PCR amplification of species specific sequences of 16S rDNA and 16S-23S rDNA intergenic spacer region for identification of Streptococcus phocae.
ABSTRACT Streptococcus phocae, a bacterial pathogen of seals, could reliably be identified by PCR amplification using oligonucleotide primers designed according to species specific segments of the previously sequenced 16S rRNA gene and the 16S-23S rDNA intergenic spacer region of this species. The PCR mediated assay allowed an identification of S. phocae isolated from harbor and gray seals and from Atlantic salmons. No cross-reaction could be observed with 13 different other streptococcal species and subspecies and with Lactococcus garvieae strains investigated for control purposes.
- SourceAvailable from: Jose R López[Show abstract] [Hide abstract]
ABSTRACT: The aims of this work were to characterize the 16S-23S internal spacer region of the fish pathogen Tenacibaculum soleae and to develop a PCR assay for its identification and detection. All T. soleae strains tested displayed a single internal spacer region class, containing tRNA(I) (le) and tRNA(A) (la) genes; nevertheless, a considerable intraspecific heterogeneity was observed. However, this region proved to be useful for differentiation of T. soleae from related and non-related species. Species-specific primers were designed targeting the 16S rRNA gene and the internal spacer region region, yielding a 1555-bp fragment. Detection limit was of 1 pg DNA per reaction (< 30 bacterial cells) when using pure cultures. The detection level in the presence of DNA from fish or other bacteria was lower; however, 10 pg were detected at a target/background ratio of 1 : 10(5) . The PCR assay proved to be more sensitive than agar cultivation for the detection of T. soleae from naturally diseased fish, offering a useful tool for diagnosis and for understanding the epidemiology of this pathogen.FEMS Microbiology Letters 11/2011; 324(2):181-8. · 2.72 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Traditional mitochondrial 16S rRNA is commonly used in many species identification studies. However, it is difficult to apply to the phylogenetic studies among the Oncorhynchus subspecies, which is a crucial need for management purposes for Oncorhynchus masou formosanus, Taiwan salmon. In this study, we have developed an improved species identification method for Taiwan salmon distinguished with other Oncorhynchus subspecies tested by exploiting PCR for growth hormone (GH) 1 gene. By comparing DNA sequences for GH1 from 11 species of Oncorhynchus subspecies we designed novel PCR primers that exploit differences between Taiwan salmon and other Oncorhynchus subspecies. Therefore, the technique is an important tool in the management of populations of the endangered land-locked Taiwan salmon preventing from their possible hybrids with other Oncorhynchus subspecies once tested.Molecular and Cellular Probes 02/2009; 23(2):103-6. · 1.87 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The 16S-23S intergenic spacers (ITS) of ribosomal DNA from ten independent isolates of Streptococcus iniae and one reference strain ATCC29178 were sequenced, aligned and used to design a polymerase chain reaction (PCR) primer set for rapid and specific detection and identification of S. iniae. This primer set amplified a 377-bp DNA fragment specifically from S. iniae, but not from other common bacterial pathogens of fish or from non-fish pathogens. The PCR conditions were optimized to allow detection of the organism from agar, broth culture or infected fish tissue. The sensitivity of the PCR assay was established by the detection of DNA as low as 0.02 ng or as few as 10 CFU bacterial cells. The establishment of the specific PCR assay provides a useful tool for the identification and diagnosis of fish infection with S. iniae.Journal of Fish Diseases 02/2011; 34(4):265-71. · 1.51 Impact Factor
Microbiological Research 163 (2008) 132—135
PCR amplification of species specific sequences of
16S rDNA and 16S–23S rDNA intergenic spacer
region for identification of Streptococcus phocae
A.A. Hassana, A. Vossenb, C. La ¨mmlerc,?,
U. Siebertd, J.F. Ferna ´ndez-Garayza ´bale
aInstitut fu ¨r Tiera ¨rztliche Nahrungsmittelkunde, Professur fu ¨r Milchwissenschaften, Justus-Liebig-Universita ¨t Gießen,
Ludwigstr. 21, 35390 Gießen, Germany
bRhein–Sieg–Kreis, Kaiser-Wilhelm-Platz 1, 53721 Siegburg, Germany
cInstitut fu ¨r Pharmakologie und Toxikologie, Justus-Liebig-Universita ¨t Gießen, Frankfurter Str. 107,
35392 Gießen, Germany
dForschung-und Technologiezentrum Westku ¨ste, Hafento ¨rn, 25761 Bu ¨sum, Germany
eDepartamento Sanidad Animal, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain
Accepted 15 January 2006
Streptococcus phocae, a bacterial pathogen of seals, could reliably be identified by
PCR amplification using oligonuclotide primers designed according to species specific
segments of the previously sequenced 16S rRNA gene and the 16S–23S rDNA
intergenic spacer region of this species. The PCR mediated assay allowed an
identification of S. phocae isolated from harbor and gray seals and from Atlantic
salmons. No cross-reaction could be observed with 13 different other streptococcal
species and subspecies and with Lactococcus garvieae strains investigated for control
& 2006 Elsevier GmbH. All rights reserved.
