Banu SANCAK, Ebru US, Z. Ceren KARAHAN, İştar DOLAPÇI, Alper TEKELİ, Gülşen HASÇELİK

Çeşitli klinik enfeksiyonlardan elde edilen Staphylococcus aureus izolatlarında insersiyon sekansı IS256’nın biofilm oluşumu üzerine etkisi

Ica (intersellüler adezyon) operonunu kodlayan enzimler tarafından polisakkarit intersellüler adezin (PIA) üretimi, stafilokoklardaki biofilm gelişiminin şu ana kadar en iyi anlaşılmış mekanizmasıdır. IS256, ica operonuna geri dönüşümlü olarak eklenmek suretiyle ica operonu ekspresyonunu ve sonrasında da biyofilm oluşumunu etkileme kapasitesine sahip mobil bir elemandır. Bu çalışmanın amacı 1998-2004 yılları arasında çeşitli klinik örneklerden elde edilen 207 Staphylococcus aureus izolatında insersiyon sekansı IS256’nın biofilm oluşumu üzerine etkisini araştırmaktır. 1998-2004 yılları arasında Ankara ve Hacettepe Üniversitelerinde yatan hastalara ait çeşitli klinik örneklerden elde edilen 207 S. aureus izolatı (kan, kateter, solunum yolu örnekleri, steril vucut sıvıları, yara, balgam, idrar, püy) çalışmaya dahil edildi. Çalışılan suşların 28’i (%13.5) metisilin duyarlı (MSSA),133’ü (%64.2) metisilin dirençli (MRSA), 46’sı (%22.2) ise hetero-VISA (vankomisine heterojen olarak orta duyarlı S. aureus) suşlarını içeren izolatlar idi. Stafilokok kültürlerinden genomik DNA ekstrakte edildikten sonra amplifikasyon kalıbı olarak kullanıldı. Biyofilm genleri (icaA, icaD) ve IS256 yı saptamak amacıyla simpleks PCR lar yapıldı. 207 suş içinde icaA pozitifliği %89.4 (185 izolat), icaD pozitifliği %97.1 (201 izolat), biofilm genlerinin her ikisinin de pozitifliği: %88.9 (184 izolat), sadece birinin pozitifliği %8.7 (18 izolat) oranında bulundu.Biyofilm genleri (ica) MRSA ve hVISA izolatlarında, MSSA izolatlarına göre daha yüksek oranda saptandı(p< 0.001). IS256 pozitifliği hVISA suşlarında MRSA’dan, MRSA suşlarında ise MSSA’dan daha yüksek oranda tesbit edildi (p

The role of insertion sequence IS256 in biofilm formation among various clinical samples of Staphylococcus aureus isolates

Biofilms consist of a cell-multilayer embedded in a slime matrix and they are the basis for production of polysaccharide adhesion by ica-operon-encoded enzymes is currently the best understood mechanism of biofilm development in staphylococci. The insertion sequence IS256, a mobile element frequently associated to transposons has the capacity to influence expression of the ica operon and subsequent biofilm formation by reversible insertion into the ica operon. We aimed to elucidate the role of insertion sequence IS256 in biofilm formation among various Staphylococcus aureus isolates from clinical specimens of hospitalized patients at Ankara and Hacettepe Universities. A total of 207 S. aureus strains isolated between 1998 and 2004 from clinical specimens were included in the study. A total of 28 of the S. aureus isolates studied were methicillin-susceptible S. aureus (MSSA), 133 were methicillin-resistant S. aureus (MRSA), and 46 were vancomycin-intermediate S. aureus (hetero-VISA) isolates. Genomic DNA was extracted from staphylococcal cultures and used as an amplification template with the primers in simplex PCRs for detection of biofilm genes (icaA, icaD), IS256. Of all S. aureus isolates studied 185 (89.4%) were positive for icaA, 201 (97.1%) were positive for icaD, 184 (88.9%) were positive for both whereas 18 (8.7%) strains were positive for only one of them. We found the positivity rates of biofilm genes (ica genes) among MRSA and hetero-VISA isolates were significantly higher than those among MSSA isolates (p< 0.001). IS256 positivity among MRSA and hetero-VISA isolates were also significantly higher than those among MSSA isolates (p

