2.1. Benzalkonium Chloride

Benzalkonium chloride (BAC; CAS 8001-54-5) (Figure 1) is a quaternary ammonium chemical that has several functions, including application as a biocide, cationic surfactant, and phase transition agent [18]. Furthermore, it serves as an antiseptic agent, employed for the preservation of pharmaceuticals, or as a disinfectant for cleaning agents. BAC consists of a blend of alkyl benzyl dimethyl ammonium chloride, wherein the alkyl chain exhibits varying numbers of carbon atoms, typically n-C12H25 (dodecyl), n-C14H29 (tetradecyl), and n-C16H33 (hexadecyl). The antimicrobial potency is directly proportional to the length of the alkyl chain. The C12-homolog demonstrates efficacy against fungal and mold species, whereas the C14 variant exhibits activity against Gram-positive bacteria, and the C16 variant targets Gram-negative bacteria. Owing to the ability of BAC to rupture the cell membranes of microbes, it is frequently included in antiseptic compositions and demonstrates efficacy against a diverse array of bacteria, fungi, and certain types of viruses at low concentrations. The introduction of novel coatings and disinfection formulations based on QACs has led to a rise in their overall usage. Consequently, there has been an accompanying spike in reservations regarding the potential environmental and health risks associated with QAC exposure.

2.2. Benzethonium Chloride

Benzethonium (CAS 121-54-0) (Figure 2) is a synthetic quaternary ammonium salt that exhibits surfactant characteristics, possesses antiseptic properties, and demonstrates a wide range of antibacterial actions. Benzethonium chloride is primarily applied in its salt form as a skin disinfectant. It is also present in cosmetic products and personal care items, including mouthwashes and ointments, and is designed to alleviate itching. The efficacy of this substance in facilitating antibacterial activity against bacteria, fungi, molds, and viruses has been previously demonstrated. The concentrations often employed for this purpose range from 0.1% to 0.2%. These concentrations have been determined to be appropriate for use, as stated by the U.S. FDA [22]. Benzethonium is a member of the cationic detergent group that disrupts lipid bilayers. It is widely used because of its broad-spectrum antimicrobial activity against bacteria, viruses, and fungi [23]. Benzethonium chloride in antiseptic solutions kills bacteria by interfering with the integrity of microbial cell membranes. This leads to the leakage of cell contents and eventual cell death [24]. Benzethonium chloride is an active component of certain hard-surface disinfection solutions listed by the Environmental Protection Agency (EPA).

2.3. Cetylpyridinium Chloride

Cetylpyridinium chloride (CAS 123-03-5) (Figure 3) is another QAC used in various oral hygiene products and disinfectants [26]. It acts as an antiseptic by disrupting the cell membranes of microorganisms, resulting in inactivation. Cetylpyridinium chloride is utilized in oral hygiene solutions to effectively manage bacterial proliferation, mitigate plaque production, and deter the occurrence of halitosis [27]. The function of this entity is vital for the preservation of oral health and prevention of oral infections. Like other QACs, the excessive and prolonged use of goods containing cetylpyridinium chloride might result in the emergence of bacterial resistance. Strains that exhibit resistance have the potential to have diminished susceptibility to other antimicrobial agents, such as antibiotics, because of the common mechanisms of resistance.

Benzethonium chloride, benzalkonium chloride, and cetylpyridinium chloride are valuable components for the formulation of antiseptics and disinfectants. Their distinct mechanisms of action, ranging from cell membrane disruption to inhibition of bacterial growth, contribute to their effectiveness in preventing infections, controlling microbial growth, and maintaining public health in various settings.

3.1. Adaptation, Tolerance, and Resistance Mechanisms of Bacterial Pathogens to QAC (Pseudomonas aeruginosa, Enterococcus Species, and Acinetobacter baumannii)

Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) pathogens frequently exhibit MDR in both developed and developing countries, indicating their ability to withstand more than three classes of antibiotics, which significantly contributes to the increasing prevalence of antibiotic resistance [62]. ESKAPE pathogens pose a significant global health challenge, complicating treatment options for severe infections, escalating the burden of disease, raising mortality rates due to treatment failures, impacting the achievement of sustainable development goals, and necessitating a unified global approach for surveillance of AMR [63].

Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterococcus spp. have become prominent nosocomial pathogens, primarily owing to their remarkable ability to rapidly acquire resistance to a wide range of antimicrobial agents, and their ability to persist in the environment, humans, and animals [64–67].

