Starch Hydrolysis Testing of Multiple Isolates for Rapid Differentiation of Streptococcus Iniae

Home , Streptococcus bovis

Bull. Eur. Ass. Fish Pathol., 24(5) 2004, 231 Starch hydrolysis testing of multiple isolates for rapid differentiation of Streptococcus iniae

J.J. Evans1, P.H. Klesius2 and C.A. Shoemaker2

United States Department of Agriculture (USDA), Agriculture Research Service (ARS), Aquatic Animal Health Research Laboratory, 151 Dixon Drive, Chestertown, Maryland 21620 USA1 and P.O. Box 952 Auburn, Alabama 36832 USA2.

Abstract Streptococcus iniae is one of the few Gram positive streptococcal species capable of hydrolyzing starch. The starch hydrolysis test is essential for its identification. The biochemical reaction for starch in multi-test systems (API 20 Strep and API CH 50) is starch acidification, not starch hydrolysis. Furthermore, API systems do not contain S. iniae in their databases, yet fish disease researchers use these systems for S. iniae identification instead of starch hydrolysis tests. We developed a modified procedure of the starch hydrolysis test and evaluated multiple pathogenic streptococcal isolates including S. iniae, S. agalactiae, Enterococcus spp. and Lactococcus spp. for their ability to hydrolyze starch on a single starch agar plate following 18 h incubation at 30 and 35°C. All of the S. iniae isolates (17/17) tested hydrolyzed starch using our modified starch hydrolysis technique. Two of eleven S. agalactiae isolates tested hydrolyzed starch. Lactococcus spp. and Enterococcus spp. did not hydrolyze starch. This method is rapid, accurate and cost effective, increasing the number of isolates that can be tested at once.

Introduction differentiate between these potential zoonotic The starch hydrolysis test is used to aid in bacterial species. Both S.iniae and Group B S. species differentiation among various Gram agalactiae (GBS), formerly associated with positive aerobic genera such as Bacillus spp., marine and terrestial mammals, respectively, Streptococcus spp., and anaerobic genera, such have become emerging fish pathogens (Austin as Clostridium spp. (MacFaddin, 2000). Starch and Austin, 1999; Evans et al., 2002). hydrolysis testing is essential for However, confusion exists in the fish disease identification of Streptococcus iniae as it is one literature on how to identify and differentiate of the few streptococcal species capable of between these species. Perhaps the difficulty hydrolyzing starch. We (Shoemaker et al., in the identification of these emerging 2001) have used this procedure extensively to streptococcal pathogens by fish researchers identify and determine the prevalence of S. stems from the lack of careful comparisons of iniae on commercial fish farms. Starch phenotypic characteristics to those hydrolysis testing can also aid in the streptococcal organisms commonly associated differentiation between streptococcal species. with mammalian infection. Initially, S. iniae Because S.iniae and S. agalactiae are and S. agalactiae were perceived as two new morphologically and phenotypically similar species of fish pathogenic streptococci, S. shiloi and cause similar disease signs in fish, and S. difficile, respectively, and were discriminating tests are needed to differentiated by hemolysis characteristics Bull. Eur. Ass. Fish Pathol., 24(5) 2004, 232 and mannitol reactions in API 50 CH and API Conventional methods of starch hydrolysis 20 Strep systems (Eldar et al., 1994). Later, testing have involved inoculating starch agar S.shiloi became synonymous with beta slants or plates from an 18-24 h pure culture hemolytic S. iniae (Eldar et al., 1995), although by making