Biocides as drivers of antibiotic resistance: A critical review of environmental implications and public health risks

Review Biocides as drivers of antibiotic resistance: A critical review of environmental implications and public health risks Mariana Sousa a , b , Idalina Machado a , b , Lúcia C. Sim~ oes c , d , Manuel Sim~ oes a , b , * a LEPABEdLaboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, Department of Chemical and Biological Engineering, University of Porto, 4200-465, Porto, Portugal b ALiCEdAssociate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto, 4200-465, Porto, Portugal c CEBdCentre of Biological Engineering, University of Minho, 4710-057, Braga, Portugal d LABBELSdAssociate Laboratory in Biotechnology and Bioengineering and Microelectromechanical Systems, Braga, Guimar~ aes, Portugal article info Article history: Received 25 November 2024 Received in revised form 18 March 2025 Accepted 19 March 2025 Keywords: Adaptive resistance Antibiotic resistance Biocides Biofilms Cross-resistance abstract The widespread and indiscriminate use of biocides poses significant threats to global health, socioeconomic development, and environmental sustainability by accelerating antibiotic resistance. Bacterial resistance development is highly complex and influenced significantly by environmental factors. Increased biocide usage in households, agriculture, livestock farming, industrial settings, and hospitals produces persistent chemical residues that pollute soil and aquatic environments. Such contaminants contribute to the selection and proliferation of resistant bacteria and antimicrobial resistance genes (ARGs), facilitating their dissemination among humans, animals, and ecosystems. In this review, we conduct a critical assessment of four significant issues pertaining to this topic. Specifically, (i) the role of biocides in exerting selective pressure within the environmental resistome, thereby promoting the proliferation of resistant microbial populations and contributing to the global spread of antimicrobial resistance genes (ARGs); (ii) the role of biocides in triggering transient phenotypic adaptations in bacteria, including efflux pump overexpression, membrane alterations, and reduced porin expression, which often result in cross-resistance to multiple antibiotics; (iii) the capacity of biocides to disrupt bacteria and make the genetic content accessible, releasing DNA into the environment that remains intact under certain conditions, facilitating horizontal gene transfer and the spread of resistance determinants; (iv) the capacity of biocides to disrupt bacterial cells, releasing intact DNA into the environment and enhancing horizontal gene transfer of resistance determinants; and (iv) the selective interactions between biocides and bacterial biofilms in the environment, strengthening biofilm cohesion, inducing resistance mechanisms, and creating reservoirs for resistant microorganisms and ARG dissemination. Collectively, this review highlights the critical environmental and public health implications of biocide use, emphasizing an urgent need for strategic interventions to mitigate their role in antibiotic resistance proliferation. ©2025 The Authors. Published by Elsevier B.V. on behalf of Chinese Society for Environmental Sciences, Harbin Institute of Technology, Chinese Research Academy of Environmental Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction Based on the “One Health”concept, which states that “the health of people is connected to the health of animals and the environment,”the measures to halt antimicrobial resistance from spreading through the environment are timely and much-needed [1,2]. Biocide pollution contributes to the evolution and dissemination of bacterial resistance among humans, animals, and the environment by creating favourable conditions for selecting resistant bacteria and facilitating the transmission of genetic material across ecological boundaries [3e5]. When released into a certain environment, biocides create a selective pressure favouring bacteria with resistance mechanisms [6,7]. For these reasons, addressing this challenge requires a “One Health”approach, incorporating sustainable biocide use, robust waste management practices, and monitoring of environmental reservoirs [8]. In addition to the *Corresponding author. at: LEPABEdLaboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, Department of Chemical Engineering, University of Porto, 4200-465 Porto, Portugal. E-mail address: [email protected].pt (M. Sim~ oes). Contents lists available at ScienceDirect Environmental Science and Ecotechnology journal homepage: www.journals.elsevier.com/environmental-science-andecotechnology/ https://doi.org/10.1016/j.ese.2025.100557 2666-4984/©2025 The Authors. Published by Elsevier B.V. on behalf of Chinese Society for Environmental Sciences, Harbin Institute of Technology, Chinese Research Academy of Environmental Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Environmental Science and Ecotechnology 25 (2025) 100557 existing monitoring programs that track unnecessary antibiotic prescriptions and misuse, it is essential to include biocides as they may also contribute to resistance spread. Additionally, the environment should be considered as a potential source of resistance [9,10]. In particular, biocides, which exert their antimicrobial activity through a multi-target mode of action, are essential to control infectious diseases and safeguard animal and human health [11,12]. Demand for biocides is expected to increase in the coming years, driven by their use in various industrial processes and for the disinfection of hospitals and public spaces [13]. A significant increase in pests and hygiene issues is expected because of global warming, climate change, and associated extreme weather events [14]. This requires effective responses to control potential pathogens while ensuring minimal negative impacts on public health and the environment. The societal dependence on biocides for disinfection was remarkable during the SARS-CoV-2 pandemic [15e18]. Understanding the intricate connections between biocides, antibiotics, and antimicrobial resistance is critical for devising strategies to mitigate the spread of resistance across humans, animals, and the environment [19,20]. This review explores the evidence linking biocide exposure to the emergence of resistant microorganisms, focusing on the mechanisms by which biocides drive resistance and the environmental pathways that facilitate the dissemination of resistance genes. This study underscores the urgent need for integrated and sustainable approaches to manage biocide use, protect public health, and preserve ecosystem integrity. 