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Genetic Analysis of Pseudomonas aeruginosa by Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (PCR) and Arbitrarily Primed PCR: Gel Analysis Compared with Microchip Gel Electrophoresis

  • Roudabeh J. Jamasbi (a1), Stephen J. Kennel (a2), Larry C. Waters (a2), Linda J. Foote (a2) and J. Michael Ramsey (a2)...

Abstract

Objectives:

To assess the applicability of a newly emerging microchip gel electrophoresis for rapid strain differentiation among clinical isolates of Pseudomonas aeruginosa, and to compare this technique with the traditional gel method for DNA separation.

Methods:

One hundred clinical strains of P. aeruginosa obtained from a hospital in northwestern Ohio were tested for reactivity to 3 serotype-specific monoclonal antibodies by enzyme-linked immunosorbent assay. Twelve strains (4 from each serogroup) were selected for DNA analysis by polymerase chain reaction (PCR)-based, single primer DNA fingerprinting methods with 3 different primers: 1 enterobacterial repetitive intergenic consensus PCR and 2 arbitrarily primed PCRs. The PCR products were analyzed by agarose slab gel and microchip gel electrophoresis.

Results:

Of the 100 clinical isolates tested, 39% (4%, 14%, and 21%) were found to be serotypes 0:3, 0:6, and 0:11, respectively. Twelve strains were chosen for DNA analysis by PCR. The PCR products were analyzed by agarose slab gel electrophoresis and on microchips to determine interspecies diversity. Both methods demonstrated that different serotypes exhibited different electrophoretic patterns. Two strains (clinical strains 6 and 7, serotype 0:6) showed identical patterns, indicating a high degree of relatedness.

Conclusion:

In all cases, there was concordance between the electrophoretic patterns detected by the two methods. The capability of conducting both PCR and microchip gel electrophoresis offers an opportunity for an automated and rapid method for genetic analysis and differentiation among strains of P. aeruginosa and other microorganisms.

Copyright

Corresponding author

Department of Public and Allied Health, 504 Life Sciences Building, Bowling Green State University, Bowling Green, OH 43403

