Prevalence of virulence genes of biofilm producing strains of Staphylococcus epidermidis isolated from clinical samples in Iran
© Solati et al. 2015
Received: 21 May 2015
Accepted: 23 July 2015
Published: 9 August 2015
Coagulase negative staphylococci are recognized as opportunistic pathogens and are widespread in the environment. It is possible to prevent and control infections due to these bacteria if their virulence factors are recognized. Eighty isolates of Staphylococcus epidermidis (S. epidermidis) including 42 from urine (52.5%), 23 from blood (28.75%), 15 from dialysis bags (18.75%) were studied for biofilm production on Congo red agar (CRA). The virulence genes in S. aureus were investigated using polymerase chain reaction (PCR) with primers. Out of 80 isolates studied, 40 isolated (50%) formed black colonies (biofilm-forming strains) on CRA. In 22 of these isolates (25%) reaction was strongly positive; in 12 isolates (15%) reaction was moderately positive, and in the remaining 6 isolates, reaction was weakly positive. In the 22 isolates that had strong positive reaction and produced black colonies on biofilm, all virulent genes (icaC, icaD, icaA icaB, icaR) were expressed. In the 12 isolates that had moderate positive reaction, 8 expressed all genes (icaC, icaD, icaA icaB, icaR) expressed while the remaining 4 expressed only ica A, and ica D genes. Of the 6 isolated which had weak positive reaction, only 1 isolate (2.5%) expressed all the genes, in the other 5 isolates no gene was observed. Urinary isolates more frequently form biofilms than the isolates from other clinical samples. Statistical analysis using Chi square test showed that there was a significant correlation between the type of sample and the biofilm production (P < 0.05). The results of biofilm production on CRA were largely in agreement with microtiter plate assay and PCR assay. The capacity of bacteria to produce biofilm is an important factor in infectivity and happens via expression of ica genes. Recognition of bacteria that produce biofilm is thus important to control infection due to these bacteria.
KeywordsStaphylococcus epidermidis Clinical isolates Biofilm Virulence genes Microtiter assay plate
Staphylococci are Gram positive non-motile, non-spore forming, facultative anaerobes, occurring as cocci in clusters, and are classified in two main groups, coagulase-positive and coagulase-negative (Oto 2009; Asadollahi Dehkordi et al. 2015). Coagulase negative staphylococci (CNS) are normal inhabitants of human skin and mucosa. Though frequently isolated from clinical specimens; they are often considered as non-pathogens (Oto 2009). However, CNS are being increasingly recognized in causing nosocomial and community infections. There are 40 recognized species of CNS (Rogers et al. 2009). In contrast to Staphylococcus aureus, virulence properties associated with Staphylococcus epidermidis are few and biofilm formation on the surface of materials is the most important virulence factor as demonstrated by animal model of animal infection (Fev and Olson 2010). Production of poly-N-acetylglucosamine (PNAG) is crucial for S. epidermidis biofilm formation and is synthesized by the gene products of the ica ADBC gene cluster. Biofilm formation protects these bacteria against the antibacterial drugs and the immune system defenses (Fev and Olson 2010). Currently S. epidermidis is the predominant cause of nosocomial infections because of its potential ability in biofilm formation and colonization in different surfaces (Uckay et al. 2009; Fev and Olson 2010). Staphylococcus epidermidis has emerged as a major nosocomial pathogen associated with infections of implanted medical devices. In the past few decades, the clinical importance, and the emergence of methicillin-resistant S. epidermidis strains have created many challenges in the treatment process (Namvar et al. 2014). Several studies have been performed on detecting virulent genes in isolates of Staphylococcus aureus and S. epidermidis (Gad et al. 2009; Kumar et al. 2009; Bien et al. 2011; Gomes et al. 2011). An extracellular polysaccharide adhesin represents a key virulence determinant in S. epidermidis and is required for biofilm formation. Production of this adhesin is encoded by the ica operon (De Silva et al. 2002). A recent study from Canada concerned virulence gene expression by S. epidermidis biofilm cells exposed to antibiotics (Gomes et al. 2011). The present investigation aims at detecting virulent genes in clinical isolates of S. epidermidis recovered from patients in Iran.
