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1 Centers for Disease Control and Prevention, Atlanta, GA 30333, USA
2 National Warmwater Aquaculture Center, 127 Experiment Station Road, Stoneville, MS 38776, USA
Correspondence
P. Lynn Shewmaker
paw3{at}cdc.gov
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ABSTRACT |
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The GenBank accession number for the 16S rRNA gene sequence of Streptococcus ictaluri 707-05T is DQ462421.
A minimum-evolution phylogenetic tree based on 16S rRNA gene sequences including Streptococcus ictaluri strains and the most closely related full-length type strains is available with the online version of this paper.
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The isolates were characterized phenotypically using conventional biochemical tests as described by Facklam & Elliott (1995)
and the Rapid ID 32 Strep system (bioMérieux) (Freney et al., 1992). Growth temperature studies at 25, 30 and 35 °C showed that the optimal temperature for growth was 30 °C; therefore, conventional biochemical and rapid tests were incubated at 30 and 35 °C. The phenotypic characteristics for S. ictaluri are listed in the species description. The Rapid ID32 Strep system was used as described by the manufacturer. The profile numbers inaccurately identified all three isolates as ‘good identification’ for Gemella haemolysans with 96 % confidence. This error in identification is somewhat expected, since the strains were pyroglutamylaminopeptidase (PYR)-positive and were relatively inert in both conventional and rapid testing, as are Gemella species. A Gram stain can readily distinguish the two genera.
Table 1 shows some of the biochemical tests that can be used to distinguish between S. ictaluri and phylogenetically related Streptococcus species. The majority of streptococci are negative for PYR; however, S. ictaluri and most of the species that are phylogenetically similar are PYR-positive. Two species, Streptococcus canis and Streptococcus dysgalactiae subsp. dysgalactiae are differentiated from S. ictaluri by a negative PYR reaction. S. ictaluri is readily distinguished from the other phylogenetically similar streptococci based on its inactivity in most carbohydrate broths except for maltose and ribose. With the exception of Streptococcus urinalis, all the other Streptococcus strains tested in this study also produce acid from maltose, and all these species (including S. urinalis) produce acid from ribose. Streptococcus iniae, Streptococcus uberis, Streptococcus parauberis, Streptococcus pyogenes, and S. dysgalactiae subsp. dysgalactiae and S. canis are distinguished from S. ictaluri by the production of acid from sucrose and trehalose. S. urinalis produces acid from trehalose and S. canis produces acid from sucrose. S. ictaluri is negative for both these carbohydrates. Haemolytic reaction, growth in 6.5 % NaCl, aesculin hydrolysis and acid production from mannitol and sorbitol are additional tests that are useful in differentiating S. ictaluri (Table 1).
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. The identical sequences obtained from strains 705-05, 706-05 and 707-05T were combined with related reference sequences from GenBank. These sequences were aligned with CLUSTAL in MEGA 3.1 (Kumar et al., 2004), trimmed to a 1377 bp consensus and a neighbour-joining tree was created (Fig. 1). The same alignment was also used to create a minimum-evolution tree (Fig. S1, available as supplementary data with the online version of this paper). Reference strain 16S sequences from the CDC Streptococcus database were aligned in BioEdit with CLUSTAL and a distance matrix was created (data not shown). This analysis showed these isolates to be phylogenetically most similar to S. iniae, S. uberis, and S. parauberis with divergence ranging from 2.0 to 2.3 %. S. pyogenes, S. urinalis, S. dysgalactiae subsp. dysgalactiae and S. canis were included in the analysis and showed even greater differences (2.5–3.2 % divergence). While the focus of this report is not an epidemiologic investigation, the identical 16S rRNA sequence suggests that this could be the same clone, although the three isolates were separated by distance and time.
