Skip to main content
NCBI home page
MicrobiologyOpen logoLink to MicrobiologyOpen
. 2024 Mar 5;13(2):e1397. doi: 10.1002/mbo3.1397

Multidrug‐resistant Enterococcus faecium strains enter the Norwegian marine environment through treated sewage

Vera Radisic 1,2, Didrik H Grevskott 1, Nadja Junghardt 1, Lise Øvreås 2, Nachiket P Marathe 1,✉
PMCID: PMC10913173  PMID: 38441345

Abstract

This study aimed to understand the antibiotic resistance prevalence among Enterococcus spp. from raw and treated sewage in Bergen city, Norway. In total, 517 Enterococcus spp. isolates were obtained from raw and treated sewage from five sewage treatment plants (STPs) over three sampling occasions, with Enterococcus faecium as the most prevalent (n = 492) species. E. faecium strains (n = 307) obtained from the influent samples, showed the highest resistance against quinupristin/dalfopristin (67.8%). We observed reduced susceptibility to erythromycin (30.6%) and tetracycline (6.2%) in these strains. E. faecium strains (n = 185) obtained from the effluent samples showed highest resistance against quinupristin/dalfopristin (68.1%) and reduced susceptibility to erythromycin (24.9%) and tetracycline (8.6%). We did not detect resistance against last‐resort antibiotics, such as linezolid, vancomycin, and tigecycline in any of the strains. Multidrug‐resistant (MDR) E. faecium strains were detected in both influent (2.3%) and effluent (2.2%) samples. Whole genome sequencing of the Enterococcus spp. strains (n = 25) showed the presence of several antibiotic resistance genes, conferring resistance against aminoglycosides, tetracyclines, and macrolides, as well as several virulence genes and plasmid replicons. Two sequenced MDR strains from the effluents belonged to the hospital‐associated clonal complex 17 and carried multiple virulence genes. Our study demonstrates that clinically relevant MDR Enterococcus spp. strains are entering the marine environment through treated sewage.

Keywords: CC17, Enterococcus faecium, last resort antibiotic, MDR strain, Norway, sewage surveillance

This study investigated the prevalence of antibiotic resistance in Enterococcus spp. from raw and treated sewage in Bergen, Norway. We analyzed 307 Enterococcus faecium isolates from raw sewage and 185 E. faecium isolates from treated sewage for antibiotic sensitivity and sequenced selected isolates (n = 25). This study found that multidrug‐resistant E. faecium clones from clonal complex 17 are entering the marine environment through treated sewage.

graphic file with name MBO3-13-e1397-g001.jpg

1. INTRODUCTION

Enterococcus spp. consists of Gram‐positive, opportunistic pathogens that are commonly found in the intestinal microbiota of healthy humans and animals (Byappanahalli et al., 2012; Iversen et al., 2002). Enterococcus spp. are a major cause of hospital‐acquired infections (HAI) and are ranked second as a cause of bloodstream‐ and urinary tract infections in the European Union/European Economic Area (EU/EEA) (ECDC, 2023; Viswanath et al., 2022). Enterococcus faecium (around 90%) and Enterococcus faecalis (around 5%–10%) are responsible for most enterococcal infections in humans (Simonsen, 2003; Top et al., 2008). Other species belonging to this genus, like the species E. durans, E. gallinarum, and E. casseliflavus, can also cause infections in humans (Arias & Murray, 2012; Liassine et al., 1998; Murray, 1990; Rahimi et al., 2007).

E. faecium is a part of the ESKAPE pathogens (consisting of E. faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), which represent pathogens that are leading causes of nosocomial infections worldwide (Santajit & Indrawattana, 2016). Vancomycin‐resistant E. faecium (VRE) is of special concern and poses a growing health problem due to limited available treatment options (Hayakawa et al., 2014). Linezolid, daptomycin, and tigecycline are last‐resort antibiotics used for the treatment of such infections (Diaz et al., 2014; Klare et al., 2015; Werner et al., 2012). Consequently, the World Health Organization (WHO) has listed this pathogen as a high priority for finding new treatments (WHO, 2023). Increasing trends of VRE, ranging from 11.6% in 2016 to 16.8% in 2020, are observed in the EU/EEA (ECDC, 2022). In Norway, only one VRE strain with clinical importance (E. faecium carrying vanB) was obtained from blood culture, while no linezolid‐resistant strains were observed in the Norwegian monitoring report (NORM/NORM‐VET) in 2021 (NORM/NORM‐VET, 2023), suggesting a low prevalence of vancomycin resistance in Enterococcus spp. in clinics in Norway.

The prevalence of resistant Enterococcus spp. within the population in Norway is largely unknown. Surveillance using a sewage‐based approach has shown to be promising for understanding antibiotic resistance in different pathogens within a studied community (Grevskott et al., 2021; Radisic et al., 2023). This study aimed at understanding the frequency of antibiotic resistance among Enterococcus spp. present within the population using sewage‐based surveillance. Further, we investigated the antibiotic resistance prevalence in Enterococcus spp. entering the marine environments through treated effluent.

2. MATERIALS AND METHODS

2.1. Sewage sample collection

The details about the collection of sewage samples used in this study have previously been described in Radisic et al. (2023). Briefly: on three different time points (1 March, 3 May, and 5 July 2021) the sewage samples (both influents and effluents) were collected from five sewage treatment plants (STPs) (Flesland, 152,000 citizens; Holen, 132,000 citizens; Knappen, 63,000 citizens; Kvernevik, 56,000 citizens; and Ytre‐Sandviken, 44,000 citizens) located in Bergen, Norway (Radisic et al., 2023), using a 24‐h time sampler. The complete overview of the different STPs, capacity, and type of treatment are presented in Table S1.

2.2. Isolation and identification of isolates

The sewage samples were serially diluted (10‐fold) with sterile saline (0.85% NaCl) and plated on Slanetz and Bartley media (Thermo Fisher Scientific) within 6 h of collection. The plates were aerobically incubated at 44°C for 48 h. The number of presumptive Enterococcus spp. was estimated by counting maroon and/or pink colonies. Up to 30 isolates were randomly picked from the Slanetz and Bartley plates for each sample and transferred to Mueller–Hinton broth (Sigma‐Aldrich), followed by aerobic incubation at 44°C for another 48 h. Afterward, 20% glycerol was added, and the samples were stored at −80°C till further use.

2.3. Antibiotic susceptibility testing (AST)

From the glycerol stocks, the strains were re‐streaked onto Slanetz and Bartley plates and incubated at 44°C for 48 h, followed by identification using MALDI‐TOF MS (Bruker Daltonics). The resistance profiles of 517 Enterococcus spp. strains were determined against 12 antibiotics using a broth microdilution assay with Sensititre® EUVENC plates (Thermo Fisher Scientific) following the manufacturer's protocol. Each strain was tested for resistance and/or reduced susceptibility against 12 antibiotics (Table S2). The plates were incubated at 37°C for 24 h. The strains were defined as susceptible and/or resistant according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints tables v.13.0 (EUCAST, 2023). For gentamicin, the minimum inhibitory concentration (MIC) breakpoint for high‐level aminoglycoside resistance (>128 mg/L) was used when defining resistance. For antibiotics with clinical breakpoints not available in EUCAST, epidemiological cut‐off (ECOFF) values were used for defining reduced susceptibility compared to wild‐type (WT) Enterococcus spp. strains with MIC above the ECOFF were classified as strains with reduced susceptibility. No ECOFF values were available for E. durans and E. casseliflavus. E. faecium CCUG 542T and E. faecalis CCUG 19916T were used as controls for the Sensititre® plates.

