SEARCH

SEARCH BY CITATION

Keywords:

  • phenotypic characterization;
  • genetic characterization;
  • BSFGE;
  • Streptococcus dysgalactiae;
  • sodA gene

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Lancefield group C Streptococcus dysgalactiae is an emerging fish pathogen, which was first isolated in 2002 in Japan. Streptococcus dysgalactiae isolates collected from diseased fish in Japan (n=12), Taiwan (n=12), China (n=2), Malaysia (n=3), and Indonesia (n=1) were characterized using biased sinusoidal field gel electrophoresis (BSFGE), sodA gene sequence analysis, and antimicrobial susceptibility. These isolates exhibited high phenotypic homogeneity irrespective of the countries from where the strains were collected. Seventeen isolates were found to be resistant to oxytetracycline and carried the tet(M) gene, except for the strains collected in Taiwan and the PP1564 strain collected in China. The sodA gene sequence analysis revealed that 23 isolates were identical, except for one Japanese isolate (KNH07902), in which a single nucleotide differed from that of the other isolates. Based on BSFGE typing by ApaI macrorestriction, the isolates – including the Japanese, Taiwanese, and Chinese isolates – could be grouped into one main cluster at a 70% similarity level. However, the macrorestriction genotypes of some isolates were apparently distinct from those of the main cluster.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

It has been reported that Streptococcus dysgalactiae belonging to Lancefield group C streptococci (GCS) (Vieira et al., 1998) was responsible for mastitis, subcutaneous cellulitis, and toxic shock-like syndrome in bovine (Aarestrup & Jensen, 1996; Chénier et al., 2008) and other animal infections (Scott, 2000; Lacasta et al., 2008). In 2002, the first epizootic outbreak caused by α-hemolytic Lancefield group C S. dysgalactiae occurred in amberjack Seriola dumerili and yellowtail Seriola quinqueradiata farms in the southern districts of Japan (Nomoto et al., 2004, 2006). During the subsequent years, many fish farms in Japan suffered huge losses due to S. dysgalactiae infection, which was characterized by high mortality and severe muscle necrosis in the caudal peduncle (Nomoto et al., 2008; Abdelsalam et al., 2009b). Since then, several comparison studies have been performed for biochemical and genetic characterizations of fish and mammalian isolates of S. dysgalactiae (Nomoto et al., 2006, 2008). The pathogen has also been isolated from the Amur sturgeon, Acipenser schrenckii, in China (Yang & Li, 2009). Recently, α-hemolytic Lancefield group C S. dysgalactiae isolated from fish was found to have caused ascending upper limb cellulitis in humans (Koh et al., 2009). Therefore, S. dysgalactiae is considered to be an emerging fish pathogen, and its clinical significance has increased in aquaculture as well as in mammalian and human health. However, the origin and infection mechanism that characterize S. dysgalactiae as a fish pathogen remain unknown (Abdelsalam et al., 2009a). Despite increased clinical significance, the characterization of S. dysgalactiae strains isolated from different fish species collected in many countries and the epidemiological relationships among them have not been studied. This study aimed to undertake the phenotypic and genetic characterizations of S. dysgalactiae strains isolated from the genus Seriola collected in Japan, and to compare the results with those of infected fish collected in other Asian countries.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial isolates

Table 1 lists the 30 S. dysgalactiae isolates used in this study. These strains were isolated from diseased fish collected from different fish farms in Kagoshima prefecture in Japan (n=12; four isolated from amberjack S. dumerili, four from yellowtail S. quinqueradiata, and four from king fish Seriola lalandi), Taiwan (n=12; 10 from gray mullet Mugil cephaleus, one from basket mullet Liza alata, and one from cobia Rachycentron canadum), Indonesia (n=1, from hybrid red tilapia Oreochromis sp.), Malaysia (n=3; two from pompano Trachinotus blochii and one from white spotted snapper Lutjanus stellatus), and China (n=2 from pompano T. blochii). Further, in this study, S. dysgalactiae ssp. dysgalactiae ATCC43078 was used as a reference strain.

