The use of cationic biocides (CBs) is critical to control and prevent the dissemination of bacterial pathogens in the most diverse environments (Wales and Davies, 2015; Fox et al., 2022). They have been a cornerstone in the improvement of hygienic practices, preventing infections and, consequently, reducing the need for antibiotic use (Wales and Davies, 2015; Fox et al., 2022). The global antiseptics and disinfectants market, including CBs, has been continuously growing and is expected to reach $13.3 billion by 2028, which is almost double the market size of $7.5 billion in 2020 (Zion Market Research, 2021). Thus, over the last decades, the extensive use of CBs and consequent high exposure of bacteria to these compounds has been raising concerns about the possibility of selection of strains resistant either to these or other antimicrobials, as antibiotics (Moore et al., 2008). The assessment of the impact of biocides in the evolution of Enterococcus populations is of current concern, as this is one of the leading multidrug-resistant (MDR) healthcare-associated pathogens worldwide (Guzman Prieto et al., 2016; Garcia-Solache and Rice, 2019). Among the more than 60 validated Enterococcus species,¹ Enterococcus faecalis and Enterococcus faecium are the two predominantly implicated in human opportunistic infections and are among the most prevalent in the human gut microbiota (Guzman Prieto et al., 2016; Garcia-Solache and Rice, 2019; Zaheer et al., 2020). They are intrinsically resistant to a broad spectrum of antibiotics and have rapidly acquired resistance to other critical ones, particularly to ampicillin or vancomycin among E. faecium (Guzman Prieto et al., 2016; Garcia-Solache and Rice, 2019; Freitas et al., 2021). Indeed, vancomycin-resistant E. faecium are categorized as high priority on the World Health Organization priority pathogens list for research and development of new antibiotics, causing infections with limited treatment options and associated with high mortality and health care costs (Guzman Prieto et al., 2016; Garcia-Solache and Rice, 2019; Freitas et al., 2021; World Health Organization [WHO], 2024). They are easily spread between patients and across their surrounding hospital environment through fecal contamination of the hands of patients, the healthcare staff and visitors, and of the medical equipment or other inanimate surfaces (Arias and Murray, 2012; Correa-Martinez et al., 2020). Furthermore, due to their remarkable ability to survive harsh conditions, such as nutrient scarcity or desiccation, Enterococcus spp. might remain on these contaminated surfaces for extended periods, even years (Arias and Murray, 2012; Dancer, 2014; Suleyman et al., 2018; Gaca and Lemos, 2019; Correa-Martinez et al., 2020). Effective antisepsis and disinfection practices are, therefore, crucial to break the chain of transmission, prevent spread of these microorganisms in the hospital environment and potential life-threatening MDR infections. Enterococcus spp. exposure to CBs, including at subinhibitory concentrations, extends beyond human clinical settings, as they are part of the natural microbiota of plants, soil, and the human and animals’ gastrointestinal tract, and cause animal infections (Gnanadhas et al., 2013; Guzman Prieto et al., 2016; Maillard, 2018; Garcia-Solache and Rice, 2019; McCarlie et al., 2020; Zaheer et al., 2020; Fox et al., 2022). Therefore, CBs also play a key role in the control of their transmission in the veterinary, food chain (e.g., food industry, farms), or human domestic contexts (Gnanadhas et al., 2013; Guzman Prieto et al., 2016; Maillard, 2018; Garcia-Solache and Rice, 2019; McCarlie et al., 2020; Zaheer et al., 2020; Fox et al., 2022). In addition, Enterococcus spp. are exposed to CBs in sewage, aquatic systems, and soil/sediments, resulting from domestic, hospital, and industrial discharges (Matsushima and Sakurai, 1984; European Medicines Evaluation Agency [EMEA], 1996a; Lucas, 2012; Cowley et al., 2015; Tezel and Pavlostathis, 2015; Ostman et al., 2017; Environment and Climate Change Canada [ECCC], 2019; Pereira and Tagkopoulos, 2019; Pati and Arnold, 2020).

Among CBs, the most common and to which the susceptibility of Enterococcus spp. has been increasingly studied in the last years, are the quaternary ammonium compounds (QACs) and the biguanides (Moore et al., 2008; Fox et al., 2022; Maillard and Pascoe, 2024). However, the dispersed and sometimes contradictory information, coupled with limitations in study designs impacting their general conclusions, underscores the need for a comprehensive literature review to establish a standpoint on current data and address existing research gaps.

Here, we reflected on CBs’ activity against Enterococcus spp. from different sources, geographical regions and years, while taking into consideration the current challenges associated with the study of biocide susceptibility. Moreover, we explore the evolution of decreased susceptibility to CBs reported for particular enterococcal populations of diverse epidemiological backgrounds as well as discuss the outcomes of in vitro exposure of Enterococcus spp. to subinhibitory concentrations of CBs. The prevalence of genes with a known or predicted role on CBs susceptibility in Enterococcus spp. and their confirmed or possible link to decreased susceptibility phenotypes are also reviewed, as well as the potential risk of co- and cross-resistance between CBs and antibiotics. Finally, this review highlights the methodological and knowledge gaps that need to be addressed in future research to better understand the implications of CBs use on Enterococcus spp. evolution and ultimately contribute to the development of appropriate interventions in diverse sectors highly exposed to CBs.