2.4. DNA extraction and sequencing

Whole genome sequencing was performed, for 24 presumptive E. faecium strains and one E. faecalis strain, selected on the basis of phenotypic resistance profiles. The strains were re‐streaked on Mueller–Hinton agar (Oxoid) at 44°C for 48 h. Genomic DNA was processed and sequenced as described in Radisic et al. (2023). Briefly: sequencing libraries were prepared using Nextera DNA Flex Library Prep kit (Illumina) and sequencing was performed using an Illumina MiSeq‐based platform (Illumina), with 2 × 300 bp chemistry.

2.5. Genome assembly and sequencing analysis

Genome assembly and annotation was accomplished as previously described in Radisic et al. (2020, 2023). Briefly, the raw reads generated by the Illumina MiSeq sequencing were quality‐filtered and assembled using SPAdes (v.3.13.0) (Bankevich et al., 2012). Annotation was performed with the NCBI Prokaryotic Genome Annotation Pipeline (Tatusova et al., 2013). Antibiotic resistance genes (ARGs) were identified using CARD v.3.1.4 (Alcock et al., 2019) and ResFinder v.4.1 (Bortolaia et al., 2020). Plasmids were identified with PlasmidFinder v.2.1 (Carattoli et al., 2014). VFanalyzer at the VFDB (Liu et al., 2019) were used to check for virulence genes. Sequence types (STs) and clonal complexes (CCs) were identified with the PubMLST database (https://pubmlst.org/bigsdb?db=pubmlst_efaecium_seqdef and https://pubmlst.org/bigsdb?db=pubmlst_efaecalis_seqdef). Identification of sequenced strains was performed, using the average nucleotide identity based on BLAST (ANIb) analysis on the JWSpecies server (http://jspecies.ribohost.com/jspeciesws/) with recommended cut‐off values of >95% identity (Chun & Rainey, 2014) for species assignment.

2.6. Core genome‐based phylogeny of the strains

A phylogenetic tree was constructed based on the core genomes of selected sequenced E. faecium strains (n = 10) belonging to CC17 to check the clade (A1, A2, and/or B) our strains are clustering with. Strain ATCC700221 (GenBank accession no. GCA_001594345.1) was used as a reference strain for clade A1, while strain NCTC7174 (GCA_900637035.1) was used as a reference for clade A2. The assembled genomes were analyzed with CSI Phylogeny v.1.4 (https://cge.food.dtu.dk/services/CSIPhylogeny/), using the parameters previously described (Radisic et al., 2023). All strains used for constructing the phylogenetic tree are presented in Table S3.

3. RESULTS

3.1. Enterococcus spp. isolates from sewage

A higher number of the presumptive Enterococcus spp. were obtained from the influent samples (n = 92; n = 99; n = 125) compared to the effluent samples (n = 41; n = 90; n = 70) for all five STPs during the three sampling points. From all samples, a total of 627 isolates were obtained and identified using MALDI‐TOF MS. Out of 627 isolates, 517 isolates (82.5%) were identified as Enterococcus spp. with E. faecium as most prevalent (n = 492) followed by E. faecalis (n = 13), E. hirae (n = 7), E. durans (n = 3), and E. casseliflavus (n = 2). Isolates identified as other genera like Streptococcus infantarius (n = 88), S. equinus (n = 12), and S. lutetiensis (n = 10), were excluded from further analysis.

3.2. Resistance rates in E. faecium strains

In total, 307 presumptive E. faecium strains were isolated from the influent samples, and 185 presumptive E. faecium strains were isolated from the effluent samples. Strains obtained from the influents showed the highest resistance against quinupristin/dalfopristin (67.8%) and ciprofloxacin (4.2%). Reduced susceptibility to erythromycin and tetracycline was observed in 30.6% and 6.2% of the strains, respectively. Strains obtained from the effluent samples also showed the highest resistance against quinupristin/dalfopristin (68.1%), followed by ciprofloxacin (3.2%), high‐level gentamicin (1.1%) and ampicillin (1.1%). Additionally, E. faecium strains recovered from the effluent samples also showed reduced susceptibility to erythromycin (24.9%) and tetracycline (8.6%). During the three sampling occasions, no resistance against vancomycin, teicoplanin, daptomycin, tigecycline, linezolid, or chloramphenicol was observed (Table S2). From a total of 492 presumptive E. faecium strains, 2.2% were classified as multidrug‐resistant (MDR), showing resistance against at least one antibiotic belonging to three or more classes. Two of these strains (E4‐112 and E5‐100) detected in the treated effluent, showed resistance against ampicillin, ciprofloxacin, and high‐level gentamicin resistance.

3.3. AST of other Enterococcus spp. strains

From both influent and treated effluent during the three sampling occasions, 25 isolates were identified as other Enterococcus spp. For E. faecalis, reduced susceptibility to tetracycline (23.1%, n = 3) and erythromycin (7.7%, n = 1) was observed. We further observed resistance against gentamicin in 7.7% (n = 1) of the strains. Resistance and/or reduced susceptibility for E. hirae, E. durans, or E. casseliflavus was not determined due to a lack of defined clinical breakpoints and/or no available ECOFF values.

3.4. Genome sequence and STs of sequenced Enterococcus spp. strains

The overview of the assembly statistics of the 25 sequenced genomes is presented in Table S4. Four strains (E4‐185, E5‐9, E5‐80, and E5‐162), which were identified as E. faecium by MALDI‐TOF MS, were after sequencing re‐identified as E. lactis (ANIb values ≥ 97.75%) and not as E. faecium. The most prevalent ST among the sequenced E. faecium strains was ST32 (20%, n = 4), while all sequenced E. lactis strains belonged to different STs. E. faecalis strain belonged to ST227. Several strains (E4‐85, E4‐150, E4‐152, E4‐162, E4‐185, E4‐223, E5‐37, E5‐79, E5‐103, E5‐112, E5‐162, and E5‐209) were not assigned to any ST and/or CC using the PubMLST database.

3.5. Single nucleotide polymorphism (SNP)‐based phylogeny of CC17 strains

The most prevalent CC among the sequenced E. faecium strains was CC17 (n = 10). The SNP‐based phylogeny showed that most of these strains (E4‐79, E4‐163, E4‐182, E4‐220, E5‐24, E5‐119, E5‐168, and E5‐175) belonging to CC17 clustered with clade A2, representing animal derived strains (Gao et al., 2018). Two MDR strains (E4‐112 and E5‐100), showing high‐level gentamicin resistance as well as ampicillin and ciprofloxacin resistance, clustered with clinical strains belonging to clade A1 (Figure 1).

