Serological Classification and Comparison of Cell Surface Hydrophobicity and Biofilm and Proteases Formation between the Clinical and Environmental Isolates of Pseudomonas Aeruoginosa

Authors

1 Graduate Student of Microbiology, Afzalipour School of Medicine, Kerman University of Medical Sciences, Kerman, Iran

2 Professor, Department of Microbiology, Afzalipour School of Medicine, Kerman University of Medical Sciences, Kerman, Iran

Abstract

Background & Aims: Pseudomonas aeruoginosa is an opportunistic pathogen and an important cause of nosocomial infections. Different factors are involved in the pathogenicity of this bacterium. This study was performed to compare some factors associated with the virulence of clinical and environmental isolates of P. aeruoginosa. Methods: The present study was performed on 25 environmental isolates (soil, water) and 100 clinical isolates (blood, urine, wound, burn wound, and body fluids). Serotyping was performed with monovalent and polyvalent antisera. Cell surface hydrophobicity (CSH) was tested through bacterial attachment to hydrocarbons, and biofilm formation was detected through crystal violet staining method. LasA protease and LasB elastase were determined using Gongo red or boiled Staphylococcus aureus suspension as the substrates, respectively. LasR gene was detected using polymerase chain reaction (PCR) method. Results: The most common serotype among the isolates was serotype B (23.52% in clinical and 12% in environmental isolates). Serotype B was more prevalent in clinical isolates, and serotype J was found with a higher frequency in environmental samples. This serotype was not found in clinical samples. Mean production of CSH, biofilm formation, and LasA protease and LasB elastase was higher in the clinical isolates than environmental isolates. The difference between clinical and environmental isolates was significant in the case of LasA protease (P = 0.010). The LasR genes was detected in all clinical and environmental isolates Conclusion: Differences in serotype prevalence and the higher prevalence of LasA in the clinical isolates is an important issue. Owing to the lack of significant differences between clinical and environmental samples in respect to other virulence factors, it seems that the expression of virulence factors could be effected by environmental conditions. Further studies with higher number of isolates and evaluation of virulence gene expression is needed to confirm these results

