Wide Distribution of Virulence Genes among Enterococcus faecium and Enterococcus faecalis Clinical Isolates

Enterococcus, a Gram-positive facultative anaerobic cocci belonging to the lactic acid bacteria of the phylum Firmicutes, is known to be able to resist a wide range of hostile conditions such as different pH levels, high concentration of NaCl (6.5%), and the extended temperatures between 5°C and 65°C. Despite being the third most common nosocomial pathogen, our understanding on its virulence factors is still poorly understood. The current study was aimed to determine the prevalence of different virulence genes in Enterococcus faecalis and Enterococcus faecium. For this purpose, 79 clinical isolates of Malaysian enterococci were evaluated for the presence of virulence genes. pilB, fms8, efaAfm, and sgrA genes are prevalent in all clinical isolates. In conclusion, the pathogenicity of E. faecalis and E. faecium could be associated with different virulence factors and these genes are widely distributed among the enterococcal species.


Introduction
Enterococci are considered to be part of normal gut microbiota in both humans and animals and capable to survive in a diverse range of harsh conditions. Among them only two, that is, Enterococcus faecium and Enterococcus faecalis, are now being increasingly recognized to be involved in human infections such as bacteremia, endocarditis, urinary tract infections, and surgical site infections [1,2]. This could be explained by their inherent resistance to various antibiotics with greater adaptability in hospital environments and has high genetic diversity as well as the presence of various virulence factors [3][4][5].
These opportunistic bacteria possess various virulence factors including enterococcal surface protein (Esp) and aggregation substance (Agg) which could enhance the colonization process in the host and binding to the host epithelium, respectively [6]. Others such as cytolysin [7], enterolysin A [8], gelatinase [9], hyaluronidase, Znmetalloendopeptidase, enhanced expression of pheromone (Eep) [10], and adhesion-associated protein EfaA (E. faecalis endocarditis antigen A) [11] have been reported to be among the most important virulence factors.
Surprisingly through NCBI database analysis, there are some similarities observed in terms of their virulence determinants among enterococci. For instance, pilA in E. faecium has 99% similarity with the cell wall surface anchor family protein of Enterococcus sp. GMD4E (Accession: EKA01662). The pilB of E. faecium has 74% similarity with the cell wall surface anchor protein of E. faecalis B318 (Accession: ETU21897). Meanwhile, the ecbA in E. faecium has 92% similarity with the cell wall surface anchor protein of E. faecalis (Accession: WP 010708790) and 91% with Cna protein B-type domain protein E. faecalis (Accession: WP 002417581) 2 The Scientific World Journal (these regions are part of a collagen binding MSCRAMM, ecbA). The cell wall adhesion (efaAfm) in E. faecium has 66% similarity with endocarditis specific antigen of E. faecalis T8 (Accession: EEU25576). With regard to the collagen binding MSCRAMM Acm (fms8) in E. faecium, 57% similarity has been observed with the collagen binding domain protein of E. faecalis (Accession: WP 016632409). The blast analysis for the collagen binding MSCRAMM scm in E. faecium has revealed 99% similarity with the collagen-binding MSCRAMM scm (Fms10) in Enterococcus sp. GMD5E. Finally, 37% similarity of the cell wall anchored protein sgrA in E. faecium has been detected when compared to the surface adhesion protein in Enterococcus sp. C1 (Accession: WP 008378446). These findings could be related to the notion that horizontal transfer of both resistance and virulence determinants is very common among enterococci [12]. Thus, this study was conducted to investigate the distribution of the diverse virulence factors and specify the dominant virulence genes among E. faecalis and E. faecium clinical isolates. Based on the similarity results and also the possibility of horizontal virulence gene transfer the pilA, pilB, hyl, ecbA, scm, fms8, efaAfm, and sgrA genes were chosen for this study.

Bacterial Isolates.
A total of 79 clinical isolates of E. faecalis (50 isolates) and E. faecium (29 isolates) were identified during May 2009 to March 2010 from a tertiary teaching hospital. Nonrepetitive isolates were collected from these samples such as urine, blood, pus, vaginal, and sterile body fluid.

DNA Extraction of Enterococcus.
The DNA wasextracted using the DNA extraction kit (Gene ALL, South Korea) according to the manufacturer's instructions.

Evaluation of Different Virulence
Genes. All isolates were subjected to the amplified pilA, pilB, hyl, ecbA, scm, fms8, efaAfm, and sgrA genes, using specific primers as listed in Table 1. These virulence genes are most strongly associated with clinical lineages of E. faecium; however, not much data has been reported among E. faecalis. The PCR amplification was carried out in a DNA thermocycler (Bio-Rad) using the amplification parameters as follows: initial denaturation at 95 ∘ C for 2 minutes, followed by 35 cycles of denaturation at 95 ∘ C for 20 seconds, annealing at 58 ∘ C for 10 seconds, and extension at 72 ∘ C for 20 seconds, with a final extension at 72 ∘ C for 5 minutes. All PCR products were analyzed by 1% agarose gel electrophoresis.

Purification of Virulence Genes from Agarose
Gel. PCR products of each virulence gene were run on 1% (w/v) molecular grade agarose gel (Sigma, USA), using a Bio-Rad mini-gel electrophoresis system at 80 V for 70 minutes. The DNA was purified using DNA purification kit (Gene ALL, South Korea) according to the manufacturer's instructions.

Sequence
Analysis. The PCR products were purified from gel agarose and then the purified products were sequenced by Sigma Company (Singapore). The results of DNA sequencing were run in Chromas Lite program to analyze the similarity to the sequenced gene in GenBank library.

