Identification of Pathogenic Fish Bacteria Using the ...

Identification of Pathogenic Fish Bacteria Using the API ZYM System Masahiro Sakai' Can. J. Fish. Aquat. Sci. Downloaded from www.nrcresearchpress.com by Renmin University of China on 05/28/13 For personal use only.

School of Fisheries Sciences, Ka'tasato University, Sanriku, h a t e 022-0 1, japan

M.K. SsOiman and T. Yoshida Department of Animal Products, Facubty of Agriculture, Miyaaaki University, Miyaaaki, 889-2 1, japan

and Masanori Kobayashi Sehssd of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-0 9 , lapan

Sakai, M., M.K. Ssliman, T. bshida, and M. Kobayashi. 1993. ldentification of pathogenic fish bacteria using the API ZYM system. Can. ). Fish. Aquat. Sci. 50: 1137-1 141. The APl ZYM system was u s 4 to identify the bacterial fish pathogens Enterococcus serislicida, P-hemolytic Streptococcus sp., Renibacterierm sadmoninarum, Aeromonas salmonicida, A. hydrophila, Pasteurelba piscicida, Edwardsiella brda, Vibrio anguillarum, Pseudomonas Nuorescens, Flexibacter columnaris, and F. maritimus. As additional tests, Gram stain and the activities of cytochrome oxidase and catalase were examined. The results of APl ZYM and the additional tests were coded for easy identification. Several biotypes were demonstrated by Streptococcus sp., V. anguiblarum, and A. Rydrophi%a.Other bacteria showed uniform profiles. this system can distinguish each fish pathogen from all other bacterial species examined. Le systPme API ZYM a servi 2 identifier des batteries pathogenes du poisson, Enterscoccus seriolicida, Streptococcus sp. p-hkmolytiques, Renibacterium salmoninarum, Aeromonas salmonicida, A. hydrophila, Pasteurella piscicida, Edwardsiella tarda, Vibrio anguillarum, Pseudsmonas fluorescens, Flexibacter columnaris et F. maritimus. Comme tests supp14rnentairesf on a examine la coloration de Gram et les activites de la cytochrome oxydase et de la catalase. kes resultats fournis par API ZYM et les tests supplkmentaires ont etk codes pour faciliter I'identification. On a pu repkrer plusieurs biotypes chez Streptococcus sp., $/. anguillarum et A. hydrophila. B'autres bactkries presentaient des profils uniformes. Ce systPme permet de distinguer chaque pathogene de toutes les autres especes bact6riennes examinees. Received May 7, 1992 Accepted December 7 7, I 992 (JB488)

erological procedures for the diagnosis of infectious diseases have been widely used in fish (Schill et al. 1989). Several serodiagnostic procedures are very sensitive and rapid; however, the presence of cross-reactive antigens has often obscured the precise diagnosis of fish disease. Thus, the confirmatory diagnosis of fish disease requires the isolation and identification of each particular pathogen. Most pathogenic fish bacteria are routinely identified using classical biochemical tests. While accurate, these procedures are often cumbersome because of the wide variety of organisms involved. Recently, kits to identify pathogenic bacteria in human and veterinary medicine have been developed, In fish, these kits were used for the identification of St~.egtococcussp. (Hashimoto 1982; Iida et al. 19911, Vibris angui&larurm(Kent 1982; Grisea et al. 199I), Pasteurelh pgischcicfha (Kent 1982), and Yersinis ruckeri (Romalde and Toranzo 1991). However, these kits can be used only for the identification of bacteria within a specific taxonomic group. Thus, it is necessary to develop a kit that can be applied to the wide range of pathogenic fish bacteria. 'Present address: Faculty of Agriculture, Miyazaki University, Miyazaki 889-21, Japan. Can. J. fish. A q ~ a t Sci., . Vof. 50, 1993

The APH ZYM system has been used to detect the activity sf 19 different enzymes in samples such as bacteria, tissues, or cells. It has been used to study the enzymatic profiles of streptococci, staphylococci (Humble et al. 19771, Mormella sp. (Frank and Gerber B981), and Pseudomonas cepacia ( h h and Loh 1988). The enzymatic characterization of the pathogenic fish bacteria Aeromonas hydrophila (Waltman et al. 1982) and Rentbacterium salmoninarum (Austin and Austin 1987) using this kit has also been reported. In this study, we attempt to identify the important pathogenic bacteria in aquaculture in Japan using the API ZYM system.

Materials and Methods Bacterial Strains Enterococcus serio&tcdda, Streptococcus sp., R. salmoninarum, Aeromonas salmonicida, A . hydrophila, P. piscicida, Edwardsie&&a tarcia, V. anguillarum, Pseudomonas fluorescens, Flexfbacter columnaris, and 6;. maritimus were used in this study (Table 1). All bacteria were suspended in physiological saline (8.85% NaCl) and adjusted to the concentration of McFarland No. 5.

TABLE1. Species and sources of fish pathogenic bacterial strains and culture media used in this study. Bacterial species

Can. J. Fish. Aquat. Sci. Downloaded from www.nrcresearchpress.com by Renmin University of China on 05/28/13 For personal use only.