Cationic biocides (CBs) are broad-spectrum antimicrobial compounds widely employed in disinfectants, antiseptics, and preservatives (Fox et al., 2022). These agents have been used in clinical and domestic settings, the food industry, agriculture, and other sectors since the 1930s (Gilbert and Moore, 2005; Moore et al., 2008). CBs antibacterial mechanism of action primarily targets the negatively charged cytoplasmic membrane (Salton, 1951; Hugo and Longworth, 1964, 1966; Denton, 1991; Gilbert and Moore, 2005; Maillard and Pascoe, 2024). However, the specific interaction of each CB with the membrane and subsequent concentration-dependent bacteriostatic or bactericidal mechanisms differ between the chemically diverse compounds (Figure 1) (Salton, 1951; Hugo and Longworth, 1964, 1966; Denton, 1991; Gilbert and Moore, 2005; Maillard and Pascoe, 2024). Indeed, these membrane-active agents fall into different classes with various chemical structures (Figure 1), whose intrinsic properties influence the CB’s activity (Salton, 1951; Hugo and Longworth, 1964, 1966; Denton, 1991; Gilbert and Moore, 2005; Maillard and Pascoe, 2024).

The in vitro assessment of Enterococcus spp. susceptibility to CBs has been performed using diverse methodologies, each selected to provide specific data relevant to the purpose of the test. The bactericidal efficacy claim of disinfectant or antiseptic products in Europe and the USA is supported by quantitative or qualitative tests simulating practical conditions specified in different standards according to their intended use (EPA - United States Environmental Protection Agency, 2012, 2018; Official Methods of Analysis of the AOAC International, 2013; CEN-CENELEC - European Committee for Standardization - European Committee for Electrotechnical Standardization, 2018). These may include different Enterococcus spp. reference strains as test organisms (EPA - United States Environmental Protection Agency, 2012; CEN-CENELEC - European Committee for Standardization - European Committee for Electrotechnical Standardization, 2018). However, such standardized tests required for assessing biocidal product efficacy may not be ideal for examining the susceptibility of Enterococcus spp. strains from diverse genomic and epidemiological backgrounds. They may also lack insights into long-term adaptation to biocide exposure within subinhibitory ranges, potentially not detecting evolving populations that remain susceptible to biocidal products. Thus, research studies assessing the susceptibility to CBs of Enterococcus spp. from diverse epidemiological and genomic backgrounds over the years have primarily relied on the in vitro determination of minimum inhibitory concentrations (MICs), mainly due to the methodology’s ease of use (Gnanadhas et al., 2013; Fox et al., 2022).

MIC is defined as the lowest antimicrobial concentration that inhibits the growth of the microorganisms, and it is usually measured in doubling dilutions (Clinical and Laboratory Standards Institute [CLSI], 1999, 2018; Gnanadhas et al., 2013; Fox et al., 2022). Additionally, some studies also include the determination of the minimal bactericidal concentrations (MBCs), corresponding to the minimum concentration that kills >99.9% of cells, which constitute a more suitable measure of susceptibility for most biocidal applications where the desired effect is to kill the microorganisms (Clinical and Laboratory Standards Institute [CLSI], 1999; Gnanadhas et al., 2013; Fox et al., 2022; Maillard and Pascoe, 2024).

Supplementary Table 1 shows several published MICs and MBCs of QACs (BC, DDAC, CE, CPC) and biguanides (CHX and PHMB), pointing to a good antimicrobial activity of these CBs against Enterococcus spp. isolates, when MICs or MBCs are compared to typical in-use concentrations. However, despite offering information on epidemiological variability and the monitoring of susceptibility trends within this genus, MICs or MBCs may not directly correlate with the bactericidal efficacy evaluated by the standards for disinfectant or antiseptic products approval (Kampf, 2022). This may occur even if the concentrations used correspond to those in the biocidal products because, in most susceptibility studies, MICs and MBCs are determined for unformulated CBs in simple aqueous solutions and biocidal products generally include other compounds that enhance CBs’ activity or stability (Cowley et al., 2015; Fox et al., 2022; Maillard and Pascoe, 2024). Some studies with biocidal formulations against Enterococcus spp. are available, showing MIC and MBC values higher or within the ranges of those in Supplementary Table 1 for unformulated CBs (McBain Andrew et al., 2004; Moore et al., 2008; Cowley et al., 2015; Günther et al., 2015; Bhardwaj et al., 2016; Ulusoy et al., 2016; López-Rojas et al., 2017; Piątkowska et al., 2021). However, these were evaluated at the endpoint of the MIC determination protocols used, corresponding typically to 24h or sometimes longer (48h), instead of the usually recommended disinfectant contact times in different contexts of 3 to 10 min (Maillard, 2005; Hong et al., 2017; Rutala et al., 2019). Additionally, the use of MIC and MBC may fail to detect tolerant persister subpopulations that are able to survive transient exposures to lethal biocide concentrations, potentially facilitating the evolution toward resistance, as observed for E. coli exposed to BC (Nordholt et al., 2021). Hence, tests to assess the efficacy of high concentrations of CBs, included or not in biocidal products, considering real exposure times are still lacking against Enterococcus spp. from diverse epidemiological and genomic backgrounds.

