Demonstrating the In Vitro and In Situ Antimicrobial Activity of Oxide Mineral Microspheres: An Innovative Technology to Be Incorporated into Porous and Nonporous Materials

The antimicrobial activity of surfaces treated with zinc and/or magnesium mineral oxide microspheres is a patented technology that has been demonstrated in vitro against bacteria and viruses. This study aims to evaluate the efficiency and sustainability of the technology in vitro, under simulation-of-use conditions, and in situ. The tests were undertaken in vitro according to the ISO 22196:2011, ISO 20473:2013, and NF S90-700:2019 standards with adapted parameters. Simulation-of-use tests evaluated the robustness of the activity under worst-case scenarios. The in situ tests were conducted on high-touch surfaces. The in vitro results show efficient antimicrobial activity against referenced strains with a log reduction of >2. The sustainability of this effect was time-dependent and detected at lower temperatures (20 ± 2.5 °C) and humidity (46%) conditions for variable inoculum concentrations and contact times. The simulation of use proved the microsphere’s efficiency under harsh mechanical and chemical tests. The in situ studies showed a higher than 90% reduction in CFU/25 cm2 per treated surface versus the untreated surfaces, reaching a targeted value of <50 CFU/cm2. Mineral oxide microspheres can be incorporated into unlimited surface types, including medical devices, to efficiently and sustainably prevent microbial contamination.


Introduction
Recent advances in antimicrobial material technologies are revolutionizing infectious disease management by inhibiting biofouling, contamination, and infection [1][2][3]. The antimicrobial surface activity is exerted (i) by killing the micro-organisms and/or (ii) by preventing their adhesion to inanimate surfaces [1,4], leading to the inhibition of microbial survival, growth, and potential biofilm formation [1,4]. Available antimicrobial surfaces rely on the modulation of surface topography, wettability, and chemistry [4]. Conventional surfaces can be (1) biocides for contact-killing [5] and (2) biocides for release-killing [6,7].
The biocides for release-killing rely on the biocidal effect of metals such as silver (Ag), copper (Cu), gold (Au), titanium (Ti), and zinc (Zn) [6,7]. The nanocomposites and nanoparticles (NPs) of these metal oxides, such as CuO, Fe 3 O 4 , ZnO, MgO, and TiO 2 , demonstrated better antimicrobial properties than the parent metal's NPs [7][8][9][10][11][12][13]. They primarily act as with the containers used in variable sectors, including the pharmaceutical and med fields. The mineral microspheres are authorized additives for pharmaceutical contain in the European, United States, and Japanese Pharmacopeia [58]. This study aims demonstrate, according to the ISO 22196:2011, JIS Z 2801, ISO 20743:2021, and NF S 700:2019 standards, the antimicrobial activity (i) of different types of surfaces with add oxide mineral microspheres in vitro, (ii) the sustainability of this effect under real-life c ditions, worst-case scenarios, and (iii) in situ in two different types of premises (ISO room and high school self-service) under real-life use conditions.

In Vitro Assays
Methodologies described in the ISO 22196:2011 (JIS Z2801:2010) [64], ISO 20743:2 [65], and NF S90-700:2019 [66] standards were applied with some modifications to stu the antibacterial activity of microspheres on nonporous and porous surfaces. The te were conducted three times on a minimum of three samples.

Tested Surfaces
Tested materials and reference materials are described in Table 1 according to standard applied.
Regarding their treatment before assay: -For nonporous surfaces, the test pieces (untreated=control=C and treated=assay were prepared by immersion in ethanol 70°, rinsed with distilled sterile water, a then dried under a microbiological safety cabinet before the test according to I 22196:2011 and NF S90-700:2019; -For porous surfaces, the test pieces (untreated=control and treated=assay) were in a round shape (diameter: 3 and 8 cm) and sterilized (121 °C, 15 min) according the ISO 20473:2021 standard.

