**3.5 Enterococcal surface protein (***esp***)**

*Esp,* a putative virulent factor is found in both *E. fl* and *E. fm*. It is located on pathogenicity island (PAI) at the surface of the bacterium [56]. It was initially

identified in a highly virulent gentamicin resistant strain of *E. fl* [69]. *Esp* shares global structural similarity with *Streptococcus agalactiae* Rib [86], *S. pyogenes* R28, C-alpha protein, and *S. aureus* Bap (biofilm associated protein) [87]. These similarities are restricted to a highly conserved region within the C repeat units of *Esp* proteins and group A and B of streptococcal proteins of streptococci and nonrepeat N-terminal region of Bap protein [71]. Bap protein from *S. aureus* is associated with biofilm formation and shares a sequence and structural similarity with *Esp*. *Esp* is also associated with *E. fl* biofilm formation on different surfaces, like polysterylene plates and hospital equipment like catheters, prosthetic heart valves, orthopedic appliances, artificial cardiac pace makers [47], ureteral stents [88], intravascular catheters [89], silicone gastrostomy devices [90], and biliary stents [91].

A variant of *Esp* is also reported in *E. fm* isolates [92]. *E. fm esp* is predominantly present in nosocomial settings in contrast to *E. fl esp* which is widely distributed among environmental strains [93, 94]. Expression of *esp* is affected by environmental conditions like temperature (maximum at 37°C) and availability of oxygen, i.e., under anaerobiosis [56]. Several research groups demonstrated the role of *E. fm esp* in pathogenesis of experimental endocarditis, UTIs, and bacteremia. While no specific role of *esp* was found in peritonitis, and colonization of GIT [95, 96]. Role of *esp* was also established by a genetic approach. In a study conducted by Tendolkar *et al.* [97], *esp*-lacking *E. fl* strains produced biofilm in large amounts after successful induction and expression of *esp* gene. In contrast, several studies suggest that *esp* is not necessary for biofilm formation [98, 99]. Study conducted by Kristich *et al*. [100] demonstrated that *E. fl* OG1RF produced biofilms not only in the absence of *esp* and entire PAI that harbors it. In other studies, conducted on clinical enterococcal isolates, majority of the *esp*-negative isolates produced biofilms and no correlation was found among *esp* gene and biofilm forming capacity [89, 101].

### **3.6 Aggregation substance (AS)**

AS is a group of proteins encoded by pheromone-induced conjugative plasmids. AS directed bacteria to aggregate which results in close cell contact between donor and recipient. Several studies showed that AS mediated internalization of *E. fl* by cultured human intestinal epithelial cells and increased in vitro adhesion to cultured renal tubular cells [102]. Among the best studied AS proteins are Asa I, Asp I, and Acs 10 encoded by *asa1*, *aspI* and *prgB* genes of conjugated plasmids *pAD1* and *pCF10*, respectively, and shows >90% sequence identity [56, 66]. These proteins contain an N-terminal domain, a central domain, a variable region and two Arg-Gly-Asp (RGD) motifs which are also found in fibronectin and associated with integrin binding proteins [102, 103]. Apart from their function in conjugation transfer, these RGD motifs are also involve in eukaryotic cell binding and binding to renal epithelial cells [102]. It is demonstrated in a study that central domain and N-terminal domain are responsible for aggregation of Asc 10 [104]. Beside its role in conjugation, AS also serves as a virulence factor in *E. fl* by promoting cell-cell contact, adhesions to host cells and ECM proteins (including thrombospondin, fibronectin, vitronectin, and collagen type I), increased vegetation in experimental endocarditis, resistance to killing by polymorphonuclear leukocytes (PMNs) by inhibition of respiratory burst (production of ROS) in the macrophages, increased cell surface hydrophobicity [71]. All the proteins aid in the pathogenesis of AS in *E. fl,* like Asa I increases adherence to human macrophages and renal tubular cells, Asc 10 facilitates internalization and intracellular survival in PMNs [74, 103, 105]. Both

