Enterococci, flagged by the WHO as a rising cause of antibiotic-resistant infections, make surveillance crucial to control resistant strains. We investigated the resistance to linezolid, quinupristin/dalfopristin (Q/D), and erythromycin in Enterococcus faecalis (n = 251) and Enterococcus faecium (n = 434) isolates collected from patients, healthy carriers, hospitals, poultry, livestock, and municipal wastewater in Ardabil, Iran. The isolates were tested for resistance using phenotypic and genotypic methods. Although none of the isolates were resistant to linezolid, 24.9% of E. faecium isolates were resistant to Q/D, particularly those from patients and poultry slaughterhouse wastewater effluent (P < 0.05). The Q/D resistance genes msrC and ermB were detected in 76.85 and 20.37% of E. faecium isolates, respectively. Erythromycin resistance was common in E. faecalis (51.8%) and E. faecium (37.5%), with no significant difference between sources. However, isolates from patients and livestock wastewater had higher erythromycin MICs. Erythromycin resistance genes, such as ermB, ermC, ermTR, and ermA, were found in 80.7, 41.2, 26.5, and 19% of E. faecium and 80.3, 51.6, 22.4, and 25.8% of E. faecalis isolates, respectively. In conclusion, linezolid is a viable treatment for enterococcal infections in Ardabil, but widespread erythromycin- and Q/D-resistant enterococci pose a public health risk.

  • All isolates were susceptible to linezolid.

  • Enterococcus faecium isolates often resist quinupristin/dalfopristin, especially in patients and poultry wastewater.

  • Erythromycin resistance from diverse sources was common in Enterococcus faecalis and E. faecium.

  • Efflux pumps mainly drive quinupristin/dalfopristin resistance in E. faecium isolates.

  • rRNA methylating enzymes mainly drive erythromycin resistance in our isolates.

Enterococci, Gram-positive cocci ubiquitously found in the gastrointestinal tracts of humans and animals, have emerged as a significant public health concern due to their remarkable adaptability and increasing antibiotic resistance (Jannati et al. 2020). Their inherent resilience to various environmental stressors, including desiccation, extreme temperatures, and antimicrobial agents has facilitated their widespread dissemination across diverse ecosystems, including healthcare settings (Ramos et al. 2020; Hasanpour et al. 2021; Namaki Kheljan et al. 2022). This ubiquitous presence, coupled with their ability to colonize humans and animals, has created a complex reservoir for the emergence and spread of antibiotic-resistant strains (Jannati et al. 2023).

Historically considered opportunistic pathogens with relatively low virulence, enterococci, particularly Enterococcus faecalis and Enterococcus faecium, have become predominant causes of nosocomial infections (Aslam et al. 2012; Wang et al. 2013; Mousavi et al. 2020). Factors such as the increased use of invasive medical devices, broad-spectrum antibiotics, and immunocompromised patient populations have contributed to the rise of enterococcal infections (Ignak et al. 2017). Furthermore, the intrinsic resistance of enterococci to certain antibiotics, such as clindamycin, trimethoprim-sulfamethoxazole, aminoglycosides, and beta-lactams, coupled with their capacity to acquire resistance genes, has exacerbated the challenge of effective treatment (Arias et al. 2023).

Vancomycin-resistant enterococci (VRE) represent a formidable threat due to their association with high mortality rates and limited therapeutic options (Maleki et al. 2021; Eichel et al. 2023). While antibiotics like linezolid, daptomycin, tigecycline, and quinupristin/dalfopristin (Q/D) are available, their effectiveness is often compromised by the emergence of resistance (Shariati et al. 2020; So 2020). The complex interplay between enterococci, their environment, and antibiotic use has led to the dissemination of antibiotic resistance genes, further limiting treatment options (Ramos et al. 2020; Li & Wang 2021; Tan et al. 2022). The choice of commonly used antibiotics for treating enterococcal infections should be based on the culture and susceptibility test results (Mousavi et al. 2020).