2. Evidence of biocides as drivers of antimicrobial resistance Biocides are far from innocuous to the environment and public health. They can persist in their target niches for two reasons [21]. Firstly, bacteria elimination by these agents has proven to be difficult, and it has become clear that biocidal efficacy may be questionable in some circumstances. Within this context, bacteria are regularly exposed to sub-bactericidal concentrations, which can lead to bacterial adaption and resistance to in-use biocides [22e24]. Secondly, antimicrobial sub-bactericidal concentrations are present in the environment since some biocides that are not consumed in the reaction of cleaning procedures are freely released in the discharge waters (for instance, oxidative species would be actively quenched by the organic load present and have no legacy effects) [25e27]. For instance, studies have shown that Pseudomonas aeruginosa can develop resistance to quaternary ammonium compounds (QACs) such as benzalkonium chloride (BZK) through mechanisms like efflux pump overexpression and alterations in membrane composition [28,29]. This exposure can also lead to cross-resistance to antibiotics like ciprofloxacin, as the same efflux systems expel both biocides and antibiotics from the bacterial cell [28,29]. Moreover, Listeria monocytogenes exposed to sub-inhibitory concentrations of BZK in food environments can adapt by altering membrane composition and surface characteristics, leading to increased resistance to BZK and other unrelated antibiotics [29,30]. Concerning triclosan, research has demonstrated that low levels of this biocide, commonly found in personal care products, can induce bacterial resistance through mutations in key enzymes involved in fatty acid synthesis, such as FabI [30]. This resistance can extend to antibiotics like isoniazid, which also targets fatty acid synthesis pathways [30]. For Acinetobacter baumannii, sub-lethal exposure to chlorhexidine has been shown to activate efflux pump systems and promote biofilm formation, making the bacteria more resistant to chlorhexidine and antibiotics like carbapenems [30]. One of the possible solutions for this lack of efficiency is an increase in the recommended in-use concentration [11]. In this environment, pollutants are released not only from industrial and hospital discharges but also from a wide range of human activities, including consumer products, personal care items, pharmaceuticals, and their metabolites. Additionally, natural sources, such as compounds secreted by bacteria, fungi, algae, and plants, contribute to the pollution [31e33]. The increased consumption of biocides and the fact that these are poorly biodegradable make them present in the environment as parent compounds or conjugates [31,34e39]. These compounds can be very persistent in the environment, in varying types and concentrations across the globe, since they adsorb strongly to environmental elements such as sludge and sediment, showing low degradation potential [40e45]. Fig. 1 presents a compilation of some studies reporting the world distribution of biocides and antibiotics with high relevance, prevalence, and use worldwide. Wastewater treatment plants (WWTPs), rivers, and basins that receive treated and untreated sewage are important contributors to the dissemination of resistant bacteria and antibiotic-resistance genes (ARGs) [46e49]. The globalization of ecosystems, along with the rising consumption of antibiotics and biocides (such as antiseptics, preservatives, and biocides), contributes to the widespread distribution and accumulation of these biopollutants in ecosystems worldwide [50e53]. Several biocides, such as QACs, are high-production volume chemicals (i.e., production >1000 tons per year) and, in many cases, can also have non-biocidal applications [54]. In 2017, a Chinese study aimed to examine the presence and elimination of nineteen biocides across ten different WWTPs [47]. Additionally, it sought to estimate the per capita usage and emissions of these 19 biocides using a mass balance analysis approach. The mass balance used was: MInfluent ¼MEffluent þMSludge þMLoss (1) where M Influent ,M Effluent , and M Sludge (g d 1 ) represent the mass loads of the target biocide in the influent, effluent, and excess sludge of each WWTP, respectively. M Loss (g d 1 ) denotes the loss of mass loads of the target biocide during the entire treatment process, primarily due to sorption and degradation. The loss mass fractions (M f,Loss% ) for each biocide were calculated using: Mf;Loss% ¼MInfluent MEffluent MSludge MInfluent 100% (2) The mass fractions in effluent (M f,Effluent%) and excess sludge (M f,Sludge% ) were calculated as: Mf;Effluent% ¼MEffluent MInfluent 100% (3) Mf;Sludge% ¼MSludge MInfluent 100% (4) The estimated pollution loads per capita of biocides in the influent, effluent, and excess sludge for each WWTP were given by: Pollution load per capita ¼ MInfluent=Effluent=Sludge Population served (5) The back-estimated usage (U, t year 1 ) and estimated emissions (E, t year 1 ) for Guangdong Province or China were determined as: U¼LInfluent PTotal 365:25 1012 (6) M. Sousa, I. Machado, L.C. Sim~ oes et al. Environmental Science and Ecotechnology 25 (2025) 100557 2 E¼ERural þEUrban (7) E¼hLInfluent PRural þLEffluent þLSludgePUrbani365:25 1012 (8) where L Influent ,L Effluent , and L Sludge ( m gd 1 person 