Antibacterial activity of solid surfaces is critically dependent on relative humidity, inoculum volume, and organic soiling.

Antimicrobial surface materials potentially prevent pathogen transfer from contaminated surfaces. Efficacy of such surfaces is assessed by standard methods using wet exposure conditions known to overestimate antimicrobial activity compared to dry exposure. Some dry test formats have been proposed but semi-dry exposure scenarios e.g. oral spray or water droplets exposed to ambient environment, are less studied. We aimed to determine the impact of environmental test conditions on antibacterial activity against the model species Escherichia coli and Staphylococcus aureus. Surfaces based on copper, silver, and quaternary ammonium with known or claimed antimicrobial properties were tested in conditions mimicking microdroplet spray or larger water droplets exposed to variable relative air humidity in the presence or absence of organic soiling. All the environmental parameters critically affected antibacterial activity of the tested surfaces from no effect in high-organic dry conditions to higher effect in low-organic humid conditions but not reaching the effect size demonstrated in the ISO 22169 wet format. Copper was the most efficient antibacterial surface followed by silver and quaternary ammonium based coating. Antimicrobial testing of surfaces using small droplet contamination in application-relevant conditions could therefore be considered as one of the worst-case exposure scenarios relevant to dry use surfaces.

Antiviral efficacy of cerium oxide nanoparticles.

Nanomaterials are prospective candidates for the elimination of viruses due to their multimodal mechanisms of action. Here, we tested the antiviral potential of a largely unexplored nanoparticle of cerium dioxide (CeO2). Two nano-CeO2 with opposing surface charge, (+) and (-), were assessed for their capability to decrease the plaque forming units (PFU) of four enveloped and two non-enveloped viruses during 1-h exposure. Statistically significant antiviral activity towards enveloped coronavirus SARS-CoV-2 and influenza virus was registered already at 20 mg Ce/l. For other two enveloped viruses, transmissible gastroenteritis virus and bacteriophage φ6, antiviral activity was evidenced at 200 mg Ce/l. As expected, the sensitivity of non-enveloped viruses towards nano-CeO2 was significantly lower. EMCV picornavirus showed no decrease in PFU until the highest tested concentration, 2000 mg Ce/l and MS2 bacteriophage showed slight non-monotonic response to high concentrations of nano-CeO2(-). Parallel testing of antiviral activity of Ce3+ ions and SiO2 nanoparticles allows to conclude that nano-CeO2 activity was neither due to released Ce-ions nor nonspecific effects of nanoparticulates. Moreover, we evidenced higher antiviral efficacy of nano-CeO2 compared with Ag nanoparticles. This result along with low antibacterial activity and non-existent cytotoxicity of nano-CeO2 allow us to propose CeO2 nanoparticles for specific antiviral applications.

Preparation and Characterization of Photocatalytically Active Antibacterial Surfaces Covered with Acrylic Matrix Embedded Nano-ZnO and Nano-ZnO/Ag.

In the context of healthcare-acquired infections, microbial cross-contamination and the spread of antibiotic resistance, additional passive measures to prevent pathogen carryover are urgently needed. Antimicrobial high-touch surfaces that kill microbes on contact or prevent their adhesion could be considered to mitigate the spread. Here, we demonstrate that photocatalytic nano-ZnO- and nano-ZnO/Ag-based antibacterial surfaces with efficacy of at least a 2.7-log reduction in Escherichia coli and Staphylococcus aureus viability in 2 h can be produced by simple measures using a commercial acrylic topcoat for wood surfaces. We characterize the surfaces taking into account cyclic wear and variable environmental conditions. The light-induced antibacterial and photocatalytic activities of the surfaces are enhanced by short-term cyclic wear, indicating their potential for prolonged effectivity in long-term use. As the produced surfaces are generally more effective at higher relative air humidity and silver-containing surfaces lost their contact-killing properties in dry conditions, it is important to critically evaluate the end-use conditions of materials and surfaces to be tested and select application-appropriate methods for their efficacy assessment.

