# Wide Distribution of Virulence Genes amongEnterococcus faecium and Enterococcus faecalis Clinical Isolates

Authors: Sara Soheili; Sobhan Ghafourian; Zamberi Sekawi; Vasanthakumari Neela; Nourkhoda Sadeghifard; Ramliza Ramli; Rukman Awang Hamat
Journal: The Scientific World Journal
 (2014)

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## Abstract

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.


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## Body

## 1. 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 andEnterococcus  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 [3–5].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, Zn-metalloendopeptidase, 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 inE. faecium has 99% similarity with the cell wall surface anchor family protein ofEnterococcus sp. GMD4E (Accession: EKA01662). The pilB ofE. faecium has 74% similarity with the cell wall surface anchor protein ofE. faecalis B318 (Accession: ETU21897). Meanwhile, the ecbA inE. faecium has 92% similarity with the cell wall surface anchor protein ofE. faecalis (Accession: WP_010708790) and 91% with Cna protein B-type domain proteinE. faecalis (Accession: WP_002417581) (these regions are part of a collagen binding MSCRAMM, ecbA).The cell wall adhesion (efaAfm) inE. faecium has 66% similarity with endocarditis specific antigen ofE. faecalis T8 (Accession: EEU25576). With regard to the collagen binding MSCRAMM Acm (fms8) inE. faecium, 57% similarity has been observed with the collagen binding domain protein ofE. faecalis (Accession: WP_016632409). The blast analysis for the collagen binding MSCRAMM scm inE. faecium has revealed 99% similarity with the collagen-binding MSCRAMM scm (Fms10) inEnterococcus sp. GMD5E. Finally, 37% similarity of the cell wall anchored protein sgrA inE. faecium has been detected when compared to the surface adhesion protein inEnterococcus 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 amongE. faecalis andE. faecium clinical isolates. Based on the similarity results and also the possibility of horizontal virulence gene transfer thepilA, pilB, hyl, ecbA, scm, fms8, efaAfm, andsgrA genes were chosen for this study.


## 2. Material and Methods

### 2.1. Bacterial Isolates

A total of 79 clinical isolates ofE. faecalis(50 isolates) andE. 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.


### 2.2. DNA Extraction ofEnterococcus

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


### 2.3. Evaluation of Different Virulence Genes

All isolates were subjected to the amplifiedpilA, pilB, hyl, ecbA, scm, fms8, efaAfm, andsgrA genes, using specific primers as listed in Table 1. These virulence genes are most strongly associated with clinical lineages ofE. faecium; however, not much data has been reported amongE. 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.Table 1
Characteristics of different primers used in the current study.
Primer name
Sequence (5′-3′)
Size (bp)
Accession number
pilA
Forward: AAAACGCCACCAGAGAAGGTReverse: CATTGGCGCAATCACAACCA
459
ENA∣ACI49671
pilB
Forward: GATACCCAGCTGACGGCTTTReverse: GGTACTGCCGAAAACGAAGC
959
EFF34880
fms8
Forward: AGACGAGCAGATGAACAGCCReverse: CCCGTCAATCGTCGTACTGT
765
YP_006376887
efaAfm
Forward: AAAAGGCAAGCGACGCAGATReverse: AGGTCTAGCCAAGCATGAGG
199
FJ609170
sgrA
Forward: CTGATCGGATTGTTTATGAReverse: AATAAACTTCCCCAATAACTT
150
AGS75503
ecbA
Forward: GGAGTGAGGCTTTTAAACCAGAReverse: GGAAACAGGGTACTTTG
182
AGS75851
hyl
Forward: CCCTGGACACATGAAATGCGReverse: AGCATCGGCCGTTGATAGAC
605
AF544400
scm
Forward: GTTTACTAGTCCTAGTTGCReverse: TCTGTACTGTCGCTTGTGTC
1015
YP_006377279


### 2.4. 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.


### 2.5. 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.


## 2.1. Bacterial Isolates

A total of 79 clinical isolates ofE. faecalis(50 isolates) andE. 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.