3.2. Pseudomonas aeruginosa

Pseudomonas is a diverse genus of bacteria that is widely distributed in environmental niches, including water, wastewater, and plant surfaces. Furthermore, Pseudomonas aeruginosa is a significant nosocomial bacterium found in tap, recreational, and surface water and has been implicated in several infectious outbreaks in immunocompromised individuals [68, 69]. It is a widely distributed bacterium that thrives in many ecological environments because of its exceptional capacity to adapt, which is supported by its genome that is flexible and highly adaptable to different diets [70]. The remarkable ability of the bacterium to evade antimicrobial drugs is attributed to a wide array of innate and acquired resistance mechanisms within its genetic diversity.

These populations have been shown to adapt to subinhibitory concentrations by changing the structure of cell membranes, increasing the expression of efflux pumps, and accelerating biofilm growth and the capacity for persistent infections in both animal and human hosts [28, 71]. These attributes render this species especially appropriate for studying the impact of consistent subinhibitory levels of QACs. Lower concentrations of QACs can selectively affect the types of microorganisms that live in an area, possibly leading to the emergence of QAC-resistant populations. The primary cause of aerobic biodegradation of QACs has been identified as a bacterial species belonging to the Pseudomonas genus. The aerobic degradation of QACs is contingent on the presence of monooxygenases, dioxygenases, amine dehydrogenases, and/or amine oxidases [36]. The enzyme amine oxidase can significantly decrease BAC toxicity to bacteria by a factor of multiple hundred-fold. Approximately a decade ago, a gene cluster that encodes transporters, namely integrase and dioxygenase, was shown to be crucial for the biotransformation of BAC in aerobic environments [35]. The presence of multidrug efflux pump genes, including sugE, PmpM, mexAB-oprM, and mexEF-oprN, in the BAC-degrading community suggests that these efflux pumps and oxidase enzymes contribute to the enhancement of microbial resistance to QACs [72, 73].

3.3. Enterococcus Species

Among the more than 60 recognized Enterococcus species, Enterococcus faecalis and Enterococcus faecium are mostly associated with opportunistic human infections and are among the most common in the human gut microbiota [74]. It is important to examine how DAs affect the evolution of the Enterococcus microbiome, because this organism is one of the most common MDR healthcare-associated infections worldwide [75]. Inadequate cleaning during disinfection, improper biocide application, or the use of adulterated DAs in veterinary, food chain (e.g., food industry and farms), or human domestic contexts may lead to the use of QACs at subinhibitory concentrations in these specific contexts. Diverse microbiomes frequently encounter varying concentrations of QACs, potentially leading to alterations in the cellular composition of pathogens and fostering the selection of populations with reduced susceptibility to QACs and other antimicrobial agents (biocides or antibiotics) via co- or cross-selection mechanisms. The diminished sensitivity of Enterococcus species to QAC after adaptation may be attributed to several factors, including alterations in membrane fatty acid content, differential expression or mutations in efflux pumps, and stress responses. These findings reveal the complex bacterial adaptation mechanisms of QACs, potentially affecting disinfection strategies in healthcare. Understanding these processes may improve antimicrobial formulation and infection control protocols, thereby enhancing food and patient safety.

3.4. Acinetobacter baumannii

Acinetobacter is ubiquitous in nature and has been isolated from both clinical and environmental samples. MDR Acinetobacter baumannii has substantial clinical importance due to its role in hospital-associated nosocomial epidemics worldwide [76]. The potential impact of QAC and its derivatives on the spread of antimicrobial-resistant genes/bacteria via co-selection processes in manure and WWTPs, or in environments where Acinetobacter baumannii and QAC come into close contact [77]. High concentrations of BZK mostly cause membrane damage, although low concentrations have been demonstrated to disturb the balance of proteins in cells, ultimately resulting in the death of A. baumannii cells. The resistance and tolerance of Acinetobacter baumannii to QACs are frequently linked to mobile genetic elements (MGEs), which facilitate the extensive spread of AMR genes and bacteria. A. baumannii’s array of multidrug efflux pumps contribute to its high disinfectant (chlorhexidine and benzalkonium chloride) tolerance, which gives it a unique capacity to persist for extended periods of time in hospital conditions [78, 79]. Currently, resistance to disinfectants is an adaptive advantage. However, many A. baumannii efflux pump genes have been found in all species or genera, suggesting that they have a common function in the past [80]. The arrays of bacterial pathogens exhibiting QAC resistance patterns are shown in Table 2.