**117**

*The Genus* Enterococcus *and Its Associated Virulent Factors*

**Virulent factors Gene Primer sequence (5′-3′) Product** 

*espTIM* CTT-TGA-TTC-TTG-GTT-GTC-GGA-TAC

*agg* AAG-AAA-AAG-AAG-TAG-ACC-AAC

*acm* GGC-CAG-AAA-CGT-AAC-CGA-TA

*efaAfm* AAC-AGA-TCC-GCA-TGA-ATA

*efaAfs* GAC-AGA-CCC-TCA-CGA-ATA

*asa* GCA-CGC-TAT-TAC-GAA-CTA-TGA

*ace* AAA-GTA-GAA-TTA-GAT-CCA-CAC

*ccf* GGG-AAT-TGA-GTA-GTG-AAG-AAG

*cpd* TGG-TGG-GTT-ATT-TTT-CAA-TTC

*cob* AAC-ATT-CAG-CAA-ACA-AAG-C

*eep* GAG-CGG-GTA-TTT-TAGTTC-GT

*gelE* ACC-CCG-TAT-CAT-TGG-TTT

*sprE* TTG-AGC-TCC-GTT-CCT-GCC-GAA-

*fsrA* ATG-AGT-GAA-CAA-ATG-GCT-ATT-TA

*fsrB* GGG-AGC-TCT-GGA-CAA-AGT-ATT-

*fsrC* ATG-ATT-TTG-TCG-TTA-TTA-GCT-ACT

*cylLL* GAT-GGA-GGG-TAA-GAA-TTA-TGG

*cylLs* GAA-GCA-CAG-TGC-TAA-ATA-AGG

*cylM* AAA-AGG-AGT-GCT-TAC-ATG-GAA-

*cylB* AAG-TAC-ACT-AGT-AGA-ACT-AAG-GGA

*cylA* ACT-CGG-GGA-TTG-ATA-GGC

TCC-AAC-TAC-CAC-GGT-TTG-TTT-ATC

AAA-CGG-CAA-GAC-AAG-TAA-ATA

CGC-TGG-GGA-AAT-CTT-GTA-AA

CAT-TTC-ATC-ATC-TGA-TAG-TA

AGT-TCA-TCA-TGC-TGT-AGT-A

TAA-GAA-AGA-ACA-TCA-CCA-CGA

TCT-ATC-ACA-TTC-GGT-TGC-G

AGC-CGC-TAA-AAT-CGG-TAA-AAT

TAC-GGC-TCT-GGC-TTA-CTA

TTG-TCA-TAA-AGA-GTG-GTC-AT

TAC-TCCAGCATTGGATGCT

ACG-CAT-TGC-TTT-TCC-ATC

AGT-CAT-TC TTG-GTA-CCG-ATT-GGG-GAA-CCA-GAT-TGA-CC

CTA-AGT-AAG-AAA-TAG-TGC-CTT-GA

ATC-TAA-CCG TTG-GTA-CCC-ACA-CCA-TCA-CTG-ACT-TTT-GC

CAT-CGT-TAA-CAA-CTT-TTT-TAC-TG

GCT-TCA-CCT-CAC-TAA-GTT-TTA-TAG

GTA-TAA-GAG-GGC-TAG-TTT-CAC

GAT CAT-AAC-CCA-CAC-CAC-TGA-TTC-C

ACA-GTG-AAC-GAT-ATA-ACT-CGC-TAT-T

GCT-GCT-AAA-GCT-GCG-CTT

**length (bp)**

705

782

1405

937

591

740

566

1343

240

2940

2020

688

253 [117]

475 [111]

1553 [112]

353 [113]

735 [92]

375 [114]

320 [115]

542 [112]

419 [116]

**Reference**

*DOI: http://dx.doi.org/10.5772/intechopen.89083*

*Biofilm associated* 

*Gelatinase operon* 

*Cytolysin operon* 

*genes*

*genes*

*genes*


*Microorganisms*

biliary stents [91].

forming capacity [89, 101].

**3.6 Aggregation substance (AS)**

identified in a highly virulent gentamicin resistant strain of *E. fl* [69]. *Esp* shares global structural similarity with *Streptococcus agalactiae* Rib [86], *S. pyogenes* R28, C-alpha protein, and *S. aureus* Bap (biofilm associated protein) [87]. These similarities are restricted to a highly conserved region within the C repeat units of *Esp* proteins and group A and B of streptococcal proteins of streptococci and nonrepeat N-terminal region of Bap protein [71]. Bap protein from *S. aureus* is associated with biofilm formation and shares a sequence and structural similarity with *Esp*. *Esp* is also associated with *E. fl* biofilm formation on different surfaces, like polysterylene plates and hospital equipment like catheters, prosthetic heart valves, orthopedic appliances, artificial cardiac pace makers [47], ureteral stents [88], intravascular catheters [89], silicone gastrostomy devices [90], and

A variant of *Esp* is also reported in *E. fm* isolates [92]. *E. fm esp* is predominantly present in nosocomial settings in contrast to *E. fl esp* which is widely distributed among environmental strains [93, 94]. Expression of *esp* is affected by environmental conditions like temperature (maximum at 37°C) and availability of oxygen, i.e., under anaerobiosis [56]. Several research groups demonstrated the role of *E. fm esp* in pathogenesis of experimental endocarditis, UTIs, and bacteremia. While no specific role of *esp* was found in peritonitis, and colonization of GIT [95, 96]. Role of *esp* was also established by a genetic approach. In a study conducted by Tendolkar *et al.* [97], *esp*-lacking *E. fl* strains produced biofilm in large amounts after successful induction and expression of *esp* gene. In contrast, several studies suggest that *esp* is not necessary for biofilm formation [98, 99]. Study conducted by Kristich *et al*. [100] demonstrated that *E. fl* OG1RF produced biofilms not only in the absence of *esp* and entire PAI that harbors it. In other studies, conducted on clinical enterococcal isolates, majority of the *esp*-negative isolates produced biofilms and no correlation was found among *esp* gene and biofilm