## 2.2. DNA Extraction ofEnterococcus

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


## 2.3. Evaluation of Different Virulence Genes

All isolates were subjected to the amplifiedpilA, pilB, hyl, ecbA, scm, fms8, efaAfm, andsgrA genes, using specific primers as listed in Table 1. These virulence genes are most strongly associated with clinical lineages ofE. faecium; however, not much data has been reported amongE. 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.Table 1
Characteristics of different primers used in the current study.
Primer name
Sequence (5′-3′)
Size (bp)
Accession number
pilA
Forward: AAAACGCCACCAGAGAAGGTReverse: CATTGGCGCAATCACAACCA
459
ENA∣ACI49671
pilB
Forward: GATACCCAGCTGACGGCTTTReverse: GGTACTGCCGAAAACGAAGC
959
EFF34880
fms8
Forward: AGACGAGCAGATGAACAGCCReverse: CCCGTCAATCGTCGTACTGT
765
YP_006376887
efaAfm
Forward: AAAAGGCAAGCGACGCAGATReverse: AGGTCTAGCCAAGCATGAGG
199
FJ609170
sgrA
Forward: CTGATCGGATTGTTTATGAReverse: AATAAACTTCCCCAATAACTT
150
AGS75503
ecbA
Forward: GGAGTGAGGCTTTTAAACCAGAReverse: GGAAACAGGGTACTTTG
182
AGS75851
hyl
Forward: CCCTGGACACATGAAATGCGReverse: AGCATCGGCCGTTGATAGAC
605
AF544400
scm
Forward: GTTTACTAGTCCTAGTTGCReverse: TCTGTACTGTCGCTTGTGTC
1015
YP_006377279


