Disinfectant-induced bacterial resistance and antibiotic cross-resistance—mechanisms and clinical relevance

Abstract

The rise of antibiotic-resistant bacteria has become an alarming global health challenge. Disinfectants, such as alcohols, aldehydes, chlorine compounds, phenols, quaternary ammonium compounds (QACs), peroxides, and chlorhexidine, are widely used in healthcare settings as a critical line of defense against infection. Nevertheless, their overuse or misuse, especially at subinhibitory concentrations, can promote the emergence of bacterial resistance, potentially leading to cross-resistance to antibiotics. Several mechanisms, including efflux pump activation, alterations in membrane permeability, and biofilm formation, drive this process. A possible concern, although currently supported by limited and sometimes conflicting evidence—is that biocide-induced resistance might contribute indirectly to adverse clinical outcomes, such as treatment challenges, prolonged hospital stays, and increased healthcare costs. This review examines the molecular mechanisms of disinfectant-induced resistance, the epidemiological impact of multidrug-resistant (MDR) pathogens, and contemporary infection control strategies. Furthermore, the review evaluates the benefits and risks associated with disinfectant use, underscoring the necessity for optimized, evidence-based disinfection protocols to minimize the development of resistance while ensuring effective infection prevention.

Introduction

Despite significant advances in medicine, the growing incidence of multidrug-resistant (MDR) bacteria continues to pose a major global challenge. An increasing number of reports document bacterial strains resistant to three or more antibiotic classes [1–3]. Although inappropriate prescription practices and the excessive use of antibiotics in animal husbandry are well-recognized contributors to this phenomenon, there is a growing concern about the role of inappropriate disinfectant use in fostering resistance. Antimicrobial resistance (AMR) refers to the ability of bacteria to withstand the effects of one or more antimicrobial agents, thereby enabling their survival and proliferation despite the presence of antimicrobial concentrations previously effective in inhibiting their growth or inducing their death [4, 5]. Mechanisms of resistance include enzymatic inactivation of antibiotics, alterations in cell membrane permeability, enhanced efflux pump activity, and biofilm production, all of which confer protection against antimicrobial agents and facilitate the persistence of resistant bacteria. In certain bacterial populations, resistance arises from chromosomal mutations that enhance survival and facilitate adaptation to novel environments, thereby ensuring their persistence [6]. Moreover, resistance can be transmitted horizontally, involving the transfer of genetic material such as plasmids or transposons between bacterial cells. Such horizontal gene transfer significantly accelerates the dissemination of resistance both within and across microbial communities.

The emergence of antibiotic-resistant bacteria, colloquially termed “superbacteria,” constitutes a growing challenge for contemporary healthcare systems [7–9]. The term “superbacteria” describes bacterial strains that resist multiple, and in some cases all, conventional antibiotics. Their proliferation and dissemination represent a major obstacle to effective antibiotic therapy, as infections caused by these microorganisms are associated with high mortality rates due to their prolonged course and limited treatment options. A critical factor in this process is the selective pressure exerted by excessive, uncontrolled, and inappropriate antibiotic use in human medicine, animal husbandry, and agriculture. This promotes the gradual expansion of resistance to additional antibiotic classes, culminating in the emergence of multidrug-resistant (MDR), extensively drug-resistant (XDR), and, in the most severe cases, pan-drug-resistant (PDR) pathogens that are unresponsive to all available antimicrobial therapies [10, 11]. Notable examples include Pseudomonas aeruginosa (Gram-negative) aerobic (AE), oxidase (ox) +), Klebsiella pneumoniae (facultative anaerobe (FA), ox−, Gram-negative), and Acinetobacter baumannii (Gram-negative, AE, ox−). The One Health concept underscores the interconnection between human, animal, and environmental health. In the context of AMR, it emphasizes coordinated surveillance, judicious antimicrobial use, and rigorous environmental hygiene across clinical, veterinary, and community settings [12].

The biological characteristics, virulence factors, clinical picture, diagnostic methods, mechanisms of antibiotic resistance, and therapeutic strategies for selected microorganisms are presented in Table 1.

Table 1.

The biological characteristics of selected microorganisms in terms of virulence factors, clinical presentation, and mechanisms of antibiotic resistance

Microorganism	Pathogenicity	Virulence factors	Resistance mechanisms
Pseudomonas aeruginosa	Pneumonia (VAP, cystic fibrosis), UTIs, sepsis, wound infections, biofilm	Cilia and pili IV, LPS, exotoxin A, elastase, phospholipase C, pyocyanin, rhamnolipids, quorum sensing, biofilm	Inherent: low permeability, efflux pumps (MexAB-OprM, MexXY), AmpC; Acquired: ESBL, MBL (VIM, IMP, NDM), OprD mutations, gyrA/parC, aminoglycoside modification [13]
Acinetobacter baumannii	VAP, UTIs, sepsis, wound and burn infections, mainly in the ICU [14]	OmPA, LPS, capsule, phospholipases, proteases, siderophores, biofilm [15]	Congenital: low permeability, efflux pumps (AdeABC); Acquired: carbapenemases OXA-23/24/58, NDM, VIM, IMP, ESBL [16]
Klebsiella pneumoniae	Pneumonia, UTIs, sepsis, wound infections; hvKp—liver abscesses [17]	Capsule (K1, K2), LPS, fimbriae, siderophores, biofilm [18]	ESBL (CTX-M, SHV, TEM), carbapenemases (KPC, OXA-48, NDM, VIM, IMP), porin loss (OmpK35/36), efflux pumps (AcrAB-TolC) [19]
Escherichia coli	UTIs, sepsis, meningitis in newborns, diarrhea (EHEC, ETEC, EPEC, EAEC, EIEC) [20]	Fimbriae, LPS, hemolysins, Shiga toxins, and enterotoxins, biofilm	ESBL (CTX-M, SHV, TEM), carbapenemases (NDM, KPC, OXA-48), gyrA/parC mutations, aminoglycoside modification, efflux pumps [21]
Enterobacter cloacae	UTIs, VAP, sepsis, wound and catheter infections [22]	LPS, adhesive fimbriae, biofilm, siderophores	AmpC (overexpression), ESBL, carbapenemases (KPC, NDM, VIM, OXA-48), porin mutations (ompC/F), efflux pumps [23]
Serratia marcescens	UTIs, VAP, sepsis, wound and endocardial infections; newborns—meningitis and sepsis [24]	LPS, hemolysins (ShlA), proteases (serralysin), lipases, nucleases, siderophores, biofilm	AmpC, ESBL (CTX-M, TEM, SHV), carbapenemases (KPC, NDM, VIM, OXA-48), efflux pumps, porin mutations [24]
Proteus mirabilis	Complicated and catheter-associated UTIs, struvite urolithiasis, sepsis, wound infections, and, less commonly, pneumonia and meningitis [25]	Fimbriae (MR/P, PMF, UCA), LPS, urease, proteases, hemolysins (HpmA), biofilm, swarm motility	Inherent: resistance to tetracyclines and polymyxins; Acquired: AmpC, ESBL (CTX-M, TEM, SHV), carbapenemases (KPC, NDM, OXA-48), gyrA/parC mutations, efflux pumps [26]
Citrobacter freundii	UTIs, VAP, sepsis, wound infections, newborns—meningitidis and sepsis [27]	LPS, fimbriae, siderophores, biofilm, cytotoxins	AmpC (overexpression), ESBL (CTX-M, TEM), carbapenemases (KPC, NDM, OXA-48), porin mutations (ompC/F), efflux pumps [28]
Morganella morganii	UTIs, bacteremia, wound infections, cholangitis, rarely meningitidis, Fournier's gangrene [28]	LPS, adhesins, urease, biofilm	AmpC, ESBL (CTX-M, SHV), carbapenemases (NDM, KPC), reduced membrane permeability, efflux pumps, aminoglycoside modifications [28]
Staphylococcus aureus	Skin and soft tissue infections, pneumonia, bacteremia, meningitidis, sepsis, and toxic shock syndrome [29]	Adhesins (MSCRAMM), hemolysins, PVL, coagulase, superantigen toxins, biofilm	Penicillinases, PBP2a (MRSA), VISA/VRSA, efflux pumps, aminoglycoside modifications [30]
Enterococcus faecalis	Surgical site infections, wound infections, bacteremia, endocarditis, abdominal infections, sepsis [31]	Adhesins (Ace, Esp), hemolysins (CylA), gelatinase (GelE), biofilm, lipoteichoic acid (LTA)	Natural resistance to cephalosporins, low-affinity PBPs, VRE (vanA, vanB), and enzymatic modification of aminoglycosides [31]
Listeria monocytogenes	Listeriosis and opportunistic infections: sepsis, meningitis, and miscarriages [32]	Internalin (InlA, InlB), Listeriolysin O, ActA proteins	Resistance to multiple antibiotics is rare. Natural resistance to cephalosporins, susceptible to ampicillin, vancomycin, gentamicin, possibility of acquiring resistance through active efflux mechanisms and enzyme modification