1 ) are the estimated pollution loads per capita of a target biocide in the influent, effluent, and excess sludge (mean of ten WWTPs). P Total ,P Rural , and P Urban are the total, rural, and urban populations. These values are based on the “China Statistical Yearbook”(2015). For Guangdong: P Total ¼10724 10 4 ,P Rural ¼3432 10 4 , and P Urban ¼7292 10 4 . For China: P Total ¼136782 10 4 ,P Rural ¼61866 10 4 , and P Urban ¼74916 10 4 . The estimated usage of the biocides under investigation was as high as 453 tons per year, corresponding to 308 m g per day per person (including effluent and excess sludge) [47]. In Minnesota (United States),1995 metric tons of QACs were sold in 2017 for nonagricultural purposes [48]. A study by Arnold et al. [54] reported that over 450000 metric tons of QACs are produced or imported annually in the United States. In 1997, the total use of QACs in Germany was 12349 metric tons per year by industry and 95.3 metric tons per year by hospitals, while in Great Britain, 28852 metric tons per year are being used [55e57]. Moreover, approximately 75 % of the QACs used each year are discharged into wastewater treatment systems, with the remainder going directly into the environment [58]. Globally, surface water and wastewater effluent contain concentrations of QACs from 1 to 100 m gL 1 , this is 0.0000001 %e0.00001 % [weight/volume (w/v)], although influent wastewater sometimes contains 10 times these concentrations [58,59]. Furthermore, the minimum inhibitory concentrations (MICs) of QACs range from 0.005 % to 0.01 % w/v, while the minimum bactericidal concentrations (MBCs) are typically between 0.02 % and 0.05 % w/v [60]. Zheng et al. [61] assessed the bioaccumulation potential of 18 QACs with alkyl chain lengths ranging from C8 to C18. That evaluation was conducted through an in vitroein vivo Fig. 1. World distribution of biocides and antibiotics (in ng L 1 ) in rivers, basins, seas or lakes, and wastewater treatment plants (influent). The investigated biocides and antibiotics were selected due to their high relevance, prevalence, and use worldwide. AMP: Ampicillin; ATMAC: Alkyltrimethylammonium compounds; AZM: Azithromycin; BZK: Benzylalkyldimethylammonium compounds; CHX: Chlorhexidine; CIP: Ciprofloxacin; CLM: Clarithromycin; CTAB: Cetyltrimethylammonium bromide;DADMAC: Dialkyldimethylammonium compounds; ERY: Erythromycin; NOR: Norfloxacin; PhP: Ortho-phenylphenol; ROX: Roxithromycin; SMZ: Sulfamethoxazole; STZ: Sulfathiazole; TCC: Triclocarban; TCS: Triclosan; TET: Tetracycline; TMP: Trimethoprim. M. Sousa, I. Machado, L.C. Sim~ oes et al. Environmental Science and Ecotechnology 25 (2025) 100557 3 extrapolation (IVIVE) model, utilizing data obtained from experiments on human hepatic metabolism and serum protein binding. Of the 18 QACs aimed for detection, 15 were identified in blood, with cumulative QAC concentrations reaching a maximum of 68.6 ng mL 1 . Blood samples were obtained from two specific periods: pre-COVID-19 pandemic outbreak (2019; n¼111) and during the pandemic (2020; n¼111). The cumulative QAC concentrations were notably elevated in samples collected during the pandemic (6.04 ng mL 1 ) compared to those gathered before (3.41 ng mL 1 ) [61]. In Europe, the regulation for BZK falls under the purview of the European Commission (EC). Recent regulations in the European market have modified the permissible maximum residual levels of BACs in food products, reducing the limit from 0.5 to 0.1 mg kg 1 .In addition, the alterations in legislation, specifically Decision [European Union (EU)) 2016/1950 and the Biocidal Products Regulation (BPR, EU) no. 528/2012 have resulted in the disapproval of BACs for application in various biocidal products. Notably, consumer hand and body wash antiseptics are affected, marking a departure from the prevailing regulations in the United States [62]. Some of these chemical compounds end up diluted in what we eat, drink, and breathe [25]. This has triggered concern because, in the environment, biocides persist and bioaccumulate, being harmful not only to humans but also to microorganisms [8]. Biocides can modify the dynamics and action of antibiotics in natural ecosystems, selecting resistant organisms [3,63,64] with increased resistance and co-resistance to other biocides as well as crossresistance to antibiotics [24,51]. The role of biocides in the selection, spread, and maintenance of resistant bacteria worldwide should be addressed [65], considering that water basins are globally polluted with several classes of biocides [27,66]. Therefore, there is an urgency to understand the mechanisms of action of biocides and resistance and if their presence in the environment, even in reduced concentrations, can cause resistance to antibiotics. This study reviews the phenomena of bacterial resistance to in-use biocides and our current knowledge of how this can trigger antibiotic resistance, with particular emphasis on biocides that are continuously released and accumulated in the environment [51]. The main question remains: Should biocide subbactericidal concentrations be considered an important driver of increased antibiotic resistance? 