## 2.4. 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.


## 2.5. 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.


## 3. Results and Discussion

A total of 79E. faecalis(50 isolates) andE. faecium(29 isolates) were analyzed for the presence of different virulence genes includingpilA, pilB, hyl, ecbA, scm, fms8, efaAfm,andsgrA. The analysis showed different prevalence of virulence genes inEnterococcuswhich ranged from 35.4% to 100%. ThepilB, fms8, efaAfm, andsgrA 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 (n
=
73/79). TheecbAwas determined as the third most common prevalent virulence gene with 81% (n
=
64/79) frequency. ThepilA showed 73.4% (n
=
58/79) prevalence in clinical isolates ofEnterococcus. The lowest prevalence detected washyl with the prevalence of 35.4% (n
=
28/79). The prevalence of different virulence genes is shown in Figure 1. With regard to both enterococcal species, the analysis showed that all selected virulence genes were positive amongE. faeciumisolates. The exception ishylwhere it was detected with 82.7% prevalence rate. AmongE. faecalis isolates,pilB, fms8, efaAfm, andsgrA genes were detected in all isolates, followed byscm (88%),ecbA (70%), andpilA (58%). The least prevalence washyl which was detected in only 4 isolates (8%). Table 2 shows the distribution of different virulent determinants amongE. faecium andE. faecalis clinical isolates. We believe that this is the first report on the prevalence of selected virulence genes amongE. faecalis which could be demonstrated by the possibility of horizontal gene transfer amongE. faeciumandE. faecalis.Table 2
Distribution of different virulence factors inE.  faeciumand E.  faecalis.
Virulence genes
E.  faecalis
E.  faecium
Total
(
n
=
50
)
(
n
=
29
)
(
N
=
79
)
pilB
50 (100%)
29 (100%)
79 (100%)
fms8
50 (100%)
29 (100%)
79 (100%)
efaAfm
50 (100%)
29 (100%)
79 (100%)
sgrA
50 (100%)
29 (100%)
79 (100%)
scm
44 (88%)
29 (100%)
73 (92.4%)
ecbA
35 (70%)
29 (100%)
64 (81%)
pilA
29 (58%)
29 (100%)
58 (73.4%)
hyl
4 (8%)
24 (82.7%)
28 (35.4%)Figure 1
Prevalence of different virulence genes in clinicalE. faecium andE. faecalisisolates. ThepilB,  fms8,  efaAfm, and sgrAwere identified in all clinical isolates.Pili which are also known as fimbriae have been detected in Gram-positive bacteria [13–15]. This surface organelle is responsible for endocarditis and biofilm formation in Gram-positive 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 inE. faecium. In our study, high prevalence rate ofpilB in clinicalE. faeciumandE. faecalis isolates (100% for each) has been observed. In comparison withpilB, 21 (26.6%) clinicalE. faecalis isolates were negative for the presence ofpilA. Figures 2 and 3 show the presence ofpilA andpilB genes in representative isolates. Hendrickx et al. [19] have demonstrated the presence ofpilA andpilB in clinical isolates ofE. faecium. Their analysis showed thatpilA 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 detectpilA in some of our clinicalE. faecalis isolates.Another possible reason could be explained by the lack of horizontal gene transfer amongE. faecalisisolates.Figure 2
PCR results ofpilA. 1 = marker = 100 bp; 2–16 =pilA = 495 bp; 17 = negative control.Figure 3
PCR results ofpilB. 1, 10 = marker = 100 bp; 2–8 =pilB = 959 bp; 9 = negative control.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 inEnterococcus such asebp(endocarditis and biofilm-associated pilus ofE. faecalis) operon,ace (adhesion of collagen fromE. faecalis), andacm (adhesion of collagen fromE. faecium) [22–24].The role of collagen-binding MSCRAMMacm(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 offms8 (100% for each) was detected amongE. faecium andE. faecalis clinical isolates and could play an important role in pathogenicity of both enterococcal species. Figure 4 shows the presence offms8 genes in representative isolates.Figure 4
PCR results offms8. 1 = marker = 100 bp; 2–8 =fms8 = 765 bp; 9 = negative control.The exact role ofefaAfm is unknown although it is believed to be involved in cell wall adherence. TheefaAfm gene was only found inE. faecium isolates [25]. A study by Barbosa et al. [26] has demonstrated 27% of  isolates harbored theefaAfm gene while the current study showed all isolates were positive forefaAfm. The presence ofefaAfm gene among representative isolates is shown in Figure 5. Our finding corroborates with Martin et al. [27] where they have demonstrated allE. faecium isolates carryingefaAfmvirulence gene.Figure 5
PCR results ofefaAfm.  1, 8 = marker = 100 bp;  2–6 =efaAfm = 199 bp; 7 = negative control.The pathogenicE. faecium is enriched with twoorf2351 andorf2430 genes which encode thesgrA andecbALPXTG-like 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. BothecbAandsgrA were reported to be prevalent in clinical strains ofE. faecium [28]. However, our study demonstrated thatsgrAwas more prevalent thanecbA (100% versus 81%). The representative isolates for the presence ofsgrA andecbA genes are shown in Figures 6 and 7.Figure 6
PCR results ofsgrA. 1 = marker = 100 bp;  2–11 =sgrA = 150 bp;  12 = negative control.Figure 7
PCR results ofecbA. 1 = marker = 100 bp;  2–11 =ecbA = 182 bp;  12 = negative control.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 ofE. 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% ofE. faeciumstrains carried thehylvirulence gene. Our results are in accordance with the previous study by Panesso et al. [30]. Thehyl gene among representative isolates is shown in Figure 8. In a research by Worth et al. [31] among Australian haematology patients, only 2.1% of isolates showed positive reaction tohyl. A study by Vankerckhoven et al. [32] revealed 71% of VRE and 29% of vancomycin sensitiveEnterococcus (VSE) harboredhyl virulence gene. Sincehyl is not prevalent in all clinical isolates in our study, we believe that this gene could has little role in pathogenicity ofEnterococcusin comparison with other prevalent virulence genes.Figure 8
PCR results ofhyl. 11 = marker = 100 bp;  2–9 =hyl = 605 bp; 1, 10 = negative control.Scm (second collagen adhesion ofE. faecium) binds to collagen type V and fibrinogen and it is commonly distributed among clinical and nonclinical isolates ofE. faecium.Scm was first described inE. faeciumin 2008 [33] but limited information on the prevalence ofscminE. faeciumhas been reported. Nonetheless, we documented a high prevalence ofscm in clinical isolates ofE. faecium(92.4%). Isolates representing for the presence ofscm gene are shown in Figure 9.Figure 9
PCR results ofscm. 7 = marker = 100 bp; 1–5 =scm = 1015 bp; 1 = negative control.


## 4. Conclusion

The wide distribution of several virulence genes that ispilB,fms8,efaAfm,andsgrA inE. faecalis andE. 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.



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*Source: 623174-2014-07-15.xml*