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ORIGINAL ARTICLE  
Year : 2013  |  Volume : 56  |  Issue : 3  |  Page : 238-242
Adhesion and virulence factor properties of Enterococci isolated from clinical samples in Iran


1 Department of Medical Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
2 Department of Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

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Date of Web Publication24-Oct-2013
 

   Abstract 

Introduction: Enterococci rank among leading causes of nosocomial bacteremia, urinary tract infections and community acquired endocarditis. The aim of the present study was to investigate the presence of virulence factors in Enterococci strains isolated from clinical samples in Iranian Educational hospitals. Methodology: Presence of aggregation substance (asa), extracellular surface protein (esp), Enterococcus faecalis antigen A (efaA), adhesin of collagen from E. faecalis (ace), endocarditis and biofilm-associated pilli (ebp) as colonization factors and cytolysin (cyl), gelatinase (gel) and hyaloronidase (hyl) as secretary factors were investigated in isolates. A total of 201 clinical isolates of Enterococci were collected in 2009-2010 from eight educational hospitals. After deoxyribonucleic acid extraction, they were examined for presence of virulence factors by polymerase chain reaction. Results: E. faecalis and Enterococcus faecium were isolated from 56.9% to 43.1%, respectively. Resistance to vancomycin and gentamicin were 33.8% and 83.9% in E. faecium isolates and 16.3% and 88.1% in E. faecalis isolates respectively. Colonization factors were found to be more prevalent in E. faecalis isolates and almost all isolates of E. faecalis had ace, ebp and efaA genes. Esp gene had a higher rate of distribution in Enterococci isolates (75.1%) in this study compared with previous studies. One of E. faecalis isolates contained hyl gene, but 38.8% of E. faecium isolates had it. Mutual exclusive were present between hyl and efaA in all E. faecium isolates and 69.7% of E. faecium hyl - positive isolates were esp positive. Conclusion: According to these results, virulence genes were more prevalent in E. faecalis isolates and E. faecalis had more potential pathogenesis for initiating an infection; however because of E. faeciums higher antibiotic resistance, we have been facing higher E. faecium infections in hospitalized patients.

Keywords: Ahesion, Enterococcus faecalis, Enterococcus faecium, virulence factor

How to cite this article:
Kafil HS, Mobarez AM, Moghadam MF. Adhesion and virulence factor properties of Enterococci isolated from clinical samples in Iran . Indian J Pathol Microbiol 2013;56:238-42

How to cite this URL:
Kafil HS, Mobarez AM, Moghadam MF. Adhesion and virulence factor properties of Enterococci isolated from clinical samples in Iran . Indian J Pathol Microbiol [serial online] 2013 [cited 2018 Aug 17];56:238-42. Available from: http://www.ijpmonline.org/text.asp?2013/56/3/238/120375



   Introduction Top


Enterococci are Gram-positive bacteria considered normal inhabitants of the gastrointestinal tracts of humans and animals. In the last two decades, though, Enterococci have emerged as important hospital acquired infections, with high level resistance to antibiotics such as aminoglycosides, β-lactamase and glycopeptides. [1]

Now, Enterococci rank among leading causes of bacteremia and urinary tract infections (UTI) and have become the second most frequently reported cause of surgical wound infections and the third most frequently reported cause of nosocomial pathogens. [2] Several enterococcal pathogenic factors have been identified including adhesions and secreted virulence factors. The most important adhesion factors are aggregation substance (asa), extracellular surface protein (esp), Enterococcus faecalis antigen A (efaA), adhesin of collagen from E. faecalis (ace) and endocarditis and biofilm-associated pilli (ebp). [3] Asa has shown to form large aggregates and serves as a putative factor in favor of bacterium against the host defense. [4] Esp contributes in colonization and biofilm formation of Enterococci and leads to resistance to stresses and adhesion to eukaryotic cells such as those of endocarditis and UTI. [5],[6] EfaA was hypothesized to be functioning as an adhesion in endocarditis. [7] Ebp consists of the gene cluster associated with the formation of pilli by Enterococci, which is fundamental in causing endodontic and UTI by bacteria [8] and ace is a collagen binding protein belonging to the microbial surface components recognizing adhesive matrix molecules. [9]