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. Cultures were grown in 1 litre Todd–Hewitt broth and incubated for 24 h with the exception that S. ictaluri cultures were grown at 30 °C instead of 35 °C. Cells were harvested and lysed as described by Teixeira et al. (1995), and DNA extraction, purification and DNA–DNA reassociation using the hydroxyapatite method were performed as described by Brenner et al. (1982). DNA reassociation experiments were performed at the optimal temperature (55 °C) and at the stringent temperature (70 °C) (Table 2). The three catfish strains (705-05, 706-05 and 707-05T) showed 92 % DNA relatedness or greater at the optimal temperature, 89 % or greater at the stringent temperature and a divergence of 
0.5 % when hybridized against the labelled type strain (707-05T). The DNA relatedness of the type strain with S. canis, S. dysgalactiae subsp. dysgalactiae, S. iniae, S. parauberis, S. pyogenes, S. uberis and S. urinalis was 22 % or less. These results confirm that the three S. ictaluri isolates are genetically distinct from these phylogenetically related species and clearly represent a novel species.
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. The G+C content for strain 707-05T was 38.5 mol%.
Streptococcal species have been documented to cause disease in fish for decades (Hoshina et al., 1958; Robinson & Meyer, 1966; Plumb et al., 1974; Baya et al., 1990; Perera et al., 1994, 1998; Romalde et al., 1999; Nomoto et al., 2004; Baeck et al., 2006); however, identification and taxonomic placement of the offending organisms have often been confused or incorrect. Although Chang & Plumb (1996) investigated a 
-haemolytic Streptococcus species isolated from Channel Catfish in 1991, streptococcosis has not been previously recognized as a disease of I. punctatus. While this study confirms the presence of a streptococcal disease in Channel Catfish, the potential significance of this emerging pathogen to the commercial catfish farming industry is currently unknown. Similarly, the zoonotic potential of S. ictaluri is also unknown, but as other fish-pathogenic streptococci have been transmitted to humans (Lehane & Rawlin, 2000; Facklam et al., 2005), there is cause for concern that this organism could pose a potential health threat to aquaculture workers and recreational fisherman. Food safety is another potential human health concern (Ghittino et al., 2003). However, these questions can only be answered by accurate methods for identification of the species.
Description of Streptococcus ictaluri sp. nov.
Streptococcus ictaluri (ic.ta.lur'i. N.L. gen. masc. n. ictaluri of/from Ictalurus, the catfish from which the organism was isolated.)
Cells are Gram-positive cocci that occur in pairs or short chains, catalase-negative, do not produce haemolysis or pigment when grown on trypic soy agar supplemented with 5 % defibrinated sheep blood, and are susceptible to vancomycin. Strains are positive for PYR and leucine aminopeptidase activity, and produce acid from maltose and ribose. Strains are negative for arginine, hippurate, aesculin and urea hydrolysis, growth at 45 °C, growth in 6.5 % NaCl broth and pyruvate utilization. Acetoin is not produced and tellurite is not tolerated. Using the rapid ID32 Strep test system, alkaline phosphotase, alanine-phenylalanine-proline arylamidase and pyroglutamic acid arylamidase are produced. Arginine dihydrolase, -glucosidase, -galactosidase, -glucuronidase, 
-galactosidase, N-acetyl--glucosaminidase, glycine-L-tryptophan arylamidase, -mannosidase and urease are not produced. Hippurate is not hydrolysed. Acid is not produced from ribose, mannitol, sorbitol, lactose, trehalose, raffinose, sucrose, L-arabinose, D-arabitol, cyclodextrin, glycogen, pullulan, maltose, melibiose, melezitose, methyl--D-glucopyranoside and tagatose. The natural habitat is unknown.
The type strain is 707-05T (=S02-1108T=ATCC BAA-1300T=CCUG 52536T); G+C content is 38.5 mol%. Strains 705-05 (=S04-782-1), 706-05 (=S04-045) and 707-05T are distinguished by 16S rRNA gene sequencing.
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ACKNOWLEDGEMENTS |
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