Figure 1.

Figure 1

Open in a new tab

Phylogenetic tree based on the whole genome sequences of Enterococcus faecium strains. Strains belonging to clade A1 are highlighted in red, while strains belonging to clade A2 and B are highlighted in blue and green, respectively. Our strains (E4‐79, E4‐112, E4‐163, E4‐182, E4‐220, E5‐24, E5‐100, E5‐119, E5‐168, and E5‐175) are highlighted in bold. Two strains ATCC700221 (GenBank accession no. GCA_001594345.1) and NCTC7174 (GCA_900637035.1) were used as reference strains for clade A1 and A2, respectively.

3.6. Clinically important ARGs, virulence genes, and plasmid replicons present in sequenced Enterococcus spp. strains

All sequenced E. faecium strains (n = 20) carried the known intrinsic ARG, aac(6′)‐li while ARGs like msr(C) and aph(3′)‐IIIa were detected in 95% and 15% of the strains, respectively. We also detected ARGs such as msr(A) and msr(B), which have previously been detected in Enterococcus spp. (Hummel et al., 2007; Roberts, 2008). in all sequenced strains (Table 1). Several acquired clinically important ARGs were detected in the sequenced Enterococcus spp. strains (n = 25), conferring resistance against aminoglycosides (bifunctional acc(6′)‐le/aph(2″)‐la, aph(2″)‐la, ant(6)‐Ia, and ant(9)), tetracyclines (tet(L) and tet(M)), and macrolides (erm(B) and erm(T)) (Table 1). Two strains (E4‐112 and E5‐100) showing high ciprofloxacin resistance with MIC > 16 mg/L carried known murtations in gyrA (p.S83Y) and parC (p.S80I) (Leavis, Willems, et al., 2006).

Table 1.

List of antibiotic resistance genes (ARGs) and plasmid replicons detected in the 25 sequenced Enterococcus spp. strains.

Strain Species Sample type ARGs Plasmid replicons
E4‐79 E. faecium Influent msr(A), msr(B), msr(C), erm(T), tet(L), tet(M), aac(6)‐Ii rep2, repUS12, repUS43
E4‐85 E. faecium Influent msr(A), msr(B), msr(C), aac(6′)‐Ii rep1, repUS15
E4‐112 E. faecium Effluent msr(A), msr(B), msr(C), tet(M), aac(6′)‐Ii, bifunctional aac(6′)‐Ie/aph(2″)‐Ia rep2, rep29, repUS15
E4‐150 E. faecium Influent msr(A), msr(B), msr(C), aac(6′)‐Ii rep1, rep2, rep18a, repUS15,
E4‐152 E. faecium Influent msr(A), msr(B), msr(C), aac(6′)‐Ii ‐
E4‐162 E. faecium Influent msr(A), msr(B), msr(C), erm(B), lnu(B), lsa(E), tet(M), aac(6′)‐Ii, aph(3′)‐IIIa, ant(6)‐Ia, ant(9) rep1, repUS15, repUS43
E4‐163 E. faecium Influent msr(A), msr(B), msr(C), erm(T), tet(L), tet(M), aac(6′)‐Ii rep2, repUS12, repUS43
E4‐182 E. faecium Effluent msr(A), msr(B), msr(C), erm(B), lnu(B), lsa(E), tet(L), tet(M), aac(6′)‐Ii, aph(3′)‐IIIa, ant(9), aminoglycoside 6‐adenylyltransferase rep2, repUS43
E4‐185 E. lactis Effluent msr(A), msr(B), msr(C), erm(T), tet(L), tet(M), aac(6′)‐Ii rep1, rep2, repUS12, repUS15, repUS43
E4‐208 E. faecium Effluent msr(A), msr(B), msr(C), tet(M), aac(6′)‐Ii rep1, repUS15, repUS42
E4‐220 E. faecium Influent msr(A), msr(B), msr(C), aac(6′)‐Ii ‐
E4‐223 E. faecium Influent msr(A), msr(B), msr(C), aac(6′)‐Ii rep1, repUS15
E5‐9 E. lactis Influent msr(A), msr(B), msr(C), tet(L), aac(6′)‐Ii, str rep22, repUS15
E5‐24 E. faecium Influent msr(A), msr(B), msr(C), erm(T), tet(L), tet(M), aac(6′)‐Ii repUS12, repUS43
E5‐37 E. faecium Effluent msr(A), msr(B), msr(C), tet(M), aac(6′)‐Ii repUS15, repUS43
E5‐79 E. faecium Influent msr(A), msr(B), msr(C), aac(6′)‐Ii ‐
E5‐80 E. lactis Influent msr(A), msr(B), msr(C), tet(L), tet(M), aac(6′)‐Ii, str rep1, rep22, repUS15
E5‐100 E. faecium Effluent msr(A), msr(B), erm(B), aac(6′)‐Ii, aph(2″)‐Ia, aph(3′)‐IIIa, ant(6)‐Ia, catA‐8 rep2, repUS15
E5‐103 E. faecalis Influent msr(A), msr(B), tet(M), lsa(A) repUS43
E5‐112 E. faecium Influent msr(A), msr(B), msr(C), aac(6′)‐Ii ‐
E5‐118 E. faecium Influent msr(A), msr(B), msr(C), aac(6′)‐Ii repUS15
E5‐162 E. lactis Influent msr(A), msr(B), msr(C), tet(L), tet(M), aac(6′)‐Ii, ant(6)‐Ia, catA repUS12, repUS15, repUS43
E5‐168 E. faecium Influent msr(A), msr(B), msr(C), aac(6′)‐li repUS15
E5‐175 E. faecium Influent msr(A), msr(B), msr(Crep1′), tet(L), tet(M), aac(6′)‐li rep2, repUS12, repUS43
E5‐209 E. faecium Influent msr(A), msr(B), msr(C),aac(6′)‐li rep1
Open in a new tab

Plasmid replicons were detected in most sequenced E. faecium strains (84%), with plasmid replicon type repUS15 as most common (50%), followed by repUS43 (35%) and rep2 (35%). More than one plasmid replicon type was observed in 68% of the sequenced Enterococcus spp. strains. E. faecalis carried the plasmid replicon type repUS43, whereas repUS15 was the most frequently observed plasmid replicon type in E. lactis (100%) (Table 1).

All sequenced E. faecium strains carried multiple virulence factors involved in antiphagocytosis (capsule; cpsA/uppS, cpsB/cdsA) and surface proteins (lgt), as well as virulence factors involved in biofilm formation (bopD) (Qin et al., 2012). In addition to these, virulence factors involved in adherence like the virulence factors EfaA (100%) (Bourgogne et al., 2006), Ebp pili (95%), Acm (80%), Scm (20%), SgrA (15%), and EcbA (10%) were also detected in the sequenced strains (Hendrickx et al., 2009; Qin et al., 2012). The E. lactis strains (n = 4) carried virulence genes involved in adherence, antiphagocytosis, biofilm formation (Oxaran et al., 2012), enzymes (hyaluronidase and serine‐threonine phosphatase from Listeria) (Stedman et al., 2020) and surface protein anchoring (lgt). E. faecalis carried various virulence factors involved in adherence (Ace, Ebp pili, EfaA) (Golob et al., 2019; Kayaoglu & Ørstavik, 2004), antiphagocytosis (capsule; cpsA/uppS, cpsB/cdsA, and cpsC) (Hufnagel et al., 2004), biofilm formation (bopD, fsrA, fsrB, and fsrC), enzymes (gelE and sprE) (Engelbert et al., 2004; Hancock & Perego, 2004), and immune evasion (capsule from Staphylococcus). The complete overview of the different virulence genes is presented in Table S5.

