Bacteria with multidrug resistance (MDR) have become a serious threat in the clinic. This is especially true for opportunistic pathogens, which have high natural (intrinsic) resistance, and isolates with rapidly acquired MDR. Pseudomonas aeruginosa, a gram-negative, rod-shaped microbe, is one of the predominant pathogens in healthcare-associated infections due to its biological flexibility and can be considered a prime example of adaptability among opportunistic pathogens (Moradali et al., 2017; Sathe et al., 2023; Sanya et al., 2023; Theuretzbacher et al., 2020; Oliver et al., 2015; Pang et al., 2019; Brüggemann et al., 2018; Murray et al., 2015; Ruffin and Brochiero, 2019).

P. aeruginosa is an unpretentious, versatile, and ubiquitous microbe. It can literally survive without food, not only persisting but also multiplying in distilled water due to minimal contamination. It is omnivorous, with even antimicrobial drugs serving as a source of nutrients. It can grow and multiply within a wide temperature range, from 4 to 42 °C. Being oxidase-positive, it uses oxygen as an electron acceptor but can also grow and reproduce in the absence of oxygen, where nitrate serves as the final electron acceptor, or it retains the ability to microaerobically respire (Moradali et al., 2017; Diggle and Whiteley, 2020; Favero et al., 1971; Liao et al., 2022; Nolan and Behrends, 2021).

Its “basic settings” are perfect and universal, but its initial strategy of existence and survival is not so aggressive toward humans: P. aeruginosa is a saprophyte that lives freely in water, soil, and can be part of the human and animal microbiome. This is true as long as we have the immune status of a healthy person. As soon as P. aeruginosa is able to colonize a niche, its adaptive base allows it to implement a huge range of virulence factors, even showing contact-dependent secretion of toxins directly into target cells through the type 3 secretion system (Moradali et al., 2017; Sanya et al., 2023; Diggle and Whiteley, 2020; Liao et al., 2022; Nolan and Behrends, 2021; Dasgupta et al., 2006; Goldberg et al., 2022; Balasubramanian et al., 2013).

As an opportunistic pathogen, it is also a universal pathogen, as it is pathogenic to humans, vertebrates and invertebrates, and phytopathogenic. The epidemiologically significant reservoir of hospital-acquired blue blood cell infection is medical and service personnel and patients themselves (Nolan and Behrends, 2021).

Infections caused by P. aeruginosa are very difficult to treat, as this microbe uses resistance mechanisms (intrinsic and acquired), forms and states of existence (planktonic and biofilm forms, persistent cell), which usually cause and lead to difficult-to-treat chronic infections (Sanya et al., 2023; Theuretzbacher et al., 2020; Driscoll et al., 2007; Lewenza et al., 2018).

A saprophytic bacterium has turned into a clinical nightmare. Its resistance and minimal nutritional requirements determine the nearly universal presence of P. aeruginosa in hospital environments, creating ample opportunities for the emergence of nosocomial strains. Biofilm formation is also a key factor in the success of P. aeruginosa as a healthcare-associated pathogen. This is especially relevant in infections of the skin and soft tissues (Thuenauer et al., 2020; Behzadi et al., 2021; Kaiser et al., 2017; Rossi Gonçalves et al., 2017; Liew et al., 2019; Ruffin and Brochiero, 2019).

The situation has become significantly more complicated in Ukraine since the start of the full-scale war. The prevalence of multidrug-resistant Gram-negative bacteria is high among those with war-related injuries (Loban’ et al., 2023). War causes special injuries, including complex fractures with bone fragmentation, traumatic limb amputations, extensive deep burns, and severe soft tissue lacerations from artillery shells and mines. The situation is complicated by the rapid infection of wounds with explosive metabolites, dirt, and dust (Loban’ et al., 2023; Melwani, 2022). It is obvious that under such conditions, the patient’s life is the main priority on the front line, which requires the immediate use of antibacterial drugs without any testing. Until the wounded arrive at specialized medical facilities, medical care is provided directly in the combat zone and at all stages of temporary evacuation, which sometimes takes days and even weeks. In addition, throughout the entire evacuation chain, additional colonization of wounds by microorganisms, very often resistant to antibiotics, occurs. This forms a special group of resistant strains, characteristic specifically of war wound infections (Loban’ et al., 2023).

