline-coated fabrics. Colorimetric method based on the color change of polyaniline and fluorometric method utilizing fluorescence quenching microscopy are developed for visualizing the degree of modification of the escaped droplets by H⁺ and Cu²⁺, respectively. It is found that even fabrics with low fiber packing densities (e.g., 19%) can readily modify 49% of the escaped droplets by number, which accounts for about 82% by volume. The chemical modulation strategy could offer additional public health benefits to the use of face covering to make the sources less infectious, helping to strengthen the response to the current pandemic or future outbreaks of infectious respiratory diseases.

The rapid rise of multidrug-resistant (MDR) superbugs and the declining antibiotic pipeline are serious challenges to global health. Rational design and synthesis of therapeutics can accelerate development of effective therapies against MDR bacteria. In this talk, I will describe multi-pronged systems, synthetic biology, and nano-biotechnology based approaches being devised in our lab to rationally engineer therapeutics that can overcome antimicrobial resistance. We have developed a synthetic biology and materials-engineering based platform called Facile Accelerated Specific Therapeutic (FAST) for developing accelerated therapeutics in less than a week. This approach relies on designing, building and testing engineered antisense therapeutics that can block translation or increase transcription of any desired gene in a species-specific manner. We have used this approach to uncover and target novel genes to re-sensitize MDR clinical isolates of bacteria to antibiotics, develop new classes of antibiotics, as well as develop therapeutics effective against SARS-CoV2 in very short periods of time. I will also present a nano-biotechnology based approach involving development of a unique semiconductor material-based quantum dot-antibiotic (QD ABx) which, when activated by stimuli, release reactive oxygen species to eliminate a broad range of MDR bacterial clinical isolates. This approach has been shown to eliminate MDR clinical isolates under both planktonic and biofilm conditions. Pre-clinical animal studies evaluating the FAST and QD Abx platform demonstrate low toxicity and high efficacy, and thus promise for further translation. The FAST and QD Abx platforms and inter-disciplinary approaches presented in this talk offer novel methods for rationally engineering new therapeutics to combat disease challenges.

Many infectious diseases including viruses, bacteria, and toxins, present unique spatial patterns of antigens on their surfaces [1-3]. These specific patterns facilitate multivalent binding to host cells, resulting in enhanced pathogenic infectivity. Based on this naturally occurring multivalent binding mechanism, synthetic multivalent entry blockers were previously introduced by linking epitopes-binding ligands to a scaffold to improve multivalent binding avidity [4-7]. Recently, the anti-influenza assays by Kwon et al. has suggested that matching ligand spacing with the distance of viral epitopes is a critical factor in inhibiting viral infection, while higher ligand densities have resulted in null or much weaker inhibition of influenza infection [8]. However, existing scaffolds, which include polymers, dendrimers, nanofibers, inorganic nanoparticles and lipid nanoemulsions, have shown toxicity [9,10]. Furthermore, the complex geometric patterns of viral epitopes cannot be matched by existing scaffolds because they are not as precise in ligand spacing or provide limited control over the scaffold shape and ligand valency.

A customizable molecular scaffold strategy capable of incorporating pathogen-specific ligands and patterns may address these issues on both therapeutic and diagnostic fronts. DNA, when folded into nanostructures with a specific shape, is capable of spacing and arranging binding sites into a complex geometric pattern with nanometer-precision. Here we demonstrate designer DNA nanostructures (DDNs) that can act as templates to display multiple binding motifs with precise spatial pattern-recognition properties, and that this approach has been shown to confer exceptional potent viral inhibitory capabilities against dengue virus (DENV) and SARS-CoV-2.

As a proof-of-concept, we designed and synthesized a star-shaped DNA architecture to display 10 DENV envelope domain III (ED3)-targeting aptamers into a two-dimensional pattern precisely matching the spatial arrangement of ED3 clusters on the viral surface [11]. DENV was chosen as a representative target because its epitopes represent the most complex spatial pattern among all known viruses. The binding strength of monovalent ligand to proteins on a viral or cell surface is often relatively weak [8,11]. However, the “DNA star” allows for polyvalent and spatial pattern-matching interactions, affording dramatic improvement in DENV-binding avidity and providing highly potent DENV inhibitor in human blood with an EC₅₀ of 2 nM (~7,500-fold more effective than the monovalent aptamer). Live confocal imaging confirmed that dengue virions lost their cell internalization ability after binding by the DNA stars in blood. Our molecular-platform design strategy could be adapted to combat other disease-causing pathogens by generating the requisite ligand patterns on customized DNA nanoarchitectures. The design and characterization of our SARS-CoV-2 inhibitors will be also discussed.