AS is a group of proteins encoded by pheromone-induced conjugative plasmids. AS directed bacteria to aggregate which results in close cell contact between donor and recipient. Several studies showed that AS mediated internalization of *E. fl* by cultured human intestinal epithelial cells and increased in vitro adhesion to cultured renal tubular cells [102]. Among the best studied AS proteins are Asa I, Asp I, and Acs 10 encoded by *asa1*, *aspI* and *prgB* genes of conjugated plasmids *pAD1* and *pCF10*, respectively, and shows >90% sequence identity [56, 66]. These proteins contain an N-terminal domain, a central domain, a variable region and two Arg-Gly-Asp (RGD) motifs which are also found in fibronectin and associated with integrin binding proteins [102, 103]. Apart from their function in conjugation transfer, these RGD motifs are also involve in eukaryotic cell binding and binding to renal epithelial cells [102]. It is demonstrated in a study that central domain and N-terminal domain are responsible for aggregation of Asc 10 [104]. Beside its role in conjugation, AS also serves as a virulence factor in *E. fl* by promoting cell-cell contact, adhesions to host cells and ECM proteins (including thrombospondin, fibronectin, vitronectin, and collagen type I), increased vegetation in experimental endocarditis, resistance to killing by polymorphonuclear leukocytes (PMNs) by inhibition of respiratory burst (production of ROS) in the macrophages, increased cell surface hydrophobicity [71]. All the proteins aid in the pathogenesis of AS in *E. fl,* like Asa I increases adherence to human macrophages and renal tubular cells, Asc 10 facilitates internalization and intracellular survival in PMNs [74, 103, 105]. Both

**116**


#### **Table 1.**

*List of primers reported for the genotypic assessment of major virulence factors.*

**119**

**Table 2.**

*The Genus* Enterococcus *and Its Associated Virulent Factors*

**Thermal cycler programme Gene name (Reference)**

E *efaAfs* and *efaAfm* [92]

F *espTIM* [111]

G *acm* gene [113]

H *ace* and *asa1* gene [114, 115]

I *agg* or *AP* gene [112]

J *ccf*, *cpd*, *eep*, *cob* genes [112]

K *entP, entA, entB, ef1097* gene [118]

L *enlA* gene [118]

M *IS16* gene [111]

N *Hyl gene* [119]

*Illustrations for the PCR conditions for the amplification of various putative virulence genes.*

*DOI: http://dx.doi.org/10.5772/intechopen.89083*

*Microorganisms*

*Insertion sequence element gene*

*Hyaluronidase gene*

**Table 1.**

**Virulent factors Gene Primer sequence (5′-3′) Product** 

ATT-AAA-G TTA-GCA-CTT-CCC-TGG-AAT-TGC-TCC

GTT-GCA-TTT-AGA-GTA-TAC-ATT-TGC

TTA-GCT-CTT-ATT-GG TTA-ATG-TCC-CAT-ACC-TGC-CAA-ACC-AG

ATT-AGG CTT-AGC-CCA-CAT-TGA-ACT-GCC-CAT-AAA-GC

GTA-CAT-CTC-CAT-ATA-CTT-TTC-C

TCA-AAA-AGT-GGG-CTT-GGC

GAC-TGA-CGT-CCA-AGT-TTC-CAA

ACG-CAT-TGC-TTT-TCC-ATC

AGT-CAT-TC TTG-GTA-CCG-ATT-GGG-GAA-CCA-GAT-TGA-CC

CTA-AGT-AAG-AAA-TAG-TGC-CTT-GA

*entB* AGA-CCT-AAC-AAC-TTA-TCT-AAA-G

*entP* ATG-AGA-AAA-AAA-TTA-TTT-AGT-

*Ef1097* GGC-GAT-GGC-ATT-ACT-AAT-GAC-

*enlA* CGA-TTT-CTG-TTG-TAG-GAA-CC

*IS16* CATG-TTC-CAG-CAA-CCA-GAG

*hyl* ACA-GAA-GAG-CTG-CAG-GAA-ATG

*Lip-fl* ATG-AGT-GAA-CAA-ATG-GCT-ATT-TA

**Thermal cycler programme Gene name (Reference)** A *gelE*, *sprE*, *fsrA*, *fsrB*, *fsrC* genes [116]

B *lip-fm* and *lip-fl* genes [108]

D *cylLL*, *cylM*, *cylB*, *cylA* [117]

C *kat* gene [110]

*Catalase gene kat* ACC-CCG-TAT-CAT-TGG-TTT

*List of primers reported for the genotypic assessment of major virulence factors.*

*Lipase gene Lip-fm* TTG-AGC-TCC-GTT-CCT-GCC-GAA-

*Enterocin genes entA* ATG-AAA-CAT-TTA-AAA-ATT-TTG-TCT-

**length (bp)**

126

216

408

1405

740

547 [111]

276 [119]

419 [110]

591 [108]

1770 [118]

**Reference**

**118**

**Table 2.** *Illustrations for the PCR conditions for the amplification of various putative virulence genes.*

Asa I and Asc 10 increase virulence of *E. fl* in rabbit endocarditis model by increasing adherence to certain ECM proteins [79, 106].