Most studies use the microdilution broth protocol described by the CLSI guidelines for antibiotic susceptibility testing (Clinical and Laboratory Standards Institute [CLSI], 1999, 2018) which has the advantage of allowing the comparison of data between different studies. However, it was designed primarily to assess the therapeutical success of antibiotics for infections’ treatment, which is reflected in the bacterial growth conditions specified (37°C; pH = 7.3) (Wales and Davies, 2015; Clinical and Laboratory Standards Institute [CLSI], 2018; Maillard and Pascoe, 2024). Thus, several other factors (e.g., variable temperature or pH, oxygen limitation, starvation, lower or higher bacterial density, bacterial growth phase) that can affect the efficiency of biocides in real application contexts, namely by altering the cytoplasmic membrane or reducing the cell’s metabolic activity, are not pondered (Zhang and Rock, 2008; Saito et al., 2014; Ran et al., 2015; Wiegand et al., 2015; Yoon et al., 2015; Gaca and Lemos, 2019; Fox et al., 2022; Maillard and Pascoe, 2024). Recently, we learned that E. faecium and E. faecalis isolates from different epidemiological and clonal backgrounds exhibited decreased susceptibility (MIC and MBC increases of two to eightfold) to BC at 22°C and/or pH = 5, compared to standard conditions (37°C; pH = 7.3) (Pereira et al., 2023), confirming the influence of external growth conditions on CBs susceptibility.

Furthermore, Enterococcus are also regularly found within multicellular communities such as biofilms, both on wet environments and dry surfaces, in various settings (Ch’ng et al., 2019). Biofilms are usually associated with a decreased susceptibility to biocides via several mechanisms such as persister cells and surrounding extracellular polymeric matrix that forms a barrier to the diffusion of biocides through the biofilm (Ch’ng et al., 2019; Wicaksono et al., 2021; Maillard and Pascoe, 2024). Although a good biocidal activity remains generally described against Enterococcus spp. biofilms (Lima et al., 2001; Arias-Moliz et al., 2010; Baca et al., 2011; Ravi Chandra et al., 2015; Komiyama et al., 2016; Valverde et al., 2017; Machuca et al., 2019; Günther et al., 2021), one study reported a decreased susceptibility in enterococcal biofilms compared to planktonic cells, for BC, DDAC, CHX and PHMB, of around twofold between minimum biofilm eradication concentrations (MBECs) and MBCs (Cowley et al., 2015). Also, in a recent study, a BC concentration of 80 mg/L was not sufficient to eradicate E. faecium and E. faecalis biofilms for which respective planktonic cells’ MBCs varied from 10 to 40 mg/L (Salamandane et al., 2023).

Finally, given the absence of established standardized protocols for the study of biocide susceptibility of different bacterial strains or collections, prudence is necessary when comparing MIC/MBC data across different studies, making comprehensive epidemiological analysis difficult (Buffet-Bataillon et al., 2012; Maillard and Pascoe, 2024). A clear example of such issue is the broad range of CHX MICs available for the E. faecalis ATCC 29212, from <1 to 27 mg/L (Supplementary Table 1), determined using different methodologies. Factors such as the incubation time, bacterial growth phase (e.g., exponential vs stationary), growth culture media or the type of plate plastic can influence the susceptibility to CBs in vitro, conducting to diverse outcomes for the same strains (Clinical and Laboratory Standards Institute [CLSI], 1999; Bock et al., 2018).

Despite the limitations of data analysis related to the inconsistency of the methods used and the potential implications on the applicability and relevance of in vitro data in real environments, the diverse susceptibility testing methodologies (e.g., altered growth conditions, planktonic cells or biofilms, biocidal formulations vs unformulated CBs) may be valuable in different contexts, provided they are standardized. Meanwhile, more studies using biocidal formulations, along with the inclusion of biofilms or modified growth conditions simulating real scenarios, are crucial to ascertain the activity of CBs against Enterococcus populations and to conclude about the appropriate course of action.

Besides susceptibility assessment methodologies, another aspect needing standardization, also linked to susceptibility testing, is the terminology used to define the decreased susceptibility of bacteria to biocides (Maillard and Pascoe, 2024). When the increased MICs or MBCs do not reach in-use concentrations of a biocide, it has been described using the terms “tolerance” or “decreased susceptibility” (Maillard, 2007; Rutala et al., 2019; Wand and Sutton, 2022; Boyce, 2023). As the term “tolerance” has been used with different meanings to characterize bacterial susceptibility to biocides or antibiotics (Brauner et al., 2016), for the purpose of this review we will use the term “decreased susceptibility”. On the contrary, if the decreased susceptibility implies that the microorganisms are not inactivated by the in-use concentrations of a biocide, then the term “resistance” to the biocide is applied (Maillard, 2007; Rutala et al., 2019; Wand and Sutton, 2022; Boyce, 2023).

