Veterinary Microbiology 101 (2004) 109116

Development of a PCR assay for Streptococcus iniae based on

the lactate oxidase (lctO) gene with potential diagnostic value
Ana I. Mata, M. Mar Blanco, Lucas Domnguez, Jos F. Fernndez-Garayzbal ,
Alicia Gibello
Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense, Avda. Puerta de Hierro s/n,
28040 Madrid, Spain
Received 18 July 2003; received in revised form 13 February 2004; accepted 8 March 2004

Abstract
Streptococcus iniae is a well-known pathogen of both fish and humans that is difficult to identify by conventional biochemical
tests. A PCR assay based on the lactate oxidase (lctO) gene of S. iniae was developed for the rapid and specific detection and
identification of this pathogen from different sources. The PCR assay had a detection limit of 6231 cells, and 25 pg of DNA per
PCR reaction mixture. The PCR was also effective in detecting the bacterium from inoculated tissue homogenates, suggesting
its potential use for a rapid and accurate diagnosis of S. iniae infections.
2004 Elsevier B.V. All rights reserved.
Keywords: Streptococcus iniae; PCR; Detection; Fish

1. Introduction
Streptococcus iniae (junior synonym: Streptococcus
shiloi) is the main causative agent of streptococcosis
in wild and farmed fish worldwide. Originally isolated
from subcutaneous abscesses on Amazon freshwater
dolphins (Pier and Madin, 1976), this pathogen has
been associated with disease outbreaks in different
fresh and seawater commercial fish species such as
rainbow trout, tilapia, channel catfish, gilthead sea
bream or sea bass (Zlotkin et al., 1998; Shoemaker
Corresponding author. Tel.: +34-91-3943716;
fax: +34-91-3943908.
E-mail address: garayzab@vet.ucm.es
(J.F. Fernandez-Garayzabal).

et al., 2001; Colorni et al., 2002), with mortality rates

between 30 and 50% in affected fish (Eldar et al.,
1995). The economic losses caused by this bacterium
in aquaculture farms worldwide are estimated to be
over US$ 100 million per year (Shoemaker et al.,
2001). In addition to its importance in aquaculture, S.
iniae has recently emerged as a threat to public health
due to its zoonotic consideration, being isolated from
humans affected by bacteraemia, cellulitis, meningitis
and osteomyelitis (Weinstein et al., 1997; Durborow,
1999; Lehane and Rawlin, 2000; Lau et al., 2003).
The most significant clinical signs of S. iniae infections in fish are septicemia and meningoencephalitis
(Eldar et al., 1994, 1995), which are very similar
to those produced by other bacterial fish pathogens,
mainly Streptococcus parauberis, Streptococcus

0378-1135/$ see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2004.03.012

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A.I. Mata et al. / Veterinary Microbiology 101 (2004) 109116

difficilis or Lactococcus garvieae (Domnech et al.,

1993, 1996; Eldar et al., 1994, 1995; Bercovier et al.,
1997; Eldar and Ghittino, 1999). Therefore, definitive
diagnosis of S. iniae infections must be based on bacterial isolation and further biochemical identification
of the etiological agent (Bercovier et al., 1997; Eldar
and Ghittino, 1999). Although S. iniae is phenotypically well characterized, laboratory identification
from diseased fish can be difficult, especially when
using commercial identification systems, because no
currently available commercial system includes S.
iniae in its database. Similarly, in human medicine,
S. iniae can also be mistakenly identified as one of
the more common human pathogens, such as Streptococcus dysgalactiae subsp. equisimilis (Bert and
Lambert-Zechovsky, 1997; Lau et al., 2003), Streptococcus uberis (Weinstein et al., 1997; Bouskraoui
et al., 1999), and Streptococcus suis type 1 (Lehane
and Rawlin, 2000; Kopic et al., 2002; Vilaichone
et al., 2002).
Molecular diagnostic techniques, such as PCR assays, are increasingly used to detect and identify many
different bacterial pathogens including the most significant fish pathogens such as Yersinia ruckeri, Pseudomonas anguilliseptica, L. garvieae, Renibacterium
salmoninarum or Aeromonas salmonicida (Gustafson
et al., 1992; Miriam et al., 1997; Gibello et al., 1999a;
Aoki et al., 2000; Blanco et al., 2002). Although most
of these PCR-based assays target specific sequences
of the 16S rRNA gene (Gibello et al., 1999a; Blanco
et al., 2002), other genes can also be used as target molecules (Gustafson et al., 1992; Miriam et al.,
1997; Aoki et al., 2000). We observed that the gene
encoding lactate oxidase is very unusual among Streptococcus species and related genera (Gibello et al.,
1999b). In this work we evaluate the use of the lactate oxidase-encoding gene (lctO) from S. iniae as a
target molecule for a PCR-based method with the aim
of improving the detection and identification of this
pathogen.