Materials and methods
Eighty isolates of S. epidermidis that had been referred to the medical laboratory of Kashani Hospital, Imam Ali Hospital and Hajar Hospital in Shahrekord, Iran, including 42 from urine (52.5%) from cases of urinary tract infection, 23 from blood (28.75%) from patients of septicemia, and 15 from dialysis bags (18.75%) from kidney failure patients undergoing peritoneal dialysis. Biofilm production studied by phenotypic characterization, and microtiter plate assay. Virulence genre for biofilm formation were investigated by PCR.
The method employed was that described by Freeman et al. (1989). The Congo red agar medium comprised BHI (37 g/L), sucrose (50 g/L), No. 1 agar (10 g/L) and Congo Red stain (0.8 g/L). Plates of the medium were inoculated and incubated in aerobic environment for 24 h at 37°C. Under such condition, biofilm producers form black crusty colonies on CRA, whereas non-producers form red colonies.
Microtiter Plate Assay for detection of biofilm
Biofilm production was detected using microtiter plate assay, following the procedure described by O’ Toole (O’ Toole 2011). The isolates of S. epidermidis were inoculated in 10 mL of tryptic soy broth with 0.25% glucose and incubated overnight with shaking at 37°C. Next, the cultures were diluted 1:100, and 200 µL of the diluted cultures, per well, were inoculated into 96-well polystyrene microtiter plates. After 48 h incubation at 37°C under aerobic conditions, the plates were washed three times with 300 µL distilled water. Subsequently, the plates were stained with 200 µL of 1% crystal violet, per well, for 10 min. Excess crystal violet was removed by gently washing the plate twice with distilled water. Finally, a volume of 250 µL of 95% ethanol solution, per well, was added to the plate and the optical density was measured at 570 nm. The absorbance of destaining solution was measured at 570 nm in an Elisa reader (Stat fax-2100). A well with sterile TSB or LB served as controls, whereby their ODs were subtracted from that of the experimental strains. The mean OD 570 nm value was determined using four replicates, and was considered to be adherence positive at OD 570 nm greater than or equal to 0.300 high biofilm formation, between 0.200 and 0.299, and adherence negative at OD 570 nm less than 0.100.
Investigation of virulence genes
Primers used genes in Staphylococcus epidermidis
Primer Sequence (5′–3′)
Size of product (bp)
Ica D F: ATGGTCAAGCCCAGACAGAG
Ica D R: CGTGTTTTCAACATTTAATGCAA
The data on production of biofilms by the strains of S. epidermidis was analyzed by the statistical software SPSS® version 19.0 (SPSS Inc., USA). P values were calculated using the Chi square test. P < 0.05 was considered to be statistically significant.
Distribution of 40 biofilm forming strains of Staphylococcus epidermidis according to clinical samples
No. of strains according to degree of biofilm formation
The results of the ELISA readings on 80 isolates of Staphylococcus epidermidis for biofilms production
Number of samples
Number of samples
Number of samples
Number of samples
The capacity of S. epidermidis to produce biofilm is an important factor in infectivity and happens via expression of ica genes. The present study is the first of its kind from the Gulf region dealing with biofilm forming genes expression in clinical isolates of S. epidermidis from Iran. An earlier study from Canada concerned virulence gene expression by biofilm producing strains of S. epidermidis exposed to antibiotics (Gomes et al. 2011). From the results of our study it is evident that the genes responsible for biofilm production are present to a varying degree in the clinical isolates. As can be seen in Table 1, there was a significant relationship between the type of sample and the biofilm production, as tested by Chi square test (P < 0.05). The reactions of biofilm production on Congo red agar, and in microtiter plate assay and PCR assay were largely in agreement, though no statistical analysis was done.
In a previous study, S. epidermidis isolates recovered from catheter segments showed a higher extent of biofilm production than that isolated from urine samples (Gad et al. 2009). In our study, urinary isolates demonstrated a much higher percentage of high biofilm production than that from dialysis catheter (Table 2). However, the overall percentage of biofilm producing strains is much lower in our study than that in the one from Egypt (Gad et al. 2009). Among ica genes, icaA and icaD have been reported to play a significant role in biofilm formation in S. aureus and S. epidermidis (De Silva et al. 2002). It is significant to note that both these genes were demonstrated in our biofilm producing strains of S. epidermidis. Further research is needed to contribute to the development of biomaterials and physical electrical barriers to impede bacterial colonization, and also novel strategies for therapeutic intervention.
