ANTIBACTERIAL COATING

Technical field The present invention relates to a coating with antibacterial properties. In particular, the invention relates to a composition for obtaining an antibacterial coating obtained with the sol-gel technique, to the relative preparation method and to the articles which carry said coating.

Prior art Various pathologies are caused by the individual's exposure to microorganisms from the outside; this type of contamination, with the transmission of pathogens present on environmental surfaces, concerns the areas with large numbers of visitors, both public and private, where the objects used by many become, in fact, vehicles of contamination. Microorganisms are received and further transmitted through the touch of the hands. Particularly at risk are environments such as healthcare facilities, schools, stations, airports, shopping centers, etc., that is, large rooms designed to accommodate a large number of people.

Particular attention should be paid to the door handles of the aforementioned rooms, especially those inside the bathrooms, as they are a receptacle for various types of microorganisms. The latter, in fact, represent the last contact of the hands before and/or after washing, are often used by several people after unhygienic activities (e.g. defecation or urination) and are mostly contaminated with the bacterial flora of the skin and intestines [1].

As an example, a study conducted in Germany in 2016 on the interior handles of the bathrooms at 136 airports in 59 countries, to evaluate the impact of these frequently touched surfaces on the spread of resistant bacteria, showed the presence of a high contamination of Staphylococcus aureus, Stenotrophomonas maltophilia, Acinetobacter baumannii and Pseudomonas aeruginosa [2] Travelers can therefore acquire antimicrobial resistant bacteria or highly virulent toxins (such as Pantone Valentine-leukocidine) and can spread these bacteria to their countries of origin after returning.

Keyboards, computer mice, and mouse pads are a further documented source of the spread of opportunistic bacteria, pathogens and respiratory viruses [3-6]. In particular, computers shared by different people can constitute vehicles for the transfer of microorganisms between individuals in the public sphere as well as in the health sector. In fact, the contamination of computer keyboards, and in more recent years of the touch screens of tablets in the hospital environment, would seem to play a significant role as a vector of cross-contamination in hospitals.

A 2016 study reported by the American Journal of Infection Control, compared the presence of opportunistic pathogenic bacteria on the surfaces of touch screens for computers used in hospitals and supermarkets. Opportunistic pathogenic bacteria ( Clostridium difficile and Enterococcus vancomycin-resistant) were isolated on touch screens in hospitals, whilein supermarkets methicHlin-Staphylococcus aureus resistant was found to be the prevalent bacterium[7]. Enteric bacteria are more common on touch screens in grocery stores than on touch screens of hospital computers, which probably reflects better hand hygiene in the latter [8]. Cell phones are another important vehicle of contamination; in fact, they are often used during the day and are kept near the face and mouth, two areas that can be "colonized" by both gram-positive and gram negative bacteria. The phones are also placed on various surfaces in a variety of rooms (e.g. bedroom, bathroom and kitchen) and are used during various activities, even outside the home.

Some recent studies have shown that in about 6 cm ² of a mobile phone ten thousand microbes are contained, which is significantly more than a sole of a shoe or a door handle [9]. This can be explained by the fact that the constant heat generated by the telephones creates a fertile ground for the colonization of microorganisms.

Hand hygiene represents the main activity for preventing bacterial contamination. However, since it is not possible to control or manage this aspect for all visitors to public places, it is necessary to develop specific products, aimed at reducing, or at best, eliminating the bacterial load that settles on the surfaces constantly touched by many users. The so-called "highly-touched" surfaces, i.e. telephones, bathroom door handles, devices used in hospitals by several patients, etc., require greater attention and appropriate decontamination. Detergents and disinfectants are also often used in the form of pre-soaked wipes. The use of the aforementioned aids and adequate cleaning protocols, although able to reduce the microbial load present on the surfaces of public places, remains linked to the activity of the operators and the frequency of cleaning, often providing insufficient results. Even in the specific case of touch screens of mobile phones and tablets, effective disinfection protocols would not seem to be available yet [10].

