Urinary tract infections (UTIs) rank among the most prevalent infections acquired both in healthcare settings and within communities, with significant implications for patient health (Shahbazi et al., 2018; Shahkolahi et al., 2022). In 2019, more than 404.6 million individuals worldwide were diagnosed with a UTI, contributing to over 200,000 deaths globally (Codelia-Anjum et al., 2023; Shivaee and Mirshekar, 2019). UTIs are particularly common in vulnerable populations, such as pregnant women, the elderly, and sexually active individuals, who are prone to both community-acquired and healthcare-associated UTIs (CAUTIs and HAIs) (Shivaee and Mirshekar, 2019; Govindarajan and Kandaswamy, 2022). While Escherichia coli strains account for 80 to 90% of UTI cases, the rising incidence of Enterococcus faecalis strains in up to 20% of cases has garnered considerable attention (Taati Moghadam et al., 2021; Abdullah et al., 2023; Shahbazi et al., 2023). In Tehran, Iran, recent studies underscore the growing concern regarding UTIs. Research conducted at local hospitals has highlighted the high prevalence of E. faecalis among UTI patients, with significant resistance to commonly used antibiotics (Minaeian et al., 2020; Dadashi et al., 2021; Samani et al., 2021; Ma et al., 2021).

The emergence of E. faecalis as a prominent UTI pathogen is alarming, particularly due to its intrinsic resistance to a broad range of antibiotics, including aminoglycosides, cephalosporins, trimethoprim-sulfamethoxazole, and macrolides (Wojnicz et al., 2016). Furthermore, E. faecalis can acquire resistance to clinically relevant antibiotics such as vancomycin, linezolid, and kanamycin, complicating treatment options (Govindarajan et al., 2022). Recent studies have highlighted a troubling increase in linezolid-resistant E. faecalis strains, with mechanisms of resistance linked to genes such as erm(A) and optrA, and mutations like G2576U in the 23S rRNA (Ma et al., 2021; Yang et al., 2024). This trend is corroborated by research in China, which reported a 22.61% prevalence of linezolid-resistant E. faecalis isolates, primarily associated with the presence of the erm(A) gene and risk factors such as indwelling catheters (Ma et al., 2021). Similarly, a study in India identified high rates of optrA gene-mediated resistance among E. faecium strains, illustrating the widespread nature of this issue (Rani et al., 2023).

The ability of E. faecalis to form biofilms, particularly in catheter-associated urinary tract infections (CAUTIs), further exacerbates its antibiotic resistance (Govindarajan and Kandaswamy, 2022). Biofilm formation is a key virulence mechanism, allowing the bacteria to evade host immune responses and enhancing their survival in harsh conditions (Rahimzadeh et al., 2023; Șchiopu et al., 2023). Recent research highlights the crucial role of virulence factors in E. faecalis infections. These include secreted factors like cytolysin (cylA), gelatinase (gelE), and hyaluronidase (hyl), as well as cell surface proteins such as aggregation substances (asa1), enterococcal surface protein (esp), endocarditis antigen (efaA), and collagen-binding protein (ace) (Aung et al., 2023; Comerlato et al., 2013; Zhang et al., 2017). A critical enzyme involved in anchoring many of these surface proteins is sortase. Sortase plays a pivotal role in the assembly of pili, which are essential for bacterial adhesion and biofilm formation. By recognizing a cell-wall sorting (CWS) motif, sortase cleaves and anchors surface proteins to the cell wall, contributing to bacterial virulence (Sivaramalingam et al., 2024). Sortase and its associated pili assembly are attractive targets for antimicrobial interventions due to their vital role in infection and biofilm development (Sivaramalingam et al., 2024).

Moreover, biofilms formed by E. faecalis often involve interactions with other species, such as E. coli. This dual-species biofilm formation enhances virulence and antibiotic resistance, driven in part by mechanisms like iron metabolism. E. faecalis biofilms have been shown to increase iron uptake via ferrous iron transporter proteins, which promotes the survival of both E. faecalis and E. coli under iron-supplemented conditions, enhancing biofilm resilience and antibiotic resistance (Govindarajan and Kandaswamy, 2022). This symbiotic relationship complicates treatment, as biofilms provide a protective environment that shields bacteria from both the immune system and antibiotics (Govindarajan and Kandaswamy, 2022).