4.1. HGT of QAC and Antibiotic Genes

Drug-susceptible bacteria can acquire antibiotic resistance, primarily through genetic mutations or gene transfer, with HGT being the predominant mechanism. Mutations occur randomly and are typically spread through vertical inheritance; however, HGT is more concerning because it allows for the phylogenetic jumps of genes [7]. This suggests that the majority of the resistance genes found in microbiomes are part of the intrinsic resistome. The primary concern regarding HGT mechanisms in disinfectants and the spread of antiseptic resistance is conjugation. QACs facilitate the transfer of genetic material between bacteria via direct cell-to-cell contact [7]. Hu et al. [50] systematically investigated the effects of various QAC with differing structures and carbon chain homology on the conjugative transfer of antibiotic resistance genes (ARGs). Their findings indicated that most QACs at environmental concentrations could facilitate the horizontal transfer of ARGs from E. coli DH5α to E. coli MG1655 and S. sonnei through various mechanisms. The reported concentrations of QACs in aqueous environments range from 10⁻³ to 10⁻¹ mg/L. Without accounting for the synergistic or antagonistic effects of other environmental factors, it is important to highlight the potential risks of QACs facilitating the horizontal transfer of ARGs in the environment. S. sonnei is a pathogen that affects the human intestine, with its infection rate rising annually. This study demonstrated that S. sonnei can obtain ARGs from environmental bacteria through HGT, with exposure to QACs that enhance the transfer rate.

4.2. Efflux Pumps as a QAC Resistance Mechanism

Efflux pumps are the basic mechanisms of resistance to DAs. The concentrations of antimicrobial agents within cells are reduced by actively transporting agents out of the cells [101], as described by [99]. Efflux pumps can exhibit either particularity towards a substrate or possess a broad spectrum of substrates, as seen by MDR pumps. Efflux-mediated intrinsic resistance is a mechanism that enables tolerance to QACs in amounts that are considered environmentally harmful, as shown in Figure 4. Efflux-mediated QAC resistance has received attention because of its genetic basis, ability to create simultaneous resistance to antibiotics, and potential for HGT across different microbial species [103]. Proteins facilitate active expulsion of antimicrobials within bacterial cells. Efflux systems can actively transport a diverse array of chemically and structurally dissimilar substances, such as QACs, using energy- or proton-dependent mechanisms, while maintaining the integrity of transported chemicals [104, 105]. There are various categories of multidrug efflux systems, including (i) resistance-nodulation-division (RND), (ii) major facilitator superfamily (MFS), (iii) adenosine triphosphate (ATP)-binding cassette (ABC) family, (iv) small multidrug resistance (SMR) family, (v) multidrug and toxic compound extrusion (MATE) family, and (vi) proteobacterial antimicrobial compound efflux (PACE) [104, 106] MDR efflux systems, including the MFS, ATP-ABC, and SMR systems, are frequently observed in Gram-positive bacteria. Conversely, the RND, NorA, and QacA/A families of efflux pumps are more commonly associated with Gram-negative bacteria. The MATE family pump, known as PmPM, is present in several species of bacterial, including P. aeruginosa, Acinetobacter baumannii, Haemophilus influenzae, Clostridium difficile, Vibrio parahaemolyticus, Bacteroides thetaiotaomicron, and Staphylococcus aureus [107]. The upregulation of efflux pumps results in a 2- to 8-fold increase in resistance to QAC and other associated substrates transported by the pump. ABC transporters belong to the basic family class, whereas the remaining families are regarded as the advanced family class.

RND efflux pumps are mostly found in Gram-negative bacteria. They have three parts: a cytoplasmic membrane pump, a periplasmic protein, and an outer membrane protein channel. The AcrAB-TolC system, which belongs to the RND family, is prevalent in Gram-negative bacteria, including Escherichia coli. This system has a broad substrate specificity, including dyes, fluoroquinolones, and BAC. Proportion of subpopulations persisting significantly decreased by 1000-fold when the tolC gene was knocked out. This observation suggests that TolC facilitation of the efflux mechanism is crucial for the persistence of BAC tolerance in subpopulations. These results are in line with those of other studies that have found links between differences in the location of the AcrAB-TolC efflux pump, differences in tolC expression, and antibiotic resistance. The results of a recent study suggested that persistent cells exhibit greater tolerance to bactericidal antibiotics because of enhanced efflux mechanisms [100]. Further studies are required to validate this mechanism. This hypothesis posits that the random expression of the marRAB system, which serves as a regulator of acrAB and tolC51, can potentially contribute to the survival of artificial chromosomes in bacteria by inducing heterogeneous gene expression.

