Antiseptics: Their Characteristics, Application and Challenges in the 21St Century Resulting from the Spread of Antimicrobial Resistance (AMR)

Chlorhexidine (CHX) belongs to the bisbiguanide, a class of cationic antimicrobial agents. It’s structure consists of two symmetrically arranged 4-chlorophenol rings and two guanidine groups connected by a hexamethylene chain (Thangavelu et al. 2020). CHX can be obtained in a number of different forms, including acetate, dihydrochloride salts, and digluconate. However, chlorhexidine digluconate (CXG) is the most commonly used due to its high solubility. It exhibits a wide range of efficacy against both Gram-positive and Gram-negative bacteria, although higher CXG concentrations are required in order to combat Gram-negative bacteria. A 4% solution of CXG demonstrates bactericidal activity within just 5 minutes of contact against both Gram-positive and Gram-negative bacteria (Ekizoglu et al. 2016). It also effectively eliminates fungi, yeast, dermatophytes, and certain lipophilic viruses. However, its sporicidal properties is only achieved at elevated temperatures (Łukomska-Szymańska et al. 2017).

Chlorhexidine at low concentrations exhibits bacteriostatic activity, while at high concentrations it demonstrates bactericidal effects. Its mechanism of action is based on direct interaction with the bacterial cytoplasmic membrane. As a cationic surfactant, CHX binds to the negatively charged cell surface, disrupting the organization of the outer phospholipid layer. It displaces stabilizing divalent cations, leading to decreased membrane fluidity and the formation of hydrophilic domains in its structure. At higher concentrations, increased membrane permeability is observed, resulting in leakage of cytoplasmic contents and ultimately denaturation and precipitation of proteins and nucleic acids (Cieplik et al. 2019). This molecular mechanism of action correlates with observed morphological changes induced by chlorhexidine in bacterial cells. Studies have demonstrated that its action leads to the deformation and degradation of the cell wall in both Gram-negative bacteria, such as Escherichia coli, and Gram-positive bacteria, such as Bacillus subtilis. Upon exposure to chlorhexidine, characteristic indentations were observed on the bacterial cell surfaces, particularly in the tip or cap region of B. subtilis and along the trunk of E. coli cells, as revealed by scanning electron microscopy (Cheung et al. 2012). Furthermore, the number of these indentations increased proportionally with CHX concentration. Transmission electron microscopy (TEM) also showed the formation of “ghost cells” following prolonged CHX exposure (Cheung et al. 2012).

Chlorhexidine has also been evaluated for its efficacy against microbial biofilms, which are often less susceptible to antimicrobial substances than planktonic cells. Kean et al. (2018) studied the impact of CXG on the biofilm of Candida spp., including Candida auris. Currently, C. auris strains are the most multidrug-resistant pathogenic yeast causing healthcare-associated infections. It has been shown that CHX at a concentration of 0.05% showed high efficacy against planktonic C. auris cells, but yeast biofilms, especially mature ones, showed tolerance to such CHX solutions. Increasing the CHX concentration to 2% resulted in complete destruction of early-stage biofilms, as well as a reduction of mature ones. The antiseptic efficacy of 2% chlorhexidine was also tested on interspecies biofilms. The analysis showed that this agent effectively reduced the number of viable cells in single-species biofilms, including C. auris NCPF 8973, S. aureus NCTC 10,833 and Staphylococcus epidermidis RP62A (ATCC 35984). Similar efficacy was observed in dual-species biofilms (C. auris with S. aureus, and C. auris with S. epidermidis), where the reduction in cell numbers exceeded 4 log₁₀ (Gülmez et al. 2022). The effectiveness of CHX against the biofilm of Gram-negative bacteria, i.e. Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii, has also been demonstrated. Depending on the strain, the ability of CHX to inhibit biofilm formation and reduce mature biofilm was observed (Hubner et al. 2010a; Machuca et al. 2019; Perez-Palacios et al. 2022)

Antiseptic products with chlorhexidine have been widely used for a long time. Therefore, studies on the basic antimicrobial efficacy of CHX according to EN phase 1 are rarely published. Based on tests conducted in a miniaturized assay according to EN 1040, after 5 minutes of exposure P. aeruginosa to different concentrations of CXG, it was shown the highest efficacy at concentrations of 4% and 0.12%, where the bacterial cells reduction was 5.34 log₁₀ for both concentrations. For K. pneumoniae, a 4% CXG solution achieved ≥ 5 log₁₀ reduction, while efficacy dropped below 5 log₁₀ at 0.12% and 0.06%. E. coli showed the greatest sensitivity to CXG, with a log₁₀ reduction of 5.69 at 4% concentration, but less than 5 log₁₀ at lower concentrations (Hornschuh et al. 2021).

Unlike phase 1 EN, phase 2 tests are dedicated to a specific area of product application. In the medical area, in phase 2, step 1 of EN 13727 is used to test antibacterial activity, and in phase 2, step 2, several standards are recommended (Tyski et al. 2022). In scientific research, modifications are introduced to the methodology according to EN and studies are conducted on wider panels of strains. It has been shown that changes in the chlorhexidine efficacy depending on the presence of isopropyl alcohol. In a study conducted using the quantitative suspension test (EN 13624), a chlorhexidine-based skin antiseptic [2% (w/v) CXG in 70% (v/v) isopropyl alcohol (IPA)] was found to meet the full fungicidal requirements, achieving > 4 log₁₀ cells reductions for Candida albicans and C. auris in both clean and dirty conditions after 2 minutes of a contact time. In contrast, hand and body wash antiseptic [4% CXG (v/v)] showed limited efficacy against C. auris, achieving reductions in the range of 1.55–2.63 log₁₀ after 2 minutes of exposure in clean conditions and 1.15–2.45 log₁₀ in dirty conditions. For C. albicans, the effect was more pronounced, with reductions of 2.83 log₁₀ in clean conditions and 2.78 log₁₀ in dirty conditions after 1 minute, which increased to 3.57 log₁₀ and 3.36 log₁₀, respectively, after 2 minutes. Still, the 4% chlorhexidine gluconate (v/v) met the EN 13624 for hygienic hand washing, requiring a ≥ 2 log₁₀ reduction in 1 minute in dirty conditions (Moore et al. 2017).

CXG-based impregnated antiseptic wash-mitts [100 g contains 2% CXG and 0.04% benzalkonium chloride] were tested at concentrations of 10%, 50%, 80%, and 97% to evaluate their antifungal efficacy. However, quantitative suspension tests performed according to the EN 13624 demonstrated that none of the concentrations achieved ≥ 4 log₁₀ reduction in C. albicans ATCC 10231 or two C. auris strains (DSM 21092 and DSM 105986) after a 30-second contact time (Gugsch et al. 2024).

In the conducted study, following the EN 13727 and EN 13624, the bactericidal and fungicidal efficacy of a 2% CXG solution was evaluated. The results demonstrated that the efficacy of the preparation increased with prolonged exposure time. After 1 minute of contact with P. aeruginosa ATCC 15442, a reduction lover than 5 log₁₀ was observed. However, after 5 minutes of exposure, the cells reduction exceeded 5.38 log₁₀. In the case of E. coli NCTC 10538, after 1 minute of exposure, the reduction was above 5.52 log₁₀ in clean conditions, while in dirty conditions, it was below 5 log₁₀. After 5 minutes, regardless of the conditions, the reduction exceeded 5.52 log₁₀, indicating full efficacy of the preparation after a longer exposure time. In contrast, C. albicans ATCC 10231 displayed a lower sensitivity to 2% CXG, achieving a reduction of 3.52 log₁₀ in clean conditions and 3.27 log₁₀ in dirty conditions after 1 minute. After 5 minutes of exposure, the cells reduction exceeded 4.52 log₁₀, thereby meeting the fungicidal standard (reduction ≥ 4 log₁₀). In the case of Aspergillus brasiliensis, no fungicidal activity was observed, as the reduction remained below the required threshold in both clean and dirty conditions after 1 and 5 minutes of exposure (Şahiner et al. 2019).