Streptococcus phocae was first reported by Skaar
et al. (1994) in Norway as bacterial pathogen
causing pneumonia in harbor seals (Phoca vitulina).
In further studies this species was recovered from
Cape fur seals (Arctocephalus pusillus pusillus) in
Namibia (Henton et al., 1999), from harbor seals,
ringed seal pups (Phoca hispida) and harbor
porpoises (Phocoena phocoena) in Washington
state, British Columbia and in Arctic Canada
(Raverty and Fiessel, 2001; Raverty et al., 2004)
and from specimens of harbor and gray seals
ARTICLE IN PRESS
0944-5013/$-see front matter & 2006 Elsevier GmbH. All rights reserved.
?Corresponding author. Tel.: +496419938406;
E-mail address: email@example.com
(C. La ¨mmler).
(Halichoerus grypus) in the German North and
Baltic Seas (Vossen et al., 2004). Gibello et al.
(2005) isolated S. phocae from Atlantic salmons
(Salmo salar) farmed in Chile.
S. phocae as gram positive, non-encapsulated,
non-motile bacterium, grows on sheep blood
agar with a wide zone of complete (b)-haemolysis
and is biochemically characterized by acid produc-
tion from fructose, glucose, mannose, maltose,
N-acetylglucosamine and ribose and negative tre-
halose, salicin, arginin, b-glucuronidase and a-
galactosidase reactions. The species could serolo-
gically be classified into Lancefield’s serogroups C
and F (Skaar et al., 1994; Vossen et al., 2004;
Gibello et al., 2005). A molecular characterization
of S. phocae was performed by sequencing the 16S
rRNA gene (Gibello et al., 2005; Vossen et al.,
2004), the 16S–23S rDNA intergenic spacer region
(ISR) (Hassan et al., 2003a–c) and the genes sodA,
cpn60 (Alber et al., 2004) and rnpB (Ta ¨pp et al.,
In the present study a PCR mediated identifica-
tion assay was designed using species-specific
segments of 16S rDNA and 16S–23S rDNA ISR as
Materials and methods
The bacteria used in the present investigation
included the two S. phocae reference strains 8190
R2 and 8399 H1 (NCTC 12719), kindly provided by I.
Skaar (Central Veterinary Laboratory, State Veter-
inary Laboratories of Norway, Oslo, Norway), the
two S. phocae strains MT 2467 and MT 2469 isolated
from Atlantic salmons (Gibello et al., 2005) and 58
S. phocae isolates from harbor and gray seals
(Vossen et al., 2004). For control purposes strains
of the species S. pyogenes (n ¼ 2), S. agalactiae
(n ¼ 15), S. equi subsp. equi (n ¼ 3), S. equi subsp.
(n ¼ 4), S. dysgalactiae subsp.
equisimilis (serogroup G) (n ¼ 11), S. dysgalactiae
subsp. dysgalactiae (serogroup C) (n ¼ 6), S.
dysgalactiae subsp. dysgalactiae (serogroup L)
(n ¼ 6), S. canis (n ¼ 10), S. uberis (n ¼ 15), S.
parauberis (n ¼ 5), S. porcinus (n ¼ 4), S. suis
(n ¼ 4), S. iniae (n ¼ 5), S. difficilis (n ¼ 2) and
Lactococcus garvieae (n ¼ 4) were used. The
control strains were obtained from the strain
collection of the Institut fu ¨r Tiera ¨rztliche Nah-
rungsmittelkunde, Professur fu ¨r Milchwissenschaf-
ten, der Justus-Liebig-Universita ¨t Gießen, Germany
and Departamento de Sanidad Animal, Facultad
de Veterinaria, Universidad Complutense, Madrid,
A molecular identification of the S. phocae
strains was performed by amplifying a S. phocae
specific part of the 16S rDNA using the species
specific oligonucleotide primers PX1 and PX2. The
16S rDNA specific oligonucleotide primers were
designed according to the 16S rDNA sequence of S.
