Abstract
Hospital wastewater can contaminate the environment with antibiotic-resistant and virulent bacteria. We analyzed wastewater samples from four hospitals in Ardabil province, Iran for Enterococcus faecium and Enterococcus faecalis using culture and molecular methods. We also performed antimicrobial susceptibility testing and polymerase chain reaction testing for resistance and virulence genes. Out of 141 enterococci isolates, 68.8% were E. faecium and 23.4% were E. faecalis. Ciprofloxacin and rifampicin showed the highest level of resistance against E. faecalis and E. faecium isolates at 65%. High-level gentamicin resistance (HLGR), high-level streptomycin resistance (HLSR), ampicillin, and vancomycin resistance were observed in 25, 5, 10, and 5.15% of E. faecium, and 15, 6, 15, and 3.03% of E. faecalis isolates, respectively. The ant(6′)-Ia and ant(3′)-Ia genes that were responsible for streptomycin resistance were observed in HLSR isolates and aph(3′)-IIIa and aac(6′) Ie-aph(2″)-Ia genes accounting for gentamicin resistance were detected in HLGR isolates. vanA was the predominant gene detected in vancomycin-resistant isolates. The majority of isolates were positive for gelE, asa1, esp, cylA, and hyl virulence genes. We found that drug-resistant and virulent E. faecalis and E. faecium isolates were prevalent in hospital wastewater. Proper treatment strategies are required to prevent their dissemination into the environment.
HIGHLIGHTS
The prevalence of E. faecium isolates in hospital wastewater was three times higher than that of E. faecalis isolates.
Five percent of E. faecium and 3% of E. faecalis isolates were vancomycin-resistant.
The rate of HLSR was almost four times the rate of HLGR among Enterococcus spp.
Almost all of the isolates were multidrug-resistant.
The majority of E. faecalis and E. faecium isolates contained multiple virulence genes simultaneously.
INTRODUCTION
Enterococci are known to belong to the commensal microbiota of humans and animals (Jannati et al. 2020). Various species of Enterococcus are the natural inhabitants of the oral cavity and the gastrointestinal and genitourinary tracts in both humans and animals (Ramos et al. 2020). Enterococcus faecium and Enterococcus faecalis are the main enterococcal species collected from clinical specimens. These species are the causative agents of approximately 80 and 20% of enterococcal infections, respectively (Wang et al. 2013). They are associated with several important hospital-acquired infections, such as peritoneal infections, wound infections, soft tissue and skin infections, urinary tract infections, central nervous system infections, bacteremia, and endocarditis (Hasanpour et al. 2021). Enterococcal infections including vancomycin-resistant enterococci (VRE) have high mortality rates (25–50%) since they often develop in compromised hosts (Zhang et al. 2017). Enterococci have several virulence factors that contribute to their pathogenesis. These factors include adhesions, enterococcal surface protein (Esp), and collagen-binding protein (Ace), which allow the bacteria to attach to host cells; aggregation substances, which promote bacterial colonization; lytic enzymes, gelatinase (GelE), and hyaluronidase (Hyl), which cause the dissemination of infection; and cytolysin (CylA) which causes cell lysis (Heidari et al. 2017). Enterococcal infections are difficult to treat because the bacteria have the potential for resistance to virtually all clinically useful antibiotics. Enterococci express intrinsic resistance to antibiotics such as clindamycin and trimethoprim-sulfamethoxazole and show a low-level resistance to aminoglycosides and penicillins. They are capable of acquiring new resistance genes including vancomycin resistance (Sparo et al. 2018).
Enterococcus spp. are known for their inherent resilience to harsh physicochemical conditions in addition to their resistance to antibiotics. They can grow at pH 9.6, in 6.5% NaCl, and at 10–45 °C and withstand drying conditions for a prolonged time period (Weber & Rutala 1997; Blanch et al. 2003). They have also been shown to display tolerance to antimicrobial biocides (Namaki et al. 2022). Hence, they are found in various environments, especially the hospital environment (Wang et al. 2013; Strateva et al. 2016). Hospital settings can act as major reservoirs for the spread of highly virulent and drug-resistant Enterococcus spp. (Iweriebor et al. 2015). The dissemination of antibiotic-resistant bacteria from hospitals can occur through discharged patients, healthcare workers, and hospital wastewater effluent (Hocquet et al. 2016).
Hospital wastewater is one of the major sources of antibiotic-resistant bacteria and antibiotic-resistance genes (Mackuľak et al. 2021). Wastewater from healthcare facilities is often discharged into the sewage system without prior treatment and may act as a reservoir of antibiotic-resistant bacteria and antibiotic-resistance gene dissemination into the environment (Mackuľak et al. 2021). Understanding the presence of antibiotic-resistant bacteria in hospital wastewater can help mitigate the environmental and public health risks posed by these contaminants through the use of appropriate treatment strategies (Liu et al. 2023). In Iran, little is known about the antimicrobial characteristics of Enterococcus spp. in hospital wastewater.
Therefore, this study aimed to (i) investigate the prevalence of clinically significant species of enterococci E. faecalis and E. faecium in the influent of four teaching hospitals in Ardabil, Iran; (ii) assess the resistance profile of isolates against aminoglycosides, vancomycin, ampicillin, and other common antibiotics; (iii) determine the prevalence of the most common virulence genes; and (iv) evaluate the clonal relatedness of the isolates using the Enterobacterial repetitive intergenic consensus (ERIC)-polymerase chain reaction (PCR) assay.
MATERIALS AND METHODS
Sampling
Twenty-five samples of raw wastewater were collected from the wastewater influent of four teaching hospitals affiliated with the Ardabil University of Medical Science over a 10-month period between July 2017 and May 2018. The hospitals were Imam (a referral and general hospital with 500 beds and 700 m3 daily wastewater discharge), Fatemi (a trauma hospital with 220 beds and 72 m3 daily wastewater discharge), Alavi (a women's hospital with 220 beds and 65 m3 daily wastewater discharge), and Bouali (a children's hospital with 150 beds and 60 m3 daily wastewater discharge). The treatment process in these hospitals only includes the primary treatment stage. This stage involves the removal of large solids and debris, fat, and sedimentable organic matter from the wastewater through physical processes. The treated wastewater is then discharged into the municipal wastewater system.
The samples were collected in accordance with the standard operating procedures for the wastewater sampling set by the United States Environmental Protection Agency (U.S. EPA 2013). The liquid grab samples were collected from the main manhole discharging mixed wastewater from the entire hospital in sterile 250 mL bottles and immediately transferred to the microbiology laboratory in cold box containers. The samples were kept at 4 °C and microbiological analysis was performed within 3 h after sample collection.