Studies on the efficacy of CHX against SARS-CoV-2 have yielded inconsistent findings. Some laboratory investigations report that CHX-containing mouthwashes are ineffective at inactivating the viruses, (Komine et al. 2021) while others show that they can reduce viral load temporarily but not permanently. A study comparing 0.05% CHX with 0.05% cetylpyridinium chloride demonstrated a modest but statistically significant decrease in viral load among SARS-CoV-2-positive patients. Interestingly, a similar reduction was observed in patients using placebo irrigation (0.9% NaCl), suggesting that this reduction may be primarily due to the effect of mechanical irrigation (Bonn et al. 2023). Another study demonstrated that a 0.12% CHX mouthwash temporarily suppressed the SARS-CoV-2 viral load in saliva, reducing it to undetectable levels for up to two hours. However, after four hours, the viral load increased again, indicating a short duration of this effect (Yoon et al. 2020). Although some studies suggest limited effectiveness of chlorhexidine in reducing viral load, other research has shown that CHX mouthwashes and throat sprays can offer a promising method for eliminating SARS-CoV-2 from the throat in COVID-19 patients. The combination of a 0.12% CHX mouthwash and throat spray demonstrated the highest efficacy, with 86.0% of patients achieving viral clearance from the throat, compared to 62.1% in the group using mouthwash alone. This was significantly higher than the 5.5% of patients in the control group using only mouthwash and 6.3% in the control group using both mouthwash and spray (Huang and Huang 2021).

Chlorhexidine, may result in various adverse effects. Commonly reported side effects include contact skin irritation and taste disturbance. In rare cases, allergic reactions such as occupational asthma, skin rash, photodermatosis or anaphylaxis may occur. Prolonged use may also lead to tooth and tongue staining (Łukomska-Szymańska et al. 2017). Surfaces covered with plaque tend to exhibit more intense staining and a greater extent of calculus formation compared to those that are plaque-free. This suggests that performing an initial professional teeth cleaning before the use of CHX can help mitigate its undesirable side effects, especially with long-term use (Zanatta et al. 2010).

CHX is widely used as an active ingredient in various products, acting as an antiseptic either alone or in combination with other substances. A summary of commercially available products, including their concentrations and indications, is presented in Table I. It is extensively utilized in dentistry as a mouthwash to aid in the management of gingivitis during dental interventions and to reduce plaque accumulation. There are additional mouthcare gels with strengths of 1% and 0.2%, toothpastes with 0.05% CHX, and biodegradable “chips” of CXG that can be put into periodontal pockets in conjunction with subgingival debridement (Brookes et al. 2020). Higher concentrations of CHX (0.2%) demonstrate better plaque-inhibiting effects compared to lower concentrations (0.12% and 0.06%). CHX at a 0.2% concentration is an effective agent used as a mouth rinse, demonstrating efficacy in reducing Streptococcus mutans and Lactobacillus. However, they are associated with a higher risk of adverse effects, such as loss of taste and numbness (Haydari et al. 2017). Consequently, increasing attention has been directed toward natural alternatives, such as cocoa bean husk and ginger-based rinses, which have shown potential in reducing S. mutans and Lactobacillus counts with a lower risk of adverse effects.

Daily bathing with CHX has been proven effective in preventing infections, especially in the hospital setting. The use of CHX baths in intensive care unit reduces the risk of healthcare-associated infections (HAI), in particular central line-associated bloodstream infections (CLABSI) and infections caused by methicillin-resistant S. aureus (MRSA) (Frost et al. 2016). Regularly bathing patients with 2% CHX-impregnated washcloths can lower bloodstream infection rates in hospitals by 30% compared to non-CHX methods (Climo et al. 2013). Higher CHX concentrations, such as 4%, have shown even greater efficacy. One study observed a 40.4% reduction in HAIs when patients were bathed with 4% CHX followed by rinsing with water (Pallotto et al. 2019).

It is well known that bacteria acquire resistance or develop tolerance to biocides. Efflux pumps are one of the key mechanisms by which bacteria acquire resistance to antiseptics. Genes encoding efflux pumps can be located in chromosomes as well as in mobile element such as plasmids, integrons, and transposons. The six main classes of efflux pumps are the major facilitator (MFS) superfamily, the ATP-binding cassette (ABC) superfamily, the resistance-nodulation-division (RND) superfamily, the small multidrug resistance (SMR) family, the multidrug and toxic compound extrusion (MATE) superfamily and the proteobacterial antimicrobial compound efflux (PACE) superfamily (Kuznetsova et al. 2025). Efflux pumps that actively remove disinfectants from bacterial cells to the outside are summarized in Table II. The MFS family includes chlorhexidine extruded pumps such as QacA, QacB and SmvA. The ABC family consists of transporters like AdeABC. The RND family includes pumps such as AcrAB-TolC, EfrAB, MexAB-OprM, MexCD-OprJ, MexXY, and SdeAB. The SMR family primarily contains the KpnEF and Smr pumps. The MATE family includes the MepA pump, while the PACE family features the AceI transporter involved in CHX extrusion.

Efflux pump Smr from SMR family and MepA pump from MATE family play an main role in the mechanisms of S. aureus resistance to antiseptic, including CHX (Noguchi et al. 1999; Costa et al. 2013). Additionally, qacA and qacC genes, which are located in plasmids, have been shown to increase CHX resistance in S. aureus. Moreover, exposure to benzalkonium chloride can induce qacC expression, thereby enhancing CHX tolerance (LaBreck et al. 2020).

In a study analyzing 1050 S. epidermidis isolates, 63 exhibited reduced sensitivity to CHX (MIC ≥ 4 μg/ml) (Addetia et al. 2019). Among these, 9 isolates carried the qacA gene, while qacB was absent. In addition, the smr gene was present in 51 isolates. Notably, a novel qacA allele was identified, encoding a modified QacA protein with two amino acid substitutions. This new allele, designated qacA4, was located in plasmid pAQZ1 and found in the highly resistant and pathogenic ST2 clone. The qacA4 gene has been shown to play an important role in increasing CHX resistance, as loss of this gene resulted in a 4-fold reduction in the CHX MIC values, from 4 μg/ml to 1 μg/ml.

Exposure to 4% CHX through daily bathing has been linked to increased CHX tolerance in MRSA isolates. Using the modified broth microdilution method in line with the Clinical and Laboratory Standards Institute (CLSI) guidelines, isolates from CHX-exposed patients showed MIC values ranging from 1 to 8 μg/ml, with MIC ≥ 4 μg/ml occurring three times more frequently than among strains isolated from unexposed patients. Further, the possibility of transferring qac genes was analyzed. It was shown that long-term exposure to CHX predisposes to the acquisition of qacA/B genes, depending on the clone. This phenomenon was particularly pronounced in the case of clone ST22, in which the frequency of qacA/B genes in CHX-exposed strains was significantly higher compared to the unexposed group. A similar trend was also observed in the case of clone ST45, but the increase in the frequency of gene occurrence was less pronounced (Htun et al. 2019). Transferring the qacA gene between bacteria via plasmids is one potential method for the spread of CHX resistance. The qacA gene was discovered to be transferable from a CHX-resistant MRSA strain to an E. coli strain that had previously been CHX-sensitive. Transfer increased the CHX MIC values in E. coli from ≤ 0.25 to ≥ 16 μg/ml. Genetic studies confirmed the existence of the qacA gene in the recipient strain, indicating that CHX resistance genes can be transferred between bacterial species (Bes et al. 2021). Unlike Gram-positive, Gram-negative bacteria extrude CHX mainly via the RND family of efflux pumps, including the AcrAB-TolC (E. coli, K. pneumoniae, Salmonella enterica subsp. enterica), MexCD-OprJ (P. aeruginosa), SdeAB (Serratia marcescens), AdeABC (A. baumannii) systems (Table II). In addition, the occurrence of the biocide resistance genes (BRGs) such as cepA, qacEΔ1 and qacE, which encode efflux pumps, has been described in Gram-negative bacteria (Zhang et al. 2019). These genes are transferred by plasmids and transposons. It has been shown that the CepA pump is associated with K. pneumoniae resistance to CHX (Fang et al. 2002).