References:
1. Science 2010, 329 (5995), 1026-1027.
2. Science 2005, 309 (5735), 777-781.
3. J Am Chem Soc 2017, 139 (45), 16389-16397.
4. Nat Biotechnol 2001, 19 (10), 958-961.
5. Nat Biotechnol 2006, 24 (5), 582-586.
6. Nat Nanotechnol 2008, 3 (1), 41-45.
7. Nature 2000, 403 (6770), 669-672.
8. Nat Nanotechnol 2017, 12 (1), 48-54.
9. Rev Environ Health 1989, 8 (1-4), 3-16.
10. J Control Release 2000, 65 (1-2), 133-148.
11. Nat Chem 2020, 12 (1), 26-35.

The attachment and accumulation of organic substances and subsequent microbe attaching on submerged solid surfaces can cause substantial energy waste and severe damage. In addition to conventional antifouling coatings, alternating strong electric fields have also been investigated as a promising anti-biofouling strategy via the effects of killing microbes by irreversible electroporation of cell membranes or to repel microbes via the dielectrophoresis effect. We recently investigated nanogenerator technology as an effective approach to generate alternative electric fields by harvesting mechanical energy from ambient environment. With appropriate design, the electric potential generated directly from water agitation can be utilized as a self-sustainable energy source to effectively to prevent microbe adhesion by changing surface charge distribution on microbes. We demonstrated an efficient and eco-friendly anti-biofouling system using an alternating low-intensity and discrete electric field generated by a water-driven nanogenerator. The anti-biofouling mechanism was attributed to the electric field-induced disturbance to the double layer, which impairs the stable adsorption of organic substances and the subsequent microbe attachments. The anti-biofouling efficacy was directly related to the strength of the electric field but less dependent to the alternating frequency. A long-time on-site demonstration in lake demonstrated superior performance compared to copper based surfaces and commercial coatings. This development brings a novel, effective and eco-friendly solution for protecting a broad range of surfaces against biofouling, including underwater surface and maybe even for implanted medical devices.

Usual approaches to treat skin infections are based on the use of antimicrobial products such as disinfectants or antibiotics. However, these compounds are rarely fully selective so that they act not only on pathogens responsible for the skin infection but also against commensal bacteria living on the human skin and which are now recognized as a part of the host defense system. Indeed, some bacteria involved in the skin microbiota play a major role against proliferation of pathogens by secreting active substances such as antimicrobial peptides, quorum sensing molecules, etc. These microorganisms produce also substances which stimulate the wound repair and limit the inflammation related to the infection.
As a consequence, treatments impacting this community can give rise to serious skin disorders leading to pathologies even more serious than the initial infection to be treated. Therefore, it is now well-admitted that skin treatments need to be targeted to preserve the bacterial community composing the skin microbiota. In this context, recent approaches explored the use of commensal bacteria known for their beneficial activity against pathogens to treat skin diseases. However, applying directly such bacteria on the patient’s skin is not without risk considering their tremendous ability to mute or to become pathogenic in certain conditions. Therefore, an approach allowing to manipulate such bacteria to treat skin disorders while avoiding their uncontrolled proliferation could be a promising approach for topical applications.
In this presentation, we present different approaches to entrap Staphylococcus epidermidis, a predominant commensal skin bacterium known for its active role in the host defense system but also reputed to be an opportunistic agent being a major source of hospital-acquired infections, mostly associated with the use of invasive medical devices. The objective is to develop polymer envelops which preserve the viability and the activity of bacteria and allow the diffusion of secreted molecules but also avoid the release of cells to prevent their uncontrolled growth on human skin. Different strategies based on encapsulation of individual cells in a polymer shell, entrapment of cells in mats of polymer nanotubes, in polymer microtubes and in pores of polymer membranes will be presented. The advantages and the limitations of these different approaches towards the final application, i.e., the development of new therapeutic strategies to treat skin disorders, will be discussed.

This talk will present our recent work on the fabrication of antifouling surface using DNA-based nanofabrication. We used DNA triangle nanostructures as templates to produce triangular-shaped trenches ca. 100 nm in size on a SiO₂ surface. Using B.subtilis as a bacterial model, we found that such nanopatterned surface showed a 75% reduction in bacterial adhesion and 74% reduction in biofilm density at only 35% surface coverage of the nanoscale triangle trenches. Our work demonstrates the potential of DNA-based nanofabrication in antifouling and other surface engineering applications.