Bacterial isolation and identification
The specimens were diluted 50 times with phosphate-buffered saline (PBS) and then filtered by 0.45 μm membranes (Millipore Corporation, USA) as described previously (Rahimi et al. 2007). The membranes were transferred onto Brain Heart Infusion (BHI) agar (SRL, Mumbai, India), incubated at 37 °C for 2 h, and subsequently subcultured onto Enterococcus agar (Becton Dickinson and Co., Sparks, MD, USA) containing 6.5% NaCl (Merck, Germany) at 37 °C for 48 h (Rahimi et al. 2007). Five pink/red colonies were randomly selected and transferred to bile esculin agar plates, followed by incubation at 44 °C for 24 h. The black colonies suspected to be Enterococcus species were confirmed using Gram-staining, catalase, and PYR (pyrrolidonyl aminopeptidase) tests (Deasy et al. 2000; Martín-Platero et al. 2009; Jannati et al. 2020). E. faecalis and E. faecium species were identified by the PCR using specific primer sequences (Table 1) targeting the D-alanine: D-alanine ligase (ddl) gene (Dutka-Malen et al. 1995; Kariyama et al. 2000; Jannati et al. 2020). Briefly, genomic DNA was extracted with a commercial DNA isolation kit (DNP™, Sinaclon, Tehran, Iran) according to the manufacturer's recommendation. Amplification was conducted in a DNA thermal cycler (Bio-Rad, Hercules, CA, USA) using commercially available PCR premix (Premix Taq® mix, CinnaGen, Tehran, Iran) and temperature conditions described previously (Jannati et al. 2020). PCR products were analyzed via electrophoresis at 100 V for 1 h in a 1.5% agarose gel (Sinaclon, Tehran, Iran) and stained with DNA-safe stain (Sinaclon, Tehran, Iran), and DNA bands were visualized using UV illumination (Uvi Tec, Cambridge, UK). To ensure that any amplification failure was not due to poor DNA quality or to failure of the PCR itself, and the 16 s ribosomal ribonucleic acid (16s rRNA) gene amplification was used as a positive control in PCR testing (Table 1) (Kariyama et al. 2000). Furthermore, E. faecium ATCC 19434 and E. faecalis ATCC 29212 were used as control strains. The identified isolates were kept in BHI broth along with 15% glycerol (Merck, Germany) at −80 °C for the next analyses.
Oligonucleotide sequence of primers used in this study
Gene . | Oligonucleotide sequence (5′ to 3′) . | Product size (bp) . | Annealing temperatures (°C) . | Reference . |
---|---|---|---|---|
16S rRNA | GGATTAGATACCCTGGTAGTCC GGATTAGATACCCTGGTAGTCC | 320 | 56 | Kariyama et al. (2000) |
ddl E. faecalis | ATCAAGTACAGTTAGTCT′ ATCAAGTACAGTTAGTCT′ | 941 | 54 | Dutka-Malen et al. (1995) |
ddl E. faecium | TAGAGACATTGAATATGCC TCGAATGTGCTACAATC | 550 | 54 | Dutka-Malen et al. (1995) |
vanA | GGGAAAACGACAATTGC GTACAATGCGGCCGTTA | 732 | 55 | Dutka-Malen et al. (1995) |
vanB | ATGGGAAGCCGATAGTC GATTTCGTTCCTCGACC | 635 | 55 | Dutka-Malen et al. (1995) |
aac(6′) Ie-aph(2″)-Ia | GAGCAATAAGGGCATACCAAA GTTCCTATTTCTTCTTCACTATCTTCA | 829 | 55 | Leelaporn et al. (2008) |
aph(2″)-Ib | TCA AAT CCC TGC GGT AGT GTA CGCCAAAATCAATAACTCCAA | 428 | 54 | Leelaporn et al. (2008) |
aph(2″)-Ic | GAGGGCTTTAGGAATTACGC ACACAACCGACCAACAGAGG | 125 | 54 | Leelaporn et al. (2008) |
aph(2″)Id | TAATCTGCCGAAGCAATTCA TAATCCCTCTTCATACCAATCC | 550 | 54 | Leelaporn et al. (2008) |
ant(3″)-Ia | ACC GTA AGG CTT GAT GAA ACA GCCGACTACCTTGGTGATCTC | 624 | 56 | Leelaporn et al. (2008) |
ant(6′)-Ia | GCC CTT GGA AGA GTT AGA TAA TT CGGCACAATCCTTTAATAACA | 198 | 56 | Leelaporn et al. (2008) |
aph(3′)-IIIa | GGCTAAAATGAGAATATCACCGG CTTTAAAAAATCATACAGCTCGCG | 523 | 57 | Padmasini et al. (2014) |
asa1 | GCACGCTATTACGAACTATGA TAAGAAAGAACATCACCACGA | 375 | 56 | Vankerckhoven et al. (2004) |
gelE | TATGACAATGCTTTTTGGGAT AGATGCACCCGAAATAATATA | 213 | 56 | Vankerckhoven et al. (2004) |
cylA | ACTCGGGGATTGATAGGC GCTGCTAAAGCTGCGCTT | 688 | 56 | Vankerckhoven et al. (2004) |
esp | AGATTTCATCTTTGATTCTTGG AATTGATTCTTTAGCATCTGG | 510 | 56 | Vankerckhoven et al. (2004) |
hyl | ACAGAAGAGCTGCAGGAAATG GACTGACGTCCAAGTTTCCAA | 276 | 56 | Vankerckhoven et al. (2004) |
Gene . | Oligonucleotide sequence (5′ to 3′) . | Product size (bp) . | Annealing temperatures (°C) . | Reference . |
---|---|---|---|---|
16S rRNA | GGATTAGATACCCTGGTAGTCC GGATTAGATACCCTGGTAGTCC | 320 | 56 | Kariyama et al. (2000) |
ddl E. faecalis | ATCAAGTACAGTTAGTCT′ ATCAAGTACAGTTAGTCT′ | 941 | 54 | Dutka-Malen et al. (1995) |
ddl E. faecium | TAGAGACATTGAATATGCC TCGAATGTGCTACAATC | 550 | 54 | Dutka-Malen et al. (1995) |
vanA | GGGAAAACGACAATTGC GTACAATGCGGCCGTTA | 732 | 55 | Dutka-Malen et al. (1995) |
vanB | ATGGGAAGCCGATAGTC GATTTCGTTCCTCGACC | 635 | 55 | Dutka-Malen et al. (1995) |
aac(6′) Ie-aph(2″)-Ia | GAGCAATAAGGGCATACCAAA GTTCCTATTTCTTCTTCACTATCTTCA | 829 | 55 | Leelaporn et al. (2008) |
aph(2″)-Ib | TCA AAT CCC TGC GGT AGT GTA CGCCAAAATCAATAACTCCAA | 428 | 54 | Leelaporn et al. (2008) |
aph(2″)-Ic | GAGGGCTTTAGGAATTACGC ACACAACCGACCAACAGAGG | 125 | 54 | Leelaporn et al. (2008) |
aph(2″)Id | TAATCTGCCGAAGCAATTCA TAATCCCTCTTCATACCAATCC | 550 | 54 | Leelaporn et al. (2008) |