Results and Discussion
A total of 79 E. faecalis (50 isolates) and E. faecium (29 isolates) were analyzed for the presence of different virulence genes including pilA, pilB, hyl, ecbA, scm, fms8, efaAfm, and sgrA. The analysis showed different prevalence of virulence genes in Enterococcus which ranged from 35.4% to 100%. The pilB, fms8, efaAfm, and sgrA were identified as the dominant virulence genes in all isolates. The second most prevalent virulence gene, scm, was found in 92.4% of the isolates  Figure 1. With regard to both enterococcal species, the analysis showed that all selected virulence genes were positive among E. faecium isolates. The exception is hyl where it was detected with 82.7% prevalence rate. Among E. faecalis isolates, pilB, fms8, efaAfm, and sgrA genes were detected in all isolates, followed by scm (88%), ecbA (70%), and pilA (58%). The least prevalence was hyl which was detected in only 4 isolates (8%). Table 2 shows the distribution of different virulent determinants among E. faecium and E. faecalis clinical isolates. We believe that this is the first report on the prevalence of selected virulence genes among E. faecalis which could be demonstrated by the possibility of horizontal gene transfer among E. faecium and E. faecalis.
Pili which are also known as fimbriae have been detected in Gram-positive bacteria [13][14][15]. This surface organelle is responsible for endocarditis and biofilm formation in Grampositive bacteria [16], and mediates attachment to human epithelium and skin [17] and confers resistance against macrophages [18].
So far, limited data has been documented on the prevalence of pili in E. faecium. In our study, high prevalence rate of pilB in clinical E. faecium and E. faecalis isolates (100% for each) has been observed. In comparison with pilB, 21 (26.6%) clinical E. faecalis isolates were negative for the presence of pilA. Figures 2 and 3 show the presence of pilA and pilB genes in representative isolates. Hendrickx et al. [19] have demonstrated the presence of pilA and pilB in clinical isolates of E. faecium. Their analysis showed that pilA could not be expressed in broth condition and the best temperature for their expression was 37 ∘ C. This could probably be a reason as to why we could not detect pilA in some of our clinical E. faecalis isolates. Another possible reason could be explained by the lack of horizontal gene transfer among E. faecalis isolates.
Nowadays, it is obvious that in pathogenic bacteria several proteins have evolved to adhere to and invade into the host cells and subsequently escape and resist host defense   [20]. MSCRAMMs which are known as microbial surface components recognizing adhesive matrix molecules have an important role for cell adhesion and involve in pathogenicity of bacteria [21]. In silico analysis has revealed a family of genes that encode MSCRAMM-like proteins in Enterococcus such as ebp (endocarditis and biofilm-associated pilus of E. faecalis) operon, ace (adhesion of collagen from E. faecalis), and acm (adhesion of collagen from E. faecium) [22][23][24]. The role of collagen-binding MSCRAMM acm (Fms8) is to bind to the collagen types I and IV. These types of collagen are also an important antigen involved in human during endocarditis. In the current research, high prevalence rate of fms8 (100% for each) was detected among E. faecium and E. faecalis clinical isolates and could play an important role in pathogenicity of both enterococcal species. Figure 4 shows the presence of fms8 genes in representative isolates.
The exact role of efaAfm is unknown although it is believed to be involved in cell wall adherence. The efaAfm gene was only found in E. faecium isolates [25]. A study by Barbosa et al. [26] has demonstrated 27% of isolates harbored the efaAfm gene while the current study showed all isolates were positive for efaAfm. The presence of efaAfm gene among representative isolates is shown in Figure 5. Our finding corroborates with Martin et al. [27] where they have demonstrated all E. faecium isolates carrying efaAfm virulence gene.
The pathogenic E. faecium is enriched with two orf2351 and orf2430 genes which encode the sgrA and ecbA LPXTGlike cell wall anchored proteins, respectively. sgrA is a surface adhesion that can bind to the extracellular matrix molecules nidogen 1 and nidogen 2 and is also involved in biofilm formation. Meanwhile, ecbA binds to the collagen type V and   fibrinogen. Both ecbA and sgrA were reported to be prevalent in clinical strains of E. faecium [28]. However, our study demonstrated that sgrA was more prevalent than ecbA (100% versus 81%). The representative isolates for the presence of sgrA and ecbA genes are shown in Figures 6 and 7.
In addition, hyl virulence gene encodes for a putative glycosyl hydrolase that is considered as a plasmid harboring gene which has colonized the gastrointestinal tracts of mice subsequently caused an increase of pathogenicity of E. faecium in experimental peritonitis [29]. In a study by Panesso et al. [30] that subjected 32 hospitals in Colombia, Ecuador, Peru, and Venezuela, the results revealed that 23% of E. faecium strains carried the hyl virulence gene. Our results   are in accordance with the previous study by Panesso et al. [30]. The hyl gene among representative isolates is shown in Figure 8.  Scm (second collagen adhesion of E. faecium) binds to collagen type V and fibrinogen and it is commonly distributed among clinical and nonclinical isolates of E. faecium. Scm was first described in E. faecium in 2008 [33] but limited information on the prevalence of scm in E. faecium has been reported. Nonetheless, we documented a high prevalence of scm in clinical isolates of E. faecium (92.4%). Isolates representing for the presence of scm gene are shown in Figure 9.

Conclusion
The wide distribution of several virulence genes that is pilB, fms8, efaAfm, and sgrA in E. faecalis and E. faecium clinical isolates could give a clue that these virulence genes might play an important role in the pathogenicity of both enterococcal species. This could also be explained that the horizontal virulence gene transfer is common among the clinical isolates.