Table 1.   The α-hemolytic Lancefield group C Streptococcus dysgalactiae isolates used in this study
IsolateSourceCountryYear of isolationtet(M)SodA accession no.BSFGE profile
  1. tet(M), tetracycline-resistant gene; ND, not determined.

Kdys0707YellowtailJapan2007+AB470097B
Kdys0728YellowtailJapan2007+AB520738A
Kdys0719YellowtailJapan2007+AB520741B
KGB07804YellowtailJapan2007+AB470098C
Kdys0716AmberjackJapan2007+AB470095C
Kdys0717AmberjackJapan2007+NDB
Kdys07905AmberjackJapan2007+AB470096B
KG07906AmberjackJapan2007+AB520739C
KNH07807King fishJapan2007+AB440302C
KNH07901King fishJapan2007+AB440303B
KNH07902King fishJapan2007+AB440305C
KNH07903King fishJapan2007+AB440304B
94377MulletTaiwan2005AB440299D
94414MulletTaiwan2005AB440300D
94455MulletTaiwan2005AB440301D
94485MulletTaiwan2005NDD
95542MulletTaiwan2007NDD
95900MulletTaiwan2007NDD
95921MulletTaiwan2007NDD
95980MulletTaiwan2007NDD
95985MulletTaiwan2007AB520740E
951001MulletTaiwan2007NDD
951003Basket mulletTaiwan2007AB470090D
AOD-96086-KCobiaTaiwan2007AB470091F
PF880PompanoMalaysia2003+AB470089G
PP1398PompanoMalaysia2005+AB470092H
PP1564PompanoChina2005AB470088D
PP1635PompanoChina2005+AB470093D
WSSN1609SnapperMalaysia2004+AB470087B
T11358TilapiaIndonesia2004+AB470094I
ATCC43078Cow  NDZ95900ND

Growth conditions and genomic DNA preparation

Stock cultures of S. dysgalactiae isolates were maintained at −80 °C in Todd Hewitt broth (Difco, Sparks, MD). All the isolates were routinely aerobically grown on Todd Hewitt agar (THA; Difco) or blood agar (Columbia agar base; Becton Dickinson, Cockeysville, MD) containing 5% sheep blood (Nippon Bio-Test Laboratories, Japan) and incubated at 37 °C for 24 h. Genomic DNA was extracted from bacterial colonies using a DNAzol® reagent (Invitrogen, Carlsbad) according to the manufacturer's protocol. The identification of the S. dysgalactiae isolates was performed by the PCR assay targeting the intergenic 16S–23S rRNA gene spacer region and the sodA gene according to the method described by Nomoto et al. (2004, 2008).

Phenotypic characterization

Lancefield serotyping (Lancefield, 1933) was performed using Pastorex Strep (Bio-Rad, Marnes-la-Coquette, France) according to the manufacturer's protocol. Biochemical and enzymatic characterizations were performed using the API 20 STREP® and the API ZYM® systems (bioMerieux, Marcy-l'Etoile, France), respectively. All the isolates were cultured on blood agar (Columbia agar base; Becton Dickinson) containing 5% sheep blood (Nippon Bio-Test Laboratories) at 37 °C for 24 h, and fresh colonies were evaluated according to the manufacturer's instructions.

Antimicrobial susceptibility and detection of tetracycline-resistant genes

The antimicrobial susceptibility of the strains was determined using the disk diffusion method on Muller–Hinton agar (Difco Laboratories, Detroit, MI). The following chemotherapeutic agents (microgram per disk) were used in the disk diffusion method: oxytetracycline (30) (Eiken Chemical Co. Ltd, Tokyo, Japan), erythromycin (15) (Oxoid, UK), florfenicol (30) (Oxoid), lincomycin (10) (Oxoid), and ampicillin (10) (Oxoid). The strains were considered resistant to oxytetracycline if the diameter of the inhibition zone around the disk was less than 19 mm (Constable & Morin, 2002). The presence of tet(L), tet(O), tet(S), and tet(M) genes that encode tetracycline resistance was investigated for all the resistant isolates by PCR according to the method reported previously (Agersøet al., 2002).