a fishtail slant or a single starch hydrolysis was not performed, and horizontal streak across the center of the plate non-hemolytic S. difficile was indistin- (Facklam and Washington, II, 1991; quishable from Group B S.agalactiae MacFaddin, 2000). Unlike broth and slant (Vandamme et al., 1997). All previously media, starch agar plates can be divided into reported isolates of GBS from fish have been four quadrants to accommodate four separate non-hemolytic and are considered to be determinations. Generally, higher incubation “atypical” compared to the more typically temperatures (35°C) produce results between beta hemolytic mammalian isolates. Evans 18-48 h while lower temperatures (20-30°C) et al. (2002), however, isolated beta hemolytic have been reported to require five days S. agalactiae isolates from infected mullet. incubation (Bullock, 1978). The purpose of Without additional specific tests beyond this study was to develop a rapid, accurate, hemolysis, identification of S.agalactiae and cost effective, differential test (technique) for S.iniae could be confused. identifying fish pathogens using a modified procedure of the starch hydrolysis test. In Starch hydrolysis by conventional methods addition to providing information on starch was used to characterize the S. iniae Ameri- hydolysis of streptococcal isolates, starch can Tissue Culture Collection (ATCC) type acidification of isolates were examined in API isolates from Amazon dolphins, Inia systems to determine the differences in starch geoffrensis, (Pier and Madin, 1976; Pier et al., reactions for the same isolate and between 1978) and is the gold standard for S. iniae iden- isolates. We also compared the results of our tification to which all other suspected S. iniae starch hydrolysis and starch acidification isolates should be compared. Streptococcus testing of streptococcal isolates to what has iniae cannot be identified based solely on been previously reported for these isolates. acidification of starch. Despite this, starch hydrolysis testing is lacking from many S.iniae Materials and methods characterizations. Fish researchers began and Pathogenic streptococcal isolates originated continue utilizing API multi-test systems to from mammalian and aquatic animal sources identify S. iniae, an organism not in the (Table 1) and were previously characterized databases of these API systems, and many using conventional starch hydrolysis and bio- substitute acidification of starch for hydroly- chemical techniques (Pier and Madin, 1976; sis in publications. Although API systems Pier et al., 1978; Facklam and Washington, II, may give the same starch reaction end points, 1991; Shoemaker et al., 2001; Evans et al., i.e. positive for S. iniae or negative for 2002). Isolates were grown on tryptic soy agar S.agalactiae, starch acidification is a different (TSA, Difco, Detroit, MI, USA) supplemented biochemical reaction than starch hydrolysis. with 5% sheep blood for 24 h at 30°C. Single Furthermore, other streptococcal organisms bacterial colonies were point inoculated have been reported to acidify starch. around the perimeter of eight 2% soluble Bull. Eur. Ass. Fish Pathol., 24(5) 2004, 233

Geenus and species Designation and sourc Incubation temperature of bacterial isolate 30°C 35°C

Starch hydrolysis positive (+) or negative (-) & % times positive b

