Background
Over the past decades, enterococci have emerged as the most prevalent nosocomial pathogens. Once viewed as part of the normal gut flora with little clinical significance, and they are now recognized as the cause of several types of community- and hospital-acquired infections, including life-threating bloodstream infections, endocarditis, meningitis, and urinary tract infections [
1]. Disease outbreaks from antimicrobial-resistant enterococci have occurred in the United States and Europe [
2,
3], and the European Centre for Disease Prevention and Control (ECDC) has reported that enterococci are the most commonly isolated bacteria after
Escherichia coli and
Staphylococcus aureus from healthcare-associated infections in Europe [
4]. Furthermore, nosocomial infections caused by vancomycin-resistant enterococci (VRE) represent a serious clinical problem in healthcare facilities in many countries worldwide [
5,
6].
Enterococcus faecalis is the most common enterococcal species associated with nosocomial infections, accounting for 80–90% of the infections, followed by
Enterococcus faecium (5–10%) [
7]. Other species of enterococci rarely cause infection [
2]. Genotyping of
E. faecalis and
E. faecium by multilocus sequence typing (MLST) and other methods revealed that distinct clones of these species are associated with hospital infections/outbreaks; these clones are referred to as high-risk enterococcal clonal complexes [
8,
9]. In recent years, these species have shown increasing resistance to several antibiotics, including penicillin, aminoglycosides, and glycopeptides, which thus limits antimicrobial therapeutic options [
10]. Glycopeptide resistance may be due to the acquisition of
van genes, whereas
vanC1 and
vanC2/3 are responsible for intrinsic resistance in enterococci [
11]. Vancomycin resistance that mainly arises from the
vanA gene cluster is commonly identified on the mobile genetic element Tn1546 [
12]. The mobile genetic elements from enterococci have also recently been shown to be able to transfer vancomycin resistance to more pathogenic bacteria such as
S. aureus [
13]. Monitoring the antimicrobial resistance (AMR) in enterococci from clinical specimens is essential for controlling the spread of resistance genes against vancomycin and other antibiotics.
Available literature has highlighted an increasing prevalence of multi-drug resistant (MDR)
E. faecalis in the eastern region of Saudi Arabia [
14]. However, no molecular characterization of MDR
E. faecalis isolates from Saudi Arabia, and neighboring countries has been performed. An active molecular epidemiology program is critical for creating basic knowledge about local microorganisms and their resistance to refine policies on controlling infections from antimicrobial-resistant bacteria in hospitals and other healthcare facilities within the country. The main objective of this study was to perform antibiotic susceptibility, and genomic analysis of clinical
E. faecalis isolates from the western part of the country. The isolates were evaluated for the presence of virulence and antimicrobial resistance genes (ARGs). MLST analysis was performed to track the global distribution of the
E. faecalis sequence types (STs) identified in this study.
Discussion
Consistent with the international trend, the prevalence of enterococcal infections is increasing in Saudi Arabia, and
E. faecalis is one of the most common enterococcal species isolated from hospital-associated infections. The ECDC has estimated that enterococci are responsible for 8% of the healthcare-associated infections on average in Europe, and the agency has placed them in the category of pathogens posing a major threat to healthcare systems [
4]. Enterococcal species are intrinsically resistant to a broad range of antibiotics, such as cephalosporins and sulfonamides [
28]. They represent a major infection control challenge because of their ability to acquire additional resistance through the transfer of plasmids and transposons and because they can disseminate easily in the hospital environment.
In this study, linezolid, tigecycline, and vancomycin demonstrated > 95% activity against
E. faecalis isolates in vitro. The antibiotics quinupristin/dalfopristin, clindamycin, and erythromycin demonstrated almost no coverage, and other antibiotics such as high-dose streptomycin, gentamycin, and ciprofloxacin demonstrated suboptimal coverage. Previous studies from Saudi Arabia revealed 21–25% resistance to high-dose gentamycin and 11–13% resistance to high-dose streptomycin [
29,
30]. The high level of aminoglycoside resistance observed in this study is highly concerning, given that aminoglycosides are used in combination with other active molecules, mainly β-lactams, to treat enterococcal infections such as enterococcal endocarditis [
28]. In particular, gentamycin is used as a synergistic antibiotic with ampicillin [
31], and amoxicillin is the first choice of treatment for
E. faecalis causing UTIs [
32]. In contrast to a study from the eastern region of Saudi Arabia, comparatively lower resistance to ampicillin and higher resistance to erythromycin, gentamycin, streptomycin, and tetracycline were observed in this study, suggesting the diverse geographical distribution of MDR
E. faecalis isolates in Saudi Arabia [
30,
33].
