Development and In Vitro Validation of Antibacterial Paints Containing Chloroxylenol and Terpineol.

The establishment of self-disinfecting surfaces is an important method to avoid surface contamination. Recently, paints with antimicrobial properties have been developed to be applied on different surfaces, avoiding contamination with pathogens. In this work, self-disinfecting paints containing Chloroxylenol (CLX), Terpineol (TRP), and a mixture of both substances were developed. The goal was to evaluate and validate these paints using international standards for eventual commercialization and application in scenarios where surface contamination represents a problem. The paints were challenged with five different bacteria, Gram-positive and Gram-negative, before and after a scrub resistance test, where the long-term efficacy of the paints was evaluated. The antibacterial activity assessment was performed following ISO 22196 and JIS Z2801. In general, the paints showed very promising results, demonstrating their antibacterial activity, before and after scrub resistance test. The paint incorporating the mixture of CLX and TRP (CLX+TRP) stood out by revealing consistent results of antibacterial activity both before and after the scrub resistance test for most of the tested bacteria. The cytotoxicity of the developed paints was assessed in vitro by performing tests by direct contact with a human skin cell line, HaCaT, and testes on extracts with HaCaT and a pulmonary cell line, A549. The methodologies for cytotoxicity assessment were developed based in ISO 10993. For genotoxicity assessment, alkaline comet assay was conducted on both cell lines. The cytotoxicity assessment revealed promising results with the paints, demonstrating values of cellular viability above 70% and values of lactate dehydrogenase (LDH) leakage below 30%. The genotoxic assessment also revealed acceptable values of primary DNA damage for the developed antibacterial paints. In general, the selected methodologies presented good potential to be applied in the validation of both efficacy and safety of the antimicrobial paints, aiming to be applied in real scenarios.

In Vitro Cyto- and Genotoxicity Assessment of Antibacterial Paints with Triclosan and Isoborneol.

Surfaces with antimicrobial properties are gaining notoriety as an efficient method to avoid surface contamination. Self-disinfecting paints are a promising strategy towards cleaner indoor environments by preventing the colonization of walls with microorganisms. However, its widespread use needs an appropriate toxicological safety evaluation due to the potential for biological disturbance associated to its biocidal activity. In this work, the cyto- and genotoxic assessment of two self-disinfecting paints containing the antimicrobial substances triclosan (TCS) and isoborneol (ISB) is performed. HaCaT and A549 cell lines models were selected for the in vitro assessment. To evaluate the cytotoxicity, tests by direct contact and on extracts obtained from leaching were performed following ISO 10993, whereas the genotoxicity was assessed by comet assay and cytokinesis-block micronucleus (CBMN) assay. The results showed low levels of cyto- and genotoxicity under the models and conditions tested, indicating that these substances have commercial potential.

Development of a new multiplex PCR to detect prevalent species of house dust mites in house dust.

Dermatophagoides pteronyssinus and Dermatophagoides farinae are the most common House Dust Mite (HDM) species in home environments worldwide and responsible for HDM allergy. Since the prevalence of HDM-related clinical conditions is linked to exposure to the mite itself, the detection of HDM in the human households gains importance. We aimed to develop a fast and accessible multiplex PCR to detect and distinguish two relevant HDM species in house dust. New primers were designed, and sensitivity analysis was performed. Sequencing of PCR products was also performed to confirm the method's specificity. The limit of detection of the multiplex PCR for both species was as low as 30 pg µL-1. The application of the multiplex PCR to dust samples also resulted in the identification of both species with high sensitivity. The protocol required small amount of template, reagents and a short reaction time thus presenting an alternative to classically used techniques for HDM identification.

Self-Disinfecting Paints with the Natural Antimicrobial Substances: Colophony and Curcumin.

