Introduction

The increase in antimicrobial resistance (AMR) has become a great concern all over the world. Since 2015, a global plan for the control of bacterial resistance was agreed by the World Health Organization (WHO), the World Organization for Animal Health (WOAH), and the Food and Agriculture Organization of the United Nations (FAO) [1, 2]. In 2022, the United Nations Environment Program (UNEP) became part of this quadripartite alliance [2, 3]. Bacterial resistance is a problem that affects worldwide health, causing failure in human and animal therapy.

Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) are two microorganisms classified as high-priority pathogens in 2024 by the WHO due to their global threat to public health [4]. They belong to the ESKAPE group (acronym that includes Enterococcus faecium, S. aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, and Enterobacter spp.), six genera of gram-negative and gram-positive bacteria that are of global concern due to their resistance to multiple antibiotics and their ability to cause serious infections, especially in hospital settings [5]. P. aeruginosa is an opportunistic pathogen found both in the environment and in patients (humans and animals), which constitutes a significant threat due to its high antibiotic resistance (natural and acquired) and its virulence. In veterinary medicine, P. aeruginosa is frequently found in dogs, and multidrug-resistant (MDR) phenotypes are mainly reported in cases of chronic otitis externa [6]. S. aureus is a major opportunistic pathogen in humans and one of the most important in veterinary medicine. It can be transmitted not only from animals to humans but also from humans to animals [7, 8]. This microorganism can select resistance to several antibiotics, and the methicillin-resistance phenotype has been identified and increasingly reported in dogs since 2006 in S. aureus [methicillin-resistant S. aureus (MRSA)], and in methicillin-resistant Staphylococcus pseudintermedius (MRSP) species [9]. This last fact has been associated with colonization and infections due to close interaction between humans and pet dogs, which is a great concern [10]. Both Staphylococcus spp. and P. aeruginosa possess multiple virulence factors, including the ability to produce biofilms. Biofilms are composed of microorganisms embedded in an extracellular matrix of exopolysaccharides, proteins, and nucleic acids. This protective barrier hinders the entry of molecules, such as antimicrobials or disinfectants, into the microorganism, as well as the effect of the immune system itself, and they can be formed on animal and human bodies, and on inert surfaces [11].

Consequently, environmental biosecurity is a key tool to reduce the spread of resistant bacteria. Several studies have shown that hygiene practices in clinical and hospital settings, along with the proper use of disinfectants, are essential in preventing bacterial resistance and its spread [12].

Dermosedan MRSA Nano AG^® (Instituto de Dermatología Veterinaria, Argentina) is a topical preparation formulated to prevent and treat skin infections caused by resistant bacteria such as MRSA. Its composition includes silver nanoparticles (AgNP) (monodisperse nanoparticle size between 20–30 nm) combined with chlorhexidine (CHX). CHX is a cationic molecule belonging to the biguanide group, composed of two 4-chlorophenyl rings and two biguanide units linked by a hexamethylene chain [13, 14]. It is widely used as an antiseptic, disinfectant, and preservative in pharmaceuticals and cosmetics, as well as for the control of bacterial plaque in dentistry, both in human and veterinary medicine. Due to its positive charge, CHX binds to the negatively charged extracellular components of the bacterial membrane, causing its destabilization and altering osmotic balance. This leads to the loss of essential intracellular substances, such as ions, nucleic acids, and proteins, and, consequently, the emptying of cellular contents. It also inhibits the activity of key enzymes such as the phosphoenolpyruvate phosphotransferase system and ATPase. At high concentrations, it can precipitate bacterial cytoplasm, causing cell death [15]. This substance has a broad antimicrobial spectrum, being effective against numerous microorganisms, including gram-positive and gram-negative bacteria, as well as fungi and yeasts [16].

AgNPs have garnered significant attention for their antimicrobial capabilities, which have been recognized since ancient medicine and are currently utilized in various commercial products containing silver in ionic or nanoparticulate form. AgNP exhibits broad-spectrum antimicrobial activity against bacteria, fungi, viruses, and mycobacteria. Additionally, they can enhance the effectiveness of certain antibiotics through synergistic interactions. Their antimicrobial mechanisms involve the production of reactive oxygen species, interference with DNA, disruption of microbial cell membranes, and inhibition of protein synthesis [17]. These antimicrobial mechanisms are based on the formation of Ag⁺ ions in aqueous solution, which are also responsible for the bactericidal activity of other silver-based compounds such as silver oxide [18]. These are highly reactive and capable of interacting with various bacterial structures such as nucleic acids, cell membranes, and proteins. Ag⁺ ions can bind to thiol groups of proteins and enzymes, altering the structure of the bacterial cell wall [19]. The use of AgNP has advantages over the direct use of Ag⁺ solutions such as silver nitrate, allowing for a prolonged and controlled release of Ag⁺ through gradual dissolution, thus maintaining antimicrobial efficacy over time [20]. Recent studies have highlighted the efficacy of AgNP against various clinically relevant bacterial strains, including S. aureus and P. aeruginosa [21].