Bacterial infections of patients often initiate at the surface of medical devices. In consequence, biofilms form, often with life-threatening consequences. It is estimated that by 2050, up to 10 million people will die every year due to bacterial infections if the current trends cannot be reverted. Thus, antimicrobial polymers are currently experiencing a renaissance both as drugs and materials. Polycationic materials have long been known for their antimicrobial activity; however, they fail once they are fully covered by bacterial debris. To overcome this problem, we followed different strategies. First, we designed micro- and nanostructured polymer surfaces from a protein-repellent and an antimicrobial poly(oxanorbornene) by colloidal lithography and microcontact printing. By varying the polymer patch sizes, we obtained structure-property relationships for the interaction of these patterned polymer surfaces with proteins, bacteria, and human cells, and found that they were simultaneously protein-repellent, antimicrobial, and cell-compatible at a spacing of 1-2 µm, a size matching bacterial dimensions. We also serendipitously found a stimulus-responsive poly(oxanorbornenes) that was protein-repellent and cell adhesive, yet antimicrobial when in contact with bacteria, and thus makes ideal material for implant coatings. Finally, we investigated interfaces that can shed their functional skin when contaminated, like a reptile, and thereby regenerate their original surface functionality.

Microbial keratitis has ~1 million new cases annually worldwide and is the principal cause of blindness in Asia. The most frequent cases occur in Nepal and South India, which have an incidence rate of 799 and 113 per 100,000 individuals, respectively. One study in India found up to 44% of these infections were caused by fungi. These fungal infections are usually caused by injuries contaminated by soil, allowing invasive molds into the wound area. In humid regions across the world, keratitis is often caused by the invasive mold Fusarium oxysporum, while in the United States, keratitis is often caused by the opportunistic bacteria Pseudomonas aeruginosa.

The current method to prevent microbial infection is overused chemical solutions that lead to antimicrobial resistance. This leads to the development of antimicrobial surfaces which aim to limit the spread of microbes in the first place. The two main methods of antimicrobial surfaces are inherently antimicrobial materials, and physical nanopattern topography. Inherently antimicrobial materials include cationic polymers and chemical tethering of antimicrobial agents. These are simple, effective, but may suffer from leaching. Natural, nanotextured surfaces such as those on cicada wings cause bacterial and fungal cell rupture and death. These textures are promising as a long term solution, but many times the fabrication steps use harsh chemicals. This leads to our work that harnesses the synergistic antimicrobial activity of the inherently antimicrobial biopolymer chitosan and a nanopillar coating. We examined the growth of pathogenic fungi and bacteria on these hydrogel surfaces. After 24 hours, we found fewer viable fungi on all of the hydrogel films compared to a control. While dense biofilm was observed on the control surface, the surface without nanopillars inhibited biofilm development due to the antimicrobial activity of the chitosan material. The addition of nanopillars onto the chitosan surfaces appear to stunt the germination and germ tube development even further compared to the film without nanotextures. Additionally, there was a decrease in viable bacteria on the hydrogel surfaces compared to a control. We conclude that the nanopillars act synergistically with the inherently antimicrobial chitosan material to inhibit disease causing microbial growth. This study may inspire the design of future antimicrobial systems that harness multiple antimicrobial effects.

This research presents bio-friendly and cost-effective antibiofilm coating formulations based on Pickering emulsion templating. The coating does not contain any active material, where its antibiofilm function is based on passive mechanisms, laying solely on the superhydrophobic nature of the coating, and thus highly suitable for food and medical applications.
The coating formulation is based on water in toluene or xylene emulsions that are stabilized by commercial hydrophobic silica, with Polydimethylsiloxane (PDMS) that is dissolved in the organic phase. The stability of the emulsions and their structure were studied by microscopy methods. The most stable emulsions were applied on polypropylene surfaces and dried in an oven to form PDMS/silica rough coatings. The surface morphology of the coatings shows a honeycomb-like structure that exhibits a combination of micron-scale and nano-scale roughness resulting in a Superhydrophobic property.
The superhydrophobicity of the resulting coatings has been tuned to meet the demands of highly efficient antibiofilm passive activity. The obtained coatings have shown a decrease of one order of magnitude in the E-coli accumulation on the surface, that is a significant value for coating with a passive based antibiofilm coating.