2. Materials and methods

2.1. Bacterial strains and growth conditions
The source of the isolation and the geographic origin of the S. iniae strains, other Streptococcus species

and other bacteria used in this study are indicated in

Table 1. All bacterial strains were grown on Columbia
blood agar plates (bioMrieux Espaa S.A.), and incubated for 2448 h, at 30 or 22 C, according to the
bacterial characteristics.
All the S. iniae strains were biochemically characterized with the Rapid ID32 STREP system
(bioMrieux Espaa S.A.) and further compared with
the characteristics described for this species (Pier and
Madin, 1976; Colorni et al., 2002).
2.2. Presence of lactate oxidase-encoding gene
The presence of a gene homologous to lctO in the
bacterial taxa listed in Table 1 was examined by PCR
amplification with the biotin FWL and RVL primers,
which amplified a region of the lactate oxidase
gene, and by Southern blot hybridization, against the
biotin-labeled lactate oxidase PCR product from S.
iniae, as described previously (Gibello et al., 1999b).
2.3. Isolation of bacterial DNA for PCR amplification
Bacterial chromosomal DNA used in PCR assays
and Southern blot was extracted by the method of
Lawson et al. (1989). Purified DNA was suspended
in 100 l of distilled water and then stored at 20 C
until use.
2.4. Primer design and PCR amplification
The forward primer LOX-1 (5 -AAGGGGAAATCGCAAGTGCC-3 ) and the reverse primers LOX-R
(5 -TGGTACACGGCGATCAACAG-3 ) and LOX-2
(5 -ATATCTGATTGGGCCGTCTAA-3 ), were derived from non-conserved regions of the S. iniae
lactate oxidase-encoding gene sequence (accession
number Y07622), compared to the homologous sequence in Aerococcus viridans (accession number
D50611). These LOX primers were designed from
positions 3088 to 3107, 3634 to 3653 and 3936 to
3957, respectively, using the primer design program
OLIGO Primer Analysis Software version 4.0 (MBI,
USA), and were synthesized by ISOGEN, Bioscience
B.V. (Maarssen, The Netherlands). Primer combinations (LOX-1/LOX-2 and LOX-1/LOX-R) were tested
for partial PCR amplification of lctO, at 55 and 60 C
annealing temperatures, with DNA from S. iniae, and

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111

Table 1
Bacterial species assayed in the PCR experiments
Species
Streptococcus spp.
S. iniaea

S. agalactiae
S. difficilis
S. equi subsp. equi
S. equi subsp. zooepidemicus
S. parauberis
S.
S.
S.
S.
S.
S.

phocae
pneumoniae
pyogenes
salivarius
suis
uberis

Other genera
A. viridans
C. piscicola

L. garvieae

Lactococcus lactis subsp. lactis

Lactococcus piscium
Vagococcus fluvialis
Vagococcus salmoninarum

Strain

Source and/or geographic origin

ATCC 29178T
ATCC 29177
CIP 103769T
MT 2375
MT 2376
MT 2377
MT 2378
P1
P2
9116
9033
9066
9098
STR 34
CIP 103768T
CIP 103853
CECT 989T
1428
94/16
NCDO 2020T
MT 2468
MT 1907
CECT 985T
CECT 805T
CECT 958T
CECT 994T
STR 164