Ongoing surveillance and genomic studies are crucial for understanding and mitigating antibiotic resistance in enterococci. Antibiotic resistance in enterococci, particularly to linezolid, streptogramins, and erythromycin, is a growing concern in clinical settings. Surveillance programs have been established globally to monitor resistance trends and inform treatment strategies.

The Zyvox® Annual Appraisal of Potency and Spectrum (ZAAPS) and Linezolid Experience and Accurate Determination of Resistance (LEADER) programs have been instrumental in monitoring linezolid resistance among Gram-positive pathogens, including enterococci. These programs have consistently reported high susceptibility rates for linezolid, with rare resistance (Ross et al. 2007; Flamm et al. 2016). Resistance rates have remained low, with some regional variations. For instance, the European surveillance reported a 1.6% resistance rate in vancomycin-resistant E. faecium (Markwart et al. 2021). In the US, resistance was 0.3% in E. faecalis and 0.6% in E. faecium (Gargis et al. 2022).

Erythromycin resistance is more common among enterococci, with significant rates reported in various studies. For example, a study in Iran found that 64% of Enterococcus isolates were resistant to erythromycin (Ahmadpoor et al. 2021). Resistance genes for erythromycin and other macrolides have been detected in enterococci from livestock, meat samples, and clinical settings, highlighting the role of environmental reservoirs in the spread of resistance (Amuasi et al. 2023).

Streptogramin resistance is less frequently reported compared with linezolid and erythromycin. However, resistance genes are present in both clinical and environmental isolates, indicating the potential for spread (Messele et al. 2022; Amuasi et al. 2023).

While previous studies have explored the antibiotic resistance profiles of enterococci in Iran (Jannati et al. 2020, 2023; Mousavi et al. 2020; Maleki et al. 2021), data on the resistance patterns of linezolid, Q/D, and erythromycin in enterococci from Ardabil, Iran, are lacking. This study addresses this knowledge gap by investigating the prevalence of resistance to these critical antibiotics in enterococcal isolates from diverse regional sources. The advantage of this study is that isolates from clinical and environmental sources were studied in the context of the One Health approach. By characterizing the antibiotic resistance landscape in Ardabil, this research contributes to the broader understanding of enterococcal epidemiology and informs targeted interventions to mitigate the threat of these pathogens.

Isolates

This study included 685 Enterococcus spp. isolates (251 E. faecalis and 434 E. faecium) from Ardabil City, Iran, collected and characterized during 2019–2021. The isolates came from various sources, such as clinical specimens (this study and Maleki et al. 2021), healthy carriers (Jannati et al. 2020), untreated hospital wastewater (Jannati et al. 2023), municipal wastewater, and poultry and livestock slaughterhouse wastewater. Table 1 shows the relative abundance of Enterococcus spp. in different sources. The isolates were plated onto 5% sheep blood agar medium after being revived from stock cultures stored at −80 °C.

Table 1

Relative abundance of Enterococcus spp. according to isolation sources

Isolation sourceE. faecium N = 434 n (%)E. faecalis N = 251 n (%)
Clinical specimens 25 (22.7) 85 (77.3) 
Healthy carriers 220 (83.3) 44 (16.6) 
Poultry slaughterhouse wastewater 39 (50.6) 38 (49.3) 
Municipal wastewater 42 (66.6) 21 (33.3) 
Livestock slaughterhouse wastewater 45 (60.8) 29 (39.2) 
Hospital wastewater 63 (64.9) 34 (35) 
Total 434 (61.6) 251 (38.3) 
Isolation sourceE. faecium N = 434 n (%)E. faecalis N = 251 n (%)
Clinical specimens 25 (22.7) 85 (77.3) 
Healthy carriers 220 (83.3) 44 (16.6) 
Poultry slaughterhouse wastewater 39 (50.6) 38 (49.3) 
Municipal wastewater 42 (66.6) 21 (33.3) 
Livestock slaughterhouse wastewater 45 (60.8) 29 (39.2) 
Hospital wastewater 63 (64.9) 34 (35) 
Total 434 (61.6) 251 (38.3) 

Antimicrobial susceptibility testing

The antibiotic susceptibility profiles of the isolates to linezolid and Q/D (Padtan Teb, Tehran, Iran) were determined using the disk diffusion method (CLSI 2023). Erythromycin resistance was assessed using the minimum inhibitory concentration assay through the agar dilution method (Schwalbe et al. 2007), with isolates considered erythromycin-resistant and intermediate-resistant if their MIC values were ≥8 and 1–4 μg/mL, respectively (CLSI 2023). Standard strains of E. faecalis (ATCC 29212) and E. faecium (ATCC 19434) served as control strains. The tests were performed and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI 2023).