ant(3″)-Ia | ACC GTA AGG CTT GAT GAA ACA GCCGACTACCTTGGTGATCTC | 624 | 56 | Leelaporn et al. (2008) |
ant(6′)-Ia | GCC CTT GGA AGA GTT AGA TAA TT CGGCACAATCCTTTAATAACA | 198 | 56 | Leelaporn et al. (2008) |
aph(3′)-IIIa | GGCTAAAATGAGAATATCACCGG CTTTAAAAAATCATACAGCTCGCG | 523 | 57 | Padmasini et al. (2014) |
asa1 | GCACGCTATTACGAACTATGA TAAGAAAGAACATCACCACGA | 375 | 56 | Vankerckhoven et al. (2004) |
gelE | TATGACAATGCTTTTTGGGAT AGATGCACCCGAAATAATATA | 213 | 56 | Vankerckhoven et al. (2004) |
cylA | ACTCGGGGATTGATAGGC GCTGCTAAAGCTGCGCTT | 688 | 56 | Vankerckhoven et al. (2004) |
esp | AGATTTCATCTTTGATTCTTGG AATTGATTCTTTAGCATCTGG | 510 | 56 | Vankerckhoven et al. (2004) |
hyl | ACAGAAGAGCTGCAGGAAATG GACTGACGTCCAAGTTTCCAA | 276 | 56 | Vankerckhoven et al. (2004) |
Antimicrobial susceptibility testing
BHI agar with 6 μg/mL of vancomycin antibiotic (Bio Basic, Canada) was used for the screening of vancomycin resistance. The isolates with growth on BHI agar were subjected to minimum inhibitory concentration (MIC) testing using the agar dilution technique (vancomycin concentration; 0.125–512 μg/mL). The isolates with an MIC of ≥32 μg/mL were regarded as vancomycin-resistant isolates (VRE). The MIC of ampicillin was also determined using the agar dilution method, and isolates with MIC values of ≥16 μg/mL were considered ampicillin-resistant (AR). High-level resistance to gentamicin (HLGR) and streptomycin (HLSR) were assessed by the agar dilution technique. In brief, a 0.5 McFarland bacterial suspension was spotted onto a BHI agar (SRL Diagnostics, India) with 2,000 μg/mL streptomycin and 500 μg/mL gentamicin, individually. The plates were incubated at 37 °C for 24–48 h and then assessed in terms of bacterial growth. Growth of more than one colony in a spotted region was regarded as HLGR/HLSR enterococci. Furthermore, Antimicrobial sensitivity testing of the seven other antibiotics (Padtan Teb, Tehran, Iran) was done by the disk diffusion method. The tested antibiotics were rifampicin (5 μg), ciprofloxacin (5 μg), chloramphenicol (30 μg), penicillin G (10 μg), nitrofurantoin (300 μg), tetracycline (30 μg), and teicoplanin (30 μg).
All antibiotic susceptibility testings were performed and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI 2023).
Determination of resistance and virulence genes
The presence of genes encoding for HLGR, i.e. aac(6′) Ie-aph(2″)-Ia, aph(2″)-Ic, aph(2″)-Ib, aph(2″)-Id and those for HLSR, i.e. ant(3″)-Ia and ant(6′)-Ia, were examined using the multiplex PCR, and the amplification of aph(3″)-IIIa was done using the singleplex PCR assay as described, previously (Leelaporn et al. 2008; Padmasini et al. 2014; Jannati et al. 2020). The vancomycin resistance encoding genes (vanA and vanB) were also identified according to the previous report (Dutka-Malen et al. 1995). Moreover, the presence of genes encoding for five common enterococcal virulence determinants, such as aggregation substance (asa1), cytolysin (cylA), hyaluronidase (hyl), esp, and gelatinase (gelE), was determined by specific primer sequences using the multiplex PCR as described previously (Vankerckhoven et al. 2004).
The sequence of primers used in this study along with annealing temperatures is listed in Table 1.
DNA from previously identified isolates carrying the corresponding antibiotic-resistance and -virulence genes was used as a positive control in all PCR tests (Jannati et al. 2020).
ERIC-PCR analysis
This method was used for the genotyping of the isolates using specific primers (Table 1) and procedures described earlier (Espigares et al. 2006; Jannati et al. 2020). ERIC patterns were analyzed by Bionumeric II 7.0 (Applied Maths, Belgium), and the resemblance between ERIC-PCR profiles was measured by the dice coefficient as well as the unweighted pair group approach with arithmetic (UPGMA). Isolates that had 80% similarity were categorized in the same clusters and regarded as clonally related.
Statistical analysis
We used descriptive statistics to summarize and present our data.
RESULTS
Distribution of Enterococcus species
A total of 141 Enterococcus isolates were obtained from hospital wastewater in Ardabil City. Through the standardized genotypic identification of the isolates, it was found that 68.8% (n = 97/141) of the species were E. faecium and 23.4% (n = 33/141) were E. faecalis.
Antibiotic-resistance pattern
Table 2 shows that the total of E. faecium and E. faecalis isolates had the highest level of resistance to ciprofloxacin and rifampicin at 65% and the lowest level of resistance to teicoplanin (1%). In the BHI–vancomycin screening agar (6 μg/mL) test, 24% (n = 31/130) of E. faecium and E. faecalis showed colony growth, while in MIC testing only 5% (n = 5/97) of E. faecium isolates and 3% (n = 1/33) of E. faecalis isolates were confirmed as VRE strains (vancomycin MIC = 512 μg/mL). The vanA gene was found in 60% (n = 3/5) of vancomycin-resistant E. faecium and 100% (n = 1/1) of vancomycin-resistant E. faecalis isolates, while the vanB gene was not found in VRE isolates.