Dolphin, North America

Dolphin, North America
Tilapia, Israel
Unknown fish, Scotland
Tilapia, USA
Unknown fish, Scotland
Hybrid striped bass, USA
Human cellulitis, China
Human osteomyelitis, China
Human cellulitis, Canada
Brain fish, Canada
Fish swab, Canada
Tilapia swab, Canada
Ovine mastitis, Spain
Tilapia brain, Israel
Fish brain, Israel
Submaxillary gland of horse
Pig clinical sample, Spain
Turbot, Spain
Mastitis milk
Salmon, South America
Unknown, Scotland
Human
Human rheumatism, USA
Pig
Austria
Ovine mastitis, Spain

NCDO 1225
01/5423
01/5685
ATCC 35586
ATCC 43921T
1925
1935
CECT 185T
CECT 4493T
NCDO 2497
NCDO 2777

Air sample
Rainbow trout, Italy
Rainbow trout, Spain
Cutthroat trout, USA
Bovine mastitis, UK
Rainbow trout, Spain
Rainbow trout, Spain
Cow milk
Salmonid fish, UK
Chicken feces
Rainbow trout, USA

a Clinical strains of S. iniae were all -hemolytic except the strains MT 2375 and 9116 that were -hemolytic. All clinical strains were
ADH-positive (serotype I), except the strains MT 2375 and MT 2378 that were ADH-negative (serotype II).

the bacterial species included in Table 1. The primer

pair LOX-1/LOX-2 gives a 870 bp fragment product
and the pair LOX-1/LOX-R gives a DNA amplification product of 565 bp.
The PCR amplifications were performed in 50 l
reaction volumes containing DNA template (5070 ng
of chromosomal bacterial DNA, 20 l of boiled cells,
or 10 l of DNA extracted from fish tissue, see be-

low), 1 M of each primer (LOX-1 and LOX-2, or

LOX-1 and LOX-R), 0.2 mM of each deoxynucleotide
triphosphate, and 2 U of DNA polymerase (Biotools B
& M Labs. S.A, Madrid, Spain) in 1 reaction buffer.
After a denaturation step of 95 C for 1 min, 35 serial
cycles of a denaturation step of 92 C for 1 min, annealing at the selected temperature for 1 min and extension at 72 C for 2 min were performed, followed

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A.I. Mata et al. / Veterinary Microbiology 101 (2004) 109116

by a final extension step of 72 C for 5 min. Negative (no template DNA) and positive (50 ng of purified DNA from S. iniae strain ATCC 29178T ) controls
were included in each batch of reactions. The amplifications were carried out in a Mastercycler gradient
(Eppendorf). PCR-generated products were detected
by electrophoresis of 20 l of each amplification mixture in 1.5% agarose gels in 1 Tris-acetate-EDTA
buffer. Gels were stained with 0.5 g ml1 ethidium
bromide.
2.5. PCR specificity and sensitivity
Specificity of the PCR assay was tested by using
DNA extracted of all collection strains and the clinical
isolates listed in Table 1 as a template. For screening
the clinical isolates of S. iniae, one bacterial colony
was resuspended in 0.2 ml of sterile water and boiled
for 10 min, and 20 l of this suspension was added
directly to 80 l of PCR mixture.
The lowest limit of detection of S. iniae purified
DNA or bacterial cells by PCR was examined for
the strain ATCC 29178T . Purified genomic DNA
of 100 ng were serially two-fold diluted in sterile
distilled water to 3 pg per PCR reaction. Bacterial
suspensions containing 4 106 cells/ml were serially
diluted 10-fold to 4 104 and further diluted two-fold
to 1.5 102 cells/ml, in 0.9% saline solution. Fifty
microliters of each dilution were processed for DNA
extraction by the method of Casas et al. (1995), and
the obtained DNA was added directly to the PCR mixture. The bacterial concentration was determined by
plating 0.1 ml of each dilution onto Columbia blood
agar plates (bioMrieux Espaa S.A.). All m-PCR reactions assessing limits of detection were performed
in triplicates.
2.6. Preparation of artificially inoculated fish tissue
homogenates
Samples of liver, kidney and brain from five rainbow trout and five sea bream were obtained aseptically. These organs were weighed and blended with
the appropriate volume of 0.9% saline solution to obtain a 1/10 dilution of each organ. The absence of S.
iniae in the tissue homogenates was determined by
plating 0.1 ml onto Columbia blood agar plates that
were incubated at 22 C for 2 days.