The risk of infection arising from indirect sources-namely, contaminated surfaces-has been proved, particularly in healthcare facilities. In the attempt to minimize this problem, innumerable research projects involving the development of surfaces with self-disinfecting properties are being conducted. In this work, wall-paints with self-disinfecting properties were developed with the scope of being applied in environments prone to contamination, such as those at healthcare settings. Our approach was to develop new paint formulations containing two natural plant-based products with known antimicrobial activity-colophony (CLF) and curcumin (CUR). The natural substances were separately incorporated on a commercial paint and their antibacterial activity was evaluated with several bacterial species following ISO 22196. To assess the paints' safety, cytotoxicity tests were performed on HaCaT and A549 cell lines, using tests on extracts and direct contact tests, as suggested by the standardized protocol ISO 10993. In general, both paints containing CLF and CUR were able to reduce the bacterial growth after 24 h, compared with the control, the commercial unmodified paint. Colophony was even able to reduce the number of culturable bacteria by over 2 log for Staphylococcus aureus, Escherichia coli, and Bacillus cereus. Regarding the cytotoxicity tests performed (WST-1, NRU, and LDH), both formulations revealed promising results regardless of the methodology used.

Auto-Disinfectant Acrylic Paints Functionalised with Triclosan and Isoborneol-Antibacterial Assessment.

Environmental surface contamination with microorganisms is a serious concern worldwide. Triclosan and isoborneol present good antimicrobial activity. Their immobilisation to paint substrates allows for development of a material that stays effective over a longer time. In this work, we disclosed the preliminary studies to evaluate the antimicrobial activity of the active molecule after being functionalised with isocyanates for further immobilisation on the paint substrate. Overall, the newly developed non-release antimicrobial coating provides an effective way of preventing the spread of diseases and has been proven to inhibit bacterial growth and with a considerable antimicrobial activity towards S. aureus, E. coli, and K. variicola at the tested concentrations.

Optimization of the harvesting and freezing conditions of human cell lines for DNA damage analysis by the alkaline comet assay.

The comet assay is a commonly used method for in vitro and in vivo genotoxicity assessment. This versatile assay can be performed in a wide range of tissues and cell types. Although most of the studies use samples immediately processed after collection, frozen biological samples can also be used. The present study aimed to optimize a collection and freezing protocol to minimize the DNA damage associated with these procedures in human cell line samples for comet assay analysis. This study was conducted in glial A172 and lung alveolar epithelial A549 cells. Two cell detachment methods (mechanical vs enzymatic) and two cryoprotective media [FBS + 10% DMSO vs Cell Culture Media (CCM) + 10% DMSO] were tested, and DNA damage assessed at four time points following storage at -80 °C (one, two, four and eight weeks). In both cell lines, no differences in % tail intensity were detected between fresh and frozen cells up to eight weeks, irrespective of the harvesting method and freezing medium used. However, freshly isolated A172 cells exhibited a significant lower DNA damage when resuspended in CCM + 10% DMSO, while for A549 fresh cells the preferable harvesting method was the enzymatic one since it induced less DNA damage. Although both harvesting methods and cryoprotective media tested were found suitable, our data indicate that enzymatic harvesting and cryopreservation in CCM + 10% DMSO is a preferable method for DNA integrity preservation of human cell line samples for comet assay analysis. Our data also suggest that CCM is a preferable and cost-effective alternative to FBS in cryopreservation media. This optimized protocol allows the analysis of in vitro cell samples collected and frozen at different locations, with minimal interference on the basal DNA strand break levels in samples kept frozen up to eight weeks.

Self-disinfecting surfaces and infection control.

According to World Health Organization, every year in the European Union, 4 million patients acquire a healthcare associated infection. Even though some microorganisms represent no threat to healthy people, hospitals harbor different levels of immunocompetent individuals, namely patients receiving immunosuppressors, with previous infections, or those with extremes of age (young children and elderly), requiring the implementation of effective control measures. Public spaces have also been found an important source of infectious disease outbreaks due to poor or none infection control measures applied. In both places, surfaces play a major role on microorganisms' propagation, yet they are very often neglected, with very few guidelines about efficient cleaning measures and microbiological assessment available. To overcome surface contamination problems, new strategies are being designed to limit the microorganisms' ability to survive over surfaces and materials. Surface modification and/or functionalization to prevent contamination is a hot-topic of research and several different approaches have been developed lately. Surfaces with anti-adhesive properties, with incorporated antimicrobial substances or modified with biological active metals are some of the strategies recently proposed. This review intends to summarize the problems associated with contaminated surfaces and their importance on infection spreading, and to present some of the strategies developed to prevent this public health problem, namely some already being commercialized.