Antibiotic susceptibility profiles of Enterococcus species isolated from hospital wastewater
Antimicrobial agent . | . | |||||
---|---|---|---|---|---|---|
E. faecium (N = 97) n (%) . | E. faecalis (N = 33) n (%) . | Total (N = 130) n (%) . | ||||
R + I . | S . | R + I . | S . | R + I . | S . | |
Ciprofloxacin | 60 (47) | 35 (36) | 24 (73) | 9 (27) | 84 (65) | 44 (37) |
Chloramphenicol | 8 (7) | 89 (92) | 7 (21) | 26 (79) | 15 (11) | 115 (88) |
Nitrofurantoin | 23 (15) | 74 (76) | 6 (18) | 27 (82) | 29 (22) | 101 (78) |
Penicillin | 22 (23) | 75 (77) | 7 (21) | 26 (79) | 29 (22) | 101 (78) |
Ampicillina | 11 (11) | 87 (90) | 5 (15) | 28 (87) | 16 (12) | 115 (88) |
Rifampicin | 54 (53) | 43 (44) | 30 (91) | 3 (9) | 84 (65) | 46 (35) |
Tetracycline | 5 (5) | 92 (95) | 22 (67) | 11 (33) | 27 (20) | 103 (79) |
Vancomycina | 5 (5) | 93 (96) | 1 (3) | 32 (97) | 6 (5) | 125 (96) |
Teicoplanin | 1 (1) | 96 (99) | – | 33 (100) | 1 (1) | 129 (99) |
Antimicrobial agent . | . | |||||
---|---|---|---|---|---|---|
E. faecium (N = 97) n (%) . | E. faecalis (N = 33) n (%) . | Total (N = 130) n (%) . | ||||
R + I . | S . | R + I . | S . | R + I . | S . | |
Ciprofloxacin | 60 (47) | 35 (36) | 24 (73) | 9 (27) | 84 (65) | 44 (37) |
Chloramphenicol | 8 (7) | 89 (92) | 7 (21) | 26 (79) | 15 (11) | 115 (88) |
Nitrofurantoin | 23 (15) | 74 (76) | 6 (18) | 27 (82) | 29 (22) | 101 (78) |
Penicillin | 22 (23) | 75 (77) | 7 (21) | 26 (79) | 29 (22) | 101 (78) |
Ampicillina | 11 (11) | 87 (90) | 5 (15) | 28 (87) | 16 (12) | 115 (88) |
Rifampicin | 54 (53) | 43 (44) | 30 (91) | 3 (9) | 84 (65) | 46 (35) |
Tetracycline | 5 (5) | 92 (95) | 22 (67) | 11 (33) | 27 (20) | 103 (79) |
Vancomycina | 5 (5) | 93 (96) | 1 (3) | 32 (97) | 6 (5) | 125 (96) |
Teicoplanin | 1 (1) | 96 (99) | – | 33 (100) | 1 (1) | 129 (99) |
R, resistant; I, intermediate; S, susceptible.
aA susceptibility profile was determined using the agar dilution method.
The resistance rate to ampicillin was 10% (n = 10/97) for E. faecium and 15% (n = 5/33) for E. faecalis isolates (MIC ≥ 16 μg/mL). In AR isolates, the MICs of ampicillin ranged from 16 to 512 μg/mL (Table 3).
Frequency and range of ampicillin MICs of enterococci isolates using the agar dilution method
MIC (μg/mL) . | E. faecium (N = 97) n (%) . | E. faecalis (N = 33) n (%) . | Total (N = 130) n (%) . |
---|---|---|---|
<0.12 | 5 (5) | – | 5 (4) |
0.25 | 16 (16.5) | 1 (3) | 17 (13) |
0.5 | 31 (32) | 9 (27) | 40 (31) |
1 | 24 (25) | 16 (48.5) | 40 (31) |
2 | 3 (3) | 2 (6) | 5 (4) |
4 | 6 (6) | – | 6 (5) |
8 | 1 (1) | – | 1 (1) |
16a | 1 (1) | 2 (6) | 3 (2) |
32 | 2 (2) | 2 (6) | 4 (3) |
64 | 2 (2) | – | 2 (1) |
128 | 1 (1) | – | 1 (1) |
256 | 2 (2) | 1 (3) | 3 (2) |
512 | 2 (2) | – | 2 (1) |
MIC50 | 0.5 | 1 | 0.5 |
MIC90 | 32 | 32 | 32 |
MIC (μg/mL) . | E. faecium (N = 97) n (%) . | E. faecalis (N = 33) n (%) . | Total (N = 130) n (%) . |
---|---|---|---|
<0.12 | 5 (5) | – | 5 (4) |
0.25 | 16 (16.5) | 1 (3) | 17 (13) |
0.5 | 31 (32) | 9 (27) | 40 (31) |
1 | 24 (25) | 16 (48.5) | 40 (31) |
2 | 3 (3) | 2 (6) | 5 (4) |
4 | 6 (6) | – | 6 (5) |
8 | 1 (1) | – | 1 (1) |
16a | 1 (1) | 2 (6) | 3 (2) |
32 | 2 (2) | 2 (6) | 4 (3) |
64 | 2 (2) | – | 2 (1) |
128 | 1 (1) | – | 1 (1) |
256 | 2 (2) | 1 (3) | 3 (2) |
512 | 2 (2) | – | 2 (1) |
MIC50 | 0.5 | 1 | 0.5 |
MIC90 | 32 | 32 | 32 |
aMIC ≥16 μg/mL indicates resistance to ampicillin.
In this study, 25% (n = 24/97) of E. faecium and 15% (n = 5/33) of E. faecalis isolates exhibited high-level streptomycin resistance (HLSR), while high-level gentamicin resistance (HLGR) was found in 5% (n = 5/97) and 6% (n = 2/33) of E. faecium and E. faecalis isolates, respectively. Three percent (n = 3/97) of E. faecium isolates and 6% (n = 2/33) of E. faecalis isolates showed both HLGR and HLSR phenotypes (Table 4).