Congo red agar
coagulase negative staphylococci
production of poly-N-acetylglucosamine
- S. epidermidis :
polymerase chain reaction
All authors had participated equally. All authors read and approved the final manuscript.
This research was supported by Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.
Compliance with ethical guidelines
Competing interests The authors declared that they have no competing interests.
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- Arciola CR, Gamberini S, Campoccia D, Visai L, Speziale P, Baldassarri L et al (2005) A multiplex PCR method for the detection of all five individual genes of ica locus in Staphylococcus epidermidis. A survey on 400 clinical isolates from prosthesis-associated infections. J Biomed Mater Res A 75:408–413PubMedView ArticleGoogle Scholar
- Asadollahi Dehkordi A, Tajbakhsh E, Tajbakhsh F, Khamesipour F, Momeni Shahraki M, Momeni H (2015) Molecular typing of staphylococcus aureus strains from Iranian raw milk and dairy products by coagulase gene polymorphisms. Adv Stud Biol 7:169–177Google Scholar
- Bien J, Sokolova O, Boxko P (2011) Characterization of virulence factors of Staphylococcus aureus: novel function of known virulence factors that are implicated in activation of airway epithelial proinflammatory response. J Pathogens Article ID 601905: p 13
- Fev PD, Olson ME (2010) Current concepts in biofilm formation of Staphylococcus epidermidis. Future Microbiol 5:917–933View ArticleGoogle Scholar
- Freeman DJ, Falkiner FR, Keane CT (1989) New method for detecting slime production by coagulase negative staphylococci. J Clin Pathol 42:872–874PubMed CentralPubMedView ArticleGoogle Scholar
- Gad GFM, El-Feky MA, El-Rehewy MS, Amin M, Hassan I, Abolella H et al (2009) Detection of icaA, icaD genes and biofilm production by Staphylococcus aureus and Staphylococcus epidermidis isolated from urinary tract catheterized patients. J Infect Dev Ctries 3:342–351PubMedGoogle Scholar
- Gomes F, Teixeira P, Cerca N, Ceri H, Oliveira R (2011) Virulence gene expression by Staphylococcus epidermidis biofilm cells exposed to antibiotics. Microb Drug Resist 17:191–196. doi:10.1089/mdr.2010.0149
- Kumar JD, Negi YK, Gaur A, Khanna D (2009) Detection of virulence genes in Staphylococcus aureus isolated from paper currency. Int J Infect Dis 13:e450PubMedView ArticleGoogle Scholar
- Namvar AE, Bastarahang S, Abbasi N, Ghehi GS, Farhadbakhtiarian S, Arezi P et al (2014) Clinical characteristics of Staphylococcus epidermidis: a systematic review. GMS Hyg Infect Control 9: doi:10.3205/dgkh000243.eCollection2014
- O’ Toole GA (2011) Microtiter dish biofilm formation assay. J Vis Exp 2011:2437–2438Google Scholar
- Oto M (2009) Staphylococcus epidermidis—the “accidental” pathogen. Nat Rev Micobiol 7:555–567View ArticleGoogle Scholar
- Rogers KL, Fey PD, Rupp ME (2009) Coagulase-negative staphylococcal infections. Infect Dis Clin North Am 23:73–98PubMedView ArticleGoogle Scholar
- Sambrook J, Russell DW (2001) Molecular Cloning. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- Silva De, Silva GDI, Kantzanou M, Justice A, Massey RC, Wilkinson AR et al (2002) The ica operon and biofilm production in coagulase-negative staphylococci associated with carriage and disease in a neonatal intensive care unit. J Clin Microbiol 40:382–388PubMed CentralPubMedView ArticleGoogle Scholar
- Uckay I, Pittet D, Vaudaux P, Sax H, Lew D, Waldvogel F (2009) Foreign body infections due to Staphylococcus epidermidis. Ann Med 41:109–119PubMedView ArticleGoogle Scholar