Starch hydrolysis testing of multiple isolates for rapid differentiation of Streptococcus iniae

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 The starch hydrolysis test is used to aid in species differentiation among various Gram positive aerobic genera such as Bacillus spp., Streptococcus spp., and anaerobic genera, such as Clostridium spp. (MacFaddin, 2000). Starch hydrolysis testing is essential for identification of Streptococcus iniae as it is one of the few streptococcal species capable of hydrolyzing starch. We (Shoemaker et al., 2001) have used this procedure extensively to identify and determine the prevalence of S. iniae on commercial fish farms. Starch hydrolysis testing can also aid in the differentiation between streptococcal species. Because S.iniae and S. agalactiae are morphologically and phenotypically similar and cause similar disease signs in fish, discriminating tests are needed to differentiate between these potential zoonotic bacterial species. Both S.iniae and Group B S. agalactiae (GBS), formerly associated with marine and terrestial mammals, respectively, have become emerging fish pathogens (Austin and Austin, 1999; Evans et al., 2002). However, confusion exists in the fish disease literature on how to identify and differentiate between these species. Perhaps the difficulty in the identification of these emerging streptococcal pathogens by fish researchers stems from the lack of careful comparisons of phenotypic characteristics to those streptococcal organisms commonly associated with mammalian infection. Initially, S. iniae and S. agalactiae were perceived as two new species of fish pathogenic streptococci, S. shiloi and S. difficile, respectively, and were differentiated by hemolysis characteristics Bull. Eur. Ass. Fish Pathol., 24(5) 2004, 232 and mannitol reactions in API 50 CH and API 20 Strep systems (Eldar et al., 1994). Later, S.shiloi became synonymous with beta hemolytic S. iniae (Eldar et al., 1995), although starch hydrolysis was not performed, and non-hemolytic S. difficile was indistinquishable from Group B S.agalactiae (Vandamme et al., 1997). All previously reported isolates of GBS from fish have been non-hemolytic and are considered to be “atypical” compared to the more typically beta hemolytic mammalian isolates. Evans et al. (2002), however, isolated beta hemolytic S. agalactiae isolates from infected mullet. Without additional specific tests beyond hemolysis, identification of S.agalactiae and S.iniae could be confused. Starch hydrolysis by conventional methods was used to characterize the S. iniae American Tissue Culture Collection (ATCC) type isolates from Amazon dolphins, Inia geoffrensis, (Pier and Madin, 1976; Pier et al., 1978) and is the gold standard for S. iniae identification to which all other suspected S. iniae isolates should be compared. Streptococcus iniae cannot be identified based solely on acidification of starch. Despite this, starch hydrolysis testing is lacking from many S.iniae characterizations. Fish researchers began and continue utilizing API multi-test systems to identify S. iniae, an organism not in the databases of these API systems, and many substitute acidification of starch for hydrolysis in publications. Although API systems may give the same starch reaction end points, i.e. positive for S. iniae or negative for S.agalactiae, starch acidification is a different biochemical reaction than starch hydrolysis. Furthermore, other streptococcal organisms have been reported to acidify starch. Conventional methods of starch hydrolysis testing have involved inoculating starch agar slants or plates from an 18-24 h pure culture by making a fishtail slant or a single horizontal streak across the center of the plate (Facklam and Washington, II, 1991; MacFaddin, 2000). Unlike broth and slant media, starch agar plates can be divided into four quadrants to accommodate four separate determinations. Generally, higher incubation temperatures (35°C) produce