Partial sequences of the sodA gene

Internal fragments representing 85% of the sodA gene of 23 fish isolates were amplified using the universal primer set and sequenced according to the method reported by Nomoto et al. (2008). The nucleotide sequences were analyzed using bioedit version 7.0 (Hall, 1999). The phylogenetic analysis was carried out using the neighbor-joining method using mega version 3 (Kumar et al., 2004).

Biased sinusoidal field gel electrophoresis (BSFGE)

The restriction enzyme-digested chromosomal DNA was analyzed by BSFGE, a modified pulsed-field gel electrophoresis (PFGE) technique (Madinabeitia et al., 2009). Streptococcus dysgalactiae strains were cultured on THA at 37 °C for 24 h, and the preparation of genomic DNA and DNA digestion with the restriction enzyme ApaI were carried out according to the previously described method (Nomoto et al., 2006). Macrorestriction fragments digested by ApaI were separated using a 1% agarose horizontal gel using the BSFGE system (Genofield; Atto, Tokyo, Japan). The biased sinusoidal electric field was applied for 20 h at DC 48 V and AC 288 V at a frequency of 0.005 Hz (initial) and 0.330 Hz (final). After gel electrophoresis, the gel was stained and visualized under UV light. The macrorestriction patterns were then calibrated and analyzed using the gene profiler software package along with treecon software (version 4.05; Scanalytics Inc., Fairfax, VA). Similarities among the strains were determined based on the genetic distance, which was calculated using the formula proposed by Nei & Li (1979). The generated matrix was subjected to clustering using the unweighted pair-group method with arithmetic means.

Nucleotide sequence accession numbers

The nucleotide sequences determined in this study were submitted to the DNA Data Bank of Japan nucleotide sequence database, and the accession numbers were given as shown in Table 1.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Phenotypic characterization

From all the given cultures, we recovered colonies with a consistent morphological characteristic, i.e., α-hemolysis colonies on the Columbia blood agar. Gram-stained smears obtained from the colonies revealed the presence of chains formed by Gram-positive cocci, and isolates were positively reacted to the intergenic 16S–23S rRNA gene spacer region and sodA gene primers specific to S. dysgalactiae. The results from the Lancefield typing revealed that all the fish isolates belonged to the Lancefield group C. In the API 20 STREP® and API ZYM® systems, complete phenotypic homogeneity was observed among the fish isolates, in the hydrolyses of arginine, and in the acidifications of ribose, trehalose, amygdaline, and in the existence of the enzymes of alkaline phosphatase, leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-glucuronidase, and α-glucosidase, except for the T11358 and Kdys0716 strains, which could acidify both arabinose and mannitol, the Kdys0728 strain, which could acidify glycogen, and the Kdys0707 strain, which could acidify raffinose. Valine arylamidase was not found to exist in any of the strains of S. dysgalactiae, except AOD-96086-K, PP1398, and T11358. The result of ATCC43078 was acidifications of ribose, lactose, trehalose, and amygdaline and the existence of enzymes of alkaline phosphatase, leucine arylamidase, acid phosphatase, β-glucuronidase, and α-glucosidase.

Antimicrobial susceptibility

All the strains were susceptible to all the chemotherapeutic agents used in this study, except oxytetracycline. Seventeen strains were found to be resistant to oxytetracycline; these did not include the strains collected in Taiwan and the PP1564 strain collected in China. The presence of the tet(M) gene was confirmed in all the resistant strains using PCR (Table 1).