Secreted pathogenic factors of Enterococci with a value in pathogenesis are cytolysin (cyl), gelatinase (gelE) and hyaloronidase (hyl). [4] Gelatinase is an extracellular zinc-containing metalloproteinase, which has a role in providing nutrients for the bacteria by degrading host tissue. It has some functions in biofilm formation as well. [10],[11] Cyl (also hemolysin) has β hemolytic properties in human and is bactericidal against other Gram-positive bacteria. [12] Hyl acts on hyaloronic acid and mainly consists of degredative enzymes which is associated with tissue damage and facilitates spread of Enterococci and their toxins through host tissue. [4] These virulence factors may play a role in promoting persistence of Enterococci. The aim of the present study was to investigate distribution of virulence factors in Enterococci strains isolated from several educational hospitals in Iran.


   Methodology Top


Two hundred and one clinical isolates of Enterococci were collected in 2009-2010 from eight educational hospitals containing Tehran, Shiraz, Tabriz, Zahedan and Mashhad [Figure 1]. All isolates were preserved in Tryptocase soy broth with 30% glycerol in –70°C.
Figure 1: Iran Map: Centers participated for samples collection

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The isolates were identified as Enterococci by mass spectrophotometer (MALDI-TOF MS microflex, bruker, Germany) and confirmed by biochemical tests and polymerase chain reaction (PCR). [13],[14] Antibiotic resistance properties of strains were examined by Kerby-bauer method according to Clinical and Laboratory Standards Institute (CLSI) guideline (CLSI M100-S22). Antibiotic discs were provided by Mast Group LTD (United Kingdom) and Staphylococcus aureus ATCC 25923 was used as quality control for disc diffusion.

Genomic PCR

Deoxyribonucleic acid (DNA) extraction was done by Cinnapure TM DNA extraction kit (Cinnagen, Iran). Briefly speaking, 1 μl of bacterial fresh culture in BBL TM Enterococcosel Agar (Becton, France) was resuspended in 100 μl G + pre-lysis buffer by inoculation loops and 20 μl lysosyme was added and incubated at 37°C for at least 30 min. Having added lysis buffer and precipitation solution, the solution was transferred to a spin column, and after washing the spin, DNA was eluted by elution buffer in 65°C. PCR was performed in 25 μl volumes that contained 20-200 ng DNA, 0.5 μM of specific primers for each gene [Table 1], 1.5 mM MgCl 2 and 200 μM of each 2'-deoxynucleoside 5'-triphosphate, 1X PCR buffer and 2U DNA polymerase (Cinnagene Iran). DNA was amplified by general PCR. An initial 10 min denaturation at 94°C was followed by 35 cycles of 1 min denaturation at 94°C, annealing at 58°C (for esp, gelE, cyl, hyl, efaA and ace) and 52°C (for ebp and asa) for 1 min and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min. Positive control for PCR were E. faecalis MMH594 (gelE, asa, esp, cyl, ebp positive), E. faecalis 29212 (gelE, asa positive), Enterococcus faecium C38 and C68 and E. faecalis 217. [16] PCR products were analyzed in agarose gels and visualized under ultraviolet after staining with 0.5 μg/ml ethidium bromide.
Table 1: Target genes and primers used in study

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Chi-square test (or Fisher exact test) was performed for data analysis. P values below 0.05 were considered to be significant.


   Results Top


In 2009-2010, we collected 201 isolates of E. faecium and E. faecalis from eight educational hospitals of Iran. Among 114 (56.7%) were E. faecalis and 87 (43.2%) were E. faecium. 40.3% samples were collected from males and 59.7% from females. The samples were 82.1% urine, 7.5% wound, 6% blood, 2.5% phlegm, 1% asit, 0.5% trachea and 0.5% stool. The samples were obtained from 37.9% to 62.1% of outpatients and hospitalized patients, respectively. In outpatients, 78.3% of isolates were E. faecalis and 21.7% were E. faecium. While, in hospitalized patients 58.4% of isolates belonged to E. faecium and 41.6% of isolates to E. faecalis. The sections from which isolates were collected were internist and infectious disease, pediatrics, surgery, nephrology, intensive care unit, neurology and heart section, respectively. Antibiotic resistance pattern of isolates are shown in [Table 2]. All isolates were studied for the presence of virulence factors and E. faecium and E. faecalis isolates were compared for the presence of these virulence factors [Table 3]. We couldn't find any significant differences comparing isolates with different sources, but something noteworthy is that cly and gelE genes were found to be more prevalent in urine samples than isolates from other sources (P = 0.01, P = 0.01 respectively) [Table 4].
Table 2: Antibiotic resistance patterns of isolates