results between 18-48 h while lower temperatures (20-30°C) have been reported to require five days incubation (Bullock, 1978). The purpose of this study was to develop a rapid, accurate, cost effective, differential test (technique) for identifying fish pathogens using a modified procedure of the starch hydrolysis test. In addition to providing information on starch hydolysis of streptococcal isolates, starch acidification of isolates were examined in API systems to determine the differences in starch reactions for the same isolate and between isolates. We also compared the results of our starch hydrolysis and starch acidification testing of streptococcal isolates to what has been previously reported for these isolates. Materials and methods Pathogenic streptococcal isolates originated from mammalian and aquatic animal sources (Table 1) and were previously characterized using conventional starch hydrolysis and biochemical techniques (Pier and Madin, 1976; Pier et al., 1978; Facklam and Washington, II, 1991; Shoemaker et al., 2001; Evans et al., 2002). Isolates were grown on tryptic soy agar (TSA, Difco, Detroit, MI, USA) supplemented with 5% sheep blood for 24 h at 30°C. Single bacterial colonies were point inoculated around the perimeter of eight 2% soluble Bull. Eur. Ass. Fish Pathol., 24(5) 2004, 233 s e i c e p s d n a s u n e G e c r u o s d n a n o i t a n g i s e D e t a l o s i l a i r e t c a b f o e r u t a r e p m e t n o i t a b u c n I C ° 5 3 C ° 0 3 r o ) + ( e v i t i s o p s i s y l o r d y h h c r a t S e v i t i s o p s e m i t % & ) ( e v i t a g e n b e a i n i . S S R A c 0 6 s s a b d e p i r t s d i r b y H d + 0 0 1 + 0 0 1 e a i n i . S 9 1 7 4 T S R A s s a b d e p i r t s d i r b y H + 0 0 1 + 0 0 1 e a i n i . S 6 1 M S R A s s a b d e p i r t s d i r b y H + 0 0 1 + 0 0 1 e a i n i . S 9 1 M S R A s s a b d e p i r t s d i r b y H + 0 0 1 + 0 0 1 e a i n i . S 3 2 T 9 9 S R A s s a b d e p i r t s d i r b y H + 0 0 1 + 0 0 1 e a i n i . S 8 1 D 3 2 T S R A s s a b d e p i r t s d i r b y H + 0 0 1 + 0 0 1 e a i n i . S a 2 0 2 0 T E s s a b d e p i r t s d i r b y H e + 0 0 1 + 0 0 1 e a i n i . S B 1 S D N S R A a i p a l i t e l i N f + 0 0 1 + 0 0 1 e a i n i . S 0 1 S D N S R A a i p a l i t e l i N + 0 0 1 + 0 0 1 e a i n i . S 1 0 5 0 O M a i p a l i t d i r b y H g + 0 0 1 + 0 0 1 e a i n i . S 1 0 9 0 O M a i p a l i t d i r b y H + 0 0 1 + 0 0 1 e a i n i . S 2 0 2 1 L D a i p a l i t d i r b y H + 0 0 1 + 0 0 1 e a i n i . S 4 3 9 6 1 4 / 5 9 I P D Q i d n u m a r r a B h + 0 0 1 + 0 0 1 e a i n i . S C C T A i 7 7 1 9 2 n i h p l o d n o z a m A j + 0 0 1 + 0 0 1 e a i n i . S 8 7 1 9 2 C C T A n i h p l o d n o z a m A + 0 0 1 + 0 0 1 e a i n i . S C D C k 2 3 0 2 n a m u H + 0 0 1 + 0 0 1 e a i n i . S 6 3 0 2 C D C n a m u H + 0 0 1 + 0 0 1 e a i t c a l a g a . S 1 F K U K S R A t e l l u m i r e g n i z n u l K l 0 0 e a i t c a l a g a . S 3 2 U K S R A t e l l u m i r e g n i z n u l K 0 0 e a i t c a l a g a . S 4 3 U K S R A t e l l u m i r e g n i z n u l K 0 0 e a i t c a l a g a . S 1 1 U K S R A t e l l u m i r e g n i z n u l K 0 0 e a i t c a l a g a . S 7 1 U K S R A t e l l u m i r e g n i z n u l K 0 0 e a i t c a l a g a . S 9 1 U K S R A t e l l u m i r e g n i z n u l K + 5 2 0 e a i t c a l a g a . S 4 2 U K S R A t e l l u m i r e g n i z n u l K + 0 0 1 5 2 e a i t c a l a g a . S 1 U K S R A m a e r b a e s d a e h t l i G m 0 0 e a i t c a l a g a . S 8 3 U K S R A m a e r b a e s d a e h t l i G 0 0 e a i t c a l a g a . S 3 1 8 3 1 C C T A n a m u H 0 0 e a i t c a l a g a . S 6 5 9 7 2 C C T A e n i v o B 0 0 s i v o b . S 3 3 1 9 4 C C T A e n i v o B 0 0 s i l a c e a f s u c c o c o r e t n E 2 6 8 3 3 C C T A n a m u H 0 0 s n a r u d . E ) 1 6 6 S S ( C D C n a m u H 0 0 s u e r u a s u c c o c o l y h p a t S 2 6 8 3 3 C C T A n a m u H 0 0 Table 1. Starch hydrolysis reactions of Streptococcus iniae, S. agalactiae, and other Gram positive coccus isolates from different animal species.a 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. Bull. Eur. Ass. Fish Pathol., 24(5) 2004, 234 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. 