Distribution of HLSR, HLGR, AR, and VRE phenotypes among Enterococcus species isolated from hospital wastewater
Resistance phenotype . | E. faecium (N = 97) n (%) . | E. faecalis (N = 33) n (%) . | Total (N = 130) n (%) . |
---|---|---|---|
No resistant | 67 (69) | 25 (76) | 92 (71) |
AR | 2 (2) | 3 (9) | 5 (4) |
AR, VRE | 1 (1) | – | 1 (1) |
HLGR, AR | 2 (2) | – | 2 (1) |
HLSR | 19 (17) | 2 (6) | 21 (16) |
HLSR, AR | 2 (2) | 1 (3) | 3 (2) |
HLSR, HLGR | – | 1 (3) | 1 (1) |
HLSR, HLGR, AR, VRE | 2 (2) | 1 (3) | 3 (2) |
HLSR, HLGR, VRE | 1 (1) | – | 1 (1) |
AR, VRE | 1 (1) | – | 1 (1) |
Total | 97 (69) | 33 (23) | 130 |
Resistance phenotype . | E. faecium (N = 97) n (%) . | E. faecalis (N = 33) n (%) . | Total (N = 130) n (%) . |
---|---|---|---|
No resistant | 67 (69) | 25 (76) | 92 (71) |
AR | 2 (2) | 3 (9) | 5 (4) |
AR, VRE | 1 (1) | – | 1 (1) |
HLGR, AR | 2 (2) | – | 2 (1) |
HLSR | 19 (17) | 2 (6) | 21 (16) |
HLSR, AR | 2 (2) | 1 (3) | 3 (2) |
HLSR, HLGR | – | 1 (3) | 1 (1) |
HLSR, HLGR, AR, VRE | 2 (2) | 1 (3) | 3 (2) |
HLSR, HLGR, VRE | 1 (1) | – | 1 (1) |
AR, VRE | 1 (1) | – | 1 (1) |
Total | 97 (69) | 33 (23) | 130 |
HLSR, high-level streptomycin-resistant; HLGR, high-level gentamicin-resistant; AR, ampicillin-resistant; VRE, vancomycin-resistant enterococci.
All of the AR (100%), HLSR (96.5%), and HLGR (100%) isolates were multiple drug-resistant (MDR; resistant to ≥3 antibiotic classes) (Table 5).
Antibiotic resistance (intermediate resistance + resistance) profile of HLSR-, HLGR-, VR- and AR-Enterococcus species
Enterococcus species . | Phenotypic resistance combination pattern . | Isolates n (%) . | Antibiotic number . | Antibiotic class number . | Totala n (%) . | MDR (%) . |
---|---|---|---|---|---|---|
AR N = 15 | P/CIP/V/TE/FM/C/RA/S/G/Amp | 2 (13) | 10 | 8 | 2 (13) | 100 |
P/CIP/TE/FM/C/RA/S/Amp | 1 (7) | 8 | 7 | 1 (7) | ||
P/CIP/V/TE/RA/S/G/Amp | 1 (7) | 8 | 6 | 4 (27) | ||
P/CIP/TE/FM /RA/S/Amp | 1 (7) | 7 | 6 | 4 (27) | ||
P/CIP/TE/FM /RA/G/Amp | 1 (7) | 7 | 6 | 4 (27) | ||
P/CIP/V/TE/FM/RA/Amp | 1 (7) | 7 | 6 | 4 (27) | ||
P/CIP/TE/RA/G/Amp | 1 (7) | 6 | 5 | 5 (36) | ||
P/CIP/V/TE/RA/Amp | 1 (7) | 6 | 5 | 5 (36) | ||
P/CIP/TE/RA/S/Amp | 1 (7) | 6 | 5 | 5 (36) | ||
P/CIP/TE/FM/RA/Amp | 2 (1) | 6 | 5 | 5 (36) | ||
P/TE/FM/RA/Amp | 1 (7) | 5 | 4 | 1 (7) | ||
P/CIP/RA/Amp | 1 (7) | 4 | 3 | 1 (7) | ||
TE/RA/Amp | 1 (7) | 4 | 3 | 1 (7) | ||
HLGR N = 7 | P/CIP/V/TE/TEI/C/RA/G | 1 (14) | 8 | 7 | 2 (29) | 100 |
P/CIP/V/TE/FM/RA/G | 1 (14) | 7 | 7 | 2 (29) | ||
P/CIP/V/TE/TEI/RA/G | 1 (1) | 7 | 6 | 3 (43) | ||
P/CIP/TE/FM/RA/G | 2 (29) | 6 | 6 | 3 (43) | ||
P/CIP/TE/RA/G | 2 (29) | 5 | 5 | 2 (29) | ||
HLSR N = 29 | P/CIP/V/TE/FM/C/RA/G/S | 1 (3) | 9 | 8 | 2 (6) | 96.5 |
P/CIP/V/TE/TEI/C/RA/G/S | 1 (3) | 9 | 7 | 2 (6) | ||
P/CIP/TE/FM/C/RA/Amp/S | 1 (3) | 8 | 7 | 4 (13) | ||
P/CIP/TE/FM/RA/G/S | 2 (6) | 7 | 6 | 4 (13) | ||
P/CIP/V/TE/RA/G/S | 1 (3) | 7 | 6 | 4 (13) | ||
P/CIP/TE/FM/RA/Amp/S | 1 (3) | 7 | 6 | 8 (26) | ||
P/CIP/TE/FM/RA/S | 7 (23) | 7 | 6 | 8 (26) | ||
P/CIP/TE/RA/Amp/S | 1 (3) | 7 | 5 | 3 (10) | ||
CIP/TE/C/RA/S | 2 (6) | 6 | 5 | 3 (10) | ||
CIP/TE/RA/S | 3 (10) | 6 | 4 | 5 (26) | ||
CIP/TE/C/S | 1 (3) | 5 | 4 | 5 (26) | ||
CIP/C/RA/S | 1 (3) | 5 | 4 | 5 (26) | ||
CIP/RA/S | 1 (3) | 5 | 3 | 6 (19) | ||
CIP/TE/S | 3 (10) | 4 | 3 | 6 (19) | ||
TE/RA/S | 2 (6) | 4 | 3 | 6 (19) | ||
S | 1 (3) | 1 | 1 | 1 (3) | ||
VRE N = 6 | P/CIP/V/TE/FM/C/RA/S/G/Amp | 2 (33) | 11 | 8 | 2 (33) | 100 |
P/CIP/V/TE/RA/S/G/AMP | 1 (17) | 9 | 6 | 2 (33) | ||
P/CIP/V/TE/FM/RA/S/G | 1 (17) | 8 | 7 | 2 (33) | ||
