The introduction of antibiotics marked a pivotal advancement in contemporary clinical infection control for humans. However, owing to the prevalent misuse or overuse of antibiotics coupled with inadequate supervision, the emergence of antibiotic-resistant pathogens has evolved into a formidable challenge in the clinical management of infectious diseases and public health [1,2]. Despite concerted endeavors to develop novel drugs or antibiotic alternatives, only a few new antibiotics, such as daptomycin, have been approved by the Food and Drug Administration (FDA) in recent decades [3]. The pace of new antibiotic development significantly lags behind the proliferation of antibiotic-resistant bacteria. Although alternative antibacterial agents, including antibodies, non-antibiotic antibacterial agents, probiotics, and vaccines, have been proposed, their efficacy and safety remain elusive [[4], [5], [6], [7], [8]]. Consequently, they are primarily recommended as adjuncts or prophylactic measures in clinical practice, with traditional antibiotics persisting as the predominant fundamental antibacterial agents for combating infectious diseases.

The antibacterial mechanism of aminoglycoside antibiotics involves the inhibition of protein synthesis and disruption of bacterial cell membrane integrity. Bacterial resistance to aminoglycosides stems from enzymatic modification of 16S rRNA, activity of antibiotics-inactivating enzymes, enzymatic modification of antibiotics, or alterations in bacterial membrane permeability to antibiotics [9]. Resistance to clinically achievable aminoglycoside concentrations primarily results from their limited permeability through the cell envelope of Enterococci [10]. In strains exhibiting high-level gentamicin resistance, the aminoglycoside-modifying enzyme gene aac(6′)-Ie-aph(2′')-Ia contributes to their resistance [11]. Studies have explored the use of cell wall disruptors such as penicillin combined with aminoglycosides to achieve synergistic effects against low-level aminoglycoside-resistant Enterococci. However, combination therapy appears ineffective against pathogens harboring acquired high-level resistance to aminoglycosides [12].

Quinolone antibiotics impede bacterial DNA replication by interacting with DNA gyrase, type II topoisomerase, and topoisomerase IV, thereby rapidly halting bacterial proliferation and exerting a bactericidal effect. Topoisomerase IV, comprised of two subunits, parC and parE, is the primary target of quinolones in gram-positive bacteria. Mutations in gyrA and parC have been observed in highly quinolone-resistant strains [9]. Additionally, efflux pumps have been implicated in conferring resistance of Enterococci to fluoroquinolones [10].

Triton X-100 (TX-100), a non-ionic surfactant without significant antibacterial activity, is commonly employed to enhance cell membrane permeability to antibodies [13]. TX-100 has been widely used in combination with other antibacterial agents, including in modifying nanomaterials to augment its antibacterial effects [14] or combined with other materials to elicit synergistic antibacterial outcomes [15,16]. TX-100 can modulate methicillin resistance in Staphylococcus aureus [17,18], indicating its potential in addressing antibiotic resistance issues. However, the molecular mechanisms underlying the role of TX-100 as an adjuvant to enhance antibacterial activity or counteract antibiotic resistance remain unclear.

E. faecalis, an infectious bacterium frequently isolated from refractory root canals and failed endodontically-treated teeth [[19], [20], [21], [22], [23]], exhibits resilience in harsh environments characterized by nutrient deficiency or high alkalinity. Its ability to penetrate deeply into various canal irregularities or dentinal tubules poses challenges for its eradication. Various root canal disinfectants, including calcium hydroxide (Ca(OH)2) and antibiotic pastes, have been employed to manage E. faecalis infection within the root canal system. However, E. faecalis shows resistance to the alkaline properties of Ca(OH)2 and could not be eliminated [24]. What's more, studies have confirmed that repeated or long-term use of antibiotics would facilitate antibiotic-tolerant persisters or resistance mutations [[25], [26], [27], [28]]. Therefore, intra-canal repeated or long-term use of antibiotic pastes might also induce the antibiotic resistance of bacteria. These unresolved issues compromise the infection control efficacy of these root canal disinfectants, thereby impacting the long-term outcomes of root canal treatments.

To address these gaps, we investigated the efficacy of TX-100 as an antibiotic adjuvant against the antibiotic resistance of E. faecalis and elucidated their underlying mechanisms.