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, ARSKU11, 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) a r e n e G b s l l e C o m e H s i s y l d l e i f e c n a L h c r a t S s i s y l o r d y H t a h t w o r G C ° 0 1 C ° 5 4 % 5 . 6 l C a N s u c c o c o t p e r t S h c α/β n / , N K , L , H A g n V , U , O / + v v s u c c o c o r e t n E h c α/β n / D / + + + + s u c c o c o t c a L h c α n / N + -c v s u c c o c o i d e P e t / l c α n / g N / + + v s e i c e p S d s i s y l o m e H d l e i f e c n a L n o i t c a e r h c r a t S t a h t w o r G o r d y H s i s y l 0 2 I P A 0 5 H C C ° 0 1 C ° 5 4 R O R O R O R O R O R O R O e a i n i . S e α/β β g n g n + + + + + * + / + + / + e a i t c a l a g a . S f § α/β n / β B B / + / + s i v o b . S g α n / α D g n + r n r n * + + + a d i c i l o i r e s . E h α/β α D t o n g n r n + + + s i l a c e a f . E i α/β n / α D t n + + + + e a e i v r a g . L j ¥ α/β n / α N g n + + / + + s i t c a l . L k r n t n N t n r n t n r n t n / + t n + t n t n s i t c a l o n i f f a r . L l r n t n N t n r n t n r n t n + t n + t n t n m u i c s i p . L m β t n N t n r n t n r n t n + t n + t n r n t n s u c i n i r t x e d . P n α n / n g n g n + r n r n + / + + a l o c i c s i p . C o r n d n r n d n r n t n r n t n + t n + t n r n t n Bull. Eur. Ass. Fish Pathol., 24(5) 2004, 235 starch plates (Remel, Lenexa, KS, USA) using a pipette tip (10 μl or 200 μl) to make a circular impression and incubated at 30 or 35°C for 18 h. Following incubation and growth assessment, plates were covered with 10 ml of 5% or 10% Gram’s iodine for 5-8 min and checked for clear zones (disappearance of starch) indicative of positive starch hydrolysis after pouring off the iodine. Positive and negative starch reactions of four treatments (200 μl, 5% iodine; 200 μl, 10% iodine; 10 μl, 5% iodine; 10 μl, 10% iodine) per isolate at each temperature were visually evaluated for optimal conditions to perform starch hydrolysis testing. Staphylococcus aureus served as a negative control. In the second experiment, isolates were chosen for starch reaction testing based on their reported positive starch acidification in API systems or positive starch hydrolysis in conventional tests (Table 2). Because infected mammals and fish are potential reservoirs of group B streptococci, S.iniae, and other similar catalase negative cocci that might be transmitted to humans, additional mammalian and piscine isolates with either reported negative starch reactions or non-reported starch reactions were also included (Table 2). Cell morphology, hemolysis on 5% SBA plates, growth at 10 and 45°C, Lancefield antigen detection, and starch hydrolysis of selected streptococcal and related Gram positive isolates were assessed by conventional methodologies as described by Evans et al. (2002). Starch acidification was performed using the API 20 Strep and API 50 CH systems (bioMérieux, Inc., Hazelwood, MO, USA), according to manufacturer’s instructions. Tests were incubated at 35°C and readings were made at 4, 24, and 48 h. Results Starch hydrolysis testing aided in the differentiation between morphologically similar streptococcal species, such as S. iniae and S.agalactiae, and in the identification of S. iniae . Optimal conditions for starch hydrolysis testing were bacterial inoculation of starch plate with a 10 μl pipette tip, which was easier to manipulate, incubation at 35°C, which provided better bacterial growth, and flooding the plate with 5% iodine, which gave better visualization of the zone of hydrolysis. The clear zone produced by starch hydrolysis faded in approximately 5-10 min after the 5% iodine was removed, although clear zones could still be distinguished for up to 1.5 h after the removal of iodine. Streptococcus