Recently Corning™ Inc has developed a tempered glass capable of exchanging silver ions, which are slowly released from the surface of the glass with consequent antimicrobial action. There are currently no detailed data on antimicrobial efficacy, but from what is reported in the literature, it would seem to reduce the number of bacteria 1000 times after 24 hours

[11]. The use of silver ions has been widely exploited in the field of mobile telephony. Silver ions incorporated within zeolites (Agion® technology)

[12], were in fact used by Motorola™ for the production of telephones with antibacterial properties; LG™ has developed a coating based on silver nanoparticles [13]; while Biomaster® has produced the Ecoskin ™ cover with antibacterial properties for the Apple™ iPhone 4 [14] Despite the proven antibacterial efficacy of these technologies based on the incorporation of silver ions, the latter are unfortunately not applicable to other components other than plastic covers. Furthermore, from a chemical point of view, the presence of silver ions incorporated into additives, in turn dispersed in the final plastic material, limits their mechanical and thermal stability. Further studies report about the incorporation of light-activated antimocrobial agents (LAAA) into polymeric films, such as T1O2 nanoparticlesor photosensitive organic dyes. The resulting material would have an antibacterial effect following the exposure of LAAA to radiation of an appropriate wavelength, capable of generating radical species [15-17] Also with regard to this technology there are important limits, such as the fact that the antimicrobial activity is linked to the application of photosensitizers which, in some cases, require the use of high temperatures.

It is therefore evident the need to create "self-disinfecting" surfaces that require very low user compliance and which, at the same time, are able to reduce the microbial contamination found in public places. The currently unsolved technical problem is that of the lack of an ideal antibacterial material that has maximum efficiency (100% active for a long time on a wide spectrum of microorganisms), biocompatible, inexpensive and easy to make, which presents itself as a solution to the evolving resistance of microorganisms to existing drugs. This technical problem has been solved by the coating according to the present invention.

Summary of the invention

The inventors have in fact found that a sol-gel formulation according to the present invention can be used to prepare coatings with excellent antibacterial properties with a simple and effective methodology.

It is therefore an object of the present invention to provide a formulation with antibacterial activity for a sol-gel coating of manufactured articles.

Another object of the present invention is to provide a composition and a method for preparing a silica-based formulation that has antibacterial characteristics.

Still another object of the present invention is to provide a simple and inexpensive preparation method for a silica-based formulation with antibacterial activity.

Still another object of the present invention is to provide a silica-based formulation for coating a surface subject to contact with bacteria. Still another object of the present invention is to provide a coating with antibacterial properties.

Still another object of the present invention is to provide manufactured articles comprising a coating or coating with antibacterial properties. Still another object of the present invention is the method for carrying out the formulation of the invention which provides for the following basic stages: mixing an alkoxysilane such as tetraethyl orthosilicate (TEOS) with an ionic liquid under conditions of acid hydrolysis to form a film; - in the presence of an inorganic acid which favors the hydrolysis of the silicon-containing compound by acid catalysis.

Another object of the invention is the method for preparing the coating according to the sol-gel technique which provides the basic steps of:

- preparing a mixture by mixing: a compound containing silicon, an inorganic acid and an ionic liquid in which the compound containing silicon and the ionic liquid are set to react in a molar ratio between 8 - 100 at room temperature (range 20-30°C) for a time between 2 and 10h, with an optimal value of 3 hours (so that the hydrolysis process and an adequate degree of condensation is obtained) under acid hydrolysis conditions. The molar ratio 100 is the preferred one since it provides the least amount of ionic liquid.

Apply the mixture thus obtained on the surface of a product with a technique such as dip-coating, spin-coating, spray or brush;

Allow to air dry at room temperature (temperature range: 20 - 30°C).

Still another object of the invention is an antibacterial coating containing the silicon-based compound and the ionic liquid.

Still another object of the invention are the articles which carry the antibacterial coating obtained with the sol-gel technique. Still another objective is the development of a protocol for the verification of antibacterial activity suitable for porous materials. The one reported by the Japanese standard JISZ2801 and the European ISO 22196: 2011 refer to non-porous materials. The preferred acid reaction medium of the method is an aqueous solution of a 37% hydrochloric acid by weight at a pH between 1 and 2.

Further objects and advantages of the present invention will become clear from the following detailed description of a currently preferred embodiment which is schematically illustrated in the attached drawings. Brief description of the figures

Figure 1. X-ray diffraction pattern of powders of the sample containing 10vol% of the ionic liquid BmimPF6 and prepared with an extraction speed of the object to be coated of 300 mm/min.