Infections caused by P. aeruginosa require special empirical and targeted antibiotic regimens, given its innate resistance to many classes of drugs and the ability to rapidly acquire resistance to current treatments (Karruli et al., 2023). And the treatment of biofilm infections is a serious problem, as there is currently no targeted therapy that can completely destroy biofilms in vivo (Sathe et al., 2023; Sanya et al., 2023; Kaiser et al., 2017; Rossi Gonçalves et al., 2017).

To overcome the growing problem of resistance and taking into account the biofilm status of the pathogen, an approach involving the use of a combination of antibiotics with alternative therapies will be necessary. Combination therapy has significant advantages over conventional antibiotic therapy, as the former exerts minimal selective pressure on P. aeruginosa and is therefore less likely to cause drug resistance. In wound care, comprehensive topical treatment is extremely important, with antiseptic agents being a key element (Liao et al., 2022; Goldberg et al., 2022; Kramer et al., 2018; Babalska et al., 2021; Murray et al., 2015; Schultz et al., 2017; Alves et al., 2023).

This article focuses on the activity of antiseptics as important means of combating P. aeruginosa MDR, their ability to effectively counteract the formation of biofilm and promote its eradication.

The emergence of multidrug resistance in bacteria has become one of the most dauntingchallenges of this century: the prevalence of infections that are difficult to treat is increasing, and there are no appropriate therapeutic alternatives. The scale of the problem has been identified by the political leaders of the G7 countries, who have expressed strong support for the first World Health Organization (WHO) Global Action Plan on Antimicrobial Resistance (AMR) [Rossi Gonçalves et al., 2017; Global Antibiotic Research and Development Partnership (GARDP), n.d.]. The global collaborative organization Joint Programming Initiative on Antimicrobial Resistance (JPIAMR) has engaged 29 countries in the fight against antimicrobial resistance, based on the One Health approach (JPIAMR, n.d.; CDC, 2019).

Infections caused by antibiotic-resistant P. aeruginosa are spreading steadily around the world due to its high internal resistance and ability to rapidly acquire resistance to all classes of antibiotics. The emergence of a specific resistance type of P. aeruginosa, namely the emergence of carbapenem-resistant (CRPA) strains, has attracted considerable attention from clinical microbiologists and infection control specialists (Moradali et al., 2017; Nolan and Behrends, 2021; Rossi Gonçalves et al., 2017; Rossolini et al., 2014; Oliver et al., 2015; Kovalchuk et al., 2024). The WHO recognizes carbapenem-resistant P. aeruginosa (CRPA) as a high priority pathogen for which antibiotic development is urgently needed (World Health Organization, 2024; Gergova et al., 2024). The emergence, spread, and persistence of multidrug-resistant bacteria, or “superbugs,” threatens human, animal, and environmental health as interconnected components of a single ecosystem (Davies and Davies, 2010; Aslam et al., 2021). P. aeruginosa MDR exists in a triangle-reservoir of animals, humans, and the environment, and there is interconnected coexistence of these pathogens within this triad. Numerous causes of “global resistance” contribute to the pressure of genetic selection and the emergence of bacterial MDR infections in society (CDC, 2019; Gergova et al., 2024; Van Boeckel et al., 2015; Aslam et al., 2018; Ahmad I et al., 2021; Crone et al., 2019; Balcázar et al., 2015; Abd El-Ghany, 2021).

P. aeruginosa is striking in the variety of pathology it causes, being the cause of a wide range of diseases - from intoxication to extensive purulent inflammatory processes and septic shock. Purulent complications of wound processes are very significant. In the general structure of wound infections, the P. aeruginosa bacterium occupies a significant place, being one of the most common bacteria (Ruffin and Brochiero, 2019; Wolcott et al., 2016; Phan et al., 2023). Pseudomonas aeruginosa infections in the epithelium of the skin, cornea and respiratory tract are the main cause of hospitalizations, disability and deaths worldwide (Ruffin and Brochiero, 2019). Along with S. aureus, K. pneumoniae, E. coli, and Acinetobacter spp., P. aeruginosa is among the leading superbugs that complicate the course of combat trauma (Mende et al., 2022; Weintrob et al., 2018; Petersen et al., 2007; Ford et al., 2017; Kvasnevska et al., 2024). One of the predictors of high mortality P. aeruginosa infections is multidrug resistance of the causative strain (Ruffin and Brochiero, 2019; Oliver et al., 2015), and infections caused by antibiotic-resistant P. aeruginosa are increasing worldwide (Nolan and Behrends, 2021; Rossolini et al., 2014; Oliver et al., 2015; Galdino et al., 2019; Pogue et al., 2020; Buehrle et al., 2016; Rubio et al., 2021; Kyaw et al., 2015). For example, Mareș, C. and colleagues reported an increase in antibiotic resistance in opportunistic pathogens due to the COVID-19 pandemic, and some of the highest rates of increase were observed for P. aeruginosa (Mareș et al., 2022).

