Enterococcus faecium (E. faecium) is a Gram-positive member of the gut microbiota in humans. Commensal intestinal E. faecium is not pathogenic in healthy hosts but can cause infection in susceptible ones (Ch’ng et al., 2019). E. faecium is a leading cause of hospital-acquired infections that are often difficult to treat due to multiple antimicrobial resistances (Santajit and Indrawattana, 2016; Panda et al., 2022). In recent years, new variants of E. faecium have emerged that are better adapted to the healthcare environment, which are prone to outbreaks of infection (O’Toole et al., 2023). Infections caused by vancomycin-resistant E. faecium (VRE-fm) have further reduced treatment options due to increased resistance to a wide range of antibiotics (Lagatolla et al., 2022; Cairns et al., 2023). In addition, the ability of E. faecium to form biofilms is its remarkable pathogenicity characteristic, and the biofilm greatly enhances antibiotic resistance compared to planktonic cells (Ch’ng et al., 2019; Patil et al., 2021). Therefore, the infection caused by VRE-fm is a serious challenge worldwide, and it is urgent to develop new antimicrobial agents to combat VRE-fm, as well as to inhibit and eradicate the biofilm formed by VRE-fm.

Unsaturated fatty acids are well known to display antimicrobial activity (Desbois, 2012; Yoon et al., 2018). However, due to the discovery and development of traditional antibiotics, research on the antimicrobial properties of unsaturated fatty acids has been neglected for a long time. With the increasing severity of traditional antibiotic resistance, unsaturated fatty acids have been reconsidered as antimicrobial agents. Recently, numerous studies indicated that unsaturated fatty acids could also inhibit or eradicate biofilms formed by various microbial pathogens (Kim et al., 2018; Ramanathan et al., 2018; Cui et al., 2019; Wang et al., 2022). Consequently, unsaturated fatty acids could represent the next generation of antimicrobial agents to treat and prevent biofilm-associated infections.

Essential fatty acids (EFAs) that the body needs but cannot make and must get from food, belong to unsaturated fatty acids and play an important role in the body. EFAs are based on α-linolenic acid (ω-3 group) and linoleic acid (ω-6 group), and correlated with infant development, reduction of cardiovascular morbidity and mortality, cancer prevention, brain and vision functioning, etc. (Kaur et al., 2014) EFAs are not only nontoxic, but also essential nutrients for the human body. Therefore, it is a good model to carry out research on antimicrobial and antibiofilm using EFAs as representative of novel antimicrobial agents.

In the context, three ω-3 group including α-linolenic acid (ALA, 18:3), eicosapentaenoic acid (EPA, 20:5), and docosahexaenoic acid (DHA, 22:6), and three ω-6 group members including linoleic acid (LOA, 18:2), γ-linolenic acid (GLA; 18:3) and arachidonic acid (AA, 20:4) were used to evaluate the antimicrobial and antibiofilm effects against VRE-fm in vitro. In addition, for the first time, we study the antibiofilm mechanism of GLA against VRE-fm. Our study indicates that GLA may be developed as a novel therapeutic agent to treat VRE-fm and biofilm-related infections.

The emergence of VRE-fm poses a significant threat to public health. Additionally, the ability of E. faecium to form biofilms further enhances its resilience and makes it challenging to eradicate infections caused by this pathogen. In recent years, there has been growing interest in the potential antimicrobial and antibiofilm effects of EFAs against clinically isolated VRE-fm. In this study, we not only evaluated the antimicrobial and antibiofilm abilities of EFAs against VRE-fm but also first analyzed the possible molecular mechanisms of GLA eradication of VRE-fm biofilms.

