Background

Enterococci are commensal organisms responsible for hospital-acquired infections in immunosuppressed patients [1]. Sources of infection are diverse, whereas these organisms may be transferred from environmental sources to animals and humans [2]. Enterococcus faecalis and Enterococcus faecium are predominant Gram-positive cocci in human clinical samples [1, 3]. Both organisms can be virulent to humans, but E. faecalis is more prevalent than E. faecium [4]. They are able to acquire new antibiotic resistance genes through a variety of mechanisms, which complicates the treatment of infections caused by these organisms [3]. However, E. faecalis and E. faecium are naturally resistant to clindamycin, trimethoprim-sulfamethoxazole, and gentamicin (low-level resistance) [5]. Furthermore, they can withstand all the antibiotics used to treat human infections [6]. However, a major concern is the emergence of vancomycin and teicoplanin-resistant organisms [6, 7]. Moreover, biofilm formation is recognized as a key factor in the development of enterococcal infections [8]. Biofilm can tolerate antimicrobial concentrations 100–1000 times greater than those needed to kill planktonic cells [9]. Biofilm-associated infections are difficult to treat because bacteria living in biofilms are resistant to antibiotics, environmental stress, and phagocytosis [10]. Microorganism adhesion to host cell surfaces is critical for the pathogenesis of infections and biofilm formation [8]. The most important virulence factors in Enterococci include the collagen-binding protein (ace), E. faecalis endocarditis specific antigen (efaA), and enterococcal surface protein (esp) [11, 12]. Ace, EfaA, and Esp are adhesion proteins that have an important role in adhesion to eukaryotic cells and surfaces along with the colonization of host tissues [12, 13]. For these reasons, this study aimed to evaluate the antibiotic susceptibility pattern, in vitro biofilm formation ability, and the prevalence of virulence genes (esp, ace, and efaA) among fecal normal-flora and environmental isolates of E. faecalis and E. faecium.

Methods

Sample collection

In this cross-sectional study, clinical and environmental samples were collected from hospital environments, healthy volunteers, and health staff of 4 educational hospitals affiliated with Mazandaran University of Medical Sciences, Sari, Iran, from October 2018 to August 2019. Participants had not taken any antibiotics for at least three weeks before sampling. The sample size was calculated according to the following formula: where n is the sample size, \({z}_{1-\frac{a}{2}}\) is the Z statistic for confidence level at 95%, p is the estimated prevalence of E. faecalis and E. faecium infections, and \({\varepsilon }^{2}\) is the precision [14].

$$n=\frac{{(z_{1-{\displaystyle\frac a2}})}^2\left[P\left(1-P\right)\right]}{\varepsilon^2}$$

Isolation and identification of E. faecalis and E. faecium

This study strictly adhered to the principles outlined in the Declaration of Helsinki, ensuring ethical conduct throughout the research process. Approval for the study was obtained from the Iran National Committee for Ethics in Biomedical Research, with the national ethical code (consent ref number) IR.MAZUMS.REC.1398.416. Additionally, informed consent was ethically obtained from all study participants or their guardians, emphasizing our commitment to ethical standards and participant welfare. This study was approved by Biosafety committee of Mazandaran University of Medical Sciences (#1397.3490). A total of 145 clinical (stool samples, n = 100) and environmental samples (n = 45) were cultivated from four hospitals in Sari, North Iran. The samples were cultured on Slanetz and Bartley (M-Enterococcus) agar (Sigma, Germany) and blood agar (Merck, Germany) at 37°C for 24 h to isolation Enterococcus strains. Enterococcal species identification was done by using conventional tests (morphology of colonies, Gram staining, growth and blacken of bile-esculin agar, growth at 6.5% NaCl, 0.04% tellurite reduction, catalase test, Pyrrolidonyl arylamidase (PYR) test, arginine dehydrolase activity, motility, and some carbohydrate fermentation tests, especially arabinose [15]. The E. faecalis and E. faecium strains were confirmed by polymerase chain reaction (PCR) assay using species-specific primers for the ddl (D-alanine-D-alanine ligase) encoding genes.