In Vitro Assays
Methodologies described in the ISO 22196:2011 (JIS Z2801:2010) [64], ISO 20743:2021 [65], and NF S90-700:2019 [66] standards were applied with some modifications to study the antibacterial activity of microspheres on nonporous and porous surfaces. The tests were conducted three times on a minimum of three samples.

Tested Surfaces
Tested materials and reference materials are described in Table 1   Tests were repeated three times during the same assay. Controls (C0 and C24 h) were tested using test pieces without microsphere incorporation (C) to check the lack of antimicrobial activity and to calculate the log reduction.

Validation
Validation of each assay followed the indication of the ISO 22196:2011 and ISO 20473:2021 standards, considering that the logarithmic value of the number of viable bacteria recovered immediately after inoculation (T0) from the untreated pieces shall satisfy the following equation: where -Lmax is the maximum logarithmic number of viable bacteria; -Lmin is the minimum logarithmic number of viable bacteria; -Lmean is the average logarithmic number of viable bacteria of three untreated test pieces.
Validation of each assay followed the indication for NF 90-700 standard, considering that the suspension is in the range 5.18-5.70 log, that the logarithmic value of the number of viable bacteria for the control of dilution-neutralization is not more than 2 log from the deposition with a difference ≤0.3 logs between the two controls of dilution-neutralization and (Lmax − Lmin)/(Lmean) ≤ 0.3 for the three values obtained at T0 for the reference surface during the same assay.

Antimicrobial Surface Activity Calculation
The antibacterial activity (R) represents the logarithmic reduction/cm 2 in the number of bacteria between the Control and Assay surfaces after 24 h of contact according to the following matrix: R is the logarithmic reduction; -C0 is the average of logarithmic numbers (CFU per cm 2 ) of viable micro-organisms recovered from the untreated pieces (controls) immediately after inoculation; -C24h is the average of logarithmic numbers (CFU per cm 2 ) of viable micro-organisms recovered from the untreated test pieces (controls) after 24 h; -A24h is the average logarithmic numbers (CFU per cm 2 ) of viable micro-organisms recovered from the treated test pieces (microsphere-added) after 24 h.

In Vitro Antimicrobial Surface Activity Characterization
Antimicrobial surface activity tests, as previously described, were performed in different assay conditions to characterize the antimicrobial properties of the innovative process of homogeneous incorporation of oxide mineral microspheres into materials.

Spectrum of Activity
In the first step, the antimicrobial activity of the microsphere-added (2.5% w/w) nonporous surfaces was tested against (1) the two reference bacteria recommended by the ISO 22196:2011 standard (E. coli CIP 53.126 and S. aureus CIP 4.83) and expanded to antibioticresistant bacteria, and (2) against S. aureus CIP 4.83 recommended by NF S90-700:2019. The antimicrobial activity of microsphere-added (2.5% w/w) porous surfaces was examined against S. aureus CIP 4.83, the reference bacteria recommended by the ISO 20743 standard.
Nonporous Surfaces: Impact of Contact Time, Relative Humidity, and Temperature Antimicrobial activity assays were undertaken by varying the contact time (0.5-24 h) between the plastic as well as other nonporous surfaces and E. coli CIP 53.126 to determine the minimum time required to obtain a significant effect using different compositions and forms of plastic surfaces (polyethylene (PE), polypropylene (PP), and PP film) containing various concentrations of microspheres (0.5-4% w/w).

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The temperature varied from 36 ± 1 • C to 20 ± 2.5 • C and 22 ± 2 • C and the humidity from >90% to between 24% and >80%; - The contact time varied between 30 min and 1-6 h depending on the tested surface; - The size of the inoculum ranged between 10 2 CFU/cm 2 and 10 5 CFU/cm 2 ; - The volume of the inoculum was also tested (50 µL versus 400 µL in the standard) to evaluate the drying time impact.
According to the conditions NF S90-700:2019 standard and following the method of ISO 22196:2011, the testing conditions against S. aureus CIP 4.83 were changed to 20 ± 2.5 • C at an RH of 50% and a contact time of 24 h.

