An investigation of type 3 secretion toxins encoding-genes of Pseudomonas aeruginosa isolates in a University Hospital in Egypt

Yıl 2013, Cilt: 3 Sayı: 03, 116 - 122, 01.09.2013

Awny A. Gawish Nahla A Mohamed Gehan A El-shennawy Heba A. Mohamed

https://doi.org/10.5799/ahinjs.02.2013.03.0093

Cited By: 3

Öz

Objective: Pseudomonas aeruginosa uses type 3 secretion system (T3SS) to directly inject four secretion toxins into host cells. The aim of the study was to determine the prevalence of thee secretion toxins-encoding genes among P. aeruginosa isolates collected from nosocomial infections and environmental sources. Methods: Clonal relatedness of the studied P. aeruginosa isolates (n=85) was studied by RAPD typing. Multiplex PCR was performed on the 85 P. aeruginosa isolates (68 nosocomial isolates obtained from hospitalized patients present in different wards and departments, and 17 environmental isolates including 2 isolates from health-care worker hands from ICU) to detect the secretion toxins-encoding genes. Results: RAPD fingerprinting demonstrated that most strains were of distinct genotypes and determined the presence of 54 RAPD patterns. The prevalence of the genes among all isolates was as follows; exoT-100%, exoS- 40%, exoY- 83.5% and exoU- 62.4%. No significant differences in the prevalence of these genes were observed between nosocomial and environmental isolates or between isolates cultured from different sites of infection. The part of P. aeruginosa strains harboring either exoS (37.6 %) or exoU (60 %) gene was significantly higher (P<0.001) than that contained both genes (2.4 %). Concolusion: ExoT was present in all isolates and exoS, exoY and exoU are variable traits with exoU gene was the third in prevalence after exoT and exoY. No significant differences in exoS, exoY and exoU prevalence were observed between nosocomial and environmental isolates or between isolates cultured from different sites of infection. ExoS and exoU genes are mutually exclusive that almost all isolates contain either of both genes not both.

Anahtar Kelimeler

Pseudomonas aeruginosa, type 3 secretion toxins, multiplex PCR

An investigation of type 3 secretion toxins encoding-genes of Pseudomonas aeruginosa isolates in a University Hospital in Egypt

Öz

Amaç: Pseudomonas aeruginosa tip 3 sekresyon sistemiyle (T3SS ) direk dokuya salınan 4 sekretuar toksin kullanır. Bu çalışmanın amacı hastane enfeksiyonlarından ve çevresel kaynaklardan izole edilen P. aeruginosa izolatlarında sekretuar toksin kodlayan üç genin prevalansını saptamaktır. Yöntemler: Çalışılan P. aeruginosa izolatlarının klonal yakınlığı RAPD tiplendirme metodu ile bakılmıştır. Sekretuar toksin kodlayan genleri saptamak için 85 P. aeruginosa izolatında (farklı klinik ve bölümlerde yatan hastalardan izole edilen 68 hastane kaynaklı izolatı ve ikisi yoğun bakım ünitesindeki sağlık çalışanının ellerinden izole edilen 17 çevresel izolat) multiplex PCR çalışıldı. Bulgular: RAPD fingerprint analizi ile varlığı tespit edilen 54 RAPD paterninin varlığı suşların çoğunun uzak genotiplere sahip olduğunu gösterdi. Tüm izolatlarda saptanan genlerin prevalansı sırasıyla exoT (% 100), exoS (% 40), exoY (% 83,5) ve exoU (% 62,4) şeklindeydi. Farklı enfeksiyon bölgelerinden üretilen izolatlar arasında yada hastane ve çevresel kaynaklardan elde edilen izolatlar arasında bu genlerin prevalansı açısından anlamlı bir fark yoktu. P. aeruginosa suşlarından exoS (% 37,6) veya exoU (% 60) genlerinden birini taşıyanlar her iki geni taşıyanlardan (% 2,4) anlamlı olarak daha fazlaydı (p<0.001). Sonuç: ExoT tüm izolatlarda bulunurken exoS, exoY ve exoU genlerinin varlığı değişkenlik gösteriyordu. ExoU geni exoT ve exoY geninden sonra üçüncü sıklıkta görülmekteydi. exoS, ExoY ve exoU görülme prevalansı hastane kaynaklı ve çevre kaynaklı suşlar arasında veya değişik bölge veya enfeksiyonlar arasında anlamlı bir farklılık göstermedi. ExoS ve exoU genleri hemen tüm izolatlarda ya birlikte bulunmakta veya iki de bulunmamakta idiler

