Novel antimicrobial efficacy on inert surfaces of chlorhexidine-silver nanoparticle veterinary formulation

Abstract

Aim: This study aimed to establish the in vitro efficacy of chlorhexidine (CHX)-silver nanoparticles (AgNP) based preparation, against Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) in planktonic cultures, biofilms, and on inert stainless-steel surfaces.

Methods: The in vitro antimicrobial activity of the CHX-AgNP formulation (Dermosedan MRSA Nano AG^®) was evaluated against reference and multidrug-resistant (MDR) strains of S. aureus and P. aeruginosa by determining the minimum inhibitory and bactericidal concentrations (MIC and MBC), as well as the minimum biofilm inhibitory and eradication concentrations (MBIC and MBEC). Also, the residual bactericidal activity on inert stainless-steel surfaces was evaluated.

Results: MIC against methicillin-resistant S. aureus (MRSA) and MDR P. aeruginosa (MDR-PA) isolates ranged between 5/2.5–20/10 µg/mL, up to 8,000 times lower than the manufacturer’s recommended concentration, while MBC ranged from 500 to 2,000 times lower. MBIC matched the MBC, while higher concentrations were needed to eradicate preformed biofilms. On stainless-steel surfaces, high antimicrobial activity was observed for both pathogens. No bacterial survival was detected even after 6 hours at 4% CHX + 2% AgNP concentration. The CHX-AgNP combination demonstrated strong antimicrobial and antibiofilm activity against both S. aureus and P. aeruginosa.

Conclusions: These results support the potential application of Dermosedan MRSA Nano AG^® as a strategy for managing topical resistant infections and for environmental disinfection in both veterinary and clinical settings.

Keywords

antimicrobial resistance / chlorhexidine / silver nanoparticles / persistence effect / Staphylococcus aureus / Pseudomonas aeruginosa

References

[1] Global action plan on antimicrobial resistance [Internet]. Geneva: World Health Organization; c2015 [cited 2025 Nov 15]. Available from: https://iris.who.int/server/api/core/bitstreams/1a487887—e162—46a0—8aef—802907c66070/content

[2] Implementing the global action plan on antimicrobial resistance: First quadripartite biennial report [Internet]. Geneva: World Health Organization, Food and Agriculture Organization of the United Nations, United Nations Environment Programme and World Organization for Animal Health; c2023 [cited 2025 Jun 13]. Available from: https://iris.who.int/server/api/core/bitstreams/af8a5f2b—70a8—4bcb—92b4—5f6f2bf9f0d6/content

[3] Kumar M, Sarma DK, Shubham S, Kumawat M, Verma V, Nina PB, et al. Futuristic Non—antibiotic Therapies to Combat Antibiotic Resistance: A Review. Front Microbiol. 2021; 12: 609459.

[4] WHO Bacterial Priority Pathogens List 2024: Bacterial pathogens of public health importance to guide research, development and strategies to prevent and control antimicrobial resistance [Internet]. Geneva: WHO; c2024 [cited 2025 Jun 13]. Available from: https://iris.who.int/server/api/core/bitstreams/1a41ef7e—dd24—4ce6—a9a6—1573562e7f37/content

[5] Chávez—Jacobo VM. The battle against the superbugs: No more antimicrobials, there is no ESKAPE. TIP Rev Esp Cienc Quim Biol. 2020; 23: 1-11.

[6] Broglia G, Buchamer A, Mestorino N, Marchetti L. Pseudomonas aeruginosa in canine otitis externa: current situation. Analecta Vet. 2020; 40: 13-24.

[7] Krueger M, Bajric S, Godden S, Bender JB, Mani R, Sreevatsan S. Phylogenomic associations among methicillin—resistant Staphylococcus aureus isolates derived from pets, dairies, and humans. Microbiol Spectr. 2025; 13: e0199524.

[8] Iverson SA, Brazil AM, Ferguson JM, Nelson K, Lautenbach E, Rankin SC, et al. Anatomical patterns of colonization of pets with staphylococcal species in homes of people with methicillin—resistant Staphylococcus aureus (MRSA) skin or soft tissue infection (SSTI). Vet Microbiol. 2015; 176: 202—8.