Antibiotic susceptibility testing

Susceptibility testing was performed using the standard Kirby Bauer disk agar diffusion method in accordance with Clinical and Laboratory Standards Institute (CLSI, 2020) guidelines. Antimicrobial agents (HiMedia, India) in this study were ampicillin (10μg), vancomycin (30μg), teicoplanin (30μg), erythromycin (15μg), tetracycline (30μg), ciprofloxacin (5μg), levofloxacin (5μg), nitrofurantoin (300μg), chloramphenicol (30μg), linezolid (300μg), gentamicin (120μg), streptomycin (300μg), and quinupristin-dalfopristin (15μg) [16]. The results of the test were interpreted according to the CLSI; M100 criteria E. faecalis ATCC 29212 was used as a control strain in the disk agar diffusion test.

Biofilm formation capacity

Enterococcus isolates were tested for their ability to produce biofilms using a quantitative microplate assay [17]. Briefly, a 0.5 McFarland suspension of the overnight cultures of Enterococcus strains was prepared. To each well of 96-well micro titer plates, 180 μl of Trypticase Soy Broth (TSB; Merck, Germany) + 0.5% glucose was added along with 20 μl of 0.5 McFarland suspension of the isolates, and then incubated at 37 ˚C and 5% CO2 for 24 h. Next, the medium was discarded, and micro titer plates were gently washed three times with 300μl of sterile phosphate buffer saline (PBS) (Merck, Germany) to remove planktonic cells. Then, 150μl of 99% methanol was added to each well for 20 min to fix the biofilm biomass. Later, the methanol was removed, and the plates were left to dry in room temperature and then, 100μl of 2% crystal violet was added to each well for 20 min. Excess stains were removed from the plates using sterile distilled water and the plates were located at room temperature for 30 min. The dye bounded to the adherent cells was dissolved using 150 µL of 33% (v/v) glacial acetic acid for each well. The optical density (OD) was measured using an ELISA reader (Bio-Rad, USA) at a wavelength of 595 nm. Uninoculated TSB medium + 0.5% glucose was used as a negative control. The ability to form biofilm in these isolates was categorized based on the OD values of the strains compared to the OD cutoff (ODC) value of the control strain (E. faecalis ATCC 29212) into 4 separate groups: non-biofilm-formers (OD ≤ ODC), weak (ODC < OD ≤ 2 × ODC), medium (2 × ODC < OD ≤ 4 × ODC) and strong biofilm formers (4 × ODC < OD) [18].

Polymerase chain reaction

DNAs were extracted by the alkaline lysis method following the standard protocols [19]. The distribution of esp, ace, and efaA genes were investigated in all Enterococcus isolates by PCR assay. The primer sequences used in this work are listed in Additional file 1 [20,21,22]. The PCR reactions contained 7.5 μl of master mix (Ampliqon, Denmark) and 0.5 μl of each primer for all genes, 100 ng of the extracted DNA for esp and ddl genes, and 200 ng DNA for ace and efaA genes. The PCR condition was as follows: an initial denaturation step at 95°C for 5 min followed by 34 cycles of denaturation at 95°C for 30 s, annealing at 54°C for E. faecalis ddl (45 s), 56°C for E. faecium ddl (45 s), 65°C for esp (45 s), 65°C for efaA and ace (30 s), and an extension at 72°C for esp and ddl genes (90 s) and for ace and efaA genes (60 s), with a final extension step at 72°C for 10 min (BioRad, USA). The PCR products were electrophoresed on a 1% (w/v) agarose gel (Wizbiosolutions, South Korea). Then, a UV trans-illuminator (UVITEC Gel documentation System, Cambridge, UK) was used for the documentation of the PCR products.

Statistical analysis

Statistical analysis of results was performed with SPSS version 22 software (SPSS Chicago, IL). The Chi-square (χ2) and Fisher’s exact test were used for statistical analysis. A P value < 0.05 was used for statistical significance.

Results

Bacterial isolation

Out of 145 samples, 84 (57.9%) E. faecalis and 61 (42.1%) E. faecium were isolated. The majority of E. faecalis strains (36/84, 42.8%) were isolated from hospital staff, while the majority of E. faecium strains (24/61, 39.3%) were isolated from hospital environments (Fig. 1).