References

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1.Boukadida, J, Montolenbert, M, Gaillard, JL, Berche, P. Outbreak of gut colonization by Pseudomonas aeruginosa in immunocompromised children undergoing total digestive decontamination: analysis by pulsed-field electrophoresis. J Clin Microbiol 1991;29:20682071.
2.Kielhofner, M, Almar, RL, Hamill, RJ, Musher, DM. Life-threatening Pseudomonas aeruginosa infections in patients with human immunodeficiency virus infections. Clin Infect Dis 1992;14:403411.
3.Shepp, DH, Tang, IT, Ramundo, MB, Kaplan, MH. Serious Pseudomonas aeruginosa infection in AIDS. J Acquir Immune Defic Syndr 1994;7:823831.
4.Wiblin, RT. Nosocomial pneumonia. In: Wenzel, RP, ed. Prevention and Contrat of Nosocomial Infections, ed. 3. Baltimore: Williams & Wilkins; 1997:807819.
5.Pollack, M. Pseudomonas aeruginosa. In: Mandell, GL, Benet, JE, Dolin, R, eds. Principles and Practice of Infections Diseases, ed. 4. New York: Churchill Livingston; 1995:19802003.
6.Gordon, SM, Serkey, JM, Keys, TF, et al.Secular trends in nosocomial bloodstream infections in a 55-bed cardiothoracic intensive care unit. Ann Thorac Surg 1998;65:95100.
7.Kluytmans, J. Surgical infections including burns. In: Wenzel, RP, ed. Prevention and Control of Nosocomial Infections, ed. 3. Baltimore: Williams & Wilkins; 1997:841865.
8.Masuda, N, Sakagawa, E, Ohya, S. Outer membrane proteins responsible for multiple drug resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 1995;39:645649.
9.Liu, PV. Changes in somatic antigens of P. aeruginosa induced by bacteriophages. J Infect Dis 1969;119:237246.
10.Pitt, TL. Epidemiological typing of Pseudomonas aeruginosa. Eur J Clin Microbiol Infect Dis 1988;7:238247.
11.Poh, CL, Yeo, CC. Recent advances in typing of Pseudomonas aeruginosa. J Hosp Infect 1993;24:175181.
12.Liu, PV, Matsumoto, H, Kusama, H, Bergan, T. Survey of heat-stable major somatic antigens of Pseudomonas aeruginosa. Int J Syst Bacteriol 1983;33:256264.
13.Liu, PV, Wang, S. Three new major somatic antigens of Pseudomonas aeruginosa. J Clin Microbiol 1990;28:922925.
14.Jamasbi, RJ. Frequency and distribution of Pseudomonas aeruginosa serotypes 0:3, 0:6, 0:11 in three northwestern Ohio hospitals as determined by ELISA using specifie monoclonal antibodies. Ohio Journal of Science 1999;99:1015.
15.Lam, JS, MacDonald, LA, Lam, MYC. Production of monoclonal antibodies against serotype strains of Pseudomonas aeruginosa. Infect Immun 1987;55:28542856.
16.Welsh, J, McClelland, M. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res 1990;18:72137218.
17.Hernandez, J, Ferrus, MA, Hernandez, M, Owen, RJ. Arbitrary primed PCR fingerprinting and serotyping of clinical Pseudomonas aeruginosa strains. FEMS Immunol Med Microbiol 1997;17:3747.
18.Lau, YJ, Liu, PYF, Hu, BS, et al.DNA fingerprinting of Pseudomonas aeruginosa serotype 0:11 by enterobacterial repetitive intergenic con-sensus-polymerase chain reaction and pulsed-field gel electrophoresis. J Hosp Infect 1995;31:6166.
19.Nociari, MM, Catalano, M, Garcia, DC, Copenhaver, SC, Vasil, ML, Sordelli, DO. Comparative usefulness of ribotyping, exotoxin A genotyping, and SalI restriction fragment length polymorphism analysis for Pseudomonas aeruginosa lineage assessment. Diagn Microbiol Infect Dis 1996;24:179190.
20.Waters, LC, Jacobson, SC, Kroutchinina, N, Khandurina, J, Foote, RS, Ramsey, JM. Microchip device for cell lysis, multiplex PCR amplification, and electrophoretic sizing. Anal Chem 1998;70:158162.
21.Waters, LC, Jacobson, SC, Kroutchinina, N, Khandurina, J, Foote, RS, Ramsey, JM. Multiple sample PCR amplification and electrophoretic analysis on a microchip. Anal Chem 1998;70:51725176.
22.Hjerten, S. Capillary coating with linear polyacrylamide for protein separation. J Chromatogr 1985;347:191.
23.Jacobson, SC, Hergenröder, R, Koutny, LB, Warmack, RJ, Ramsey, JM. Effects of injection schemes and column geometry on the performance of microchip electrophoresis devices. Anal Chem 1994;66:11071113.
24.Jacobson, SC, Ramsey, JM. Integrated microdevice for DNA restriction fragment analysis. Anal Chem 1996;68:720723.
25.Pitt, TL, MacDougal, TLJ, Penkothm, ARL, Cooke, EM. Poly-agglutinating and non-typable strains of Pseudomonas aeruginosa in cystic fibrosis. J Med Microbiol 1996;21:179186.
26.Kersulyte, D, Struelens, MJ, Deplano, A, Berg, DE. Comparison of arbitrarily primed PCR and macrorestriction (pulsed-field gel electrophoresis) typing of Pseudomonas aeruginosa strains from cystic fibrosis patients. J Clin Microbiol 1995;33:22162219.
27.Olive, DM, Bean, P. Principal and application of methods for DNA-based typing of microbial organisms. J Clin Microbiol 1999;37:16611669.
28.Webster, CA, Towner, KJ, Humphreys, H, Ehrenstein, B, Harutng, D, Grundman, H. Comparison of rapid automated laser fluorescence analysis of DNA fingerprints with four other computer-assisted approaches for studying relationships between Acinetobacter baumannii isolates. J Med Microbiol 1996;44:185194.
29.Bej, AK, Mahbubani, MH, Dicesare, JL, Atlas, RM. Polymerase chain reactions-gene probe detection of microorganisms by using filter-concentrated samples. Appl Environ Microbiol 1991;57:35293534.
30.Way, JS, Josephson, KL, Pillai, SD, Abbazadegan, M, Gerba, CP, Pepper, IL. Specifie detection of Salmonella spp. by multiplex polymerase chain reaction. Appl Environ Microbiol 1993;59:14731479.
31.Ronai, Z, Barta, C, Sasvari-Szekely, M, Guttman, A. DNA analysis on electrophoretic microchips: effect of operational variables. Electrophoresis 2001;22:294299.
32.Tian, H, Hiihmer, AFR, Landers, JP. Evaluation of silica resins for direct and efficient extraction of DNA from complex biological matrices in a miniaturized format. Anal Biochem 2000;283:175191.
33.Tian, H, Janquins-Gerst, A, Munro, N, Turcco, M, Brody, LC, Landers, JP. Single-strand conformation polymorphism analysis by capillary and microchip electrophoresis: a fast, simple method for detection of com-mon mutation in BRACA1, and BRAC2. Genomics 2000;63:2534.

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