Molecular characterization of resistance genes

To identify erythromycin and Q/D resistance genes in corresponding resistant isolates, the isolated bacteria were cultured on blood agar plates and incubated at 37 °C overnight. Subsequently, total genomic DNA was extracted using the DNP Genomic DNA Extraction Kit (Cinagen Co., Tehran, Iran) following the manufacturer's instructions. The presence of erythromycin resistance genes (ermA, ermB, ermC, ermTR, and msrA) and Q/D resistance genes (ermB, msrC, vatD, vatE, vgaA, and vgaB) was determined using specific primers via the polymerase chain reaction (PCR) method. The primer sequences and their annealing temperatures are detailed in Supplementary Table S1. The PCR reaction was conducted in a thermocycler (Bio-Rad, USA), with a final volume of 25 μL. The PCR mixture consisted of 12.5 μL of Premix Taq® mix (Sinaclon Co., Tehran Iran), 2 μL of template DNA, 1 μL (10 pmol) of each forward and reverse primer, and 8.5 μL of deionized nuclease-free water. The PCR thermal cycling protocol employed the following steps: initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 52–60 °C (Supplementary Table S1) for 35 s, and extension at 72 °C for 1 min, with a final extension at 72 °C for 5 min. The PCR products were analyzed on 1.5% agarose gels stained with DNA-safe stain (Sinaclon Co., Tehran, Iran) and visualized under ultraviolet light.

Ensuring reliability and managing uncertainties

To ensure reliable results, antimicrobial susceptibility testing adhered to CLSI guidelines. Quality control included testing standard strains of E. faecalis and E. faecium. Molecular tests employed positive controls (DNA from known isolates) whenever available. For genes lacking positive controls, PCR products were sequenced for confirmation. Both parametric (e.g., measurement errors) and non-parametric (e.g., gene presence/absence) uncertainties were addressed through robust controls and established protocols. However, external factors influencing resistance, such as local antimicrobial use patterns and geographic variations, were beyond the scope of this study and are discussed further in the discussion section.

Statistical analysis

We examined the difference in the occurrence of erythromycin and Q/D resistance and the distribution of the resistance-encoding genes based on the isolates' source of isolation. To assess this, we employed the chi-squared test. A p-value below 0.05 was considered statistically significant.

Of the 685 Enterococcus spp. isolates (251 E. faecalis and 434 E. faecium) none exhibited resistance to linezolid. However, 108 (24.9%) E. faecium isolates demonstrated resistance to the Q/D combination. Table 2 presents the Q/D resistance pattern of E. faecium isolates by source. The highest Q/D resistance rates were observed in isolates from poultry wastewater effluent (48.7%) and clinical specimens (48%). Isolates from municipal and hospital wastewater effluent displayed lower Q/D resistance rates, at 11.90 and 11.10%, respectively. Statistical analysis revealed significantly higher (P < 0.05) Q/D resistance in isolates from clinical specimens than those from healthy carriers. Similarly, Q/D resistance was significantly higher (P < 0.05) among isolates obtained from poultry slaughterhouse wastewater effluent than other wastewater types. Genetic analysis identified msrC and ermB as the predominant Q/D resistance-encoding genes in 76.85 and 20.37% of Q/D-resistant isolates. No other Q/D resistance genes were detected in this study.