More and more the wide use of antiseptics and disinfectants in particular environments (e.g., hospitals, the food chain) has been a cause of concern given the possibility that repeated exposure to subinhibitory concentrations of these agents may progressively select for populations with decreased susceptibility to these antimicrobials (Fraise, 2002; Meyer and Cookson, 2010; Kampf, 2018e; Maillard and Pascoe, 2024). As a baseline to monitor the susceptibility evolution trends to CBs over the years or among strains from diverse sources under a gradient of selective subinhibitory pressures, setting epidemiological cut-off (ECOFF) values could be a useful tool (Turnidge et al., 2006; Morrissey et al., 2014; Kahlmeter and Turnidge, 2022). ECOFFs are established based on the MIC or MBC distributions of an antimicrobial for each bacterial species, and correspond to the minimum concentration above which bacterial strains have phenotypically detectable acquired reduced susceptibility mechanisms (Turnidge et al., 2006; Morrissey et al., 2014; Kahlmeter and Turnidge, 2022). Although the methods used to determine MICs or MBCs may not accurately reflect resistance to biocides under real-world application conditions, as previously discussed, the purpose of setting ECOFF values is not to separate between resistant or susceptible isolates to biocide products, but rather to distinguish non-wild-type (those with acquired reduced susceptibility mechanisms) from wild-type strains (Turnidge et al., 2006; Morrissey et al., 2014; Kahlmeter and Turnidge, 2022).

Few individual analyses have proposed CBs’ ECOFF values based on their Enterococcus spp. collection’s MIC and MBC distributions, including isolates from different sources, geographical regions and time frames (Morrissey et al., 2014; Duarte et al., 2019; Kheljan et al., 2022; Pereira et al., 2022). For CHX, MIC ECOFFs of 8 mg/L and 32 mg/L and an MBC ECOFF of 64 mg/L have been proposed for E. faecium, whereas, for E. faecalis, MIC ECOFFs of 8 mg/L, 16 mg/L and 64 mg/L and MBC ECOFFs of 64 mg/L or higher have been recommended (Morrissey et al., 2014; Kheljan et al., 2022; Pereira et al., 2022). For BC, MIC ECOFFs of 8 mg/L and 16 mg/L and an MBC ECOFF of 16 mg/L have been estimated for both E. faecium and E. faecalis (Morrissey et al., 2014; Kheljan et al., 2022). However, the variation in ECOFFs proposed by different studies underscores the need for comprehensive analyses of MIC and MBC distributions across the various species-biocide pairs. These must be conducted using diverse Enterococcus spp. collections and laboratories to address potential biological, methodological, and interlaboratory variations. Such an approach aligns with EUCAST recommendations for antibiotics and is crucial for establishing definitive ECOFFs for biocides (Kahlmeter and Turnidge, 2022).

In one of the studies proposing ECOFFs to CHX for E. faecalis, even though the whole population was considered wild type by the statistical model recommended for the ECOFF estimation, differences in the susceptibility were detected among isolates from diverse sources and years (Pereira et al., 2022). Similarly, other authors have found significant differences across their Enterococcus spp. collections (Schwaiger et al., 2014; Duarte et al., 2019; Sobhanipoor et al., 2021; Kheljan et al., 2022; Pereira et al., 2022, 2023). Most found a higher occurrence of strains with decreased susceptibility to CHX, DDAC, or BC among clinical isolates comparing to Enterococcus from other origins included in the same study (Schwaiger et al., 2014; Guzman Prieto et al., 2017; Duarte et al., 2019; Sobhanipoor et al., 2021; Pereira et al., 2023), although contradictory data has also been reported for BC (Sobhanipoor et al., 2021; Kheljan et al., 2022). Moreover, an increase in the mean CHX MICs and MBCs of human infection E. faecalis isolates over the years, between 2001 and 2020, has been recently reported (Pereira et al., 2022). Beyond the clinical environment, a significant increasing trend in the BC MICs over time has also been detected in E. faecium isolated from the food chain, including food-animal production settings, meat of animal origin, and other food products (Pereira et al., 2023). Furthermore, within populations of E. faecalis and E. faecium from these settings, higher average CHX MICs and MBCs have been identified compared to other sources (Kheljan et al., 2022; Pereira et al., 2022). Of note, most of these studies did not find any particular genotype justifying the evolution of phenotypes between sources or time frames (Schwaiger et al., 2014; Kheljan et al., 2022; Pereira et al., 2022, 2023).

Available data suggest an adaptation of Enterococcus populations in settings where they are exposed to CBs. Of note, an increase in the CHX MIC₅₀ (minimum concentration that inhibits the growth of 50% of the isolates), from 2 to 8 mg/L, and MIC₉₀ (minimum concentration that inhibits the growth of 90% of the isolates), from 16 to 32 mg/L, was detected in vancomycin-resistant E. faecium recovered from patients’ infections or colonization after daily CHX bathing was instituted in the hospital ward, compared to isolates recovered before the intervention, suggesting that prolonged exposure to this biocide might indeed select for decreased susceptibility (Mendes et al., 2016). Nonetheless, large longitudinal metadata analyses of the populations’ dynamics in such contexts are critically needed for more supported conclusions. Currently, most available CBs susceptibility studies lack objective data on local biocide consumption (e.g., type of compound, amount used, compliance with effective biocide application practices, “during use” concentrations) or on the occurrence of subinhibitory concentrations (Maillard and Pascoe, 2024). This hinders the establishment of a clear cause-and-effect relationship of biocide use and Enterococcus spp. evolved phenotypes. Most studies also use a limited number of Enterococcus strains, lack clonal or genotypic characterization, and show a low source diversity, making it challenging to have a global perspective on the evolution of susceptibility to CBs in particular Enterococcus spp. populations or environments.

