Molecular Diagnosis of Clinically Isolated Klebsiella pneumoniae Strains by PCR-ELISA

Document Type : Original Article

Authors

1 Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran

2 Department of Biology, Damghan Azad University, Damghan, Iran

3 Imam Hossain University, Faculty of Science, Department of Biology, Tehran, Iran

Abstract

Klebsiella pneumoniae is the most important infectious bacteria in Enterobacteriaceae family and the most common bacteria causing Urinary Tract Infection (UTI) after Escherichia coli. Therefore, accurate and rapid identification of this bacterium in hospital infection is very important.In this study, PCR-ELISA method was used for detecting Klebsiella pneumonia clinical strains. For this purpose, 16S rDNA gene based specific primers were designed and the DIG-labeled PCR products were bound to streptoavidin-coated wells of a microtiter plate and detected by anti-DIG–peroxidase conjugate. Biotin-labeled DNA probe specific for 16S rDNA gene was used in PCR-ELISA.Sensitivity and specificity of PCR-ELISA method were determined by using Enterobacteria strains. 16S rDNA of Klebsiella pneumoniae was amplified using gene specific primers resulted in a fragment of the 260 bp. The results of PCR-ELISA showed that this technique does not cross-react with the bacteria in their families as well as the sensitivity of 6.0 ng were evaluated.PCR-ELISA is known as an accurate and rapid method for detection of the infectious agents and therefore can be used as a suitable substitute for all the above aspects because it is quite a sensitive, specific, and rapid method for detection of the Klebsiella pneumoniae strains.

Keywords

References

  1. De Souza Lopes, A.C., Falcao, R.J., de Morais Junior, M.A. Molecular typing of Klebsiella pneumoniae isolates from public hospitals in Recife, Brazil. Microbiol Res, 2005,Vol. 160, pp.37-46.
  2. Podschun, R., Ullmann, U. Klebsiella spp. as Nosocomial Pathogens. Epidemiology, Taxonomy, Typing Methods, and Pathogenicity Factors. Clin Microbiol Rev, 1998, Vol. 11, pp. 589-603.
  3. Feizabadi, M.M., Raji, N., Delfani,  S. Identification of Klebsiella pneumoniae K1 and K2 Capsular Types by PCR and Quellung Test. Jundishapur J Microbiol, 2013, Vol. 6, pp .e7585.
  4. Jerassy, Z., Yinnon, A.M., Mazouz-Cohen, S., Benenson, S., Schlesinger, Y., Rudensky, B.Prospective hospital-wide studies of 505 patients with nosocomial bacteraemia in 1997 and 2002. J Hosp Infect, 2006, Vol. 62, pp.230-236.
  5. Onori, R., Gaiarsa, S., Comandatore, F., Pongolini, S., Brisse, S., Colombo, A. Tracking Nosocomial Klebsiella pneumoniae Infections and Outbreaks by Whole-Genome Analysis: Small-Scale Italian Scenario within a Single Hospital. J Clin Microbiol, 2015, Vol. 53, pp. 2861-2868.
  6. Lawlor, M.S., Hsu, J., Rick, P.D., Miller, V.L. Identification of Klebsiella pneumoniae virulence determinants using an intranasal infection model. Mol Microbiol , 2005, Vol. 58, pp. 1054-1073.
  7. Wu, K.M., Li, L.H., Yan, J.J., Tsao, N., Liao, T.L., Tsai, H.C. Genome sequencing and comparative analysis of Klebsiella pneumoniae NTUH-K2044, a strain causing liver abscess and meningitis. J Bacteriol, 2009, Vol. 191, pp. 4492-4501.
  8. Liu, P., Li, P., Jiang, X., Bi, D., Xie, Y., Tai, C. Complete genome sequence of Klebsiella pneumoniae subsp. pneumoniae HS11286, a multidrug-resistant strain isolated from human sputum. J Bacteriol, 2012, Vol. 194, pp. 1841-1842.
  9. Coudron, P.E., Moland, E.S., Thomson, K.S.Occurrence and detection of AmpC beta-lactamases among Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates at a veterans medical center. J Clin Microbiol, 2000, Vol. 38, pp. 1791-1796.
  10. Gholipour, A., Soleimani, N., Shokri, D., Mobasherizadeh, S., Kardi, M., Baradaran, A. Phenotypic and Molecular Characterization of Extended-Spectrum beta-Lactamase Produced by Escherichia coli, and Klebsiella pneumoniae Isolates in an Educational Hospital. Jundishapur J Microbiol , 2014, Vol. 7, pp. e11758.
  11. Yong, D., Toleman, M.A., Giske, C.G., Cho, H.S., Sundman, K., Lee, K. Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother, 2009, Vol. 53, pp. 5046-5054.
  12. Kurupati, P., Chow, C., Kumarasinghe, G., Poh, C.L.Rapid detection of Klebsiella pneumoniae from blood culture bottles by real-time PCR. J Clin Microbiol, 2004, Vol. 42, pp. 1337-1340.
  13. Chen, L., Mediavilla, J.R., Endimiani, A., Rosenthal, M.E., Zhao, Y., Bonomo, R.A. Multiplex real-time PCR assay for detection and classification of Klebsiella pneumoniae carbapenemase gene (bla KPC) variants. J Clin Microbiol, 2011, Vol. 49, pp. 579-585.
  14. Mousavi, S.L., Nazarian, S., Amani, J., Rahgerdi, A.K.Rapid screening of toxigenic Vibrio cholerae O1 strains from south Iran by PCR-ELISA. Iran Biomed J , 2008, Vol. 12, pp. 15-21.
  15. Amani, J., Ahmadpour, A., Imani Fooladi, A.A., Nazarian, S. Detection of E. coli O157:H7 and Shigella dysenteriae toxins in clinical samples by PCR-ELISA. Braz J Infect Dis, 2015, Vol. 19, pp. 278-284.
  16. Kim, O.S., Cho, Y.J., Lee, K., Yoon, S.H., Kim, M., Na, H. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol, 2012, Vol. 62, pp. 716-721.
  17. Sue, M.J., Yeap, S.K., Omar, A.R., Tan, S.W. Application of PCR-ELISA in molecular diagnosis. Biomed Res Int, 2014, Vol. 2014, pp. 653014-563020.
  18. Kuo, J.T., Cheng, C.Y,, Huang, H.H., Tsao, C.F., Chung, Y.C. A rapid method for the detection of representative coliforms in water samples: polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA). J Ind Microbiol Biotechnol, 2010, Vol. 37, pp. 237-244.
Journal of Applied Biotechnology Reports
Volume 3, Issue 4
Autumn 2016
Pages 501-505

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History

  • Receive Date: 16 September 2016
  • Accept Date: 16 September 2016

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