iniae was an active starch hydrolyzing organism (Figure 1) and hydrolyzed starch 100% of the times tested regardless of host origin or incubation temperature (Table 1). Repetitive testing of selected beta hemolytic Group B S.agalactiae mullet isolates (Evans et al., 2002) in this study indicated variability in starch hydrolysis. Although the majority of S.agalactiae isolates gave negative starch reactions (Figure 1), two S. agalactiae isolates, obtained from mullet blood (ARS-KU19) and head kidney (ARSKU24), gave positive starch hydrolysis reactions (Table 1). Starch hydrolysis of these S. agalactiae isolates occurred more frequently at 30°C than at 35°C. The ARS-KU24 isolate was starch positive 100% of the times tested at 30°C and positive 25% of the times tested at 35°C. The ARS-KU19 isolate was starch positive 25% of the times tested at 30°C and was positive 0% of the times tested at 35°C. Streptococcus bovis, Enterococcus faecalis, E. durans, E. seriolicida, Lactococcus garvieae, Pediococcus dextrinicus, and S. aureus did not Bull. Eur. Ass. Fish Pathol., 24(5) 2004, 236 hydrolyze starch in our testing (Tables 1 & 2). The testing of selected streptococcus isolates for starch hydrolysis by our method and starch acidification by API 20 strep and API 50 CH systems indicated consistent positive starch reactions for S. iniae, although starch acidification was weak in the API 50 CH strip. In contrast, S. agalactiae, E.faecalis, E. seriolicida, and L. garvieae did not acidify starch. Variability was noted in starch acidification for S. bovis and P. dextrinicus in different API systems. Our results indicated negative reactions for S. bovis and P. dextrinicus in API 20 strep and positive reactions in API CH 50. 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 and not starch hydrolysis. MacFaddin (2000) provides a detailed description of the biochemistry involved in starch hydrolysis. Furthermore, the interpretation of these colorimetric reactions is semi-quantitative (bioMérieux, Inc., Hazelwood, MO, USA). The acidification of starch is frequently weaker than that of other sugars in the test strip such that intermediate reactions are difficult to assign as either positive or negative. We noted weak starch acidification for S. iniae and S. bovis in the API CH 50 system. Unfortunately, starch reactions from these systems are often equated with or taken to mean starch hydrolysis and are erroneously reported as such. As a result, there is considerable confusion and misinterpretation in the literature as to whether an isolate is truly positive by starch hydrolysis. Rapid Strept 32 (bioMérieux, Hazelwood, MO) does not contain a starch test and does not include S. iniae in the database, yet it is widely used Figure 1. Clear zones of starch hydrolysis produced by five isolates of Streptococcus iniae (ARS #60, ATCC 29178, ATCC 2917, CDC 2036, CDC2032) (A) and absence of starch hydrolysis by five isolates of S. agalactiae (ARS KU-KF1, ARS KU 11, 17, ATCC 13813, ATCC 27956) (B) in 2% soluble starch agar plates. Single bacterial colonies were point inoculated around the perimeter of the plate using 10 μl pipette tip and incubated at 35°C for 18 h. Plate was flooded with 5% iodine for 5-8 min prior to observing the zones of starch hydrolysis. Bull. Eur. Ass. Fish Pathol., 24(5) 2004, 237 to identify and characterize streptococci. Positive starch acidification has been reported for S. iniae, L. piscium, Carnobacterium piscicola, L.lactis, L. raffinolactis, and P. detrinicus, and negative starch acidification has been reported for S. agalactiae , E. seriolicida, E.faecalis, and L. garvieae in API systems (Table 2) (Collins et al., 1983; Williams et al., 1990; Kusuda et al., 1991; Doménech et al., 1993; Eldar et al., 1994; 1995; 1996; MacFaddin 2000; Evans et al., 2002; Ravelo et al., 2001). Our results were in agreement with previous reports of positive starch acidification for S. iniae and negative starch acidification for