P/CIP/V/TE/RA/Amp | 1 (17) | 7 | 5 | 1 (17) | ||
P/CIP/V/RA/Amp | 1 (17) | 6 | 4 | 1(17) |
Enterococcus species . | Phenotypic resistance combination pattern . | Isolates n (%) . | Antibiotic number . | Antibiotic class number . | Totala n (%) . | MDR (%) . |
---|---|---|---|---|---|---|
AR N = 15 | P/CIP/V/TE/FM/C/RA/S/G/Amp | 2 (13) | 10 | 8 | 2 (13) | 100 |
P/CIP/TE/FM/C/RA/S/Amp | 1 (7) | 8 | 7 | 1 (7) | ||
P/CIP/V/TE/RA/S/G/Amp | 1 (7) | 8 | 6 | 4 (27) | ||
P/CIP/TE/FM /RA/S/Amp | 1 (7) | 7 | 6 | 4 (27) | ||
P/CIP/TE/FM /RA/G/Amp | 1 (7) | 7 | 6 | 4 (27) | ||
P/CIP/V/TE/FM/RA/Amp | 1 (7) | 7 | 6 | 4 (27) | ||
P/CIP/TE/RA/G/Amp | 1 (7) | 6 | 5 | 5 (36) | ||
P/CIP/V/TE/RA/Amp | 1 (7) | 6 | 5 | 5 (36) | ||
P/CIP/TE/RA/S/Amp | 1 (7) | 6 | 5 | 5 (36) | ||
P/CIP/TE/FM/RA/Amp | 2 (1) | 6 | 5 | 5 (36) | ||
P/TE/FM/RA/Amp | 1 (7) | 5 | 4 | 1 (7) | ||
P/CIP/RA/Amp | 1 (7) | 4 | 3 | 1 (7) | ||
TE/RA/Amp | 1 (7) | 4 | 3 | 1 (7) | ||
HLGR N = 7 | P/CIP/V/TE/TEI/C/RA/G | 1 (14) | 8 | 7 | 2 (29) | 100 |
P/CIP/V/TE/FM/RA/G | 1 (14) | 7 | 7 | 2 (29) | ||
P/CIP/V/TE/TEI/RA/G | 1 (1) | 7 | 6 | 3 (43) | ||
P/CIP/TE/FM/RA/G | 2 (29) | 6 | 6 | 3 (43) | ||
P/CIP/TE/RA/G | 2 (29) | 5 | 5 | 2 (29) | ||
HLSR N = 29 | P/CIP/V/TE/FM/C/RA/G/S | 1 (3) | 9 | 8 | 2 (6) | 96.5 |
P/CIP/V/TE/TEI/C/RA/G/S | 1 (3) | 9 | 7 | 2 (6) | ||
P/CIP/TE/FM/C/RA/Amp/S | 1 (3) | 8 | 7 | 4 (13) | ||
P/CIP/TE/FM/RA/G/S | 2 (6) | 7 | 6 | 4 (13) | ||
P/CIP/V/TE/RA/G/S | 1 (3) | 7 | 6 | 4 (13) | ||
P/CIP/TE/FM/RA/Amp/S | 1 (3) | 7 | 6 | 8 (26) | ||
P/CIP/TE/FM/RA/S | 7 (23) | 7 | 6 | 8 (26) | ||
P/CIP/TE/RA/Amp/S | 1 (3) | 7 | 5 | 3 (10) | ||
CIP/TE/C/RA/S | 2 (6) | 6 | 5 | 3 (10) | ||
CIP/TE/RA/S | 3 (10) | 6 | 4 | 5 (26) | ||
CIP/TE/C/S | 1 (3) | 5 | 4 | 5 (26) | ||
CIP/C/RA/S | 1 (3) | 5 | 4 | 5 (26) | ||
CIP/RA/S | 1 (3) | 5 | 3 | 6 (19) | ||
CIP/TE/S | 3 (10) | 4 | 3 | 6 (19) | ||
TE/RA/S | 2 (6) | 4 | 3 | 6 (19) | ||
S | 1 (3) | 1 | 1 | 1 (3) | ||
VRE N = 6 | P/CIP/V/TE/FM/C/RA/S/G/Amp | 2 (33) | 11 | 8 | 2 (33) | 100 |
P/CIP/V/TE/RA/S/G/AMP | 1 (17) | 9 | 6 | 2 (33) | ||
P/CIP/V/TE/FM/RA/S/G | 1 (17) | 8 | 7 | 2 (33) | ||
P/CIP/V/TE/RA/Amp | 1 (17) | 7 | 5 | 1 (17) | ||
P/CIP/V/RA/Amp | 1 (17) | 6 | 4 | 1(17) |
HLSR, high-level streptomycin-resistant; HLGR, high-Level gentamicin-resistant; AR, ampicillin-resistant; VRE, vancomycin-resistant enterococci.
P, penicillin G′; CIP, ciprofloxacin; TE, tetracycline; FM, nitrofurantoin; C, chloramphenicol; RA, rifampicin.
aTotal number of isolates resistant to the same number of antibiotic classes.
Frequency of gentamicin resistance (HLGR) encoding genes in E. faecalis and E. faecium isolated from hospital wastewater.
Frequency of gentamicin resistance (HLGR) encoding genes in E. faecalis and E. faecium isolated from hospital wastewater.
Frequency of streptomycin resistance (HLSR) encoding genes in E. faecalis and E. faecium isolated from hospital wastewater.
Frequency of streptomycin resistance (HLSR) encoding genes in E. faecalis and E. faecium isolated from hospital wastewater.
Virulence genes profile
Out of 97 E. faecium isolates, 88% (n = 85) were positive for gelE, 30% (n = 29) for asa1, 20% (n = 19) for esp, and 5% (n = 5) for cylA and negative for hyl genes. Similarly, out of 33 E. faecalis isolates, 91% (n = 30) were positive for gelE, 39% (n = 13) for asa1, 39% (n = 13) for esp, 33% (n = 11) for cylA, and 3% (n = 1) for hyl genes.
The virulence gene profile analysis showed that 87% (n = 86/97) of E. faecium isolates and 97% (n = 32/33) of E. faecalis isolates harbored at least one virulence determinant encoding gene (Table 6).