Simulation of Use
A worst-case scenario simulation test undertaken under indicated chemical, physical and mechanical conditions depending on the material is finally used to evaluate the stability of the antimicrobial activity in vitro. The antimicrobial surface activity of the microsphereadded plastic and other nonporous surfaces was checked after 7 and 12 weeks at 50 • C according to the ASTM F 1980-7 standard (E. coli CIP 53.126 according to the ISO 22196:2011 standard). The antimicrobial activity was also tested after 100 washing cycles with selected detergents/disinfectants frequently used in hospitals. The detergents/disinfectants were diluted at the recommended doses of use in hospitals: Sodium hypochlorite solution (NaClO) at 0.9%, Primactyl (dodecyl dimethyl ammonium, CAS: 7173-51-5, 2.5 g/kg, ethyl alcohol, CAS: 64-17-5, 125 g/kg-propane-2-ol, CAS: 67-63-0, 104 g/kg.) à 2.4%, isopropyl alcohol, Surfanios Premium (51 mg g −1 of N-(3-aminopropyl)-N-dodecylpropane-1,3diamine and 5 mg g −1 of didecyldimethylammonium chloride, Laboratoires Anios, France) diluted at 0025%, and ready-to-use product.

In Situ Assays
Two tests were conducted in public places to examine the CFU/25 cm 2 of surfaces (high-touch areas) covered versus noncovered with treated polyethylene film. We compared the covered treated surfaces with none to represent the actual in situ characteristics of high-touch areas. (Table 2) Cleaning of the tested surfaces was not undertaken prior to sampling to avoid any interference and inaccuracy in the results.
Sampling between 8 h and 9 h AM Maintain the cleaning disinfection process with Surfanios premium (monthly) Cleaning and disinfection of the tested surfaces were not undertaken prior to sampling to avoid any interference and inaccuracy in the results. Cultures were carried out once (11 June 2020) at the end of the last shift

Testing conditions
The temperature and humidity were recorded. Depending on the season, the temperature varied between 20.8 • C to 27 • C and the humidity between 25% and 63% Not recorded The first test was undertaken in an ISO 8 cleanroom during regular working hours. Sampling was performed according to ISO 144698-2 standard on five high-touch areas, including a bench, door handle, mouse tablet, remote control device, and hydroalcoholic gel button. The microbial burden was examined by taking samples weekly for seven consecutive weeks, every Monday or Tuesday from April to June 2021. Routine cleaning and disinfection procedures were maintained. Count-tact ® plates (Biomérieux) were used with the corresponding applicator (500 g precision during 10 s) to obtain samples between 8:00 a.m. and 9:00 a.m. during regular working hours and under normal environmental conditions of temperature and humidity (temperature between 20.8 • C to 27.0 • C and relative humidity between 25% and 63%). Cleaning of the tested surfaces was not undertaken before sampling to avoid any interference with the accuracy of the results. The incubation time was 72 h at 32.5 • C. The quantitative analyses aimed to determine the colony count of aerobic mesophilic microorganisms, including yeasts and some moulds, expressed as CFU/25 cm 2 .
The second test was conducted in a high school on high-touch surfaces. The microbial burden was examined at normal environmental conditions of temperature and humidity. A One-time sampling was performed on high-touch critical zones, including the table surface and the entrance door handle interior and exterior, just after the last service of the day and before the current cleaning. Cultures were undertaken using a petri dish type PCA and swabbing the indicated surface (25 cm 2 ). Incubation time was five days at 30 • C. The quantitative analyses, expressed as CFU/25 cm 2 , aimed to measure the colony count of aerobic mesophilic microorganisms, including fungi. Results are expressed in percentages of CFU/25 cm 2 reduction per type of surface using the following formula: Total number o f CFU 25cm 2 in sur f aces not covered with polyethylene adhesive f ilm − total number o f CFU 25cm 2 in sur f aces covered with polyethylene adhesive f ilm totalnumberof CFU 25cm 2 insurfacesnotcoveredwithpolyethyleneadhesivefilm × 100

