Årsrapport 2011 - Region Sjælland

KMA_0811
Application of DNA sequence analysis of 16S-23S Intergenic Spacer (ITS) region for
species identification of strains belonging to the genera Abiotrophia, Aerococcus,
Alloiococcus, Dolocicoccus, Dolosigranulum, Facklamia, Gemella, Globicatella,
Granulicatella, Ignavigranum, Leuconostoc, and Rothia.
X. C. Nielsen 1 , R. Dargis 2 , M. Hammer 2 , A. Hesselbjerg 2 , L. Hannecke 1 , U. S. Justesen 3 , M. Kemp 3 , J. J. Christensen 1
Department of Clinical Microbiology, Slagelse Hospital, Slagelse 1 ; Department of Microbiological Surveillance and Research, Statens Serum Institut,
Copenhagen 2 ; Department of Clinical Microbiology, Odense University Hosptial, Odense 3 , Denmark.
Introduction and Purpose
The 16S-23S Intergenic Spacer (ITS) region has been found
useful in separating most of the clinically relevant nonhaemolytic
streptococci (NHS)1. The group of catalasenegative
gram-positive cocci not belonging to the
Streptococcus genus cause endocarditis and can easily be
misidentified with conventional methods as well2. In this
study we have included 40 species belonging to the genera of
Abiotrophia (1), Aerococcus (7), Alloiococcus (1), Dolocicoccus
(1), Dolosigranulum (1), Facklamia (6), Gemella (7), Globicatella
(2), Granulicatella (3), Ignavigranum (1), Leuconostoc (4), and
Rothia (6). The purpose was to investigate the possibility of
using the ITS sequence analysis for species identification of
the strains belonging to these genera.
Methods
39 type strains were purchased from CCUG.
Six pairs of primers were designed based on the available
16S and 23S sequences from the included species from
GenBank.
Optimization of PCR conditions for the six primer pairs.
ITS PCR performed on all 39 strains with the optimal primer
pair.
PCR product analysis using 2% agarose gel electrophoresis
and automatic capillary electrophoresis system Qiaxcel
(Qiagen)
Sequencing of the PCR products using ABI Prism 3100
Avant genetic analyzer (Applied Biosystems, USA).
Sequence editing and analysis using CLC-DNA workbench
(Aarhus, Denmark) and MultAlin (http://multalin.toulouse.
inra.fr/multalin/)
A. M 1 2 3 4 5 6 7 8 9
B. M 1 2 3 4 5 6 7 8 9
108 ÅRSRAPPORT 2011
Results
1. The primer pair with the best PCR result is:
6R-IGS 5’-GGG TTC CCC CAT TCG GAH AT- 3’
Strep16S_1471F 5’-GTG GGA TAG ATG ATT GGG GTG AAG T-3’
2. PCR from all strains generated one
major product (Figure 1.)
3. ITS sequences were generated for 33 out of 40 strains
(Table 1). Sizes of the edited ITS sequences ranged from
195- 384 bp. The two species belonging to Globicatella did
not generate a reasonable sequence.
4. Alignment of the ITS sequences showed that there was
great interspecies variation among the different species,
including those belonging to the same genus (>5%).
Conclusions
ITS gene sequence analysis seems to be a good candidate
for species identification of strains belonging to the genera
Abiotrophia, Aerococcus, Alloiococcus, Dolocicoccus,
Dolosigranulum, Facklamia, Gemella, Granulicatella,
Ignavigranum, Leuconostoc, and Rothia. Also, the potential
usefulness of ITS for identification for Globicatella has to be
explored further as, if possible, ITS can be used as the initial
step in identifying this large group of genera, including NHS.
References
Nielsen, X.C., U. S. Justesen, R. Dargis, M. Kemp, and J. J. Chrsitensen. 2009. J. Clin. Microbiol. 47: 932-939
Brouqui, P., and D. Raoult. 2001. Clin. Microbiology. Rev. 14:177-207
Results Table 1. ITS sequencing results.
Figure 1. Detection of PCR
products after ITS PCR.
A. 2% agarose gel
B. Qiaxcel
M. DNA ladder
1. Abiotrophia defectiva
2. Aerococcus christensenii
3. Aerococcus sanuinicola
4. Aerococcus suis
5. Aerococcus urinae
6. Aerococcus urinaeequi
7. Aerococcus urinaehomonis
8. Aerococcus viridans
9. Alloiococcus otitis
Correspondence author: Xiaohui Chen Nielsen, Department of Clinical Microbiology, Slagelse Hospital,
Region Zealand, Denmark. Phone: 0045 5855 9404. E-mail: xcn@regionsjaelland.dk
Genus
Included species Species achieved ITS
(n)
sequence (n)
ITS size (bp)
Species achieved ITS sequence
(taxon)
Species NOT achieved ITS
sequence (taxon)
Abiotrophia 1 1 242 A. defectiva
A. christensenii, A. sanguinicola, A.
Aerococcus 7 7 218-242 suis, A.urinae, A. urinaehominis, A.
viridans
Alloiococcus 1 1 238 A. otitis
Dolosicoccus 1 1 264 D. paucivorans
Dolosigranulum 1 1 249 D. pigrum
Facklamia 6 4 226-280
F. hominis, F. languida, F.
miroungae, F. sourekii
F. ignava, F. tabacinasalis
G. bergeri, G. cuniculi, G.
Gemella 7 6 195-217 morbillorum, G. palaticanis, G.
sanguinis
G. asaccharolytica,
Granulicatella 3 3 209-228
G. elegans, G. balaenopterae,G.
adiascens
Globicatella 2 0 G. sanguinis, G. sulfidificiens
Ignavigranum 1 1 208 I. ruoffiae
Leuconostoc 4 3 382
Rothia 6 5 317-384
L. mensenteroides (subspp.
Mesenteroides, cremoris,
dextranicum)
R. amarae, R. dentocariosa, R.
mucilaginosa, R. terrae, R.
nasimurium
L. lactis
R. aeria