S.agalactiae, E. seriolicida, E. faecalis, and L.garvieae in API systems. Mammalian and piscine isolates of S. iniae (Pier and Madin, 1976; Shoemaker et al., 2001) and clinical bovine isolates of S. agalactiae, S. bovis, nonenterococci (Group D), and P. dextrinicus have been reported as starch hydrolyzing organisms (Wilkinson et al. 1973; Facklam and Washington, II, 1991; Facklam and Elliott, 1995; MacFaddin, 2000) (Table 2). Starch hydrolysis testing of clinical and fish isolates by the method reported in this paper demonstrated positive starch hydrolysis results for S. iniae and negative starch hydrolysis for E.faecalis, E. seriolicida, E. durans, and L.garvieae. Ravelo et al. (2001), in characterizations of L. garvieae and E. faecalis, also reported negative starch reactions by a conventional starch hydrolysis technique. Negative starch hydrolysis for S. bovis and P. dextrinicus by our testing, however, was in disagreement with what has been previously reported for these organisms (Facklam and Washington, II, 1991; Ruoff et al., 1999; MacFaddin, 2000). Although S. iniae was positive for both starch acidification and hydrolysis it cannot be assumed that other streptococcal organisms will behave similarly and that results can be extrapolated from one test system to another. For example, S. bovis and P. dextrinicus were previously reported as starch hydrolyzing, yet we found these isolates incapable of hydrolyzing starch or acidifying starch (API 20 strep). Conversely, S. bovis and P. dextrinicus acidified starch in the API CH 50 test. Repetitive testing of S. agalactiae fish isolates showed the majority of isolates incapable of hydrolyzing starch, although positive starch hydrolysis was noted in two mullet isolates tested at 30°C (Table 1). In previous studies, Wilkinson et al. (1973) found non-hemolytic, Group B S. agalactiae isolates obtained from fish and humans to be negative for starch hydrolysis, although five of seven (71%) isolates tested from cow’s milk were positive for starch hydrolysis. However, details of the starch hydrolysis testing procedure and temperature at which the test was performed was not given. These results indicate that although S. agalactiae from fish, human, and bovine sources is primarily starch hydrolysis negative, variants may exist or the temperature at which starch hydrolysis testing is performed may alter hydrolysis reaction, necessitating additional tests such as Lancefield serological grouping to discriminate between, at present, nontypeable S. iniae and typeable Group B S. agalactiae. Ruoff et al. (1999) suggested that starch hydrolysis testing should be supplemental to serological testing of some Streptococcus spp. Epidemiological considerations as to the source, origin, potential for zoonotic infection by streptococcal organisms, and effective treatment against these organisms emphasize Bull. Eur. Ass. Fish Pathol., 24(5) 2004, 238 the importance for accurate bacterial speciation. We encourage the use of the starch hydrolysis test at 35°C and Lancefield grouping to identify and better differentiate between S. iniae and S. agalactiae. We also recommend using the starch hydrolysis test presented here to rapidly test multiple isolates on a single starch agar plate, thereby minimizing supplies needed and time required for results and increasing the number of isolates that can be tested costeffectively. AcknowledgementsWe thank Dr. Dina Zilberg (Ben-Gurion Uni-versity of the Negev, Sede-Boker Campus, Is-rael) for the Israeli isolates, Sherry Pattersonfor technical assistance, and Lisa Biggar forediting the manuscript. ReferencesAustin B & Austin DA (1999). Characteristicsof the pathogens: Gram-positive bacteria. In“Bacterial Fish Pathogens: Diseases ofFarmed and Wild Fish”. pp. 35-62. SpringerPublisher, New York, N.Y. Barnes AC & Ellis AE (2003). Variation in ar-ginine dihydrolase activity in Streptococcusiniae may be an artifact of the assay. 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