Figure 2. SEM images at different magnifications (500 c and 1000 x) of the specimens prepared with 10vol% of the ionic liquid BmimPF6 at different extraction rates indicated in brackets. In the order 2a and 2b (300 mm/min), 2c and 2d (500 mm/min), 2e and 2f (1450 mm/min).

Figure 3. TG (solid line) and dTG (dashed line) curves of the silica-coated samples (R_0) and with the antibacterial coating containing 10vol% of ionic liquid (FM0) respectively.

Figure 4. Plates of C+ positive controls (top) and C- negative controls immediately after inoculation (bottom) of specimen R_10a (coating at 10vol% of the ionic liquid BmimPFe). No bacterial growth is observed in the C+ plates, while the C- plates are uncountable. Figure 5. Negative control plates C- immediately after inoculation of the specimens R_1 Od, R_1 Oe and R_1 Of (coating with 10vol% of Ionic Liquid).

The plates are uncountable.

Figure 6. C- negative control plates immediately after inoculation of the specimens R_1 Og, R_1 Oh and R_1 Oi (coating at 10vol% of the ionic liquid BmimPFe). The plates are uncountable. Figure 7. Positive control wash medium plates (C+) for the specimens at decreasing concentrations of the ionic liquid BmimPF6 (10, 7.5, 5, 2.5, 1 and 0vol% respectively). Bacterial growth is observed only in the absence of an antibacterial molecule (specimen R_0, uncountable plate). Figure 8. C- negative control plates immediately after inoculation of the specimen at 1vol% of the ionic liquid BmimPF6 (R_1), using an inoculum concentration equal to 2 x 10 ⁴ cells/mL and an inoculum quantity per specimen equal to 200 mI_. The plates are countable.

Figure 9. Cell viability of fibroblasts at different growth times (24, 48 and 96h) in multiwell without specimens (NoGlass), uncoated specimens and specimens with 10vol% coating (coated).

Detailed description

As used herein, "ionic liquid" with its acronym LI, indicates a salt comprising a cation and an anion. The salt is generally liquid at room temperature with a melting point or melting range of less than about 100°C. Typically the ionic liquids are based on phosphonium, imidazolium or pyridinium and are alkyl-substituted.

Particularly preferred is 1-Butyl-3-methylimidazole hexafluorophosphate (hereinafter BmimPFe). A new approach has been developed by the inventors for controlling the contamination of surfaces constantly touched by a single or a large number of users. In particular, a coating with antibacterial properties was created able to overcome the limits still presented by current technologies.

The inventors have developed a sol-gel coating with antibacterial activity that can be applied on various surfaces with a high rate of contamination such as, by way of non-limiting example, handles, touch-screens, mobile phones, tablets and devices used in the medical-health field.

The coating according to the invention, unlike the products known to date which only lead to a reduction in the microbial load, has a high bactericidal activity which allows the microbial load to be zeroed on the treated surfaces. The coating components are perfectly mixed together and the final product obtained is totally colorless and transparent, adapting to the application on different types of materials, which will favor its subsequent use both in the field of mobile telephony and computers, and in the medical one - Sanitary.

The coating according to the invention consists of a composition based on silica and an ionic liquid, prepared with the sol-gel technique and having antibacterial properties.

The coating can be applied with any known technique, such as dipping, brush, spray. The technique particularly suitable is dip-coating. The thickness obtained is between 10 and 70 pm.

The dip-coating technique is often used to lay thin films on a solid substrate by immersing the same substrate in a container containing the coating mixture, followed by subsequent removal by extraction of the solvent and finally drying.

The starting mixture is prepared through the sol-gel technique by which it is possible to produce a wide variety of inorganic networks using silicon alkoxides as precursor monomers. The term "silicon alkoxides" means organic silicon oxides of the Si (OR)4type, where R indicates the generic organic group. During the gelation process the silicon alkoxide solution is subject to reactions of:

1) hydrolysis

Si (OR)4 + H 0 ^± Si (OR) ₃ (OH) + ROH and 2) condensation

Si (OR)4 + Si (OR) ₃ (OH) «= ^► (OR) ₃ Si - O - Si (OR) ₃ + ROH Si (OR) ₃ (OH) + Si (OR) ₃ (OH) (OR) ₃ Si - O - Si (OR) ₃ + H ₂ 0

Preparation of the sol-gel: a mixture of the precursors of the coating is prepared (compound containing silicon, inorganic acid and ionic liquid), mixing under stirring at room temperature, and according to the following order, the compound containing silicon, inorganic acid and the ionic liquid. Once the silicon-containing compound is stirred in the beaker, the inorganic acid and then the ionic liquid are immediately added. The mixture is stirred successively for 3 hours at the rate of 10Orpm.