Despite unclear cause-and-effect relationships in the previously mentioned studies that detected CBs phenotypic evolutions among field Enterococcus spp. isolates from diverse sources or dates, the hypothesis that exposure to diverse subinhibitory concentration gradients of CBs could lead to decreased susceptibility has been tested in vitro (Cowley et al., 2015; Tezel and Pavlostathis, 2015). These tests, in which diverse Enterococcus spp. are exposed to low CBs concentrations similar to those found in different environments, such as residual concentrations in treated surfaces (e.g., skin, food products, abiotic surfaces) or in surface or residual waters as a result of indirect contamination, contribute to identifying the cellular mechanisms involved in Enterococcus spp. response or adaptation to CBs as well as to other antimicrobials, including antibiotics (Cowley et al., 2015; Tezel and Pavlostathis, 2015).

Enterococcus spp. passages with BC, DDAC, CPC, CE, CHX, and PHMB have resulted in MIC increases of 1.2 to more than 100-fold, that were strain-specific within each species (Supplementary Table 2). In most cases, MICs and MBCs of Enterococcus spp. adapted strains remained below the in-use concentrations of CBs. However, for one E. casseliflavus and one E. faecalis treated with increasing CE and PHMB concentrations, respectively, MICs and, for the E. faecalis, MBCs reached the in-use concentrations range and were stable or only partially reversed after several biocide-free subcultures (Supplementary Table 2) (European Medicines Evaluation Agency [EMEA], 1996b; Cowley et al., 2015; Gadea et al., 2017b; Fox et al., 2022). Also, for most CHX experiments, including different species, MICs reached the concentrations typically used for preservation (25–100 mg/L) after exposure (Supplementary Table 2; Maillard, 2005; Kampf, 2018c; Fox et al., 2022).

The decreased bacterial susceptibility following the adaptation protocols may be explained by several factors such as changes in membrane fatty acid composition, differential expression or mutations in efflux pumps, induction of stress responses, among others (Cowley et al., 2015). Although some of the analyses revealed stable phenotypes, suggesting genotypic adaptation rather than noninheritable physiological or metabolic adaptation mechanisms (Baquero and Coque, 2014), these were scarcely investigated. Bhardwaj et al. (2017) identified significant changes in membrane phospholipids and mutations in several genes with previously predicted or experimentally confirmed roles in decreased susceptibility to CHX, among CHX-adapted E. faecium strains. In particular, all of them shared a mutation (A290V) in the gene efrE, which encodes one subunit of the heterodimeric ATP-binding cassette (ABC) transporter EfrEF whose deletion resulted in increased CHX susceptibility (Bhardwaj et al., 2017). Additionally, increased surface hydrophobicity was detected in E. faecalis passaged in the presence of CPC as well as in those serially exposed to CHX for which a change in the protein profile was also found (Kitagawa et al., 2016). Findings such as these suggest the occurrence of complex bacterial adaptation mechanisms to CBs and underscore the importance of more in-depth analyses employing advanced technologies, like whole-genome sequencing (WGS) and transcriptomics, to identify possible drivers of Enterococcus spp. decreased susceptibility.

The effects of in vitro serial exposure to subinhibitory concentrations of CBs in Enterococcus spp. were not limited to the decreased susceptibility to that specific biocide. Considerable increases of several fold in the MICs of other biocides (2 to >100 fold), reaching in-use concentrations in many cases, have also been detected (Supplementary Table 2; Bhardwaj et al., 2017; Gadea et al., 2017a,b). On the other hand, an increase in susceptibility to CE occurred in CPC-adapted Enterococcus spp. and BC-adapted E. faecium, E. faecalis, and Enterococcus spp., to BC in CPC-adapted Enterococcus spp., to didecyldimethylammonium bromide in CPC-adapted Enterococcus spp., to CPC in BC-adapted Enterococcus spp. and CHX-adapted Enterococcus spp., and to triclosan in CHX-adapted E. casseliflavus and CPC-adapted Enterococcus spp. (Gadea et al., 2017a,b).

All in all, despite the in vitro research suggesting that bacteria can adapt to CBs exposure, evidence of such rapid adaptation in the environment is scarce (Cowley et al., 2015; Maillard and Pascoe, 2024). Multiple external factors specific of each setting (e.g., physicochemical, wide range of CBs concentrations, presence of additional compounds with antimicrobial properties, presence of organic matter), the different physiological state of bacterial populations (e.g., changes in metabolic activity and gene expression), among others, may not allow for the conditions required for Enterococcus bacteria to adapt (Fox et al., 2022; Maillard and Pascoe, 2024). Also, another hypothesis that might explain the limited correlation data between biocide use and an evolution toward decreased susceptibility in real environmental contexts are the loss of the adaptation mechanism or the struggle of adapted populations to compete in their microbial communities when the CB stress is removed (Cowley et al., 2015). This once again supports that field studies are a critical research gap, as mentioned throughout this review, namely longitudinal analyses with appropriate controls and with collection of data on the biocide concentrations used, exposure times and time intervals between exposures, simultaneous application of other compounds, among others.