*UTIs urinary track infections, VAP ventilator-associated pneumonia, LPS lipopolysaccharides, ICU intensive care unit, COPD chronic obstructive pulmonary disease, EHEC enterohemorrhagic E. coli, ETEC enterotoxic E. coli, EPEC enteropathogenic E. coli, EAEC enteroaggregative E. coli, EIEC enteroinvasive E. coli, ESBL Extended-spectrum beta-lactamases, MRSA methicillin-resistant S. aureus, VRE vancomycin-resistant Enterococcus

Given the escalating threat posed by antibiotic-resistant strains, preventive strategies and rational antimicrobial use are imperative.

This article aims to present the current state of knowledge regarding the potential relationship between the use of disinfectants and the development of bacterial cross-resistance to antibiotics. Particular emphasis is placed on the molecular mechanisms of resistance, including efflux pump activation, alterations in cell membrane structure, biofilm formation, and horizontal gene transfer. The study also explores the clinical and epidemiological consequences of MDR strains in healthcare settings, with a focus on treatment efficacy, prolonged hospitalization, and increased healthcare costs. Moreover, the article reviews contemporary infection control strategies aimed at limiting the spread of resistant pathogens and assesses both the benefits and potential risks associated with the use of disinfectants in medical environments. Ultimately, this work seeks to provide a foundation for optimizing disinfection practices as part of a broader effort to combat global AMR.

Implications for modern hospital infection control and prevention strategies

Disinfectants are widely employed in medical facilities to reduce the transmission of infections [33]. However, their inappropriate application, particularly in conjunction with antibiotics, may promote the development of microbial resistance. Consequently, resistant strains become increasingly difficult to eradicate using standard therapeutic approaches [34]. In Romania, at the National Clinical Hospital, Zlatian et al. conducted a study to assess the prevalence of invasive bacterial infections and the microbial resistance profile in hospitalized patients between September 2016 and July 2017 [35]. The results revealed that hospital-acquired infections exhibited a different etiological profile compared to out-of-hospital infections, with S. aureus (Gram-positive, FA) strains and non-fermenting bacilli such as A. baumannii and Burkholderia cepacia (Gram-negative, ox + , AE) predominating.By contrast, community-acquired infections exhibited a different distribution of etiological agents.

The diagnosis of infections caused by resistant bacteria is also challenging [36]. Traditional microbiological tests can be insufficiently sensitive and specific, delaying the implementation of appropriate treatment. For this reason, rapid molecular tests that allow precise identification of strains and selection of effective therapy are becoming increasingly important.

Antibiotic-resistant infections significantly prolong hospitalization [37, 38]. A prolonged hospital stay increases the risk of acquiring nosocomial infections and other complications. This is especially true for elderly patients, those with chronic diseases, or those undergoing immunosuppression. For these patients, infections can lead to severe complications, such as sepsis or multi-organ failure, which can be immediately life-threatening.

Extended contact with hospital infrastructure and staff also promotes pathogen transmission and the occurrence of cross-infection [39–41]. Hospital-acquired infections, such as pneumonia, surgical wound infections, or urinary tract infections, are often caused by bacteria resistant to standard antibiotics, making them even more challenging to treat and heightening the risk of severe complications [42, 43]. Antibiotic-resistant infections pose a major challenge both medically and economically [44–46]. Analyses conducted by the National Academies of Sciences, Engineering, and Medicine indicate that the increase in the incidence of treatment-resistant infections led to a significant increase in hospitalizations, longer average hospital stays, increased mortality, and a significant burden on medical infrastructure [47]. Globally, AMR is responsible for over 1.27 million deaths annually, and the total number of disability-adjusted life years (DALYs) lost due to it reaches many millions [48, 49].

The economic consequences of AMR are multifaceted (Table 2). They include not only the direct costs of treatment and hospitalization, but also indirect costs such as loss of workforce productivity, absenteeism among medical staff, the need to reorganize wards, and disruptions in the agri-food sector. Macroeconomic models, such as G Cubed and GTAP, indicate that uncontrolled growth of AMR could result in global gross domestic product (GDP) losses amounting to trillions of dollars annually.

Table 2.

Estimated economic impact of AMR and effectiveness of IPC strategies [48–50]

Aspect	EU	USA
Annual number of AMR infections	~ 0.7–0.9 million	~ 2.8 million
Annual number of deaths attributable to AMR	~ 31–39 k	~ 35 k (up to 48 k incl. C. difficile)
DALYs (disability-adjusted life years)	~ 0.9–1.1 million	no official Centers for Disease Control and Prevention (CDC) figure; Global Burden of Disease estimates several million in North America
Extension of hospital stay (LOS)	+ 6–20 days (depending on pathogen)	similar: several to > 10 days
Annual direct costs (health system)	~ 26–29 billion USD PPP	~ 20 billion USD (medical costs), ~ 55 billion incl. productivity losses
Estimated macroeconomic losses/GDP impact	tens of billions USD annually; scenarios to 2050 → significant GDP decline	tens to hundreds of billions USD annually by 2050 scenarios
Return on investment in IPC (ROI)	$1 →  ~ $28 saved (global/regional models)	similar magnitude (PASTEUR analyses, CDC models)

The Organization for Economic Co-operation and Development (OECD) (2024) estimates that the lack of effective prevention and control of infections could cost member states between 8.7 and 13% of current healthcare spending, equivalent to approximately $606 billion per year, not including social costs, which could exceed $1 trillion over the long term [50].

The implementation of integrated hospital infection control strategies (infection prevention and control, IPC) as part of the One Health approach brings both measurable health benefits and significant economic returns [48, 50]. Studies show that every USD 1 invested in prevention—including hand hygiene, environmental decontamination, active microbiological surveillance, antibiotic stewardship, and rapid molecular diagnostics—generates an average of $28 in savings resulting from reduced hospital stays, decreased use of expensive last-line antibiotics, and reduced staff absenteeism (WOAH, 2024; OECD, 2024).

The effectiveness of IPC interventions is particularly evident in the prevention of infections caused by methicillin-resistant S. aureus (MRSA), vancomycin-resistant Enterococcus (VRE) (Gram-positive, FA), Clostridioides difficile (Gram-positive, anaerobic (AN)), and MDR Gram-negative bacteria [47, 48]. Failure to implement, such strategies, leads to a significant increase in economic burdens: the average length of hospital stay is extended by 6–20 days, the costs of pharmacotherapy increase due to the need to use broad-spectrum antibiotics, and in extreme cases, wards are temporarily closed and hospital work is reorganized [47].

The conclusions from the above data are clear—implementing comprehensive IPC strategies is both a health necessity and a highly cost-effective measure [47, 48]. Adopting an integrated One Health approach in hospitals reduces the costs associated with treatment, hospitalization, and lost productivity, while limiting the selective pressure that promotes the development of antibiotic resistance.

An evaluation of the clinical and economic value of reducing antibiotic resistance in the context of treating hospital-acquired infections, where available treatment options are limited, was conducted in Greece [51]. Economic modeling, based on data from teaching hospitals in the country, was utilized for the study. Additionally, treatment costs, length of hospitalization, mortality, and QALYs (quality-adjusted life years) were considered, and current scenarios were compared with a hypothetical scenario of more effective treatment due to a reduction in AMR. A reduction in AMR could significantly lower mortality and hospitalization rates. Additionally, considerable savings for the health system were estimated, primarily from reduced costs for infection treatment and hospitalization. Furthermore, positioning effective antibiotics and investments in infection control offers cost-effective benefits.

Kingston et al. estimated the additional resource use costs associated with infections caused by six key resistant pathogens in Europe [52]. The most expensive cases were attributed to pathogens such as MRSA and resistant Gram-negative strains. The total costs to health systems reach hundreds of millions of euros annually on a continental scale.