The synthesis process involves two simple steps: mixing an alkoxysilane such as tetraethyl orthosilicate (TEOS) with an ionic liquid under acid hydrolysis conditions to form a colloidal solution in the presence of an inorganic acid that promotes hydrolysis of the compound containing silicon by acid catalysis; adjust the pH to 1 - 2. The acidic reaction medium is preferably hydrochloric acid. Neutralizing agents are not used. At the end of the 3 hours, the coating is applied to the surface to be treated. According to the dip-coating process, 3 steps are required:

(i) the specimen to be coated is immersed inside the container containing the gel, it is left for 1 minute, sufficient time to adhere completely to the specimen;

(ii) the specimen is extracted using a controlled speed (on which the thickness of the coating depends, the thickness being greater using high extraction speeds);

(iii) the specimen dries.

The drying of the coating can take place, for example, in natural conditions, leaving the product coated in the air at room temperature for at least 2-3 days (depending on the temperature and humidity conditions of the environment). A possible alternative could be drying in a stove at a temperature not higher than 50°C for a time sufficient to avoid any cracking of the coating.

The gelling of the sol particles will begin during the 3 hours of mixing and will continue over time until complete drying. The viscosity of the resulting gel can be easily controlled by adjusting the pH and the times between the two simple synthesis steps. This is a simple synthesis technique of a few hours that does not require purification. Thanks to this simplicity, large- scale production of the mixture is possible. The process is a synthesis technique that does not use organic solvents. The idea behind the invention is to confine the ionic liquid in a silicon oxide lattice with a relatively simple process, such as a sol-gel reaction.

In sol-gel processes the liquid phase is removed by evaporation and the solid obtained has a porous matrix. Flowever the presence of the ionic liquid allows to consider the gel as a stable solid-liquid structure called ionogel.

The matrix obtained is stable to solvents and to heating.

In the following, reference will be made to a preferred, non-limiting embodiment of the scope of the invention and to the relative application results.

According to the present invention, decreasing concentrations of a ionic liquid selected from the ionic liquids based on imidazole, pyridinium, pyrrolidinium, piperidinium, quaternary ammonium, preferably 1 -Butyl-3- methylimidazole hexafluorophosphate (hereinafter BmimPFe) have been added to TEOS (precursor of silica - tetraethyl orthosilicate) and HCI 37% by weight with the function of acid catalyst, so as to determine its minimum effective concentration. A series of syntheses was therefore carried out in which the concentration of BmimPFwas varied6 (10, 7.5, 5, 2.5 and 1vol%)as described in the table below. The reactants making up the starting mixture were left to stir (magnetic stirring) inside a Teflon beaker for 3 hours at the speed of 100 rpm.

Following the formation of the gel, the specimens consisting of coverslips with a size of 22 mm x 22 mm less than one mm in thickness were previously coated, previously subjected to cleaning and degreasing treatment before coating. The above treatment can be carried out with the 4 steps indicated below to be carried out in the following order:

First washing to eliminate grease residues on the surface, for example with dishwashing detergent - second washing with distilled water third washing with acetone

Drying, for example in an oven at 40°C for 24h.

The subsequent coating process can be schematized in three essential phases: - Immersion. The specimens were immersed in the starting mixture at constant speed.

- Permanence in contact with the solution. The specimens remained completely immersed in the starting mixture for 1 minute so as to allow the adhesion of the coating material. - Extraction. The specimens were extracted from the mixture at constant speed. The faster the extraction takes place, the greater the thickness of the final deposit obtained. In this regard, 4 different extraction speeds were tested: 200, 300, 500 and 1450 mm/min.

Finally, the coated specimens were left to air dry for a few days protected from dust.