Partial sequences of the sodA gene

The sodA gene sequences of the 23 isolates collected from the different fish species and countries were identical (100% sequence identity), except for KNH07902, in which a single nucleotide differed from that of the other isolates. The nucleotide sequences of the sodA gene were submitted to the GenBank sequence database (Table 1). Figure 1 shows a phylogenetic tree generated based on the sodA gene sequences of fish isolates of S. dysgalactiae and the sodA gene of other related Streptococcus species. This tree revealed that all the fish strains clearly belonged to only one cluster, and they were separated from other related Streptococcus species.

image

Figure 1.  Phylogenetic tree generated based on the comparative analysis of the sodA gene sequences, showing the relationship among the fish strains of Streptococcus dysgalactiae, which were identified in this study, and related species of the genus Streptococcus.

Download figure to PowerPoint

BSFGE profiles of isolates

All the fish isolates were typeable using BSFGE. The macrorestriction patterns of the genomic DNAs of fish isolates (n=30) digested by ApaI were classified into nine genotypes: A, B, C, D, E, F, G, H, and I (Fig. 2 and Table 1). The computer-generated dendrogram revealed that a total of 25 isolates belonged to one main cluster (Cluster I) at a 70% similarity level (Fig. 3). All the Taiwanese strains (except 95985 and AOD-96086-K) and the Chinese strains PP1564 and PP1635 showed an identical genotype (D). All the Japanese isolates were grouped into genotypes A, B, and C, while the genotype of the Malaysian strain WSSN1609 was B. On the other hand, the AOD-96086-K (E), 95985 (F), PP1398 (G), and PF880 (H) strains and the tilapia strain of T11358 (I) had unique profiles.

image

Figure 2.  The macrorestriction fragment profiles of DNAs digested with ApaI in nine representative isolates of Streptococcus dysgalactiae collected from fish. Lane M, marker DNA; lane A, Kdys0728 (Japan); lane B, Kdys0717 (Japan); lane C, KGB7804 (Japan); lane D, 94485 (Taiwan); lane E, 95985 (Taiwan); lane F, AOD-96086-K (Taiwan); lane G, PF880 (Malaysia); lane H, PP1398 (Malaysia); lane I, T11358 (Indonesia).

Download figure to PowerPoint

image

Figure 3.  Dendrogram showing clusters generated based on the analysis of the BSFGE data obtained from Streptococcus dysgalactiae fish isolates.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This paper presents the first epidemiological comparison study comprising a total of 30 strains of S. dysgalactiae isolated from diseased fish species in different Asian countries. The epidemiological study was conducted based on phenotypic characterization in addition to both sequencing of the sodA gene and BSFGE due to their high discriminative power. Most of the studies on the phenotypic characterization of streptococci, which have been reported thus far, used the API 20 STREP® and API ZYM® systems (Tillotson, 1982; Gruner et al., 1992). The biochemical and enzymatic characterizations performed in this study revealed that all the fish isolates exhibited a high phenotypic homogeneity irrespective of their country of origin as well as the fish species, and their comparison with the reference strain ATCC43078 revealed that all tested fish isolates could hydrolyze arginine, but could not acidify lactose. Therefore, the phenotypic homogeneity should be taken into account when these systems are used for routine identification of clinical isolates of S. dysgalactiae. All the isolates carried the tet(M) gene, except for the Taiwanese isolates and the PP1564 isolate collected in China, resulting in resistance to oxytetracycline. This finding suggested that the oxytetracycline-resistance gene tet(M) prevailed in the majority of fish S. dysgalactiae isolates collected in various Asian countries. This fact concurred with the results obtained by Kim et al. (2004), who suggested that the tet(M) gene was present in fish intestinal and seawater bacteria at aquaculture sites, and these bacteria could be important reservoirs of tetracycline-resistance genes in the marine environment.