The expression of efflux genes is dependent on local as well as global regulators. Recently, it has been shown that exposure to biocides can cause mutations in these regulatory genes or in the regions surrounding efflux pump genes. In K. pneumoniae, a single DNA mutation was found in the intergenic region between smvR and smvA after exposure to CHX. SmvA pump extruded two main antiseptic CHX and OCT. The SmvR protein plays a regulatory role and inhibits the expression of smvA. This mutation probably interferes with the mechanism of this regulation, weakening inhibition and leading to increased smvA expression. Exposure to antiseptics also leads to the accumulation of multiple mutations in different locations of the bacterial genome. Additionally, E359K substitutions or deletions were detected in the malT2 gene, which encodes an HTH-type transcriptional activator regulating the maltose operon. However, exposure of Enterobacter cloacae to CHX caused mutations in the bamE gene, which is an assembly factor for outer membrane proteins, and in the betI gene, which is a member of the TetR/AcrR family of transcriptional regulators (Lescat et al. 2021). The overproduction of the AcrAB-TolC efflux pump in Enterobacterales depends on both local and global regulators. Among the global regulators, increased overproduction of MarA, SoxR, and RamA has been associated with the overexpression of acrAB-TolC operon contributing to CHX resistance (Curiao et al. 2015).

Octenidine dihydrochloride (OCT) is a positively charged surfactant belonging to the bispyridine group. Its structure contains two independent cationic active centers connected by a long aliphatic hydrocarbon chain. OCT demonstrates a wide antimicrobial spectrum, being effective against Gram-positive cocci, including MRSA strains, and Gram-negative bacteria. It also targets plaque-forming bacteria, including Actinomyces and Streptococcus spp., as well as Chlamydia, Mycoplasma, and various fungi (Hubner et al. 2010b). It shows limited virucidal effectiveness against hepatitis B and herpes simplex viruses (Sathiyamurthy et al. 2016). Recently, the OCT-based formulation was found to be effective against SARS-CoV-2 virus (Smeets et al. 2021, Steinhauer 2022 et al. 2022). Moreover, OCT is a potentially active against Acanthamoeba trophozoites and cysts at concentrations used in commercially available products (Hamad 2023, Wekerle et al. 2020).

Studies on OTC’s mechanism of action have shown that first point of its attachment in Gram-negative bacterial cells is the outer membrane. As a cationic molecule, OCT has a high affinity for anionic bacterial surface components, e.g. lipopolysaccharides. Due to electrostatic interactions, OCT binds to the surface of E. coli cells and then penetrates through the lipopolysaccharide (LPS) layer into the interior of the outer membrane. Analysis of zeta potential changes in response to increasing OCT concentrations revealed that neutralization of the negative surface charge of cells occurs already at a very low concentration of octenidine (10⁻⁶ %). At this stage, no inhibition of bacterial growth was observed, which indicates that surface neutralization is the first step of action, but not sufficient to kill the cell. The hydrocarbon chains of OCT rapidly interact with the hydrophobic core of the outer membrane, leading to its significant disruption through the so-called hydrophobic mismatch. As a result, subsequent OCT molecules can penetrate deep into the bacterial cell, reaching the inner membrane. To confirm that OCT also interacts with the inner membrane, a depolarization assay with the membrane potential-sensitive dye, i.e. 3,3′-dipropylthiadicarbocyanine iodide (DiS-C₃-5), was used. Upon disruption of the membrane integrity, the dye is released, which causes an increase in the fluorescence signal. Application of 0.0001% OCT induced a rapid increase in fluorescence, indicating that OCT effectively depolarizes the inner membrane. As a consequence, the integrity of both the outer and inner membranes is disrupted by OCT, leading to cell lysis (Malanovic et al. 2020).

Ponnachan et al. (2019) investigated the effect of OCT on yeast cells. They showed that OCT affects C. auris cell integrity in a concentration-dependent manner, leading to its damage and, at higher concentrations, full disintegration. Electron microscopy studies showed that after 6 hours of incubation with 1 μg/ml octenidine resulted in a reduction of the cell envelope of C. auris (clinical isolates), which suggests the beginning of cell disintegration. As the octenidine concentration increased to 2 μg/ml and 5 μg/ml, the cell structure was increasingly damaged. More serious damage to yeast cells, leading to leakage of their contents, was visible after 24 hours of incubation at a concentration of 2 μg/ml, and complete lysis occurred at a concentration of 5 μg/ml. OCT also exhibits antifungal activity against C. albicans (Fang et al. 2023). At 1 μM, a cells reduction of 3.22 log₁₀ was observed, while 2 μM caused a 5.32 log₁₀ reduction. At 4 μM, OCT completely eliminated C. albicans cells and inhibited biofilm formation by 92.54%, but mature biofilms were eradicated by 71.88%. At 8 μM, mature biofilms were completely removed.

In the case of S. aureus, 1 mM OCT reduced planktonic cells by > 3 log₁₀ and 2 mM OCT led to complete eradication. It also inhibited biofilm formation and removed mature biofilms at 5 mM and 10 mM concentrations (Amalaradjou and Venkitanarayanan 2014). OCT was also effective against P. aeruginosa biofilms after 30 minutes of exposure to a 0.1% solution (Junka et al. 2014). A. baumannii biofilm was completely eliminated by OCT after 5–10 minutes of exposure to a 0.9% (15 mM) and 0.6% (10 mM) solutions, respectively (Narayanan et al. 2016).

OCT effectiveness tests were also carried out in accordance with EN standards including additional pathogenic species. In the medical area, ENs require testing only on C. albicans (effectiveness against yeast), and on S. aureus, P. aeruginosa and E. coli (effectiveness against bacteria). The fungicidal assay by quantitative suspension tests were performed according to EN 13624 (a phase 1 step 1), including MDR yeast C. auris (Gugsch et al. 2024). The yeast-killing efficacy of OCT-impregnated washing mitts was demonstrated at concentrations of 80%, 50% and 10% against three Candida species tested (C. auris DSM 21092, C. auris DSM 105986 and C. albicans ATCC 10231) after a 30-second contact time under low organic load conditions. At lower concentrations, C. albicans showed greater resistance compared to C. auris. At 1% concentration, C. auris strains achieved > 4 log₁₀ reductions, with C. albicans showing 2.19 log₁₀ reductions. An OCT concentration of 0.5% proved to be ineffective against both C. auris strains (Gugsch et al. 2024).

In a study conducted in accordance with EN 13727, a bactericidal effect of OCT was demonstrated. Exposure to a 0.01% OCT solution led to a > 5 log₁₀ reduction in clinically relevant bacterial species, including E. coli, K. pneumoniae, E. cloacae, A. baumannii, and P. aeruginosa, within just 1 minute of contact in both clean and dirty conditions. In addition, a concentration of 0.0001% OCT required a longer exposure time of 2.5 minutes to achieve a reduction of > 5 log₁₀ (Alvarez-Marin et al. 2017).