Virulence genes' profile in Enterococcus species isolated from hospital wastewater
Enterococcus spp. . | Virulance genes' profile . | Isolates n (%) . | Gene number n (%) . | Totala n (%) . |
---|---|---|---|---|
E. faecium (N = 97) | cylA esp asa geleE | 2 (2) | 4 | 2 (2) |
esp asa geleE | 10 (10) | 3 | 10 (10) | |
asa geleE | 17 (17) | 2 | 26 (27) | |
esp geleE | 6 (6) | 2 | ||
cylA geleE | 3 (3) | 2 | ||
geleE | 46 (47) | 1 | 47 (48) | |
Esp | 1 (1) | 1 | ||
– | 11 (11) | 0 | 11 (11) | |
E. faecalis (N = 33) | cylA esp asa geleE | 4 (12) | 4 | 4 (12) |
cylA asa geleE | 5 (15) | 3 | 7 (21) | |
esp, asa geleE | 1 (3) | 3 | ||
cylA esp asa | 1 (3) | 3 | ||
esp geleE | 8 (27) | 2 | 11 (33) | |
asa geleE | 2 (2) | 2 | ||
cylA hyl | 1 (3) | 2 | ||
geleE | 10 (33) | 1 | 10 (33) | |
– | 1 (3) | 0 | 1 (3) |
Enterococcus spp. . | Virulance genes' profile . | Isolates n (%) . | Gene number n (%) . | Totala n (%) . |
---|---|---|---|---|
E. faecium (N = 97) | cylA esp asa geleE | 2 (2) | 4 | 2 (2) |
esp asa geleE | 10 (10) | 3 | 10 (10) | |
asa geleE | 17 (17) | 2 | 26 (27) | |
esp geleE | 6 (6) | 2 | ||
cylA geleE | 3 (3) | 2 | ||
geleE | 46 (47) | 1 | 47 (48) | |
Esp | 1 (1) | 1 | ||
– | 11 (11) | 0 | 11 (11) | |
E. faecalis (N = 33) | cylA esp asa geleE | 4 (12) | 4 | 4 (12) |
cylA asa geleE | 5 (15) | 3 | 7 (21) | |
esp, asa geleE | 1 (3) | 3 | ||
cylA esp asa | 1 (3) | 3 | ||
esp geleE | 8 (27) | 2 | 11 (33) | |
asa geleE | 2 (2) | 2 | ||
cylA hyl | 1 (3) | 2 | ||
geleE | 10 (33) | 1 | 10 (33) | |
– | 1 (3) | 0 | 1 (3) |
aTotal number of isolates harboring the same number of virulence genes.
ERIC-PCR analysis
Dendrogram of ERIC-PCR patterns showing the genetic relationship among the 53 E. faecium isolates collected from hospital wastewater. Similarities of more than 80% were considered for the clustering of the isolates.
Dendrogram of ERIC-PCR patterns showing the genetic relationship among the 53 E. faecium isolates collected from hospital wastewater. Similarities of more than 80% were considered for the clustering of the isolates.
Dendrogram of ERIC-PCR patterns showing the genetic relationship among the 18 E. faecalis isolates collected from hospital wastewater. Similarities of more than 80% were considered for the clustering of the isolates.
Dendrogram of ERIC-PCR patterns showing the genetic relationship among the 18 E. faecalis isolates collected from hospital wastewater. Similarities of more than 80% were considered for the clustering of the isolates.
DISCUSSION
The dissemination of antimicrobial-resistant bacteria and antibiotic-resistance genes through untreated hospital wastewater is a growing health concern (Liu et al. 2023). Studies have shown that the increased use of antibiotics in hospitals admitting Covid-19 patients is associated with a higher relative abundance of antibiotic-resistant bacteria and antibiotic-resistance genes in untreated hospital wastewater (Wang et al. 2022). A One Health approach is needed to address this potential threat (Despotovic et al. 2023). In the context of the One Health approach, studying antibiotic-resistant bacteria in hospital wastewater is important because it helps to understand the growth of particular pathogenic microbes, the development and spread of antibiotic resistance in microbes, and subsequent changes in treatment efficiencies (Liu et al. 2023; Despotovic et al. 2023). In the current study, E. faecium and E. faecalis were found to compose 68.8 and 23.4% of Enterococcus spp. isolated from Ardabil hospital wastewater. Similar reports have been declared in other cities in Iran such as Tehran (E. faecium 80% and E. faecalis 17%) (Rahimi et al. 2019) and Sari (E. faecium 53.3% and E. faecalis 46.6%) (Asgharzadeh et al. 2021). This result was also in agreement with studies reported from other countries such as Poland, which showed that the occurrence of E. faecium isolates (42.9%) was higher than that of E. faecalis isolates (31%) in hospital wastewater (Gotkowska-Płachta 2021). However, the rate of E. faecalis isolates in clinical specimens is usually higher almost two or three times more than that of E. faecium isolates (Maleki et al. 2021; Ebrahimi et al. 2022). The high incidence of E. faecium in wastewater may be due to the higher frequency of intestinal colonization with E. faecium compared to E. faecalis in people, which their fecal material is discharged into the wastewater (Jannati et al. 2020).
Invasive enterococcal infections are typically treated with a combination of vancomycin and ampicillin along with aminoglycoside antibiotics. However, resistance to these antibiotics can make it challenging to treat such infections (Goldstein et al. 2003). The emergence of vancomycin resistance has been increasingly reported in clinical Enterococcus isolates all over the world since 1986 (Novais et al. 2005; Sood et al. 2008; Emaneini et al. 2016; Maleki et al. 2021). Nowadays, VRE isolates are reported beyond infected patients and can be found in various environments. According to a meta-analysis study conducted by Hasanpour et al., the prevalence of VRE strains in non-clinical settings in Iran was found to be 33.4% in foods, 26.4% in hospital environments, and 13.1% in wastewater and surface water (Hasanpour et al. 2021). In comparison, in our study, the prevalence of VRE strains in hospital wastewaters in Ardabil was lower than the average reported above. However, similar results were reported from some regions in Iran (3.6%) (Talebi et al. 2008) and Portugal (3.4%) (Araújo et al. 2010). In contrast, a higher prevalence of VRE was reported in Sweden (36%) (Iversen et al. 2002). However, the extent of vancomycin usage and hence selective pressure imposed on bacteria and laboratory methods used to study vancomycin resistance can also affect the prevalence of VRE isolates in different geographical regions (Zakaria et al. 2023).
Several genes encoding D-Ala–D-Ala ligases have been identified as responsible for vancomycin resistance. Among them, vanA and vanB are the most common genes that convey high-level resistance in Enterococcus spp. (Eliopoulos & Gold 2001).