Resistance against ATMACs through efflux pumps might be attributed to two distinct processes: First, efflux pumps are overexpressed in response to exposure to high concentrations of QACs, which then results in resistance against QACs; Second, internally generated QAC stress can trigger overexpression of efflux pumps, resulting in resistance against QACs. Exposure to such stressors can activate a regulatory mechanism responsible for modulating the expression of efflux determinants. Alternatively, stress can cause one or more mutations in the genetic elements, which results in increased production of the efflux driver or makes its outflow more effective [108, 109]. In this context, genes encoding proteins associated with efflux pumps are often encoded within chromosomes or acquired through MGEs and exhibit activities against a diverse range of antimicrobial drugs. QAC resistance genes, such as class-1 integrons are frequently located in the same genetic elements as the antibiotic resistance genes. This colocalization potentially facilitates the emergence of co-resistance between QACs and antibiotics because they might possess comparable modes of action [52, 110]. Exposure to comparable sub-inhibitory concentrations of QACs or DAs can lead to the survival of potentially pathogenic microbes. Survival could result in the emergence of cross-resistance against medications that share structural or functional similarities, including antibiotics that are often used in medical interventions [111]. This phenomenon presents a plausible mechanism for producing antibiotic-resistant bacteria in the surroundings contaminated with QACs. It is possible for QAC efflux pump genes to be moved from one cell to another through conjugation, which can be accomplished by integrons, plasmids, transposons, and conjugative elements [112]. The qac gene provides an initial selection advantage by conferring resistance to DAs. Hence, the increasing use of QACs facilitates the fixation of additional genetic elements that are new and distinct. Integrons serve as reservoirs for resistance cassettes and can efficiently acquire supplementary resistance determinants through plasmids. The present vehicle technology demonstrates high efficiency in facilitating interspecies mobility. It is common for Gram-negative bacteria to become resistant to QACs by having qac genes, and for Gram-positive bacteria to become resistant by having class 1 integrons [113]. It is important to acknowledge that reduced sensitivity and sometimes resistance against QACs is facilitated by efflux pumps, among other possible mechanisms. Numerous studies have indicated that efflux pumps might facilitate biofilm formation either through direct or indirect mechanisms [114, 115]. Empirical and genetic evidence supports the idea that QAC environmental residues might potentially have a significant impact on the evolution of AMR, and the transfer of genes linked to AMR [98]. Because of its extensive array of efflux pumps, which can expel many antimicrobial agents, including QACs and antibiotics, the genus Pseudomonas has significant relevance in the context of AMR, owing to its extensive distribution in many environments. Its inclusion as one of the most notorious drug-resistant bacteria poses significant challenges in the treatment of bacterial infections [28, 116]. QACs facilitated the transfer of RP4-resistant plasmids into E. coli at low doses. The quantities of QACs that maximized the enhancement of trans-conjugate efficiency were comparable to those observed in the environment. This indicates that the presence of residual QACs in the environment might worsen environmentally induced resistance of bacteria to antimicrobials and/or antibiotics [117].

4.3. Biofilm Formation as a QAC-Resistant Mechanism

Compounds such as benzalkonium chloride and chlorhexidine can potentially induce biofilm development. Biofilms, which are intricate assemblies of microorganisms surrounded by a protective extracellular matrix, serve as physical barriers that can confer protection against antimicrobial agents, such as antibiotics. It has been suggested that EPS matrices enhance recalcitrance by impeding the diffusion of disinfection solutes into biofilms. This hindrance can occur through mechanisms such as size exclusion, interactions, and reactions with solutes [118]. Biofilm formation contributes to a decrease in the vulnerability of both Gram-positive and Gram-negative bacteria to disinfectants. Gram-negative bacteria have a greater propensity to acquire resistance against QACs because of the composition of their cell walls [119]. Furthermore, the extracellular matrix contains other constituents, including enzymes, that can contribute to the detoxification of harmful substances. For example, P. aeruginosa can withstand exposure to disinfectants through the formation of persistent cells, effectively hindering the manufacture of antibiotic targets [120, 121]. According to Qin et al. [122], the presence of molecules derived from persistent cells can sustain their viability and replenish biofilms. In the absence of disinfectants, bacteria regain growth and viability, resulting in the development of chronic infections. The effectiveness of disinfectants against biofilms is significantly lower than that of planktonic organisms [123].