Increasing rates of antibiotic-resistant infections also have long-term social consequences. Patients who survive resistant infections often face long-term health problems such as chronic organ failure, loss of organ function or permanent tissue damage [46, 53]. The use of disinfectants that can result in the occurrence of increased bacterial resistance to antibiotics can lead to a reduced quality of life for patients, as they often require years of treatment and monitoring. Resistance-related health problems can also affect patients’ ability to work, resulting in lost productivity.

However, modern approaches to infection control that use advanced technology, precise analysis of epidemiological data, rationality of antibiotic therapy, and control of the hospital environment are increasingly being used. Automated epidemiological surveillance systems (ESS) are among the most important developments in recent years. By integrating clinical, pharmacological, and microbiological data, they enable ongoing monitoring of infections [54].

Compared to traditional manual surveillance, electronic systems are more sensitive—in some centers, the time to detect bacteremia outbreaks has been reduced from three to four days to several hours [55]. This allows for faster isolation, detection of the source of infection, and protection of employees and patients of the medical entity. The systems operate based on rule-based logic or machine learning-automated detection of patterns of collected data related to the presence of infection. They actively analyze source data from laboratory systems, electronic medical record systems, pharmacy systems, and epidemiological and hospital statistics systems.

Automated environmental decontamination using noncontact technologies, such as UV–C radiation and hydrogen peroxide vapor, represents an emerging approach in hospital infection prevention. The results of clinical studies indicate that ultraviolet C (UV–C) rays effectively remove 99.9% of pathogens from hospital surfaces, such as Clostridioides difficile, MRSA and Enterococcus VRE + , with exposure times of 15–30 min [56]. On the other hand, gaseous hydrogen peroxide sprayed in confined spaces can also destroy pathogens present in hard-to-reach areas, such as hinges or capillary perforated interiors of medical equipment [57].

The application of ATP (adenosine triphosphate) bioluminescence assays supports contemporary methods of surface cleanliness monitoring [58, 59]. They allow an immediate assessment of the effectiveness of sanitization, which is a key element of the decontamination chain. Advantages of ATP analysis include repeatability, speed of execution, and the ability to perform the test immediately after the cleaning and disinfection process. The test is a functional tool for environmental cleanliness audits, employee education, and internal sanitary inspections in medical facilities [58, 60].

Daily patient antisepsis also plays an important role in the contemporary approach to the prevention of infection. A recommendation from the US Center for Infection Control and Prevention is the use of regular chlorhexidine-containing bathing (CHG bathing) in intensive care and hematology units [61]. The recommendations are based on studies showing a 31% decrease in the incidence of bacteremia and a 23% reduction in infections because of regular antiseptic bathing in intensive care units.

Another key component of modern infection control is a strategy to decolonize carriers of alert pathogens such as MRSA, Enterococcus VRE + , ESBL (Extended-Spectrum Beta-Lactamases), and KPC (K. pneumoniae Carbapenemase) [62, 63]. The screen-and-treat protocol identifies the infected patient and immediately implements a procedure to decolonize the pathogen. The procedure is one of the currently recommended preventive methods used before surgical procedures. The procedure includes screening, and if the result is positive, eradication treatment is initiated. A study by Bode et al. showed that decolonizing MRSA-infected patients before surgery reduced the rate of perioperative infections by 60%.

Using digital technology, modern infection control methods also monitor hand hygiene compliance. Each time a disinfectant is used, the duration of disinfection and washing is recorded [64]. Furthermore, the stage of the medical procedure in which the dispenser was activated is also recorded. The data are recorded and collected by built-in RFID (radio-frequency identification) systems in automatic dispensers. This makes it possible to report and analyze the behavior of employees and patients of the facility. The system reports directly on detected errors during the procedure. The introduction of such systems has been shown to increase hand hygiene compliance from 40% to as high as nearly 90%.

Cohorting and zoning are other important method that reduces cross-transmission and are particularly effective in intensive care and hematology units, where patients are more susceptible to infection due to impaired immune function [65]. This strategy involves assigning patients to zones with a certain level of risk of exposure to biological material and transmission of infection.

Current infection control systems emphasize staff education through regular training sessions and systematic audits [66]. Short training sessions, based on analysis of specific clinical cases, are a more effective didactic method than lengthy theoretical lectures. Equally important are unannounced hospitalizations, real-time observations, and daily certification of the sanitization process. It has been observed that the use of this model of action leads to adherence to current sanitation procedures and a reduction in the number of transmissions of infections associated with the provision of health care. The infection control strategies described above act synergistically. None of the above methods is sufficient as a single intervention. An effective infection prevention and control system requires an integrated, multifaceted and individual approach that considers the medical entity’s environment, available technology, infrastructure, pharmacotherapy, and the organizational structure of the hospital. The feasibility of implementing these strategies depends on appropriate resource allocation, staff engagement, and institutional commitment to continuous quality improvement.

Common mechanisms associated with increased resistance to disinfectants and the co-occurrence of resistance to disinfectants and antibiotics

Disinfectants are chemical agents capable of killing most pathogenic microorganisms under defined conditions, although not necessarily bacterial spores [67]. The main purpose of their use is to reduce the number of microorganisms to a level considered safe, which helps prevent the spread of infectious diseases. They are widely recognized for their ability to kill or inactivate microorganisms on object surfaces and within transmission media. Disinfectants are generally applied at concentrations much higher than their minimum inhibitory concentrations (MICs) to maximize bacterial killing. At such levels, which are up to 1,000 times above the MIC, it becomes impossible for bacteria to overcome such a rapid onslaught of mass damage and develop resistance. However, bacteria possess a remarkable ability to adapt to biocide-induced chemical stress. Overuse and misuse of disinfectants can therefore lead to reduced microbial susceptibility [33, 68] (Fig. 1). More concerning is that commonly used disinfectants are contributing to the problem of antibiotic resistance. In healthcare facilities, the choice of an appropriate disinfectant is crucial to ensure the hygiene and safety of patients and medical staff. The decision should be based on an analysis of several important factors, such as the type of surface to be disinfected, the degree of contamination, and the specific pathogens to be neutralized. Among the most common types of disinfectants are agents based on alcohols, aldehydes, chlorine and its compounds, phenols and phenol derivatives, quaternary amine compounds, or peroxides [69].

Fig. 1.

Effect of disinfectants on the co-occurrence of drug resistance

Due to their widespread use, disinfectants exert selective pressure on microorganisms, potentially contributing to the emergence of resistant strains [70, 71]. Furthermore, the rapid evolution of bacterial genomes under the selective pressures imposed by antibiotics and environmental factors facilitates the development of antibiotic resistance.

Suboptimal concentration may be ineffective, as they do not induce bacterial cell death [72]. Cells can recover their functional processes after biocide application. They can recover from the dormant state (e.g., persister (shallow dormancy state) and VBNC (Viable but nonculturable) (deeper dormancy state cells)) through a process called resuscitation. These processes can lead to the survival of bacteria with a specific phenotype and genotype, often already with acquired mutations because of the biocide, which the co-occurrence of resistance can also accompany a given antibiotic. Reduced susceptibility and even resistance to biocides are thought to be due to internal and external mechanisms of bacterial cells that result from genotypic and/or phenotypic adaptations [5].

One of the primary mechanisms by which bacteria resist disinfectants is by altering the composition of the cell wall or cytoplasmic membrane, thereby reducing biocide penetration [73] (Fig. 2). An example of this is strains of P. aeruginosa, which show changes in the composition of membrane lipids, which increase their resistance to disinfectant quaternary ammonium compounds (QAC). Moreover, the changes may involve not only lipids, but the resistance may also be related to modifications in outer membrane proteins [74]. In the case of this bacterium, changes in the proteins of the outer membrane, its fatty acid content, as well as changes in the hydrophobicity and surface charge of the cells were also observed when benzalkonium chloride (BKC) was used [75]. In addition, it is thought that ethanol can cause changes in the morphology and shape of E. coli (Gram-negative, ox−, FA) cells [76, 77]. A mutation in a gene that decreases susceptibility to alcohol has been shown to cause changes in cell membrane composition in bacteria. Alterations in the composition of bacterial cell membranes are also observed after exposure to organic solvents [78]. It is considered that they can cause cis–trans isomerization of membrane fatty acids by cis-isomerase and decrease the hydrophobicity of the cell surface, which reduces its permeability. In Bacillus cereus (Gram-positive, FA), changes in genes involved in fatty acid metabolism were observed after exposure to BKC, which in turn contributed to changes in cell membrane composition [79].

