A PCR-mediated method for discrimination of Klebsiella oxytoca between closely related bacteria in environmental and clinical specimens

Kovtunovych, G. L.; Lytvynenko, T.; Negrutska, V. V.; Lar, O. V.; Koltukova, N. V.; Kozyrovska, N. O.

doi:10.7124/bc.00067D

Biopolym. Cell. 2003; 19(6):520-524.

http://dx.doi.org/10.7124/bc.00067D

Molecular and Cell Biotechnologies

A PCR-mediated method for discrimination of Klebsiella oxytoca between closely related bacteria in environmental and clinical specimens

1Kovtunovych G. L., 1Lytvynenko T., 1Negrutska V. V., 1Lar O. V., 2Koltukova N. V., 1Kozyrovska N. O.

Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
Gromashevsky L.V. Institute of Epidemiology and Infection Diseases, AMS of Ukraine
5, Amosova Str., Kyiv, Ukraine, 03038

Abstract

A specific detection method was developed to discriminate Klebsiella oxytoca between other species of the genus Klebsiella on the basis of PCR amplification of the unique DNA sequences within the polygalacturonase-encoding (pehX) gene. Four primers have been designed for performing PCRs gaining amplicons of 282, 344, 451 and 513 bp. The specificity of the test was verified by the lack of PCR products in case of related K. pneumoniae, K. planticola, and polygalacturonate-degrading species of the genus Erwinia. The PCR-mediated test gives a rapid answer, concerning the presence of K. oxytoca in a sample, or in differentiating this bacterium from other species, such as K. pneumoniae, with which they can be confused. The diagnostic test can be used in ecological monitoring of K. oxytoca as well as in medical laboratories.

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References

[1] Mahl MC, Wilson PW, Fife MA, Ewing WH. Nitrogen fixation by members of the tribe Klebsielleae. J Bacteriol. 1965;89:1482-7.

[2] Dixon RA, Postgate JR. Transfer of nitrogen-fixation genes by conjugation in Klebsiella pneumoniae. Nature. 1971;234(5323):47-8.

[3] Streicher S, Gurney E, Valentine RC. Transduction of the nitrogen-fixation genes in Klebsiella pneumoniae. Proc Natl Acad Sci U S A. 1971;68(6):1174-7.

[4] Eady RR, Smith BE, Cook KA, Postgate JR. Nitrogenase of Klebsiella pneumoniae. Purification and properties of the component proteins. Biochem J. 1972;128(3):655-75.

[5] Hirota Y, Fujii T, Sano Y, Iyama S. Nitrogen fixation in the rhizosphere of rice. Nature. 1978;276(5686):416–7.

[6] Kozyrovska N, Alexeyev M, Kovtunovych G, Gunkovska N, Kordyum V. Survival of Klebsiella oxytoca VN13 engineered to bioluminescence on barley roots during plant vegetation. Microbial Releases. 1994. 2: 262-5.

[7] Reiter B, B?rgmann H, Burg K, Sessitsch A. Endophytic nifH gene diversity in African sweet potato. Can J Microbiol. 2003;49(9):549-55.

[8] Chelius MK, Triplett EW. Immunolocalization of dinitrogenase reductase produced by Klebsiella pneumoniae in association with Zea mays L. Appl Environ Microbiol. 2000;66(2):783-7.

[9] Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev. 1998;11(4):589-603.

[10] Orskov I. Genus Klebsiella II Bergey's manual of determinative bacteriology. Eds R. E. Buchanau, N. E. Gibson. Baltimore: Williams and Wilkins Co. publ., 1974: 321-4.

[11] Jain K, Radsak K, Mannheim W. Differentiation of the Oxytocum Group from Klebsiella by Deoxyribonucleic Acid-Deoxyribonucleic Acid Hybridization. Int J Syst Bacteriol. 1974;24(4):402–7.

[12] Ohkusu K. Cost-effective and rapid presumptive identification of gram-negative bacilli in routine urine, pus, and stool cultures: evaluation of the use of CHROMagar orientation medium in conjunction with simple biochemical tests. J Clin Microbiol. 2000;38(12):4586-92.

[13] Hibbert-Rogers LC, Heritage J, Gascoyne-Binzi DM, Hawkey PM, Todd N, Lewis IJ, Bailey C. Molecular epidemiology of ceftazidime resistant Enterobacteriaceae from patients on a paediatric oncology ward. J Antimicrob Chemother. 1995;36(1):65-82.

[14] Pat. USA WO 0123604-A 1929 05.04. Highly conserved genes and their use to generate probes and primers for detection of microorganisms. M. G. Bergeron, M. Boissinot, A. Huletsky, C. Nard, M. Ouellette, J. Picard, P. H. Roy. Publ. 2001.

[15] Steffensen SA, Poulsen AB, Mortensen KK, Sperling-Petersen HU. E. coli translation initiation factor IF2--an extremely conserved protein. Comparative sequence analysis of the infB gene in clinical isolates of E. coli. FEBS Lett. 1997;419(2-3):281-4.

[16] Pat. USA WO 0123606-A 144 05.04. Nucleic acid molecules for detecting bacteria and phylogenetic units of bacteria. R. Grabowski, K. Berghof. Publ. 2001.

[17] Anthony RM, Brown TJ, French GL. Rapid diagnosis of bacteremia by universal amplification of 23S ribosomal DNA followed by hybridization to an oligonucleotide array. J Clin Microbiol. 2000;38(2):781-8.

[18] Brisse S, Verhoef J. Phylogenetic diversity of Klebsiella pneumoniae and Klebsiella oxytoca clinical isolates revealed by randomly amplified polymorphic DNA, gyrA and parC genes sequencing and automated ribotyping. Int J Syst Evol Microbiol. 2001;51(Pt 3):915-24.

[19] Monnet D, Freney J. Method for differentiating Klebsiella planticola and Klebsiella terrigena from other Klebsiella species. J Clin Microbiol. 1994;32(4):1121-2.

[20] Starr MP, Chatterjee AK, Starr PB, Buchanan GE. Enzymatic degradation of polygalacturonic acid by Yersinia and Klebsiella species in relation to clinical laboratory procedures. J Clin Microbiol. 1977;6(4):379-86.

[21] Kovtunovich GV, Lar OV, Kozyrovska NO. Cloning and structural analysis of the Klebsiella oxytoca VN13 peh gene. Biopolym Cell. 2000;16(5):356-362.