The problem of P. aeruginosa infections requires a joint international interdisciplinary effort to translate current knowledge into strategies to prevent and treat P. aeruginosa infections, while reducing antibiotic resistance and avoiding the spread of resistant strains in nature, as patient sanitation is one of the key measures in efforts to break the epidemic chain by acting on the source of infection, thus preventing the spread of MDR strains (Moradali et al., 2017; Sanya et al., 2023; Theuretzbacher et al., 2020; Pang et al., 2019; Brüggemann et al., 2018; Rossi Gonçalves et al., 2017; Loban’ et al., 2023; Babalska et al., 2021). As correctly summarized by Kramer, A. and colleagues, wound antisepsis has experienced a renaissance due to the development of effective wound-compatible antiseptic agents, their bactericidal effect instead of bacteriostatic, the relatively high level of sensitization to topically applied antibiotics, also due to the pandemic spread of multidrug-resistant microorganisms, and, to the advantage, the absence (rarely) of resistance to those antiseptics that irreversibly damage pathogens (Kramer et al., 2018).

There are no generally accepted recommendations for the use of antiseptics for wounds. Regular monitoring (control) of sensitivity, correction of initial antiseptic concentrations with adjustment for multidrug-resistant strains of pathogens, and especially given the potential presence of such a widespread and resistant pathogen as pseudomonas are important and necessary. Suppression of the associated microflora with prolonged use of antibiotics sometimes leads to the fact that P. aeruginosa remains the only bacterial species in the infection site, impeding wound healing (Betchen et al., 2022; Kawamura et al., 2019). The results of our study indicate the high efficiency of modern antiseptics against MDR strains of P. aeruginosa. The MIC values of antiseptics (except for miramistin) against P. aeruginosa strains were always lower than the initial commercial concentrations. Certainly, the MBC for all microbicides were higher than their respective MICs, but the ratio of MBC/ MBS was less than 4, indicating that the products exhibit predominantly bactericidal properties (Betchen et al., 2022; Levison, 2004).

In recent studies by Barrigah-Benissan and colleagues, the MIC values for polyhexanide, povidone-iodine, and octenidine were also always lower than the original commercial concentrations (Barrigah-Benissan et al., 2022). Grzegorz Krasowski and colleagues determined the bactericidal concentrations of polyhexanide and octenidine at dilutions several tens of times below the threshold of the initial solution (Krasowski et al., 2021). Similarly, Rafael López-Rojas and colleagues previously found high activity of polyhexanide against clinical isolates of P. aeruginosa with the MDR phenotype at much lower concentrations than the initial ones (López-Rojas et al., 2017). Studies by Tomasz M. Karpiński characterized octenidine as a very effective drug against clinical wound isolates and reference strains of P. aeruginosa. However, for their sample of isolates, the MIC values for octenidine and polyhexanide did not differ from previous studies (López-Rojas et al., 2017; Karpiński, 2019; Koburger et al., 2010; Murray et al., 2015). We selected strains with the MDR phenotype, and the MIC and MIC values for the antiseptics studied were higher than they were in previous studies. The same trend, for example, was observed by Gupta, P. et al. for MDR of P. aeruginosa and povidone-iodine (Gupta et al., 2018). Vásquez, Daniel and colleagues also found high MICs and MBCs of chlohexidine in many home and hospital isolates of P. aeruginosa (Vásquez et al., 2017). In our previous similar studies concerning other MDR opportunistic pathogens (Ljungquist et al., 2023; Kovalchuk et al., 2024; Nazarchuk et al., 2024), we referred to the review by Jean-Yves Maillard and colleagues, whose analysis convincingly confirmed the decrease in the sensitivity of wound pathogens, including P. aeruginosa, to all biocides, which is associated with the spread of resistance (Maillard et al., 2021). At the same time, antibiotics have more resistance determinants than antiseptics and disinfectants, and gene expression under the influence of antimicrobial agents is not a good predictor of these resistance determinants (Murray et al., 2015; Schultz et al., 2017). Antiseptics act on multiple targets, inside and on the surface of the bacterial cell, unlike antibiotics (Krasowski et al., 2021; Assadian, 2016). The antiseptic activity index allows you to assess the effectiveness and appropriateness of the drug, and compare antiseptics with each other. The antiseptic concentration should be at least 4 MIC. We interpreted the results using the differential IAA index, focusing on the cidal activity of the antiseptic (Kramer et al., 2018). According to the results of the evaluation of the activity of drugs based on bacteriostatic IAA, povidone-iodine 10%, decamethoxine 0.1%, octenidine 0.1%, polyhexanide 0.1%, decamethoxine 0.02%, chlorhexidine 0.5%, povidone-iodine 2%, povidone-iodine 1% are effective. The concentration of miramistin 0.01% is not sufficient for use against MDR strains of P. aeruginosa. The concentration of the active ingredient of this drug is the lowest among those studied here. According to the cidal activity index, the most effective are povidone-iodine 10%, decamethoxine 0.1%, octenidine 0.1%, polyhexanide 0.1% (BC IAA = 14.05), chlorhexidine 0.5%, povidone-iodine 2%. The highest values of the indices were taken for povidone-iodine 10%. The ratio of BC IAA to BS IAA in favor of cidal action was the highest for polyhexanide. Our research and that of colleagues from other countries shows that antiseptics, including those tested in this study, are effective against planktonic bacteria. However, pathogenic bacteria are mostly found in biofilms, as this is their natural state (Rossi Gonçalves et al., 2017; Günther et al., 2021; Nazarchuk et al., 2019).

