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Journal of Clinical Microbiology, July 1998, p. 2164-2166, Vol. 36, No. 7
Department of Pathology and Laboratory
Medicine, University of British Columbia,1 and
the Provincial Laboratory, British Columbia Centre for Disease
Control Society,2 Vancouver, British Columbia,
University of Toronto, Toronto,
Ontario,3
National Research Council
Canada, Plant Biotechnology Institute, Saskatoon,
Saskatchewan,4 and
National Centre for
Streptococcus, Edmonton, Alberta,5 Canada
Received 26 November 1997/Returned for modification 13 March
1998/Accepted 23 April 1998
It was recently reported that Streptococcus iniae, a
bacterial pathogen of aquatic animals, can cause serious disease in
humans. Using the chaperonin 60 (Cpn60) gene identification method with reverse checkerboard hybridization and chemiluminescent detection, we
identified correctly each of 12 S. iniae samples among 34 aerobic gram-positive isolates from animal and clinical human sources.
Streptococcus iniae was
first isolated from skin lesions of freshwater dolphins (Inia
geoffrensis) in the mid-1970s (8). In the 1980s, a new
Streptococcus species, causing acute meningoencephalitis with mortalities as high as 50%, was isolated from infected rainbow trout (Onchorynchus mykiss) and diseased tilapia
(Oreochromis species) farmed in Israel, Taiwan, and the
United States (3, 4). This pathogen, which was named
Streptococcus shiloi, was subsequently shown to be
genetically and phenotypically identical to S. iniae. It has
been suggested that the original name, S. iniae, be retained
(2). The first reported case of human disease caused by this
Streptococcus species was in Texas in 1991, and a second
case was found in Ottawa in 1994 (1). During the winter of
1995 to 1996, four patients were diagnosed as having acute cellulitis
due to a viridans group Streptococcus (1, 10). Further diagnostic tests subsequently confirmed the identity of this
common infecting pathogen as S. iniae and not a viridans group Streptococcus (1, 10). Common to these
patients was their handling of fresh tilapias, a popular freshwater
food fish, prior to the onset of disease. Another five cases were
subsequently identified in Toronto (10). Recently, because
of the awareness of this disease in Canada (1, 10), two
strains (isolates 582 and 608) were isolated from patients in
Vancouver, British Columbia, Canada. Thus, there is clear evidence that
this pathogen is capable not only of causing serious disease in fish
and dolphins but also of being transferred to and infecting humans. It
is possible that other human cases have occurred but that in such
instances the causative pathogen was misidentified as one of the
viridans group streptococci. Of the various Streptococcus
species that can be found as normal flora on humans and animals, some
species can cause disease in their hosts (9). Current
conventional phenotypic identification methods for
Streptococcus can easily identify opportunistic pathogens
such as Streptococcus pyogenes and Streptococcus
agalactiae. However, the unequivocal identification of some of the
other species of streptococci such as the viridans group streptococci
and S. iniae can be problematic.
Phenotyping methods for the preliminary identification of S. iniae have been described previously (1), but accurate
species identification requires further extensive biochemical testing which is often available only in reference laboratories. Like other
streptococci, this organism appears in smears as gram-positive cocci in
chains, is catalase negative and leucine aminopeptidase positive, and
is susceptible to vancomycin. When the organism is incubated
anaerobically, betahemolysis is clearly demonstrated. Like S. pyogenes, this species is pyrrolidonylarylamidase positive and may
be susceptible to bacitracin. However, S. iniae has not been
demonstrated to carry any known Lancefield group antigen. S. iniae may grow in 6.5% NaCl, but only after prolonged incubation, and does not grow on bile esculin agar. Esculin and arginine are hydrolyzed and the CAMP test is positive; the Voges-Proskauer, urease,
and hippurate tests are negative. Typical strains ferment glucose,
salicin, sucrose, and starch, while tests for the fermentation of
arabinose, inulin, lactose, melibiose, raffinose and sorbitol are
negative. Mannitol fermentation is variable. We previously provided
evidence that PCR amplification of the universal chaperonin 60 (Cpn60)
gene (5) with a pair of degenerate Cpn60-specific primers
followed by reverse checkerboard hybridization may be a useful
alternative nucleic acid-based method for identifying microbes (6,
7). The method, described in detail in reference 7, consists essentially of evenly depositing each
species-specific PCR-generated 600-bp Cpn60 fragment as a 13-cm-long
slot onto a nylon membrane with a minislot apparatus (Immunetics Inc.,
Cambridge, Mass.). The filter, following heat fixation of the DNA and
prehybridization, is loaded into the miniblot setup (Immunetics Inc.)
