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 patho- genic 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.