Therefore, biofilm elimination is important from a therapeutic point of view and for infection control (Rossi Gonçalves et al., 2017; Maurice et al., 2018). An effective antiseptic used for the treatment of colonized/infected chronic wounds should exhibit biofilm control properties (Krasowski et al., 2021; Schultz et al., 2017). The data are not yet clear on whether the MDR phenotype correlates with biofilm-forming properties (Rossi Gonçalves et al., 2017). Some researchers have noted an increased ability to form biofilm by P. aeruginosa strains (Magiorakos et al., 2012; Kaiser et al., 2017; Karballaei Mirzahosseini et al., 2020; Gurung et al., 2013). In any case, the biofilm is an important factor in the virulence of P. aeruginosa, the main form of its existence, which protects against the harmful effects of environmental factors, including biocides, and also contributes to the persistence and spread of MDR strains (Yin et al., 2019; Uddin et al., 2021). This was not the aim of our study, but it should be noted that the P. aeruginosa strains tested by MDR were characterized by high biofilm-forming capacity. The same was observed by Rossi Gonçalves I. et al., Bakht, M. et al., Sanchez, C et al. Behzadi, P. et al., Cepas, V. et al. point out that, indeed, strong biofilm producers are more common among clinical isolates, but MDR status or resistance to individual antibiotics does not imply an increased ability to form biofilms (Rossi Gonçalves et al., 2017; Bakht et al., 2022). This selection is logical, since biofilm-forming strains survive better and have a better chance of acquiring the determinants of acquired resistance. However, these are most likely not genetically linked traits.

Our studies of the effect of antiseptics on immature biofilm, i.e., their effectiveness in inhibiting biofilm formation, showed that all antiseptics have a high level of inhibitory capacity. Octenidine in sub-MIC concentrations showed the strongest effect on immature biofilm. Decamethoxine, polyhexanide, chlorhexidine, and povidone-iodine were next on the scale of effectiveness. A negative correlation was found between the ability of MDR strains of P. aeruginosa to form biofilms in the presence of subbacteriostatic concentrations of antiseptics and the susceptibility of these isolates to antiseptics. Thus, for the tested antiseptics from the group of detergents and halogen-containing compounds, the ability to inhibit biofilm formation depended on the concentration of the antiseptic, not on the sensitivity of P. aeruginosa isolates to them.

