Enterococcus corresponds to Gram-positive microorganisms, whereupon 49 species are distributed in diverse environments, such as marine waters, plants, animal intestines, and others [1] . Among identified species, E. faecalis and E. faecium are those commonly found in the human intestinal tract. However, they are considered opportunistic pathogens being able to cause urinary tract infections, bacteremia, endocarditis, and neonatal meningitis [1] [2] [3] [4] [5] .

Different factors contribute to the bacteria permanence in the hospital environment, mainly resistance [6] and virulence factors [7] . Vancomycin Resistance in Enterococcus spp. (VRE) was first reported in 1986 and has been associated with an increased mortality rate in patients suffering from bacteremia [8] [9] [10] . E. faecium and E. faecalis are the main species of this taxonomic group associated with resistance to vancomycin. There are reports of this type of resistance in other species of the genus (E. gallinarum, E. casseliflavus, E. avium and E. raffinosus); however, this resistance profile is less frequent [10] [11] .

Virulence factors are related to the invasion of the pathogen in host tissue, persistence of infection, and biofilm formation potential. The biofilm architecture is influenced by several factors including hydrodynamic conditions, nutrient concentration, bacterial motility, and intercellular communication. Bacterial adhesion to the formation site is influenced by cell movement, electrostatic, and hydrophobic interactions, also adhesin expression [12] [13] .

Over the years, a number of virulence genes have been described for Enterococcus, such as: aggregation substance, gelatinase, enterococcal surface protein, cytolysin, pheromones, and hyaluronidase [14] [16] . Gelatinase is a protein secreted by E. faecalis that potentially contributes to the virulence of this species, as well as the enterococcal adhesion encoded by the esp gene that contributes to colonization and persistence of E. faecalis during infection of ascending urinary tract [17] . The aggregation substance, on the other hand, corresponds to a pheromone-induced surface protein, which promotes the formation of conjugation aggregates during bacterial conjugation, contact between cell-cell, and between cell-host cell [18] .

The aim of study was to evaluate and compare resistance profile and biofilm formation between E. faecalis and E. faecium clinical isolates in different culture media and adhesion substrate, and also to identify cell hydrophobicity and the presence of virulence genes.

The taxonomic identity of all isolates was confirmed by the MALDI-TOF-MS technique. Of the 35 isolates analyzed, 12 (34.3%) corresponded to E. faecalis and 23 (65.7%) to E. faecium (Table 2). It is possible observed in Table 2 that identified isolates were obtained from different origins and sectors of a public hospital in Recife, Pernambuco, Brazil. Most isolates were collected from the Coronary Units (COU = 40.0%) and the Intensive Care Unit (ICU = 31.4%). Others were obtained from the medical clinic (14.3%), cardiology (11.4%) and emergency (2.9%). Regarding the colonization site, it was observed that most bacteria (71.4%) were isolated from rectal swab. However, other sites of infection were also reported, but with a lower percentage. 14.3% were collected from blood, 11.4% from urine, and 2.9% from catheter. E. faecalis was most obtained

UFPEDA—Department of Antibiotics of Federal University of Pernambuco, ICU—Intensive care unit, COU—Coronary unit, HFL—hydrophilic, M HFB—Moderately hydrophobic, MFB—Hydrophobic. In biofilm formation: Weak (+), Moderate (++) e Strong (+++) biofilm producers and no biofilm producers (−). In virulence genes: Presence (+) e Absence (−).

from blood cultures (11.4%) and ICUs (14.3%), while isolates of E. faecium were more frequent from rectal swab (51.4%) and COUs (31.4%).

Resistance profile showed a higher resistant to ampicillin and penicillin G, 62.9% were resistant to ampicillin, but only one E. faecalis isolate was resistant to this antibiotic (19185), 77.1% were resistant to penicillin G, mainly in E. faecium isolates (62.8%). All isolates were resistant to vancomycin, 34.3% were resistant to daptomycin, and 14.3% were resistant to linezolid (Table 3).

Ability to form biofilm is showed in Table 2 and Figure 1. It was observed that E. faecium had a lower potential when compared to E. faecalis. All E. faecalis isolates were able to produce biofilm. However, only seven of 23 E. faecium isolates produced biofilm under the conditions tested. From the investigated substrates (plate and tube), it was observed that isolates of both species showed better performance for biofilm production on the hydrophobic surface (Plate = 48.6%) when compared to the hydrophilic surface (Tube = 5.7%). Regarding the culture media, it was found that in the TSB the isolates had higher formation potential, regardless of the species analyzed (Figure 1).

Table 4 shows the general characteristics of the isolates in relation to total samples, source, origin, biofilm formation, cellular hydrophobicity, and virulence genes (distribution and frequency). From this table, it is possible to compare the resistance profile and virulence potential of each species.