The antiseptic preparation containing 0.1% OCT and 2% phenoxyethanol showed significant antimicrobial efficacy in the test conducted using the quantitative suspension method based on EN 13727. After 30 minutes of exposure, a 4.77 log₁₀ reduction in P. aeruginosa DSM-939 was observed with 0.3 ml of the solution, as well as a 6.18 log₁₀ reduction in both S. aureus DSM-799 and the clinical MRSA strain. Additionally, when the tested volume of the antiseptic solution was increased to 1 ml, complete elimination of MRSA and P. aeruginosa biofilm was observed within 72 hours (Rembe et al. 2020). The study conducted according to EN 13727 showed that the application of a solution containing 0.1% OCT and 2% phenoxyethanol (Octenisept) to wound exudate reduced the number of microorganisms by at least 5 log₁₀ in just 15 seconds. Meanwhile, a wound irrigation solution with 0.05% OCT (Octenilin) also demonstrated antimicrobial efficacy but required 30 seconds of contact to achieve a similar level of bacterial reduction (Augustin et al. 2023).

OCT is commonly available as a 0.1% solution or aerosol, usually in combination with 2% phenoxyethanol, e.g. on the product Octenisept. It’s used for skin disinfection before surgical procedures, as well as for managing wounds, mucous membranes, and conditions in the oral cavity. The antimicrobial efficacy of Octenisept is the result of the activity of both ingredients. OCT can also be formulated with 1-propanol or 2-propanol. Lozenges containing 2.6 mg of OCT are used in the treatment of inflammatory conditions of the oral cavity. A summary of commercially available products, including their concentrations and indications, is presented in Table I. In the case of chronic wounds, it is recommended to use products containing 0.05% OCT, which are widely available in the form of gels or rinsing solutions, often enriched with a surfactant such as ethylhexylglycerin. The gel formulation is especially useful for antiseptic treatment in burn victims, exhibiting greater efficiency than silver and iodophores in these instances. A solution of 0.1% OCT and 2% phenoxyethanol is efficacious for the management of acute, contaminated, and traumatic wounds, including those colonized by MRSA (Kramer et al. 2018). The alcohol-based skin disinfectant containing OCT (propan-1-ol 30%, propan-2-ol 45%, octenidine dihydrochloride 0.1%) demonstrates greater effectiveness in reducing and preventing microbial recolonization around the insertion sites of central venous catheters and extracorporeal catheters compared to the disinfectant containing propan-2-ol (63%) and benzalkonium chloride (Lutz et al. 2016). OCT is an important component of strategies to prevent hospital infections and improve patient safety in intensive care units (ICUs). The use of 0.08% OCT impregnated wipes for patient bathing has been shown to be effective in the prevention of primary bacteremia associated with ICU stay (Schaumburg et al. 2024).

OCT-based antiseptics are used for a much shorter period of time than CHX-based antiseptics. Therefore, the molecular basis for the decrease in bacterial and fungal susceptibility to OCT is less well understood. Recently, such analyses have been conducted on strains from the Enterobacterales order. Studies have shown that K. pneumoniae can adapt to increased exposure to OCT by mutations in the SmvA pump (A363V, L364Q, Y391N, A363T, A368T, A474V) belonging to the MFS family (Wand et al. 2019). The AdeABC efflux pump of the RND family has been reported to extrude OCT from the bacterial cell in A. baumannii (Meyer et al. 2022). Efflux pumps that actively remove disinfectants from bacterial cells to the outside are summarized in Table II. A single nucleotide deletion in K. pneumoniae was also found in the genes encoding the RamR protein, which belongs to the TetR/AcrR family of transcriptional regulators. In Klebsiella oxytoca, mutations were identified in the gene encoding the Bm3R1 protein, which also belongs to the TetR/AcrR family and in E. cloacae in the gene encoding OmpX, a precursor of the outer membrane protein (Lescat et al. 2021).

Iodophors are compounds that release iodine, created by combining iodine with a solubilizing agent in water-based solutions, as iodine itself is unstable in water. One common example of an iodophor is povidone-iodine (PVP-I). Povidone-iodine is created by combining iodine molecules with polyvinylpyrrolidone (PVP), which makes it water-soluble. In this compound, PVP functions as a carrier for iodine, allowing it to absorb and transport iodine without chemically reacting with it. The iodine itself is the active ingredient in PVP-I (Babalska et al. 2021). PVP facilitates the release of free iodine near the cell membranes of microorganisms, which then penetrates the membrane, causing its damage and loss of structural integrity. After entering the cell, iodine denaturants the structure of nucleic acids and disrupts the basic energy processes of the cell, such as electron transport, cellular respiration, and protein synthesis. These cell function disorders ultimately lead to cell death (Williamson et al. 2017). The more diluted the PVP-I solution, the higher the concentration of free iodine in it. This occurs because dilution weakens the binding of iodine to its carrier. Consequently, solutions with lower concentrations (around 0.1–1%) tend to act faster and are more effective at killing bacteria compared to those with higher concentrations, such as the 10% solution (Babalska et al. 2021). Determination of the efficacy of PVP-I at different pH according to EN 27027 and the National Committee for Clinical Laboratory Standards M27-A2 showed that with increasing pH, the antibacterial efficacy of 10% PVP-I was significantly reduced against S. aureus and P. aeruginosa (Wiegand et al. 2015). The presence of organic compounds, such as bovine serum albumin, can also diminish the effectiveness of PVP-I as a disinfectant. Studies have shown that the presence of albumin leads to a reduction in the antibacterial efficacy of PVP-I. Specifically, an albumin concentration of 0.01875% caused a decline in the antibacterial activity by PVP-I (Kapalschinski et al. 2017).

PVP-I exhibits broad-spectrum antimicrobial activity, targeting a wide range of microorganisms, including both Gram-positive and Gram-negative bacteria, Mycobacterium, fungi (i.e. Candida and Trichophyton species), and protozoa. With prolonged exposure, it also demonstrates activity against spores and various viruses, such as multiple strains of the Influenza virus and Ebola virus (Lachapelle et al. 2013; Williamson et al. 2017).

In the case of a variety of clinical Candida spp. isolates, including C. albicans associated with vulvovaginal candidiasis, PVP-I at an 8% concentration demonstrated significant fungicidal activity in a test conducted according to EN 1275 (phase 1). After 60 minutes of exposure, 8% PVP-I eliminated all Candida spp. isolates, achieving a reduction of ≥ 4 log₁₀ (Hacioglu et al. 2022). Furthermore, a quantitative suspension test by EN 13624 (phase 2, step 1) was used to evaluate yeasticidal activity, showing that 10% PVP-I demonstrated very high efficacy against C. auris, reducing the yeast count to > 4.5 log₁₀ within 2 minutes of contact. However, when tested against C. albicans ATCC 10231, this time was not sufficient (Moore et al. 2017). Şahiner et al. (2019) also evaluated the bactericidal and fungicidal efficacy of 7.5% PVP-I solution according to EN 13727 and EN 13624. P. aeruginosa ATCC 15442 and E. coli K12 NCTC 10538 showed high sensitivity, reaching a bacterial cells reduction of more than 5 log₁₀ after 1 minute of exposure, regardless of conditions. In contrast, S. aureus ATCC 6538 required 5 minutes to reach the same reduction level in dirty conditions, while in clean conditions, effectiveness was observed after 1 minute. C. albicans ATCC 10231 showed a similar trend: the required 4 log₁₀ reduction was reached after 5 minutes of exposure under dirty conditions, and after 1 minute under clean settings. Further, no fungicidal activity was observed against A. brasiliensis in either clean or dirty conditions, as the log reduction remained below the required threshold after 1 and 5 minutes of exposure.

PVP-I is available in various formulations, including antiseptic ointments, solutions, and gels, most commonly in 7.5% and 10% concentrations. These preparations are used for the treatment of burns and wounds, as well as for preoperative skin disinfection and surgical hand scrubbing. A summary of commercially available products, including their concentrations and indications, is presented in Table I.