The sequencing of the sodA gene was performed for the genetic comparison characterization between Japanese fish and mammalian isolates of S. dysgalactiae (Nomoto et al., 2008). In this study, sequencing of the sodA gene was performed in order to compare different fish isolates collected from various Asian countries. As a result, a 100% sequence identity was observed among the fish isolates irrespective of their country of origin, except for KNH07902, in which a single nucleotide differed from that of the other isolates. This finding revealed the homogeneity among fish isolates irrespective of the country of origin as well as the fish species. Thus, the phylogenetic analysis demonstrated that all the fish isolates investigated in this study belonged to a single cluster and were distinct from the other GCS strains. Therefore, we consider the possibility that fish isolates of S. dysgalactiae might be differentiated from the traditional strains of GCS at the subspecies level in future studies.

In this study, we were particularly interested in whether the strains were geographically localized or clonally related to each other at the multinational level. The most common method used for typing streptococci consists of the restriction of genomic DNA with ApaI and SmaI endonucleases, followed by PFGE analysis (Green et al., 2006; Bacciaglia et al., 2007). During the course of this study, the restriction endonucleases of ApaI and SmaI were investigated to determine their suitability for usage in the BSFGE analysis of S. dysgalactiae. Unfortunately, the SmaI genotypes comprised fragments, the number of which was too few to allow effective discrimination between isolates, at least under the operating conditions used in this study. In general, isolates whose BSFGE genotypes are highly similar to each other, as indicated by a Dice coefficient ≥0.90, are likely to be closely related to each other genetically and epidemiologically. Moreover, the correlation of the BSFGE genotype similarity to the genomic relatedness rapidly decreases to below 70% similarity values (Struelens et al., 2001). The results obtained using the computer-generated dendrogram revealed that fingerprint variations obtained by digestion with ApaI could classify most of the isolates, including the Japanese, Taiwanese, and Chinese isolates, into one main cluster at a 70% similarity level. However, the macrorestriction genotypes of the 95985, AOD-96086-K, PP1398, PF880, and T11358 fish isolates apparently differed from those of the main cluster. In this study, we demonstrated that the genotypes of S. dysgalactiae isolates collected from different fish species could be related to each other at the multinational level for the first time. To improve understanding of the epidemiology of and medical therapy for S. dysgalactiae infections, all fish streptococci should be identified to the species level and accurately tested for antimicrobial susceptibility.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors are grateful to Dr Lauke Labrie and her aquatic animal health team of Schering-Plough Animal Health, Singapore, for kindly providing S. dysgalactiae isolates. This study was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports, Japan (21580229). The first author appreciates financial support received from the Ministry of High Education, Egypt.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Aarestrup FM & Jensen NE (1996) Genotypic and phenotypic diversity of Streptococcus dysgalactiae strains isolated from clinical and subclinical cases of bovine mastitis. Vet Microbiol 53: 315323.
  • Abdelsalam M, Chen SC & Yoshida T (2009a) Surface properties of Streptococcus dysgalactiae strains isolated from marine fish. Bull Eur Assn Fish P 29: 1523.
  • Abdelsalam M, Nakanishi K, Yonemura K, Itami T, Chen SC & Yoshida T (2009b) Application of Congo Red agar for detection of Streptococcus dysgalactiae isolated from diseased fish. J Appl Ichthyol 25: 442446.