Fig. 2.

Common mechanisms associated with increased resistance to disinfectants and the co-occurrence of resistance to disinfectants and antibiotics

Among the physiological adaptations of bacteria, biofilm formation should be highlighted. According to the definition, a biofilm is a stationary, complex structure consisting of one or more species of bacteria and products of living cells that are irreversibly attached to a substrate and surrounded by an extracellular polymeric substance produced by bacteria [80]. They consist of 10% microorganism mass and 90% water. It is the bacteria in the biofilm that are responsible for most of the physiological processes in their environment. The formation of a biofilm consists of several stages, including adsorption, adhesion, microcolony formation, maturation, and dispersion.

Biofilms can hinder disinfectant penetration, promote genetic exchange between cells, modulate the surrounding environment, and produce enzymes that degrade biocides [81]. Moreover, there may also be chemical interactions between the biofilm and the disinfectant. For example, for chlorine-based disinfectants, the organic components of the biofilm consume the element before it reacts with the cellular components [74]. Microorganisms in planktonic form exhibit higher metabolic activity than those in biofilms and possess the characteristics necessary to colonize new niches, but with a lower chance of survival [82]. For this reason, biofilm formation is more beneficial for bacteria in terms of acquiring resistance to antibiotics and biocides. Another example is ethanol, under the influence of which IcaR-mediated biofilm formation was observed in staphylococci [83, 84].

Biofilm formation and increased tolerance to biocides can also depend on the species composition of the biofilm [85]. It has been shown that multispecies biofilms that are more heterogeneous can increase biocide inactivation. In the case of multispecies biofilms, if one species does not produce particles that can inactivate disinfectants, that species may be resistant due to the presence of biomolecules produced by the other species. Among the examples is P. aeruginosa, which can produce SdsA1, a sodium dodecyl sulfate hydrolase enzyme, which can protect all species that are in the biofilm. Biofilms also increase the likelihood of horizontal gene transfer due to higher cell density and close packing compared to planktonic cultures [85, 86]. This may be because if one bacterium is resistant to a particular agent in a biofilm, it is more likely to pass it on to other species.

One study showed that chlorine disinfection promoted horizontal transfer of plasmids through natural transformation [87]. This process promoted the exchange of antibiotic resistance genes between bacteria. Moreover, selection pressure after biocide application is strongly enhanced by species association [88]. It was shown that dual-species biofilms (B. cereus and P. fluorescens (Gram-negative, ox + , AE) exhibited greater tolerance to glutaraldehyde and cetyltrimethylammonium bromide than single-species biofilms.

Also, bacteria can acquire resistance to disinfectants via genes carried on plasmids, as is the case with E. coli and the resistance of these bacteria to formaldehyde donors [89]. Another example is staphylococci, where the resistance is due to genes lying on plasmids qacAB and qacCD [81]. Chromosomal mutations are also thought to be related to bacterial resistance to disinfectants. In the case of glutaraldehyde, overexpression of the yqhD gene for aldehyde reductase in E. coli led to resistance to the agent after its use [90]. It has been shown that in staphylococci, qac genes responsible for QAC resistance can be carried on plasmids [91]. In another study, S. aureus mutants showing increased resistance to triclosan inherited it despite the absence of triclosan [92]. In the case of triclosan, mutations in the fabI (efflux pump) genes are associated with resistance to this agent, and they may be associated with MDR, which can also be acquired via horizontal gene transfer.

Nother critical mechanism involves efflux pumps, which actively expel toxic substances from bacterial cells [81]. These membrane proteins actively remove toxic substances from inside the cell. They transport the chemicals out of the cell, thus reducing their concentration inside, allowing the bacteria to survive. Most efflux pumps associated with disinfectant resistance belong to the RND family [93]. Some can only pump specific substances outside, such as TtgDEF in P. putida. However, some have a wide range of substrates that can pump outside.

A well-characterized example is the AcrAB-TolC system in E. coli, which confers resistance to antibiotics, detergents, and dyes. Similarly, the MexAB-OprM pump in P. aeruginosa plays a key role in MDR [81]. It is suggested that efflux pumps AcrB and MexB may have low substrate specificity, making them capable of pumping both disinfectants and antibiotics [93]. n the case of glutaraldehyde resistance in P. fluorescens and P. aeruginosa biofilms, efflux pumps (MexE and MexF) play a major role [94].

Moreover, in P. fluorescens biofilms exposed to hypochlorite, increased transcription of genes encoding superoxide-scavenging enzymes, oxidative stress repair enzymes, and efflux pump components (MexEF-OprN) was observed [95]. These pumps are believed to be involved in biocide and drug resistance mechanisms. Exposure to BKC has also been associated with changes and mutations in efflux pumps [96, 97]. It is worth noting that one of the substances used as a substrate in most efflux pumps is triclosan [98]. For example, in the case of E. coli, it is the AcrAB-TolC pump, while for P. aeruginosa, it is MexAB-OprM.

Furthermore, biocides enhance the SOS response in bacteria, a stress-induced mechanism [97]. The results of this process can lead to DNA repair or induction of DNA polymerase errors, which can promote mutations and the acquisition of resistance to disinfectants.

Another issue is that the occurrence of co-resistance to biocides and antibiotics may be encoded on the same genetic elements, e.g., plasmids, transposons, or integrons [99]. Acquired mutations resulting from the use of either agent can lead to cross-resistance. Bacteria can acquire resistance through the exchange of genetic material by vertical inheritance (from parent to offspring), but also by horizontal gene transfer (between organisms) [100]. The three main mechanisms involved in horizontal gene transfer are conjugation, transformation, and transduction. These are considered particularly dangerous in the spread of new traits in bacteria because they allow genes to cross phylogenetic boundaries. Bacteria can also acquire new traits through mutations, which occur randomly but are already vertically inherited. One example of such agents is triclosan. It has been shown that unrestricted use of triclosan can affect antibiotic resistance in bacteria by promoting chromosomal as well as horizontally acquired mechanisms of antibiotic resistance [101].

This subsection discusses the above mechanisms by type of agent used.

Selected disinfectants and growing antibiotic resistance

Alcohol-based agents

Alcohols are among the most popular disinfectants. Among them, we can distinguish ethanol, methanol, and isopropanol, among others [102]. Hand sanitizer and other alcohol-based disinfectants are considered a key way to control hospital infections around the world. Their mechanism of action involves denaturing proteins after dissolving the phospholipid membrane. However, there are reports that tolerance to these agents can develop among bacteria. One example is B. cereus, which was previously sensitive to low concentrations of alcohols [103]. Currently, it is believed that only a 90% concentration inhibits long-term spore survival (2–12 months) [104]. Another example is E. faecium (Gram-positive, FA) [105]. It has been shown that over 18 years, their tolerance to isopropanol killing has increased. However, this study used a 23% alcohol concentration, which may not be clinically relevant, given that higher concentrations are used [106]. These reports raise the question of whether the widespread use of alcohol-based agents may not only increase pathogen tolerance to alcohol but also contribute to drug resistance.

In one study, E. faecium and S. aureus were exposed to 50% concentrations of ethanol for 20 days [107]. It was observed that after this time, they increased their tolerance to alcohol. It was shown that this was probably related to mutations in the genes responsible for proteoglycan synthesis and two-component regulatory systems and responses to environmental stresses. Furthermore, the colonization rate of dispensers with alcohol-based hand sanitizer (HSD) was assessed [108]. The most common pathogens were B. cereus (29%) and E. cloacae (Gram-negative, ox−, FA) (2%), which were alcohol tolerant with survival rates of up to 70%. Moreover, these strains had resistance to various classes of antibiotics, with higher virulence than laboratory strains. Evaluation of low concentrations of commercial alcohol-based disinfectants showed that A. baumannii exhibited increased tolerance [109]. The addition of disinfectants to the culture medium of the bacteria caused the secretion of proteins into the culture supernatant, among others, OmpA, which are identified as those associated with the pathogenicity of these bacteria.

