Detection of Esp gene for the presence of extracellular surface protein among clinical isolates of Enterococcus species

 

Sisira Padavala¹, Dr. Gopinath P²

¹BDS 3rd Year, Saveetha Dental College, Saveetha University, Chennai.

 

ABSTRACT:

Esp gene is a high molecular weight surface protein whose frequency is significantly increased among infection derived Enterococcus faecalis isolates. Esp gene is an Enterococcal surface protein gene which is present in the opportunistic pathogens Enterococcus faecalis and Enterococcus faecium. It has been proposed that esp gene can be used as a marker of human pollution in environmental waters. This gene has been shown to be a part of a 150-in putative pathogenicity island. A sum of 20 isolates of Enterococci were assessed for the presence of esp gene that encodes for extracellular surface protein. We have observed 5% positivity in our study. This indicates that, the pathogenicity of urinary tract infections by these pathogens might be crucially played by this gene encoding for extra cellular surface protein.

 

 

 

INTRODUCTION:

Enterococci are commensals of the gastrointestinal tract. They can act as opportunistic pathogens and are the leading cause of nosocomial infections^[1]. They have been isolated from the bloodstream, surgical sites and urinary tract infections. Enterococcus faecalis accounts for approximately 75% of all enterococcal infections with Enterococcus faecium accounting for the rest^[2]. The increased incidence of E.faecalis infection has been related to the innate resistance of this organism to many commonly used antimicrobial agents and its ability to become resistant to most and in some cases to all of the presently available antibiotics, either by mutation or by incorporation of foreign genetic material^[3].

 

There are several virulence factors associated with the infections caused by E. faecalis and E. faecium. The major virulence factor is the presence of esp gene. Esp is an Enterococcal surface protein of high molecular mass of approximately 202kDa.This gene has been shown to be a part of a 150-in putative pathogenicity island^[4,5]. This gene originally found in Enterococcus faecalis has been associated with increased virulence, colonization and biofilm formation^[6]. With this background, we have taken this study to detect the gene responsible for expression of extracellular surface protein  in Enterococci isolates.

 

MATERIALS AND METHODS:

Bacterial isolates:

A total of 20 different non-repetitive clinical isolates of Enterococci were collected from different clinical specimens from Saveetha Medical College and Hospitals, Chennai. These isolates were identified by standard biochemical parameters as described by Elsevier. Isolates were preserved in semi-solid brain heart infusion medium and stored at 4 ºC until further use.

 

Antibiotic susceptibility testing:

Antibiotic susceptibility test was determined for these isolates to routinely used antibiotics such as ampicillin (10µ), vancomycin (30µ), teicoplanin (30µ), erythromycin (15µ), ciprofloxacin (5µ), amikacin (200µ), gentamycin (10µ), tetracycline (30µ) and linezolid (30µ) (Hi Media, Mumbai) by Kirby-Bauer disc diffusion method^[7].

 

Detection of esp gene in Enterococcus species:

Enterococcus isolates were detected for the presence esp gene by PCR analysis. Detection of the gene was carried out using primer as depicted in table 1. Bacterial DNA was extracted by boiling lysis method. 1 µL of DNA extract was used as template for PCR reaction. The reaction mixture contained 1mM of MgCl 20.2mM dNTP mix and 0.8µM of blaNDM-1 gene with 1U of Taq polymerase (New England Biolabs) in a 1x PCR buffered reaction. PCR amplification was carried out using thermal cycler (Eppendorf) with the following cycling condition. Initial denaturation at 96°C for 3 minutes, 30 cycles of denaturation at 95°C for 1 minute, primer annealing at 54°C for 40 seconds and primer extension at 75°C for 1 minute and final extension at 72°C for 5 minutes were used. PCR products were resolved in 1.5% agarose gel. A 100bp ladder was included in all the gel analysis^[3].

 

Table 1:Gene sequencing of esp gene

 

 

RESULTS:

Sample wise distribution of clinical isolates of Enterococci:

Of the 20 clinical isolates of Enterococcus species, 12/20 (60%) were from urinary tract infections and 4/20 (20%) were from blood, 2/20(10%) were from stool and 2/20 (10%) were from wound swabs. Figure 1 depicts the sample wise distribution of clinical isolates of Enterococcus species.  

  

Figure 1: Pie chart showing the sample wise distribution of clinical isolates of Enterococcus species.

 

Bacterial isolates:

Of the 20 clinical isolates of Enterococcus species, 14/20(70%) were Enterococcus faecalis and 6/20(30%) were Enterococcus faecium. Figure 2 depicts the species distribution of clinical isolates of Enterococcus.