  • Agersø Y, Jensen LB, Givskov M & Roberts MC (2002) The identification of a tetracycline resistance gene tet(M), on a Tn916-like transposon, in the Bacillus cereus group. FEMS Microbiol Lett 214: 251256.
  • Bacciaglia A, Brenciani A, Varaldo PE & Giovanetti E (2007) SmaI typeability and tetracycline susceptibility and resistance in Streptococcus pyogenes isolates with efflux-mediated erythromycin resistance. Antimicrob Agents Ch 51: 30423043.
  • Chénier S, Leclère M, Messier S & Fecteau G (2008) Streptococcus dysgalactiae cellulitis and toxic shock like syndrome in a Brown Swiss cow. J Vet Diagn Invest 20: 99103.
  • Constable PD & Morin DE (2002) Use of antimicrobial susceptibility testing of bacterial pathogens isolated from the milk of dairy cows with clinical mastitis to predict response to treatment with cephapirin and oxytetracycline. J Am Vet Med Assoc 221: 103108.
  • Green MD, Beall BB, Marcon MJ et al. (2006) Multicentre surveillance of the prevalence and molecular epidemiology of macrolide resistance among pharyngeal isolates of group A streptococci in the USA. J Antimicrob Chemoth 57: 12401243.
  • Gruner E, Graevenitz A & Altwegg M (1992) The API ZYM system: a tabulated review from 1977 to date. J Microbiol Meth 16: 101118.
  • Hall TA (1999) Bioedit; a user friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41: 9598.
  • Kim S, Nonaka L & Suzuki S (2004) Occurrence of tetracycline resistance genes tet(M) and tet(S) in bacteria from marine aquaculture sites. FEMS Microbiol Lett 237: 147156.
  • Koh TH, Sng LH, Yuen SM, Thomas CK, Tan PL, Tan SH & Wang NS (2009) Streptococcal cellulitis following preparation of fresh raw seafood. Zoonoses Public Health 56: 206208.
  • Kumar S, Tamura K & Nei M (2004) MEGA3; integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5: 150163.
  • Lacasta D, Ferrer LM, Ramos JJ, Loste A & Bueso JP (2008) Digestive pathway of infection in Streptococcus dysgalactiae polyarthritis in lambs. Small Ruminant Res 78: 202205.
  • Lancefield R (1933) A serological differentiation of human and other groups of hemolytic Streptococci. J Exp Med 59: 571591.
  • Madinabeitia I, Ohtsuka S, Okuda J, Iwamoto E, Yoshida T, Furukawa M, Nakaoka N & Nakai T (2009) Homogeneity among Lactococcus garvieae isolates from striped jack, Pseudocaranx dentex (Bloch & Schneider), and its ectoparasites. J Fish Dis 32: 901905.
  • Nei M & Li WH (1979) Mathematical model for studying genetic variation in terms of restriction endonucleases. P Natl Acad Sci 76: 52695273.
  • Nomoto R, Munasinghe L, Jin D, Shimahara Y, Yasuda H, Nakamura A, Misawa N, Itami T & Yoshida T (2004) Lancefield group C Streptococcus dysgalactiae infection responsible for fish mortalities in Japan. J Fish Dis 27: 679686.
  • Nomoto R, Unose N, Shimahara Y et al. (2006) Characterization of Lancefield group C Streptococcus dysgalactiae isolated from fish. J Fish Dis 29: 673682.
  • Nomoto R, Kagawa H & Yoshida T (2008) Partial sequencing of sodA gene and its application to identification of Streptococcus dysgalactiae subsp. dysgalactiae isolated from farmed fish. Lett Appl Microbiol 46: 95100.
  • Scott PR (2000) Extensive fibrinous pleurisy associated with Streptococcus dysgalactiae mastitis in two ewes. Vet Rec 146: 347349.
  • Struelens MJ, De Ryck R & Deplano A (2001) Analysis of microbial genomic macrorestriction patterns by pulsed-field gel electrophoresis typing. New Approaches for the Generation and Analysis of Microbial Typing Data (DijkshoornL, TownerK & StruelensM, eds), pp. 159176. Elsevier, Amsterdam.
  • Tillotson GS (1982) An evaluation of the API-20 STREP system. J Clin Pathol 35: 468472.
  • Vieira VV, Teixeira LM, Zahner V, Momen H, Facklam RR & Steigerwalt AG (1998) Genetic relationships among the different phenotypes of Streptococcus dysgalactiae strains. Int J Syst Bacteriol 48: 12311243.
  • Yang W & Li A (2009) Isolation and characterization of Streptococcus dysgalactiae from diseased Acipenser schrenckii. Aquaculture 294: 1417.