CBs’ mechanism of action is complex and comprises multiple targets, including the cytoplasmic membrane as well as intracellular components such as proteins and nucleic acids, in a concentration-dependent manner, as previously discussed (Salton, 1951; Hugo and Longworth, 1964, 1966; Harold et al., 1969; Denton, 1991; Gilbert and Moore, 2005; Maillard and Pascoe, 2024). Decreased susceptibility to CBs in Gram-positive bacteria has been mainly attributed to efflux pumps (Tezel and Pavlostathis, 2015; Fox et al., 2022), which may prevent or reduce CB’s antibacterial action by exporting them from the cytoplasm or the cytoplasmic membrane, up to a certain CB concentration (Putman et al., 2000; Boyce, 2023). Enterococcus spp. have been found to harbor several acquired genes encoding efflux pumps located on the cytoplasmic membrane, including the well-known qac, bcrABC and oqxAB genes, all demonstrated to be implicated in the decreased susceptibility to QACs by functional studies in Gram-positive or Gram-negative bacteria, and, in the case of some qac genes, also to CHX. Intrinsic heterodimeric ABC transporters, like EfrEF in E. faecium and E. faecalis, and mutations in regulatory genes, such as the DNA-binding response regulator (ChtR), have also been shown to impact CHX susceptibility.

However, as will be detailed throughout this chapter, for most genotypes of decreased susceptibility to CBs, their role in Enterococcus spp. antimicrobial susceptibility is hypothesized based on functional assays in other bacterial genus and/or epidemiological studies in Enterococcus spp. in which genotypes and phenotypes are correlated without molecular support. Further characterization of the functionality of such genes, through, for example, gene deletion and complementation studies, is required to completely elucidate their role in Enterococcus spp. susceptibility to CBs. Nonetheless, the potential gene exchange with diverse phyla, both of Gram-positive and Gram-negative bacteria, in contexts where CBs are present in a wide range of concentrations, is noteworthy and needs further exploration as the same genotypes in diverse genetic or epidemiological backgrounds may be associated with diverse outcomes of CBs susceptibility.

Although there are several differences between CBs and antibiotics (e.g., spectrum and mechanism of action, ratio of in-use concentration per microorganisms’ MICs, commercialized formulations), both have been used for their antimicrobial activity for decades now and CBs have been critical for limiting the need of antibiotic use (Fox et al., 2022). However, research has been suggesting that exposure to biocides may directly or indirectly select for bacterial populations with particular genotypes or phenotypes leading to co- or cross-resistance to antibiotics (Maillard, 2018). Enterococcus’ response after exposure to CBs has been shown not only to alter the expression of genes involved in multidrug efflux, bacterial metabolism, and cell wall permeability, which may affect the susceptibility of the cells to very diverse antimicrobials, but also to upregulate genes directly associated with resistance to antibiotics (Bhardwaj et al., 2016; Li and Palmer, 2018). Bhardwaj et al. (2017) found that CHX stress induced the expression of the VanA-type vancomycin resistance genes and genes associated with decreased susceptibility to daptomycin (liaXYZ) in E. faecium. However, vancomycin susceptibility was actually increased for the VanA-positive E. faecium in the presence of subinhibitory concentrations of CHX, revealing a CHX-vancomycin synergy (Bhardwaj et al., 2017, 2021). Excess of D-lactate contributed to this synergism, whereas the deletion of the gene ddcP, encoding a membrane-bound carboxypeptidase, and a mutation (S199L) on an ATPase of phosphate-specific transporter, encoded by gene pstB, conferred a survival advantage in the presence of both antimicrobials (Bhardwaj et al., 2017, 2021). Nonetheless, the occurrence of a possible cross-resistance between vancomycin and CBs, including CHX, is still controversial. MICs and MBCs of CHX and BC of vancomycin-resistant Enterococcus isolates significantly higher than those of vancomycin-susceptible Enterococcus spp. have been described among diverse collections (Alotaibi et al., 2017; Ignak et al., 2017; Sobhanipoor et al., 2021). On the contrary, a decreased susceptibility in vancomycin-susceptible Enterococcus compared to vancomycin-resistant Enterococcus as well as no significant difference between the two groups has also been found for the biocides BC, CHX, DDAC or CPC (Baillie et al., 1992; Barry et al., 1999; Suller and Russell, 1999; Kõljalg et al., 2002; Braga et al., 2013; Roedel et al., 2020; Sobhanipoor et al., 2021). Such disparities among studies might be associated with the different methodologies used, various levels of previous exposure to CBs by the bacteria, or with other genetic or metabolic properties specific to each of the local bacterial populations studied.

A possible link between antibiotic resistance and CBs susceptibility has also been observed for other antibiotics. A decreased susceptibility to CHX and DDAC was detected in Enterococcus that were ampicillin resistant or with a high-level of resistance to the aminoglycosides gentamycin and streptomycin (Schwaiger et al., 2014; Wieland et al., 2017; Sobhanipoor et al., 2021). Despite the suggestion of a correlation by these results, no genetic or cellular changes supporting such co-occurring phenotypes have been explored yet.