In this study, the VRE-fm isolates were highly resistant to most traditional antibiotics, only highly susceptible to quinupristin-dalfopristin (90.0%), tigecycline (100%), and linezolid (100%). Therefore, infections caused by VRE-fm are difficult to treat due to the limited choice of antibiotics. The intrinsic resistance to β-lactams (penicillin and ampicillin) of VRE-fm strains is due to the high expression of penicillin-binding proteins that have low affinity to β-lactams, allowing peptidoglycan synthesis and bacterial growth (Montealegre et al., 2017), which makes enterococci about 100 times more resistant than streptococci (Murray, 1997). resistance to fluoroquinolones (ciprofloxacin and levofloxacin) is mediated by alteration of target enzymes, including DNA gyrase and topoisomerase IV, as well as by decreasing intracellular accumulation (Piddock, 1999). Resistance to gentamicin is due to the low permeability of the E. faecium cell wall to the large gentamicin molecules, but resistance to high-level gentamicin is conferred by alteration of the binding site in the 30S ribosomal subunit or production of modifying enzymes that lead to inactivation of gentamicin (Osuka et al., 2016). Although both vancomycin and teicoplanin belong to glycopeptide antibiotics, teicoplanin still maintains activity against some VRE-fm isolates in the study. It may be caused by the carrying vanB gene, making the isolate lower level resistant to vancomycin and susceptible to teicoplanin (Kuzma et al., 2022).

Because E. faecium is becoming increasingly resistant to traditional antibiotics, it is urgent to find new antimicrobial agents as a supplement to traditional antibiotics. One promising agent with antimicrobial activity is unsaturated fatty acids for the prevention and treatment of microbial infections (Chanda et al., 2018). Several studies have shown that unsaturated fatty acids can fight against a wide range of bacteria species (Sun et al., 2016; Churchward et al., 2017; Pushparaj Selvadoss et al., 2018). In the study, six EFAs are used to test antimicrobial activity against VRE-fm. In terms of the chemical structure of EFAs (for example, the number of double bonds, the relative position of the double bonds and the carboxyl group) which was shown in our previous work (Wang et al., 2022), it is difficult to find a pattern to explain the different antibacterial activities displayed by them. The antibacterial effect of EFAs may depend mainly on the interaction between EFAs and the cell membrane of VRE-fm strains, so it is not possible to find a pattern solely from the chemical structure of EFAs. The cell wall of strain V27 adsorbed a large number of DHA molecules was observed by SEM. It suggests that DHA may play an inhibitory role in interfering with the growth by preventing the nutrients transportation into the cell, which is also discovered by Dasa et al. (Dasa et al., 2016) No bacteria with significantly damaged cell membranes were observed, which indicates that direct destruction of the cell membrane of VRE-fm is not the primary antibacterial mechanism of DHA. This result is different from that observed by Sun et al. (Sun et al., 2016) Besides, Cartron et al. found that cis-6-hexadecanoic acid could disrupt the proton motive force and the electron transfer pathways of Staphylococcus aureus (Cartron et al., 2014), while Won et al. reported that oleic acid could inhibit the membrane enzymes of Streptococcus mutans (Won et al., 2007), which may be also involved in the antibacterial effects of DHA in the current study.

Biofilms play a crucial role in the pathogenesis of E. faecium infections, as they protect bacteria from the host’s immune response and improve their resistance to antibiotics. According to the reports by Maurya et al. (Maurya et al., 2021), the maximum biofilm formation of E. faecium was achieved in the TSB medium, followed by brain heart infusion broth and Luria Bertani broth. Thus, TSB was selected as the basis in the current study. Since glucose has a significant impact on E. faecalis biofilm formation (Tendolkar et al., 2004), different concentrations of glucose were added to TSB to test the biofilm formation of VRE-fm isolates. Finally, TSB+2% glucose medium was selected for the subsequent biofilm study. It is well known that biofilm cells are generally more resistant to antibiotics than their planktonic counterparts, due to the thick mature biofilm, slower metabolic rate, and higher rates of horizontal gene transfer of antibiotic resistance genes within the biofilm community (Komiyama et al., 2016; Lim et al., 2017). Therefore, biofilm-associated E. faecium infections are often difficult to eradicate (Ch’ng et al., 2019). Multidrug resistance and biofilm make the treatment of VRE-fm strain infection even more challenging. Numerous studies indicate that unsaturated fatty acids have the activity of inhibiting or eradicating of biofilms formed by a wide range of bacteria, including even fungi (Kim et al., 2018; Ramanathan et al., 2018; Cui et al., 2019; Wang et al., 2022). These findings are similar to those in the current study. It suggests that EFAs may play a role in the clinical treatment of infections in the future.