Statistical Analysis
Means and standard deviation (SD) were calculated at T0 and T24 to define log reduction, and the Student t-test was performed on the three independent experiments conducted for each tested sample treated with microspheres (test) versus untreated test pieces (control) according to the ISO 22196:2011, ISO 20473:2021 and S 90-700 standards. The paired sample t-test was undertaken to calculate the significance of the colony count reduction between surfaces treated with polyethylene adhesive film versus none covered surfaces on high-touch areas for in situ results. p < 0.005 was considered significant. The results show that the antimicrobial activity of different nonporous surfaces treated with microspheres is significant compared with untreated test pieces, with a log reduction R > 2. The nonporous treated surfaces included polyethylene film, polypropylene plates, latex gloves, and a beauty blender. The diversity of materials used demonstrates the efficiency of this technology applied to vast material types, including medical and pharmaceutical devices, biomaterial coatings, and fabrics used in the health settings, such as personal protective equipment (PPE) ( Table 3). The tests were conducted on S. aureus CIP 53.156 under ISO 20743 standard conditions at a temperature of 35 ± 1 • C, relative humidity (RH > 90%), contact time of at least 24 h, and an inoculum size of 10 6 CFU/mL. The results show that the antimicrobial activity (A) of different porous surfaces treated with microspheres is considered significant compared with untreated test pieces, with a log reduction A > 2 for the polycotton tested samples, over-gown polyethylene, and tested gloves, with a > 4 logs reduction. The porous-treated surfaces included nitrile gloves, latex gloves, and polyester and polycotton fabrics (thick and light) ( Table 4).

In Vitro Testing of the Antimicrobial Activity of Microsphere-Added Nonporous Surfaces under Real-Life Conditions
The tests were conducted on E. coli CIP 53.126 and S. aureus CIP 53.156 under the ISO 22196:2011 and NF S90-700:2019 standards by varying the temperature, RH, contact time, and inoculum size. The results show that the increase in the antimicrobial activity of different nonporous surfaces treated with microspheres is time-dependent, with a log reduction R > 2 starting after three hours according to the surface type (Table 5).

In Vitro Simulation of Use (Robustness)
The results of the worst-case scenario simulation tests undertaken under the JIS Z 2801:2010 and ISO 20743 standards indicate that the harsh chemical, physical, and mechanical conditions did not affect the antimicrobial activity of the surface-added microspheres that remained significant with a log reduction >2. (Table 6)

In Situ Testing in High-Touch ISO 8 Room Areas
The level of contamination of noncovered surfaces reached 2-3 logs at baseline. The highest colony count of surfaces without the treated polyethylene film was detected on the remote control (640 CFU/25 cm 2 ), followed by the door handle (570 CFU/25 cm 2 ), the bench (446 CFU/25 cm 2 ), mouse tablet (396 CFU/2 cm 2 ), and finally the hydro-alcoholic gel button (331 CFU/25 cm 2 ). The results show that the average percentage of CFU/25 cm 2 reduction in the levels of contamination between the surfaces covered with treated polyethylene adhesive film compared with noncovered surfaces exceeded 90% (p = 0.001). The antimicrobial effect was sustainable throughout the duration of the experiment (Figure 2). Details of the tests are shown in Supplementary Table S1.
The level of contamination of noncovered surfaces reached 2-3 logs at baseline. The highest colony count of surfaces without the treated polyethylene film was detected on the remote control (640 CFU/25 cm 2 ), followed by the door handle (570 CFU/25 cm 2 ), the bench (446 CFU/25 cm 2 ), mouse tablet (396 CFU/2 cm 2 ), and finally the hydro-alcoholic gel button (331 CFU/25 cm 2 ). The results show that the average percentage of CFU/25 cm 2 reduction in the levels of contamination between the surfaces covered with treated polyethylene adhesive film compared with noncovered surfaces exceeded 90% (p = 0.001). The antimicrobial effect was sustainable throughout the duration of the experiment (Figure 2). Details of the tests are shown in Supplementary Table S1.