3. Mechanisms of action of biocides and their impact on bacterial resistance Generally, antimicrobial agents can be classified as any molecule with biochemical properties that kill or prevent the growth of microorganisms, including bacteria, fungi, and algae, on a biotic or abiotic surface [67]. Antimicrobial agents can be divided into antiseptics (agents operating on living tissues), disinfectants (products applied to inanimate objects and surfaces), and preservatives [68,69]. However, they can also be classified according to the type of microorganisms against which they are effective: antibiotics, antivirals, antifungals, and antiparasitics [70,71]. Furthermore, these molecules target crucial processes in cellular metabolism, including the production of biological macromolecules, cellular enzyme activity, and cellular components [71,72]. Consequently, in contrast to antibiotics that are used to treat specific bacterial infections, biocides are a diverse group of antimicrobial compounds that, due to their versatility and broadspectrum action, are used in personal care products (soaps, handwashes, toothpaste, mouthwashes, and cosmetics) [73e75] and as part of detergent formulations for surface cleaning, the process of removing all foreign material from objects by using water and detergents, and disinfection eelimination of most or all pathogenic microorganisms (except spores) [68,76]. Industrially, biocides are used in high doses to clean hard surfaces efloors, walls, equipment eand food surfaces. Additionally, in most production processes, the water used often has a residual concentration of biocides to ensure its low microbial load [77,78]. In clinical environments, biocides are fundamental for the antisepsis of the patient's skin as well as for the disinfection of water and hard surfaces and sterilization of medical instruments and rooms [77]. Typical biocides used for cleaning and disinfection purposes are QACs, alcohols, aldehydes (glutaraldehyde, ortho-phthalaldehyde [OPA]), bisbiguanides (chlorhexidine), bisphenols (triclosan), diamidines, halogen-releasing agents, halophenols, heavy-metal derivatives and peroxygens (hydrogen peroxide) [68,79]. Many factors affect the efficacy of biocides in both actions ecleaning and disinfection ethe stability of the active molecule, the contact time, the concentration of biocide, the age and metabolic status of the microbial community, the existence of adhered bacteria or biofilms, and the presence of organic load and other environmental factors such as temperature and pH [68,80]. Biocides play an important role in limiting bacteria presence and reducing potential sources of contamination and infection and are effective due to their broad spectrum of activity and ability to act on multiple targets [11,68]. Moreover, the majority of biocides disrupt the cytoplasmic membrane [68], dissipating the proton motive force and inhibiting membrane-associated enzymes [81]. Unlike antibiotics, which act selectively against specific cell targets, the mechanism of action of biocides occurs at one or several other sites within the cell [82]. Biocides can interact with the cell envelope targeting, for instance, the cytoplasmic membrane (which is a component of the cell envelope) (e.g., chlorhexidine and QACs) [83]; cross-link with other macromolecules (e.g., glutaraldehyde) [84]; intercalate (e.g. acridines) and interact with deoxyribonucleic acid (DNA; e.g. halogens, silver ions, and oxidizing agents) [81]; and interact with thiol groups in enzymes and proteins (e.g. organomercurials and silver compounds) [81]. Although the outer cell envelope is the first target, as it is the initial point of contact, the inner cell components are also often affected. However, the cell is frequently inactivated through this initial surface interaction [85,86]. Other targets inside the cell, such as nucleic acids, enzymes, and ribosomes, are more difficult to reach since the biocide needs to penetrate the cell [81]. Understanding these interactions is essential for developing effective antimicrobial treatments and addressing the challenge of bacterial resistance. Table 1 presents an overview of various biocides, their mechanisms of action against bacteria, and corresponding bacterial resistance mechanisms. This table highlights how different chemical agents target bacterial cells and the strategies employed by bacteria to resist their effects [68,87]. Biocides are usually applied in high concentrations to improve their efficiency, making it impossible for bacteria to overcome the damage caused and develop resistance [88]. In fact, in hospitals and clinical settings, the antimicrobial in-use concentration is frequently two-to four-fold the MIC of wild-type strains, meaning that the antimicrobials are effective against 99 % of the environmental microorganisms [89,90]. Furthermore, as disinfectants generally contain more than one type of active component, each with a different antimicrobial mode of action, and as they have no specific target, the development of resistance at the level of in-use concentrations is thought to be highly unlikely [68,89]. Nevertheless, two features can contribute to the resistance to biocides and subsequent resistance: (1) the 1 % of bacteria that are not killed by the in-use concentration can present naturally reduced susceptibility due to the presence of resistant genetic information [89]; (2) a cleaning solution at a sub-inhibitory concentration can occur, for example, when organic matter is present (since the organic matter M. Sousa, I. Machado, L.C. Sim~ oes et al. Environmental Science and Ecotechnology 25 (2025) 100557 4 may physically dilute the cleaning solution, reducing the concentration of active ingredients available to act on microorganisms [91]; can react chemically with disinfectants [91]; and can create a physical barrier that protects microorganisms from direct exposure to the cleaning agent [92]), if insufficient cleaning is applied, or even in the rinsing water [89]. In the environment, we may think both situations happen: water and land accumulate a multiplicity of resistant strains and genetic material, and sub-inhibitory concentrations of biocides are naturally present as persistent organic pollutants [93,94](Fig. 1). 