phocae 8399 H1 described previously (Vossen et al.,
2004; accession number: AF235052) by the compu-
ter program OLIGO 4.0. The primer PX1 had the
sequence 50GCTAATACCGCATAAGAAGAG 30, which
corresponds to bases 157–177 of the S. phocae 16S
rRNA gene. The primer PX2 had the sequence 50
GAGCAGAAGTGACAGGGTG 30, which corresponds to
bases 1034–1053 of the gene. As second target for
the design of species specific oligonuclotide pri-
mers the previously sequenced 16S–23S rDNA ISR of
S. phocae 8399 H1 was used (Hassan et al.,
2003a–c; accession number: AF489596). Primer
cae1 had the sequence 50AAGCACGTTAGGA AAATG
30corresponding to bases 11–30, primer cae2
the sequence 50CGAAGTCATA AAAATAGTATG 30
corresponding to bases 67–87 of the 16S–23S rDNA
were synthesized by MWG-Biotech (Ebersberg,
DNA extraction of the streptococcal strains used
in this study was performed as described (Hassan et
al., 2000). PCR amplifications of the 16S rRNA gene
and the 16S–23S rDNA ISR were performed in 30ml
reaction mixtures with
(10pmol/ml), 0.6ml dNTP (10mmol, MBI Fermentas,
St. Leon-Rot, Germany), 3ml 10? thermophilic-
buffer (Promega, Mannheim, Germany), 1.8ml
MgCl2 (25mmol; Promega), 0.2ml Taq DNA poly-
merase (5U/ml, Promega) and 19.9ml aqua dest.
Finally, 2.5ml DNA template was added to each
reaction tube. The PCR reactions were performed
using the thermalcycler Techne-Progene (Thermo-
dux, Wertheim, Germany) with the following
program for the 16S rRNA gene: one denaturation
cycle at 941C for 4min followed by 35 cycles at
941C for 90s, 601C for 90s and 721C for 90s
terminated by one cycle at 721C for 5min. For
amplification of 16S–23S rDNA ISR the temperature
program consisted after a denaturation cycle at
941C for 4min of 30 cycles: 921C for 30s, 481C for
30s and 721C for 30s. The final cycle was followed
by an extension at 721C for 5min. PCR products
(12ml) were separated by agarose gel electrophor-
esis (2% agarose; Sigma, Deisenhofen, Germany) or
1ml foreach primer
ARTICLE IN PRESS
PCR amplification of species specific sequences133
3% Metaphors(MBI Fermentas) with Tris acetate-
0.001mol/l EDTA, pH 7.8). As molecular marker
the GeneRularTM50bp DNA Ladder (MBI Fermentas)
was used. The agarose gel was stained by ethidium
bromide (5mg/ml, Sigma), and visualized under a
UV transilluminator system (ImagerMaster VDS,
Pharmacia Biotech, Freiburg, Germany).
Results and discussion
Molecular biology has been widely used for identi-
fication of micro-organisms at species, genus or family
levels. Targets containing conserved regions and areas
of variability for specific identification of bacteria are
the genes encoding 16S and 23S rRNA. In addition, a
stretch of DNA between the 16S and 23S rDNA, the
16S–23S rDNA ISR, proved to be a comparably species
specific segment. These three targets have already
been used for molecular identification and differen-
tiation of various streptococcal species (Jayarao
et al., 1992; Eldar et al., 1997; Forsman et al.,
1997; Abdulmawjood et al., 1998; La ¨mmler et al.,
1998; Barsotti et al., 2002; Hassan et al., 2001,
2003a–c, 2005; Khan et al., 2003).
In the present study the previously sequenced
targets 16S rDNA and 16S–23S rDNA ISR of S. phocae
were used to design species specific oligonuclotide
primers. The 16S rRNA gene had already been used
for restriction fragment length polymorphism ana-
lysis of S. phocae indicating no significant sequence
variation in this gene (Vossen et al., 2004).