[9] Krapf M, Müller E, Reissig A, Slickers P, Braun SD, Müller E, et al. Molecular characterisation of methicillin—resistant Staphylococcus pseudintermedius from dogs and the description of their SCCmec elements. Vet Microbiol. 2019; 233: 196-203.

[10] Nocera FP, De Martino L. Methicillin—resistant Staphylococcus pseudintermedius: epidemiological changes, antibiotic resistance, and alternative therapeutic strategies. Vet Res Commun. 2024; 48: 3505-15.

[11] Amable V. Pseudomonas. In: Stanchi NO, editor. Microbiología Veterinaria. 2nd ed. Buenos Aires: Inter—Médicas; 2019. pp. 650—6.

[12] Dewulf J, van Immerseel F. Biosecurity in Animal Production and Veterinary Medicine: From Principles to Practice. Wallingford: CAB International; 2019.

[13] Block SS. Disinfection, sterilization, and preservation. 5th ed. Philadelphia: Lippincott Williams & Wilkins; 2001. pp. 321-36.

[14] Mohammadi Z, Abbott PV. The properties and applications of chlorhexidine in endodontics. Int Endod J. 2009; 42: 288-302.

[15] Williamson DA, Carter GP, Howden BP. Current and Emerging Topical Antibacterials and Antiseptics: Agents, Action, and Resistance Patterns. Clin Microbiol Rev. 2017; 30: 827-60.

[16] Günther F, Kaiser SJ, Fries T, Frank U, Mutters NT. Susceptibility of multidrug resistant clinical pathogens to a chlorhexidine formulation. J Prev Med Hyg. 2015; 56: E176—9.

[17] Rodrigues AS, Batista JGS, Rodrigues MÁV, Thipe VC, Minarini LAR, Lopes PS, et al. Advances in silver nanoparticles: a comprehensive review on their potential as antimicrobial agents and their mechanisms of action elucidated by proteomics. Front Microbiol. 2024; 15: 1440065.

[18] Ghatti V, Chapi S, Kumar Kumarswamy Y, Nandihalli N, Kasai DR. Strontium—Decorated Ag₂O Nanoparticles Obtained via Green Synthesis/Polyvinyl Alcohol Films for Wound Dressing Applications. Materials (Basel). 2025; 18: 3568.

[19] Sondi I, Salopek—Sondi B. Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram—negative bacteria. J Colloid Interface Sci. 2004; 275: 177-82.

[20] Morones JR, Elechiguerra JL, Camacho A, Holt K, Kouri JB, Ramírez JT, et al. The bactericidal effect of silver nanoparticles. Nanotechnology. 2005; 16: 2346—53.

[21] Bondarenko OM, Sihtmäe M, Kuzmičiova J, Ragelienė L, Kahru A, Daugelavičius R. Plasma membrane is the target of rapid antibacterial action of silver nanoparticles inEscherichia coli and Pseudomonas aeruginosa. Int J Nanomedicine. 2018; 13: 6779—90.

[22] Lu MM, Wang QJ, Chang ZM, Wang Z, Zheng X, Shao D, et al. Synergistic bactericidal activity of chlorhexidine—loaded, silver—decorated mesoporous silica nanoparticles. Int J Nanomedicine. 2017; 12: 3577-89.

[23] Ivanova N, Ermenlieva N, Simeonova L, Kolev I, Slavov I, Karashanova D, et al. Chlorhexidine—Silver Nanoparticle Conjugation Leading to Antimicrobial Synergism but Enhanced Cytotoxicity. Pharmaceutics. 2023; 15: 2298.

[24] Abbaszadegan A, Tayebikhorami E, Gholami A, Bonyanpour A, Asheghi B, Nikmanesh S. Synergistic bactericidal activity of chlorhexidine loaded on positively charged ionic liquid—protected silver nanoparticles as a root canal disinfectant against Enterococcus faecalis: an ex vivo study. J Ionic Liq. 2024; 4: 100117.

