16S PCR

KMA 9301

A Rapid and More Sensitive Method to Detect and Identify Bacteria

in Samples that Needs Specific Attention.
Conventional Method Versus 16S PCR for Identification of Bacteria in Spinal Fluids and Heart Valves.
Lasse A. Kvich, Pia Poss, Maria K. Bjrnsdottir, Paul Rammer, Steffen R. E. Srensen, Claus Moser, Thomas Bjarnsholt og Niels Hiby.
Department of Microbiology, Rigshospitalet, Denmark.

Introduction: Identification of microorganisms usually takes 1-2

days. Using PCR and 16S rDNA sequencing, identification can be
obtained the same day and patients can get a specific treatment
faster. 16S is a small part of the ribosomal genome in bacteria, which
is a conserved part with minimal changes through evolution, allowing
differentiation between species after sequencing. In spinal fluids and
heart valves the numbers of microorganisms are usually low, making
these samples more difficult to cultivate, especially if the patients are
in an antibiotic treatment. In the present project we optimized the
bacterial identification in heart valves and spinal fluids by PCR and
sequencing of the 16S rDNA.

Results: 21 heart valves and 33 spinal fluids were collected. Out of

the 21 heart valves both cultivation and 16S identification found 11 of
the samples negative and 10 of them positive. From the perspective
of clinical relevance (pathogenesis and etiological), 16S identification
found 10 bacteria which were possible pathogens causing
endocarditis, whereas cultivation found 1. Fig. 2.
Fig. 2 Bacteria in heart valves
Number of
10
9
8
7
6
5
4
3
2
1
0

16S
Cultivation

*Streptococcus dys. equisimilis, pneumoniae, agalactiae and

members from the mitis and viridans groups

Of the 33 spinal fluids 16S identification found 12 positives, 20

negatives and 1 result where 16S rDNA were demonstrated without
possible further identification. By cultivation the distribution were 7
positives and 26 negatives. From the perspective of clinical
relevance, 16S identification found 8 which were possible pathogens
causing meningitis, whereas cultivation found 4. Fig. 3.

Genetic Analyser 3130xl

Method: The 16S genome spans approximately 1500bp. We only

use a part of the genome counting for 500bp. The rDNA was isolated
through a modified purification method from Qiagen. We added an
extra step with lysozyme and the purifications steps were adjusted
the changed volume. The 16S rDNA is amplified by PCR
(TherminalCycler) and subsequently sequenced (ABI 3130xl genetic
analyzer) to detect the composition of bases in the amplified
sequence. The sequences are compared with a database (Microseq
ID v 2.1.1). During a period of 13 months, 33 spinal fluids and 21
heart valves were collected. All samples were analyzes by 16S
idenification and cultivation. Fig. 1.

Fig. 3 Bacteria in spinal fluids

Number of
10
9
8
7
6
5
4
3
2
1
0

16S
Cultivation

Fig. 1. Method

Sample

*Streptococcus agalactiae and pneumoniae.

Tabel 1 shows the bacteria that where assumed not to have a clinical
relation to either endocarditis or meningitis.
rDNA purification
Data

Cultivation

Tabel 1. Assumed contaminants

Sequencing

PCR

Purification

16S

Heart valves

Spinal fluids

Staphylococcus epidermidis

Staphylococcus warnerii

Staphylococcus haemolyticus

Staphylococcus saphrophyticus

Corynebacterium sp.

Propionebacterium acnes

Methylobacterium sp.

Heart valves

Spinal fluids
2
1

1
1

Discussion: In the present study we have shown that 16S rDNA identification is a rapid and sensitive method to detect and identify bacteria in
samples where cultivation is difficult. From the perspective of clinical relevance, 16S versus cultivation also showed that the specificity was improved
by using 16S. Therefore 16S PCR and sequencing enables faster targeted antibiotic therapy in a higher number of patients.