In the case of E. faecium strains, the rpoB gene was identified as the gene associated with alcohol resistance [107]. Significantly, this gene has also been linked to rifampicin resistance in these bacteria [110]. One mechanism for increasing bacterial tolerance to alcohol is a change in the composition of the cell membrane.For example, in E. coli, this involves an increase in unsaturated fatty acids [111]. It is noteworthy that many antibiotics work by interacting with bacterial cell membranes and also affect fatty acid synthesis [112, 113]. Considering the changes in its structure that the bacterium can acquire, it is likely that the use of alcoholic agents can lead to a reduction in the effectiveness of antibiotic therapy due to these modifications. It should be emphasized that alcohol-based biocides remain an indispensable and effective component of infection prevention, and their use is crucial for public health protection, despite certain limitations such as the lack of sporicidal activity.

Current epidemiological and clinical data do not indicate that resistance to alcohol-based disinfectants is a significant problem, with only a few isolates reported to date. However, further, more extensive research is needed, particularly in the context of disinfectant dispensers, which are widely used and may also be used by nonmedical personnel. It is also worth paying attention to bacterial strains selected for alcohol tolerance, for example, in industrial alcohol production processes, and analyzing the genes involved in these mechanisms to assess their possible presence in clinical isolates [114, 115].

Aldehyde-based agents

Aldehydes, such as glutaraldehyde, formaldehyde, and ortho-phthalaldehyde, are effective disinfectant and sterilizing agents [116–119]. Their mechanism of action is the denaturation of proteins and nucleic acids, which leads to the inactivation of microorganisms. Aldehydes are highly effective against bacteria, viruses, fungi, and bacterial spores, making them broad-spectrum agents. For this reason, aldehydes are widely used in medical facilities and industry as decontamination agents. However, these compounds create challenges for medical personnel. Their toxicity to humans and potential negative environmental impacts make it necessary to use appropriate personal protective equipment and room ventilation when using them. Another challenge is the growing number of reports linking aldehyde-based agents to bacterial drug resistance.

One of these is glutaraldehyde, which is used to clean bronchoscopes and endoscopes. A correlation was shown between the tolerance of glutaraldehyde by Mycobacterium chelonae (Gram-positive, AE) isolated from bronchoscope cleaning machines and their antibiotic resistance [117]. It was shown that all isolates that tolerated this agent were resistant or moderately resistant to two or three classes of antibiotics. Among mycobacteria sensitive to this agent, only 11% of isolates showed such resistance. The sensitivity to glutaraldehyde of this mycobacterium isolated from endoscopes and endoscope water was also evaluated in another study [120]. Two strains were tested and showed that increased sensitivity to this agent was associated with a slight increase in MIC for rifampicin and ethambutol. Furthermore, both strains showed increased surface hydrophobicity, and an increased reduction in the arabinogalactan/arabinomannan portion of the cell wall was detected.

The problem of cross-resistance associated with this agent concerns not only mycobacteria but also Gram-negative bacteria. E. coli strains treated with glutaraldehyde have also shown cross-resistance to ampicillin, chloramphenicol, and norfloxacin, among others. Several genes associated with this resistance have also been identified, including yqhC and aes [121]. In another study, glutaraldehyde-resistant bacterial strains were isolated from stream water [122]. Three RND efflux pumps (two deF and one rsmA) were identified in Marinobacter sp. strain G11 (Gram-negative, ox + , AE). In Halomonas sp. strain G15 (Gram-negative, ox + , FA), one RND efflux pump (rsmA) was detected. In the case of Bacillus sp. G16. two antibiotic inactivation genes and two RND efflux pumps were observed. Also, in P. fluorescens and P. aeruginosa, exposure to glutaraldehyde resulted in activation of efflux pumps and metabolic pathways of phosphate degradation, lipid biosynthesis, and polyamine biosynthesis [94]. It is worth noting that inhibitors of these pumps can be one of the strategies of antibiotic therapies, and their presence and properties often determine MDR [123].

In the case of Stenotrophomonas maltophilia (Gram-negative, ox−, AE), a role for the FadRACB system was found in mitigating formaldehyde toxicity, while cross-resistance to quinolones was found [124]. Notably, one study found that Enterobacter spp. strains with reduced sensitivity to formaldehyde also showed reduced sensitivity to β-lactam antibiotics (penicillins and cephalosporins), tetracycline, and ciprofloxacin [125]. However, this work focuses on preservatives used in cosmetics that are formaldehyde donors. Another study, in turn, showed that the so-called formaldehyde sensor EfgA as well as pathways of translation inhibition by kanamycin, may be common in Methylorubrum extorquens (Gram-negative, ox + , FA) [126].

Chlorine-based agents and their derivatives

Chlorine compounds, such as sodium hypochlorite, are well known for their broad-spectrum antimicrobial activity, including in the treatment of skin wounds [127–129]. They are used as antiseptics for patients to irrigate wounds and burns because of their efficacy against a wide range of planktonic and biofilm microorganisms. Moreover, they are often formulated as isotonic, physiologically compatible solutions that do not cause osmotic stress to damaged tissues.

Their mechanism of action on microorganisms is related to their effect on the structure of the cell membrane and changes in its permeability, including through oxidation [130, 131]. Against bacteria, sodium hypochlorite inhibits the rate of biofilm production. However, there are reports of its role in the spread of antibiotic resistance.

One study incubated P. aeruginosa isolates with subinhibitory concentrations of this biocide and observed increases in MICs for colistin, ceftazidime, amikacin, meropenem, gentamicin, piperacillin-tazobactam, and ciprofloxacin [132]. Incubation of isolates with subinhibitory concentrations of didecyldimonium chloride also resulted in increased MICs for amikacin, gentamicin, meropenem, and ciprofloxacin.

In another study, treatment of P. aeruginosa isolates was shown to increase antibiotic resistance to ceftazidime, chloramphenicol, and ampicillin. The mechanism responsible for this effect is increased expression of MexEF-OprN genes responsible for efflux pumps [133]. This pump and its mutations have also been shown to be involved in MDR in these bacteria [134, 135].

Furthermore, exposure to 100 mg/L sodium hypochlorite increased the resistance of P. aeruginosa to this agent [136]. Moreover, the strains showed resistance to ampicillin, tetracycline, chloramphenicol, and kanamycin. Sodium hypochlorite was shown to trigger the SOS (conservative DNA damage response) response in these microorganisms, as well as to induce an increase in membrane permeability, increased expression of the MuxABC-OpmB pump, beta-lactamase and antioxidant enzymes in them. As before, mutations in this pump are also associated with increased antibiotic resistance of P. aeruginosa [137, 138]. Also, overproduction/overexpression of beta-lactamases may be one of the failures of antibiotic therapy [139, 140].

Evidence of the effect of subinhibitory doses of sodium hypochlorite comes from a study in which a mutation in the GdpP gene and morphological changes in the cell wall were observed in S. aureus strains, contributing to oxacillin resistance [141]. Ox resistance is related to MRSA [142, 143]. These strains are usually resistant to most beta-lactam agents, including cephalosporins and carbapenems. Confirming these results is a study in which L. monocytogenes (Gram-positive, FA) exposed to subinhibitory concentrations of hypochlorite and chloramine T showed changes in the cell wall and increased catalase and superoxide dismutase activity [144]. It is believed that the presence of catalases may be considered as a virulence enhancer, since they may be resistant to the oxygen burst of host inflammatory cells [145–147]. Similar observations were also made in a study in which this bacterium showing tolerance to sodium hypochlorite, also showed reduced MIC values for streptomycin, gentamicin and ceftriaxone, but they were still within the susceptibility limits [148].

In another of the studies, bacteria with varying degrees of sensitivity to chlorine and antibiotics were isolated from chlorinated water [149]. It was shown that chlorine-resistant bacteria had higher MIC values for tetracycline, sulfamethoxazole and amoxicillin. The effect of chlorine-containing compounds was also confirmed in another study, in which chlorination increased the abundance of bacteria resistant to macrolides (ermB), tetracyclines (tetA, tetB and tetC), sulfonamides (sul1, sul2, and sul3), β-lactams (ampC), aminoglycosides (aph(2′)-Id), rifampicin (katG), and vancomycin (vanA) [150].

Interestingly, one study evaluated samples taken from a wholesale pork market in China [150]. Sodium hypochlorite is one of the agents most commonly used there in the pork supply chain. The bacterial strains Salmonella enterica (Gram-negative, ox−, FA), Salmonella bongori (Gram-negative, ox−, FA), E. coli, K. pneumoniae, and P. aeruginosa were shown to be resistant to the agent.