According to Facklam (2002) the 16S rDNA sequence
of S. phocae differed clearly from the 16S rDNA
sequence of 55 streptococcal species and subspe-
cies and allowed a classification of S. phocae in the
pyogenic group of genus Streptococcus. Gibello
et al. (2005) compared the 16S rDNA gene sequence
of nine S. phocae isolated from clinical specimens
of diseased Atlantic salmons and the sequence of
S. phocae CCUG 35103, revealing a 100% sequence
similarity. Comparing the 16S rRNA gene sequence
of S. phocae 8399 H1 (Vossen et al., 2004;
AF235052) with the sequence of S. phocae CCUG
35103 described by (Gibello et al., 2005, AJ621053)
using the computer program DNASTAR-MegAlign
(DNASTAR Inc., Konstanz, Germany) yielded a
sequence similarity of 95.1% (alignment data not
Sequencing the 16S–23S rDNA ISR of S. phocae
and various other pyogenic streptococci revealed
the apparently unique sequence of S. phocae
(Hassan et al., 2003a–c) and could also be used as
target for the design of species specific oligonuclo-
tide primers. In the present study the oligonuclo-
tide primers PX1 and PX2 and cae1 and cae2
displayed positive PCR reactions with amplicon
sizes of approximately 900 and 180bp, respec-
tively, for all 62 investigated S. phocae isolates
(Figs. 1a and b). No non-target amplifications could
be observed with any of the control strains.
A comparable molecular identification of S. phocae
was performed previously using the target genes
sodA and cpn60. Both genes of S. phocae also
allowed the design of species specific oligonuclo-
tide primers and the classification of S. phocae in
the pyogenic group of the genus Streptococcus
(Alber et al., 2004).
ARTICLE IN PRESS
Figure 1. Typical amplicons of S. phocae (lanes 1, 2, 3) with sizes of approximately 900bp (a) and 180bp (b) using the
S. phocae 16S rRNA gene and the 16S–23S rDNA ISR specific oligonuclotide primers, respectively. No reactions were
obtained for control strains of S. agalactiae (4), S. dysgalactiae subsp. dysgalactiae (serogroup C) (5) and S. uberis (6).
M – GeneRularTM50bp DNA Ladder (MBI Fermentas).
A.A. Hassan et al.134
The use of the species specific oligonuclotide
primers shown in the present study helps to identify
this bacterial pathogen and might elucidate the
role S. phocae plays in infections of marine and
Abdulmawjood, A., Weiß, R., La ¨mmler, C., 1998. Species
identification of Streptococcus porcinus by restriction
fragment length polymorphism analysis of 16S riboso-
mal DNA. Res. Vet. Sci. 65, 85–86.
Alber, J., El-Sayed, A., La ¨mmler, C., Hassan, A.A.,
Vossen, A., Siebert, U., 2004. Determination of
species-specific sequences of superoxide dismutase A
encoding gene sodA and chaperonin 60 encoding gene
cpn60 for identification and phylogenetic analysis of
Streptococcus phocae. Vet. Microbiol. 101, 117–122.
Barsotti, O., Decoret, D., Renaud, F.N., 2002. Identifica-
tion of Streptococcus mitis group species by RFLP of
the PCR-amplified 16S–23S rDNA intergenic spacer.
Res. Microbiol. 153, 687–691.
Eldar, A., Lawhon, S., Frelier, P.F., Assenta, L., Simpson,
B.R., Varner, P.W., Bercovier, H., 1997. Restriction
fragment length polymorphisms of 16S rDNA and of
whole rRNA genes (ribotyping) of Streptococcus iniae
strains from the United States and Israel. FEMS
Microbiol. Lett. 151, 155–162.
Facklam, R., 2002. What happened to the streptococci:
overview of taxonomic and nomenclature changes.
Clin. Microbiol. Rev. 15, 613–630.
Forsman, P., Tilsala-Timisja ¨rvi, A., Alatossava, T., 1997.
Identification of staphylococcal and streptococcal
causes of bovine mastitis using 16S–23S rRNA spacer
regions. Microbiol. 143, 3491–3500.
Gibello, A., Mata, A., Blanco, M., Casamayor, A.,
Domı ´nguez, L., Ferna ´ndez-Garayza ´bal, J.F., 2005.