 

Figure 2: Pie chart showing species distribution of Enterococcus

 

Antibiotic susceptibility testing:

In our isolates, we have found increased percentage 18/20(90%) of isolates showed sensitivity to linezolid followed by vancomycin, which showed sensitivity of 15/20(75%). 80-90% of Enterococcus isolates showed resistance to aminoglycoside group of drugs. 5(25%) were found to be resistant to teicoplanin. However, we have observed an elevated level of resistance to other routinely used antibiotics. The detailed resistant pattern of Enterococci isolates is shown in table 2.

 

Table 2: Results of antibiotic sensitivity pattern of Enterococci

Result of esp gene in Enterococcus species:

Of the 20 clinical isolates of Enterococcus species, 1/20 (5%) was found to harbour esp gene.

 

Figure 3: Representative gel picture showing positive for esp gene

L4- 315bp Esp gene;  L2-100bp ladder

 

DISCUSSION:

In our study we have observed 5% of our Enterococci isolates from patients affected with urinary tract infections showed positive for esp gene. This gene codes for extracellular surface protein. This protein is known to regulate several functions in our system, among which resistance to phagocytosis is one of the main virulence function exhibited by this trait. A novel feature of this protein is the presence of large, repeating units. Homologous recombination within these units at the genetic level leads to addition or deletion of repeat units resulting in alteration in size of the encoded protein. This size variation of esp gene at the cell surface maybe responsible for the environment specific function. An extended form of the esp protein maybe responsible for adhesion during the initial stages of infection, facilitating interaction with host receptors while the less extended form escapes the immune response^[7].Shankar et al. stated that the structural changes in the N-terminal region of the esp gene will help in evading the immune response. Esp gene is also responsible of the formation of biofilm on medical devices like catheters, artificial heart valves and ocular lenses^[8].

 

CONCLUSION:

Enterococcus species are frequently being encountered from clinical specimens with high degree of resistance. There are several virulence factors that play a crucial role in determining the pathogenicity.  In this study, only one isolate was found to be positive and hence it is important to include more number of isolates to validate the result.

 

ACKNOWLEDGMENT:

We thank Dr. Kalyani, Professor and Head of the Department of Microbiology, Saveetha Medical College, Chennai for kindly providing the clinical isolates to carry out our research work fruitfully.

 

REFERENCES:

1.     Upadhyaya GPM, Lingadevaru UP, Lingegowda RK. Comparative study among clinical and commensal isolates of Enterococcus faecalis for presence of esp gene and biofilm production. J Infect Dev Ctries.2011;5(5):365-369.

2.     Shankar V, Baghdayan AS, Huycke MM, Lindahl G, Gilmore MS. Infection-Derived Enterococcus faecalis Strains Are Enriched in esp, a Gene Encoding a Novel Surface Protein. Infection And Immunity.1999;67(1):193-200.

3.     Toledo-Ariana A, Valle J, Salono C, Arrizubieta MJ, Cucarella C, Lamata M, Amorena B, Leiva J, Penades JR, Lasa I. The Enterococcus Surface Protein, Esp, Is Involved in Enterococcus faecalis Biofilm Formation. Applied And Environmental Microbiology. 2001;67(10):4538-4545.

4.     Oancea C, Klare I, Witte W, Werner G. Conjugative transfer of the virulence gene, esp, among isolates of Enterococcus faecium and Enterococcus faecalis. Journal of Antimicrobial Chemotherapy. 2004;54:232-235.

5.     Leavis H, Top J, Shankar N, Borgen K, Bonten M, Embden JV, Willemstad RJL. Journal of Bacteriology. 2004;186(3):672-682.

6.     Hammerum AM, Jensen LB. Prevalence of esp, Encoding the Enterococcal Surface Protein, in Enterococcus faecalis and Enterococcus faecium Isolates from Hospital Patients, Poultry, and Pigs in Denmark. Journal of Clinical Microbiology. 2002;40(11):4396.

7.     Shankar N, Lockatell CV, Baghdayan AS, Drachenberg C, Gilmore MS, Johnson DE. Role of Enterococcus faecalis Surface Protein Esp in the Pathogenesis of Ascending Urinary Tract Infection. Infection and Immunity. 2001;69(7):4366-4372.

8.     Gapelah F, Mehrabi MR, Labibzadeh M. Biofilm Formation and Presence of Esp and cylA Genes Enterococcus faecalis Isolated from Hospital Infection. Clinical Microbiology & Case Reports.2015;1(3):018.

 

 

 

 

Accepted on 16.03.2017           © RJPT All right reserved