Bacteria communities are essential components of the skin ecosystem, whose disbalance can be related to several skin disorders, such as acne, eczema or psoriasis. Therefore, exogenously supplying skin-beneficial bacteria to dysbiotic skin is increasingly considered as a way to restore immune response, inhibit infection and treat skin inflammation. This requires encapsulation to control the dispersion and proliferation of bacteria while keeping their benefits. Encapsulation of bacteria was previously studied in spherical particles, microcapsules, gels and electrospun fibers; here, we investigate the encapsulation and growth of bacteria in the micropores of track-etched membranes, aiming at checking the role of channel confinement on bacteria proliferation and at producing soft patches for direct topical application.
Well-defined cylindrical micropores of polycarbonate (PC) membranes were modified by a biocompatible coating based on chitosan and alginate. Staphylococcus epidermidis (S. epidermidis), a Gram-positive commensal skin bacterium that is known to have potential benefits to human hosts, was then introduced in the modified micropores. The metabolic activity of S. epidermidis was then tested, showing the proliferation of encapsulated bacteria in the microchannels. However, the release of S. epidermidis from the microchannels is not desirable for practical skincare applications, since S. epidermidis proves to be a dangerous pathogen when it passes the skin barrier. Therefore, to prevent the free growth of S. epidermidis and maintain its metabolic activity, the modified PC membrane was coated with a layer of agarose gel, followed by coating of this gel with layer-by-layer assembled multilayers, either based on alginate/chitosan, or on polyethylenimine/poly(styrenesulfonate) (PEI/PSS). These barrier layers were demonstrated to efficiently delay the leaking of bacteria from the membrane-in-gel patch, with the lag time depending on bilayer number (coating thickness). In this respect, PEI/PSS is more efficient than alginate/chitosan multilayer in controlling the release of bacteria, with only four bilayers sufficient to effectively prevent bacterial escape over long times. The polyelectrolyte multilayers coatings act as barriers preventing bacteria from escaping, while the agarose gel protects them from direct contact with this antibacterial coating and keeps them alive. The resulting soft patches are thus ideally suited to topical applications of S. epidermidis.

Synthesis and characterization of silver halides (AgX) have drawn much attention due to its specific properties and promising application [1,2]. Particularly, AgX have the potential to be nanoantimicrobials (NAMs) by providing a constant concentration of biocidal Ag⁺ ions in aqueous medium and tailoring control release of Ag⁺ ions into the surrounding environment. Nonetheless, it is known that AgX salts in pure crystalline form are unstable [3], whereas AgX salts in a dispersed state are considered stable [4]. Therefore, it is worth pursuing to study the convenient ways to prepare a stable dispersion of AgX with intrinsic antimicrobial activity. In the present study, nano colloidal dispersion of silver chloride (AgCl) in aqueous medium is prepared by using AgNO₃ as precursor and quaternary ammonium chlorides (QAC) as both source of chloride, and stabilizer. Tetra-octyl-ammonium chloride (TOAC) and Benzyl-hexadecyl-dimetyl-ammonium chloride (BAC) were chosen as model QAC systems, holding a symmetric or asymmetric molecular structure. The synthetic approach resulted to be scalable and green. Morphology and stability of AgCl nanocolloids were investigated as a function of different molar fractions of the reagents. Size distribution and kinetics of the particle growth were monitored by dynamic light scattering, which predicted the formation of QAC bilayered structures associated with the AgCl nanoparticles (NPs). Nanocolloids were further characterized by transmission electron microscopy, X-ray photoelectron and infrared spectroscopies. Zeta potential measurements revealed a highly positive potential value at every stage of synthesis. Experimental evidences support the morphological stability of the nanocolloids, along with their antimicrobial property. Application of the antimicrobial NPs is being investigated as slow-releasing active phases in the Food Packaging industry, mainly aiming at bacteriostatic, long term effects.

References
[1] Physica E 33, (2006), 308−314
[2] Mater. Sci. Eng. C 29, (2009), 1216–1219
[3] Spectrochim. Acta, Part A 77, (2010), 1108−1114
[4] J. Phys. Chem. B 103, (1999), 5917−5919

Acknowledgements
Financial support is acknowledged from European Union’s 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement No. 813439.