Clinical trials have shown that a combination of 1% PVP-I (containing 10% free iodine) and 50% isopropyl alcohol is as effective as 2% CXG in 70% ethanol in preventing surgical site infections following cardiac and abdominal surgeries (Widmer et al. 2024). Similarly, no significant difference was observed in infection risk reduction between 5% PVP-I in 69% ethanol and 2% CHX in 70% isopropanol for cardiac surgery patients (Boisson et al. 2024).

PVP-I-based mouth rinses are considered a valuable protective tool against infections in the oral cavity and respiratory tract. Tests conducted according to the bactericidal quantitative suspension test EN 13727 demonstrated that a 0.7% povidone-iodine solution, diluted to 0.23% (1:30 dilution), exhibited significant bactericidal activity against K. pneumoniae DSM 16609 and Streptococcus pneumoniae ATCC 49619, achieving a cells reduction of over 5 log₁₀ within just 15 seconds of exposure. Compared to plain soft soap, the scalp and skin cleanser containing 7.5% PVP-I is proven to be more effective in eliminating E. coli and mouse norovirus (MNV) (Eggers et al. 2018b).

PVP-I-based antiseptic products are also effective in preventing and eradicating microbial biofilms. Research has demonstrated that C. auris biofilms exhibit increased tolerance to PVP-I as compared to planktonic cells. PVP-I concentrations in the range of 1.25–2.5% were required to inhibit the biofilms growth after 5 min of exposure. Prolonged exposure to 10–30 minutes reduced required concentrations to 0.625–1.25%. The highest efficacy in eliminating biofilms was demonstrated by a 10% PVP-I, which completely destroyed all stages of the biofilm (Kean et al. 2018). Also, a 10% PVP-I solution demonstrated high efficacy in eliminating S. aureus biofilm, achieving a 99% reduction after 30 minutes of exposure (Guimarães et al. 2012). The efficacy of PVP-I in eliminating MSSA and MRSA biofilm from titanium surfaces was assessed. Irrigation for 3 minutes with a PVP-I solution at a concentration of 0.8% for MSSA and 1.6% for MRSA resulted in a ≥ 99.9% reduction of biofilm (Semeshchenko et al. 2025). Overnight incubation with subinhibitory concentrations of PVP-I (0.17%, 0.35%, 0.7%) suppressed the ability of S. epidermidis 1457 and S. aureus RN4220 to form biofilms. In S. epidermidis, this inhibition was due to an increase in the level of icaR, a transcriptional repressor of the icaADBC operon, which is responsible for the production of polysaccharide intercellular adhesin (PIA). In S. aureus, no correlation was found between reduced icaADBC operon and icaR gene expression (Oduwole et al. 2010). A 10% PVP-I effectively reduced the number of viable cells in both single-species biofilms (C. auris NCPF 8973, S. aureus NCTC 10,833, S. epidermidis ATCC 35984) and multi-species biofilms (C. auris + S. aureus, and C. auris + S. epidermidis), reducing their numbers by more than 4 log₁₀. The presence of Staphylococcus spp. in mixed biofilms did not improve the ability of C. auris to persist under PVP-I exposure, indicating its high efficacy against multi-species biofilms (Gülmez et al. 2022). On the other hand, 7.5% PVP-I did not demonstrate full efficacy in eradicating P. aeruginosa biofilm (Junka et al. 2014). After 15 minutes of exposure, a 15% reduction in biofilm was noted, while after 30 minutes the efficacy increased to 66%.

The activity of PVP-I against viruses is extremely important. PVP-I can be employed as a nasal spray or nasal irrigation for the nasopharyngeal clearance of the SARS-CoV-2 virus in patients with COVID-19. Among various concentrations, a 0.5% solution used for nasal irrigation has shown the greatest effectiveness, while among nasal sprays, the best results were observed with the 0.6% solution (Arefin et al. 2022). PVP-I demonstrates excellent virucidal activity against the Ebola virus. PVP-I formulations, including 4% skin cleanser, 7.5% surgical scrub, 10% PVP-I solution, and 3.2% PVP-I in 78% alcohol, significantly decreased EBOV virus titers, achieving a cells reduction ranging from 5.66 to 6.84 log₁₀ after 15 seconds of application (Eggers et al. 2015). Furthermore, inactivation tests conducted according to the virucidal quantitative suspension test EN 14476 demonstrated that 0.23% PVP-I solution effectively inactivated SARS-CoV, MERS-CoV and influenza A virus (H1N1) (Eggers et al. 2018a).

Among alcohols, ethanol and isopropanol (propan-2-ol, 2-propanol) are most commonly used as antiseptics. They are effective against Gram-positive and Gram-negative bacteria, Mycobacterium, yeasts, and molds (Williamson et al. 2017; Stauf et al. 2019). Ethanol is capable of inactivating all enveloped viruses, including Coronaviridae, Herpes, Vaccinia, and Influenza viruses, as well as several non-enveloped viruses such as Adenovirus and Rotavirus. In contrast, isopropyl alcohol is ineffective against non-enveloped viruses like Adenovirus but remains effective against lipid-enveloped viruses, including coronaviruses (Parikh and Parikh 2021). However, neither ethanol nor isopropanol eliminate bacterial spores. The optimal bactericidal efficacy is noted during the 60%-90% concentration ranges, with a significant reduction in effectiveness occurring when concentrations fall below 50% (Williamson et al. 2017). Alcohols exert their antimicrobial effects by denaturing and coagulating proteins, which leads to a loss of structural integrity of cell membranes. This results in increased membrane permeability, which is manifested by leakage of intracellular components. As a result, cellular processes, including metabolic functions and enzyme activity, are impaired. Ultimately, this cascade of events causes cell lysis (Elekhnawy et al. 2020).

The antimicrobial efficacy of alcohol depends on the specific conditions under which it is used. The presence of viscosity-increasing substances can hinder alcohol penetration into microbial cells, reducing its disinfectant effectiveness. For example, in mucus samples (both artificial and sputum), the bacterial survival rate exceeded 10% after application of an alcohol-based disinfectant, indicating significantly compromised antibacterial effectiveness. Additionally, ethanol diffusion ability into mucus was inversely related to its viscosity, which was associated with increased bacterial resistance (Hirose et al. 2017). Ethanol is widely used in professional disinfection practices in both healthcare and veterinary settings. A summary of commercially available products, including their concentrations and indications, is presented in Table I. In healthcare facilities, a solution of 69% ethanol combined with 5% PVP-I has been shown to be effective for skin antisepsis prior to surgical procedures, for example cardiac surgery (Boisson et al. 2024). In veterinary medicine, for instance, 74.1% ethanol mixed with 10% propan-2-ol is used for skin antisepsis in dogs prior to medical procedures (Eigner et al. 2023). No instances of alcohol tolerance have been observed in bacteria like staphylococci and streptococci, nor have any mechanisms of acquired alcohol resistance been discovered (Williamson et al. 2017).

Hand sanitizer gel and foam containing 70% ethanol demonstrated high antimicrobial efficacy in in vitro time-kill tests according to ASTM E2783-10. At 15 seconds of contact, S. marcescens reduction was > 5.8 log₁₀ (gel) and > 4.7 log₁₀ (foam), and MRSA reduction was > 5.8 log₁₀ (gel) and > 4.2 log₁₀ (foam). ASTM E1174 testing has confirmed the effectiveness of these products. After the first application, a reduction of at least 2 log₁₀ in microorganism count was observed, and after the tenth application, the reduction reached at least 3 log₁₀, for both 5 ml and 2 ml volumes (Edmonds et al. 2012).