4. DISCUSSION

To the best of our knowledge, this is the first study performing population‐based surveillance of antibiotic resistance in Enterococcus spp. on a city scale in Norway. Although we did not detect any resistance against last‐resort antibiotics, such as linezolid, vancomycin, and tigecycline, we did detect MDR Enterococcus spp. strains in our study. Our findings are in accordance with the prevalence of resistance against last‐resort antibiotics reported from the clinics and the animal sector in Norway (NORM/NORM‐VET, 2022).

Quinupristin/dalfopristin is often used to treat MDR E. faecium infections. Quinupristin/dalfopristin resistance in clinical E. faecium strains has been reported as a concern in several countries (Wang et al., 2016). From a total of 307 presumptive E. faecium strains obtained from the influent samples during the three sampling points, 67.8% of these strains showed phenotypic resistance against quinupristin/dalfopristin. A similar resistance rate was observed against quinupristin/dalfopristin (68.1%) in 185 presumptive E. faecium strains obtained from effluents. Our results are in accordance with the resistance prevalence detected in E. faecium from fattening pigs in Norway, where 67.3% of the strains were resistant against quinupristin/dalfopristin (NORM/NORM‐VET, 2022). Our results are also in accordance with the high resistance prevalence against quinupristin/dalfopristin in E. faecium from healthy food‐producing animals in Europe (de Jong et al., 2019). Quinupristin/dalfopristin resistance in E. faecium can be achieved through several different mechanisms, including the presence of acquired ARGs like vat(D), vat(E), vgb(A), vgb(B), and eat(A), as well as the presence of the intrinsic gene, msr(C) (Boodaghi Malidareh et al., 2022; Miller et al., 2014). Almost all sequenced E. faecium strains in this study, except strain E5‐100, carried the msr(C) gene. Although this gene was detected in all strains, some strains did not show phenotypic resistance against quinupristin/dalfopristin. In addition to E. faecium, we also detected some E. faecalis strains in our samples. E. faecalis is intrinsically resistant to quinupristin/dalfopristin due to the chromosomal gene lsa (Stedman et al., 2020). However, all of the E. faecalis strains obtained in this study were sensitive to quinupristin/dalfopristin. The lack of phenotypic resistance in these strains carrying ARGs might be due to low or no expression of these genes. Further studies on transcriptomics and/or proteomics are needed to confirm these findings.

Apart from resistance against quinupristin/dalfopristin, ciprofloxacin resistance was also observed in E. faecium strains from both influents (4.2%) and treated effluents (3.2%). This is similar to the observed prevalence of ciprofloxacin resistance in E. faecium strains (5.9%) from cecal samples from fattening pigs in Norway in 2021 (NORM/NORM‐VET, 2022). High‐level ciprofloxacin resistance in E. faecium has previously been linked to gyrA and parC mutations (Rathnayake et al., 2012; Werner et al., 2010). We detected mutations in both gyrA (p.S83Y) and parC (p.S80I) in strains showing high ciprofloxacin resistance (MIC > 16 mg/L), thus explaining the observed resistance against ciprofloxacin. Furthermore, reduced susceptibility (MIC > ECOFF) against several antibiotics, including erythromycin and tetracycline, was detected in some of our strains. For erythromycin, the reduced susceptibility in E. faecium strains obtained from the influent samples (30.6%) was higher compared to the effluent samples (24.9%). In contrast, the reported reduced susceptibility to erythromycin from the veterinary setting in Norway was only 2.9% (NORM/NORM‐VET, 2022). Additionally, higher reduced susceptibility to tetracycline was observed in E. faecium strains from the effluent samples (8.6%) compared to strains from the influent samples (6.2%). This is much lower than the reported reduced susceptibility to tetracycline (33%) in E. faecium strains from pigs in Norway (NORM/NORM‐VET, 2022). We detected the presence of erm(B), erm(T), tet(M), and tet(L) in several sequenced Enterococcus spp. strains, thus explaining the observed phenotypic resistance against the respective antibiotics (Aarestrup et al., 2000; Cauwerts et al., 2007; Chajęcka‐Wierzchowska et al., 2016). These ARGs have also been shown to be prevalent in Enterococcus spp. strains from sewage from different countries like South Africa (Mbanga et al., 2021), Tunisia (Ben Said et al., 2015), and Canada (Zaheer et al., 2020).

Clinically relevant E. faecium belonging to CC17 can be characterized based on resistance against ampicillin, quinolones, and sometimes high‐level resistance against gentamicin (Leavis, Bonten, et al., 2006; Leavis, Willems, et al., 2006; Quiñones et al., 2018). We detected two MDR strains (E4‐112 and E5‐100) with resistance against ampicillin, ciprofloxacin, and gentamicin (high‐level) in the treated effluent in this study. Additionally, reduced susceptibility to erythromycin was also observed in one of the MDR strains (E5‐100). Whole genome phylogeny showed that the MDR strains belonged to the clinically relevant CC17 clade A1 (Lee et al., 2019), which represents a high‐risk epidemic clonal linage of E. faecium (Leavis, Bonten, et al., 2006) known for causing severe morbidity and mortality (Lee et al., 2019). Although no β‐lactamase was detected in our strains, several penicillin‐binding proteins (PBPs) were present. The high resistance against ampicillin observed in our study may be due to the presence of several PBPs and/or increased production of PBP5 (Jureen et al., 2003; Sifaoui et al., 2001). Although this is highly likely, further studies are needed to confirm the expression of PBPs in our strains. Apart from carrying several ARGs, the strains also carried important virulence factors involved in adherence (ebpBC, srtC, ecbA, esp, and pavA) (Leclercq et al., 2013), antiphagocytosis (cpsB/cdsA and cpsC), biofilm formation (fsrA) (Leclercq et al., 2013), immune evasion (cps2K and epsE), protease (tig/ropA), and serum resistance/immune evasion (LPS rfb locus from Klebsiella), suggesting pathogenic potential of these MDR strains. E. faecium CC17 can persist in aquatic environments polluted by sewage (Leclercq et al., 2013), including marine environments (Vignaroli et al., 2013). Hence, there is a risk that these MDR strains can persist in the marine environments in Norway (Cherak et al., 2022). Our study thus demonstrates that clinically important MDR strains of E. faecium carrying several ARGs and virulence genes are entering the marine environment in Norway through treated effluents.