In Situ Testing in High-Touch Public Zones (High School Self-Service)
The highest colony count of surfaces without the treated polyethylene film was detected on the interior door handle (>100 CFU/25 cm 2 ), followed by the table surface (79 CFU/25 cm 2 ) and the exterior door handle (41 CFU/25 cm 2 ). The difference in colony counts between surfaces covered with treated polyethylene film compared with noncovered surfaces was 72% (interior door handle), 54% (table surface), and 66% (exterior door handle). These results show that all samples from the treated polyethylene film-added surfaces reached the targeted values set in this experiment of <50 CFU/cm 2 per tested surface type. A total of 66.7% of noncovered high-touch surfaces had, at baseline, a microbial load higher than 50 CFU/cm 2 (Figure 3).

In Situ Testing in High-Touch Public Zones (High School Self-Service)
The highest colony count of surfaces without the treated polyethylene film was detected on the interior door handle (>100 CFU/25 cm 2 ), followed by the table surface (79 CFU/25 cm 2 ) and the exterior door handle (41 CFU/25 cm 2 ). The difference in colony counts between surfaces covered with treated polyethylene film compared with noncovered surfaces was 72% (interior door handle), 54% (table surface), and 66% (exterior door handle). These results show that all samples from the treated polyethylene film-added surfaces reached the targeted values set in this experiment of <50 CFU/cm 2 per tested surface type. A total of 66.7% of noncovered high-touch surfaces had, at baseline, a microbial load higher than 50 CFU/cm 2 (Figure 3).

Discussion
The recent COVID-19 pandemic, the spread of antimicrobial resistance [67,68], emerging and re-emerging disease outbreaks [69], and the variable global prevalence of healthcare-associated infections (HAIs) [70,71] mandate a drastic shift in the management of infectious diseases. The microbial contamination of high-touch surfaces in the community [72] and various surfaces and biomedical devices in the healthcare settings, such as catheters, medical instruments, and pharmaceutical outer and inner packages, are poten-

Discussion
The recent COVID-19 pandemic, the spread of antimicrobial resistance [67,68], emerging and re-emerging disease outbreaks [69], and the variable global prevalence of healthcareassociated infections (HAIs) [70,71] mandate a drastic shift in the management of infectious diseases. The microbial contamination of high-touch surfaces in the community [72] and various surfaces and biomedical devices in the healthcare settings, such as catheters, medical instruments, and pharmaceutical outer and inner packages, are potential sources of infection and the leading cause of morbidity, mortality, and healthcare expenditures [71,73]. Microbial persistence in the environment is affected by the conditions of temperature, humidity, initial titer, surface material, and the type of micro-organism, including its ability to form a biofilm [74,75]. These micro-organisms, including resistant strains, can survive for months and sometimes years on dry surfaces [74][75][76]. Studies demonstrated their viability on numerous materials [75], such as fabrics [77][78][79][80], plastics [80][81][82][83][84], steel [85][86][87][88], glass [89], and wood [45]. However, the survival of numerous species on inanimate surfaces remains poorly explored [75]. The efficacy of conventional and automated surface disinfection is influenced by multiple factors [90] and balanced by the exposure of humans to hazards as a result of direct contact with the skin and mucous membranes and airborne inhalation of the chemical residues [1,91] or oxygen-free radicals [92,93]. Recent advances in antimicrobial surfaces are game changers in the management of infectious diseases. However, their efficiency, sustainability and safety need to be demonstrated in vitro and in situ.
This study introduced a patented technology based on oxide mineral microspheres and showed that it could be incorporated into any porous and nonporous surfaces, allowing for a wide array of applicability in different settings and fields, including the pharmaceutical field. The results show the efficiency and sustainability of Pylote-patented oxide mineral microspheres in vitro (1)  The results showed that the oxide mineral microspheres incorporated into numerous materials exhibit significant antimicrobial activity in vitro under standard testing conditions (Log reduction > 2). The efficiency of this technology is related to two main features: (i) The homogenous dispersion of the ceramic particles in various materials owing to their spherical shape and being initially totally non-agglomerated; (ii) The close contact between the contaminating micro-organisms, the surface-embedded microspheres, and the microsphere-generated ROS. This feature was proven using a scanning microscope that showed a narrow distance between two microspheres ranging between 0.2 to 1 µm, which is nearly identical to the average size of bacteria (1 µm) [75].
In order to best evaluate the actual efficiency of the antimicrobial activity, previous studies recommend testing under real-life conditions and preferably setting a new ISO standard for more realistic test outcomes [94][95][96][97].