4. Cross-resistance to antibiotics Although bacterial recalcitrance to biocides (resistance) was described early in the 1950s or 1960s, it appears to be increasing, and nowadays, we are still debating on the possible emergence of biocide resistance and its association with the emergence of antibiotic recalcitrance [95]. The term “cross-resistance”can be defined as a phenomenon that occurs when a resistance mechanism to a certain antimicrobial enables a specific strain to resist several other antimicrobials with similar mechanisms of action [96]. In the case of co-resistance, the mechanisms conferring resistance to both agents are unrelated but are genetically linked, e.g., located on the same genetic element [97]. Some studies in vitro confirmed this cross-resistance concept (exceptionally clarified in C^ andido et al. [98]). Sonbol et al. [99] examined the effectiveness of several antibiotics, such as b -lactams, cephalosporins, macrolides, aminoglycosides, quinolones, sulfonamides, and tetracycline against Escherichia coli isolates that have been exposed to sub-bactericidal doses of the biocide triclosan. The outcomes showed that apart from amikacin and trimethoprim/ sulfamethoxazole, cells adapted to triclosan became more resistant to the antibiotics tested and remained susceptibility to the biocide, associated with decreased permeability of outer and inner membranes, increased depolarisation of membranes, higher negative net charge of membranes, and higher efflux activity [84]. As triclosan acts as a substrate for bacterial multidrug efflux pumps, E. coli [99] and P.aeruginosa [100] exposed to triclosan at subbactericidal doses displayed a larger operation of efflux pumps, likely associated with cross-resistance. A study by Wand et al. [101] demonstrated that, when exposed to chlorhexidine, Klebsiella pneumoniae acquired cross-resistance to the antibiotic colistin. The authors reported that the adaptation of K. pneumoniae to chlorhexidine occurs through regulating genes associated with efflux pumps, namely smvR and smvA. Moreover, the operon pmrK, which is responsible for reducing the negative charge of lipid A, was upregulated by chlorhexidine. As a result, a decreased binding affinity of colistin to lipid A occurred, resulting in cross-resistance. However, the resistance to chlorhexidine was not increased by exposure to colistin [101]. A study developed by Curiao et al. [102] showed that mutants of Salmonella enterica Typhimurium created after exposure to triclosan, chlorhexidine, and BZK exhibited different phenotypes against a broad range of antibiotics: ampicillin, ceftazidime, ciprofloxacin, erythromycin, gentamicin, chloramphenicol, and tetracycline. Most of the mutants that were confirmed to be resistant to biocides presented an increased susceptibility to molecules whose site of action is the cell wall ( b - lactams) or the cell membranes (poly-L-lysine, polymyxin B, colistin, and toxic anions). Moreover, both biocide mutants selected in vitro and field isolates with resistance to biocides exhibited overexpression of genes related to cold-shock response (cpeE), ribosomal and transcription proteins, efflux pumps (sugE), and enzymes of microaerobic metabolism, particularly those of the phosphotransferase system [102]. In Sanchez et al. [103], triclosan proved to selectively induce the expression of the efflux pump SmeDEF in Stenotrophomonas maltophilia, which in turn triggers a transient low-level resistance to the biocide and cross-resistance. Compared to the wild-type S. maltophilia,five out of the 12 triclosan-selected mutants were more resistant to the antibiotics tetracycline, chloramphenicol, ciprofloxacin, tobramycin, and triclosan [103]. Pereira et al. [104] conducted an adaptive laboratory evolution of E. coli using constant and sub-inhibitory concentrations of ten widespread biocides: chlorophene, BZK, glutaraldehyde, chlorhexidine, peroxide, povidone-iodine, isopropanol, ethanol, sodium hypochlorite, and peracetic acid. Interestingly, and as a result of evolution, 43 % of strains became resistant to three representative antibiotics with diverse cellular modes of action: ampicillin, chloramphenicol, and norfloxacin. The authors also found mutations in genes encoding for multidrug efflux proteins (mdfA and acrR), porins (envZ and ompR), and subunits of E. coli RNA polymerase (rpoA and rpoBC). The biocides chlorophene, BZK, glutaraldehyde, and chlorhexidine were the ones instigating higher antibiotic susceptibility (probably due to the occurrence of mutations in membrane proteins and their regulators, including those responsible for transporting chemicals like antibiotics into and out of the cell), while hydrogen peroxide and povidone-iodine were the ones contributing least to an increase in susceptibility to antibiotics. No evidence of antibiotic cross-resistance for isopropanol, ethanol, sodium hypochlorite, and peracetic acid was observed [104]. Salmonella spp. exhibited overexpression of AcrAB-TolC and Table 1 Overview of various biocides, their mechanisms of action against bacteria, and corresponding bacterial resistance mechanisms, focusing on how different chemical agents target bacterial cells and the strategies bacteria employ to resist their effects [68,87]. Biocide Mechanism of action Bacterial resistance mechanisms Quaternary ammonium compounds Disrupts cell membrane integrity Efflux pumps: Expel quaternary ammonium compounds out of the cell Membrane modification: Reduces permeability Alcohols (e.g. Ethanol, Isopropanol) Denatures proteins, disrupts membranes Biofilm formation: Limits penetration Membrane modification: Alters lipid composition Chlorine/Chlorine-based compounds Oxidizes proteins, lipids, and DNA Enzymatic degradation: Produces enzymes to neutralize oxidative damage Reduced uptake: Alters porin proteins Peroxides (e.g. Hydrogen peroxide) Produces reactive oxygen species, causing cellular damage Enzymatic degradation: Catalase and peroxidase break down peroxides Biofilm formation: Protects against oxidative damage Phenolics Disrupts cell walls and membranes denatures proteins Efflux pumps: Actively expel phenolics Biofilm formation: Shields bacteria from penetration Heavy metals (e.g. Silver, Copper) Binds to proteins and enzymes, disrupting cellular function Membrane modification: Reduces metal ion permeability Efflux pumps: Actively remove metal ions Biguanides (e.g. Chlorhexidine) Disrupts cell membrane, leading to leakage Efflux pumps: Expel biguanides Membrane modification: Alters membrane composition to resist disruption M. Sousa, I. Machado, L.C. Sim~ oes et al. Environmental Science and Ecotechnology 25 (2025) 100557 5 underexpression of outer membrane porins after exposure to a quaternary ammonium