S. iniae ARS c 6s0 Hybrid striped bas d +01+0 10 0 S. iniae AsRS T4719 H+0ybrid striped bas 1+0 10 0 S. iniae AsRS M16 H+0ybrid striped bas 1+0 10 0 S. iniae AsRS M19 H+0ybrid striped bas 1+0 10 0 S. iniae AsRS-99-T23 H+0ybrid striped bas 1+0 10 0 S. iniae AsRS T23 D18 H+0ybrid striped bas 1+0 10 0 S. iniae EsT-02-02a Hybrid striped bas e +01+0 10 0 S. iniae AaRS NDS-1B Nile tilapi f +01+0 10 0 S. iniae AaRS NDS-10 N+0ile tilapi 1+0 10 0 S. iniae MaO-05-01 Hybrid tilapi g +01+0 10 0 S. iniae MaO-09-01 H+0ybrid tilapi 1+0 10 0 S. iniae DaL-12-02 H+0ybrid tilapi 1+0 10 0 S. iniae QiDPI95/41693-4 Barramund h +01+0 10 0 S. iniae ATCC i 2n9177 Amazon dolphi j +01+0 10 0 S. iniae AnTCC 29178 A+0mazon dolphi 1+0 10 0 S. iniae CDC k 2n032 H+0uma 1+0 10 0 S. iniae CnDC 2036 H+0uma 1+0 10 0 S. agalactiae AtRS KU-KF1 Klunzingeri mulle l -0-0 S.agalactiae AtRS-KU23 K-0-0lunzingeri mulle S.agalactiae AtRS-KU34 K-0-0lunzingeri mulle S.agalactiae AtRS-KU11 K-0-0lunzingeri mulle S.agalactiae AtRS-KU17 K-0-0lunzingeri mulle S. agalactiae AtRS-KU19 K+5lunzingeri mulle 2-0 S.agalactiae AtRS-KU24 K+0lunzingeri mulle 1-50 2 S.agalactiae AmRS-KU1 Gilthead seabrea m -0-0 S.agalactiae AmRS-KU38 G-0-0ilthead seabrea S.agalactiae AnTCC 13813 H-0-0uma S.agalactiae AeTCC 27956 B-0-0ovin S. bovis AeTCC 49133 B-0-0ovin Enterococcus faecalis AnTCC 33862 H-0-0uma E. durans CnDC (SS-661) H-0-0uma Staphylococcus aureus AnTCC 33862 H-0-0uma aClear zone of hydrolyzed starch surrounding the growth of a single bacterial colony stabbed into 2% starch agar plate using 200 or 10 µL pipette tips following incubation at 30 or 35°C for 18 h and flooding the plates with 5 or 10% iodine solutions. b Positive and negative reactions and percentage of times an isolate was positive were the result of four treatments (200 µL, 5% iodine; 200 µL, 10% iodine; 10 µL, 5% iodine; 10 µL, 10% iodine) per isolate at each temperature. c Agricultural Research Service (ARS), Auburn, Alabama, USA.; d Morone chrysops ´ M. saxatilis isolate; e Israeli isolate; f Oreochromis niloticus; g Israeli isolates from Oreochromis sp.; h Australian isolate from Lates calcarifer (Queensland Department of Primary Industry); i American Type Culture Collection (ATCC), Rockville, MD, USA; j Inia geoffrensis; k Center for Disease Control (CDC), Atlanta, GA, USA;l Liza klunzingeri; m Sparus auratus. Table 1. Starch hydrolysis reactions of Streptococcus iniae, S. agalactiae, and other Gram positive coccus isolates from different animal species.a Bull. Eur. Ass. Fish Pathol., 24(5) 2004, 234

Genera b C-ells Hemo Lhancefield Starc Growth at lysis Hydrolysis 1C0°C 4%5° 6.5 NaCl Streptococcus ch α/β/n A-H, L, K-N, +vv-/- O, U, V ng Enterococcus ch α/β/n D ++/- ++ Lactococcus ch α/n N -+-c v Pediococcus cl/te α/n Ng +-/- +v Species d Hdemolysis Lnancefiel Sttarch reactio Growth a Hydro-- A0PI20 CCH5 1C0° 45° lysis R O R O ROROROROR O S. iniae e α/ββ ngg n + +++++- *++/ +-/ - S. agalactiae f § α/β/n β BB+-/ -+------/ - S. bovis g α/n α Dgn+-rn-n* r+- -+ + E. seriolicida h α/βα ngot D nrn-----+++ - E. faecalis i α/β/n α Dtn ------+++ + L. garvieae j¥ α/β/n α Ngn ------++++/ - L. lactis k ntr nNtnrntnrntn-+t/ n+n- t n t L. raffinolactis l ntr nNtnrntnrntn+n+ t n- t n t L. piscium m β nNtt nrntnrntn+n+ t nr tntn P. dextrinicus n α/n ngngn+-rn-n+- r -++/ - C. piscicola o ndr nrndnrntnrntn+n+ t nr tntn a Abbreviations and symbols: cells, cell arrangement; ch, chains; cl, clumps; te, tetrads; hemolysis, hemolysis on blood agar containing 5% sheep blood; α, alpha-hemolysis; β, beta-hemolysis; n, no hemolysis; +, positive; -, negative; +/-, either positive or negative; ?, unknown; nr, not reported; ng, not groupable; nt, not tested; * weak. b Data on clinical isolates from Facklam and Washington (1991); Facklam and Elliott (1995); MacFaddin (2000); Ruoff et al. (1999) c Most species grow poorly or not at all in contrast to enterococci which grow very well. d Species’ designations used in our testing of clinical and fish isolates: Streptococcus iniae, (ATCC 29178T) and ARS # 60; S. agalactiae, ARS- KU11, ARS-KU24 (Evans et al. 2002); S. bovis, (ATCC 49133); Enterococcus seriolicida, YT-3 (ATCC 49156T); E. faecalis, NCDO 581 (ATCC 27792); Lactococcus garvieae, NCDO 2155 (ATCC 43921T); Pediococcus dextrinicus (ATCC 33087T). § Vandamme et al. (1997) corrected original characterization of Eldar et al. (1994) from S. difficile to S. agalactiae.