5. CONCLUSIONS

All the Enterococcus spp. strains in our study were sensitive to last‐resort antibiotics like linezolid, vancomycin, and tigecycline, which is in accordance with the clinical prevalence in Norway (NORM/NORM‐VET, 2022). Our findings along with clinical prevalence and studies from the animal sector show that resistance against last‐resort antibiotics in E. faecium in Norway, in general, is very low (NORM/NORM‐VET, 2022). Although true, we did detect clinically important ARGs and clinically relevant MDR strains in both influent and treated effluent entering the receiving marine environment. Our study highlights the importance of sewage‐based surveillance to monitor and understand the resistance in pathogens within the population as well as demonstrates that treated sewage is a source of clinically important MDR pathogens entering the receiving marine environment.

AUTHOR CONTRIBUTIONS

Vera Radisic: Methodology (lead); investigation (equal); validation (equal); visualization (lead); formal analysis (equal); bioinformatic analysis (equal); analysis of data (lead); writing—original draft (lead); writing—review and editing (lead). Didrik H. Grevskott: Methodology (supporting); visualization (supporting); analysis of data (supporting); writing—review and editing (supporting). Nadja Junghardt: Methodology (supporting); investigation (supporting); bioinformatic analysis (equal); writing—review and editing (supporting). Lise Øvreås: Critical inputs (supporting); writing—review and editing (supporting). Nachiket P. Marathe: Funding acquisition (lead); project administration (lead); supervision (lead); conceptualization (lead); methodology (supporting); investigation (equal); supervision (lead); validation (equal); visualization (supporting); writing—review and editing (supporting).

CONFLICT OF INTEREST STATEMENT

None declared.

ETHICS STATEMENT

None required.

Supporting information

Supporting information.

MBO3-13-e1397-s001.xlsx (9.4KB, xlsx)

Supporting information.

MBO3-13-e1397-s004.xlsx (49.8KB, xlsx)

Supporting information.

MBO3-13-e1397-s003.xlsx (19KB, xlsx)

Supporting information.

MBO3-13-e1397-s002.xlsx (12.2KB, xlsx)

Supporting information.

MBO3-13-e1397-s005.xlsx (22.4KB, xlsx)

ACKNOWLEDGMENTS

This research was funded by the Research Council of Norway under the ResMarine project, grant number 315266. We wish to thank Kristine S. Akervold and Ingvild Fenne (municipality of Bergen), as well as workers at the different treatment plants for sewage sample collection. The sequencing service was provided by the Norwegian Sequencing Centre (www.sequencing.uio.no), a national technology platform hosted by the University of Oslo and supported by the “Functional Genomics” and “Infrastructure” programs of the Research Council and the Southeastern Regional Health Authorities of Norway.

Radisic, V. , Grevskott, D. H. , Junghardt, N. , Øvreås, L. , & Marathe, N. P. (2024). Multidrug‐resistant Enterococcus faecium strains enter the Norwegian marine environment through treated sewage. MicrobiologyOpen, 13, e1397. 10.1002/mbo3.1397

DATA AVAILABILITY STATEMENT

All data are provided in full in the results section and supporting information of this paper, except the assembled genome sequences which are available in GenBank under the following accession numbers: JAPMLK000000000, JAPMLJ000000000, JAPRGL000000000, JAPRGK000000000, JAPRGJ000000000, JAPRGI000000000, JAPRGH000000000, JAPRGG000000000, JAPMLI000000000, JAPRGF000000000, JAPRGE000000000, JAPMLH000000000, JAPRGD000000000, JAPRGC000000000, JAPMLG000000000, JAPRGB000000000, JAPRGA000000000, JAPRFZ000000000, JAPRFY000000000, JAPRFX000000000, JAPRFW000000000, JAPRFV000000000, JAPRFU000000000, JAPRFT000000000, and JAPRFS000000000.