The overall estimate of the vanA and vanB genes in non-clinical Enterococcus isolates in Iran has been reported to be 35.1 and 6.6%, respectively (Hasanpour et al. 2021). In the current study, 66.6% of VRE strains in hospital wastewaters were positive for vanA and negative for vanB genes. Previously, we also observed that 100% of VRE clinical Enterococcus isolates in Ardabil were positive for the vanA gene (Maleki et al. 2021). Similar findings were reported from other countries showing the predominance of the vanA gene in VRE isolates (Sood et al. 2008; Gotkowska-Płachta 2021).
Ampicillin resistance is significantly high in clinical enterococci isolates (Hidron et al. 2008). However, higher rates of ampicillin resistance have also been reported in non-clinical enterococci isolates from countries such as Portugal (45%) and Iran (30.4%) (Novais et al. 2005; Hasanpour et al. 2021). In contrast, our isolates exhibited low rates of resistance to ampicillin. It is generally accepted that E. faecium is more resistant to penicillin and ampicillin than E. faecalis (Gagetti et al. 2019). However, in our study, ampicillin resistance was higher in E. faecalis (15%) than in E. faecium (10%) isolates. Similar results were reported by Gouliouris et al. on E. faecalis isolates collected from untreated municipal wastewater in England (Gouliouris et al. 2019).
In our study, we found that 15% of E. faecalis and 25% of E. faecium isolates had a HLGR phenotype and, to a lesser extent, 5% of E. faecalis and 6% of E. faecium isolates had a HLSR phenotype. Few studies have reported the occurrence of HLGR and HLSR Enterococcus spp. in non-clinical settings, but our frequency of HLGR isolates was lower than the rates reported for clinical isolates in Iran [67% for E. faecalis and 33% for E. faecium (Mousavi et al. 2020)], Italy [71% for E. faecalis and 29% for E. faecium (Zarrilli et al. 2005)], and South Korea [23% for E. faecalis and 77% for E. faecium (Jang et al. 2010)]. Similarly, higher rates of HLSR have been reported for clinical enterococci isolates worldwide (Sahm & Gilmore 1994; Jang et al. 2010; Dadfarma et al. 2013), compared to our study and a similar study from Poland, where 20.7% of E. faecium and 16% of E. faecalis isolates from marine outflow of a wastewater treatment plant (WWTP) were HLSR (Sahm & Gilmore 1994).
Resistance against aminoglycosides is commonly observed due to enzymatic changes in the drugs by aminoglycoside-modifying enzymes (Ramirez & Tolmasky 2010). However, high-level resistance to gentamicin and streptomycin is caused by different mechanisms. These antibiotics can be used interchangeably in the treatment of enterococcal infections. We found that the commonest aminoglycoside-modifying encoding genes in HLGR and HLSR isolates were aac (6′) Ie-aph (2″) Ia and ant(6′)-Ia, respectively. This is consistent with global reports, suggesting that these genes commonly encode high-level resistance to gentamicin and streptomycin (Ida et al. 2001).
Almost all of the HLGR and HLSR isolates were MDR. Akin to our study, high rates of MDR Enterococci in hospital sewage were reported in Poland (94.0 and 88%) and Portugal (61.5%) in the literature (Varela et al. 2013; Sadowy & Luczkiewicz 2014; Gotkowska-Płachta 2021). In addition to the aforementioned resistance traits, our E. faecalis and E. faecium isolates also showed significant rates of resistance to ciprofloxacin and rifampicin. Similar results have been reported in enterococci collected from hospital sewage samples in South Africa and Greece (Kotzamanidis et al. 2009; Iweriebor et al. 2015).
Enterococci were once considered microorganisms of minimal clinical impact but have now emerged as common opportunistic pathogens of humans (Ben Braiek & Smaoui 2019). The virulence factors play a significant role in the pathogenicity of enterococcal strains (Heidari et al. 2017). Some studies reported a lesser frequency of virulence determinants in Enterococcus spp. from wastewater effluent (Ferguson et al. 2016). However, our isolates contained multiple virulence encoding genes (gelE and asa1, esp, and cyl) simultaneously. In contrast to a general assumption that E. faecalis is more virulent and associated with more virulence factors than E. faecium isolates (Noskin et al. 1995), our E. faecium isolates harbored several virulence factors as well.
The Enterococcus spp. isolates in our hospital sewages showed high genotypic diversity among HLGR, HLSR, and VRE isolates as revealed by ERIC-PCR. This indicates that there is no clonal dissemination of Enterococcus spp. in our hospital sewages. ERIC-PCR was used to study the clonality of Enterococcus isolates previously (Bachtiar et al. 2015; Xie et al. 2019); however, it is generally accepted that PCR-based methods have less discriminatory power in comparison with nucleotide sequence-based methods and pulsed-field gel electrophoresis. Unfortunately, due to limited resources, we were unable to examine our isolates using these methods.
CONCLUSION
In summary, the results of this study indicate that the clinically significant Enterococcus spp. E. faecalis and E. faecium are prevalent in our hospital's wastewater. A significant proportion of isolates were found to be resistant to key antibiotics used for the treatment of enterococcal infections and harbor several virulence genes. Our findings show that hospital wastewater could act as a reservoir for antibiotic-resistant and virulent enterococci enabling the distribution of these organisms to the environment. Therefore, we propose that hospital effluent should be strictly examined to ensure treatment efficacy before disposal into municipal wastewater systems to manage the public health and environmental risks of antibiotic-resistant enterococci. We also suggest conducting deep molecular studies to establish the relationship between wastewater isolates and clinical isolates and identify the circulating antibiotic-resistant clones in hospital settings.
ACKNOWLEDGEMENTS
This work was conducted by E. Jannati in Microbiology as part of her Ph.D. thesis requirements. We would like to express our gratitude to the Ardabil University of Medical Sciences for providing laboratory facilities for this work.
FUNDING
The authors state that there was no specific funding for this work.
AUTHOR CONTRIBUTIONS
E.J. performed the experiments, analyzed the results, and prepared the initial manuscript draft. M.M. performed the experiments. F.K. contributed to writing, reviewing, and editing the manuscript. N.A. contributed to the analysis of the results. V.S.N. contributed to molecular analysis. M.A. conceived the study, led the project, and critically revised the manuscript.
CONSENT TO PUBLICATION
All authors have read and agreed to the published version of the manuscript.
DATA AVAILABILITY STATEMENT
All relevant data are included in the paper or its Supplementary Information.
CONFLICT OF INTEREST
The authors declare there is no conflict.