All these challenges have driven the search for new strategies to combat AMR, developing new antimicrobial alternatives to treat infections and control environmental transference of AMR genetic determinants. Several reports have shown that combining CHX with AgNP results in synergistic antimicrobial effects, enhancing membrane disruption and bactericidal activity compared to each agent alone. Such synergy has been demonstrated against S. aureus, Escherichia coli (E. coli), and Enterococcus faecalis (E. faecalis) biofilms [22–24], supporting the potential of CHX-AgNP formulations, although studies focusing on S. aureus and P. aeruginosa biofilms remain scarce. The aim of this work was to determine the in vitro efficacy on planktonic forms, biofilm, and inert surface of a disinfectant/antiseptic Dermosedan MRSA Nano AG^® (Instituto de Dermatología Veterinaria, Argentina), against reference strains of S. aureus ATCC 29213 and P. aeruginosa ATCC 27853, and multi-resistant isolates [MRSA, and MDR P. aeruginosa (MDR-PA)].

Materials and methods

Determination of persistence time on an inert surface

To evaluate the residual bactericidal effect of disinfectant products on non-porous surfaces, the UNE-EN 13697 standard was adapted [30]. The strains used were MRSA and MDR-PA. Two concentrations of the formulation were used: the manufacturer-recommended concentration of use (40,000 µg/mL of CHX with 20,000 µg/mL of AgNP) and a 25% dilution of the pure formulation, obtaining a final concentration of 10,000 µg/mL of CHX with 5,000 µg/mL of AgNP. Briefly, the test procedure consisted of streaking the inoculum onto a sterile stainless-steel plate previously marked in 2.5 cm × 2.5 cm squares (Figure 1), and exposing the microorganisms to known concentrations of the formulation in order to evaluate the persistence effect and the bactericidal activity. A volume of 100 μL of two formulation concentrations (40,000/20,000 μg/mL; 10,000/5,000 μg/mL) and an untreated control were placed in these quadrants and exposed to the bacterial inoculum at different times (0, 2, 4, and 6 hours after application of the formulation on the plate).

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Figure 1. Biosafety cabinet with stainless-steel plates. Each plate had 2.5 cm × 2.5 cm square markings to evaluate the formulation against MRSA and MDR-PA strains at 0, 2, 4, and 6 hours after product application. CHX: chlorhexidine; AgNP: silver nanoparticles; MRSA: methicillin-resistant Staphylococcus aureus; MDR-PA: multidrug-resistant Pseudomonas aeruginosa.

The plates were kept throughout the test in a sterile environment (Biosafety Cabinet Class II, Biobase, China) at a temperature between 18°C and 25°C. The selection of the concentrations evaluated arose from the results obtained in the previous MIC, MBC, MBIC, and MBEC evaluation, and considering the concentrations recommended by the manufacturer. After 30 minutes of placing the formulation (time zero) on the surface to be evaluated, 50 μL of a bacterial suspension of the MDR isolates (MRSA and MDR-PA) was inoculated at 0, 2, 4, and 6 hours after the initial application of the formulations on the surface at a concentration of 1 × 10⁸ CFU/mL. After applying the bacterial inoculum, it was left in contact for 30 minutes. Subsequently, 100 μL of neutralizing solution [0.3% lecithin + 3% tween 80 (Biopack, Argentina)] was added to each quadrant to inactivate the effect of the formulation, leaving it in contact for 10 minutes. Each quadrant of the surface was swabbed with a sterile swab, scraping it completely in three directions. The swab contents were suspended by twirling the swab in physiological solution, and 6 serial 10-fold dilutions were made to count colonies after plating 100 μL of each one onto nutrient agar plates. To obtain a homogeneous colony count after plating, sterile 2-mm diameter glass beads were used per plate, and the plates were moved in three directions for 1 minute to allow inoculum dispersion across the entire agar surface. They were incubated for 24 hours at 37°C. Finally, bacterial counts were performed under each condition in duplicate.

Based on the information presented in the literature and considering the possible residual bactericidal activity of dry disinfectant solutions, the residual bactericidal effect was established when the disinfectant showed a bactericidal activity greater than or equal to 3 Log₁₀ at each time tested [31].