Moreover, the researchers identified correlations between antibiotic resistance genes—associated with aminoglycosides (aph(3″)-I, aph(6″)-I), quinolones (qnrB, abaQ), and polymyxins (arnA, mcr-4)—and disinfectant resistance genes (emrA/BD, mdtA/B/C/E/F). Additional correlations were found with aminoglycoside (aph(3′)-I), tetracycline (tetH), β-lactam (TEM-171), and disinfectant resistance genes (mdtB/C/E/F, emrA, acrB, qacG).

Among the isolates, the main hosts of genes associated with disinfectant resistance were A. baumannii and Salmonella spp. Moreover, the percentage of genes encoding efflux pumps increased after disinfection with sodium hypochlorite.

Phenol-based agents and their derivatives

Phenols and derivatives are powerful disinfectants [151–154]. They are mainly used for disinfecting surfaces, medical equipment, and as antiseptics. They effectively eliminate bacteria, fungi, and some viruses. The mechanism of action of phenols involves protein denaturation and disruption of cell membrane function, ultimately leading to microbial cell lysis. However, phenol itself is toxic. Therefore, it has been replaced by less irritating derivatives, such as chlorocresol, which have a better safety profile. Nevertheless, even these less irritating derivatives, such as hexachlorophene and triclosan, still have potential toxic effects on mucous membranes and skin. Notably, triclosan is a substrate of most RND efflux pumps, and exposure to it can induce mutations in bacteria [98]. These pumps are also antibiotic transporters, and mutations and changes in their expression can be linked to antibiotic resistance [155].

One study identified the effect of a phenolic disinfectant compound and triclosan on the development of antibiotic resistance in S. enteritica [156]. Under the influence of the phenolic compound, resistance to ampicillin, chloramphenicol, ciprofloxacin, and tetracycline was observed to increase in these bacteria, while under the influence of triclosan, their resistance to ampicillin increased. The researchers suggest that the use of these agents may exert selection pressure on the bacteria to acquire resistance to antibiotics and biocides. A triclosan concentration of 0.2 mg/L also induced drug resistance in E. coli [157]. The compound increased oxidative stress in bacterial cells, caused mutations in the fabI, frdD, marR, and acrR/soxR genes, upregulated genes encoding β-lactamases and efflux pumps, and downregulated genes responsible for membrane permeability. As previously mentioned, these changes may entail an increase in antibiotic resistance.

Another study showed that clinically relevant concentrations of triclosan increased tolerance of MRSA and E. Coli to bactericidal antibiotics in vitro and in vivo models [151]. In addition, triclosan-dependent antibiotic tolerance was shown to require the synthesis of guanosine tetraphosphate (ppGpp) (associated with drug resistance). Furthermore, the effect of triclosan and triclocarban on antibiotic resistance in bacteria was confirmed in another study [158]. It has been shown that low doses of triclosan can cause resistance to this agent but also induce reversible antibiotic resistance. It was also observed that both agents can cause the spread of antibiotic resistance genes.

Another study supported these findings, showing that chronic exposure of E. coli to triclosan led to persistent resistance associated with a fabI mutation, as well as reversible antibiotic tolerance due to reduced membrane permeability and biofilm formation [158]. The mechanism responsible for triclosan’s effect on drug resistance may be that the compound causes increased levels of free radicals in bacterial cells, damaging the cell membrane [159]. These processes are key to the spread of extracellular antibiotic resistance genes through transformation. In addition, it is also thought that triclosan may increase conjugative transfer of antibiotic resistance genes. This effect is attributed to the role of oxidative stress caused by this agent, which is associated with changes in membrane permeability and induction of the SOS response [160].

It was shown that exposure of bacteria to triclosan significantly stimulated conjugative transfer of plasmid-encoded MDR genes within and between genera [161]. As in other studies, triclosan solidified free radical generation and induced microbial membrane damage, which in turn contributed to increased expression of genes regulating the SOS response (umuC, dinB, and dinD). Moreover, higher expression levels of genes encoding ATP synthesis were observed in E. coli and P. putida (Gram-negative, ox, AE). ATP synthase inhibitors are one of the therapeutic strategies for antimicrobial therapies [162]. On the other hand, in the case of P. aeruginosa, after developing tolerance to triclosan, the microorganism was characterized by increased resistance to six antibiotics: tetracycline, ciprofloxacin, amikacin, levofloxacin, carbenicillin, and chloramphenicol [163]. These antibiotics are typical substrates for the efflux pump family division of resistance–nodulation.

In one study, triclosan caused cross-resistance to trimethoprim in E. coli, but only in one of the strains tested [164]. Antibiotic resistance in triclosan-tolerant strains was also demonstrated in C. freundii (Gram-negative, ox−, FA) [165] and in the case of S. maltophilia [166]. These strains were less sensitive to tetracycline, chloramphenicol, and ciprofloxacin, a phenotype linked to SmeDEF overexpression.

Significantly, SmeDEF has been shown to contribute to intrinsic antibiotic resistance in this organism [167]. Moreover, the effect of triclosan on the selection of drug-resistant strains of A. baumannii has also been demonstrated [168]. The triclosan-resistant microorganism also showed resistance to a variety of antibiotics including piperacillin-tazobactam, doxycycline, moxifloxacin, ceftriaxone, cefepime, meropenem, doripenem, ertapenem, ciprofloxacin, aztreonam, tigecycline and trimethoprim–sulfamethoxazole. It has also been shown that this MDR may be due to the induction of overexpression of the AdeIJK efflux pump by triclosan. Also, increased antibiotic resistance of triclosan-tolerant A. baumannii strains was found in another study [169].

Notably, triclosan was also shown to increase cross-resistance to ciprofloxacin in anaerobic bacteria [170]. This was demonstrated, also in another study, in which the agent increased the abundance of the mexB gene associated with antibiotic resistance in these microorganisms [171]. It was also observed that triclosan influenced the selection of clades that contained pathogenic and commensal bacteria.

Furthermore, studying the effect of triclosan on the nitrification process as well as the spread of resistance genes, it was shown that the addition of triclosan to the microbial community (activated sludge) increased the total abundance of antibiotic-resistant genes and mobile genetic elements by 33.1% [172]. In addition, tetracycline and multidrug resistance genes increased by 54.75% and 103.42%, respectively. Similar results were obtained in a study in which bacteria were isolated from surface water near a wastewater treatment plant [173]. It was found that 89.6% of the triclosan-resistant isolates were resistant to four classes of antibiotics, and all were identified as C. freundii.

QAC

BKC and cetylpyridinium chloride, classified as QAC, are widely used disinfectants with antimicrobial activity [77]. They show high efficacy against Gram-positive bacteria and some viruses and fungi.

Their mechanism of action involves interaction with microbial cell membranes, destabilization of lipid structures, leakage of intracellular components, and ultimately cell death. However, reports of bacterial resistance to these biocides are becoming a problem.

The widespread use and accumulation of BKCs in various environments are believed to exert selection pressure favoring bacteria with reduced antibiotic sensitivity [174]. The typical response to bacterial exposure to BKCs involves the expression of genes involved in general and oxidative stress responses [175]. This manifests itself, among other things, as the expression of genes involved in fatty acid metabolism. For example, exposure of L. monocytogenes to 50 mg/L BKC for 10 min resulted in elevated c-di-GMP levels and synthesis of an exopolysaccharide that promoted cell aggregation, inhibited motility in semi-solid media, and enhanced desiccation tolerance.

BKC has been shown to induce biofilm formation for granulomas such as Staphylococcus epidermidis (Gram-positive, FA) and L. monocytogenes, as well as Gram-negative bacilli: K. pneumoniae, E. coli, P. aeruginosa, and A. baumannii. This may contribute to their survival in the environment [176–180]. It is also worth noting that increased biofilm formation can be one of the causes of therapeutic failure.

In one study, BKC was added to a mass of river sediment, which resulted in the selection of P. aeruginosa, which is resistant to several antibiotics [181]. It is suggested that the main reason for this phenomenon is the co-occurrence of BKC tolerance and antibiotic resistance genes on the same mobile DNA molecule. In addition, mutations were also observed in the pmrB gene, which is the gene responsible for resistance to polymyxin. The isolate was also resistant to other antibiotics such as tetracycline and ciprofloxacin. Moreover, subinhibitory concentrations of BKC induced overexpression of the mexCD-oprJ efflux pump, which contributes to resistance to fluoroquinolones and tetracycline.