The employment of bioactive nanomaterials is one of the most strategical approach to fight antimicrobial resistance and biofilm formation. In particular, metal and metal oxide nanoparticles with controlled ion release can show a noteworthy antimicrobial activity [1]. The use of zinc oxide (ZnO) nanostructures for these purposes is continuously expanding, due to its biocompatibility and low toxicity. In our research group we are exploring electrochemical strategies for the preparation of ZnO nanostructures based on the use of a sacrificial Zn anode in an aqueous electrolytic bath [2] as alternative approach to conventional methods. By tuning the synthesis parameters and selecting the proper stabilizer, spheroidal and flower-like ZnO nanostructures are synthesized. Particularly, poly-sodium-4-styrenesulfonate (PSS), cetyltrimethylammonium bromide (CTAB), benzyl-hexadecyl-dimethylammonium chloride (BAC) and poly-diallyl-(dimethylammonium) chloride (PDDA) have been tested as capping agents [3–5]. Novel hybrid coatings were developed by dispersing the as-synthesized ZnO into commercially-available consolidating agents. The nanostructured coatings were deposited on stone monuments as multifunctional films, providing antimicrobial and consolidating properties [6,7]. More recently, flower-like nanostructures have been successfully tested against Bacillus subtilis as a Gram-positive model microorganism [5]. Morphological analyses carried out on the ZnO-based nanomaterials will be presented. The combination of UV–Vis, FTIR and XPS spectroscopies afforded for the univocal assessment of the material composition as a function of different processing and deposition conditions. A critical comparison of the different materials will be presented, outlining the effects of the stabilizer.
1. Sportelli, M.C.; Picca, R.A.; Cioffi, N. Nano-Antimicrobials Based on Metals. In Novel Antimicrobial Agents and Strategies; Wiley-Blackwell, 2014; pp. 181–218 ISBN 978-3-527-67613-2.
2. Izzi, M.; Sportelli, M.C.; Ditaranto, N.; Picca, R.A.; Innocenti, M.; Sabbatini, L.; Cioffi, N. Pros and Cons of Sacrificial Anode Electrolysis for the Preparation of Transition Metal Colloids: A Review. ChemElectroChem 2020, 7, 386–394, doi:10.1002/celc.201901837.
3. Picca, R.A.; Sportelli, M.C.; Hötger, D.; Manoli, K.; Kranz, C.; Mizaikoff, B.; Torsi, L.; Cioffi, N. Electrosynthesis and Characterization of ZnO Nanoparticles as Inorganic Component in Organic Thin-Film Transistor Active Layers. Electrochimica Acta 2015, 178, 45–54, doi:10.1016/j.electacta.2015.07.122.
4. Picca, R.A.; Sportelli, M.C.; Lopetuso, R.; Cioffi, N. Electrosynthesis of ZnO Nanomaterials in Aqueous Medium with CTAB Cationic Stabilizer. J. Sol-Gel Sci. Technol. 2017, 81, 338–345, doi:10.1007/s10971-016-4268-9.
5. Sportelli, M.C.; Picca, R.A.; Izzi, M.; Palazzo, G.; Gristina, R.; Innocenti, M.; Torsi, L.; Cioffi, N. ZnO Nanostructures with Antibacterial Properties Prepared by a Green Electrochemical-Thermal Approach. Nanomaterials 2020, 10, 473, doi:10.3390/nano10030473.
6. Ditaranto, N.; Werf, I.D. van der; Picca, R.A.; Sportelli, M.C.; Giannossa, L.C.; Bonerba, E.; Tantillo, G.; Sabbatini, L. Characterization and Behaviour of ZnO-Based Nanocomposites Designed for the Control of Biodeterioration of Patrimonial Stoneworks. New J. Chem. 2015, 39, 6836–6843, doi:10.1039/C5NJ00527B.
7. van der Werf, I.D.; Ditaranto, N.; Picca, R.A.; Sportelli, M.C.; Sabbatini, L. Development of a Novel Conservation Treatment of Stone Monuments with Bioactive Nanocomposites. Herit. Sci. 2015, 3, 29, doi:10.1186/s40494-015-0060-3.

Copper is one of the most promising agents to fight antimicrobial resistance (AMR) and growth of biofilms: it is commonly incorporated as part of the novel composite systems with synergistic effects [1]. In this study, organic/inorganic Cu-based nanohybrids were prepared by Sacrificial Anode Electrolysis (SAE) technique in organic medium. Several quaternary ammonium compounds (QAC) were used as cationic stabilizers and supplementary organic components with intrinsic antimicrobial properties [2]. These organic species, carrying positive charge, are well-known for their ability to bind to a negatively-charged outer surface of majority of pathogens. In particular, Benzyl-hexadecyl-dimethyl-ammonium chloride (BAC) and Tetra-butyl-ammonium perchlorate (TBAP) were used as a “shell” for comprising a hybrid composite: these organic compounds are widely used as disinfectants with ability to prevent formation of biofilms [3].
Morphological and spectroscopic characterization of these nanocomposites was performed: UV-vis spectroscopy, transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and Fourier Transform infrared spectroscopy (FTIR) were used. Stability and kinetics were studied at different conditions: organic to inorganic component ratio, time and applied potential to the working electrode among other parameters. Stability of the colloids and surface potential were studied by dynamic light scattering (DLS) and Zeta potential measurements. These hybrid nanoantimicrobials (NAMs) are subjects for further microbiological and toxicological tests as potential materials for controlling and inhibiting biofilm and pathogen growth. Inclusion of the NAMs in biodegradable polymers and their use as active coatings for Food Packaging applications are being investigated, as well.

[1] Nanomaterials 2020, 10, 2491
[2] ACS Infect. Dis. 2015, 1, 7, 288–303
[3] Molecules 2020, 25, 49

Acknowledgements

Financial support is acknowledged from European Union’s 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement No. 813439.