First identification of Streptococcus phocae isolated
from Atlantic salmon (Salmo salar). J. Clin. Microbiol.
Hassan, A.A., Abdulmawjood, A., Yildirim, A.O ¨., Fink, K.,
La ¨mmler, C., Schlenstedt, R., 2000. Identification of
streptococci isolated from various sources by deter-
mination of cfb gene and other CAMP-factor genes.
Can. J. Microbiol. 46, 946–951.
Hassan, A.A., Khan, I.U., Abdulmawjood, A., La ¨mmler,
C., 2001. Evaluation of PCR methods for the rapid
identification and differentiation of Streptococcus
uberis and Streptococcus parauberis. J. Clin. Micro-
biol. 39, 1618–1621.
Hassan, A.A., Khan, I.U., Abdulmawjood, A., La ¨mmler,
C., 2003a. Development of PCR assays for detection of
Streptococcus canis. FEMS Microbiol. Lett. 219,
Hassan, A.A., Khan, I.U., La ¨mmler, C., 2003b. Identifica-
tion of Streptococcus dysgalactiae strains of Lance-
field’s group C, G and L by polymerase chain reaction.
J. Vet. Med. 50, 161–165.
Hassan, A.A., Khan, I.U., La ¨mmler, C., 2003c. Inter- and
intraspecies variations of the 16S–23S rDNA intergenic
spacer region of various streptococcal species. Syst.
Appl. Microbiol. 23, 97–103.
Hassan, A.A., Akineden, O ¨., Usleber, E., 2005. Identifica-
tion of Streptococcus canis isolated from milk of dairy
cows with subclinical mastitis. J. Clin. Microbiol. 43,
Henton, M.M., Zapke, O., Basson, P.A., 1999. Strepto-
coccus phocae infection associated with starvation in
Cape fur seals. J. S. Afr. Vet. Assoc. 2, 98–99.
Jayarao, B.M., Dore ´, J.J.E., Oliver, S.P., 1992. Restriction
fragment length polymorphism analysis of 16S riboso-
mal DNA of Streptococcus and Enterococcus species of
bovine origin. J. Clin. Microbiol. 30, 2235–2240.
Khan, I.U., Hassan., A.A., Abdulmawjood, A., La ¨mmler,
C., Wolter, W., Zscho ¨ck, M., 2003. Identification and
epidemiological characterization of Streptococcus
uberis isolated from bovine mastitis using conven-
tional and molecular methods. J. Vet. Sci. 3, 213–223.
La ¨mmler, C., Abdulmawjood, A., Danic, G., Vaillant, S.,
Weiß, R., 1998. Differentiation of Streptococcus
uberis and Streptococcus parauberis by restriction
fragment length polymorphism analysis of the 16S
ribosomal RNA gene and further studies on serological
properties. Med. Sci. Res. 26, 177–179.
Raverty, S., Fiessel, W., 2001. Pneumonia in neonatal and
juvenile harbor seals (Phoca vitulina) due to Strepto-
coccus phocae. Animal Health Center. Newsletter.
Diagn. Diary 11, 11–12.
Raverty, S.A., Gaydos, J.K., Nielsen, O., Ross, P., 2004.
Pathologic and clinical implications of Streptococcus
phocae isolated from pinnipeds along coastal Wa-
shington state, British Columbia, and Arctic Canada.
35th Annual Conference of the International Associa-
tion of Aquatic Animal Medicine, Galveston, TX, April
Skaar, I., Gaustad, P., Tonjum, T., Holm, B., Stenwig, H.,
1994. Streptococcus phocae sp. nov., a new species
isolated from clinical specimens from seals. Int.
J. Syst. Bacteriol. 44, 646–650.
Ta ¨pp, J., Thollesson, M., Herrmann, B., 2003. Phyloge-
netic relationships and genotyping of the genus
Streptococcus by sequence determination of the
RNase P RNA gene, rnpB. Int. J. Syst. Evol. Microbiol.
Vossen, A., Abdulmawjood, A., La ¨mmler, C., Weiß, R.,
Siebert, U., 2004. Identification and molecular char-
acterization of beta-hemolytic streptococci isolated
from harbour seals (Phoca vitulina) and grey seals
(Halichoerus grypus) of the German North and Baltic
Seas. J. Clin. Microbiol. 42, 469–473.
ARTICLE IN PRESS
PCR amplification of species specific sequences135