Streptococcus suis, a major porcine pathogen and an important zoonotic agent, is considered to be composed of phenotypically and genetically diverse strains. However, recent studies reported several “S. suis-like strains” that were identified as S. suis by commonly used methods for the identification of this bacterium, but were regarded as distinct species from S. suis according to the standards of several taxonomic analyses. Furthermore, it has been suggested that some S. suis-like strains can be assigned to several novel species. In this review, we discuss the current taxonomical situation of S. suis with a focus on (1) the classification history of the taxon of S. suis; (2) S. suis-like strains revealed by taxonomic analyses; (3) methods for detecting and identifying this species, including a novel method that can distinguish S. suis isolates from S. suis-like strains; and (4) current topics on the reclassification of S. suis-like strains.

A Lancefield serological group C Streptococcus sp. was isolated from cultured amberjack, Seriola dumerili Risso, and yellowtail, Seriola quinqueradiata Temminck and Schlegel, immunized with Lactococcus garvieae commercial vaccines in Japan. The isolated bacteria were Gram-positive cocci, auto-aggregating in saline, morphologically long chains in growth medium, catalase negative and alpha-haemolytic on blood agar. An almost complete gene sequence of the 16S rDNA of two isolates was determined and compared with that of bacterial strains in the database. The isolates were identified as Streptococcus dysgalactiae based on the results of the 16S rDNA sequence, the bacteriological properties and the Lancefield serological grouping. Oligonucleotide primers specifically designed for the 16S-23S rDNA intergenic spacer region of S. dysgalactiae amplified a gene from all the fish isolates, as well as the type strains alpha-haemolytic S. dysgalactiae subsp. dysgalactiae ATCC430738 and beta-haemolytic S. dysgalactiae subsp. equisimilis ATCC35666, but not those of S. equi ATCC33398, Lactococcus garvieae ATCC43921 and L. garvieae KG9408. The severe necrotic lesions of the caudal peduncle seen in experimentally infected fish were similar to those seen in naturally infected fish.

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The terminal step of 4-hydroxy-3-nitrosobenzamide biosynthesis in Streptomyces murayamaensis is performed by NspF, a mono-oxygenase that converts o -aminophenols to the corresponding nitroso product (hydroxyanilinase activity). Previous biochemical characterization of the resting form of NspF suggested that this enzyme belonged to the coupled binuclear copper enzyme (CBC) family. Another member of this enzyme family, tyrosinase, is able to mono-oxygenate monophenols (monophenolase activity) but not o -aminophenols. To gain insight into the unique reactivity of NspF, we have generated and characterized the oxy form of its active site. The observation of spectral features identical to those of oxy-tyrosinase indicates that oxy-NspF contains a Cu 2 O 2 core where peroxide is coordinated in a μ - η 2 ∶  η 2 mode, confirming that NspF is a CBC enzyme. This oxy form is found to react with monophenols, indicating that, like tyrosinase, NspF also possesses monophenolase activity. A comparison of the two electrophilic mechanisms for the monophenolase and hydroxyanilinase activity indicates a large geometric change between their respective transition states. The potential for specific interactions between the protein pocket and the substrate in each transition state is discussed within the context of the differential reactivity of this family of enzymes with equivalent μ - η 2 ∶ η 2 peroxy bridged coupled binuclear copper active sites.