¥ Eldar et al. (1996) proposed L. garvieae as a junior synonym for E. seriolicida. e API 20, CH 50 data from Eldar et al. (1994;1995); Growth data from Perera et al. (1994); Starch hydrolysis from Pier and Madin (1976); Shoemaker et al. (2001) f Data from Evans et al. (2002); MacFaddin (2000); Vandamme et al. (1997); Wilkinson et al. (1973) g Data from Facklam and Washington (1991); McFaddin (2000); Ruoff et al. (1999) h Data from Doménech et al. (1993); Eldar et al. (1996); Kusuda et al. (1991); i Data from Kusuda et al. (1991); Ravelo et al. (2001) j Data from Collins et al. (1983); Eldar et al. (1996; 1999b); Ravelo et al. (2001) k l Data from Collins et al. (1983) m Data from Collins et al. (1983); Williams et al. (1990) n Data from Facklam and Elliott (1995); MacFaddin (2000) o Data from Doménech et al. (1993)

Table 2. Characteristics of selected streptococcal and related Gram positive, catalase negative species as reported in the literature (R) and from our testing (O) a. Bull. Eur. Ass. Fish Pathol., 24(5) 2004, 235 starch plates (Remel, Lenexa, KS, USA) using Results a pipette tip (10 µl or 200 µl) to make a circu- Starch hydrolysis testing aided in the lar impression and incubated at 30 or 35°C differentiation between morphologically for 18 h. Following incubation and growth similar streptococcal species, such as S. iniae assessment, plates were covered with 10 ml and S.agalactiae, and in the identification of S. of 5% or 10% Gram’s iodine for 5-8 min and iniae. Optimal conditions for starch checked for clear zones (disappearance of hydrolysis testing were bacterial inoculation starch) indicative of positive starch hydroly- of starch plate with a 10 µl pipette tip, which sis after pouring off the iodine. Positive and was easier to manipulate, incubation at 35°C, negative starch reactions of four treatments which provided better bacterial growth, and (200 µl, 5% iodine; 200 µl, 10% iodine; 10 µl, flooding the plate with 5% iodine, which gave 5% iodine; 10 µl, 10% iodine) per isolate at better visualization of the zone of hydrolysis. each temperature were visually evaluated for The clear zone produced by starch hydrolysis optimal conditions to perform starch hydroly- faded in approximately 5-10 min after the 5% sis testing. Staphylococcus aureus served as a iodine was removed, although clear zones negative control. In the second experiment, could still be distinguished for up to 1.5 h after isolates were chosen for starch reaction test- the removal of iodine. Streptococcus iniae was ing based on their reported positive starch an active starch hydrolyzing organism (Figure acidification in API systems or positive starch 1) and hydrolyzed starch 100% of the times hydrolysis in conventional tests (Table 2). tested regardless of host origin or incubation Because infected mammals and fish are po- temperature (Table 1). Repetitive testing of tential reservoirs of group B streptococci, selected beta hemolytic Group B S.agalactiae S.iniae, and other similar catalase negative mullet isolates (Evans et al., 2002) in this study cocci that might be transmitted to humans, indicated variability in starch hydrolysis. additional mammalian and piscine isolates Although the majority of S.agalactiae isolates