Advantages and Limitations of Ribosomal RNA PCR and DNA Sequencing for Identification of Bacteria in Cardiac Valves of Danish Patients



Michael Kemp*, 1, 2, Jette Bangsborg 3, Anne Kjerulf 3, Thomas Andersen Schmidt 4, John Christensen 5, Akhmadjon 6 Irmukhamedov , Niels Eske Bruun 6, Rimtas Dargis 1, 7, Keld Andresen 1, Jens Jørgen Christensen 1, 7
1 Microbiological Surveillance & Research, Statens Serum Institut, Copenhagen, Denmark
2 Department of Clinical Microbiology, Odense University Hospital, Odense, Denmark
3 Department of Clinical Microbiology, Herlev University hospital, Copenhagen, Denmark
4 The Emergency Department, Holbaek University Hospital, Holbaek, Denmark
5 Department of Cardiothoracic Surgery, Gentofte University Hospital, Copenhagen, Denmark
6 Department of Cardiology, Gentofte University Hospital, Copenhagen, Denmark
7 Department of Clinical Microbiology, Slagelse Hospital, Slagelse, Denmark


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© Kemp et al.; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

* Address correspondence to this author at the Dept. of Clinical Microbiology, Odense University Hospital, J.B. Winsløwsvej 21,2, 5000 Odense, DENMARK; Tel: (+45) 6541 3161, Fax: (+45) 6541 4785; E-mail: michael.kemp@ouh.regionsyddanmark.dk


Abstract

Studies on the value of culture-independent molecular identification of bacteria in cardiac valves are mostly restricted to comparing agreement of identification to what is obtained by culture to the number of identified bacteria in culture-negative cases. However, evaluation of the usefulness of direct molecular identification should also address weaknesses, their relevance in the given setting, and possible improvements.

In this study cardiac valves from 56 Danish patients referred for surgery for infective endocarditis were analysed by microscopy and culture as well as by PCR targeting part of the bacterial 16S rRNA gene followed by DNA sequencing of the PCR product. PCR and DNA sequencing identified significant bacteria in 49 samples from 43 patients, including five out of 13 culture-negative cases. No rare, exotic, or intracellular bacteria were identified. There was a general agreement between bacterial identity obtained by ribosomal PCR and DNA sequencing from the valves and bacterial isolates from blood culture. However, DNA sequencing of the 16S rRNA gene did not discriminate well among non-haemolytic streptococci, especially within the Streptococcus mitis group.

Ribosomal PCR with subsequent DNA sequencing is an efficient and reliable method of identifying the cause of IE, but exact species identification of some of the most common causes, i.e. non-haemolytic streptococci, may be improved with other molecular methods.

Keywords: 16S rRNA gene, BLAST, culture independent identification, NCBI database, Streptococcus mitis group, viridans streptococci.