Table 2

The Q/D resistance pattern in E. faecium isolates by source

Isolation source (N)Resistant n (%)Susceptible n (%)
Clinical specimens (25) 12 (48) 13 (52) 
Healthy carriers (220) 59 (26.8) 161 (73.1) 
Poultry slaughterhouse wastewater (39) 19 (48.7) 20 (51.3) 
Municipal wastewater (42) 5 (11.9) 37 (88.1) 
Livestock slaughterhouse wastewater (45) 6 (13.3) 39 (86.7) 
Hospital wastewater (63) 7 (11.1) 56 (88.9) 
Total (434) 108 (24.9) 326 (75.1) 
Isolation source (N)Resistant n (%)Susceptible n (%)
Clinical specimens (25) 12 (48) 13 (52) 
Healthy carriers (220) 59 (26.8) 161 (73.1) 
Poultry slaughterhouse wastewater (39) 19 (48.7) 20 (51.3) 
Municipal wastewater (42) 5 (11.9) 37 (88.1) 
Livestock slaughterhouse wastewater (45) 6 (13.3) 39 (86.7) 
Hospital wastewater (63) 7 (11.1) 56 (88.9) 
Total (434) 108 (24.9) 326 (75.1) 

Overall, erythromycin resistance was observed in 51.8% (n = 130/251) of E. faecalis and 37.5% (n = 163/434) of E. faecium isolates. Notably, a significant proportion of isolates also exhibited intermediate resistance to erythromycin. Table 3 presents the prevalence of erythromycin resistance among E. faecium isolates from various sources. All sources harbored resistant isolates, with the highest resistance rate observed in clinical isolates (64%). Furthermore, the MIC50 for erythromycin was elevated in clinical isolates (1,024 μg/mL) and isolates from poultry slaughterhouse wastewater effluent (512 μg/mL) (Table 3).

Table 3

Erythromycin resistance profile in E. faecium isolates according to isolation sources

Isolation source (N)Profile
MIC50 (μg/mL)
Resistant n (%)Intermediate n (%)Susceptible n (%)
Clinical specimens (25) 16 (64) 7 (28) 2 (8) 1,024 
Healthy carriers (220) 63 (28.6) 119 (54.1) 38 (17.3) 
Poultry slaughterhouse wastewater (39) 24 (61.5) 4 (10.2) 11 (28.2) 512 
Municipal wastewater (42) 19 (45.2) 14 (33.3) 9 (21.4) 
Livestock slaughterhouse wastewater (45) 24 (53.3) 13 (28.9) 8 (17.8) 
Hospital wastewater (63) 17 (27) 27 (42.8) 19 (30.2) 
Total (434) 163 (37.5) 184 (42.5) 87 (20) 
Isolation source (N)Profile
MIC50 (μg/mL)
Resistant n (%)Intermediate n (%)Susceptible n (%)
Clinical specimens (25) 16 (64) 7 (28) 2 (8) 1,024 
Healthy carriers (220) 63 (28.6) 119 (54.1) 38 (17.3) 
Poultry slaughterhouse wastewater (39) 24 (61.5) 4 (10.2) 11 (28.2) 512 
Municipal wastewater (42) 19 (45.2) 14 (33.3) 9 (21.4) 
Livestock slaughterhouse wastewater (45) 24 (53.3) 13 (28.9) 8 (17.8) 
Hospital wastewater (63) 17 (27) 27 (42.8) 19 (30.2) 
Total (434) 163 (37.5) 184 (42.5) 87 (20) 

Statistical analysis revealed a significantly higher erythromycin resistance rate in clinical isolates than in those from healthy carriers (P < 0.05). However, no significant relationship was found between resistance rates among different wastewater types (P > 0.05).

Table 4 demonstrates widespread erythromycin resistance among E. faecalis isolates from diverse sources. Isolates from livestock slaughterhouse wastewater effluent exhibited the highest resistance rate at 69%, while isolates from healthy carriers displayed the lowest resistance rate at 27.3%. Notably, no statistically significant association was found between the resistance rates of E. faecalis isolates from different sources (P > 0.05). Furthermore, elevated erythromycin MIC50 were observed in isolates from clinical specimens (1,024 μg/mL), livestock slaughterhouse wastewater effluent (1,024 μg/mL), and poultry slaughterhouse wastewater effluent (256 μg/mL) (Table 4).