with either reported negative starch reactions gave negative starch reactions (Figure 1), two or non-reported starch reactions were also in- S. agalactiae isolates, obtained from mullet cluded (Table 2). Cell morphology, hemolysis blood (ARS-KU19) and head kidney (ARS- on 5% SBA plates, growth at 10 and 45°C, KU24), gave positive starch hydrolysis Lancefield antigen detection, and starch hy- reactions (Table 1). Starch hydrolysis of these drolysis of selected streptococcal and related S. agalactiae isolates occurred more frequently Gram positive isolates were assessed by con- at 30°C than at 35°C. The ARS-KU24 isolate ventional methodologies as described by was starch positive 100% of the times tested Evans et al. (2002). Starch acidification was at 30°C and positive 25% of the times tested performed using the API 20 Strep and API 50 at 35°C. The ARS-KU19 isolate was starch CH systems (bioMérieux, Inc., Hazelwood, positive 25% of the times tested at 30°C and MO, USA), according to manufacturer’s in- was positive 0% of the times tested at 35°C. structions. Tests were incubated at 35°C and Streptococcus bovis, Enterococcus faecalis, E. readings were made at 4, 24, and 48 h. durans, E. seriolicida, Lactococcus garvieae, Pediococcus dextrinicus, and S. aureus did not Bull. Eur. Ass. Fish Pathol., 24(5) 2004, 236

Discussion Starch hydrolysis testing is not routinely performed or reported in characterizations of S.iniae and related similar catalase negative, Gram positive cocci. Starch reactions for S.iniae, when reported, are frequently not starch hydrolysis but are starch acidification reactions that were performed using commercial bacterial identification test kits (API 20 strep, API CH 50) (Eldar et al., 1994; 1995; 1999a; Stoffregen et al., 1996; Bowser et al., 1998; Zlotkin et al., 1998; Bromage et al., 1999; Yuasa et al., 1999) of which many fish pathogens such as S. iniae are lacking from the API database. Starch reactions in API systems are indicative of acid production or acidification (Barnes and Ellis, 2003) by either fermentative or oxidative metabolic pathways Figure 1. Clear zones of starch hydrolysis produced and not starch hydrolysis. MacFaddin (2000) by five isolates of Streptococcus iniae (ARS #60, ATCC provides a detailed description of the 29178, ATCC 2917, CDC 2036, CDC2032) (A) and absence of starch hydrolysis by five isolates of S. biochemistry involved in starch hydrolysis. agalactiae (ARS KU-KF1, ARS KU 11, 17, ATCC Furthermore, the interpretation of these 13813, ATCC 27956) (B) in 2% soluble starch agar colorimetric reactions is semi-quantitative plates. Single bacterial colonies were point inoculated around the perimeter of the plate using (bioMérieux, Inc., Hazelwood, MO, USA). 10 µl pipette tip and incubated at 35°C for 18 h. The acidification of starch is frequently Plate was flooded with 5% iodine for 5-8 min prior to observing the zones of starch hydrolysis. weaker than that of other sugars in the test strip such that intermediate reactions are hydrolyze starch in our testing (Tables 1 & 2). difficult to assign as either positive or The testing of selected streptococcus isolates negative. We noted weak starch acidification for starch hydrolysis by our method and for S. iniae and S. bovis in the API CH 50 starch acidification by API 20 strep and API system. Unfortunately, starch reactions from 50 CH systems indicated consistent positive these systems are often equated with or taken starch reactions for S. iniae, although starch to mean starch hydrolysis and are erroneously acidification was weak in the API 50 CH strip. reported as such. As a result, there is In