Samples (1 ml) of tissue homogenate were inoculated with S. iniae ATCC 29178T to obtain a final
concentration of 4 106 bacteria/ml. Then, the inoculated tissue homogenates were serially 10-fold diluted
to 4 104 and two-fold to 1.5 102 cells/ml, in 0.9%
saline buffer. One hundred microliters of the appropriate dilutions were plated onto Columbia blood agar
plates that were incubated for 2 days at 30 C, for the
enumeration of S. iniae in the inoculated tissue homogenates. Also, 50 l of these dilutions were processed for DNA extraction by the method described
by Casas et al. (1995). The total DNA extracted was
suspended in 10 l sterile distilled water and used for
PCR experiments. Non-inoculated tissue homogenates
were used as controls.

3. Results
3.1. Distribution of lactate oxidase-encoding (lctO)
gene in streptococcus-related bacteria
The presence of a homologous lactate oxidaseencoding gene in S. iniae and some other phylogenetically related bacteria was examined previously
(Gibello et al., 1999b). In this work we extended the
study to other Streptococcus species and related taxa
involved in fish and human diseases. PCR amplification products of 300 bp were observed with S. iniae,
A. viridans and Streptococcus equi subsp. zooepidemicus, thus corroborating previous results (Gibello
et al., 1999b). Additionally, PCR amplifications of
300 and 200 bp were also observed with Streptococcus pyogenes and Carnobacterium piscicola, respectively. No evidence of lctO in fish or human pathogens
other than those described above could be obtained
by Southern blot of digested bacterial DNA against
the biotin labeled PCR product of the lctO gene in S.
iniae (data not shown).
3.2. PCR assay specificity and sensitivity
The primer combination LOX-1/LOX-R gave an
amplification product of 565 bp, but non-specific
amplifications of DNA fragments, between 100 and
300 bp were obtained with Streptococcus agalactiae,
S. suis, L. garvieae and C. piscicola. Increasing in the
annealing temperature (60 C) and/or decreasing in

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113

Fig. 1. Specificity of the lctO gene-based PCR for S. iniae. Lane 1, 100 bp ladder (Biotools); lane 2, negative control; lanes 36, S. iniae
ATCC 29178T , MT 2375, MT 2378 and P1, respectively; lanes 717, S. agalactiae STR 34, S. difficilis CIP 103768T , S. suis CECT 958T ,
S. uberis CECT 994T , S. parauberis 94/16, A. viridans NCDO 1225, C. piscicola 01/5423, L. garvieae ATCC 43921T , L. piscium CECT
4493T , V. fluvialis NCDO 2497 and V. salmoninarum NCDO 2777, respectively.