Keywords


  1. Frank DW. Research Topic on Pseudomonas aeruginosa, Biology, Genetics, and Host-Pathogen Interactions. Front Microbiol 2012; 3: 20.
  2. Karatuna O, Yagci A. Analysis of quorum sensing-dependent virulence factor production and its relationship with antimicrobial susceptibility in Pseudomonas aeruginosa respiratory isolates. Clin Microbiol Infect 2010; 16(12): 1770-5.
  3. Kessler E, Safrin M, Gustin JK, Ohman DE. Elastase and the LasA protease of Pseudomonas aeruginosa are secreted with their propeptides. J Biol Chem 1998; 273(46): 30225-31.
  4. Deptula A, Gospodarek E. Reduced expression of virulence factors in multidrug-resistant Pseudomonas aeruginosa strains. Arch Microbiol 2010; 192(1): 79-84.
  5. Farshad S, Emamghoraishi F, Japoni A. Association of virulent genes hly, sfa, cnf-1 and pap with antibiotic sensitivity in Escherichia coli strains isolated from children with community-acquired UTI. Iran Red Crescent Med J 2012; 12(1): 33-7. [In Persian].
  6. Kustos T, Kustos I, Kilar F, Rappai G, Kocsis B. Effect of antibiotics on cell surface hydrophobicity of bacteria causing orthopedic wound infections. Chemotherapy 2003; 49(5): 237-42.
  7. Kessler E, Safrin M, Blumberg S, Ohman DE. A continuous spectrophotometric assay for Pseudomonas aeruginosa LasA protease (staphylolysin) using a two-stage enzymatic reaction. Anal Biochem 2004; 328(2): 225-32.
  8. Whiteley M, Lee KM, Greenberg EP. Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 1999; 96(24): 13904-9.
  9. Kumar R, Chhibber S, Harjai K. A comparative study of clinical and environmental isolates of Pseudomonas aeruginosa in terms of quorum sensing, outer membrane proteins and their ability to cause urinary tract infection. Am J Biomed Sci 2009; 1(3): 205-14.
  10. Perez LR, Machado AB, Barth AL. The presence of quorum-sensing genes in Pseudomonas isolates infecting cystic fibrosis and non-cystic fibrosis patients. Curr Microbiol 2013; 66(4): 418-20.
  11. Sandoz KM, Mitzimberg SM, Schuster M. Social cheating in Pseudomonas aeruginosa quorum sensing. Proc Natl Acad Sci U S A 2007; 104(40): 15876-81.
  12. Mahon CR, Manuselis G. Non fermenting and mmiscellaneous gram negative bacilli. In: Mahon CR, Manuselis G, editors. Textbook of diagnostic microbiology. 2nd ed. Saunders; 2000. p. 482-503.
  13. Kong KF, Jayawardena SR, Indulkar SD, Del PA, Koh CL, Hoiby N, et al. Pseudomonas aeruginosa AmpR is a global transcriptional factor that regulates expression of AmpC and PoxB beta-lactamases, proteases, quorum sensing, and other virulence factors. Antimicrob Agents Chemother 2005; 49(11): 4567-75.
  14. Pijanowska A, Kaczorek E, Chrzanowski L, Olszanowski A. Cell hydrophobicity of Pseudomonas spp. and Bacillus spp. bacteria and hydrocarbon biodegradation in the presence of Quillaya saponin. World Journal of Microbiology and Biotechnology 2007; 23(5): 677-82.
  15. George M, Pierce G, Gabriel M, Morris C, Ahearn D. Effects of quorum sensing molecules of Pseudomonas aeruginosa on organism growth, elastase B production, and primary adhesion to hydrogel contact lenses. Eye Contact Lens 2005; 31(2): 54-61.
  16. O'Toole GA, Kolter R. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol 1998; 30(2): 295-304.
  17. Stepanovic S, Vukovic D, Dakic I, Savic B, Svabic-Vlahovic M. A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods 2000; 40(2): 175-9.
  18. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-54.
  19. Ciofu O, Mandsberg LF, Bjarnsholt T, Wassermann T, Hoiby N. Genetic adaptation of Pseudomonas aeruginosa during chronic lung infection of patients with cystic fibrosis: strong and weak mutators with heterogeneous genetic backgrounds emerge in mucA and/or lasR mutants. Microbiology 2010; 156(Pt 4): 1108-19.
  20. Bacterial Typing Antisera Handbook Available at :denka-seiken.jp/english/pdf/.bacterial-handbook4th.pdf.version4.October 2006.
  21. Tassios PT, Gennimata V, Maniatis AN, Fock C, Legakis NJ. Emergence of multidrug resistance in ubiquitous and dominant Pseudomonas aeruginosa serogroup O:11. The Greek Pseudomonas Aeruginosa Study Group. J Clin Microbiol 1998; 36(4): 897-901.
  22. Estahbanati HK, Kashani PP, Ghanaatpisheh F. Frequency of Pseudomonas aeruginosa serotypes in burn wound infections and their resistance to antibiotics. Burns 2002; 28(4): 340-8.
  23. Shahcheraghi F, Feizabadi MM, Yamin V, Abiri R, Abedian Z. Serovar determination, drug resistance patterns and plasmid profiles of Pseudomonas aeruginosa isolated from burn patients at two hospitals of Tehran (IRAN). Burns 2003; 29(6): 547-51.
  24. Vitkauskiene A, Skrodeniene E, Jomantiene D, Macas A, Sakalauskas R. Changes in the dependence of Pseudomonas aeruginosa O serogroup strains and their resistance to antibiotics in a university hospital during a 5-year period. Medicina (Kaunas) 2011; 47(7): 361-7.
  25. Yousefi S, Nahaei MR, Farajnia S, Aghazadeh M, Iversen A, Edquist P, et al. A multiresistant clone of Pseudomonas aeruginosa sequence type 773 spreading in a burn unit in Orumieh, Iran. APMIS 2013; 121(2): 146-52. [In Persian].
  26. Bradbury RS, Roddam LF, Merritt A, Reid DW, Champion AC. Virulence gene distribution in clinical, nosocomial and environmental isolates of Pseudomonas aeruginosa. J Med Microbiol 2010; 59(Pt 8): 881-90.
  27. Finnan S, Morrissey JP, O'Gara F, Boyd EF. Genome diversity of Pseudomonas aeruginosa isolates from cystic fibrosis patients and the hospital environment. J Clin Microbiol 2004; 42(12): 5783-92.
  28. Mansouri S, Norouzi F, Moradi M, Nakhaee N. Comparison of virulence factors among clinical isolates of pseudomonas aeruginosa producing and non-producing extended spectrum â-lactamases. Currt Res Bacteriol 2011; 4(3): 85-93. [In Persian].