The selection of antibiotic resistant subpopulations after serial exposure to subinhibitory concentrations of several CBs has been identified (Supplementary Table 2; Bhardwaj et al., 2017; Gadea et al., 2017a,b). Adaptation to CHX, CPC, BC or CE of E. casseliflavus, E. durans, E. faecalis, E. faecium, E. saccharolyticus or other Enterococcus spp. led to cross-resistance to the clinically-relevant ampicillin, ciprofloxacin, daptomycin, imipenem and tetracycline (Supplementary Table 2; Bhardwaj et al., 2017; Gadea et al., 2017a,b). On the other hand, there was also evidence of loss of resistance to ampicillin in CHX-adapted E. faecalis, to ciprofloxacin in CE-adapted E. faecium, and to ceftazidime and/or cefotaxime in BC-adapted E. faecium, CPC-adapted Enterococcus spp., CE-adapted E. casseliflavus, E. faecium and Enterococcus spp., and CHX-adapted E. faecium (Gadea et al., 2017a,b). The genetic mechanisms or phenotypic expression events behind the increase or loss of antibiotic resistance detailed in these studies are mostly unknown, but they were mainly transient and may be associated with non-specific membrane permeability increase, metabolic changes (e.g., decreased growth rate), or others (Gadea et al., 2017a,b). For E. faecium 1,231,410, in which decreased susceptibility to daptomycin arose with passages in increasing CHX concentrations, Bhardwaj et al. (2017) detected physiological and genetic alterations in the adapted strains compared to the parental strain. These included significantly lower growth rates, changes in cellular membrane phospholipid and glycolipid content, overexpression of the three-component regulatory system encoded liaXYZ involved in cell envelope homeostasis, and mutations in genes associated with global nutritional stress response (relA), nucleotide metabolism (cmk), multidrug efflux (efrE), phosphate acquisition (phoU), and glycolipid biosynthesis (bgsB) (Bhardwaj et al., 2017). Daptomycin is a lipopeptide antibiotic often used to treat vancomycin-resistant Enterococcus spp. infections, that interacts primarily with the bacterial cell membrane, as part of a daptomycin-calcium complex, ultimately leading to cell death (Mishra et al., 2012; Bhardwaj et al., 2017). Thus, the selection of potential structural changes in the biophysical properties of the cell wall or membrane and in the cellular stress responses by the biocide are strongly associated with decreased susceptibility to daptomycin in Enterococcus (Mishra et al., 2012; Bhardwaj et al., 2017). Since this antibiotic is used for the treatment of serious Enterococcus infections that lack other therapeutic alternatives, and CBs are extensively used in hospitals, these results may have serious clinical implications and deserve further studies (Bhardwaj et al., 2017). However, as previously discussed, it must be taken into consideration that in vitro adaptation experiments may not accurately mimic real environments’ conditions in the different contexts.

Of note, CBs, antibiotics, and other antimicrobials such as metals may co-exist as selective agents in many ecosystems, not only in human or animal clinical contexts, the community or food production, but also in wastewater and others (Pal et al., 2015; Wales and Davies, 2015; Singer et al., 2016). In a report produced by SCENIHR (Scientific Committee on Emerging and Newly Identified Health Risks), in 2009, it was stated that ‘biocides are likely to contribute to maintaining selective pressure allowing the presence of mobile genetic elements harboring specific genes involved in the resistance to biocides and antibiotics’, and recommended the surveillance of levels of biocide resistance (Scientific Committee on Emerging and Newly Identified Health Risks [SCENIHR], 2009). Indeed, co-location of genetic determinants conferring decreased susceptibility to CBs and metals and resistance to antibiotics on the same plasmid or other mobile genetic element has been observed among diverse Enterococcus spp. strains (Pal et al., 2015; Silveira et al., 2015; Yuan et al., 2018; Pereira et al., 2023). In previous studies, the analyzed genetic contexts of several genes encoding decreased susceptibility to CBs (qacA/B, qacC, qacZ, oqxAB) in Enterococcus spp., from different sources, geographical regions, or dates of isolation, harbored genes conferring resistance to aminoglycosides, beta-lactams, macrolides, lincosamides, streptogramin of group B, or trimethoprim (Silveira et al., 2015; Yuan et al., 2018; Pereira et al., 2023). Genes coding for decreased susceptibility to metals, namely to copper and cadmium, were also detected within the vicinity of qacA/B genes in two clinical E. faecalis (Pereira et al., 2023). The genetic contexts of qacA/B, qacC, qacJ, qacZ, qrg, bcrABC and oqxAB detected in Enterococcus were compared with those from other taxa and found to be generally very diverse, probably resulting from a high number of recombination events, as suggested by the abundance of insertion sequences and recombinases detected (Pereira et al., 2023). Also, the co-location of these genes on mobile genetic elements such as plasmids, often carrying toxin-antitoxin systems that contribute to their maintenance in the bacterial populations, may facilitate their spread and, thus, mechanisms of co-selection (Pal et al., 2015; Silveira et al., 2015; Yuan et al., 2018; Fox et al., 2022; Pereira et al., 2023; Maillard and Pascoe, 2024).