Bactericidal activity against enterococci Enterococcus hirae ATCC 10541, E. faecium ATCC 6057 and Enterococcus faecalis ATCC 47077 was assessed in accordance with EN 13727. After 5 min exposure to 40% ethanol significant differences in species tolerance were observed. E. faecium and E. faecalis showed the lowest susceptibility, with reductions of only 1.24 and 4.11 log₁₀, respectively. On the other hand, E. hirae showed the highest sensitivity at 40% concentration, with cells reduction of 7.31 log₁₀. Ethanol concentrations of 50% or higher consistently resulted in reductions of at least 5 log₁₀ after just 30 seconds of exposure (Suchomel et al. 2019).

The fungicidal activity of ethanol was tested in a quantitative suspension test, according to EN 13624. Reference strains were included: C. albicans ATCC 10231, Candida tropicalis ATCC 13803, A. brasiliensis ATCC 16404, and Aspergillus niger ATCC 6275, as well as clinical antifungal-resistant isolates. After 1 minute of exposure, ethanol at 50% concentration showed efficacy against yeasts, achieving ≥ 4.0 log₁₀ reduction, while an 80% concentration was effective against molds (Stauf et al. 2019).

The effect of alcohol solutions on biofilm formation depends on the bacterial species and alcohol concentration. In one study, a comparison of 41 ethanol concentrations from 0% to 20% revealed that low concentrations stimulated S. aureus biofilm formation, with the highest biofilm stimulation noted at 7% ethanol. Biofilm formation then gradually decreased with increasing ethanol concentration up to 20%. Furthermore, extending incubation from 24 to 48 hours increased biofilm production (Vaezi et al. 2020). Importantly, higher concentrations of ethanol, starting from 30% and upwards, reduce the ability to form biofilms. Alonso et al. (2018) showed that therapy with both concentrations of 40% and 70% ethanol almost 100% reduced metabolic activity in 72-hour biofilms of S. aureus ATCC 29213, S. epidermidis (clinical isolate), E. faecalis ATCC 33186, C. albicans ATCC 14058, and E. coli ATCC 25922. However, 70% ethanol was more effective against 48-hour biofilms.

Similarly, exposure to subinhibitory concentrations of ethanol (1/4 MIC, 2.5% and 1/2 MIC, 5.0%) significantly increased the ability of Salmonella Enteritidis to form biofilm, with a stronger effect observed at 5.0%. This suggests that sublethal ethanol stress may trigger mechanisms that promote biofilm development. It was examined whether there were changes in attachment genes (adrA, csgB, csgD), quorum sensing genes (luxS, sdiA), and sRNAs (ArcZ, CsrB, OxyS, SroC). Expression analysis showed that the luxS gene was significantly upregulated, with 2.49-fold and 10.08-fold increases at 2.5% and 5% ethanol, respectively. The remaining genetic elements examined did not alter their activity in response to ethanol exposure. Similarly, in the case of P. aeruginosa, ethanol at concentrations of 1% and 2% increased biofilm formation (He et al. 2022).

In tests conducted according to EN 13727 and EN 13624, isopropanol at a concentration of 70% has been shown to have an effective bactericidal and fungicidal effect, regardless of the presence of organic substances. The preparation provided a reduction of > 5 log₁₀ for bacteria (S. aureus ATCC 6538, E. coli K12 NCTC 10538, P. aeruginosa ATCC 15442 and E. hirae ATCC 10541) and > 4 log₁₀ for fungi (C. albicans ATCC 10231 and A. brasiliensis ATCC 16404) after 1 and 5 minutes of exposure, in both clean and dirty conditions (Şahiner et al. 2019).

CHX is the most extensively studied antiseptic. Both Gram-negative and Gram-positive bacteria, as well as fungi, have been long-term exposed to CHX. CHX has sometimes been used as a reference antiseptic.

Zhang et al. (2019) found that prolonged exposure to CHX, using the increment method, increased the resistance of K. pneumoniae. In all three strains, the CHX MIC reached 128 μg/ml, and this adaptive resistance remained stable even after about 10 passages in CHX-free medium. Furthermore, the adapted to CHX strains developed cross-resistance to colistin. This CHX resistance was associated with higher expression of the cepA gene in all strains, whereas the qacE and qacE1 genes were not found. Additionally, all adapted strains carried mutations in PmrB, particularly Leu82Arg. The Leu82Arg mutation is suspected to play a key role in colistin resistance. What’s more, those strains had different growth rates than their wild-type counterparts. Similarly, another study has shown acquired cross-resistance to colistin in two CHX-exposed clinical strains of carbapenemase-producing K. pneumoniae: ST258-KPC3 and ST846-OXA48. After e xposure to chlorhexidine, the MIC of the tested strains increased 4-fold for ST258-KPC3 from 9.8 μg/ml to 39.1 μg/ml and for ST846-OXA48 from 19.5 μg/ml to 78.2 μg/ml. In addition, a 32-fold increase in the MIC of colistin was observed in strain ST846-OXA48. No differences in susceptibility were observed for the other antibiotics tested, as no changes in MIC values were detected. In the ST258-KPC3 strain, the expression of the smvA gene, which encodes the efflux pump, was increased (log₂ fold change: 3.635), while ST846-OXA48 was characterized by high expression of the pmrD (log₂ fold change: 2.36) and pmrK (log₂ fold change: 1.57) genes, which are related to lipid A synthesis. In the plasmid of the ST846-OXA48CA strain, a novel toxin/antitoxin system (PemI/PemK) was identified. It was further observed that expression of gene encoding the PemK toxin resulted in reduced biofilm formation (Bleriot et al. 2020). All microbial mutants obtained after exposure to chlorhexidine, changes in their antiseptic sensitivity and drug susceptibility profiles, and genotypic changes are listed in Table III.

Cross-resistance to colistin was found not only in K. pneumoniae but also in A. baumannii and P. aeruginosa strains using the gradient method (Hashemi et al. 2019). For K. pneumoniae the MIC value of colistin increased from 2 μg/ml to 16 μg/ml, for A. baumannii from 1 μg/ml to 16 μg/ml, and for P. aeruginosa from 1 μg/ml to 32 μg/ml. A potential mechanism of cross-resistance to colistin may result from LPS modification, which could be suggested by morphological changes in the obtained mutants. Colonies of A. baumannii and K. pneumoniae strains were characterized by irregular shape and rough surface, while colonies of P. aeruginosa had slightly rough structure and wavy edges. Differences in the protein composition of the resistant P. aeruginosa strain were detected. These involved increased overproduction of proteins including outer membrane porin F, LPS assembly protein LptD, TolPal system protein TolB, Tol-Pal system protein TolA, UDP-N-acetylmuramoylalanine-dglutamate ligase, lipid A deacylase PagL, Chaperone protein ClpB, Sec proteins, and efflux pump MexA (Hashemi et al. 2019).

Zheng et al. (2022) reported that P. aeruginosa mutants selected through exposure to CHX by gradient method exhibited 4- to 32-fold higher MICs. These mutants showed reduced susceptibility to multiple antibiotics, including imipenem, meropenem, levofloxacin, ciprofloxacin, ceftazidime, cefepime, and tobramycin. Reduced CHX susceptibility was linked to efflux pump activity. qRT-PCR showed significantly upregulated expression of mexA, mexC, mexE, and mexX, and downregulation of oprD gene (Zheng et al. 2022).