Anahtar Kelimeler

Pseudomonas aeruginosa, type-3 sekresyon toksinleri, multiplex PCR

Kaynakça

• 1. Engel J, Balachandran P. Role of Pseudomonas aeruginosa type III effectors in disease. Current Opin Microbiol 2009;12:61-66.
• 2. Bradbury RS, Roddam LF, Merritt A, et al. Virulence gene distribution in clinical, nosocomial and environmental isolates of Pseudomonas aeruginosa. J Med Microbiol 2010;59:881- 890.
• 3. Haucer A. The type III secretion system of pseudomonas aeruginosa: infection by injection. Nat Rev Microbiol 2009;7:654- 665.
• 4. Stover CK, Pham XQ, Ewin AL, et al. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 2000;406:956-964.
• 5. Engel JN. Molecular pathogenesis of acute Pseudomonas aeruginosa infections. In: Hauser AR, Rello J, eds. Severe infections caused by Pseudomonas aeruginosa. Dordrech, Kluwer Academic Publishers, 2003:201-229.
• 6. Lee VT, Smith RS, Tummler B, Lory D. Activities of Pseudomonas aeruginosa effectors secreted by the type III secretion system in vitro and during infection. Infect Immun 2005;73:1695-1705.
• 7. Vance RE, Rietsch A, mekalanos JJ. Role of the type III secreted exoenzymes S, T and Y in systemic spread of Pseudomonas aeruginosa Pa01 in vivo. Infect Immun 2005;73:1706- 1713.
• 8. Shaver CM, Haucer AR. Relative contribution of Pseudomonas aeruginosa exoU, exoS and exoT to virulence in the lung. Infect Immun 2004;72:6969-6977.
• 9. Shafikhani SH, Morales C, Engel J. The Pseudomonas aeruginosa type III secreted toxin exoT is necessary and sufficient to induce apoptosis in epithelial cells. Cell Microbiol 2008;10:994-1007.
• 10. Lin HH, Huang SP, Teng HC, et al. Presence of exoU gene of Pseudomonas aeruginosa is correlated with cytotoxicity in MDCK cells but not with colonization in BALB ̸c mice. J Clin Microbiol 2006;44:4596-4597.
• 11. Wong-Beringer R, Wiener-Kronish J, Lynch S, Flanagan J. Comparison of type III secretion system virulence among fluoroquinolon-susceptible and –resistant clinical isolates of Pseudomonas aeruginosa. Clin Microbiol Infect 2008;14:330-336.
• 12. Feltman H, Schulert G, Khan S, et al. Prevalence of type lll secretion genes in clinical and environmental isolates of Pseudomonas aeruginosa. Microbiol 2001;147:2659-2669.
• 13. Wilfinger WW, Mackey M, Chomcynski P. Effect of pH and ionic strength on the spectrophotometeric assessment of nucleic acid purity. Bio Techniques 1997;22:474.
• 14. Strateva T, Markova B, Ivanova D, Mitove I. Distribution of the type III effector proteins-encoding genes among nosocomial Pseudomonas aeruginosa isolates from Bulgaria. Ann Microbiol 2010;60:503-509.
• 15. Woods CR, Versalovic J, Koeuth T, Lupski JR. Analysis of relationships among isolates of Citrobacter diversus by using DNA fingerprints generated by repetitive sequence-based primers in the polymerase chain reaction. J Clin Microbiol 1992;30:2921-2929.
• 16. Ajayi T, Allmond LR, Sawa T, Wiener-Kronish JP. Single-nucleotide-polymorphism mapping of the Pseudomonas aeruginosa type III secretion toxins for development of a diagnostic multiplex PCR system. J Clin Microbiol 2003;41:3526-3531.
• 17. Verove J, Bernarde C, Bohn YS, et al. Injection of Pseudomonas aeruginosa exo toxins into host cells can be modulated by host factors at the level of translocon assembly and/or activity. Biol Cancer Infect 2012;7:e30488.
• 18. Lomholt JA, Poulson K, Kilian M. Epidemic population structure of Pseudomonas aeruginosa: evidence for a clone that is pathogenic to the eye and that has a distinct combination of virulence factors. Infect Immun 2001;69:6284-6295.
• 19. Rumbaugh KP, Hamood AN, Griswold JA. Analysis of Pseudomonas aeruginosa clinical isolates for possible variations within the virulence genes exotoxin A and exotoxin S. J Surg Res 1999;82:95-105.
• 20. Fleiszig SMj, Zaidi TS, Preston MJ, et al. Relationship between cytotoxicity and corneal epithelial cell invasion by clinical isolates of Pseudomonas aeruginosa. Infect Immun 1996; 64:2288-2294.
• 21. Dacheux D, Toussaint B, Richard M, et al. Pseudomonas aeruginosa cystic fibrosis isolates induce rapid , type III secretion- dependant, but exoU-independent, oncosis of macrophages and polymorphonuclear neutrophils. Infect Immun 2000;68:2916-24.
• 22. Kulasekara BR, Kulasekara HD, Wolfgang MC, et al. Acquisition and evolution of the exoU locus in Pseudomonas aeruginosa. J Bacteriol 2006;188:4037-4050.
• 23. Winstanley C, Kaye SB, Neal TJ et al. Genotypic and phenotypic characteristics of Pseudomonas aeruginosa isolates associated with ulcerative keratitis. J Med Microbiol 2005;54(Pt6):519-260.
• 24. Allewelt M, Coleman FT, Grout M, et al. Acquisition of expression of the Pseudomonas aeruginosa ExoU cytotoxin leads to increased bacterial virulence in a murine model of acute pneumonia and systemic spread. Infect Immun 2000;68:3998–4004.
• 25. Hirakata Y, Finlay BB, Simpson DA, et al. Penetration of clinical isolates of Pseudomonas aeruginosa through MDCK epithelial cell monolayers. J Infect Dis 2000;181:765-769.
• 26. Wolfgang MC, Kulasekara BR, Liang X, et al. Conservation of genome content and virulence determinants among clinical and environmental isolates of Pseudomonas aeruginosa. PNAS 2003;100:8484-8489.