REFERENCES

  1. Aarestrup, F. M. , Agerso, Y. , Gerner–Smidt, P. , Madsen, M. , & Jensen, L. B. (2000). Comparison of antimicrobial resistance phenotypes and resistance genes in Enterococcus faecalis and Enterococcus faecium from humans in the community, broilers, and pigs in Denmark. Diagnostic Microbiology and Infectious Disease, 37, 127–137. 10.1016/s0732-8893(00)00130-9 [DOI] [PubMed] [Google Scholar]
  2. Alcock, B. P. , Raphenya, A. R. , Lau, T. T. Y. , Tsang, K. K. , Bouchard, M. , Edalatmand, A. , Huynh, W. , Nguyen, A.‐L. V. , Cheng, A. A. , Liu, S. , Min, S. Y. , Miroshnichenko, A. , Tran, H.‐K. , Werfalli, R. E. , Nasir, J. A. , Oloni, M. , Speicher, D. J. , Florescu, A. , Singh, B. , … McArthur, A. G. (2019). CARD 2020: Antibiotic resistome surveillance with the comprehensive antibiotic resistance database. Nucleic Acids Research, 48, D517–D525. 10.1093/nar/gkz935 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arias, C. A. , & Murray, B. E. (2012). The rise of the Enterococcus: Beyond vancomycin resistance. Nature Reviews Microbiology, 10, 266–278. 10.1038/nrmicro2761 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bankevich, A. , Nurk, S. , Antipov, D. , Gurevich, A. A. , Dvorkin, M. , Kulikov, A. S. , Lesin, V. M. , Nikolenko, S. I. , Pham, S. , Prjibelski, A. D. , Pyshkin, A. V. , Sirotkin, A. V. , Vyahhi, N. , Tesler, G. , Alekseyev, M. A. , & Pevzner, P. A. (2012). SPAdes: A new genome assembly algorithm and its applications to single‐cell sequencing. Journal of Computational Biology, 19, 455–477. 10.1089/cmb.2012.0021 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ben Said, L. , Klibi, N. , Lozano, C. , Dziri, R. , Ben Slama, K. , Boudabous, A. , & Torres, C. (2015). Diversity of enterococcal species and characterization of high‐level aminoglycoside resistant enterococci of samples of wastewater and surface water in Tunisia. Science of the Total Environment, 530‐531, 11–17. 10.1016/j.scitotenv.2015.05.091 [DOI] [PubMed] [Google Scholar]
  6. Boodaghi Malidareh, E. , Ahanjan, M. , Asgharzadeh Marghmalek, S. , & Goli, H. R. (2022). Dissemination of quinupristin‐dalfopristin and linezolid resistance genes among hospital environmental and healthy volunteer fecal isolates of Enterococcus faecalis and Enterococcus faecium . Molecular Biology Reports, 49, 7929–7937. 10.1007/s11033-022-07627-3 [DOI] [PubMed] [Google Scholar]
  7. Bortolaia, V. , Kaas, R. S. , Ruppe, E. , Roberts, M. C. , Schwarz, S. , Cattoir, V. , Philippon, A. , Allesoe, R. L. , Rebelo, A. R. , Florensa, A. F. , Fagelhauer, L. , Chakraborty, T. , Neumann, B. , Werner, G. , Bender, J. K. , Stingl, K. , Nguyen, M. , Coppens, J. , Xavier, B. B. , … Aarestrup, F. M. (2020). ResFinder 4.0 for predictions of phenotypes from genotypes. Journal of Antimicrobial Chemotherapy, 75, 3491–3500. 10.1093/jac/dkaa345 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bourgogne, A. , Hilsenbeck, S. G. , Dunny, G. M. , & Murray, B. E. (2006). Comparison of OG1RF and an isogenic fsrB deletion mutant by transcriptional analysis: The Fsr system of Enterococcus faecalis is more than the activator of gelatinase and serine protease. Journal of Bacteriology, 188, 2875–2884. 10.1128/jb.188.8.2875-2884.2006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Byappanahalli, M. N. , Nevers, M. B. , Korajkic, A. , Staley, Z. R. , & Harwood, V. J. (2012). Enterococci in the environment. Microbiology and Molecular Biology Reviews, 76, 685–706. 10.1128/MMBR.00023-12 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Carattoli, A. , Zankari, E. , García‐Fernández, A. , Voldby Larsen, M. , Lund, O. , Villa, L. , Møller Aarestrup, F. , & Hasman, H. (2014). In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrobial Agents and Chemotherapy, 58, 3895–3903. 10.1128/AAC.02412-14 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cauwerts, K. , Decostere, A. , De Graef, E. M. , Haesebrouck, F. , & Pasmans, F. (2007). High prevalence of tetracycline resistance in Enterococcus isolates from broilers carrying the erm(B) gene. Avian Pathology, 36, 395–399. [DOI] [PubMed] [Google Scholar]
  12. Chajęcka‐Wierzchowska, W. , Zadernowska, A. , & Łaniewska‐Trokenheim, Ł. (2016). Diversity of antibiotic resistance genes in Enterococcus strains isolated from ready‐to‐eat meat products. Journal of Food Science, 81, M2799–M2807. 10.1111/1750-3841.13523 [DOI] [PubMed] [Google Scholar]
  13. Cherak, Z. , Bendjama, E. , Moussi, A. , Benbouza, A. , Grainat, N. , Rolain, J.‐M. , & Loucif, L. (2022). First detection of vanA positive Enterococcus faecium clonal complex 17 in hospital wastewater in Algeria: An epidemiological report. New Microbes and New Infections, 47, 100977. 10.1016/j.nmni.2022.100977 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chun, J. , & Rainey, F. A. (2014). Integrating genomics into the taxonomy and systematics of the bacteria and archaea. International Journal of Systematic and Evolutionary Microbiology, 64, 316–324. 10.1099/ijs.0.054171-0 [DOI] [PubMed] [Google Scholar]
  15. Diaz, L. , Tran, T. T. , Munita, J. M. , Miller, W. R. , Rincon, S. , Carvajal, L. P. , Wollam, A. , Reyes, J. , Panesso, D. , Rojas, N. L. , Shamoo, Y. , Murray, B. E. , Weinstock, G. M. , & Arias, C. A. (2014). Whole‐genome analyses of Enterococcus faecium isolates with diverse daptomycin MICs. Antimicrobial Agents and Chemotherapy, 58, 4527–4534. 10.1128/AAC.02686-14 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. ECDC . (2022). Antimicrobial resistance surveillance in Europe 2022–2020 data. ECDC.
  17. ECDC . (2023). Annual epidemiological report for 2019—Healthcare‐associated infections acquired in intensive care units. ECDC. [Google Scholar]
  18. Engelbert, M. , Mylonakis, E. , Ausubel, F. M. , Calderwood, S. B. , & Gilmore, M. S. (2004). Contribution of gelatinase, serine protease, and fsr to the pathogenesis of Enterococcus faecalis endophthalmitis. Infection and Immunity, 72, 3628–3633. 10.1128/iai.72.6.3628-3633.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. EUCAST . (2023). Breakpoint tables for interpretation of MICs and zone diameters. Version 13.0.
  20. Gao, W. , Howden, B. P. , & Stinear, T. P. (2018). Evolution of virulence in Enterococcus faecium, a hospital‐adapted opportunistic pathogen. Current Opinion in Microbiology, 41, 76–82. 10.1016/j.mib.2017.11.030 [DOI] [PubMed] [Google Scholar]
  21. Golob, M. , Pate, M. , Kušar, D. , Dermota, U. , Avberšek, J. , Papić, B. , & Zdovc, I. (2019). Antimicrobial resistance and virulence genes in Enterococcus faecium and Enterococcus faecalis from humans and retail red meat. BioMed Research International, 2019, 1–12. 10.1155/2019/2815279 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Grevskott, D. H. , Ghavidel, F. Z. , Svanevik, C. S. , & Marathe, N. P. (2021). Resistance profiles and diversity of β‐lactamases in Escherichia coli strains isolated from city‐scale sewage surveillance in Bergen, Norway mimic clinical prevalence. Ecotoxicology and Environmental Safety, 226, 112788. 10.1016/j.ecoenv.2021.112788 [DOI] [PubMed] [Google Scholar]
  23. Hancock, L. E. , & Perego, M. (2004). The Enterococcus faecalis fsr two‐component system controls biofilm development through production of gelatinase. Journal of Bacteriology, 186, 5629–5639. 10.1128/jb.186.17.5629-5639.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hayakawa, K. , Martin, E. T. , Gudur, U. M. , Marchaim, D. , Dalle, D. , Alshabani, K. , Muppavarapu, K. S. , Jaydev, F. , Bathina, P. , Sundaragiri, P. R. , Rajuri, S. D. , Khatri, J. , Pogue, J. M. , Lephart, P. R. , Rybak, M. J. , & Kaye, K. S. (2014). Impact of different antimicrobial therapies on clinical and fiscal outcomes of patients with bacteremia due to vancomycin‐resistant enterococci. Antimicrobial Agents and Chemotherapy, 58, 3968–3975. 10.1128/aac.02943-14 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hendrickx, A. P. A. , van Luit‐Asbroek, M. , Schapendonk, C. M. E. , van Wamel, W. J. B. , Braat, J. C. , Wijnands, L. M. , Bonten, M. J. M. , & Willems, R. J. L. (2009). SgrA, a nidogen‐binding LPXTG surface adhesin implicated in biofilm formation, and EcbA, a collagen‐binding MSCRAMM, are two novel adhesins of hospital‐acquired Enterococcus faecium . Infection and Immunity, 77, 5097–5106. 10.1128/iai.00275-09 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hufnagel, M. , Hancock, L. E. , Koch, S. , Theilacker, C. , Gilmore, M. S. , & Huebner, J. (2004). Serological and genetic diversity of capsular polysaccharides in Enterococcus faecalis . Journal of Clinical Microbiology, 42, 2548–2557. 10.1128/jcm.42.6.2548-2557.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hummel, A. , Holzapfel, W. H. , & Franz, C. M. A. P. (2007). Characterisation and transfer of antibiotic resistance genes from enterococci isolated from food. Systematic and Applied Microbiology, 30, 1–7. 10.1016/j.syapm.2006.02.004 [DOI] [PubMed] [Google Scholar]
  28. Iversen, A. , Kühn, I. , Franklin, A. , & Möllby, R. (2002). High prevalence of vancomycin‐resistant enterococci in Swedish sewage. Applied and Environmental Microbiology, 68, 2838–2842. 10.1128/AEM.68.6.2838-2842.2002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. de Jong, A. , Simjee, S. , Rose, M. , Moyaert, H. , El Garch, F. , Youala, M. , Marion, O. , Lin, D. , Filip, B. , Mireille, B. , Bénédicte, C. , Jeroen, D. , Sophie, G. , Szilárd, J. , Isabelle, K. , Lourdes, M. G. , Mogens, M. , Caroline, P. , Ellen, P. B. , … Thais, V. (2019). Antimicrobial resistance monitoring in commensal enterococci from healthy cattle, pigs and chickens across Europe during 2004–14 (EASSA Study). Journal of Antimicrobial Chemotherapy, 74, 921–930. 10.1093/jac/dky537 [DOI] [PubMed] [Google Scholar]
  30. Jureen, R. , Top, J. , Mohn, S. C. , Harthug, S. , Langeland, N. , & Willems, R. J. L. (2003). Molecular characterization of ampicillin‐resistant Enterococcus faecium isolates from hospitalized patients in Norway. Journal of Clinical Microbiology, 41, 2330–2336. 10.1128/JCM.41.6.2330-2336.2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kayaoglu, G. , & Ørstavik, D. (2004). Virulence factors of Enterococcus faecalis: Relationship to endodontic disease. Critical Reviews in Oral Biology and Medicine, 15, 308–320. 10.1177/154411130401500506 [DOI] [PubMed] [Google Scholar]