Fig. 1
figure 1

Frequency of the enterococci isolates collected from different sources

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Determination of antimicrobial susceptibility

The susceptibility profiles of tested strains are shown in Fig. 2A, B. Resistance to kanamycin (85.7%; 72/84) and quinupristin-dalfopristin (82.1%; 69/84) was high in E. faecalis isolates and a high prevalence of kanamycin resistance (62.3%; 38/61) was observed in the E. faecium isolates (Fig. 2). The antibiotic inhibition zones diameter (mm) of Enterococci isolated from hospital staffs, healthy volunteers and hospital environments are shown in Additional file 2 (Tables S2-S4).

Fig. 2
figure 2

Antibiotic susceptibility of Enterococcus strains A shows the antibiotic susceptibility of E. faecalis and B shows the antibiotic susceptibility of E. faecium

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Distribution of virulence genes

All the virulence genes were screened among the Enterococcal isolates based on the occurrence of expected amplicon sizes (Figs. 3, 4 and 5). The results of PCR showed that among the E. faecalis isolates, 62 (73.8%) harbored the esp gene, 26 (30.95%) isolates had the esp gene, and 51 (60.71%) isolates carried the efaA gene. Among the E. faecium samples, 35 (57.37%), 3 (4.91%) and 12 (19.67%) were positive for esp, ace, and efaA genes, respectively (Table 1).

Fig. 3
figure 3

Lane M, 100–3 kb DNA size marker; Lane P, positive control; Lane N, negative control; Lane 1—12, esp gene positive/negative strains

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Fig. 4
figure 4

Lane M, 100–3 kb DNA size marker; Lane P, positive control; Lane N, negative control; Lane 1—12, efa gene positive/negative strains

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Fig. 5
figure 5

Lane M, 100–3 kb DNA size marker; Lane P, positive control; Lane N, negative control; Lane 1—12, ace gene positive/negative strains

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Table 1 Frequency of virulence genes among Enterococcus strains
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Results of biofilm formation in Enterococcus strains

Based on the quantitative microplate method for biofilm formation, out of 145 enterococci strains from different sources, 99 isolates could form biofilms (Table 2). Also, there are statistically significant differences between the distributions of the esp gene in healthy volunteers and environmental samples of E. faecalis and environmental samples of E. faecium (p < 0.05). There was a statistically significant difference in the distribution of the efaA gene only between samples taken from healthy volunteers and environmental sources that contained E. faecalis (p < 0.05) (Table 3). The distribution of the esp gene among the moderate and strong phenotypes, as well as the distribution of the efaA gene among the moderate phenotype and ace gene in negative phenotype of E. faecalis, were statistically significant (p < 0.05), unlike other cases (Table 2). The results of the PCR assay indicated that there are statistically significant differences between the distributions of the esp gene in healthy volunteers and environmental samples of E. faecalis and environmental samples of E. faecium (p < 0.05). The distribution of the efaA gene showed a statistically significant difference only in samples from healthy volunteers and environmental samples of E. faecalis (p < 0.05) (Table 3). The results showed a statistically significant relationship between the presence of esp virulence gene and the ability of biofilm formation among E. faecalis isolates (p = 0.04). The distribution of the esp gene among the moderate and strong phenotypes, as well as the distribution of the efaA gene among the moderate phenotype and ace gene in negative phenotype of E. faecalis, were statistically significant (p < 0.05) (Table 4). Also correlation between antibiotic resistance pattern of the Enterococcal isolates and biofilm formation ability were assessed. The statistical analysis indicated a significant correlation between the Enterococcus species that form biofilms and resistance to certain antibiotics, including quinupristin/dalfopristin, streptomycin, and chloramphenicol (Table 5).