For the following variables, cell hydrophobicity and presence of virulence genes, E. faecalis and E. faecium also presented distinct profiles. Most isolates of E. faecalis were moderately hydrophobic (M HFB = 25.7%), while all of E. faecium were hydrophilic (HFL = 62.9%), excepting isolate 14872 which also presented as M HFB.

In the present study, we also investigated the presence and frequency of virulence genes: gelE, esp and asa1. The gelatinase gene, gelE (54.3%), and the Enterococcus surface protein gene, esp (54.3%), were most prevalent. Followed by the aggregation substance gene, asa1 (22.9%). E. faecalis showed higher positivity for the asa1 gene (20%), followed by gelE (5.7%) and esp (5.7%) genes. In E. faecium isolates, the frequency of gelE and esp was higher (48.6% for each gene), followed by asa1 that was detected in only one isolate (00931), see Table 2.

Only two isolates, one from E. faecalis (19185) and one from E. faecium

UFPEDA—Department of Antibiotics of Federal University of Pernambuco. Antibiotics: AMP—ampicillin, PEN G—penicillin G, VAN—Vancomycin, DAP—daptomycin, LIN—linezolid. RIS—Resistance International System, MIC—Minimum inhibitory concentration (µg/mL). R—resistant, S—susceptible. *Note: When clinical breakpoints for daptomycin were originally set there was insufficient evidence to set a susceptible-resistant breakpoint. In our study we considered daptomycin resistant with MIC ≥ 4¹⁹.

(00931) were positive for three genes tested. The other isolates presented different genetic profiles. The profile (esp + asa1) was identified in only one isolate (18576) belonging to the species E. faecalis. While the profile (gelE + esp) was detected in 14 isolates of E. faecium. Isolates containing only one gene were also found, 6 in E. faecalis and 4 in E. faecium (see Table 2).

Interestingly, five isolates, two from E. faecalis (17870, 00640), three from E. faecium (02089, 13679, 10800), and control 09 did not show any of the genes investigated. These same samples presented themselves as moderate to strong biofilm producers in hydrophobic subtracts. On the other hand, eight isolates were positive for at least two of the three genes investigated and were classified as weak or non-producer of biofilm (see Table 2).

ICU—Intensive care unit, COU—Coronary unit, AMP—ampicillin, PEN G—penicillin G, VAN—vancomycin, DAP—daptomycin, LIN—linezolid, HFL—hydrophilic, M HFB—Moderately hydrophobic, MFB—Hydrophobic. TSB—Tripyc Soy Broth media, BHI—Brain-Heart infusion media.

To confirm the potential for biofilm formation, 19185 E. faecalis isolate was selected and analyzed on two substrates (plate and tube) by fluorescence microscopy. The results confirmed that 19185 is a strong biofilm producer, regardless of the condition tested, be it a hydrophobic substrate (Figure 2(c)) or a hydrophilic substrate (Figure 2(f)). Figure 2(a) and Figure 2(d) show the microorganisms. Also, in Figure 2(b) and Figure 2(e) it is possible to notice the production of extracellular matrix secreted by bacterial cells. The Figure 2(c) and Figure 2(f) is the overlay of previous images.

Since 2017, a reduction in hospital infection rates in general has been observed. However, there is an increase in the number of infections caused by multi-resistant microorganisms. This phenomenon has been occurring not only in Brazil, but worldwide. In adult and neonatal ICU, Enterococcus spp. vancomycin resistance are among the microorganisms that cause the most health care-related infection (HAI) and increase in-patient mortality [22] [23] . According to the World Health Organization (WHO), E. faecium VRE isolates are more frequent in hospital outbreaks than E. faecalis, since they have a larger gene arsenal related to resistance and virulence factors [10] [24] . As well as E. faecium is more resistant to AMP than E. faecalis according by EUCAST and CLSI protocols. It was not different in our study [19] [25] .

Collection with rectal swab is indicated by the Agência Nacional de Vigilância Sanitária (ANVISA) as a practice of epidemiological surveillance after the patient was hospitalized, which is a concern, since most isolates of E. faecium of this study came from this collection, and all isolates were VRE. Enterococci are already in the normal microbiota, and in this case VRE isolates may be the main transmission mechanisms within the hospital sectors [26] . This is a worrying factor for immunocompromised patient. According to a study [27] , hospitalized patients have a high incidence of enterococcal infections, not only due to the virulence of isolates, but also due to the circulation of health professionals and the hospital area. This represents a risk environment, which can justify the spread of species in different sectors of a hospital or from different hospitals as previously demonstrated [28] .