Tag ElDein et al. (2021) used two methods to assess the effect of CHX on the selection of P. aeruginosa strains exhibiting cross-resistance to antibiotics. Resistant strains were obtained using gradient method and by a single exposure to a lethal concentration of CHX. Of the 28 mutants, 12 showed at least an 8-fold MIC increase of CHX compared to the parent strain. All of these mutants exhibited a 2-fold to 4-fold MIC increase of amikacin. Furthermore, seven of them became resistant to meropenem (MIC change from 4 to 8 or to 16 μg/ml), while six shifted from full susceptibility to intermediate resistance to ciprofloxacin (MIC change from 1 to 2 μg/ml). Three mutants developed intermediate resistance to cefepime (MIC change from 4 to 16 μg/ml). In addition, some strains had higher MICs of amikacin, ceftazidime, and colistin yet remained susceptible to these antibiotics. Two of the obtained mutants demonstrated significantly decreased membrane permeability, whereas the remaining mutants showed increased permeability or no change. Exposure to 0.5 MIC of CHX resulted in an increase in mexX gene expression. In 7 out of 12 isolates, this increase was high, reaching up to a 43-fold change. In contrast, 2 isolates showed no overexpression of mexX following CHX exposure.

The study conducted by Gregorchuk et al. (2021) showed that E. coli after adaptation to increasing concentrations of CHX, using increment method, did not develop cross-resistance to any of the tested antibiotics. On the contrary, it resulted in increased susceptibility to tobramycin, with a reduction in the MIC from 16 to 4 μg/ml. They also showed increased susceptibility to antimicrobials, including QAC, cetyltrimethylammonium bromide, and cetyltrimethylammonium bromide. At the same time, as a result of this adaptation, three isolates showed reduced sensitivity to CHX, with their MIC increasing from 2- to 4-fold compared to the initial MIC value: changes from 2 μg/ml to 4 μg/ml in two mutants, and to 8 μg/ml in one mutant. Proteome analysis of the strain showing the highest phenotypic stability revealed changes in the abundance of many proteins, e.g. porin OmpF, lipid synthesis/transporter MlaA, efflux pump MdfA, proteins controlling acid resistance (GadE, CdaR), and antimicrobial stress-inducible pathways Mar-Sox-Rob. Scanning electron microscopy (SEM) imaging revealed that adaptation to CHX caused a change in cell shape, resulting in narrowing, and in 2/3 of the isolates, it also reduced average cell length. In addition, changes in the cell membrane were investigated using a fluorescent dye (propidium iodide) that does not pass through the membrane. The results demonstrated that strains adapted to CHX had more permeable cell membranes than the wild-type strain. These findings suggest that E. coli adaptation to increasing concentrations of CHX results in significant phenotypic changes that may be detected using both visual and fluorescence methods.

Wu et al. (2016) investigated whether subinhibitory exposure to the antibiotics, chlorhexidine and Rhizoma coptidis extract (RCE) induced cross-resistance or reduced susceptibility in Staphylococcus spp. including 14 clinical isolates and the reference strain S. aureus ATCC 25923. After exposure to sublethal concentrations of chlorhexidine, most isolates showed no major change in susceptibility, but six isolates showed a 4- to 8-fold increase in MICs, with MIC changes from 1.56-0.78 μg/ml to 6.25 μg/ml, respectively. S. aureus ATCC 25923 exhibited cross-resistance to tetracycline and cefepime (MICs changes from ≤ 1 μg/ml to 8 μg/ml). One isolate showed a > 512-fold increase in MIC of amikacin, tetracycline, and gentamicin. No significant change in susceptibility was observed for ciprofloxacin in 4 isolates, gentamicin in 5 isolates, amikacin in 2 isolates, cefepime in 3 isolates, and meropenem in 5 isolates. Additionally, 7 strains exhibited reduced sensitivity to RCE. Most strains exposed to sub-MIC tetracycline showed a 4-fold increase in MICs, except for one strain. S. aureus ATCC 25923 also developed resistance to ciprofloxacin and cefepime. Eleven S. aureus isolates exposed to tetracycline acquired cross-resistance to five additional antibiotics, while three developed resistance to two or three others. Reduced susceptibility to chlorhexidine and resazurin was observed in strains exposed to sublethal concentrations of tetracycline. After exposure to RCE, all tested strains were found to be more resistant to RCE, with MIC values increased by 4- to 32-fold. Most of them showed no significant change in CHX susceptibility, except for three isolates that showed a 4- to 8-fold MIC increase.

Renzoni et al. (2017) used CHX as a reference antiseptic in their polyhexanide study. Applying a stepwise exposure by increment method, culturing MRSA strains with increasing concentrations of CHX every two days for 7 to 10 passages, they obtained mutants with 2- to 4-fold increased antiseptic tolerance. In one of the obtained CHX mutants, point mutations led to amino acid changes in the MepA (an efflux pump protein) and PurR (a DNA-binding transcriptional repressor that regulates the expression of several genes involved in the synthesis, metabolism, and transport of purines) proteins. In the second CHX mutant, sequencing revealed mutations in genes (mprF, pldB, and glpD) involved in lipid metabolism resulting in amino acid substitutions. For the obtained MRSA mutants with reduced CHX susceptibility neither cross-resistance with polyhexanide nor with antibiotics was observed.

A total of 177 clinical isolates from early plaque colonizers were exposed to subinhibitory levels of CXG using the gradient method (Auer et al. 2022). These isolates included 112 Streptococcus spp., 19 Actinomyces spp., 20 Rothia spp., and 26 Veillonella spp. After exposure to the antiseptic, a 2-fold MIC increase was observed for Veillonella and Rothia isolates, a 2- to 4-fold MIC increase for Actinomyces isolates, and a 2- to 8-fold MIC increase for Streptococcus isolates. Only mutants showing an 8-fold MIC increase were used for further research. Among them there were mutants resistant to erythromycin and tetracycline, intermediate resistant to penicillin G and ampicillin, and intermediate or resistant to cefuroxime and amoxicillin/clavulanic acid. These isolates were further examined for the presence of antibiotic resistance genes. The antibiotic resistance genes as MefI and tetM were detected, which correlated with their phenotypic resistance to erythromycin and tetracycline, respectively. In addition, patA and patB genes were found, which are associated with resistance to the fluoroquinolone - moxifloxacin; however, the obtained mutants did not show resistance to this antibiotic. The presence of two genes encoding proteins involved in the transposition of the Tn916 transposon was also detected, namely int-II, responsible for the production of integrase, and xis-II, responsible for the production of excisionase. In addition, these strains showed an increased capacity for biofilm formation (Auer et al. 2022).

Früh et al. (2022) using the gradient method, examined the effect of repeated exposure to subinhibitory levels of chlorhexidine digluconate on supragingival plaque samples from six healthy volunteers. After 10 sequential passages in CXG, each time selecting the highest concentration still supporting growth, Streptococcus oralis and Granulicatella adiacens were isolated from the biofilm of these samples. Furthermore, G. adiacens exhibited a 4-fold CXG MIC increase and a 2-fold CXG MBC increase, whereas S. oralis showed a 2-fold MIC increase and a 4-fold MBC increase. The antibiotic susceptibility of these mutants was then assessed, revealing that S. oralis showed decreased susceptibility to erythromycin and increased MIC for clindamycin, amoxicillin, and ampicillin. On the other hand, G. adiacens showed reduced susceptibility to erythromycin and clindamycin, as well as increased MICs for penicillin G, tetracycline, cefuroxime, and ciprofloxacin. The study also showed that exposure to CXG for 10 days had no significant effect on the ability of S. oralis isolates to form biofilm, while in G. adiacens it led to increased biofilm formation.