PDF

___

1. Petrelli D, Zampaloni C, D’Ercole S, et al. Analysis of different genetic traits and their association with biofilm formation in Staphylococcus epidermidis isolates from central venous catheter infections. Eur J Clin Microbiol Infect Dis 2006, 25:773-81.
2. Cramton SE, Gerke C, Schnell NF, et al (1999) The intercellular adhesion (ica) locus is present in Staphylococcus aureus and is required for biofilm formation. Infect Immun 1999; 67:5427–33
3. 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; 39:2151-6.
4. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science 1999;284:1318–22.
5. Conlon KM, Humphreys H, O’Gara JP. Inactivations of rsbU and sarA by IS256 represent novel mechanisms of biofilm phenotypic variation in Staphylococcus epidermidis. J Bacteriol 2004;186:6208-19.
6. Kwon AS, Park GC, Ryu SY, et al Higher biofilm formation in multidrug-resistant clinical isolates of Staphylococcus aureus. Int J Antimicrob Agents 2008; 32:68-72
7. Ziebuhr W, Dietrich K, Trautmann M, et al M. Chromosomal rearrangements affecting biofilm production and antibiotic resistance in a Staphylococcus epidermidis strain causing shunt-associated ventriculitis. Int J Med Microbiol 2000;290:115–20.
8. Kiem S, Oh WS, Peck KR, et al. Phase variation of biofilm formation in Staphylococcus aureus by IS256 insertion and its impact on the capacity adhering to polyurethane surface. J Korean Med Sci 2004;19:779–82.
9. Kozitskaya S, Cho SH, Dietrich K, et al The bacterial insertion sequence element IS256 occurs preferentially in nosocomial Staphylococcus epidermidis isolates: association with biofilm formation and resistance to aminoglycosides. Infect Immun 2004;72:1210–15.
10. Montanaro L, Campoccia D, Pirini V, Antibiotic multiresistance strictly associated with IS256 and ica genes in Staphylococcus epidermidis strains from implant orthopedic infections. J Biomed Mater Res A 2007 1;83:813-8
11.Kozitskaya S, Olson ME, Fey PD, et al Clonal analysis of Staphylococcus epidermidis isolates carrying or lacking biofilm-mediating genes by multilocus sequence typing. J Clin Microbiol 2005;43:4751-7.
12 Gu J, Li H, Li M, et al Bacterial insertion sequence IS256 as a potential molecular marker to discriminate invasive strains from commensal strains of Staphylococcus epidermidis. J Hosp Infect 2005; 61:342–348
13. Maki H, McCallum N, BischoffM,et al tcaA inactivation increases glycopeptide resistance in Staphylococcus aureus. Anti microb Agents Chemother 2004;48:1953–9.
14. Maki H, Murakami K. Formation of potent promoters of the mutant llm gene by IS256 transposition in methicillin-resistant Staphylococcus aureus. J Bacteriol 1997;179:6944– 8.
15. Vasudevan P, Nair MK, Annamalai T, et al Phenotypic and genotypic characterization of bovine mastitis isolates of Staphylococcus aureus for biofilm formation. Vet Microbiol 2003;92:179-85.
16. Ammendolia MG, Rosa RD, Montanaro L, et al Slime production and expression of the slime-associated antigen by staphylococcal isolates. J Clin Microbiol 1999;37:3235–8.
17. Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet 2001;358:135–8.
18. Frebourg NB, Lefebvre S, Baert S, et al PCRbased assay for discrimination between invasive and contaminating Staphylococcus epidermidis strains. J Clin Microbiol 2000; 38:877–80
19. Rohde H, Kalitzky M, Kröger N, et al Detection of virulence-associated genes not useful for discriminating between invasive and commensal Staphylococcus epidermidis strains from a bone marrow transplant unit. J Clin Microbiol 2004; 42:5614–19
20. Koskela A, Nilsdotter-Augustinsson A, Persson L, et al Prevalence of the ica operon and insertion sequence IS256 among Staphylococcus epidermidis prosthetic joint infection isolates. Eur J Clin Microbiol Infect Dis 2009;28:655-60.
21. Bradford R, Abdul Manan R, Daley AJ, et al Coagulase-negative staphylococci in verylow- birth-weight infants: inability of genetic markers to distinguish invasive strains from blood culture contaminants. Eur J Clin Microbiol Infect Dis 2006; 25:283–90.
22. Jones SM, Morgan M, Humphrey TJ, et al H. Effect of vancomycin and rifampicin on methicillin-resistant Staphylococcus aureus biofilms. Lancet 2001;357:40–1.
23. Götz F. Staphylococcus and biofilms. Mol Microbiol 2002;43:1367-78.