the number of PCR amplification cycles (30 and 25)

did not improve the specificity of this pair of primers
(data not shown). However, the primer combination
LOX-1/LOX-2 gave, at both annealing temperatures
(55 and 60 C), a single and specific amplification
product of 870 bp with S. iniae strains only. No amplification products were observed with any of the
other Streptococcus species or fish pathogens listed in
Table 1 (Fig. 1). Identical results were obtained when
PCR was performed with whole bacterial cells boiled
for 10 min instead of extracted DNA. The PCR assay
had a detection limit of 6231 cells per PCR reaction
mixture and 25 pg of DNA.
The lctO-gene-based PCR was compared with the
pair of primers Sin-1/Sin-2 described for the identification of S. iniae on the basis of its 16S rRNA gene
sequence (Zlotkin et al., 1998). When these primers
were tested with the bacterial fish pathogens included
in Table 1, a DNA amplification product of 300 bp was
observed with all the strains of S. iniae, but a similar
non-specific amplification product was also observed
with S. difficilis (Fig. 2, lanes 8 and 9).
3.3. Evaluation of PCR assay in inoculated fish
tissues
When the PCR assay was used for the detection
of S. iniae in experimentally inoculated fish tissues,
the 870 bp amplification product was consistently detected in all tissue homogenates, regardless of their
type (data not shown). The detection limit of the PCR
assay in fish tissues was equivalent to 4103 cells/g of

kidney and 5 103 cells/g of liver and brain tissues.

No significant differences were observed in the detection limit between the trout and sea bream tissues.
Non-inoculated tissues were always PCR negative.

4. Discussion
The PCR technique, as a molecular diagnosis tool,
has facilitated the detection and identification of an
increasing number of bacteria of clinical significance
in veterinary and human medicine. Many of the PCR
assays use the 16S rRNA gene as target molecule
(Zlotkin et al., 1998; Gibello et al., 1999a; Blanco
et al., 2002). The PCR assay described for the identification of S. iniae on the basis of its 16S rRNA gene
sequence (Zlotkin et al., 1998), gave with the strains
of S. difficilis a DNA amplification product of 300 bp
similar to that observed with all the strains of S. iniae
(Fig. 2, lanes 8 and 9). Thus, the primer combination LOX-1/LOX-2 appeared to be more specific than
the previously described 16S RNA gene PCR assay
(Zlotkin et al., 1998). Sometimes, among phylogenetically related microorganisms, it is difficult to design
a specific set of primers for the identification of bacterial species based on the 16S rDNA. Thus, the high
genetic relatedness between S. iniae and S. difficilis
(Vandamme et al., 1997; Berridge et al., 2001) may explain the non-specific amplification observed with the
strains of the later species. This lack of specificity in
the 16S rDNA PCR assay, could be especially important taking into consideration that S. difficilis is also

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A.I. Mata et al. / Veterinary Microbiology 101 (2004) 109116

Fig. 2. PCR amplification products using the primer sets LOX-1/LOX-2 (A) and Sin-1/Sin-2 (B) (Zlotkin et al., 1998). Lane 1, 100 bp
ladder (Biotools); lane 2, S. iniae ATCC 29178T ; lane 3, negative control; lanes 4 and 5, S. iniae ATCC 29177 and MT 2375, respectively;
lane 6, S. iniae ATCC 29178T ; lane 7, negative control; lanes 8 and 9, S. difficilis CIP 103768T and CIP 103853, respectively. The
non-specific PCR amplification observed with S. difficilis using the primers Sin-1/Sin-2 (Zlotkin et al., 1998) was not detected with the
primers LOX-1/LOX-2 (see Fig. 1, line 8).

a significant fish pathogen that can be found concurrently with S. iniae (Berridge et al., 2001).
We previously provided evidence that the lctO gene
was limited to a few species of streptococci and related genera (Gibello et al., 1999b). Data reported in
the present work corroborate the previous results, and
a homologous lctO gene was only observed in S. iniae,
A. viridans, S. equi subsp. zooepidemicus, S. pyogenes
and C. piscicola. These results indicate that the lctO
gene should be an appropriate target molecule for the
development of a specific S. iniae PCR assay. The
primer combination LOX-1/LOX-2 gave a single amplification product of 870 bp, which was specific for
S. iniae. This bacterium is considered -hemolytic,
but -hemolytic strains (which could be misidentified
with other viridans group streptococci) are not unusual
(Eldar et al., 1994; Colorni et al., 2002). Moreover,
two different serotypes (I and II) have been described
for this pathogen, which can be respectively differentiated by their ability to hydrolyze arginine or not
(Bachrach et al., 2001). PCR amplifications were obtained with all the S. iniae isolates tested, regardless
of their serotype, type of hemolysis, source (animal
or human), or geographical origin, indicating, therefore, that the primer combination LOX-1/LOX-2 was