The sensitivity to antiseptics of cultures in mature biofilms was much lower. An effective effect on the formed biofilm requires much higher concentrations of antiseptics. It is much easier to inhibit or prevent its formation. The tested concentrations of antiseptics do not destroy the formed biofilm by more than 42.4%. The ability of the antiseptics to eradicate the biofilm depended on the concentration: the highest tested concentrations were the most effective, equal to half the concentration of the finished commercial product. Tracing the trend of octenidine action at different concentrations at different stages of biofilm formation, it should be noted that it is most active against P. aeruginosa biofilm. But, in general, it should be noted that all tested antiseptics are effective against P. aeruginosa biofilm. Junka A et al. also noted the high activity of octenidine and povidone iodine against biofilms of nosocomial P. aeruginosa strains (Sanchez Jr et al., 2013; Cepas et al., 2019; Junka et al., 2014). The results obtained by Grzegorz Krasowski et al. also indicate a high anti-biofilm activity of antiseptics based on polyhexanide and octenidine. The researchers note that antiseptics based on polyhexanide or octenidine are very useful for treating biofilm (Levison, 2004). Gryson, L et al. recently studied the anti-biofilm activity of povidone iodine and polyhexnide and reported that PVP-I and PHMB demonstrated sustained activity against biofilms in vitro, and PVP-I led to complete eradication of 3- and 5-day-old Pseudomonas aeruginosa biofilms (in ≤0.5 h) (Gryson et al., 2023).

There have also been important advances in the development of strategies for treating infections caused by P. aeruginosa and the use of combination therapy. Elodie Lefebvre et al. used a combination of polyhexanide, EDTA, and proteases in low concentrations, which had a synergistic effect that led to the complete eradication of dense P. aeruginosa biofilms (Lefebvre et al., 2016). Ciecholewska-Juśko D investigated the phenomenon of increasing the activity of an octenidine dihydrochloride-based antiseptic against Pseudomonas aeruginosa biofilms in the presence of a rotating magnetic field of two frequencies of 5 and 50 Hz. The authors noted that the combination of a rotating magnetic field and OCT may be particularly promising for the destruction of biofilms located in areas such as wound pockets, where physical obstacles limit antiseptic activity (Ciecholewska-Juśko et al., 2022).

P. aeruginosa has an innate resistance to many classes of drugs, the ability to form biofilms and, most importantly, the ability to quickly acquire resistance after treatment. One of the obvious unfortunate consequences of increased resistance to antimicrobial drugs is that bacteria are often treated at concentrations below their minimum inhibitory concentration (Nolan and Behrends, 2021). For example, in terms of biocides in general, Daniel Vásquez and colleagues note that hospitals with highly resistant strains of P. aeruginosa and A. baumannii with high drug resistance, it is necessary to review new formulations in cleaning and disinfection protocols (Vásquez et al., 2017). Also, Rasha Gharieb and colleagues report that carbapenem-resistant P. aeruginosa (CRPA) on intensive livestock farms is a serious problem that threatens animal and human health and increases the risk of P. aeruginosa infection in the community, so it is vital to control the spread of CRPA by limiting the use of antibiotics and applying proper cleaning and disinfection protocols on livestock farms (Ciecholewska-Juśko et al., 2022; Rasha Gharieb et al., 2021).

Regular monitoring of susceptibility, development of new therapeutic strategies against multidrug-resistant P. aeruginosa, correction of initial antiseptic concentrations with adjustment for multidrug-resistant strains and bacterial bloom status are relevant and important. The potential presence of such a resistant pathogen as P. aeruginosa should always be taken into account. It should be treated and prevented by following the “One Health” strategy.

The most active antiseptics against P. aeruginosa MDR are decamethoxin 0.1%, polyhexanide 0.1%, octenidine 0.1%, povidone-iodine 10%. The efficacy of miramistin 0.01% was found to be insufficient, as the IAA was below the threshold value (<4). Octenidine in sub-MIC concentrations demonstrated the strongest effect on immature biofilm (on its formation). The minimum bacteriostatic concentration of most antiseptics stimulated the development of mature biofilm. The bacteriostatic concentration of octenidine led to the eradication of mature biofilm by 4.7%. The MBC of most antiseptics (except chlorhexidine) led to eradication of mature biofilm by 4–30.6%. Chlorhexidine stimulated mature biofilm by 17.9%. Chlorhexidine showed the lowest activity against the formed biofilm at a concentration of half the initial concentration. But its initial concentration is half that of other detergents. The tested concentrations of antiseptics do not destroy the formed biofilm by more than 42.4%.

Tracing the trend of octenidine action at different concentrations at different stages of biofilm formation, its highest activity against P. aeruginosa biofilm should be emphasized. The results indicate the possibility of wider use of octenidine and decamethoxin for treatment of surgery wounds in patients with infection caused by MDR P. aeruginosa with possible recommendation for inclusion in wound infection treatment protocols.

The results of our study emphasize the importance of careful monitoring of P. aeruginosa isolates for antiseptic susceptibility. This will ultimately help prevent the creation of selective conditions for the emergence of resistant microorganisms and prevent their spread.