The rate of vancomycin-resistant
E. faecalis in this study was slightly higher than that found in a previous study from Saudi Arabia that was conducted at King Khalid Hospital in Riyadh. That study identified the vancomycin-resistant phenotype in 0–1.8% of isolates [
30]. The resistance rates reported by the National Healthcare Safety Network during 2009–2010 was between 6.2% and 9.8% for
E. faecalis, depending on the site of infection [
34]. Despite the increasing number of reports of VRE in different geographical regions of the world, there is a distinct lack of data regarding the molecular characterization of VRE isolates originating from the Middle East region. No genotypic characterization of vancomycin-resistance genes and other acquired AMR genes in
E. faecalis has been described in the available literature from Saudi Arabia. In this study, acquired
vanA and
vanB genes were identified in the vancomycin-resistant
E. faecalis isolates from patients in the western region of Saudi Arabia. No intrinsic resistance genes (
vanC1/C2) were detected in the tested isolates. Similar results have been reported in Europe, where a mix of VRE carrying
vanA and
vanB were found [
6].
Enterococcus faecalis isolates tested in this study also possessed
gyrA, and
parC, genes conferring resistance to quinolone groups of antibiotics including ciprofloxacin, levofloxacin, and moxifloxacin that are commonly prescribed for UTIs, enteric infections, and respiratory tract infections.
Consistent with previous studies, the most common aminoglycoside-modifying enzyme genes (
aac(6′)-Ie-
aph(2″)-Ia and
ant(6′)-Ia) were found in the
E. faecalis isolates that were resistant to high-dose gentamycin and streptomycin [
35]. Two mechanisms are responsible for the cross resistance to macrolide-lincosamide-streptogramin A in
E. faecalis, including an intrinsic
lsa gene and a change in the target site of erythromycin that is mediated by the
erm(B) gene [
36,
37]. In this study, we found the
lsa gene in all genome sequence isolates, and the
erm(B) gene was found in 68.1% isolates, similar to previous studies from the United States, China, and Korea [
36,
38,
39]. The results obtained in this study are consistent with a previous report indicating that 98% of the
E. faecalis isolates possess
emeA gene and ATP-binding cassettes (ABC), which are
efrA and
efrB [
40].
The ability of enterococci to form biofilm contributes to the pathogenicity of the bacteria in nosocomial infections because mature biofilms of
E. faecalis can withstand antimicrobial agents up to 100- to 1000-fold concentrations. In contrast to previous studies, a large group of virulence factors was found in the genome sequences of
E. faecalis isolates, as seen in other Gram-positive cocci, such as β-hemolytic
Streptococcus and
S. aureus. In this study, 22 virulence genes were retrieved from the
E. faecalis isolates, including the genes associated with biofilm formation as previously mentioned. The presence of virulence determinants in enterococcal isolates assist them in acquiring adaptive elements that provide them with evolutionary benefits for relative fitness in hospital settings [
41,
42]. In comparative studies,
E. faecalis is considered to be inherently more virulent and to have greater capability to acquire virulence factors than
E. faecium strains [
1]. Overall, variation was observed in the relative distribution of virulence factors in the enterococcal species from different geographical regions [
43,
44]. Findings from the current study are consistent with a previous report that
esp and
cylA genes were mainly found in CC16 isolates. Importantly, the cCF10 gene, which activates the conjugation of pCF10 plasmid, was found in this study. This plasmid plays an important role in the dissemination of virulence factors and resistance genes among enterococci [
45].
A noticeable diversity of strains was observed in the
E. faecalis isolates from Saudi Arabia, and 17 distinct STs, including two novel STs, from the genome sequences of 44 isolates were identified. Major clusters from a phylogenetic tree based on SNPs were assigned to ST179 and ST16 from CC16. Generally, STs from CC16 are considered to be well acclimated to hospital environments. They have previously been reported to cause human infections, acquire exogenous genes via recombination, and be able to carry
vanA or
vanB genes, conjugative plasmids, and transposons involved in the genetic transfer of resistance and virulence in hospital-derived isolates [
46‐
48]. All 18 isolates belonging to CC16 from various specimen types in this study presented MDR phenotypes and genotypes. In addition, they carried virulence genes, most commonly
gelE and
asa1, which accords with a previous study [
49]. In contrast to previous reports, CC2 isolates were vancomycin sensitive, which supports the hypothesis that this clone was originally vancomycin susceptible and later subsets acquired the vancomycin-resistance gene. In addition, several STs such as ST21, ST480, and ST40 detected in this study were previously reported in China, Tunisia, France, and Spain from human subjects, hospitalized patients, and wastewater, and shared the same characteristics in terms of high-dose antibiotic-resistant phenotypes, resistance genotypes, and virulence genes [
50‐
52]. The lack of information on the population structure of
E. faecalis in neighboring countries makes it difficult to speculate about the regional spread of these novel STs and other STs detected in Saudi Arabia.