contrast, S. agalactiae, E.faecalis, E. seriolicida, considerable confusion and misinterpretation and L. garvieae did not acidify starch. in the literature as to whether an isolate is Variability was noted in starch acidification truly positive by starch hydrolysis. Rapid for S. bovis and P. dextrinicus in different API Strept 32 (bioMérieux, Hazelwood, MO) does systems. Our results indicated negative not contain a starch test and does not include reactions for S. bovis and P. dextrinicus in API S. iniae in the database, yet it is widely used 20 strep and positive reactions in API CH 50. Bull. Eur. Ass. Fish Pathol., 24(5) 2004, 237 to identify and characterize streptococci. hydrolysis it cannot be assumed that other Positive starch acidification has been reported streptococcal organisms will behave similarly for S. iniae, L. piscium, Carnobacterium piscicola, and that results can be extrapolated from one L.lactis, L. raffinolactis, and P. detrinicus, and test system to another. For example, S. bovis negative starch acidification has been and P. dextrinicus were previously reported reported for S. agalactiae, E. seriolicida, as starch hydrolyzing, yet we found these E.faecalis, and L. garvieae in API systems (Table isolates incapable of hydrolyzing starch or 2) (Collins et al., 1983; Williams et al., 1990; acidifying starch (API 20 strep). Conversely, Kusuda et al., 1991; Doménech et al., 1993; S. bovis and P. dextrinicus acidified starch in Eldar et al., 1994; 1995; 1996; MacFaddin 2000; the API CH 50 test. Repetitive testing of S. Evans et al., 2002; Ravelo et al., 2001). Our agalactiae fish isolates showed the majority of results were in agreement with previous isolates incapable of hydrolyzing starch, reports of positive starch acidification for S. although positive starch hydrolysis was noted iniae and negative starch acidification for in two mullet isolates tested at 30°C (Table 1). S.agalactiae, E. seriolicida, E. faecalis, and In previous studies, Wilkinson et al. (1973) L.garvieae in API systems. Mammalian and found non-hemolytic, Group B S. agalactiae piscine isolates of S. iniae (Pier and Madin, isolates obtained from fish and humans to be 1976; Shoemaker et al., 2001) and clinical negative for starch hydrolysis, although five bovine isolates of S. agalactiae, S. bovis, of seven (71%) isolates tested from cow’s milk nonenterococci (Group D), and P. dextrinicus were positive for starch hydrolysis. However, have been reported as starch hydrolyzing details of the starch hydrolysis testing organisms (Wilkinson et al. 1973; Facklam and procedure and temperature at which the test Washington, II, 1991; Facklam and Elliott, was performed was not given. These results 1995; MacFaddin, 2000) (Table 2). Starch indicate that although S. agalactiae from fish, hydrolysis testing of clinical and fish isolates human, and bovine sources is primarily starch by the method reported in this paper hydrolysis negative, variants may exist or the demonstrated positive starch hydrolysis temperature at which starch hydrolysis results for S. iniae and negative starch testing is performed may alter hydrolysis hydrolysis for E.faecalis, E. seriolicida, E. reaction, necessitating additional tests such durans, and L.garvieae. Ravelo et al. (2001), in as Lancefield serological grouping to characterizations of L. garvieae and E. faecalis, discriminate between, at present, non- also reported negative starch reactions by a typeable S. iniae and typeable Group B S. conventional starch hydrolysis technique. agalactiae. Ruoff et al. (1999) suggested that Negative starch hydrolysis for S. bovis and P. starch hydrolysis testing should be dextrinicus by our testing, however, was