In Vitro Efficiency of the Antimicrobial Materials with Added Oxide Mineral Microspheres under Real-Life Conditions
The efficiency of porous and nonporous surfaces treated with oxide mineral microspheres was demonstrated in vitro under real-life conditions. When lowering the temperature and humidity and varying the inoculum size, the results showed that the log reduction per cm 2 increased proportionally to the contact time, where a >2 log reduction was seen starting after 3 h. Multiple studies showed that the conditions determined by the standard assays might not accurately replicate authentic conditions [6,94,[97][98][99][100]. However, our results proved the antimicrobial activity of porous and nonporous surfaces with added oxide mineral microspheres under real-life conditions. Campos (2016) compared the efficacy of antimicrobial thin-film surfaces and highlighted that the activity of these surfaces varies depending on the adapted testing protocol [98]. The effect of temperature and humidity on the antimicrobial activity of silver compared with copper alloy metals by challenging S. aureus (MRSA) was assessed under standards and real-life conditions [95]. The results showed that under JIS Z 2801 standard conditions, silver ion-containing materials showed effective antimicrobial activity (log reduction > 5), while no significant response was detected at lower temperatures and humidity levels, which was similar to the indoor environment conditions [95]. Ojeil (2013) showed that tested copper alloy surfaces showed different antimicrobial activity depending on the testing conditions [97]. Michels (2009) demonstrated the high efficacy of antimicrobial materials containing copper alloys, favouring its use in hospital settings under real-life conditions; silver-containing materials that showed high efficacy under standard testing conditions (JIS Z 2801) did not exhibit a significant antimicrobial effect at lower temperatures and humidity [95].

In Vitro Efficiency under Simulation of Use Tests
Published studies tested the activity of antimicrobial surfaces for a duration of more than 10 weeks to replicate the real-life effect of continuous use and demonstrate the sustainability of the antimicrobial effect [99,100]. These tests were perceived as impractical for routine use because they are time-and resource-consuming [6].
In our study, the oxide mineral-added microspheres underwent harsh mechanical, chemical, and physical manipulations that simulate worst-case scenarios (robustness and ageing). According to ASTM F 1980-7, physical ageing at 6 weeks at 50 • C is equivalent to 9 months at 23 • C, and 8 weeks at 50 • C equates to 12 months at 23 • C. The nonporous and porous materials were exposed in vitro to high mechanical ageing through 50 to 100 washes and chemical ageing using Isopropyl alcohol, bleach, and Surfanios Premium ® washes. The results demonstrated the robustness and sustainability of the antimicrobial activity over an extended time. Previous assays showed a long-lasting effect of 50 months [58].
The antimicrobial activity was high after exposure to various chemically and mechanically robust conditions. The demonstrated sustainability of the oxide mineral microspheres may be related to the mechanism of action that does not need the release nor consumption of the particles to generate a permanent self-decontaminating surface.
Here, we demonstrated the in vitro efficiency applied to bacteria with acquired resistance, including E. coli ESBL and MRSA, and above all, the maintenance of antimicrobial activity when microspheres were added to various porous and nonporous materials. At the same time, we showed a lasting effect according to the assay conditions: high RH and temperature (35 or 36 • C) during the contact time (ISO 22196) and after rapid drying, simulating microdroplet surface contamination and ambient RH and temperature (NF 90-700) for nonporous materials, as well as under ISO 20743 conditions for the porous ones.