Results

Determination of persistence time on an inert surface

The evaluation of antimicrobial activity on an inert stainless-steel surface showed a significant decrease in bacterial load compared to the positive control due to the application of the formulation for both MRSA and MDR-PA (Figures 2 and 3, respectively). In the case of the pure formulation (4% CHX-2% AgNP or 40,000 µg/mL CHX-20,000 µg/mL AgNP), bacterial survival was zero for both microorganisms, even after 6 hours post-application of the product on the metal surface. However, efficacy for a 1/4 dilution of the formulation (1% CHX-0.5% AgNP or 10,000 µg/mL CHX-5,000 µg/mL AgNP) was lower and decreased with increasing residence time, but even so, the bacterial count was significantly lower than for the untreated control (p < 0.05) for 6 hours. At 0 hours, for this concentration, the microorganism count was significantly lower than the control, and remained so for both bacterial species at higher times (p < 0.05). At 4 and 6 hours, the 1/4 dilution had similar efficacy against MRSA and MDR-PA.

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Figure 2. Effect of chlorhexidine (CHX) with silver nanoparticles (AgNP) against methicillin-resistant Staphylococcus aureus (MRSA), expressed as percentage of bacterial survival (%) at 0, 2, 4, and 6 hours post-treatment on an inert metal surface. No viable bacteria were obtained with the 4% CHX-2% AgNP treatment (blue bar). Error bars indicate the standard error of the mean, and there are no statistically significant differences between bars that share the same letter after a significant (p < 0.05) two-way ANOVA (p < 0.05, Fisher’s Least Significant Difference).

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Figure 3. Effect of chlorhexidine (CHX) with silver nanoparticles (AgNP) against multi-resistant Pseudomonas aeruginosa (MDR-PA), expressed as percentage of bacterial survival (%) at 0, 2, 4, and 6 hours post-treatment on an inert metal surface. No viable bacteria were obtained with the 4% CHX-2% AgNP treatment (blue bar). Error bars indicate the standard error of the mean, and there are no statistically significant differences between bars that share the same letter after a significant (p < 0.05) two-way ANOVA (p < 0.05, Fisher’s Least Significant Difference).

For both S. aureus and P. aeruginosa, a significant interaction was demonstrated between the two factors, time and concentration of the formulation (F = 265.78 and F = 12.02, respectively; both corresponding to a p-value < 0.0001), indicating in the overall analysis that bactericidal activity varies according to exposure time. However, examination of individual single effects showed that the time factor is a determinant of the bacterial count only at the 1% CHX-0.5% AgNP concentration (Figures 4 and 5), demonstrating an initial impact with a significant reduction in bacterial load and lower efficacy over the 6-hour evaluated interval. In contrast, the other concentrations, 0% CHX-0% AgNP and 4% CHX-2% AgNP, did not show statistically significant variations between the evaluated times (Figures 4 and 5). This discrepancy confirms that the effect of time is actually conditioned by the concentration of the formulation evaluated, which is consistent with the statistically significant interaction detected in the overall analysis between both factors.

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Figure 4. Graph of interaction between the factors of time and concentration of the formulation in the antimicrobial activity on the steel surface against MRSA. MRSA: methicillin-resistant Staphylococcus aureus; CHX: chlorhexidine; AgNP: silver nanoparticles.

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Figure 5. Graph of interaction between the factors of time and concentration of the formulation in the antimicrobial activity on the steel surface against MDR-PA. MDR-PA: multi-resistant Pseudomonas aeruginosa; CHX: chlorhexidine; AgNP: silver nanoparticles.

Discussion

This study evaluated the antimicrobial activity of a combined formulation, Dermosedan MRSA Nano AG^®, which contains 4% CHX and 2% AgNP, considering its efficacy against several microorganisms in different conditions: planktonic cultures, biofilms, and inert metal surfaces. CHX is commonly used in concentrations ranging from 0.5% to 4%, depending on the specific clinical use. For example, hand sanitizers typically contain concentrations within this range, adjusting for antimicrobial efficacy needs [15].

The formulation showed similar MIC and MBC values against most of the microorganisms tested, which means similar efficacy. In the case of S. aureus, both for the reference strain and for the MRSA, the formulation showed a slightly lower MIC (5/2.5 µg/mL CHX/AgNP) compared to that obtained for P. aeruginosa (20/10 µg/mL CHX/AgNP), demonstrating greater intrinsic resistance and tolerance to the formulation, which could be due to its great adaptive capacity. P. aeruginosa shows strong intrinsic resistance to biocides due to the lack of membrane permeability, presence of efflux pumps, and other intrinsic mechanisms of natural and acquired resistance [32].

For both microorganisms, the MBC/MIC ratio was equal to 4, with the formulation behaving as a bactericidal agent [33]. Ivanova et al. [23] showed that the combination of AgNP with CHX has a MIC of 8.8 µg/mL CHX and 5.5 µg/mL AgNP against S. aureus and E. coli, showing a synergistic activity. Other authors have also demonstrated synergism between CHX and AgNP against microorganisms like E. coli and S. aureus [22].