The use of zinc oxide nanoparticles (ZnO NPs), biologically safe and compatible, has gained a great amount of attention in a wide range of biotechnological applications due to their ability to act as antimicrobial materials. That is, ZnO NPs could interact with a small cluster of bacteria like Escherichia coli (E. coli) preventing a growth of E. coli molecules. While many studies showed the antimicrobial behavior of the ZnO NPs, bioactivities of the ZnO NPs with E. Coli have not been clearly understood at a molecular level. Here, we perform reactive molecular dynamics (RMD) simulations based on ReaxFF to investigate effects of the addition of ZnO NPs on the growth of E. coli molecules. We found that the addition of ZnO NPs could successfully delay the growth of E. coli clusters. We also identified key reaction pathways for the antibacterial behaviors of ZnO NPs. As such, our work will help guide experimental design of Antimicrobial materials using ZnO NPs for a wide range of medical solutions like lotions and ointments.

R.E. and S.H. acknowledge new tenure-track faculty start-up funds from School of Natural Science, Mathematics and Engineering at California State University, Bakersfield (CSUB).

Significant interest has been directed to synthesizing topical gel formulations capable of encapsulating silver nanoparticles where the nanoparticles released can be effective against antibiotic resistant bacterial strains. The release of nanoparticles from the hydrogel nanocomposite is dictated by the interaction between nanoparticles and the crosslinked hydrogel. The objective of this study is to develop an understanding of the interactions that exist within the nanocomposite hydrogel between the bovine serum albumin stabilized silver nanoparticles (Ag/BSA) and the hydrogel (hyaluronic acid-PEG based hydrogel) using XPS and FTIR, as well as AAS to quantify the release of Ag/BSA nanoparticles from the hydrogel nanocomposite. Characteristic IR peaks in the nanocomposite were found to have broadened and/or red shifted compared to the neat hydrogel; suggesting possible hydrogen bonding or weak interactions between the nanoparticles and the hydrogel in the nanocomposite. A striking difference in the C1s XPS spectrum of the neat hydrogel and the nanocomposite suggested structural rearrangement of the hydrogel in the presence of the BSA coated nanoparticles. Specifically, a large increase in C-O bonding at the surface relative to hydrocarbon bonds in the composite was observed and is indicative of the hydrogel molecule rearranging such that PEG is pointing towards the surface while the ends of the hyaluronic acid are interacting with the BSA of nanoparticles. Additionally, only 50% and 20% of nanoparticles were desorbed from the lightly and highly crosslinked nanocomposite matrix, respectively, after a 14 day of desorption study. This suggests that the role of the nanostructure and the importance of nanoparticle-hydrogel interaction is in controlling the release of nanoparticles from the hydrogel matrix. Antimicrobial studies of the neat hydrogel against E. coli 107, L. monocytogenes, and S. sonnei showed poor antibacterial activity. All the while the nanocomposite showed excellent bactericidal activity against the three kinds of bacteria; indicating that the observed antimicrobial properties are a direct result of the nanoparticles released from the hydrogel. Future studies will explore the potential wound healing properties of the hydrogel nanocomposite when dosed with antibiotic resistant bacterial strains.

Microscale ZnO particles are known to inhibit the growth of bacteria. The fundamental mechanisms driving this process, however, are not completely understood. While there are many contributing factors to consider, we hypothesize that the antimicrobial action is most fundamentally derived from the ZnO surface and its interaction with growth media and the bacteria’s extracellular material. In this work, we implement minimum inhibition concentration and novel comparative assays to evaluate the antibacterial activity of ZnO microcrystals produced by us using a hydrothermal chemical growth method. The samples were synthesized in the range of sizes from 1µm to 5µm with varying abundances of surfaces with different polarities. This approach prevents the ZnO particles from being internalized by the bacterial cells with diameters ca. 500 nm, thus allowing one to study correlations between overall surface polarity and antibacterial action. These experiments were performed in conjunction with optoelectronic studies of ZnO crystals (photoluminescence, surface photovoltage) to characterize electronic structure and dominant charge transport mechanisms as fundamental phenomena, which could potentially govern the processes leading to an antibacterial behavior in our samples. We report on the results of these comparative studies relating antibacterial properties with surface morphology and electronic behavior.

Antibiotic resistance has been on the rise due to the overuse of antibiotics in the livestock and fishing industry. This has led to an increase in foodborne illnesses and a need for alternative modes of action to help mitigate bacterial growth. This study broaches the potential applications of hot water treated (HWT) aluminum foil for use in the food packaging industry. Through HWT, a layer of aluminum oxide nanostructures is formed on the surface of the foil, which conveys antibacterial properties. In this study, we analyze the efficacy of HWT aluminum foil in preventing bacterial growth of Escherichia coli on red meat. Aluminum foil samples were treated in hot water at different temperatures including 75°C, 85°C, and 95°C. It was found that HWT foils were on average 80% more effective than untreated foil samples at preventing bacterial colony growth. This research shows promising alternative modes of action against foodborne illnesses by offering a green and cost-effective means of curbing bacterial growth.