Efficiency of the Antimicrobial Materials with Added Oxide Mineral Microspheres In Situ
With the availability of different types of antimicrobial surfaces in the market, manufacturers need to provide evidence of their product's in situ efficacy for end-user decisionmaking [54]. A literature search showed scarce studies of the efficiency of antimicrobial surfaces undertaken in situ and primarily in healthcare settings [101]. The in situ experiments targeted high-touch surfaces, considered a reservoir of pathogens, including GNB and GPB-susceptible and resistant strains, viruses, fungi, yeasts, and parasites [102][103][104][105]. These surfaces are commonly disinfected with chemical products that can often provide limited efficacy due to perpetual and instant recontamination [104,105]. The contaminating pathogens may originate from the hands of the personnel in the community and the patient's microbiota in healthcare settings [72,106]. Micro-organisms may survive cleaning for months and accumulate, providing an additional source of contamination [72].
In this study, in situ experiments were conducted under real-life conditions during working hours. The culture protocol and laboratory testing followed standardized antimicrobial testing to ensure the findings' validity, comparability, and reliability [54]. In the first in situ experiment (ISO 8 room), the tests were repeated seven times on five different hightouch areas (five with and five without the treated polyethylene film) at specific periods over three months, while in the second (high school self-service), they were carried out once on three high-touch areas (three with and three without the treated polyethylene film).
The results show a significant reduction in the CFU/25 cm 2 on high-touch surfaces covered with treated polyethylene film compared to the noncovered surfaces. The antimicrobial activity remained sustainable under the testing conditions in situ. These findings from authentic conditions confirm those obtained in the in vitro tests and demonstrate the efficiency of the antimicrobial activity of oxide mineral microspheres under standard and environmental conditions.

Conclusions
Oxide mineral microspheres are a unique nonrelease, nonleaching, non-nanoparticlebased technology. Porous and nonporous materials with added microspheres showed efficient and sustainable antimicrobial activity in vitro and in situ under high testing standards and real-life conditions. This promising green-tech innovation is incorporated without any change in the manufacturing process regarding any material, offering countless applicability in preventing inanimate surface contamination in the pharmaceutical and medical fields and in different healthcare and community settings. The antimicrobial activity was demonstrated in vitro and in situ against the indicated bacteria according to the testing standards and has also shown effectiveness in a previously published study against susceptible and resistant bacteria, viruses, and to a lesser extent, fungi.
A new testing standard replicating real-life conditions is desirable to show the antimicrobial activity of the microspheres against a broad spectrum of micro-organisms, including additional priority pathogens. The long-term safety of humans and the environment, including the selection of resistant bacteria, is recommended for future research.

Conflicts of Interest:
A part of the financial support to conduct this study was provided by Pylote SA (Dremil-Lafage, France). K. Iskandar was in charge of the article writing and had no conflict of interest. S. Pecastaings, C. LeGac, C. Feuillolay, S. Salvatico, and C. Roques declare that they have no conflict of interest concerning this article. They respectively made and validated the tests and wrote the protocols and the article after analysing the results. Marc Verelst declared no conflict of interest and was involved in the physico-chemical characterisation and the article writing. L. Marchin is the Manager of Pylote, and Mrs. Guittard is in charge of Pylote R&D, who validated the protocols, prepared the samples and approved the article. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.