COATING CHARACTERIZATION

The following are the results of the characterizations relating to the coated specimens, identified by the initials R followed by the concentration of BmimPF6 (expressed in volume%) X-ray powder diffraction (XRPD) and Scanning Electron Microscopy (SEM)

X-ray powder diffraction (XRPD) was performed by operating with the Seifert X3000TT diffractometer (X-ray laboratory of the DSCG), which uses CuKa radiation and in which the diffracted rays are monochromatized through the use of a graphite crystal placed in front of the detector (scintillation counter). Intensity data were collected in close- step step-scan mode exploring an angular range from 10 to 80° 2Q. The accumulation time for each step has been chosen from time to time in order to optimize the signal/noise ratio. The results obtained from the X- ray analysis show for all the specimens analyzed the presence of the typical halo of amorphous silica, deriving from the hydrolysis and condensation of the TEOS in turn responsible for the formation of the gel network, within which it is hypothesized BmimPF6 is trapped during gelation and subsequent drying (figure 1).

In support of the above hypothesis, the SEM images (figure 2) show the formation of a porous silica coating hosting the BmimPF6 inside the pores and the grain edges. This structure can be found in all the samples analyzed, regardless of the speed of extraction of the specimen during the coating phase and the percentage of BmimPF6 present, as evident in the micrographs shown in figure 2. The above figures show the micrographs relating to the specimens coated with different extraction speeds (300, 500 and 1450 mm/min) and at concentrations of BmimPF6 10vol% respectively. The micrographs were obtained using a Dual Beam FEI NovaNanolab 600 instrument.

Thermogravimetric analysis (TGA). The presence of the BmimPF6 inside the coating was also confirmed by the thermogravimetric analysis, carried out using the STA 6000 Perkin- Elmer instrument at the DSCG. The set heating rate was 10°C/min in the range of 25 to 850°C in a controlled atmosphere of oxygen with a flow of 40 mL/min.

As an example, in the figure 3 shows the thermograms TG and the relative derivative curves dTG of the sample without BmimPF6 (R_0) and of the sample containing 10% of BmimPF6 (R_10). Both samples were prepared with an extraction speed of 300 mm/min.

As can be seen from the thermograms, both samples show a first loss in mass within 150°C due to the elimination of residual solvent. This loss is more evident in sample R_0 and this variability between the different samples, which is difficult to control, depends on various factors including the humidity level of the environment and the time that elapses between filling the sample holder and starting the analysis. Thermal. In the case of sample R_10, a second mass loss in the range between 150 and 400°C is also observed, not present in sample R_0 and attributable to the decomposition of BmimPF6, in accordance with what is reported in the literature. The differences between the two samples are more evident in the dTG curves, whose peaks are generally able to reveal small variations in the slope that may not be visible in the TG curves. Antibacterial activity and Calculation of the Minimum Bactericidal Concentration (CMB)

Experimental protocol for the evaluation of antibacterial properties

The activity of the coated specimens using an extraction speed of 300 mm/min, has been tested against the bacterium Escherichia coli ATCC 8739 (GRAM-). For this purpose, an experimental protocol has been defined using the Japanese standard JISZ2801 Antibacterial products - Test for antibacterial activity and Efficacy of 2010 and the corresponding ISO 22196: 2011 standard as starting points. Since the developed coating can be classified as porous material, it was necessary to make some changes to the experimental protocol indicated by the two standards, as these are aimed at evaluating the antibacterial activity of non-porous materials such as plastics, metals, ceramics, etc. These changes were made in compliance with the changes granted by the two standards. The following are the four fundamental phases of the protocol used.

Phase 1. Pre-experiment (duration: 2 days)

The first phase of the experimental protocol involves the preparation of the necessary material for the following phases, namely the mediums (nourishing medium and washing medium Soybean Casein Lecithin Polysorbate Medium (SCDLP), the solutions (phosphate buffer solution, saline solution), the nourishing agar and the agar count (used for the preparation of the plates) At the end of this first step, the starting bacterial stock is reactivated, which after hydration in LB medium is plated on blood agar, in which growth will occur after incubation at 37°C for 24 h. Finally, we proceed with the passage of one of the colonies grown in blood agar, on a plate of nourishing agar, with respective growth after incubation at 37°C for 24 h. Phase 2. Experiment (duration: 4 days).

The second phase of the protocol is divided into 4 days, each of which involves the execution of different steps of the experiment, described below.