such that the immobilized DNA slots are at right angles to the
hybridization channels on the miniblot apparatus. To produce
hybridization probes, 600-bp regions of the Cpn60 genes from bacterial
isolates are simultaneously amplified and the amplicons are labeled
with digoxigenin-11-dUTP by using the degenerate pair of PCR primers
(6, 7). Each probe, in hybridization buffer, is pipetted
into an individual hybridization channel on the miniblot apparatus.
DNA hybridization signals are detected according to Boehringer Mannheim
digoxigenin chemiluminescent detection protocols and by using Kodak
Biomax X-ray films.
The data reported herein demonstrate that S. iniae can be
accurately identified by the Cpn60 identification method. In the initial phase of this study, one putative S. iniae isolate
(isolate 288) was recovered from a swab taken from a live tilapia. It
was confirmed as S. iniae by standard phenotypic methods
(data not shown). To compare the specificity of our Cpn60
identification method against those of standard phenotypic methods for
S. iniae identification, 34 bacterial isolates were
identified by Cpn60 gene reverse checkerboard hybridization followed by
chemiluminescent detection according to previously published protocols
(7). The type strain, S. iniae ATCC 29178 (8), was used to generate a positive species-specific 600-bp
Cpn60 target. The negative-control target streptococci, S. anginosus ATCC 33397, S. mutans ATCC 25175, S. mitis ATCC 9811, S. bovis ATCC 9809, S. salivarius 25975, S. pneumoniae ATCC 27336, S. porcinus SR1387/93, and S. equi ATCC 9528, were from
the culture collection of the National Centre for
Streptococcus, Edmonton, Alberta, Canada. The other
bacterial cultures used to generate the known 600-bp Cpn60 DNA targets
shown in Fig. 1 (see the legend for Fig.
1) were from the culture collection of the Provincial Laboratory,
British Columbia Centre for Disease Control.
Digoxigenin-11-dUTP-labelled probes from a total of 34 isolates
consisting of 31 isolates obtained in the blind from humans and animals
(Table 1), S. iniae ATCC
29178, and isolates 286 and 288 were synthesized with the degenerate
Cpn60-specific PCR primers (7). The miniblot apparatus was
used to hybridize the probes against the panel of 600-bp Cpn60 gene
targets comprising S. iniae ATCC 29178 and the 27 known
bacterial negative controls (Fig. 1). Nine of the 31 isolates obtained
in the blind, ATCC 29178, and isolates 286 and 288 were positive for
S. iniae by the Cpn60 identification method (Fig. 1 and
Table 1). No cross-hybridization against any of the negative controls,
which included 10 different Streptococcus species, was
observed (Fig. 1). All 12 S. iniae isolates previously had
been confirmed as S. iniae by standard biochemical
phenotyping methods (Table 1). Probes from the remaining 22 bacterial
isolates failed to hybridize with the Cpn60 DNA targets from either
S. iniae or the negative controls (Fig. 1 and Table 1).
These isolates, which were recovered from tilapias, consisted of either
Vagococcus or Enterococcus species, with one
(9002-3) being an unidentified species (Table 1).
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Streptococcus iniae, a Human and Animal
Pathogen: Specific Identification by the Chaperonin 60 Gene
Identification Method
ABSTRACT
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FIG. 1.