Considering the growing clinical significance of E. faecalis, the aim of this study is to explore key attributes, including antibiotic resistance, virulence factors, biofilm formation capacity, and molecular typing through multilocus sequence typing (MLST). Understanding these factors will contribute to the development of more effective therapeutic strategies for combating E. faecalis infections.

E. faecalis is recognized as a significant Gram-positive pathogen in UTIs, exhibiting notable resistance to a range of commonly used antibiotics such as macrolides and cephalosporins. This resistance arises from both intrinsic factors and acquired mechanisms (Gilmore et al., 2020). Our study highlights a substantial prevalence of antimicrobial resistance among clinical isolates of E. faecalis from UTIs, with particularly pronounced resistance observed against minocycline and tetracyclines. Notably, all E. faecalis isolates, with the exception of two resistant to linezolid, maintained susceptibility to vancomycin, ampicillin, penicillin G, and nitrofurantoin.

These findings are consistent with the reports by Ma et al. (2021) and Chen et al. (2017), who also noted an increase in resistance to minocycline and tetracyclines while finding that all isolates were susceptible to vancomycin and ampicillin. This observation suggests minimal cross-resistance between linezolid and other antibiotics in the E. faecalis isolates studied.

A global perspective on linezolid resistance, as indicated by Dadash et al., reveals that while linezolid resistance is generally low, it exhibits significant regional variability, with higher prevalence observed in Asia compared to other regions (Dadashi et al., 2021). These data corroborate our findings and underscore the importance of localized surveillance in effectively understanding and addressing resistance patterns. The regional variability emphasizes the need for tailored approaches in managing antimicrobial resistance and highlights the value of region-specific data in formulating effective treatment strategies.

Recent research has highlighted the frequent use of antibiotics such as aminoglycosides and nitrofurantoin in the treatment of UTIs caused by vancomycin-resistant E. faecalis (Zhanel et al., 2001; Meena et al., 2017; Levitus et al., 2023). This prevalent exposure may exert selective pressure that contributes to the emergence and persistence of resistant E. faecalis strains. These observations underscore the critical need for stringent antibiotic stewardship to curb the development of resistance. In our study, we observed a relatively lower rate of resistance to vancomycin and nitrofurantoin compared to findings reported by Tripathi et al. (2016) and Meena et al. (2017). These studies document a troubling increase in resistance to these essential antibiotics, which are crucial for managing nosocomial enterococcal infections. The observed discrepancy in resistance patterns highlights the importance of continuous surveillance and research to adapt treatment strategies effectively and maintain the efficacy of these key antimicrobial agents. The divergence between our findings and those in the literature emphasizes the necessity for vigilant monitoring of resistance trends to vancomycin and nitrofurantoin. Such efforts are vital for ensuring the continued availability of effective therapeutic options for managing severe and complex UTIs (Rahbar et al., 2007).

The widespread use of antimicrobial agents has led to a notable rise in multidrug-resistant (MDR) Gram-positive bacteria, posing significant challenges in clinical settings (Patel et al., 2013). Linezolid, a last-resort antimicrobial for Gram-positive infections, has become a cornerstone in treating such resistant strains (Koulenti et al., 2020). However, the increasing use of linezolid has spurred the emergence of linezolid-resistant strains. Our study identified a linezolid resistance rate of 1.2% (2/160) among E. faecalis isolates, which is lower compared to the 3.5 and 3.4% reported by Chen et al. (2018) and Wang et al. (2021) respectively. Moreover, the finding that 1.8% of vancomycin-resistant E. faecalis isolates were also resistant to linezolid underscores a critical limitation in treatment options (Cho et al., 2018). Alarmingly, all linezolid-resistant isolates in our study were also vancomycin-resistant, indicating a potential crisis in managing these infections.

The detection of ST150 in our inpatients suggests its potential adaptation to the hospital environment and acquisition of multidrug resistance. The presence of ST150 in a clinical setting raises concerns about its potential as a problematic strain, especially given its broad-spectrum antibiotic resistance. Recent findings indicate that strains from high-risk clonal complexes (CCs), associated with human infections, have also been found in animals (Freitas et al., 2011). This underlines the need for targeted research into ST150’s genetic mechanisms and its impact on clinical outcomes to develop effective interventions and mitigate its dissemination in healthcare settings (Ma et al., 2021).