Day 1. During day 1 , the inoculum of 9 specimens (6 negative controls C- and 3 positive controls C+) is carried out with a known quantity of nourishing medium containing an inoculum of bacterial cells which must be between 6.2 x 10 ³ and 10.0 c 10 ⁵ cells/cm ² .

In the following, 3 of the C- and 3 C+ specimens previously subjected to inoculation are incubated at 37°C for 24h. The C- specimens used are coverslips having a coating free of BmimPF6, while C+ are coverslips having a coating containing BmimPF6. The other 3 C- specimens are instead washed immediately with the SCDLP washing medium, which is recovered and diluted (5 serial dilutions 1 :10 by volume). Finally, the plates of the washing medium and of the dilutions are prepared, subsequently incubated at 37°C for 24h.

Day 2. On day 2 we proceed with the count of the colonies grown in the plates of the washing medium and the dilutions of the 3 C- washed the previous day. Furthermore, the 3 C- and 3 C+ specimens inoculated and incubated the previous day are washed with SCDLP medium. The washing medium is recovered, diluted through 5 serial dilutions and plated. The plates are incubated at 37°C for 24h.

Day 3. On day 3, the colonies grown in the plates of the washing medium and the dilutions of the 3 C- and 3 C+ specimens washed the previous day are counted.

Day 4. Finally, on day 4 the numerical analysis of the results is carried out, as reported in the Japanese standard JISZ2801 and in the respective ISO 22196: 2011 standard. First of all, the number of bacteria is determined by using the following formula

N = (C x D x V)/A

Where N = number of bacteria recovered per cm ² of the specimen (in our case being a porous material, it must be expressed in n° of bacteria recovered per g of coating).

C = average of the colonies counted in duplicates

D = dilution factor

V = volume in ml_ of washing medium SCDLP used to wash the inoculum from the specimens A = area of the film expressed in grams of coating as it is a porous material Subsequently, the conditions of validity of the test are verified, for which:

1) the logarithmic value of the number of live bacteria recovered after inoculation in the C- specimens must satisfy the following equation: (Lmax-Lmin)/(Lmedio) < 0.2

Where:

Lmax = Logio of the maximum number of bacteria found in the sample Lmin = Logio of the minimum number of bacteria found in the sample Lmedio = Logio of the average number of bacteria found in the sample

2) The number of bacteria present immediately after inoculation of the C- sample must be between 6.2 x 10 ³ cells/cm ² and 2.5 c 10 ⁴ cells/cm ² ; while after 24h, it must not be less than 62 cells/cm ² .

In our case, our coating being porous, these reference values cannot be considered reliable, as they relate to non-porous materials. When the conditions of validity of the test are met, the antibacterial activity of the material in question (antibacterial coating) can be calculated, according to the following equation:

R = (Ut - U0) - (At - U0) = Ut - At Where

R = antibacterial activity

U0 = average of Logio of the number of bacteria expressed in cells/grams, obtained from C- immediately after inoculation

Ut = average of Logio of the number of bacteria expressed in cells/grams i obtained from C- after 24h of incubation

At = mean of Logio of the number of bacteria expressed in cells/grams obtained from C- after 24h of incubation In order for the material to be considered antibacterial, thevalue R must be > 2

Evaluation of antibacterial activity Identification of the Minimum Bactericidal Concentration (CMB) and optimal working conditions.

On the basis of the aforementioned protocol, the antibacterial activity (R) of the specimens prepared with decreasing concentrations of BmimPF6 was assessed in order to identify the Minimum Bactericidal Concentration (CMB).

During the definition of the protocol, both the quantity of bacteria of the starting inoculum (cells/mL) and the quantity of inoculum per specimen (ml_) were varied. The first tests were carried out on the specimens with a coating having 10% of BmimPF6 as shown in the table below:

^* the values belong to the limit intervals indicated in the JISZ2801 and in the ISO 22196:2011 (6.2x10 ³ - 10.0 x 10 ⁵ cell/mL) All the tests showed zero bacterial growth on the plates relating to the C+ specimens (slides coated with antibacterial coating). As an example, the images relating to the plates of the sample R_10a are shown (figure 4). Due to the abnormal growth of bacteria in the C- specimens (slides coated with the BmimPFfree coating6-), however, it was necessary to use decreasing concentrations of inoculum (cells/mL) and quantity of inoculum per specimen (ml_), the results are shown in figures 4, 5 and 6.