  32. Klare, I. , Fleige, C. , Geringer, U. , Thürmer, A. , Bender, J. , Mutters, N. T. , Mischnik, A. , & Werner, G. (2015). Increased frequency of linezolid resistance among clinical Enterococcus faecium isolates from German hospital patients. Journal of Global Antimicrobial Resistance, 3, 128–131. 10.1016/j.jgar.2015.02.007 [DOI] [PubMed] [Google Scholar]
  33. Leavis, H. L. , Bonten, M. J. , & Willems, R. J. (2006). Identification of high‐risk enterococcal clonal complexes: Global dispersion and antibiotic resistance. Current Opinion in Microbiology, 9, 454–460. 10.1016/j.mib.2006.07.001 [DOI] [PubMed] [Google Scholar]
  34. Leavis, H. L. , Willems, R. J. L. , Top, J. , & Bonten, M. J. M. (2006). High‐level ciprofloxacin resistance from point mutations in gyrA and parC confined to global hospital‐adapted clonal lineage CC17 of Enterococcus faecium . Journal of Clinical Microbiology, 44, 1059–1064. 10.1128/jcm.44.3.1059-1064.2006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Leclercq, R. , Oberlé, K. , Galopin, S. , Cattoir, V. , Budzinski, H. , & Petit, F. (2013). Changes in enterococcal populations and related antibiotic resistance along a medical center‐wastewater treatment plant‐river continuum. Applied and Environmental Microbiology, 79, 2428–2434. 10.1128/AEM.03586-12 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lee, T. , Pang, S. , Abraham, S. , & Coombs, G. W. (2019). Antimicrobial‐resistant CC17 Enterococcus faecium: The past, the present, and the future. Journal of global antimicrobial resistance, 16, 36–47. 10.1016/j.jgar.2018.08.016 [DOI] [PubMed] [Google Scholar]
  37. Liassine, N. , Frei, R. , Jan, I. , & Auckenthaler, R. (1998). Characterization of glycopeptide‐resistant enterococci from a Swiss hospital. Journal of Clinical Microbiology, 36, 1853–1858. 10.1128/jcm.36.7.1853-1858.1998 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Liu, B. , Zheng, D. , Jin, Q. , Chen, L. , & Yang, J. (2019). VFDB 2019: A comparative pathogenomic platform with an interactive web interface. Nucleic Acids Research, 47, D687–D692. 10.1093/nar/gky1080 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Mbanga, J. , Amoako, D. G. , Abia, A. L. K. , Allam, M. , Ismail, A. , & Essack, S. Y. (2021). Genomic analysis of Enterococcus spp. isolated from a wastewater treatment plant and its associated waters in Umgungundlovu district, South Africa. Frontiers in Microbiology, 12, 648454. 10.3389/fmicb.2021.648454 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Miller, W. R. , Munita, J. M. , & Arias, C. A. (2014). Mechanisms of antibiotic resistance in enterococci. Expert Review of Anti‐Infective Therapy, 12, 1221–1236. 10.1586/14787210.2014.956092 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Murray, B. E. (1990). The life and times of the Enterococcus . Clinical Microbiology Reviews, 3, 46–65. 10.1128/CMR.3.1.46 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. NORM/NORM‐VET . (2023). Usage of antimicrobial agents and occurrence of Antimicrobial Resistance in Norway (pp. 1–180). Tromsø/Oslo. ISSN:1502‐2307 (print)/1890‐9965 (electronic).
  43. Oxaran, V. , Ledue‐Clier, F. , Dieye, Y. , Herry, J.‐M. , Péchoux, C. , Meylheuc, T. , Briandet, R. , Juillard, V. , & Piard, J.‐C. (2012). Pilus biogenesis in Lactococcus lactis: Molecular characterization and role in aggregation and biofilm formation. PLoS One, 7, e50989. 10.1371/journal.pone.0050989 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Qin, X. , Galloway‐Peña, J. R. , Sillanpaa, J. , Roh, J. H. , Nallapareddy, S. R. , Chowdhury, S. , Bourgogne, A. , Choudhury, T. , Muzny, D. M. , Buhay, C. J. , Ding, Y. , Dugan‐Rocha, S. , Liu, W. , Kovar, C. , Sodergren, E. , Highlander, S. , Petrosino, J. F. , Worley, K. C. , Gibbs, R. A. , … Murray, B. E. (2012). Complete genome sequence of Enterococcus faecium strain TX16 and comparative genomic analysis of Enterococcus faecium genomes. BMC Microbiology, 12, 135. 10.1186/1471-2180-12-135 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Quiñones, D. , Aung, M. S. , Sousa Martins, J. P. , Urushibara, N. , & Kobayashi, N. (2018). Genetic characteristics of VanA‐type vancomycin‐resistant Enterococcus faecalis and Enterococcus faecium in Cuba. New Microbes and New Infections, 21, 125–127. 10.1016/j.nmni.2017.12.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Radisic, V. , Grevskott, D. H. , Lunestad, B. T. , Øvreås, L. , & Marathe, N. P. (2023). Sewage‐based surveillance shows presence of Klebsiella pneumoniae resistant against last resort antibiotics in the population in Bergen, Norway. International Journal of Hygiene and Environmental Health, 248, 114075. 10.1016/j.ijheh.2022.114075 [DOI] [PubMed] [Google Scholar]
  47. Radisic, V. , Nimje, P. S. , Bienfait, A. M. , & Marathe, N. P. (2020). Marine plastics from Norwegian west coast carry potentially virulent fish pathogens and opportunistic human pathogens harboring new variants of antibiotic resistance genes. Microorganisms, 8, 1200. 10.3390/microorganisms8081200 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Rahimi, F. , Talebi, M. , Seyfi, M. , & Pourshafiei, M. (2007). Distribution of enterococcal species and detection of vancomycin resistance genes by multiplex PCR in Tehran sewage. Iranian Biomedical Journal, 11(3), 161–167. [PubMed] [Google Scholar]
  49. Rathnayake, I. U. , Hargreaves, M. , & Huygens, F. (2012). Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates. Systematic and Applied Microbiology, 35, 326–333. 10.1016/j.syapm.2012.05.004 [DOI] [PubMed] [Google Scholar]
  50. Roberts, M. C. (2008). Update on macrolide–lincosamide–streptogramin, ketolide, and oxazolidinone resistance genes. FEMS Microbiology Letters, 282, 147–159. 10.1111/j.1574-6968.2008.01145.x [DOI] [PubMed] [Google Scholar]
  51. Santajit, S. , & Indrawattana, N. (2016). Mechanisms of antimicrobial resistance in ESKAPE pathogens. BioMed Research International, 2016, 1–8. 10.1155/2016/2475067 [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sifaoui, F. , Arthur, M. , Rice, L. , & Gutmann, L. (2001). Role of penicillin‐binding protein 5 in expression of ampicillin resistance and peptidoglycan structure inenterococcus faecium. Antimicrobial Agents and Chemotherapy, 45, 2594–2597. 10.1128/AAC.45.9.2594-2597.2001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Simonsen, G. S. (2003). Prevalence of resistance to ampicillin, gentamicin and vancomycin in Enterococcus faecalis and Enterococcus faecium isolates from clinical specimens and use of antimicrobials in five Nordic hospitals. Journal of Antimicrobial Chemotherapy, 51, 323–331. 10.1093/jac/dkg052 [DOI] [PubMed] [Google Scholar]
  54. Stedman, A. , van Vliet, A. H. M. , A. Chambers, M. , & Gutierrez‐Merino, J. (2020). Gut commensal bacteria show beneficial properties as wildlife probiotics. Annals of the New York Academy of Sciences, 1467, 112–132. 10.1111/nyas.14302 [DOI] [PubMed] [Google Scholar]
  55. Tatusova, T. , DiCuccio, M. , Badretdin, A. , Chetvernin, V. , Ciufo, S. , & Li, W. (2013). Prokaryotic genome annotation pipeline. NCBI. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Top, J. , Willems, R. , & Bonten, M. (2008). Emergence of CC17 Enterococcus faecium: From commensal to hospital‐adapted pathogen. FEMS Immunology and Medical Microbiology, 52, 297–308. 10.1111/j.1574-695X.2008.00383.x [DOI] [PubMed] [Google Scholar]
  57. Vignaroli, C. , Luna, G. M. , Pasquaroli, S. , Di Cesare, A. , Petruzzella, R. , Paroncini, P. , & Biavasco, F. (2013). Epidemic Escherichia coli ST131 and Enterococcus faecium ST17 in coastal marine sediments from an Italian beach. Environmental Science & Technology, 47, 13772–13780. 10.1021/es4019139 [DOI] [PubMed] [Google Scholar]
  58. Viswanath, L. S. , Sugumar, M. , Chandra Murthy Peela, S. , Walia, K. , & Sistla, S. (2022). Detection of vancomycin variable enterococci (VVE) among clinical isolates of Enterococcus faecium collected across India‐first report from the subcontinent. Indian Journal of Medical Microbiology, 40, 285–288. 10.1016/j.ijmmb.2021.12.011 [DOI] [PubMed] [Google Scholar]
  59. Wang, S. , Guo, Y. , Lv, J. , Qi, X. , Li, D. , Chen, Z. , Zhang, X. , Wang, L. , & Yu, F. (2016). Characteristic of Enterococcus faecium clinical isolates with quinupristin/dalfopristin resistance in China. BMC Microbiology, 16, 246. 10.1186/s12866-016-0863-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Werner, G. , Fleige, C. , Ewert, B. , Laverde‐Gomez, J. A. , Klare, I. , & Witte, W. (2010). High‐level ciprofloxacin resistance among hospital‐adapted Enterococcus faecium (CC17). International Journal of Antimicrobial Agents, 35, 119–125. 10.1016/j.ijantimicag.2009.10.012 [DOI] [PubMed] [Google Scholar]
  61. Werner, G. , Fleige, C. , Feßler, A. T. , Timke, M. , Kostrzewa, M. , Zischka, M. , Peters, T. , Kaspar, H. , & Schwarz, S. (2012). Improved identification including MALDI‐TOF mass spectrometry analysis of group D streptococci from bovine mastitis and subsequent molecular characterization of corresponding Enterococcus faecalis and Enterococcus faecium isolates. Veterinary Microbiology, 160, 162–169. 10.1016/j.vetmic.2012.05.019 [DOI] [PubMed] [Google Scholar]
  62. WHO . (2023). WHO publishes list of bacteria for which new antibiotics are urgently needed. https://www.who.int/news/item/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed
  63. Zaheer, R. , Cook, S. R. , Barbieri, R. , Goji, N. , Cameron, A. , Petkau, A. , Polo, R. O. , Tymensen, L. , Stamm, C. , Song, J. , Hannon, S. , Jones, T. , Church, D. , Booker, C. W. , Amoako, K. , Van Domselaar, G. , Read, R. R. , & McAllister, T. A. (2020). Surveillance of Enterococcus spp. reveals distinct species and antimicrobial resistance diversity across a one‐health continuum. Scientific Reports, 10, 3937. 10.1038/s41598-020-61002-5 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting information.