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Table 3: Presence of different virulence factors in isolates of E. faecium and E. faecalis

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Table 4: Presence of virulence genes in urinary tract infections isolates

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   Discussion Top


Because of a rapid growth of nosocomial infections by Enterococccus spp, in hospitals and communities, further research is needed on this pathogen. Therefore, we aimed to study the presence and importance of formerly confirmed pathogenic factors of Enterococci in clinical samples isolated from hospitals in all corners of Iran [Figure 1]. Results revealed that colonization factors were more prevalent in E. faecalis isolates; almost all isolates of E. faecalis contained ace, ebp and efaA genes. Some previous studies also found efaA in all the clinical E. faecalis strains, [7],[21] but in E. faecium only 60% of our clinical isolates had efaA gene. Particularly, isolates of E. faecium with blood and wound sources (83.3% and 75% respectively) harbored more efaA in comparison with urinary isolates (with 57.7%).

Esp gene had higher distribution in Enterococci's isolates in this study compared with previous studies investigating this gene in clinical samples [Table 3]. [21],[22]

Ebp was present in almost all of E. faecalis and E. faecium isolates [Table 3]. This operon encodes pilus components shown to be important for pathogenesis in the endocarditis and UTI. This operon is also important for biofilm formation. [8],[19] High presence of this gene may indicate the importance of biofilm formation for pathogenesis of Enterococci in clinical isolates. Particularly, in UTI, 97.9% of E. faecalis isolates had ebp [Table 4]. Asa was present in 38.8% of isolates that was consistent with previous studies on clinical isolates. [23]

Distribution of secretary virulence genes was completely different in E. faecalis and E. faecium isolates. Almost none of E. faecalis isolates had hyl gene, but 38.8% of E. faecium isolates had it [Table 3]. Interestingly, mutual exclusive was observed between presence of hyl and efaA in all E. faecium isolates. In the results of the study by Billstrom et al. [24] isolates of E. faecium which harbored efaA had no hyl gene and vice versa. In 69.7% of E. faecium also hyl - positive isolates were esp positive, which is in agreement with the findings of Rice et al. [25] and Vankerckhoven et al. [16] studies. Presence of cly and gel in E. faecalis isolates was significantly higher than E. faecium isolates [Table 3]. In antibiotic results according to [Table 2] resistance to common antibiotics was higher in E. faecium than E. faecalis isolates. With these results we can conclude that E. faecalis isolates harbored more virulence genes and more potential pathogenesis for initiating an infection, but it is unlikely that high resistance to antibiotic in E. faeciums can cause ecological changes. We faced with partial replacement of E. faecalis with E. faecium especially in nosocomial infections.

We couldn't find any significant differences comparing isolates with different sources, but something noteworthy is that cly and gelE genes were found to be more prevalent in urine samples than isolates from other sources (P = 0.01, P = 0.01 respectively). Comparing E. faecium and E. faecalis with different sources, secretary virulence genes were recognized to be different [Table 4].

In conclusion, In Enterococi strains collected from Iranian hospitals, virulence genes were more prevalent in E. faecalis isolates, especially colonization factors such as ace, efaA and asa. Moreover, E. faecalis harbored high presence of cly and gel genes. Interestingly, almost no E. faecalis had hyl gene; however, efaA and hyl genes were mutual exclusive in E. faecium isolates. According to these results, virulence genes were more prevalent in E. faecalis isolates and it had more potential pathogenesis for initiating an infection; but, because of E. faeciums higher antibiotic resistance we have been facing higher E. faecium infections in hospitalized patients.


   Acknowledgments Top


We would like thank all staffs of educational hospitals for cooperation in collecting samples and antibiogram of isolates. Also we thank Dr. Hossein Navidinia and Dr. Mohammad Momenian for their helpful comments on manuscript. This study was part of Hossein Samadi Kafils PhD dissertation and has been granted by Tarbiat Modares University.

 
   References Top

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Correspondence Address:
Ashraf Mohabati Mobarez
Department of Medical Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box: 14115-111, Tehran
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.120375

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]

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