Table 4

Erythromycin resistance profile in E. faecalis isolates according to isolation sources

Isolation source (N)Profile
MIC50 (μg/mL)
Resistant n (%)Intermediate n (%)Susceptible n (%)
Clinical specimens (85) 54 (63.5) 6 (7) 25 (29.4) 1,024 
Healthy carriers (44) 12 (27.3) 23 (52.3) 9 (20.5) 
Poultry slaughterhouse wastewater (38) 22 (57.9) 1 (2.6) 15 (39.5) 256 
Municipal wastewater (21) 7 (33.3) 4 (19) 10 (47.6) 
Livestock slaughterhouse wastewater (29) 20 (69) 3 (10.3) 6 (20.7) 1,024 
Hospital wastewater (34) 15 (44.1) 11 (32.4) 8 (23.5) 
Total (251) 130 (51.8) 48 (19.1) 73 (29.1) 32 
Isolation source (N)Profile
MIC50 (μg/mL)
Resistant n (%)Intermediate n (%)Susceptible n (%)
Clinical specimens (85) 54 (63.5) 6 (7) 25 (29.4) 1,024 
Healthy carriers (44) 12 (27.3) 23 (52.3) 9 (20.5) 
Poultry slaughterhouse wastewater (38) 22 (57.9) 1 (2.6) 15 (39.5) 256 
Municipal wastewater (21) 7 (33.3) 4 (19) 10 (47.6) 
Livestock slaughterhouse wastewater (29) 20 (69) 3 (10.3) 6 (20.7) 1,024 
Hospital wastewater (34) 15 (44.1) 11 (32.4) 8 (23.5) 
Total (251) 130 (51.8) 48 (19.1) 73 (29.1) 32 

Figures 1 and 2 illustrate the distribution of ermA, ermB, ermC, ermTR, and msrA genes among erythromycin-resistant (including both resistant and intermediate-resistant) E. faecium and E. faecalis isolates. Statistical analyses indicated significant variations (P < 0.05) in the distribution of resistance genes among isolates, depending on their source of isolation. ermB was the most prevalent gene, detected in 80.7% of E. faecium isolates and 80.3% of E. faecalis isolates. ermC, ermTR, and ermA were found in 41.2, 26.5, and 19% of E. faecium isolates and 51.6, 22.4, and 25.8% of E. faecalis isolates, respectively. None of the isolates carried the msrA gene.
Figure 1

The distribution of ermA, ermB, ermC, ermTR, and msrA genes among erythromycin-resistant E. faecium isolates by the source of isolation. CS, clinical specimens; HC, healthy carriers; HW, hospital wastewater; MW, municipal wastewater; LSW, livestock slaughterhouse wastewater; PSW, poultry slaughterhouse wastewater.

Figure 1

The distribution of ermA, ermB, ermC, ermTR, and msrA genes among erythromycin-resistant E. faecium isolates by the source of isolation. CS, clinical specimens; HC, healthy carriers; HW, hospital wastewater; MW, municipal wastewater; LSW, livestock slaughterhouse wastewater; PSW, poultry slaughterhouse wastewater.

Close modal
Figure 2

The distribution of ermA, ermB, ermC, ermTR, and msrA genes among erythromycin-resistant E. faecalis isolates by the source of isolation. CS, clinical specimens; HC, healthy carriers; HW, hospital wastewater; MW, municipal wastewater; LSW, livestock slaughterhouse wastewater; PSW, poultry slaughterhouse wastewater.

Figure 2

The distribution of ermA, ermB, ermC, ermTR, and msrA genes among erythromycin-resistant E. faecalis isolates by the source of isolation. CS, clinical specimens; HC, healthy carriers; HW, hospital wastewater; MW, municipal wastewater; LSW, livestock slaughterhouse wastewater; PSW, poultry slaughterhouse wastewater.

Close modal

Supplementary Table S2 presents the distribution of erythromycin resistance gene profiles among E. faecalis and E. faecium isolates. A total of 15 distinct profiles were identified. Notably, a large proportion of the isolates harbored multiple resistance-encoding genes.