Biofilm formation by E. faecalis in UTIs is a significant concern, especially in the context of catheter use. Our study observed that 15% of isolates showed weak, 8.8% moderate, and 1.2% strong biofilm formation. Notably, the two isolates with strong biofilm-forming abilities were ST150 and resistant to all tested antibiotics. This suggests that strong biofilm formation, coupled with extensive antibiotic resistance, could exacerbate infection management challenges.

The lower prevalence of E. faecalis biofilm formation (25%) in our study compared to previous reports (60–90%) in Europe (Sandoe et al., 2003; Arciola et al., 2008; Duprè et al., 2003) could be due to variations in strain sequence types or methodological differences in biofilm assessment. Factors such as strain variability, operational errors in the microtiter plate assay, and lack of standardized biofilm positivity criteria might contribute to these discrepancies.

The pathogenesis of E. faecalis in UTIs involves factors beyond antibiotic resistance, such as colonization, tissue destruction, and evasion of host immune responses. In this study, 93.8% of isolates possessed the efaA and ace genes. Other virulence genes were present at the following rates: esp (72.5%), asa1 (61.2%), cylA (52.5%), and gelE (88.8%). These findings align with previously reported data: 98, 100, and 92.6% for the ace gene in Poland (Łysakowska et al., 2012), and 90, 89.9, and 92.6% for the gelE gene in Italy (Creti et al., 2004) and Iran (Kafil et al., 2013).

The high prevalence of the efbA gene among our isolates underscores its importance in UTI virulence. EfbA facilitates adherence to extracellular matrix (ECM) proteins, crucial for virulence in ascending UTI models. The Ace protein also binds to ECM proteins, aiding in early-stage colonization. Gelatinase (gelE) plays a role in bacterial dissemination by degrading fibrin (Karimi et al., 2018). The esp gene was found in 72.5% of strains, comparable to rates in Iran (77.9%) (Gulhan et al., 2015), Italy (66.7%) (Creti et al., 2004), India (81%) (Singhal et al., 2014), and Japan (72.2%) (Seno et al., 2005), indicating its role as an adhesin. The asa1 gene was present in 61.2% of isolates, similar to rates reported in Iran (69.6%) (Arbabi et al., 2016) and Italy (51%) (Creti et al., 2004). The cylA gene was identified in 52.5% of strains, consistent with findings in Iran (Nasaj et al., 2016), Japan (Seno et al., 2005), and India (Gupta et al., 2014). The predominance of virulence determinants such as efaA, ace, and gelE in our isolates underscores their significant role in E. faecalis pathogenicity. The high prevalence of these factors in our study, compared to others, highlights the need for ongoing surveillance and research. The presence of multiple virulence factors in our isolates suggests a complex interplay between resistance and pathogenicity that warrants further investigation.

In summary, our findings underscore the need for continuous monitoring of antimicrobial resistance and virulence factors in E. faecalis. The identification of ST150 and its associated resistance profile, coupled with biofilm-forming capabilities, points to a critical area for future research and intervention. Addressing these challenges will be essential for improving clinical outcomes and managing resistant infections effectively.

This study highlights the emergence of the ST150 clonal lineage of Enterococcus faecalis in Tehran, Iran, with a focus on its role in urinary tract infections (UTIs). The data indicate a significant presence of E. faecalis in UTIs, with high resistance rates to tetracycline and minocycline, while maintaining high susceptibility to vancomycin, penicillin G, ampicillin, and nitrofurantoin. Notably, a small percentage of isolates demonstrated resistance to linezolid, with these resistant strains belonging to the previously unreported ST150 lineage. The presence of various virulence factors and the ability to form biofilms among these isolates underline their pathogenic potential. Although no definitive correlation between biofilm formation and antibiotic resistance was found, patterns suggest that biofilm production might be associated with resistance. The study underscores the importance of continuous surveillance and molecular characterization of E. faecalis to better understand and address emerging resistance patterns and enhance infection control strategies.