Another study showed that BKCs at concentrations ranging from 0.1 to 500 μg/L in surface water selected for ciprofloxacin- and sulfamethoxazole-resistant bacteria [182]. Moreover, it has been shown that prolonged exposure to BKC resulted in an increase in the number of microorganisms resistant to this agent, and an increase in resistance to penicillin G, tetracycline, and ciprofloxacin was observed in bacterial communities [68]. It has been suggested that resistance to the latter two drugs may be related to BKC’s effect on efflux pumps. Another study also confirmed the effect of BKC on the occurrence of microorganisms with induced broad-spectrum antibiotic resistance [183].

Furthermore, BKC has been shown to antagonize the effects of gentamicin and other aminoglycosides [184]. Moreover, it increases the frequency of A. baumannii colony formation in the presence of gentamicin and causes a decrease in intracellular antibiotic accumulation. At subinhibitory concentrations, the compound may also affect other ESKAPE pathogens (E. coli, E. cloacae, and K. pneumoniae) by reducing their sensitivity to gentamicin.

This is consistent with another study in which subinhibitory concentrations of BKC induced the development of tolerance to the agent, resulting in changes in the shape, size, and surface roughness of K. pneumoniae cells [185]. Polarization of the cell membrane and changes in the expression of efflux pump genes were also observed, both of which have been linked to increased antibiotic resistance. In the case of another Gram-negative bacterium, E. coli, the development of tolerance to BKC has also been observed, as well as an increase in cross-resistance to several antibiotics [97]. Changes in outer membrane protein profiles may be responsible for one possible mechanism for this occurrence. Also, in the case of P. aeruginosa, a similar effect was observed with the occurrence of tolerance to BKC and resistance to antibiotics [186]. In another study, small-bore strains exposed to this agent developed tolerance: one strain became resistant to tobramycin, and another to chloramphenicol and polymyxin B [75].

This relationship was confirmed in the case of S. enterica subtype Typhimurium as well [187]. In strains that developed resistance to this agent, mutations in the ramR gene (point mutations L158P, A37V, G42E, F45L, and R46H and indel type) were observed, as well as cross-resistance to quinolones, cefepime, tetracyclines, and macrolides, among others. Increased activity and expression of the efflux pump AcrAB-TolC were also noted. This pump is characterized by its ability to expel many different antibiotics from the cell, which is associated with antibiotic resistance [188]. It can also be linked to biofilm formation, pathogenicity during infection, and adaptation to environmental stresses. Similar results were obtained in another study [188]. After developing tolerance to BKC, resistance to levofloxacin, ceftazidime, and tigecycline was observed in two strains of S. enterica. Mutations in genes associated with antibiotic resistance (gyrA, parC) and mutations in genes associated with antibiotic efflux (acrB, mdsA, mdsB) were also noted.

In another study, Gram-negative bacteria (K. pneumoniae, E. coli, and P. aeruginosa) were observed to be less sensitive to BKC, but the use of the agent at subinhibitory concentrations for these pathogens resulted in the development of antibiotic resistance [189]. It is worth noting that Gram-positive bacteria were generally more sensitive to BKC; however, in S. aureus, reduced sensitivity to penicillin was observed, while in Corynebacterium xerosis (Gram-positive, AE) reduced sensitivity to neomycin and bacitracin was reported. [190]. MRSA isolates resistant to this agent showed higher resistance to various β-lactam antibiotics, including cloxacillin, moxalactam, flomoxef, and cefmetazole than the parental strain. Similar results were also observed on another QAC, cetylpyridinium chloride, in S. aureus as well [191].

For S. marcescens, it was observed that strains showing tolerance to this biocide also showed increased resistance to certain antibiotics including fluoroquinolones, tetracycline, chloramphenicol, pipemidic acid, and enoxacin [192]. Moreover, this has been linked with modifications in the efflux pump SdeAB.

Peroxides

The most widely used disinfectant from the peroxide group is hydrogen peroxide. It is a widely used disinfectant with antimicrobial activity [193, 194]. It has a broad spectrum of antimicrobial activity and a favorable safety profile compared to other biocides. Its mechanism of action is mainly related to its oxidative activity. It is also considered to have a lower risk of resistance development than other disinfectants. However, reports also indicate the simultaneous development of resistance to hydrogen peroxide and antibiotics.

One study showed that bacteria that survived disinfection with this agent exhibited increased antibiotic resistance and virulence [195]. It was noted that this mechanism is likely to be mediated by the formation of free radicals, mycobacterial responses to increased oxidative stress, as well as changes in bacterial membranes and activation of efflux pumps and biofilm compaction. Furthermore, in subinhibitory concentrations, hydrogen peroxide increased biofilm production in uropathogenic strains of E. coli [196]. Similar results were obtained in another study, which showed that depending on the concentration and duration of exposure to hydrogen peroxide, lipid peroxidation increased up to 20-fold [197]. Moreover, there was also an increase in biofilm formation and an increase in gene transfer associated with antibiotic resistance.

Bisbiguanides

Chlorhexidine is a disinfectant belonging to the bisbiguanide group, which was developed in the 1940s in the UK [198, 199]. This agent is not considered antiviral, but it does exhibit antibacterial activity. Its spectrum of activity can include Gram-positive and Gram-negative bacteria, spore-forming bacteria and lipophilic viruses, yeasts, and dermatophytes. It is used widely in ophthalmology, gynecology, dentistry, or burn treatment. Chlorhexidine binds to the cell wall of bacteria, disrupting membrane transport and precipitating cytoplasmic proteins [200].

Reports in the literature also indicate a link between chlorhexidine use and increasing drug resistance. One study showed that chlorhexidine, after serial exposure, can reduce daptomycin sensitivity in E. faecium VRE + [201]. After exposure, significant changes in membrane lipids were observed in these strains, i.e., reduction of cardiolipin, and depending on the strain, changes in monohexosyl-diacylglycerol, dihexosyl-diacylglycerol, and diglucosyl-diacylglycerol glycerophosphate. Changes were also observed in genes related to the global response to nutritional stress, nucleotide metabolism (cmk), phosphate acquisition (phoU), and glycolipid biosynthesis (bgsB).

Another study showed that chlorhexidine can lead to colistin resistance in K. pneumoniae strains [202]. An amino acid substitution in the PmrB gene that may be associated with the onset of cross-resistance to colistin has also been identified. In the case of this microorganism, another study was also carried out, which confirms the results obtained above [202].

It has also been shown that the development of antibiotic resistance depends on chlorhexidine concentration, with low concentrations resulting in stable cross-resistance to antibiotics in E. coli strains [203]. It has also been shown that the activity of the efflux pump RND may have a role in this process.

Similar results were also obtained when testing subinhibitory concentrations of chlorhexidine on Neisseria gonorrhoeae (Gram-negative, ox + , AE) [204]. Cross-resistance to azithromycin, cefixime, ceftriaxone, and ciprofloxacin was observed in this microorganism. All chlorhexidine-resistant variants (MIC = 20 mg/l) carried variants in the efflux pump promoter of MtrCDE.

Another study tested subinhibitory concentrations of chlorhexidine for the development of drug resistance in P. aeruginosa [204]. Increased MICs were found for colistin, ceftazidime, meropenem, ciprofloxacin, and amikacin, and some strains expressed cross-resistance to meropenem. Furthermore, chlorhexidine activated the efflux pump MexXY. Induction of this pump is believed to contribute to the antibiotic resistance of this pathogen [205, 206]. In Gram-negative bacteria (E. coli, K. pneumoniae, P. aeruginosa, A. baumannii) showing increased resistance to chlorhexidine, it was also noted that they could show reduced sensitivity to antibiotics such as ciprofloxacin, imipenem, cefotaxime, ceftazidime, gentamicin, and aztreonam [207]. Such a relationship was not observed in Gram-positive bacteria (S. aureus (not MRSA), Streptococcus pyogenes, and E. faecalis). Nevertheless, another study showed that exposure to low concentrations of chlorhexidine can exert selection pressure and increase MICs for all tested antibiotics (ampicillin, tetracycline, vancomycin, gentamicin, cefotaxime, cefuroxime, and ciprofloxacin) in a sensitive control strain of S. aureus [208] (Fig. 3.).

Fig. 3.