The resistance of biofilms to disinfectants is contingent on factors such as the efficacy of disinfectants, temperature, and surface characteristics [125]. In addition, bacterial biofilms formed within wastewater treatment systems can function as reservoirs of AMR. Biofilms provide a shield against antimicrobial agents, allowing bacteria to survive and potentially exchange resistance genes within the biofilm community [10]. The coexistence of various microorganisms within the surrounding ecosystem may potentially contribute to increased survival when exposed to disinfectant-induced stress as shown in Figure 4. For example, Listeria monocytogenes monocytes have been shown to protect against Pseudomonas putida when exposed to low levels of unclassified BAC [126]. The treatment selectively targeted and eradicated L. monocytogenes. Another discovery was that the addition of Pseudomonas fluorescens to dual-species biofilms made Salmonella enterica more resistant to a group of unknown QACs [121, 127].

4.4. Biotransformation as a QAC-Resistance Mechanism

Most types of antibiotics can be broken down by photo- or hydrolytic activity in the environment because of the way their molecules are structured and the physical and chemical properties they have [124]. The initial steps of QAC biodegradation are a multistep process conducted by microorganisms, which is similar to the degradation of antibiotics. Metabolism requires the recognition of the molecule and efficient binding between enzymes and substrates [129]. This phenomenon is contingent on the arrangement of the molecule and its physicochemical characteristics. The metabolic processes of QACs are influenced by their structural compositions. Consequently, the presence of hydrophobic alkyl chains has been shown to significantly affect the biodegradation and toxicity of QACs. Specifically, owing to their lower solubility, the elongation of alkyl chains has been shown to reduce biodegradability [130, 131]. Hence, reduced solubility restricts the availability of microbial species. DAs might have less of an effect on the ecosystem if there is a diverse community of microbes that include species with moderate to high tolerance and the ability to break down antimicrobial compounds. Because biotransformation reduces the concentrations of antimicrobials, exposure-susceptible species may reach subinhibitory concentrations. This could facilitate the development of resistance. Previous studies have demonstrated that certain bacteria possess the ability to metabolize disinfectants, such as QACs. These microbes respond to antimicrobial agents and may potentially acquire resistance through several metabolic pathways [132, 133].

Biotransformation is likely to be the primary method for removing disinfectants such as QACs from wastewater treatment facilities. When a community of microbes is exposed to QACs, the genera that are naturally resistant or that can biotransform QACs are given more attention [134]. For instance, a previous study reported that after exposure to BAC, microbial communities exhibited a decrease in activity and were composed of Pseudomonas sp., as described by [99, 131]. P. nitroreducens, P. aeruginosa, and P. putida were the Pseudomonas species that were most often seen after being exposed to BAC in the special enrichment medium. Additional taxa, including Citrobacter sp., Klebsiella sp., Salmonella sp., Enterobacter sp., and Achromobacter, have been identified [36]. Relative to enhanced microbial communities, bacterial artificial chromosomes (BACs) undergo degradation via dealkylation, resulting in the formation of BDMA. This is followed by successive demethylation, resulting in the production of dimethylamine and methylamine [99]. With the development of resistance, microbes may acquire the capacity to use antimicrobials as a viable source of nutrition or energy. These microbes, which are related to P. nitroreducens and P. putida, have been linked to the process of assimilation and degradation of BAC [135]. This phenomenon not only provides advantages to the destruction of microorganisms but might also offer protection to other members of the community because of the influence effect.

The continuous emergence of QAC resistance in the environment necessitates urgent development of next-generation disinfectants for human health. It is essential to balance antibiotic efficacy with environmental factors in order to successfully combat the emergence of bacterial resistance [135]. Recent research has shown that biscationic quaternary phosphonium compounds (bis-cationic QPCs) are very effective against both Gram-positive and Gram-negative pathogenic bacterial strains that can acquire QAC resistance mechanisms [135].