Among the strategies to combat antimicrobial resistance (ADR) are socio-educational interventions, monitoring access to these drugs and rational prescriptions [29] . According to Pan American Health Organization (PAHO), ADR surveillance work should be continuous and multidisciplinary [30] . Thus, the collective effort of the hospital unit investigated in our study and also of local health agencies could result in a more effective control of ADR.

Regarding of biofilm formation, the composition of the medium is probably the most important factor that influences the ability of bacteria to produce biofilm under in vitro conditions. Thus, TSB is the most widely used medium for the cultivation of Gram-positive biofilms [21] . The relationship between biofilm formation and cellular hydrophobicity is still difficult to confirm. The determination of bacterial surface hydrophobicity alone is not a sufficient factor to characterize biofilm formation. Other factors are needed to stimulate or develop this ability in microorganisms. Considering that, in enterococcal cells this trait seems to be no different [20] .

The biofilm formation is a multifactorial process related to both gene expression and interference of environmental factors, which can be aggravated, as these genes as well as resistance genes can be propagated between species through mobile DNA elements [31] . The presence of virulence genes may be correlated with biofilm formation, which has been reported over the years [22] [27] [32] . The prevalence of gelE, esp and agg (other aggregation substance) genes is high when it comes to clinical isolates [32] .

Furthermore, biofilm formation is more related to the presence of gelE gene, regardless of whether or not isolates present esp and agg genes in E. faecalis species. However, studies demonstrated biofilm formation in mutated strains for the esp gene (Esp-negative) and confirmed that neither esp nor gelE appear to be necessary to develop this factor in vitro. In addition, also confirm that the presence of these genes seems to be more related to the successful establishment of an infection [22] [33] . These data are interesting because they indicate that the presence or absence of the genes investigated in this study does not seem to be the primary or only characteristic for biofilm formation in these species. It is noteworthy that, the ability to form biofilm contributes to the pathogenicity of infections, since it enables the mediation of adhesion, colonization, and invasion of host tissue. Thereby, modulating immunity, producing enzymes, and toxins that help the installation and increase of severity of infection [13] [14] .

It was observed that in Figure 2 it is possible to evidence the formation of biofilm by fluorescence. For this, we used SYBR Green (capable of intercalating in the hydrogen bridges of double strand of bacterial DNA) and Calcofluor White dyes (which binds the β1-6 polysaccharide bonds that are formed in the exopolysaccharide matrix or EPS) [34] [35] [36] . The fluorescence technique is widely used, these dyes together in our work were capable to show the biofilm and prove that Enterococcus isolates can be able to form biofilm in different surfaces.

In general, the formation of biofilms depends on several factors such as the types of microorganisms, surfaces and environmental conditions such as pH and temperature [13] . The biofilm matrix is mainly composed of EPS, but also contains water, lipids, nucleic acids and extracellular proteins, forming a porous architecture with channels that allow the passage of nutrients [12] . In addition, electrostatic and hydrophobic attractive forces, van der Waals interactions, hydrogen bonds and covalent bonds, as well as flagella, fimbrial adhesin and polymers are considered for biofilm formation [12] [37] . Biofilms are a suitable microenvironment for microbial survival, being a protective barrier, including against antimicrobials [38] . Therefore, studies on the subject are crucial to understand the functioning of this mechanism in different microorganisms and help in the development of effective therapies.

Our findings show the importance of characterizing virulence factors and biofilm formation related to clinical isolates of Enterococcus that present resistance mainly to vancomycin, daptomycin, and linezolid. Also, it demonstrates genetic and biochemical alternatives, which facilitate the adaptation and survival of Enterococcus isolates. In addition, the presence and high frequency of gelE, esp and asa1 genes are an indicative that monitoring studies in the hospital setting should occur frequently. These factors increase the spread and severity of infection, making treatment difficult and increasing rates of infection morbidity and mortality of hospitalized patient.

We thank the Centro de Tecnologias Estratégicas do Nordeste (CETENE) for the support in taxonomic identification, the Laboratório de Genética e Biologia Vegetal (LGBV) for the support in the fluorescence microscopy and Fiocruz Pernambuco - Instituto Aggeu Magalhães (IAM) for the support in the sequencing.

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), No. 09/2018; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)/Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE), No. BCT-0019-2.08/18.

The authors declare no conflicts of interest regarding the publication of this paper.

de Azevedo Ramos, B., Manta, M.M., da Silva, S.M., de Sá, R.A.Q.C., de Souza, N.L., Coutinho, H.D.M., da Silva, M.V., da Silva, T.D., dos Santos Correia, M.T. and de Oliveira, M.B.M. (2023) Virulence Factors and Biofilm Formation in Vancomycin Resistant Enterococcus faecalis and Enterococcus faecium Isolates in Brazil. Advances in Microbiology, 13, 299-314. https://doi.org/10.4236/aim.2023.136019