MBO3-13-e1397-s001.xlsx (9.4KB, xlsx)

Supporting information.

MBO3-13-e1397-s004.xlsx (49.8KB, xlsx)

Supporting information.

MBO3-13-e1397-s003.xlsx (19KB, xlsx)

Supporting information.

MBO3-13-e1397-s002.xlsx (12.2KB, xlsx)

Supporting information.

MBO3-13-e1397-s005.xlsx (22.4KB, xlsx)

Data Availability Statement

All data are provided in full in the results section and supporting information of this paper, except the assembled genome sequences which are available in GenBank under the following accession numbers: JAPMLK000000000, JAPMLJ000000000, JAPRGL000000000, JAPRGK000000000, JAPRGJ000000000, JAPRGI000000000, JAPRGH000000000, JAPRGG000000000, JAPMLI000000000, JAPRGF000000000, JAPRGE000000000, JAPMLH000000000, JAPRGD000000000, JAPRGC000000000, JAPMLG000000000, JAPRGB000000000, JAPRGA000000000, JAPRFZ000000000, JAPRFY000000000, JAPRFX000000000, JAPRFW000000000, JAPRFV000000000, JAPRFU000000000, JAPRFT000000000, and JAPRFS000000000.

Articles from MicrobiologyOpen are provided here courtesy of Wiley

RESOURCES