The initial attachment is the first step in the formation of a biofilm. Various surface adhesins are involved in this stage. The absence of surface adhesins, including enterococcal surface protein (Esp), aggregation substance (Agg), and adhesion to collagen of E. faecium (Acm), will reduce adherence to human cells and weaken biofilm formation (Nallapareddy et al., 2007; Soares et al., 2014). Hydrolases, including secreted antigen A (SagA), autolysin (AtlA), and gelatinase (GelE), also play a crucial role in biofilm formation. SagA, which is secreted during biofilm formation, is an important component of the biofilm matrix. It binds to a broad spectrum of extracellular matrix proteins. When SagA is hydrolyzed by proteinase K, the thickness of the biofilm will decrease (Paganelli et al., 2015). autolysin (AtlA) plays a role in the stage of biofilm growth and maturation and is mainly responsible for facilitating the release of eDNA by hydrolyzing E. faecium cells (Xie et al., 2022). GelE mainly hydrolyzes gelatin, collagen, casein, and hemoglobin, which assists AtlA in the release of eDNA (Dunny et al., 2014; Kiruthiga et al., 2020). In the study, the esp, sagA, and acm genes were detected in 20/20 (100%), while the atlA gene was detected in 19/20 (95.0%) VRE-fm isolates. Only one isolate with low biofilm-forming ability did not detect the atlA gene. The results suggest that the atlA gene alone cannot determine whether an isolate can form a biofilm but may affect the ability of biofilm formation. However, agg and gelE genes were not detected in any isolates. This may be because the two genes are present mainly in E. faecalis (Kiruthiga et al., 2020), they are not crucial genes for E. faecium to form a biofilm.

In terms of eradicating biofilms, in addition to directly killing VRE-fm, further study is needed on how EFAs affect biofilm-related genes which include acm, atlA, esp, and sagA. GLA with the highest biofilm eradication rate, and six VRE-fm isolates (V05, V06, V08, V09, V22, and V27) with moderate biofilm formation ability were used to illustrate the mechanism. In this study, GLA could significantly down-regulate the expression of the atlA gene in VRE-fm strains, which inhibit the release of eDNA, may result in the eradication of biofilm. The finding suggests that GLA may offer a potential alternative to traditional antibiotics to combat E. faecium infections. The acm gene was up-regulated in two isolates under the action of GLA, which suggests that these isolates may resist EFAs eradicating or repairing biofilm through high expression of the acm gene. These inferences require further work to confirm.

In summary, DHA and GLA showed excellent antibacterial and antibiofilm activity against VRE-fm isolates in the current study, and they are promising next generation antimicrobial agents. Through analysis of relative expression levels of biofilm-related genes, we found that the expression level of atlA gene in VRE-fm strains was significantly down-regulated, which may be an important molecular mechanism for EFAs to eradicate biofilms.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

The studies involving humans were approved by Ethics Committee of Beijing Chao-Yang Hospital, Capital Medical University. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements.

MW: Conceptualization, Methodology, Project administration, Writing – original draft. PW: Methodology, Writing – original draft. TL: Investigation, Writing – original draft. QW: Validation, Writing – original draft. MS: Formal Analysis, Writing – original draft. LG: Supervision, Writing – review & editing. SW: Funding acquisition, Visualization, Writing – review & editing.