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Journal of Clinical Microbiology, December 2003, p. 5500-5510, Vol. 41, No. 12
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.12.5500-5510.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Culture-Independent Identification of Pathogenic Bacteria and Polymicrobial Infections in the Genitourinary Tract of Renal Transplant Recipients

Eugen Domann,1* George Hong,2 Can Imirzalioglu,1 Simon Turschner,1 Johannes Kühle,1 Corinna Watzel,1 Torsten Hain,1 Hamid Hossain,1 and Trinad Chakraborty1

Institute of Medical Microbiology, Hospital of the Justus Liebig University Giessen, D-35392 Giessen, Germany,1 Transgenomic Inc., Omaha, Nebraska 681642

Received 27 June 2003/ Returned for modification 26 August 2003/ Accepted 8 September 2003

Renal transplant recipients are predisposed to urinary tract infections caused by both common uropathogens and opportunistic bacteria resulting frequently in significant polymicrobial infections. In this study, a culture-independent 16S rRNA-based approach was established to identify unusual, fastidious, or anaerobic bacteria and to investigate bacterial diversity in urinary tract specimens. Similarly sized amplicons encompassing the V6 to V8 region of the 16S rRNA were analyzed with denaturing high-performance liquid chromatography (DHPLC) (WAVE System). Artificial mixtures of single amplicons from commonly encountered uropathogenic bacteria produced distinct peak profiles whose identities were confirmed by sequencing individually collected peak products. We evaluated the application of the method on 109 urinary tract specimens from renal transplant recipients; 100% correlation was found for culture-positive specimens, and DHPLC generated peak profiles. However, for culture-negative specimens, DHPLC facilitated the detection of novel peak profiles. DNA sequencing of these individual peaks was used to identify the bacteria involved. Thus, in PCR-positive but culture-negative samples the method allowed detection of previously known uropathogens such as Corynebacterium urealyticum and Gardnerella vaginalis, but also unusual agents including Anaerococcus lactolyticus, Bacteroides vulgatus, Dialister invisus, Fusobacterium nucleatum, Lactobacillus iners, Leptotrichia amnionii, Prevotella buccalis, Prevotella ruminicola, Rahnella aquatilis, and Streptococcus intermedius were detected as single pathogens or as constituents of polymicrobial infections. The method described is reproducible and rapidly and enables both DHPLC-based profiling and sequence-based investigation of microbial communities and polymicrobial infections. A detailed understanding of infections found in recipients of renal transplants will guide antibiotic therapy regimens and provide new perspectives for decreasing the risk of graft rejection.

* Corresponding author. Mailing address: Institute of Medical Microbiology, Hospital of the Justus Liebig University Giessen, Frankfurter Strasse 107, D-35392 Giessen, Germany. Phone: 49 641-9941287. Fax: 49 641-9941259. E-mail: eugen.domann{at}mikrobio.med.uni-giessen.de.

Journal of Clinical Microbiology, December 2003, p. 5500-5510, Vol. 41, No. 12
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.12.5500-5510.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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