Miniblot reverse checkerboard hybridization results for
bacterial isolates described in Table 1. The horizontal slots contained
immobilized Cpn60 600-bp target (T) DNA from S. anginosus
ATCC 29971 (2T), S. bovis ATCC 9809 (3T), S. equi
ATCC 9528 (4T), S. mitis ATCC 9811 (5T), S. mutans ATCC 25175 (6T) S. pneumoniae ATCC 27336 (7T),
S. porcinus SR1387/93 (8T), S. salivarius ATCC
25957 (9T), S. pyogenes ATCC 19615 (10T), S. agalactiae ATCC 12386 (11T), S. iniae ATCC 29178 (12T),
Staphylococcus aureus ATCC 12600 (13T), Citrobacter
freundii ATCC 8090 (14T), Enterobacter aerogenes ATCC
13048 (15T), Escherichia coli ATCC 25922 (16T),
Klebsiella pneumoniae ATCC 13883 (17T), Proteus
vulgaris ATCC 13315 (18T), Salmonella typhimurium ATCC
14028 (19T), Serratia marcescens ATCC 8100 (20T),
Shigella sonnei ATCC 25931 (21T), Neisseria
gonorrhoeae ATCC 41426 (22T), Bacillus cereus ATCC
11778 (23T), Yersinia enterocolitica ATCC 27729 (24T),
Proteus mirabilis ATCC 25933 (25T), Staphylococcus
lugdunensis ATCC 43804 (26T), Borrelia burgdorferi ATCC
53899 (27T), Streptococcus faecalis ATCC 19433 (28T), and
Staphylococcus epidermidis ATCC 14990 (29T). Slots 1T and
30T contained no DNA. The vertical hybridization channels contained
digoxigenin-labelled Cpn60 600-bp probe (P) DNA from S. iniae ATCC 29178 (1P), isolate 582 (3P), isolate 608 (5P), isolate
286 (7P), isolate 288 (9P), isolate 9001-1 (11P), isolate 9001-2 (12P),
isolate 9001-3 (13P), isolate 9001-4 (14P), isolate 9002-1 (15P),
isolate 9002-2 (16P), isolate 9002-3 (17P), isolate 9002-4 (18P),
isolate 9003-2 (19P), isolate 9003-3 (20P), isolate 9003-4 (21P),
isolate 9003-5 (22P), isolate 9004-1 (23P), isolate 9004-2 (24P),
isolate 9004-3 (25P), isolate 9005-1 (26P), isolate 9005-2 (27P),
isolate 9005-3 (28P), isolate 9005-4 (29P), isolate 9005-5 (30P),
isolate 9005-6 (31P), isolate 9005-7 (32P), isolate 9006-1 (33P),
isolate 9006-2 (34P), isolate 9006-3 (35P), isolate 9019 (36P), isolate
9020 (37P), isolate 9085 (38P), and isolate 9086 (39P). Vertical
hybridization channels 2P, 4P, 6P, 8P, 10P, and 40P contained
hybridization buffer alone.
TABLE 1.
Identification of S. iniae isolates by the
Cpn60 Gene identification method and reverse
checkerboard hybridization
No false negatives were observed despite the fact that the S. iniae-positive cultures were isolated from both infected animals and humans. DNA sequencing of the 600-bp Cpn60 fragment from isolate ATCC 29178, a dolphin isolate, isolate 286, a human isolate, and isolate 288, a fish isolate, showed identical DNA sequences (data not shown). This suggests that the targeted 600-bp Cpn60 gene fragment is probably well conserved among S. iniae isolates, an important factor in the specificity of the tested bacterial identification system. Data from previous studies further support this conclusion (6, 7). Results from this study suggest that S. iniae, a potentially serious zoonotic agent, may be accurately discriminated from related organisms by the Cpn60 gene identification method. Future tests of suspected S. iniae organisms isolated from new clinical cases and various fish species should further confirm the validity of the Cpn60 gene identification results reported in this paper.
Nucleotide sequence accession number. The sequence of the 600-bp Cpn60 DNA fragment from S. iniae ATCC 29178 has been deposited in the GenBank under accession no. AF064076.
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ACKNOWLEDGMENTS |
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We thank B. Panchuk for DNA sequencing.
Funding was from the Canadian Bacterial Diseases Network to D.L.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Pathology and Laboratory Medicine, University of British Columbia and the Provincial Laboratory, BCCDC Society, 655W 12th Ave., Vancouver, British Columbia, Canada. V5Z 4R4. Phone: (604) 660-6005. Fax: (604) 660-0403. E-mail: shgoh{at}unixg.ubc.ca.
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Journal of Clinical Microbiology, July 1998, p. 2164-2166, Vol. 36, No. 7
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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