Air quality is the most important factor in heating, ventilating, and air conditioning (HVAC) systems. This is even more important in an environment where the population is composed of an already immunocompromised segment. Nowhere is this more apparent than in hospitals and health facilities. A review of the current literature suggest that these HVAC systems increase the risk of nosocomial infections if not properly maintained and frequently scrutinized via quality checks. Bacteria, viruses, and fungi find entry into health facilities via equipment, people, and air flow. It is the latter which this research has as its focus. This airflow, once inside the health care center, recirculates throughout the facility, carrying with it the microorganisms it has within it. We utilized a novel hot water treatment (HWT) to produce aluminum oxide nanostructures on the surface of aluminum sheets which are used as ductwork for heating, ventilating, and air conditioning (HVAC) systems. We hypothesized that our HWT duct would greatly reduce the bacterial activity in the air circulating through the HVAC system as well as on the surface of the HWT duct. Air and surface analysis were done with an untreated ventilation system and compared to a ventilation system which had received the hot water treatment to produce Aluminum oxide nanostructures. This research was carried out at temperatures of 20–22 °C. Our results show the extreme effectiveness of using this novel, inexpensive, chemical-free method of producing aluminum oxide nanostructures to decrease bacteria growth in HVAC systems, and in turn significantly improving the air quality.

Optimized polymer and nanocomposite materials for additive manufacturing (3D printing) and smart coatings play an important role in improving thermo-mechanical properties, preventing corrosion, influencing the wetting properties of surfaces. And improving any process industry. Graphene nanomaterials and their unique processing methods have been reported and be optimum with a minimum percolation threshold and optimum performance peak. They have been demonstrated to have anti-microbial, anti-fungal, and anti-viral properties, including preventing biofilm formation. Nanostructuring of these materials is important to understand its efficacy. In this talk, we will describe nanostructured graphene composite materials' use to demonstrate and distinguish this anti-pathogenic effect in 3D printed materials and coatings and applications even with high-temperature environments. The use and characterization of these nanocomposite materials coatings are described. Graphene oxide (GO) additives also enhance their surface chemistry ability, having acid, hydroxy, and other oxidized species sufficient for interaction with silanes. The interest is in utilizing the capabilities of GO to form a hierarchical structure capable of anti-microbial properties, non-cytotoxicity, and preventing biofilm formation. Important surface analytical and characterization methods will also be described.

Burn wounds are a devastating form of injury that leads to substantial morbidity, mortality, and costs. One of the critical complications of burn wounds is infections, especially with Staphylococcus aureus. Rising antimicrobial resistance is contributing to the complexity of wound management. Topical antimicrobial therapy, early wound debridement and grafting, and advances in trauma care and intensive management have all contributed to the decline in burn injury mortality. Management for these burns is challenging and is dependent on many factors including but not limited to patient age, total body surface area (TBSA) involved, the cause of the burn and other aspects that shape the treatment plan. Staphylococcus aureus and Pseudomonas aeruginosa are the most common pathogens isolated from wound infections including burn wounds. These microorganisms commonly attach to any surface and start producing extracellular polysaccharides, creating a film like a matrix. Biofilms are a serious problem for public health because of the protective effect of this matrix to the microorganisms they house, shielding them from antibiotics or immune cells, and set the path for deveoplment of chronic wounds, and failure of skin grafts. Hydrogen Sulfide (H₂S) is a novel gasotransmitter that has many physiological functions and acts as a signaling molecule, with pro-inflammatory effects. It also acts as a vasodilator allowing for more blood perfusion to the burn wound, We hypothesized that the a slow releasing H₂S peptide, based on the S-aroylthiooxime (SATO) functional group (S-FE), that self-assembles into nanofibers that form into a gel, could provide benefits in an infected burn wound model. Dipeptide gels were examined in vitro to evaluate the antimicrobial effects on S. aureus. Assays showed bactericidal and bacteriostatic properties of both S-FE and control (non-H₂S releasing) hydrogels. We next evaluated the H₂S dipetides in an ex vivo burn model using porcine skin and established S. aureus biofilm. These results aligned with the in vitro results showing a decrease in bacterial burden with both S-FE and control gels compared to the bacteria only group. Building on the behavior of bacterial populations after S-FE and control treatments in vitro and ex vivo experiments the dipeptides were further in vivo on a well-established infected porcine burn model. The S-FE dipeptide hydrogels resuislted in reduced bacterial burden, improved blood perfusion to the burn area and better wound healing compared to the control hydrogels.