adequate for a specific identification of S. iniae. This

PCR assay represents an alternative to that described
by Zlotkin et al. (1998), which gave a non-specific
amplification with S. difficilis.
The detection limit of the lctO-based PCR assay is
similar to that described in PCRs for other bacterial
fish pathogens based on the amplification of either
16S rRNA (Blanco et al., 2002) or single-copy genes
(Gustafson et al., 1992; Coleman et al., 1996; Miriam
et al., 1997). Therefore, these results reinforce the idea
that the sensitivity of PCR assays based on single-copy
genes can be as good as those obtained with multicopy
genes, like the 16S rRNA gene (Brown et al., 1994;
Osorio et al., 2000; Chen et al., 2002).
Experimental challenge studies with S. iniae in fish
have been usually focused on the determination of
the DL50 of this pathogen and the mortality rates obtained after different inoculation doses and/or routes
of administration (Bromage et al., 1999; Nguyen et al.,
2001). There is only one study in which the bacterial
counts of S. iniae in the target organs during natural infections were determined (Nguyen and Kanai, 1999).
The detection limit in inoculated tissues homogenates
of the PCR assay was lower than the tissue bacterial
counts that S. iniae can reach in naturally diseased

A.I. Mata et al. / Veterinary Microbiology 101 (2004) 109116

fish suffering from streptococosis, which are, in most

cases, higher than 1 104 cells/g. Thus, it is likely hat
the PCR would be also useful for the rapid and precise
diagnosis of natural infections by S. iniae.
S. iniae has emerged during the last years as a
zoonotic microoorganism associated with aquaculture,
being related to different human infections (Weinstein
et al., 1997; Lau et al., 2003). Although the incidence
is not high, the severity of S. iniae infections makes
necessary a rapid and accurate identification of this
pathogen. Conventional biochemical methods may fail
to identify S. iniae from clinical samples, which can
be misidentified with other biochemically related bacterial pathogens or causing similar clinical presentations (Weinstein et al., 1997; Lau et al., 2003). Thus,
the PCR assay could be also a useful tool in human
medicine to verify the identity of those -hemolytic
streptococci suspected to be S. iniae.
The results from this study show that the PCR assay for the lactate oxidase (lctO) gene of S. iniae is
a reliable, specific and sensitive method for accurate
identification of this microorganism isolated from different sources. In addition, PCR was effective in detecting S. iniae from inoculated tissue homogenates.
The direct use of this PCR assay in tissue of diseased
fish, would allow the detection and identification of
this bacterium within the same day of receiving a sample, in comparison with the 23 days requirement for
its isolation and further identification by conventional
microbiological approaches. Future analysis of suspected S. iniae organisms isolated from naturally diseased fish will allow to determine the potential value
of this lctO-based PCR assay for the accurate diagnosis of S. iniae infections.

Acknowledgements
The authors wish to thank Dr. S.K.P. Lau (Department of Microbiology of the University of Hong
Kong, China), Dr. de Azavedo (Department of Laboratory Medicine and Pathobiology of the University of
Toronto, Ontario, Canada), J. Black and J. Birrell (FRS
Marine Laboratory, Aberdeen, Scotland) for supplying fish and/or human clinical strains of S. iniae. We
also thank F. Uruburu (Director of the Spanish Type
Culture Collection) for providing the collection CECT
strains. Ana I. Mata was recipient of a grant from the

115

Comunidad de Madrid. This study was supported by

project ACU00-004-C2-2 from the Spanish Ministry
of Sciences and Technology.
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