Antibacterial Activity of Positively and Negatively Charged Hematite (α-Fe2O3) Nanoparticles to Escherichia coli, Staphylococcus aureus and Vibrio fischeri.

In the current study, the antibacterial activity of positively and negatively charged spherical hematite (α-Fe2O3) nanoparticles (NPs) with primary size of 45 and 70 nm was evaluated against clinically relevant bacteria Escherichia coli (gram-negative) and Staphylococcus aureus (gram-positive) as well as against naturally bioluminescent bacteria Vibrio fischeri (an ecotoxicological model organism). α-Fe2O3 NPs were synthesized using a simple green hydrothermal method and the surface charge was altered via citrate coating. To minimize the interference of testing environment with NP's physic-chemical properties, E. coli and S. aureus were exposed to NPs in deionized water for 30 min and 24 h, covering concentrations from 1 to 1000 mg/L. The growth inhibition was evaluated following the postexposure colony-forming ability of bacteria on toxicant-free agar plates. The positively charged α-Fe2O3 at concentrations from 100 mg/L upwards showed inhibitory activity towards E. coli already after 30 min of contact. Extending the exposure to 24 h caused total inhibition of growth at 100 mg/L. Bactericidal activity of positively charged hematite NPs against S. aureus was not observed up to 1000 mg/L. Differently from positively charged hematite NPs, negatively charged citrate-coated α-Fe2O3 NPs did not exhibit any antibacterial activity against E. coli and S. aureus even at 1000 mg/L. Confocal laser scanning microscopy and flow cytometer analysis showed that bacteria were more tightly associated with positively charged α-Fe2O3 NPs than with negatively charged citrate-coated α-Fe2O3 NPs. Moreover, the observed associations were more evident in the case of E. coli than S. aureus, being coherent with the toxicity results. Vibrio fischeri bioluminescence inhibition assays (exposure medium 2% NaCl) and colony forming ability on agar plates showed no (eco)toxicity of α-Fe2O3 (EC50 and MBC > 1000 mg/L).

Propidium iodide staining underestimates viability of adherent bacterial cells.

Combining membrane impermeable DNA-binding stain propidium iodide (PI) with membrane-permeable DNA-binding counterstains is a widely used approach for bacterial viability staining. In this paper we show that PI staining of adherent cells in biofilms may significantly underestimate bacterial viability due to the presence of extracellular nucleic acids (eNA). We demonstrate that gram-positive Staphylococcus epidermidis and gram-negative Escherichia coli 24-hour initial biofilms on glass consist of 76 and 96% PI-positive red cells in situ, respectively, even though 68% the cells of either species in these aggregates are metabolically active. Furthermore, 82% of E. coli and 89% S. epidermidis are cultivable after harvesting. Confocal laser scanning microscopy (CLSM) revealed that this false dead layer of red cells is due to a subpopulation of double-stained cells that have green interiors under red coating layer which hints at eNA being stained outside intact membranes. Therefore, viability staining results of adherent cells should always be validated by an alternative method for estimating viability, preferably by cultivation.

Potential ecotoxicological effects of antimicrobial surface coatings: a literature survey backed up by analysis of market reports.