Besides co-location of diverse antimicrobial resistance genes on the same genetic contexts, the occurrence of cross-resistance, where a single resistance mechanism to a certain antimicrobial also affects additional compounds, has also been hypothesized in Enterococcus spp. (Pal et al., 2015). Genes encoding decreased susceptibility to CBs, such as emeA, efrEF, oqxAB and chtR, are known to be involved in antibiotic resistance, as previously mentioned (Jonas et al., 2001; Lee et al., 2003b; Lubelski et al., 2007; Hürlimann et al., 2016; Yuan et al., 2018). For instance, the chtR and chtS E. faecium deletion mutants showed an increased susceptibility to both CHX and the antibiotic bacitracin (Guzman Prieto et al., 2017). Additionally, Yuan et al. (2018) proposed that the extensive use of quinoxalines in animal husbandry in China could be selecting for a local high prevalence of the oqxAB genes, known to confer decreased susceptibility to such antibiotics and CBs, among Enterococcus spp.

Recently, a few studies have proposed another process in which CBs exposure could contribute to antibiotic resistance spread (Zhang et al., 2017; Jutkina et al., 2018; Han et al., 2019; Schmidt et al., 2022; Liu et al., 2023). In these, subinhibitory concentrations of different CBs, including QACs and CHX, have been shown to increase horizontal gene transfer via conjugation, through multiple cellular processes such as increased reactive oxygen species (ROS) production, upregulated stress and SOS response, enhanced cell membrane permeability, and changes in the expression of conjugative transfer genes, among others (Zhang et al., 2017; Jutkina et al., 2018; Han et al., 2019; Schmidt et al., 2022; Liu et al., 2023). However, this has not yet been studied for Enterococcus spp.

All these examples identify possible overlaps between responses to different biocides and antibiotics and the potential for the development of co- and cross-resistance among antimicrobials in various environments, with impact in diverse Public Health contexts. However, it is crucial to recognize the large limitations of the research on this topic. Although some studies support the associations detected between decreased susceptibility to CBs and resistance to specific antibiotics through robust methodological approaches, for most, caution is required in interpreting the correlations made as they may be linked to independent, co-occurring events of cellular response to diverse antimicrobials. Thus, the underlying processes by which exposure to one substance may lead to decreased susceptibility to another remain unclear.

Currently, the available literature offers valuable insights concerning the state of the genotypic and phenotypic Enterococcus spp. susceptibility to CBs, pointing to an effective biocidal activity with still no descriptions of resistance to CB’s typical in-use concentrations. However, it also reveals several key research gaps that need to be tackled in future investigations in Enterococcus spp., that could extend to other bacterial species.

Urgent priorities include standardizing biocide susceptibility methodologies that are of relevance to real-world scenarios, facilitating direct study comparisons and the establishment of surveillance protocols applicable across diverse environments. Designing evidence-supported methodologies for this purpose is currently difficult, as the influence of several factors mentioned throughout this review on the activity of biocides against Enterococcus spp. or other bacterial species is scarcely studied. Thus, future studies should primarily elucidate the impact of such factors, including different environmental parameters (e.g., temperature, pH, oxygen or nutrient availability), times of biocide exposure, presence of other compounds usually included in biocidal formulations or in the environment, phase of bacterial growth, planktonic cells or biofilms, among others. Furthermore, the integration of cutting-edge technologies into future studies or surveillance programs could facilitate the monitoring of CBs efficacy and susceptibility trends, enabling timely interventions if needed, particularly in settings where CBs are heavily used. Specifically, large longitudinal metadata analyses incorporating WGS and transcriptomic approaches will be critical for clarifying the dynamics of Enterococcus spp. populations exposed to CBs, representative of multiple clones and epidemiological backgrounds, and the long-term consequences for biocidal or antibiotic resistance.

Another priority concerns the characterization of the mechanisms involved in Enterococcus adaptation to CBs or in the co-selection or cross-resistance with antibiotics. Conducting functional genetic assays, such as the deletion and complementation of genes accompanied by the observation of phenotype changes, as well as the use of high-throughput screening platforms or advanced bioinformatic tools, will be crucial for elucidating the role of genes with a predicted effect on decreased susceptibility to CBs in Enterococcus spp. or to better identify still undetected molecular processes. These data will determine the need for strategies aimed at identifying and controlling bacteria harboring particular genotypes in critical contexts.

Fulfilling these methodological and knowledge gaps while taking into account interdisciplinary data from fields such as environmental (e.g., ecotoxicology) and social (e.g., health economics) sciences will provide holistic insights into the intricate dynamics of CBs use and Enterococcus’ antimicrobial resistance development. Collaborative efforts among diverse stakeholders at local and global levels across sectors can enable the development of effective One Health strategies that ensure the continued efficacy of these critical agents in safeguarding Public Health.

All authors are active members of the ESCMID Study Group on Food- and Water-borne Infections (EFWISG).

AP: Conceptualization, Writing – original draft, Writing – review and editing, Visualization. PA: Writing – review and editing, Funding acquisition. LP: Supervision, Writing – review and editing, Funding acquisition. AF: Writing – review and editing, Supervision, Funding acquisition. CN: Conceptualization, Writing – original draft, Writing – review and editing, Supervision, Funding acquisition.