disinfectantdcontaining formaldehyde and glutaraldehyde. This phenomenon resulted in antibiotic resistance to ciprofloxacin, chloramphenicol, tetracycline, and ampicillin [105,106]. Tabata et al. [107] reported that the expression level of OprR was correlated with the resistance level of P. aeruginosa to QACs. A QACresistant P. aeruginosa strain exhibited a higher expression level of OprR compared with a knock-out mutant [107]. Machado et al. [108] demonstrated that P. aeruginosa adapted to BZK showed differences regarding the expression of outer membrane proteins (OMPs) compared to a non-adapted strain. Some of these proteins are involved in ciprofloxacin resistance [108]. One example of target alteration was demonstrated by Sheridan et al. [109], where a triclosan-tolerant E. coli strain exhibited an amino acid substitution in the FabI protein, where a glycine was replaced by a valine. This point mutation prevents the formation of the FabI-NAD þ complex, reducing the effect of triclosan in E. coli [109]. A stable chlorhexidine-resistant Pseudomonas stutzeri, obtained after exposure to chlorhexidine, showed resistance to triclosan and BZK and antibioticsdpolymyxin B, gentamicin, and ampicillin [110]. Nevertheless, cross-resistance phenomena do not occur exclusively in cells in the planktonic state, having also been observed in biofilms, as demonstrated in Tabak et al. [111]. The results clarified that the expression of degradative enzymes, changes in the bacterial cell envelope, activation of the efflux pump, and mutations in the enoyl reductase enzyme are all associated with the recalcitrance observed in planktonic S. Typhimurium after exposure to triclosan. Additionally, the authors found that, regarding the cells in biofilms of S. Typhimurium, their susceptibility to triclosan is associated with the low extracellular matrix diffusion of the substrate, overactivity of efflux pumps, and exopolysaccharide production [111]. In that sense, the underlying message of the previous studies is the same: there is a direct relationship between the presence of biocides in the environment and antibiotic resistance [112]. However, other authors are not certain that this phenomenon can occur, as studies addressing this in vivo are still missing [89,97]. The main mechanisms of resistance of conventional classes of biocides and the possible relationship between biocide and antibiotic crossresistance described in the literature are summarized in Table 2. Resistance to antibiotics due to exposure to biocides is of public health relevance when it concerns pathogenic or opportunistic bacteria [24]. Co-resistance can also be a problem with nonpathogenic and commensal bacteria when there is a risk of horizontal gene transfer of resistance determinants to (opportunistic) pathogens [113,114]. Moreover, the awareness of risks related to sub-inhibitory biocide concentrations triggering cross and coresistance in bacteria has substantially increased [97]. In addition, cross and co-resistance appear when the biocide and the antibiotic act on the same cellular target; the biocide and the antibiotic have the same transport mechanism; biocide and antibiotic can be accommodated by the same resistance mechanism, and in situations where genes contributing toward biocide resistance and antibiotic resistance are carried on the same mobile genetic element [22]. Biocides and antibiotics may have similar and common interactions and target sites in bacteria (e.g., efflux pumps, permeability changes, and biofilms), which might express shared resistance mechanisms [93]. When only sub-bactericidal concentrations of biocides are present, minor cell damages can occur, and the expression of multidrug efflux pumps can be induced, selecting clones that confer resistant phenotypes located on the same mobile genetic element such as a plasmid, transposon, or integrons [88,115,116]. Table 3 presents a compilation of the biocides referred to in this study, organized by their chemical category and common applications: QACs [54], bisbiguanides [117], phenolics compounds [118], aldehydes [119], alcohols [120], peroxygens [121], halogenreleasing agents [122], bisphenols [123], heavy-metal derivatives [124], and others (povidone-iodine and diamidines) [125]. 5. Mechanisms induced by biocides in the development of resistance Recent literature [15,84,126,127] provides clear scientific evidence demonstrating that the presence of biocides in the environment can be associated with various types of resistance, namely. (i) the natural selection of most resistant strainsdintrinsic property [15,126]; (ii) the phenotypic adaptation to biocides [84,127]; (iii) the acquisition and/or release of genetic elements from resistant strains; chromosomal gene mutation or genetic material acquisition of (plasmids or transposons) [126]; (iv) the role of biofilm communities in resistance development [127]. In the following sections, we will critically examine these different types of resistance to biocides. Fig. 2 provides a compilation of the mechanisms by which biocides promote antibiotic resistance, which this study addresses. 5.1. Biocides as natural selectors of resistant strains Intrinsic resistance is the naturally greater resistance related to certain microbial species than others. Some microorganisms are already genetically predisposed to resist biocides and antibiotics [68,84,128e130](Fig. 3). A feature that is extensively present within several bacterial species, unrelated to prior antibiotic exposure and unconnected to horizontal gene transfer is referred to as intrinsic resistance [128,131]. Reduced outer membrane permeability and efflux pump activity, namely those associated with multidrug efflux pumps, are the two most frequent bacterial mechanisms resulting in intrinsic resistance [128,132]. The cytoplasmic membrane is probably the major target for most classes of biocides, and since different microorganisms have different membrane structures, it is easy to understand that the activity of biocides varies between different types of microorganisms and