In order to clarify the taxonomic position of serotypes 20, 22 and 26 of Streptococcus suis, biochemical and molecular genetic studies were performed on isolates (SUT-7, SUT-286 T , SUT-319, SUT-328 and SUT-380) reacted with specific antisera of serotypes 20, 22 or 26 from the saliva of healthy pigs as well as reference strains of serotypes 20, 22 and 26. Comparative recN gene sequencing showed high genetic relatedness among our isolates, but marked differences from the type strain S. suis NCTC 10234 T , i.e. 74.8-75.7 % sequence similarity. The genomic relatedness between the isolates and other strains of species of the genus Streptococcus, including S. suis, was calculated using the average nucleotide identity values of whole genome sequences, which indicated that serotypes 20, 22 and 26 should be removed taxonomically from S. suis and treated as a novel genomic species. Comparative sequence analysis revealed 99.0-100 % sequence similarities for the 16S rRNA genes between the reference strains of serotypes 20, 22 and 26, and our isolates. Isolate STU-286 T had relatively high 16S rRNA gene sequence similarity with S. suis NCTC 10234 T (98.8 %). SUT-286 T could be distinguished from S. suis and other closely related species of the genus Streptococcus using biochemical tests. Due to its phylogenetic and phenotypic similarities to S. suis we propose naming the novel species Streptococcus parasuis sp. nov., with SUT-286 T (5JCM 30273 T 5DSM 29126 T ) as the type strain.Streptococcus suis is a major pathogen of swine and has also been isolated from a variety of animals such as ruminants, cats, dogs, deer and horses (Staats et al., 1997). Moreover, human S. suis infection is considered to be one of the most important emerging zoonotic diseases in Asian countries (Gottschalk et al., 2010). On the basis of their polysaccharide capsular antigens, a total of 33 serotypes (types 1-31, 33 and type 1/2) of S. suis have been described elsewhere (Gottschalk et al., 1989(Gottschalk et al., , 1991Higgins et al., 1995), in which serotype 2 was most frequently associated with disease in both pigs and humans (Gottschalk et al., 2007). Genetic relatedness among S. suis serotypes has been described previously by several investigators. However, a phylogenetic analysis based on 16S rRNA gene and chaperonin-60 gene sequences comparison (Chatellier et al., 1998; Brousseau et al., 2001) indicated a marked genetic discrepancy between serotypes 20, 22, 26 and 33 and other serotypes of S. suis. Moreover, our previous study (Tien et al. 2013) suggested that the reference strains of serotypes 20, 22, 26 and 33 should be removed taxonomically from S. suis. This was based on DNA-DNA relatedness 3Present address:

A Lancefield group C streptococcal (GCS) infection caused by Streptococcus dysgalactiae that is characterized by severe necrotic lesions of the caudal peduncle has been an increasing cause of mortality in farmed fish such as amberjack, Seriola dumerili, and yellowtail, Seriola quinqueradiata, in the southern part of Kyushu, Japan. In this study, enzymatic profiles of GCS strains from fish and mammals were investigated using the API ZYM system, and genotypic characterization of GCS strains was performed using biased sinusoidal field gel electrophoresis (BSFGE). The partial sequence of the 16S-23S rDNA intergenic spacer region of the GCS strain isolates from fish and mammals was also compared. The API ZYM test indicated that it is difficult to differentiate isolates of S. dysgalactiae from fish and animals based on enzymological variations. In the BSFGE analysis, the macrorestriction profiles, which were obtained using SmaI or ApaI as a restriction enzyme, revealed variations between the fish and animal isolates. The partial sequence of the 16S-23S rDNA intergenic spacer region of all the tested fish isolates differed from all mammalian isolates in one or two nucleotides. The possibility of a clonal expansion of S. dysgalactiae strains in farmed fish was also suggested by the BSFGE profiles of fish isolates.

The phenotypic and genetic characterizations of 58 isolates of the fish pathogen Nocardia seriolae, from amberjack, Seriolae dumerili, yellowtail, Seriola quinqueradiata, Japanese flounder, Paralichthys olivaceus, and chub mackerel, Scomber japonicus, in Japan from 1970-2005, were examined to investigate the epidemiological relationship between isolates. The phenotypic and genetic characterizations were determined by alpha-glucosidase activity and biased sinusoidal field gel electrophoresis (BSFGE) analysis, respectively. There was no alpha-glucosidase activity in strains isolated from 2000-05 (n = 50) with a few exceptions (n = 3), while all strains isolated from 1970-90 (n = 8) were positive. In BSFGE analysis, digestions with restriction enzymes Xba I and Ase I produced 15 and 16 restriction patterns, respectively. All restriction patterns obtained from 50 strains isolated during 2000-05 were unrelated to those obtained from eight strains isolated during 1970-90, with the exception of two strains isolated during recent outbreaks. Based on the phenotypic and genetic characterizations, recent outbreaks of nocardiosis in Japan are suggested to be epidemiologically unrelated to earlier outbreaks in Japan. Although a low genetic relationship was observed in the restriction pattern between recent and earlier isolates, identity was confirmed between these groups of isolates because five representative strains showed 99.9% homology with N. seriolae ATCC43993(T) in the 16S rRNA sequence.