[25] CLSI VET01S: Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals, Ed7 [Internet]. Wayne: Clinical and Laboratory Standards Institute; c2024 [cited 2025 Jun 10]. Available from: https://clsi.org/shop/standards/vet01s/

[26] Watts JL ; National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals: Approved Standard. Wayne: NCCLS; 1999.

[27] Stepanović S, Vuković D, Hola V, Di Bonaventura G, Djukić S, Cirković I, et al. Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. APMIS. 2007; 115: 891-9.

[28] Provenzani R, San—Martin—Galindo P, Hassan G, Legehar A, Kallio A, Xhaard H, et al. Multisubstituted pyrimidines effectively inhibit bacterial growth and biofilm formation of Staphylococcus aureus. Sci Rep. 2021; 11: 7931.

[29] Buldain D, Gortari Castillo L, Marchetti L, Buchamer A, Honor Irala F, Islan G, et al. Antimicrobial activity of nanostructured lipid carriers loaded with Melaleuca armillaris essential oil against Staphylococcus aureus sensitive and resistant to methicillin. Front Nanotechnol. 2024; 6: 1476423.

[30] Asociación Española de Normalización y Certificación (AENOR). UNE—EN 13697: Chemical antiseptics and disinfectants. Quantitative non—porous surface test for the evaluation of bactericidal, yeasticidal and/or fungicidal activity of chemical disinfectants used in food, industrial, domestic and institutional areas. Test method without mechanical action and requirements (phase 2/step 2). UNE. 2024.

[31] Mosteller TM, Bishop JR. Sanitizer Efficacy Against Attached Bacteria in a Milk Biofilm. J Food Prot. 1993; 56: 34-41.

[32] Virieux—Petit M, Hammer—Dedet F, Aujoulat F, Jumas—Bilak E, Romano—Bertrand S. From Copper Tolerance to Resistance in Pseudomonas aeruginosa towards Patho—Adaptation and Hospital Success. Genes (Basel). 2022; 13: 301.

[33] Pankey GA, Sabath LD. Clinical relevance of bacteriostatic versus bactericidal mechanisms of action in the treatment of Gram—positive bacterial infections. Clin Infect Dis. 2004; 38: 864-70.

[34] Le Ouay B, Stellacci F. Antibacterial activity of silver nanoparticles: A surface science insight. Nano Today. 2015; 10: 339-54.

[35] Joshi AS, Singh P, Mijakovic I. Interactions of Gold and Silver Nanoparticles with Bacterial Biofilms: Molecular Interactions behind Inhibition and Resistance. Int J Mol Sci. 2020; 21: 7658.

[36] Suller MT, Russell AD. Antibiotic and biocide resistance in methicillin—resistant Staphylococcus aureus and vancomycin—resistant enterococcus. J Hosp Infect. 1999; 43: 281-91.

[37] Bonez PC, Dos Santos Alves CF, Dalmolin TV, Agertt VA, Mizdal CR, Flores Vda C, et al. Chlorhexidine activity against bacterial biofilms. Am J Infect Control. 2013; 41: e119—22.

[38] Leng D, Li Y, Zhu J, Liang R, Zhang C, Zhou Y, et al. The Antibiofilm Activity and Mechanism of Nanosilver— and Nanozinc—Incorporated Mesoporous Calcium—Silicate Nanoparticles. Int J Nanomedicine. 2020; 15: 3921-36.

[39] Saeki EK, Martins HM, Camargo LC, Anversa L, Tavares ER, Yamada—Ogatta SF, et al. Effect of Biogenic Silver Nanoparticles on the Quorum—Sensing System of Pseudomonas aeruginosa PAO1 and PA14. Microorganisms. 2022; 10: 1755.

[40] Qais FA, Ahmad I, Altaf M, Manoharadas S, Al—Rayes BF, Ali Abuhasil MS, et al. Biofabricated silver nanoparticles exhibit broad—spectrum antibiofilm and antiquorum sensing activity against Gram—negative bacteria. RSC Adv. 2021; 11: 13700—10.