This review was initiated by the COST action CA15114 AMICI "Anti-Microbial Coating Innovations to prevent infectious diseases," where one important aspect is to analyze ecotoxicological impacts of antimicrobial coatings (AMCs) to ensure their sustainable use. Scopus database was used to collect scientific literature on the types and uses of AMCs, while market reports were used to collect data on production volumes. Special attention was paid on data obtained for the release of the most prevalent ingredients of AMCs into the aqueous phase that was used as the proxy for their possible ecotoxicological effects. Based on the critical analysis of 2,720 papers, it can be concluded that silver-based AMCs are by far the most studied and used coatings followed by those based on titanium, copper, zinc, chitosan and quaternary ammonium compounds. The literature analysis pointed to biomedicine, followed by marine industry, construction industry (paints), food industry and textiles as the main fields of application of AMCs. The published data on ecotoxicological effects of AMCs was scarce, and also only a small number of the papers provided information on release of antimicrobial ingredients from AMCs. The available release data allowed to conclude that silver, copper and zinc are often released in substantial amounts (up to 100%) from the coatings to the aqueous environment. Chitosan and titanium were mostly not used as active released ingredients in AMCs, but rather as carriers for other release-based antimicrobial ingredients (e.g., conventional antibiotics). While minimizing the prevalence of healthcare-associated infections appeared to be the most prosperous field of AMCs application, the release of environmentally hazardous ingredients of AMCs into hospital wastewaters and thus, also the environmental risks associated with AMCs, comprise currently only a fraction of the release and risks of traditional disinfectants. However, being proactive, while the use of antimicrobial/antifouling coatings could currently pose ecotoxicological effects mainly in marine applications, the broad use of AMCs in other applications like medicine, food packaging and textiles should be postponed until reaching evidences on the (i) profound efficiency of these materials in controlling the spread of pathogenic microbes and (ii) safety of AMCs for the human and ecosystems.

Rapid in situ assessment of Cu-ion mediated effects and antibacterial efficacy of copper surfaces.

Release of metal ions from metal-based surfaces has been considered one of the main drivers of their antimicrobial activity. Here we describe a method that enables parallel assessment of metal ion release from solid metallic surfaces and antimicrobial efficacy of these surfaces in a short time period. The protocol involves placement of a small volume of bioluminescent bacteria onto the tested surface and direct measurement of bioluminescence at various time points. In this study, two recombinant Escherichia coli strains, one expressing bioluminescence constitutively and applicable for general antimicrobial testing, and the other induced by Cu ions, were selected. Decrease in bioluminescence of constitutive E. coli on the surfaces showed a good correlation with the decrease in bacterial viability. Response of Cu-inducible E. coli showed a correlation with Cu content in the tested surfaces but not with Cu dissolution suggesting the role of direct bacteria-surface contact in Cu ion-driven antibacterial effects. In summary, the presented protocol enables the analysis of microbial toxicity and bioavailability of surface-released metal ions directly on solid surfaces within 30-60 min. Although optimized for copper and copper alloy surfaces and E. coli, the method can be extended to other types of metallic surfaces and bacterial strains.

UVA-induced antimicrobial activity of ZnO/Ag nanocomposite covered surfaces.

Application of efficient antimicrobial surfaces has been estimated to decrease both, the healthcare-associated infections and the spread of antibiotic-resistant bacteria. In this paper, we prepared ZnO and ZnO/Ag nanoparticle covered surfaces and evaluated their antimicrobial efficacy towards a Gram-negative bacterial model (Escherichia coli), a Gram-positive bacterial model (Staphylococcus aureus) and a fungal model (Candida albicans) in the dark and under UVA illumination. The surfaces were prepared by spin coating aliquots of ZnO and ZnO/Ag nanoparticle suspensions onto glass substrates. Surfaces contained 2 or 20 µg Zn/cm2 and 0-0.02 µg Ag/cm2. No significant antimicrobial activity of the surfaces, except of those with the highest Ag or Zn content was observed in the dark. On the other hand, UVA illuminated surfaces containing 20 µg Zn/cm2 and 2 µg Zn plus 0.02 µg Ag/cm2 caused >3 log decrease in the viable counts of E. coli and S. aureus in 30 min. As proven by brilliant blue FCF dye degradation and elemental analysis of the surfaces, this remarkable antimicrobial activity was a combined result of photocatalytic effect and release of Zn and Ag ions from surfaces. Surfaces retained significant antibacterial and photocatalytic properties after several usage cycles. Compared to bacteria, yeast C. albicans was significantly less sensitive to the prepared surfaces and only about 1 log reduction of viable count was observed after 60 min UVA illumination. In conclusion, the developed ZnO/Ag surfaces exhibit not only high antibacterial activity but also some antifungal activity.