Spettel et al. (2025) performed an in vitro study on the effects of long-term exposure of three biocides, CHX, OCT and triclosan, on 96 isolates of C. albicans and Nakaseomyces glabratus (formerly Candida glabrata) using the high-throughput modified increment method. These strains were exposed to increasing concentrations of each biocide for 60 days. No C. albicans strain showed changes in sensitivity to CHX, OCT and triclosan after long-term biocide exposure. However, for several N. galbratus strains, mutants with reduced sensitivity to CHX (4-fold increase in MIC values) and triclosan (from 4- to 16-fold increase in MIC values) were generated. Furthermore, long-term exposure to CHX, OCT, or triclosan did not induce antiseptic cross-resistance. On the other hand, after prolonged exposure to CHX and triclosan, N. glabratus mutants developed resistance to following azoles: fluconazole, posaconazole, voriconazole, itraconazole and isavuconazole, with a 4- to 512-fold increase in MIC values. Whole-genome sequencing of the azole-resistant N. glabratus mutants genomes revealed potential gain-of-function mutations in the transcription factor PDR1, which is responsible for the control of efflux pump genes expression, including Cdr1p, Cdr2p, and Snq2p genes. These mutations identified at positions D261Y, C469R, L936S, G943A, D1082G, and G1088E. Overexpression of the genes encoding these efflux pumps, Cdr1/2p and Snq2p, has previously been implicated as one of the mechanisms responsible for azole resistance. Furthermore, Spettel et al. (2025) demonstrated overexpression of the CDR1 efflux pump gene in these mutants. In other seven azole-resistant N. glabratus mutants that did not have changes in PDR1, mutations in the PMA1 gene were demonstrated. It is known that PMA1 plays a role of a major regulator of intracellular pH in fungi. The detected mutations may therefore lead to the loss of PDR1 functionality and, further, to a reduction in the intracellular cytosolic pH, which may result in a decrease in the fungal susceptibility to azoles. However, 4 of the 7 PDR1-mutants also showed overexpression of the efflux pump CDR1 gene.

Despite the studies undertaken on the exposure of bacterial and fungal strains to octenidine, only in a few cases mutants with reduced sensitivity to this antiseptic or showing cross-resistance to antibiotics were generated.

Garratt et al. (2021) examined how long-term exposure to OCT affects the sensitivity and development of resistance in Gram-negative bacteria present in the waste trap of a hospital sink. During the experiment, water samples were collected from the trap successively at time points T0, T28, T62, T97, and T118 days. After 28 days of exposure to OCT, an increase in tolerance was observed in P. aeruginosa (the MIC and MBC values increased from 4 μg/ml to > 64 μg/ml). In Citrobacter spp. isolates, a constant 2-fold increase in the MIC and MBC values was observed at subsequent time points starting at T62. Enterobacter spp. exhibited cross-resistance to ciprofloxacin, chloramphenicol and ceftazidime, while Citrobacter spp. were cross-resistant to ampicillin, piperacillin, ceftazidime, ciprofloxacin, chloramphenicol and meropenem. Additionally, it was examined whether these strains developed tolerance to cetylpyridinium chloride, hexadecylpyridinium chloride monohydrate, benzalkonium chloride, cetyltrimethylammonium bromide, triclosan, chlorhexidine digluconate, and cetrimide. The results showed that Enterobacter spp. became more tolerant to all tested biocides except benzalkonium chloride. Citrobacter spp. showed increased resistance to most tested biocides, while P. aeruginosa developed the greatest resistance to chlorhexidine. It was also analyzed whether exposure to octenidine influenced the growth rate. A reduction in growth was observed in Citrobacter strains isolated at later time points, while the growth rate of P. aeruginosa and Enterobacter spp. remained unchanged. Virulence testing in the Galleria mellonella model revealed a loss of virulence in Citrobacter spp., which was not observed in Enterobacter spp. and P. aeruginosa (Garratt et al. 2021). Table III lists and characterizes the microbial mutants obtained after exposure to octenidine.

Shepherd et al. (2018) assessed the effectiveness and implications of OCT exposure of P. aeruginosa strains in both laboratory and hospital settings. The first study group contained P. aeruginosa strains isolated from clinical materials of hospital patients. Adaptation of strains to increasing OCT concentrations was performed in laboratory conditions by the increment method, transferring the obtained cultures to new media with 2-fold higher OCT concentrations every two days for 2 weeks. The second group contained P. aeruginosa isolated from a hospital drain trap, exposed to 0.3% OCT bodywash solution four times a day for three months. Water samples for isolation of bacteria were taken from the drain trap at regular intervals. Only one of the first group of clinical strains exposed to OCT exhibited significant changes in antibiotic resistance: a 4-fold increase in gentamicin MIC (up to 32 μg/ml), a 2-fold increase for amikacin (up to 32 μg/ml), a 2-fold increase for tobramycin (up to 8 μg/ml), and a 4-fold increase for colistin (up to 4 μg/ml). This strain also showed an 8-fold increase in OCT MIC and increased tolerance to CHX. In a simulated hospital environment using an automated sink and drain system, an 8-fold increase in OCT MIC (from 4 μg/ml to 32 μg/ml) for the second group of P. aeruginosa isolates was recorded. However, after 10 days without biocide bodywash exposure, the OCT MIC values decreased and then returned to 32 μg/ml after 5 days of re-exposure (Shepherd et al. 2018).

On the other hand, Spettel et al. (2025) did not obtain fungal mutants with altered sensitivity to OCT when they exposed 96 strains of C. albicans and N. glabratus to this antiseptic for 60 days. Also, none of the strains changed the level of sensitivity to the tested azoles.

Shepherd and Parker (2023) investigated how repeated exposure to an antibacterial liquid handwash containing ethyl alcohol (Lifebuoy) can affect bacterial resistance to antimicrobials and potential cross-resistance to antibiotics. The test was conducted according with EN 1276. Exposure steps were performed repeatedly, reflecting consumer handwash use, over a 4–5 day period. The tested strains included S. aureus ATCC 6538, S. epidermidis ATCC 14990, E. coli ATCC 10536, E. hirae ATCC 10541 and P. aeruginosa ATCC 15442. It was shown that, even at a 1/100 dilution and a brief handwashing contact time of 10 seconds, the tested strains were unable to survive eight repeat-exposures. In general, repeated exposure to liquid soap did not cause significant changes in antibiotic susceptibility (Table III).

Barakat et al. (2022) studied long-term exposure to two commercially available biocidal preparations on the potential development of antiseptic and antibiotic resistance in S. aureus ATCC 25923. The first product contained 10% w/v PVP-I and the second named PBM was a mix containing 45% w/w 1-propanol, 30% w/w 2-propanol, and 0.2% w/w mecetronium ethyl sulphate. The exposures were performed using increment method. After 10 passages, only a 2-fold increase in PVP-I MIC was noted (from 5,000 μg/ml to 10,000 μg/ml), which decreased to 5,000 μg/ml after another five passages in medium without antiseptic. When the strain was exposed to PBM, a obtained mutant showed a 128-fold increase in PBM MIC (from 664 μg/ml to 85,000 μg/ml) and was still stable after five subsequent passages in medium without biocide. This mutant acquired cross-resistance to cefoxitin, penicillin, ciprofloxacin, and intermediate-level resistance to clindamycin (Table III). Furthermore, the vancomycin MIC value of the PBM-resistant mutant increased 4-fold but the mutant still remained sensitive to this antibiotic.

In additionally, Barakat et al. (2022) the effects of short-term exposure to subinhibitory concentrations (1/4 and 1/2 MIC) of two commercially available biocidal preparations on the potential development of virulence in both S. aureus ATCC 25923 and PBM-resistant mutant was investigated. Subinhibitory concentrations of PVP-I (1/4 and 1/2 MIC) significantly reduced hemolysin activity (by 7% and 0.28%, respectively) and completely inhibited biofilm formation only in the case of S. aureus ATCC 25923. In contrast, subinhibitory concentrations of PBM led to a non-significant decrease in hemolysin activity and a moderate reduction in biofilm activity in both strains. Moreover, the 1/2 PVP-I MIC value significantly downregulated in S. aureus ATCC 25923 the expression of hla gene responsible for alpha-hemolysin activity, and the following biofilm formulation genes: ebps, eno, fib, icaA, and icaD.