Summary of the mechanisms of action of disinfectants in the context of antibiotic resistance

Disinfectants—pros and cons

Given the growing concern about MDR, the question arises: Should disinfectants be used in healthcare facilities? While there is a growing body of evidence addressing the relationship between disinfectant use and the development of MDR, the data remain insufficient, particularly when considering the wide variety of disinfectants and bacterial species involved. Many studies focus on a single disinfectant and a single bacterial strain, which makes it difficult to extrapolate the findings to broader clinical settings. Although no definitive clinical recommendations exist to avoid specific disinfectants, rational use and systematic monitoring of their microbiological impact are essential, particularly in high-risk clinical environments (Table 3). Implementing evidence-based disinfection protocols and routine microbiological surveillance may reduce the risk of cross-resistance development, thereby preserving the effectiveness of disinfectants and antibiotics in infection control. Interestingly, few studies address the potential effects of antiseptic dyes, such as quinosol and ethacridine, or disinfectants based on nitrofuran derivatives. Additionally, while much attention has been given to agents like triclosan and BKC, some reports have suggested no significant link between these disinfectants and the development of cross-resistance to antibiotics [209–212]. This indicates that the relationship between disinfectant exposure and antibiotic resistance may depend on various factors, including the disinfectant concentration. Notably, subinhibitory concentrations of disinfectants have been shown to promote the development of MDR in several bacterial species [132, 196, 213]. Another critical factor is the frequency and duration of exposure, which has been linked to an increased risk of resistance development [197].

Table 3.

Disinfectants—pros and cons

	Pros	Cons
Alcohol-based agents	rapid action; broad antibacterial spectrum; essential for hand hygiene	not sporicidal; can dry the skin; tolerance may develop at low concentrations
Aldehyde-based agents	broad spectrum, including spores; effective sterilant	toxic to humans; irritating fumes; potential cross-resistance
Chlorine-based agents and their derivatives	strong oxidizer; inexpensive; effective against bacteria, viruses, and biofilms	corrosive; inactivated by organic matter; may promote resistance at low doses
Phenol-based agents and their derivatives	effective against bacteria and some fungi; stable on surfaces	skin irritation; environmental toxicity; strongly promotes antibiotic resistance
Quaternary Ammonium Compounds	good surface disinfectant; effective against Gram-positive bacteria and enveloped viruses	ineffective against spores; induces biofilm production; associated with multidrug resistance
Peroxides	broad spectrum; environmentally friendly (decomposes to water and oxygen)	less stable in light/heat; may stimulate biofilm and resistance at sublethal doses
Bisbiguanides	long-lasting action on skin; effective against many bacteria and fungi	possible cross-resistance to last-resort antibiotics; reduced activity against some Gram-negatives

From an epidemiological perspective, prolonged exposure to certain disinfectants has been associated with resistance to last-resort antibiotics, such as carbapenems and colistin, significantly limiting treatment options for healthcare-associated infections [202, 202, 214]. Despite these concerns, the use of disinfectants remains essential in healthcare settings due to their high bactericidal and bacteriostatic efficacy [215, 216]. When used properly, disinfectants can effectively eliminate highly virulent and MDR pathogens, such as A. baumannii and S. maltophilia. In these cases, the benefits of disinfection outweigh the risks associated with resistance development. However, it is crucial that disinfectants are used according to established guidelines, tailored to specific medical environments and surfaces, and applied correctly to maximize their effectiveness while minimizing the risk of resistance.

Implications for practice

To mitigate the risks associated with disinfectant-induced cross-resistance, healthcare professionals must implement stricter protocols regarding the selection, concentration, and frequency of disinfectant use. Training programs should emphasize the importance of proper application techniques, adherence to manufacturer instructions, and the need to avoid underdosing. Infection control teams should integrate routine environmental monitoring, including ATP testing and microbiological surface assessments, into standard procedures. Moreover, hospitals should consider adopting advanced technologies, such as automated disinfection systems and electronic surveillance tools, to ensure consistent and effective hygiene practices.

Future directions

Without compromising the vital role disinfectants play in infection prevention, future research should focus on expanding our knowledge of the intricate relationship between disinfectant use and AMR. Well-designed, longitudinal, multicenter studies are required to determine whether—and under what circumstances—exposure to disinfectants may lead to cross-resistance in bacterial populations. Furthermore, there is also a need for research to answer the question of whether bacteria that have developed cross-resistance because of exposure to biocides can regain their sensitivity to antibiotics after the selection factor has been eliminated. As the cornerstone of infection control, proper and evidence-based disinfectant usage should be promoted continuously in tandem with these initiatives. Determining concentration levels that preserve bactericidal effectiveness while reducing possible selective pressure is a crucial objective for future research.

Additionally, studies on novel biocidal formulations, such as synergistic combinations or alternative compounds with lower resistance potential, could provide valuable supplements to current strategies. Establishing defined and generally recognized procedures for evaluating the establishment of disinfectant-associated resistance in clinical isolates is equally important. To ensure that these vital instruments continue to be both successful and sustainable in the battle against illnesses linked to hospital settings and AMR, such research will enable ongoing optimization of disinfection methods in healthcare settings by concentrating on both safety and efficacy.

An important regulatory consideration is that there are no universal concentrations recommended for each chemical class of disinfectants that would be valid across all products and use scenarios. According to the European Medical Device Regulation (2017/745), healthcare facilities must strictly follow the manufacturer’s instructions for use (IFU) when preparing disinfectant solutions for medical devices and reprocessing reusable instruments [12, 217]. These instructions specify concentration, contact time, temperature, and compatibility requirements that ensure the targeted microbiological reduction without compromising the integrity, coating, or functionality of medical instruments and other reusable devices. Deviating from these validated parameters may result not only in insufficient decontamination but also in material damage and regulatory noncompliance. Therefore, the optimal approach—according to both regulatory and infection prevention standards—is to implement evidence-based, procedure-driven protocols that adhere precisely to the IFU, with continuous staff training and periodic auditing to verify compliance.

Conclusion

Inappropriate use of disinfectants, particularly at subinhibitory concentrations, can promote the development of bacterial tolerance and mechanisms leading to cross-resistance with antibiotics. The scientific literature describes mechanisms such as efflux pump activation, alterations in cell membrane permeability, and biofilm formation, all of which can limit the effectiveness of both biocides and antibiotics. However, current clinical data on the impact of disinfectant use on bacterial resistance and antibiotic therapy remain inconclusive and require further rigorous investigation. Therefore, it is crucial to optimize disinfection protocols based on available, reliable scientific knowledge and ensure responsible and rational use of disinfectants. A comprehensive approach to the problem should include strict infection control procedures, ongoing monitoring of microbial susceptibility, systematic training of medical personnel, and the development of new biocidal products with minimal potential to induce resistance. In the future, it will be important to define biocide concentration thresholds that do not exert selective pressure promoting tolerance, and to standardize methods for assessing disinfectant resistance in clinical isolates. Implementing these measures will ensure the effectiveness of both disinfection and antibiotic treatment, which is crucial for patient protection and the sustainable development of healthcare systems.

Abbreviations

AER

Aerobic

AMR

Antimicrobial resistance

AN

Anaerobic

ATP

Adenosine triphosphate

BKC

Benzalkonium chloride

CDC

Centers for disease control and prevention

CHG

Chlorhexidine-containing

DALY

Disability-adjusted life years

ESBL

Extended-spectrum beta-lactamases

ESS

Epidemiological surveillance systems

FA

Facultative anaerobe

GDP

Gross domestic product

HPV

Hydrogen peroxide gas

IFU

Instructions for use

IPC

Infection prevention and control

KPC

K. pneumoniae Carbapenemase

MDR

Multidrug resistance

MIC

Minimum inhibitory concentration

MRSA

Methicillin-resistant S. aureus

OECD

Organization for economic co-operation and development

ox

Oxidase

PDR

Pan-drug-resistant

QAC

Quaternary ammonium compounds

QALY

Quality-adjusted life years

RFID

Radio-frequency identification

VBNC

Viable but nonculturable

VRE

Vancomycin-resistant Enterococcus

XDR

Extensively drug-resistant

Authors contribution

**Ł.W-** writing, original draft preparation, drafted the manuscript and designed the figures, the main conceptual idea; **M.K-** writing, original draft preparation, conceptualization; **B.T.-** writing, designed the figures; **M.K-** writing, designed the figures; **J.P-** designed the figures; **M.H** review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors declare no competing interests.