in supplemental to serological testing of some disagreement with what has been previously Streptococcus spp. Epidemiological reported for these organisms (Facklam and considerations as to the source, origin, Washington, II, 1991; Ruoff et al., 1999; potential for zoonotic infection by MacFaddin, 2000). Although S. iniae was streptococcal organisms, and effective positive for both starch acidification and treatment against these organisms emphasize Bull. Eur. Ass. Fish Pathol., 24(5) 2004, 238 the importance for accurate bacterial Collins MD, Farrow JAE, Phillips BA & Kandler O (1983). Streptococcus garvieae sp. speciation. We encourage the use of the starch nov. and Streptococcus plantarum sp. nov. Jour- hydrolysis test at 35°C and Lancefield nal of General Microbiology 129: 3427-3431. grouping to identify and better differentiate between S. iniae and S. agalactiae. We also Doménech A, Prieta J, Fernández-Garayzábal JF, Collins MD, Jones D & Domínguez L recommend using the starch hydrolysis test (1993). Phenotypic and phylogenetic evidence presented here to rapidly test multiple isolates for a close relationship between Lactococcus on a single starch agar plate, thereby garvieae and Enterococcus seriolicida. minimizing supplies needed and time Microbiologia (SEM) 9, 63-68. required for results and increasing the Eldar A, Bejerano Y and Bercovier H (1994). number of isolates that can be tested cost- Streptococcus shiloi & Streptococcus difficile: Two effectively. new streptococcal species causing meningoencephalitis in fish. Current Microbiology 28, 139-143. Acknowledgements Eldar A, Frelier PF, Asanta L, Varner PW, We thank Dr. Dina Zilberg (Ben-Gurion Uni- Lawhon S & Bercovier H (1995). Streptococcus versity of the Negev, Sede-Boker Campus, Is- shiloi, the name for an agent causing rael) for the Israeli isolates, Sherry Patterson septicemic infection in fish, is a junior for technical assistance, and Lisa Biggar for synonym of Streptococcus iniae. International Journal ofSystematic Bacteriology 45, 840-842. editing the manuscript. Eldar A, Ghittino C, Asanta L, Bozzetta E, Goria M, Prearo M & Bercovier H (1996). References Enterococcus seriolicida is a junior synonym of Austin B & Austin DA (1999). Characteristics Lactococcus garvieae, a causative agent of of the pathogens: Gram-positive bacteria. In septicemia and meningoencephalitis in fish. “Bacterial Fish Pathogens: Diseases of Current Microbiology 32, 85-88. Farmed and Wild Fish”. pp. 35-62. Springer Publisher, New York, N.Y. Eldar A, Perl S, Frelier PF & Bercovier H (1999a.) Red drum Sciaenops ocellatus Barnes AC & Ellis AE (2003). Variation in ar- mortalities associated with Streptococcus iniae ginine dihydrolase activity in Streptococcus infection. Diseases of Aquatic Organisms 36, iniae may be an artifact of the assay. Bulletin 121-127. of the European Association of Fish Pathologists 23, 163-166. Eldar A, Goria M, Ghittino C, Zlotkin A & Bercovier H (1999b). Biodiversity of Bowser PR, Wooster GA, Getchell RG & Lactococcus garvieae isolated from fish in Timmons MB (1998). Streptococcus iniae infec- Europe, Asia, and Australia. Applied tion of tilapia Oreochromis niloticus in a Environmental Microbiology 65, 1005-1008. recirculation production facility. Journal of World Aquaculture Society 29, 335-339. Evans JJ, Klesius PH, Glibert PM, Shoemaker CA, Al Sarawi MA, Landsberg J, Duremdez Bromage ES, Thomas A & Owens L (1999). R, Al Marzouk A & Al Zenki S (2002). Streptococcus iniae, a bacterial infection in Characterization of beta-haemolytic Group B barramundi Lates calcarifer. Diseases of Aquatic Streptococcus agalactiae in cultured seabream, Organisms 36, 177-181. Sparus auratus (L.) and wild mullet, Liza klunzingeri (Day), in Kuwait. Journal of Fish Bullock AM (1978). Laboratory methods. In Diseases 25, 505-513. “Fish Pathology” (R.J. Roberts, Ed.) pp. 235- 236. Bailliere, Tindall, London. Bull. Eur. Ass. Fish Pathol., 24(5) 2004, 239