Table 2 Frequency of biofilm phenotypes in Enterococcus based on the source of samples
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Table 3 Frequency of virulence genes among Enterococcus strains based on the source of samples
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Table 4 Frequency of biofilm phenotypes in Enterococcus isolates based on the distribution of virulence genes
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Table 5 Correlation between antibiotic resistance pattern of the Enterococcal isolates and biofilm formation ability
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Discussion

A number of severe and life-threatening diseases can be caused by enterococci [23]. E. faecalis and E. faecium are the most commonly detected species of enterococci in human clinical samples [24]. Among the 145 Enterococcus isolates in this study, 57.9% were E. faecalis and 42.1% were E. faecium. In several other studies, E. faecalis was the predominant strain. The incidence of E. faecalis as a predominani enterococci strains has been reported to vary from 70% (in Tehran, Iran), 69% (in Zanjan, Iran), and 41.99% (in China) [25,26,27]. The difference in the prevalence of E. faecalis and E. faecium can be due to differences in the type of samples, methods of detection, or geographical location. Enterococci are innately resistant to antibiotics, but can acquire resistance genes and new mutations from other bacteria as well [28]. Several studies in Iran have reported high rates of antibiotic resistance among Enterococcus strains [29, 30]. A high level of kanamycin resistance was detected in 85.7% and 62.3% of E. faecium and E. faecalis isolates, respectively. Although intrinsic resistance mechanisms may result in low levels of aminoglycoside resistance, acquiring mobile genetic elements usually leads to high levels of aminoglycoside resistance in these isolates [31]. Additionally, ampicillin resistance in E. faecium isolates of the present study was considerable, similar to a previous study in Kenya [32]. On the other hand, several virulence genes (efaA, asa1, ebpA, esp, and ace) have been identified as effective genes for biofilm formation in Enterococci [33]. In our study, the prevalence of ace, esp, and efaA genes among E. faecalis isolates, were 74%, 31%, and 31.1%, respectively, while 57%, 5%, and 31.1% of E. faecium isolates contained these genes, respectively. A number of virulence genes were found in our study to be consistent with those found in previous studies conducted on food, animal, and medical isolates [22, 34, 35]. Among these two common enterococci species, the prevalence of the esp gene varies from one country to the next [12, 36]. However, enterococcal surface protein (Esp) is one of the most important factors in colonization and persistence of E. faecalis in human urinary tract infections and its biofilm formation [12, 37]. The esp gene has been detected in clinical and environmental samples in the past [22, 39], but they are more commonly adopted in clinical isolates [39]. There is a wide variation in the distribution of the esp gene in enterococci even within the same geographic region [37]. Lenz et al. report that efaA plays a significant role in response to bile salt stress in E. faecalis strains [38]. Biofilm formation in enterococci is directly affected by esp, efaA, and ace genes, based on the phenotypic results and the presense of these selected genes. According to the findings of the study, esp and efaA genes were more frequently found among E. faecalis strains with moderate and strong biofilm forming capability. Several studies have also reported similar findings [36]. It has been demonstrated that the esp gene plays an important role in the formation of biofilm [39].

One limitation inherent in these studies is the potential impact of the surface, culture medium, and duration chosen for biofilm formation on the resulting strength of biofilm production. In future investigations, it is imperative to thoroughly explore and address this limitation to enhance our overall comprehension of the factors influencing biofilm formation.

Conclusion

The results of this study revealed a notable increase in resistance levels to kanamycin, tetracycline, and streptogramin. Our interpretation suggests a potential correlation between this elevated resistance and the intensive use of tetracycline and kanamycin for various purposes within the studied region. The widespread application of these antibiotics, whether in medical, agricultural, or other contexts, may contribute to the emergence and persistence of resistance patterns observed in this study. This correlation underscores the need for a comprehensive understanding of antibiotic usage practices and their impact on antibiotic resistance within the specific geographic context of our study. Also, the increase in resistance to streptogramin showed the importance of MLSB (macrolide-lincosamide-streptogramin B) resistance phenotypes in enterococci. Eventually, we showed that the presense of esp, ace, and efaA genes in E. faecalis was higher than in E. faecium, which could be due to the high expression of these genes in E. faecalis. The control of enterococcal infections in hospitals may be affected by the presence of the esp, efaA, and ace genes in E. faecium and E. faecalis isolates, which would maintain their establishment and growth in hospital settings.

Suggestions

The presence of other genes related to the biofilm production should be investigated. Study on clinical isolates collected from hospitalized patients infected with enterococcus isolates should be performed. Also, in order to achieve better results, the mulecular typing techniques, such as RAPD-PCR and PFGE, are necessary to assess the sources and/or diversity of the strains.