Biofilm Formation and Detection of IcaAB Genes in Clinical Isolates of Methicillin Resistant Staphylococcus aureus

Document Type: Original Article

Authors

Microbiology Department, Faculty of Biological Sciences, Shahid Beheshti University, G.C., Evin, Tehran, Iran

Abstract

Objective(s)
Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of nosocomial and community
infections. Biofilm formation, mediated by a polysaccharide intercellular adhesin (PIA) and encoded by the ica operon, is considered to be an important virulence factor in both S. epidermidis and S. aureus. However, the clinical impact of the ica locus and PIA production is less well described in S. aureus. We studied biofilm formation in clinical isolates of MRSA in relation to the presence of the ica operon.
Materials and Methods
Forty five MRSA were studied for biofilm formation by colony morphology on Congo red agar (CRA) and the microtitre plate assay (MtP). Presence of the ica genes was detected by PCR and specific primers.
Results
The results showed that 53.3% of the isolates had the potential to form biofilm by colony morphology of which, 75% carried the ica operon. Weak biofilm production was observed in the MtP assay by 57.8%, of which 53.8% harbored the ica operon. However, about 70% of biofilm non-producers also carried the ica operon.
Conclusion
Overall, there was no agreement between the icaAB gene carriage and biofilm phenotype by either of the two phenotypic methods. However, 91% of biofilm formers on CRA also produced biofilm in the MtP assay.

Keywords


1. Tiemersma EW, Bronzwear LAM, Lyytikainen O, Degener JE, Schrijnemakers P Bruinsma N, et al. Methicillin resistant Staphylococcus aureus in Europe, 1999-2002. Emerg Infect Dis 2004; 10:1627-134.

2. Cramton SE, Gerke C, Schnell NF, Nichols WW, Gotz F. The intercellular adhesin locus (ica) is present in Staphylococcus aureus and is required for biofilm formation. Infect Immun 1999; 67:5427-5433.

3. Gotz F. Staphylococcus and biofilms. Mol Microbiol 2002; 43:1367-1378.

4. Heilmann C, Schweitzer O, Gerke C, Vanittanakom N, Mack D, Götz F. Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis. Mol Microbiol 1996; 20:1083-1091.

5. Arciola CR, Baldassarri L, Montanaro L. Presence of icaA and icaD genes and slime production in a collection of staphylococcal strains from catheter-associated infections. J Clin Microbiol 2001; 39:2151-2156.

6. Fowler VG Jr, Fey PD, Reller LB, Chamis AL, Corey GR, Rupp ME. The intercellular adhesin locus ica is present in clinical isolates of Staphylococcus aureus from bacteremic patients with infected and uninfected prosthetic joints. Med Microbiol Immunol 2001; 189:127-131.

7. Rohde H, Knobloch JK, Horstkotte MA, Mack D. Correlation of biofilm expression types of Staphylococcus epidermidis with polysaccharide intercellular adhesin synthesis: evidence for involvement of icaADBC genotypeindependent factors. Med Microbiol Immun 2001; 190:105-112.

8. Ziebuhr W, Krimmer V, Rachid S, Lössner I, Götz F, Hacker J. A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: evidence for control of the polysaccharide intercellular adhesion synthesis by alternating insertion and excision of the insertion sequence element IS256. Mol Microbiol 1999; 32:345-356.

9. McKenney D, Pouliot K L, Wang Y, Murthy V, Ulrich M, Doring G, Lee JC, Goldmann DA, Pier GB. Broadly protective vaccine for Staphylococcus aureus based on an in vivo-expressed antigen. Science 1999; 284:1523- 1527.

10. Beenken KE, Blevins JS, Smeltzer MS. Mutation of sarA in Staphylococcus aureus limits biofilm formation. Infect Immun 2003; 71:4206-4211.

11. National Committee for Clinical Laboratory Standards (NCCLS). Performance standards for antimicrobial disk susceptibility test. 6th ed. Approved Standard. M100-A6. Wayne, PA, 1997.

12. National Committee for Clinical Laboratory Standard (NCCLS). Performance standards for antimicrobial susceptibility testing. 9th ed. International Supplement. M100-S9. Wayne, PA, 1999.

13. Handke LD, Conlon KM, Slater SR, Elbaruni S, Fitzpatrick F, Humphreys H, et al. Genetic and phenotypic analysis of biofilm phenotypic variation in multiple Staphylococcus epidermidis strains. J Med Microbiol 2004; 53:367-374.

14. Aricola CR, Compoccia D, Baldassarri L, Donati ME, Pirini V, Gamberini S, et al. Detection of biofilm formation in Staphylococcus epidermidis from implant infections. Comparison of a PCR method that recognizes the presence of ica genes with two classic phenotypic methods. J Biomed Mater Res 2005; 76:425-430.

15. Eftekhar F, Speert DP. Biofilm formation by persistent and non-persistent isolates of Staphylococcus epidermidis from a neonatal intensive care unit. J Hosp Infect 2009; 71:112-116.

16. Pérez-Roth E, Claverie-Martín F, Villar J, Méndez-Álvarez S. Multiplex PCR for simultaneous identification of Staphylococcus aureus and detection of methicillin and mupirocin resistance. J Clin Microbiol 2001; 39:4037-4041.

17. Martin-Lopez JV, Perez-Roth E, Claverie-Martin F, Diez-Gil O, Batista N, MoralesM, et al. Detection of Staphylococcus aureus clinical isolates harboring the ica gene cluster needed for biofilm establishment. J Clin Microbiol 2002; 40:1569-1570.

18. Grinholc M, Wegrzyn G, Kurlenda J. Evaluation of biofilm production and prevalence of the icaD gene in methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains isolated from patients with nosocomialinfections and carriers. FEMS Immunol Med Microbiol 2007; 50:375-379.

19. Yazdani R, Oshaghi M, Havayi A, Pishva E, Salehi R, Sadeghizadeh M, et al. Detection of icaAD gene and biofilm formation in Staphylococcus aureus isolates from wound infections. Iran J Publ Health 2006; 35:25-28.

20. Fitzpatrick F, Humphreys H O’Gara JP. Evidence for icaADBC independent biofilm development mechanism in methicillin-resistant Staphylococcus aureus clinical isolates. J Clin Microbiol 2005; 43:1973-1976.

21. Rachid S, Ohlsen K, Wallner U, Hacker J, Hecker M, Ziebuhr W. Alternative transcription factor B is involved in regulation of biofilm expression in a Staphylococcus aureus mucosal isolate. J Bacteriol 2000; 182:6824-6826.

22. Cramton SE, Ulrich M, Götz F, Döring G. Anaerobic conditions induce expression of polysaccharide intercellular adhesin in Staphylococcus aureus and Staphylococcus epidermidis. Infect Immun 2001; 69:4079-4085.

23. O’Neill E, Pozzi C, Houston P, Smyth D, Humphreys H, Robinson DA, et al. Association between methicillin susceptibility and biofilm regulation in Staphylococcus aureus isolates from device-related infections. J Clin Microbiol 2007; 45:1379-1388.

24. Eftekhar F, Mirmohamadi Z. Biofilm formation by clinical and normal skin isolates of Staphylococcus epidermidis. Int J Med Med Sci 2009; 10:438-441.