SU-8 is an epoxy-based photo resist which has been used as a novel platform for biomedical applications due to its chemically tunable and biocompatible surface in addition to its relatively high stiffness, chemical resistance, optical transparency and ease of processing properties. Such properties can have a deep impact on the adhesion of single prokaryote cells and subsequent biofilm formation. In this work, we tailor SU-8 surface properties to investigate single cell motility and adhesion of the bacteria Xylella fastidiosa.

Different SU-8 samples have been prepared using UV illumination, thermal processing and oxygen plasma treatment. In addition, flat InP substrates were used for reference control since adhesion on InP surfaces has been well studied in our group. Atomic Force Microscopy and X-Ray Photoelectron Spectroscopy were used to determine nanoscale surface properties; ex-vivo studies at the level from single cell to biofilm formation were carried out with Confocal Laser Scanning Microscopy (CLSM) for different bacterial growth times. The mean velocity and displacement of single cells have been extracted from CLSM tracking information data and the size and quantity of biofilms are compared for different samples. We observed a significant difference in bacterial cell motility, adhesion and biofilm architecture on SU-8 as nanoscale surface property changes. Larger density of carboxyl groups in treated SU-8 surfaces provide enhanced cell motility, while denser biofilms are found in pristine SU-8. Our results can improve understanding of the role of nanoscale properties on bacteria-surface interaction and thereby create strategies to prevent microbial adhesion and consequently, biofilm development of pathogenic species.

Biofilm infections are suggested to be associated with over 99,000 deaths annually and are thought to be responsible for over 80% of bacterial infections. It has been reported that over 90% of chronic wounds become colonized by biofilm, like that caused by Staphylococcus aureus. These ubiquitous biofilms are also a well-known cause of medical device implant infections and are thought to be responsible for 80% of limb amputations. These biofilms consist of an extracellular matrix (ECM) of extracellular polymeric substances (EPS), composed of polysaccharides, various proteins and glycoproteins, extracellular DNA (eDNA), and other host-derived factors and substances. These aspects of the biofilm help to confer resistance to the host immune response and antimicrobial resistance, as well as tolerance to other stressors like heat. Research demonstrates that antibiotic efficacy can be increased against biofilms after exposure to both mildly hyperthermic (T < 45°C) and ablative (T > 45°C) temperatures. Studies indicate that hyperthermia alone can alter biofilm structural integrity and viability, but the lack of localization of hyperthermia upon the biofilm matrix creates a challenge for clinical application. This study investigates the impact of near-infrared photothermal ablation (NIR-PTA), via polymer nanoparticles and combined antibiotic treatment, on S. aureus biofilms.

We have synthesized biocompatible polymer dynamic organic theranostic spheres (PolyDOTS) composed of two polymers: Poly[4,4-bis(2-ethylhexyl)-cyclopenta[2,1-b;3,4-b']dithiophene-2,6-diyl-alt−2,1,3-benzoselenadiazole-4,7-diyl] (PCPDTBSe) for heat generation and Poly(3-hexylthiophene-2,5-diyl) (P3HT) for fluorescence. PolyDOTS are capable of repeated photothermal generation under NIR irradiation and exhibit no photobleaching. They absorb in the NIR range, peaking at 760 nm. Thermocouple measurements determined the photothermal generating capacity of PolyDOTS at different concentrations, under 5 W, 60 secs 800 nm irradiation. They exhibited increasing heat generation at increasing concentrations, with an approximate plateau of 80°C.

Biofilms were challenged with a one-log (90%) effective minimal bacterial eradication concentration (MBEC-90) of clindamycin and a similarly effective dose (100 µg/mL) of PolyDOTS NIR-PTA. Clindamycin was administered either simultaneously with, 24-hours before or immediately after PolyDOTS NIR-PTA. Results showed that simultaneous administration was the most effective, up to a clinically significant three-log (99.9%) reduction in biofilm viability, compared to controls. The least efficacy occurred when clindamycin was added after NIR-PTA, suggesting that the observed ECM changes can inhibit antibiotic efficacy. Confocal microscopy using LIVE/DEAD stain surprisingly showed no confirmation of viability reduction, which is suggestive of a hyperthermia-induced loss of the biofilm layers. Biofilm biomass and structural changes were analyzed via an ECM confocal stain, scanning electron microscopy, and crystal violet assay. S. aureus biofilms showed increasingly pronounced aggregation on SEM and uncharacteristic coalescence, suggestive of compaction of the ECM. The crystal violet assay and spectrophotometry seemed to support the suspected ECM compaction of the PolyDOTS NIR-PTA-treated S. aureus biofilms. The results of this study provide evidence that PolyDOTS NIR-PTA alone, and in combination with an antibiotic, can significantly mitigate biofilm viability. While further investigation into the ECM structural changes resulting from localized biofilm hyperthermia would be merited, this study suggests that the combination treatment may improve existing biofilm infection therapy by enhancing antimicrobial efficacy.