Antimicrobial resistance is a complex and urgent global problem affecting human and animal health (White & Hughes 2019). To effectively combat this challenge, adopting a One Health approach is imperative. The One Health framework highlights the interconnectedness of human, animal, and environmental health, which is essential for understanding antibiotic resistance (White & Hughes 2019). Humans can acquire resistant bacteria through direct contact with animals, consuming contaminated food and water, or environmental routes (Pandey et al. 2024). Antibiotics are widely used in livestock and poultry farming for disease prevention and growth promotion. This can lead to the selection of resistant bacteria which can be transmitted to humans (Ibekwe et al. 2023). Wastewater from hospitals, farms, and municipalities spreads resistant bacteria and genes into the environment, affecting wildlife and entering the human food chain (Pandey et al. 2024). Addressing these interconnected domains is essential for developing effective strategies to mitigate the spread of resistance. Our findings contribute to a holistic understanding of resistance patterns involving linezolid, Q/D, and erythromycin in Enterococcus isolates obtained from patients, healthy individuals, and wastewater sources.

Linezolid is an oxazolidinone antibiotic that blocks protein synthesis in bacteria. It differs from other protein synthesis inhibitors by affecting the first step of protein synthesis and preventing the formation of the initiation complex. It does this by binding to the 23srRNA portion of the ribosomal 50s subunit and changing its shape, preventing tRNA from attaching. This unique mechanism of action makes cross-resistance between linezolid and other protein synthesis inhibitors extremely rare (Hashemian et al. 2018). In this study, we did not find resistance to linezolid in any of the isolates we collected from patients, stool samples from healthy individuals, or different types of wastewater. This is probably because linezolid is a relatively new antibiotic that is only prescribed in hospitals and is not widely used like other antibiotics. In general, linezolid resistance is low in non-clinical enterococcal isolates worldwide. Resistance was reported in E. faecalis and E. faecium isolates at varying levels in some areas where linezolid has been used for extended periods. Similar to our study, Rahimi et al. (2008) in Iran, Novais et al. (2005) in Portugal, and Hölzel et al. (2010) in Germany also reported zero resistance to linezolid in enterococcal isolates from environmental samples. In contrast, in Poland and Iraq, linezolid resistance in environmental E. faecalis isolates accounted for 4.5 and 14% and in E. faecium isolates 6.3 and 0%, respectively (Łuczkiewicz et al. 2010; Alduhaidhawi et al. 2022). In Greece, linezolid resistance in Enterococcus isolates from environmental sources was 85.7% (Sakkas et al. 2019). As mentioned above, linezolid resistance in clinical enterococci isolates is higher worldwide. In Iran, linezolid resistance in clinical E. faecalis and E. faecium isolates ranged from 0 to 46% (Yasliani et al. 2009; Ghaffarpasand & Moniri 2010; Heidari et al. 2017; Arshadi et al. 2018; Jahansepas et al. 2018; Azimi et al. 2019; Haghi et al. 2019; Shahi et al. 2020). In Korea and China, linezolid resistance in clinical E. faecalis isolates was 18.8 and 58.5% and in E. faecium isolates was 39 and 42.3%, respectively (Lee et al. 2017; Cai et al. 2019). The lack of resistance in the clinical isolates in this study may be due to the minimal use of this antibiotic in hospitals in Ardabil.

Q/D (Synrecid) is a combination drug that synergistically inhibits protein synthesis in bacterial cells. It is used to treat infections caused by staphylococci and E. faecium. E. faecalis is intrinsically resistant to this antibiotic due to the presence of the lsa gene (Smith et al. 2018). In the present study, the resistance rate to Q/D was significantly higher in isolates from clinical specimens, poultry wastewater effluent, and stool from healthy carriers than in other samples. Previous studies have also reported higher rates of resistance to this antibiotic in E. faecium isolates in clinical settings. For example, studies in Zanjan, Tehran, and Tabriz found that resistance rates ranged from 53 to 100% (Jahansepas et al. 2018; Azimi et al. 2019; Haghi et al. 2019). Poultry wastewater effluent isolates also showed high resistance to Q/D (48.7%), which aligns with findings from a study conducted in Zambia where 53.2% resistance was observed (Mudenda et al. 2022). This increased resistance is probably due to the widespread use of antibiotics in poultry farming. Antibiotics are often used to prevent disease and promote growth in poultry, selecting for potentially resistant bacteria. Although Q/D is not directly used in poultry farms, resistance observed in isolates from poultry wastewater effluent can be explained by cross-resistance with other antibiotics, such as virginiamycin, used as a feed additive in food animals (Hershberger et al. 2004). Similar trends can explain the relatively high resistance rate in healthy carriers because streptogramin antibiotics are often used without a doctor's prescription in developing countries (Hershberger et al. 2004). In contrast, municipal wastewater isolates exhibited a lower resistance rate. This finding is consistent with other studies in Iran and Portugal that reported low levels of Q/D resistance in E. faecium isolates from municipal wastewater effluent (Da Costa et al. 2006; Rahimi et al. 2008).