The above figures show the images relating to the washing and dilution plates 1 of the C- immediately after inoculation for the specimens R_10a (figure 4), R_10d, R_10e and R_1 Of (figure 5) , R_10g, R_10h and R_10i (figure 6). Although the growth in the plates relating to the C+ specimens is zero, demonstrating the antibacterial activity of the same coating and despite the reduction in the concentration and quantity of inoculum, the inconsistency of the plates relating to the C- specimens makes it impossible to overcome the conditions of validity of the test as indicated in the Japanese standard JISZ2801 and ISO 22196: 2011

The results obtained from the above tests confirmed the bactericidal action of the coating containing 10% of BmimPFinoculum6 at concentrations (cells/mL) and decreasing quantity of inoculum per specimen, but did not however allow to identify the working conditions ideal for the validation of the test and the subsequent identification of the antibacterial activity value (R) due to the inconsistency of C-. It was therefore decided to continue with the search for the optimal concentration and quantity of inoculum to meet the validity conditions of the test in accordance with the standards, in parallel with the identification of the CMB.

In this regard, using an inoculum concentration of 2.5 c 10 ⁴ cells/mL and an inoculum quantity of 400 ml per specimen (conditions used with the R_10g specimen, which gave the best results in the previous series of tests), the antibacterial activity of the specimens having a decreasing concentration of BmimPF6, was tested as shown in the following table: All the specimens analyzed showed 100% bactericidal action, thus demonstrating that even at low concentrations of BmimPF6 (1%) no growth is observed (figure 7), defining 1% of BmimPF6 as CMB. Once again, however, due to the incountability of the C- plates immediately after inoculation, it was not possible to satisfy the validity condition of the test, according to the provisions of the protocol, for which:

(Lmax-Lmin)/(Lmedio) < 0.2

Subsequently, further tests were carried out, using only the 1% (CMB) specimens, which we remember to be the minimum concentration of BmimPF6 having 100% bactericidal activity. Therefore, the optimal inoculum concentration, found to be 2.0 c 10 ⁴ cells/mL and the optimal amount of inoculum per specimen, found to be 200 ml_, were identified and defined. In these conditions, in fact, the plates relating to the C- are countable, as can be seen in figure 8 which, by way of example, shows the images relating to C- washes immediately after inoculation. It was therefore possible to demonstrate the bactericidal action of the coating at decreasing concentrations of BmimPF6 and to satisfy the validity condition of the test for which

(Lmax-Lmin)/(Lmedio) < 0.2 A value of 0.09 was in fact obtained.

As for the other validity conditions, i.e. the number of bacteria recovered immediately after the inoculum and those recovered after 24 hours, starting from the above concentration (2.0 ^c 10 ⁴ cells/mL) and quantity of inoculum (200 ml_ per specimen ), for the 1% coating of BmimPF6 an average number of bacteria equal to 1.02 c 10 ⁶ cells/g of coating was obtained immediately after inoculation of the sample C- and equal to 6.44 x 10 ⁵ cells/g of coating after 24 hours.

From the results obtained it was therefore possible to define that the CMB is equal to 1% of BmimPF6 with an antibacterial activity R = 2.49.

Since the R value is greater than 2, the coating has antibacterial activity.

The table below summarizes the results obtained from the numerical analysis relating to the test conducted on specimen R_1, using an inoculum concentration equal to 2.0 ^c 10 ⁴ cells/mL and a quantity of inoculum per specimen equal to 200 pL.

^* Number of cells per gram of coating,

^* Mean number of cells per gram of coating immediately after inoculation of the untreated sample at t=0h,

^*** Mean number of cells per gram of coating for the untreated sample at t=24h.

The antibacterial activity of specimen R_1 was also tested against thebacterium Staphylococcus Aureus ATCC6538P(GRAM +), using the optimal operating conditions (inoculum concentration equal to 2.0 c 10 ⁴ cells/mL and an inoculum quantity per specimen equal to 200 ml_). It was possible to satisfy the validity condition of the test, in accordance with the protocol, whereby

(Lmax-Lmin)/(Lmedio) < 0.2

A value of 0.06 was in fact obtained.