Facklam RR & Elliott JA (1995). Identification, Ruoff KL, Whiley RA & Beighton B (1999). classification, and clinical relevance of cata- Gram positive cocci Streptococcus. In lase-negative, Gram-positive cocci, excluding “Manual of Clinical Microbiology”, 7th ed. the Streptococci and Enterococci. Clinical (P.R. Murray,E.J. Baron,M.A. Pfailer,F.C. Microbiology Reviews 8, 479-495. Tenover & R.H. Yolken, Eds.) pp. 282-296. American Society of Microbiology, Facklam RR & Washington JA, II (1991). Strep- Washington, DC. tococcus and related catalase-negative, gram- positive cocci. In “Manual of Clinical Micro- Shoemaker CA, Klesius PH & Evans JJ (2001). biology”, 5th ed. (A. Balows, W.J. Hausler Jr., Prevalence of Streptococcus iniae in tilapia, K.L. Hermann, H.D. Lsenberg & H.J. Sadomy, hybrid striped bass and channel catfish from Eds.) pp. 238-257. American Society of Micro- fish farms in the United States. American Jour- biology, Washington, D.C. nal of Veterinary Research 62, 174-177.

Kusuda R, Kawai K, Salati F, Banner CR & Stoffregen DA, Backman SC, Perham RE, Fryer JL (1991). Enterococcus seriolicida sp. Bowser PR & Babish JG (1996). Initial disease nov., a fish pathogen. International Journal of report of Streptococcus iniae infection in hybrid Systematic Bacteriology 41, 406-409. striped (sunshine) bass and successful thera- peutic intervention with the fluoroquinolone MacFaddin JF (2000). Individual biochemical antibacterial enrofloxacin. Journal of World tests. Starch hydrolysis test. In “Biochemical Aquaculture Society 27, 420-434. Tests for Indentification of Medical Bacte- ria”, 3rd ed. pp. 412-423. Lippincott Williams Vandamme P, Devriese LA, Pot B, Kersters K & Wilkins, Philadelphia, PA. & Melin P (1997). Streptococcus difficile is a nonhemolytic group B, type Ib Streptococcus. Perera RP, Collins MD, Johnson SK & Lewis International Journal of Systematic Bacteriology DH (1994). Streptococcus iniae associated with 47, 81-85. mortality of Tilapia nilotica x T.aurea hybrids. Journal of Aquatic Animal Health 6, 335-340. Wilkinson HW, Thacker LG & Facklam RR (1973). Nonhemolytic group B Streptococci of Pier GB & Madin SH (1976). Streptococcus iniae human, bovine, and ichthyic origin. Infection sp. nov., a beta-hemolytic streptococcus & Immunity 7, 496-498. isolated from an Amazon freshwater dolphin, Inia geoffrensis. International Journal of Williams AM, Fryer JL & Collins MD (1990). Systematic Bacteriology 26, 545-553. Lactococcus piscium sp. nov., a new Lactococcus species from salmonid fishes. FEMS Micro- Pier GB, Madin SH & Al-Nakeeb S (1978). biology Letters 68, 109-114. Isolation and characterization of a second isolate of Streptococcus iniae. International Yuasa K, Kitancharoen N, Kataoka Y & Al- Journal of Systematic Bacteriology 28, 311-314. Murbaty FA (1999). Streptococcus iniae, the causative agent of mass mortality in rabbitfish Ravelo C, Mararinos B, Romalde JL, & Siganus canaliculatus in Bahrain. Journal of Toranzo AE (2001). Conventional versus Aquatic Animal Health 11, 87-93. miniaturized systems for the phenotypic caharacteization pf Lactococcus garvieae starins. Zlotkin A, Hershko H & Eldar A (1998). Bulletin of the European Association of Fish Possible transmission of Streptococcus iniae Pathologists 21, 136-143. from wild fish to cultured marine fish. Applied Environmental Microbiology 64, 4065-4067.