In this study, a substantial proportion of E. faecium and E. faecalis isolates showed resistance to erythromycin. Clinical isolates and isolates from livestock and poultry slaughterhouse wastewater effluents showed higher resistance rates than isolates from other sources.

In clinical samples, 64% of E. faecium and 63.5% of E. faecalis isolates were resistant to erythromycin. Similar studies conducted in Iran (Ahmadpoor et al. 2021) and China (Cai et al. 2019) also reported elevated rates of erythromycin resistance among clinical Enterococcus isolates. Isolates obtained from healthy carriers generally exhibited lower resistance rates than clinical isolates. However, it is noteworthy that a significant proportion of isolates (28.6% for E. faecium and 27.3% for E. faecalis) remained resistant to erythromycin within this group. The emergence of antibiotic resistance in isolates from healthy individuals may be attributed to widespread antibiotic usage within the community and the potential overprescription of these drugs by physicians (Hosseinzadeh et al. 2016).

In this study, a significant percentage of isolates from livestock (E. faecalis 69% and E. faecium 53.3%) and poultry (E. faecalis 57.9% and E. faecium 61.5%) slaughterhouse wastewater effluent were resistant to erythromycin. Similar studies from Portugal and Germany have also reported high rates of erythromycin resistance in enterococcal isolates obtained from poultry wastewater effluent (Martins da Costa et al. 2006; Hölzel et al. 2010). Macrolides are among the most commonly used antibiotics for livestock (Shrestha et al. 2023). Erythromycin is employed for treating bacterial infections in livestock and poultry and at subtherapeutic levels to promote growth and enhance productivity (Shrestha et al. 2023). This could explain the high incidence of erythromycin-resistant bacteria in livestock and poultry.

The present study also found that erythromycin-resistant Enterococcus isolates were widely distributed in municipal and hospital wastewater. In municipal wastewater, 33.3% of E. faecalis and 45.2% of E. faecium isolates were resistant to erythromycin. Similar to this study, other studies conducted by Da Silva et al. (2006), Łuczkiewicz et al. (2010), Sadowy & Luczkiewicz (2014), Talebi et al. (2007), and Rahimi et al. (2008) found that 35, 61.4, 65.1, 8, and 75% of the isolates of E. faecalis and 40, 44.6, 51.2, 55, and 97% of the isolates of E. faecium from municipal wastewater were identified as erythromycin-resistant. Furthermore, in the present study, 44.1% of E. faecalis and 27% of E. faecium isolates from hospital wastewater were also erythromycin-resistant. Similar to our findings in studies conducted in Portugal and Greece, a substantial proportion of isolates obtained from hospital wastewater were resistant to erythromycin (Novais et al. 2005; Sakkas et al. 2019).