An average number of bacteria recovered immediately after the inoculum of the C- sample was obtained equal to 3.47 x 10 ⁶ cells/g of coating and equal to 2.09 c 10 ⁶ cells/g of coating after 24 hours.

From the obtained results, it was therefore possible to confirm that the CMB is equal to 1% of BmimPF6 also for the gram positive S. aureus with an antibacterial activity R = 2.69. Since the R value is greater than 2, the coating also exhibits antibacterial activity against the positivegram S. aureus.

The table below summarizes the results obtained from the numerical analysis relating to the test conducted:

* Number of cells per gram of coating

** Average number of cells per gram of coating immediately after inoculation of the untreated sample at t = Oh *** Average number of cells per gram of coating of the untreated sample at = 24h

Preliminary tests of cytotoxicity.

Preliminary cytotoxicity studies on fibroblasts showed cell viability at different growth times (24, 48 and 96h) in the presence of the 10vol% coating of BmimPF6 similar to that of the controls (multiwell without specimens and uncoated specimens) as shown in figure 9.

BIBLIOGRAPHY

[1 ] G. E. Flores et al., “Microbial Biogeography of Public Restroom Surfaces,” vol. 6, no. 11 , 2011.

[2] F. Schaumburg, R. Kock, F. H. Leendertz, and K. Becker, “Airport door handles and the global spread of antimicrobial-resistant bacteria: a cross sectional study,” Clin. Microbiol. Infect., 2016.

[3] G. Anderson and E. A. Palombo, “Microbial contamination of computer keyboards in a university setting,” Am. J. Infect. Control, vol. 37, no. 6, pp. 507-

509, 2009.

[4] S. A. Boone and C. P. Gerba, “The Prevalence of Human Parainfluenza Virus 1 on Indoor Office Fomites,” pp. 41^6, 2010.

[5] S. A. Boone and C. P. Gerba, “The occurrence of influenza A virus on household and day care center fomites,” pp. 103-109, 2005.

[6] R. Duszak. Jr, B. Lanier, and J. A. Tubbs, “Bacterial Contamination of Radiologist Workstations JACR, vol. 11 , no. 2, pp. 176-179, 2014. [7] C. P. Gerba, A. L. Wuollet, P. Raisanen, G. U. Lopez, and K. Words, “American Journal of Infection Control Bacterial contamination of computer touch screens,” AJIC Am. J. Infect. Control, vol. 44, no. 3, pp. 358-360, 2016.

[8] S. F. Bloomfield, A. E. Aiello, B. Cookson, and C. O. Boyle, “The effectiveness of hand hygiene procedures in reducing the risks of infections in home and community settings including handwashing and alcohol-based hand sanitizers,” 2007.

[9] M. Canales, G. Craig, J. Boyd, M. Markovic, and R. Chmielewski, “Dissemination of Pathogens by Mobile Phones in a Single Hospital,” vol. 7, no. 3, 2017.

[10] P. Pal et al., “Keypad mobile phones are associated with a significant increased risk of microbial contamination compared to touch screen phones,” vol. 14, no. 2, pp. 65-68, 2013.

[11] P. Information, “Antimicrobial Corning ® Gorilla ® Glass Product Information,” 2014.

[12] “http://www.agion-tech.com.”

[13]“http://www.nanotechproject.org/inventories/consume r/browse/products/ Ig antibacterial mobile phone.” .

[14] “http://www.ethical-junction.org/blogs/2010/09/13/anokimob i- launchesecoskin%E2%84%A2-iphone-4-cover-with-antibacterial-p rotection/.” .

[15] J. C. Ireland, P. Klostermann, E. W. Rice, and R. M. Clark, “Inactivation of Escherichia coli by Titanium Dioxide Photocatalytic Oxidation,” vol. 59, no. 5, pp. 1668-1670, 1993.

[16] S. Noimark, C. W. Dunnill, and I. P. Parkin, “Shining light on materials — A self-sterilising revolution Adv. Drug Deliv. Rev., 2012.

[17] K. Page, A. Correia, M. Wilson, E. Allan, and I. P. Parkin, “Journal of Photochemistry and Photobiology A : Chemistry Light-activated antibacterial screen protectors for mobile telephones and tablet computers,” "Journal Photochem. Photobiol. A Chem., vol. 296, pp. 19-24, 2014.