even between different strains of the same species [81,128,133,134]. Based on the membrane structure, among vegetative bacteria, mycobacteria are considered the most resistant to biocides, followed by Gram-negative bacteria, and vegetative Gram-positive the most susceptible [81,135]. Some bacterial species are also innately more resistant to biocides than others due to the presence of a low level of efflux pump systems that decrease intracellular concentration, reduce biocide penetration, enzymatic degradation of biocides, and physiological, structural, and metabolic changes [84,136e139]. In addition, it is conveniently accepted that most of the in-use biocides, at sub-inhibitory concentrations, are naturally selecting bacteria according to their susceptibility and intrinsic resistance [130,140,141](Fig. 3). The specific growth rate is another significant factor associated with antimicrobial resistance as it is a determinant factor of how bacterial populations respond to molecules, influences the expression of resistance mechanisms, and can affect the outcome of bacterial treatment [142e144]. Rapidly growing bacteria are often more susceptible to molecules that target active cellular processes, such as cell wall synthesis and DNA replication, because these cells can experience more mutations in a given period due to more M. Sousa, I. Machado, L.C. Sim~ oes et al. Environmental Science and Ecotechnology 25 (2025) 100557 6 Table 2 Compilation of studies establishing a possible connection between several biocides and their resistance mechanisms with cross-resistance to antibiotics in various bacterial strains. Biocides Antibiotics Microorganism Mechanism of resistance/Cross-resistance References Triclosan Amikacin Ampicillin Ampicillin/ sulbactam Azithromycin Cefaclor Cefazoline Cefepime Cefotaxime Chloramphenicol Ciprofloxacin Erythromycin Gentamicin Imipenem Lomefloxacin Tetracycline Trimethoprim/ sulfamethoxazole Escherichia coli clinical isolates The cells adapted to triclosan exhibited extended susceptibility to the biocide and recalcitrance to the investigated antibiotics associated with: Decreased permeability of outer and inner membranes Increased depolarisation of membranes Higher negative net charge of membranes Higher efflux activity [99] Triclosan ePseudomonas aeruginosa Triclosan is a substrate for Pseudomonas aeruginosa efflux pumps, namely MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY, associated with cross-resistance to as antibiotics [100] Chlorhexidine Colistin Klebsiella pneumoniae clinical isolates The adaptation of Klebsiella pneumoniae to chlorhexidine occurs through the regulation of genes associated with efflux pumps, namely smvR and smvA The operon pmrK is upregulated by chlorhexidine, resulting in a decreased binding affinity of colistin to lipid A, associated with cross-resistance [101] Benzalkonium chloride Chlorhexidine Triclosan Ampicillin Ceftazidime Chloramphenicol Ciprofloxacin Erythromycin Gentamicin Tetracycline Salmonella enterica Typhimurium Resistance to biocides and cross-resistance is associated with the expression/overexpression of: Genes related to cold-shock response (cpeE) Ribosomal and transcription proteins Efflux pumps (sugE) Enzymes of microaerobic metabolism [102] Triclosan Chloramphenicol Ciprofloxacin Tetracycline Tobramycin Triclosan Stenotrophomonas maltophilia Triclosan selectively induces the expression of the efflux pump SmeDEF, which in turn triggers a transient low-level resistance to the biocide and cross-resistance to antibiotics Compared to the wild-type, five out of the twelve triclosan-selected mutants were more resistant to antibiotics [103] Benzalkonium chloride Chlorhexidine Chlorophene Ethanol Glutaraldehyde Isopropanol Peracetic acid Peroxide Povidone-iodine Sodium hypochlorite Ampicillin Chloramphenicol Norfloxacin Escherichia coli 43 % of strains became resistant to the antibiotics Resistance and cross-resistance with antibiotics is associated with the expression of: Multidrug efflux proteins (mdfA and acrR) Porins (envZ and ompR) Subunits of Escherichia coli RNA polymerase (rpoA and rpoBC) For isopropanol, ethanol, sodium hypochlorite, and peracetic acid, there was no evidence of antibiotic cross-resistance [104] Quaternary ammonium compounds disinfectant containing formaldehyde and glutaraldehyde Ampicillin Chloramphenicol Ciprofloxacin Tetracycline Salmonella spp. After exposure to a quaternary ammonium disinfectant containing formaldehyde and glutaraldehyde, Salmonella spp. exhibited overexpression of AcrAB-TolC and underexpression of outer membrane porins, resulting in resistance to ciprofloxacin, chloramphenicol, tetracycline, and ampicillin [105,106] Quaternary ammonium compounds ePseudomonas aeruginosa A Quaternary ammonium compound-resistant Pseudomonas aeruginosa strain exhibited a higher expression level of OprR compared with a knockout mutant, showing that the expression level of OprR was correlated with the level of resistance to quaternary ammonium compounds. [107] Benzalkonium Chloride Ciprofloxacin Pseudomonas aeruginosa Pseudomonas aeruginosa adapted to benzalkonium chloride showed differences regarding the expression of outer membrane proteins when compared with a non-adapted strain, and some of these proteins are involved in ciprofloxacin resistance [108] Triclosan eEscherichia coli A triclosan-tolerant Escherichia coli strain exhibited an amino acid substitution in the FabI protein, where a glycine was replaced by a valine, preventing the formation of the FabI-NAD þ complex and reducing the effect of triclosan in Escherichia coli [109] Chlorhexidine Ampicillin Gentamicin Polymyxin B Pseudomonas stutzeri A chlorhexidine-resistant Pseudomonas stutzeri, obtained after exposure to chlorhexidine, showed resistance to triclosan, benzalkonium chloride, polymyxin B, gentamicin, and ampicillin [110] Triclosan eSalmonella enterica Typhimurium The recalcitrance of planktonic cells of Salmonella enterica Typhimurium after exposure to triclosan is associated with: The expression of degradative enzymes