From the mechanistic point of view, ribosomal modification is the most common mechanism of macrolides–lincosamides–streptogramins (MLS) resistance in Gram-positive cocci, involving erm (ermA, ermB, ermC, and ermTR), msr (A, C, D, and E), vat (A to F) and vga (A to C) type genes. erm genes encode methyltransferase enzymes that methylate the 23S rRNA component of the bacterial ribosome, preventing MLS antibiotics from binding to their target site. This modification renders the antibiotics ineffective, leading to resistance (Saribas et al. 2006; Pourmand et al. 2014). msr and vga genes encode efflux pumps that actively transport antibiotics out of the bacterial cell. vat type genes encode for acetyltransferase enzymes which modify antibiotic chemical structure and render resistance to streptogramins A antibiotics (CVMP 2011). These mechanisms can lead to cross-resistance, as the same resistance genes, such as erm, confer protection against macrolides and streptogramins (Cattoir & Leclercq 2017). The interconnectedness of these resistance pathways underscores the importance of comprehensive strategies to address antibiotic resistance in clinical and agricultural settings (CVMP 2011).

In this study, msrC and ermB genes were present in 76.85 and 20.37% of Q/D-resistant E. faecium isolates, respectively. Other genes such as vgaA, vgaB, vatD, and vatE were not detected. These results are consistent with previous findings that msrC and ermB are the most common genes associated with Q/D resistance. A similar report from England identified msrC and ermB genes in Q/D-resistant E. faecium isolates, while no other genes were detected (Da Silva et al. 2006). In another study from China, the frequency of msrC and ermB genes was 88%, vatE 11%, and vatD 0% (Wang et al. 2016).

Furthermore, in this study, all subtypes of erm genes were identified alone or coexistent with others in erythromycin-resistant Enterococcus isolates. However, ermB was the most prominent gene observed in E. faecalis (80.3%) and E. faecium (80.7%) isolates. In other similar studies, ermB has been reported with the highest frequency as the coding factor for erythromycin resistance (Portillo et al. 2000).

The broad distribution of MLS resistance-encoding genes among our isolates from various sources indicates that resistance can be acquired and spread through horizontal gene transfer. This is especially alarming in antibiotic-intensive environments like hospitals and farms, which can accelerate the spread of resistance within microbial communities (CVMP 2011).

As a limitation, the results of this study may not be directly applicable to other geographical regions with different environmental and clinical contexts. However, with the use of advanced machine learning algorithms, these findings can contribute to global data. Machine learning principles, including pattern recognition and predictive modeling, enable long-term outbreak predictions and regularization of optimal policies (Tutsoy & Tanrikulu 2022). These algorithms analyze extensive datasets to predict resistance patterns, optimize treatment strategies, and develop novel anti-infectives (Anahtar et al. 2021). Integrating these advanced techniques enhances our ability to mitigate resistance propagation and curb the spread of antibiotic-resistant Enterococcus spp., ultimately supporting a One Health approach to global health resilience.

The study concludes that linezolid resistance is absent in both clinical and non-clinical enterococcal isolates in Ardabil, Iran. However, it is important to restrict the use of this antibiotic to prevent the emergence of resistant strains. On the other hand, Q/D- and erythromycin-resistant E. faecalis and E. faecium isolates are prevalent in both clinical and non-clinical settings in Iran. This highlights the need for multilateral action in the context of a One Health approach to prevent the spread of resistant strains.

In this study, all experimental protocols received approval from the regional ethics committee of Ardabil University of Medical Sciences (reference number ‘IR.ARUMS.REC.1399.245’). The methods strictly adhered to relevant guidelines and regulations. Clinical isolates were obtained from the hospital's bacterial collection for research purposes, without using patient samples or data. Informed consent was obtained from parents or legal guardians to collect samples from healthy children. Additionally, permissions were secured from the Ardabil Water and Wastewater Company for municipal wastewater samples, from the Ardabil Provincial Veterinary Organization office for poultry and livestock slaughterhouse wastewater samples, and from hospital authorities for hospital wastewater samples.

All authors have read and agreed to the published version of the manuscript.

The research leading to these results received funding from the Vice Chancellor for Research of Ardabil University of Medical Sciences under Grant Agreement No. 1002955.

F.H.: Methodology, Investigation, Formal analysis, and Original draft preparation. F.K.: Conceptualization and Revising the manuscript. B.M.G.: Review and Revise the manuscript. E.J.: Investigation. M.A.: Conceptualization, Supervision, Project administration, Critically reading and revising the manuscript.

All relevant data are provided in the paper and its supplementary information.

The authors declare there is no conflict.

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