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Rapid Identification of Bacteria from Positive Blood Cultures by Terminal Restriction Fragment Length Polymorphism Profile Analysis of the 16S rRNA Gene

Jeffrey E. Christensen, Jennifer A. Stencil, and Kurt D. Reed^*

Clinical Research Center, Marshfield Clinic Research Foundation, Marshfield, Wisconsin 54449

Received 24 February 2003/ Returned for modification 13 April 2003/ Accepted 24 May 2003

Bacteremia results in significant morbidity and mortality, especially among patient populations that are immunocompromised. Broad-spectrum antibiotics are administered to patients suspected to have bloodstream infections that are awaiting diagnosis that depends on blood culture analysis. Significant delays in identification of pathogens can result, primarily due to the dependence on growth-based identification systems. To address these limitations, we took advantage of terminal restriction fragment (TRF) length polymorphisms (T-RFLP) due to 16S ribosomal DNA (rDNA) sequence diversity to rapidly identify bacterial pathogens directly from positive blood culture. TRF profiles for each organism were determined by sizing fragments from restriction digests of PCR products derived from two sets of 16S rDNA-specific fluorescent dye-labeled primers. In addition, we created a TRF profile database (TRFPD) with 5,899 predicted TRF profiles from sequence information representing 2,860 different bacterial species. TRF profiles were experimentally determined for 69 reference organisms and 32 clinical isolates and then compared against the predicted profiles in the TRFPD. The predictive value of the profiles was found to be accurate to the species level with most organisms tested. In addition, identification of 10 different genera was possible with profiles comprising two or three TRFs. Although it was possible to identify Enterobacteriaceae by using a profile of three TRFs, the similarity of the TRF profiles of these organisms makes differentiation of species less reliable with the current method. The ability to rapidly (i.e., within 8 h) identify bacteria from blood cultures has potential for reducing unnecessary use of broad-spectrum antibiotics and promoting more timely prescription of appropriate antibiotics.

Present address: Department of Microbiology, University of Colorado Health Sciences Center, Denver, CO 80262.

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