Regarding the ability to MBIC, for the reference S. aureus strain and the MRSA strain, the MBIC values were similar, being 10/5 µg/mL of CHX/AgNP for both. For P. aeruginosa, there was a slight difference between the ATCC reference strain (40/20 µg/mL of CHX/AgNP) and MDR-PA isolate (80/40 µg/mL of CHX/AgNP). Although the antibacterial effect of AgNP on planktonic bacteria is largely attributed to the release of Ag⁺ ions through dissolution, their role in biofilm disinfection appears particularly advantageous [34]. As individual nanoparticles penetrate deeper into the biofilm, they can gradually dissolve and emit thousands of silver ions, resulting in potent antibacterial activity even in the innermost biofilm layers [35].

Regarding the eradication of already formed biofilms (MBEC), the values were considerably higher than the MBIC, reflecting the greater structural resistance of biofilms once built on the polycylinder surface against both microorganisms. P. aeruginosa again required higher concentrations compared to S. aureus. This is expected due to the complexity of the biofilm already formed. In line with our results, a report informs that CHX was also significantly less effective against P. aeruginosa biofilm bacteria compared to S. aureus, likely due to differences in their cell wall structures [36]. However, it is important to note that the concentrations mentioned for each of the susceptibility tests on planktonic forms or biofilms are clearly lower than those recommended for commercial use (40,000/20,000 µg/mL or 4%/2% of CHX/AgNP), demonstrating that the desired chemotherapeutic effect will be achieved at the manufacturer’s recommended doses. Abbaszadegan et al. [24] found loading AgNP with CHX significantly enhanced its antibacterial activity against E. faecalis biofilms.

CHX had presented in other studies a lower activity in biofilms with respect to planktonic cultures, probably being unable to penetrate biofilm [37]. On the other hand, there are studies in which the activity of AgNP against bacterial biofilms was high and established as concentration-dependent. Biofilms are structures that provide a protective environment to bacteria that improves survival, facilitates nutrient acquisition, and increases resistance to antimicrobial agents and the host immune response [38]. It has been reported that this type of nanoparticle interferes with bacterial quorum sensing by altering the signaling mechanisms that control bacterial communication [39]. This alteration of the quorum-sensing system can weaken bacterial pathogenicity, making AgNP an important tool to control bacterial infections and reduce the effectiveness of bacterial communication mechanisms, particularly in the treatment of topical infections or the coating of medical implants to prevent bacterial adhesion and biofilm development [40].

On stainless-steel surfaces, antimicrobial activity against MRSA and MDR-PA was high. Comparing the 4%/2% (CHX-AgNP) and 1%/0.5% (CHX-AgNP) formulations, the latter was less effective. However, the diluted formulation showed a significant decrease in bacterial count compared to the untreated control. Regarding the formulation’s efficacy after being placed on the metal surface and exposed to microorganisms, we observed that the formulation maintained its efficacy against MRSA and MDR-PA even after 6 hours. In the case of the diluted formulation, efficacy decreased over time, being highest at shorter times; however, even after 6 hours, the recovered bacterial colonies were lower than in the untreated control. The literature on the use of AgNP combined with CHX on inert surfaces is scarce; no works have been found on the subject. CHX is commonly used in the form of a solution for disinfecting skin and mucous membranes, although it is also applied to inert, non-porous surfaces, such as metals, making it a valuable tool for environmental biosecurity. It can adsorb to these surfaces and maintain its antibacterial action for an important period of time, helping to prevent the presence of microorganisms. AgNP can act in different ways. One of the mechanisms of antimicrobial action reported in the literature is based on the formation of Ag⁺ ions in aqueous solution. These are highly reactive and are capable of interacting with various bacterial structures such as nucleic acids, cell membranes, and proteins. Ag⁺ ions can bind to thiol groups of proteins and enzymes, altering the structure of the bacterial wall [19], a mechanism that also underlies the potent bactericidal activity described for other silver-based compounds such as silver oxide [18].

This study has some limitations that should be acknowledged. All experiments were performed under controlled in vitro conditions, which may not fully represent the complexity of clinical or environmental settings. The antimicrobial activity observed on inert stainless-steel surfaces could vary in the presence of organic matter such as blood, exudates, or tissue debris, where protein binding and pH fluctuations can interfere with disinfectant efficacy. In addition, although S. aureus is a relevant zoonotic and opportunistic pathogen, its use as a model organism may not completely reflect the behavior of strictly animal-associated species, such as S. pseudintermedius, that are commonly isolated in veterinary infections. Future studies should therefore include in vivo evaluations in animal models and clinical cases, as well as testing under conditions that simulate organic contamination. Further research will also focus on the efficacy of CHX-AgNP formulations on diverse veterinary surfaces and materials, their long-term persistence, and their integration into hygiene protocols for clinical and environmental disinfection in veterinary practice.