Changes in the bacterial cell envelope The activation of the efflux pump [111] (continued on next page) M. Sousa, I. Machado, L.C. Sim~ oes et al. Environmental Science and Ecotechnology 25 (2025) 100557 7 frequent DNA replication [142,145,146]. In contrast, slow-growing or dormant bacteria may evade, leading to tolerance and the development of resistance mechanisms, like persister cells [147,148]. Slow-growing or dormant cells within a bacterial population can act as highly tolerant persister cells. These cells can survive the treatment and then resume growth once the molecule is removed [147,149]. In addition, slow-growing or stressed bacteria may activate stress-response pathways, which can lead to an increased mutation rate and the acquisition of resistance elements [147,150]. The specific growth rate can also modulate bacterial resistance mechanisms through the expression of resistance genes and affect the evolution of resistance [151,152]. 5.2. Biocides as inducers of resistant phenotypes The concentrations of biocides in natural environments are unlikely to be sufficient to kill large numbers of bacteria [25]. Moreover, chronic sub-bactericidal exposure to biocides can cause Table 2 (continued ) Biocides Antibiotics Microorganism Mechanism of resistance/Cross-resistance References Mutations in the enoyl reductase enzyme The susceptibility of biofilms of Salmonella enterica Typhimurium to triclosan is associated with: The substance's low extracellular matrix diffusion Overactivity of efflux pumps, Exopolysaccharide production Table 3 Compilation of the biocides referred to in this study, organized by their chemical category and common applications. Category Biocides Common Applications References Quaternary ammonium compounds Benzalkonium chloride Cetyltrimethylammonium bromide Alkyltrimethylammonium compounds Dialkyldimethylammonium compounds Disinfection in healthcare, personal care products, and industrial cleaning [54] Bisbiguanides Chlorhexidine Hospital antisepsis, dental care, and household cleaning [117] Phenolics compounds Triclosan Ortho-phenylphenol Personal care products (e.g., soaps, toothpaste, cosmetics) [118] Aldehydes Glutaraldehyde Ortho-phthalaldehyde Industrial disinfection, hospital sterilization [119] Alcohols Isopropanol Ethanol Hand sanitizers, surface disinfectants [120] Peroxygens Hydrogen peroxide Surface disinfection, water treatment [121] Halogen-releasing agents Sodium hypochlorite Peracetic acid Water disinfection, industrial cleaning, and surface sterilization [122] Bisphenols Triclosan Triclocarban Antimicrobial agents in personal care products [123] Heavy-metal derivatives Silver compounds Medical devices and surface coatings [124] Povidone-iodine Diamidines Antisepsis in healthcare, personal hygiene products. [125] Fig. 2. Compilation of the mechanisms by which biocides promote antibiotic resistance addressed in this study [15,84,126,127]. M. Sousa, I. Machado, L.C. Sim~ oes et al. Environmental Science and Ecotechnology 25 (2025) 100557 8 transient mutations in these microorganisms [25,153,154]. Such mutations are usually not enough to kill the cell, but more likely, they just cause a minor phenotypic change in the bacteria that will make it more or less fit for its environment and more or less susceptible [155]. These phenotypic changes are temporary, most of the time, but can sometimes become permanent when mutants arise [156,157]. These responses have evolved to permit bacteria to adapt rapidly to environmental stresses and may drive selective enrichment of antimicrobial-resistant strains of bacteria [156,158]. The induction of such bacterial resistance mechanisms is often linked with the over-expression of efflux pumps [159], the overexpression of multigene and protein systems [128], and altered exopolysaccharide production [151]. Also, alterations in surface charge that result in less affinity for cationic antimicrobials and alteration in lipid content that complicates the diffusion of lipidsoluble antimicrobials across the cellular membrane [160]; reduced expression of porins, which limits antimicrobials uptake [161]; high expression of efflux pumps that expel a wide range of toxic molecules reduces antimicrobials accumulation inside the cell [162]. This topic has been comprehensively reviewed recently and was graphically summarized in Fig. 4 [130,159,163,164]. Furthermore, alterations in membrane properties can lead to QAC resistance and cross-resistance to membrane-active antibiotics [29,84,165,166]. In E. coli, spontaneous acquisition of resistance to cetyltrimethylammonium bromide (CTAB) was reported to be due to alteration in membrane lipopolysaccharide composition and reduction in OmpF, which reduces bacteria permeability. These changes also conferred resistance to a wide variety of antibiotics [167]. Concerning the role of OMP expression associated with QAC resistance, some studies [105,168,169] showed that resistance to QACs was accompanied by reduced levels of OmpC,OmpF, and OmpA.InE. coli, the loss of OmpF and OmpC genes makes cells resistant to some antibiotics, namely to hydrophilic antibiotics, such as b -lactams [85,170]. Macrolides, aminoglycosides, rifamycins, novobiocin, fusidic acid, and cationic peptides diffuse across the lipid bilayer, so alterations in lipid content can be related to resistance to these antibiotics [170]. One of the main controversial issues in antimicrobial research is the fact that in vitro conditions and laboratory findings do not mimic perfectly the environmental concentrations of such compounds and so, adaptive resistance and its role in cross-resistance to antibiotics must be studied and reported with some precaution [171,172]. The majority of susceptibility tests are based on bacterial inoculum, which is prepared in a very rich medium, and it is very unlikely that bacteria in situ have such readiness of nutrients [171]. Fig. 3. Ascending order of resistance to biocides and antibiotics (based on McDonnell and Russell [68]). Fig. 4. 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