En~erococcusserkolicida Streptococcus sp.

Source Yellowtail Yeiiowtail Rainbow trout A Y ~ Tilapia Sea bream (Archosargus probarocephalus) Japanese flounder Coho salmon Coho salmon Charr (Swkvekinus kecicomaenis) Japanese eel (Anguillw jqonicu) Coho salmon AY'J Japanese flounder Yellowtail Rainbow trout Wain bow $rout Sea bream

Location

No. of strains

Kagoshima Kagoshima Miyazaki Shiga Kagoshima Kagoshima

Culture media"

Temperature

("el

BH% BHI

Kagoshima Hwate Hwde Hwate

Miyagi Shiga Kagoshima Kagoshima Miyazaki Iwate Iwate Tokushirna

TS TY MTY

"BHI, brain heart infusion agar; TS, trypts-soya agar; ITS, trypto-soya agar with 1% NaCI; 2TS, trypto-soya agar with 2% NaC1; MTY, TY agar with 50% seawater.

API ZYM The API ZYM system (La Balme les Grottes, 38390 Montalieu, France) is capable of detecting and measuring the presence of 19 enzymes. Two drops of each bacterial suspension were added to each of the 20 cupules of one panel. Each panel was incubated wtihin its humidification chamber for 6 h. The incubation temperature for each bacterial suspension is shown in Table 1. After incubation, one drop of reagent A (250 g of Tris, 110 mL of 37% HCl, amad 100 g of sodium dodecyl sulfate made up to a final volume of 1000 mL of distilled water) and one drop of reagent B (3.5 g of Fast Blue BB per 1080 mL of 2-methoxyethanol) were added to each cupule. Color reactions were allowed to develop for 5 min, after which the cupules were exposed to a 500-W high-intensity light source for 18 s. The presence of enzymatic activity was determined by comparing the color of the bacterial samples with the manufacturer's reading scale. All test strains were examined twice.

TABLE2. Code method used for the API ZYM profiles and additional tests (e.g . PasceureEh piscicida). Characteristic

Results

No. of codes

Total No. of codes

-

0

0

Rod Gram stain Cytochrome oxidase Catalase Alkaline phosphatase Butyrate esterase (C4) Caprylate esterase (C8) Myristate lipase (C 14) Leucine arylamidase Valine arylamidase Cystine arylamidase Trypsin Chymotrypsin Acid phosphatase Phosphoarnidase

Additional Tests Gram stain, cytochrome oxidase, and catalase tests were performed as additional tests. Gram stain was conducted using Hucker's method (Mendrickson and Krenz 19911. The cvtochrome oxidase and catalase tests were performed usinithe methods of Cowan (1974). Bacterial Identification Each API ZYM result was scored as follows. Nineteen enzymes were divided into seven groups, each composed of three enzymes, with the exception of at-fucosidase (Table 2). If the activity of the first enzyme in each group is positive, the score is 1. If the activity of the second enzyme in each group is positive, the score is 2. If the activity of the third enzyme in 1138

p-Glucuronidase ci-GlucosiQase p-Gluco"dase N-Acetyl-Pgiucosaminidase a-Mannosidase a-Fucosidase Bt~steurr!lapiscicida

Rod

037203020

each group is positive, the score is 4. No score is given for negative reactions. Finally, the scores for each group are totalled and expressed as a seven-digit number. Thus, the maximum score for each enzymatic test is 7 and the minimum is 0. Can. J. Fish. Aquar. Sci., Vul. 58, I993

TABLE3. Enzymatic activities of Gram-positive pathogenic fish bacteria detected with API ZYM. + , 90% or more strains positive; - , 90% or more strains negative; V, 11-89% of strains positive. ES, Enberococcus seriolicid~;SS, Serepeococcus sp .; RS , Re~abhucteri~~n salmoninarum.


Enzyme

ES

SS

RS

No. of strains Alkaline phosphatase Butyrate esterase (C4) Caprylate esterase (C8) Myristate lipase (C14) Leucine arylarnidase Valine arylamidase Cystine arylamidase Trypsin Chyrnotrypsin Acid phosphatase Phosphoamidase a-Galactosidase p-Calaetosidase

~~Glucuronidase

a-Glucosidase P-Glucosidase N-Acetyl-Pglucosarninidase a-Mannosidase a-Fucosidase The results of the additional tests were scored by the same method. The Gram stain is treated as one group; the score is 1 for Gram-positive bacteria and 0 for Gram-negative bacteria. The cytochrome oxidase and catalase tests were in the same group. Cytochrome-oxidase-positivebacteria are given a score of 1; negatives are given a score of 0. For catalase, the score for a positive reaction is 2 and for a negative reaction is 0. Adding in the scores from the additional tests results in a ninedigit number to characterize each bacterial strain.

Results and Discussion The API ZYM system was useful for identifying many pathogenic bacteria of fishes. Enterococcus seriokicida infects yellowtail (Seriolw quinqueradiata) and was recently described by Kusuda et al. (1991). The API Z Y M activities of this bacterium had only two patterns (106243010 and 106243410) (Tables 3 and 4) and the code number was not close to those for other bacteria. Kitao (1982) also examined the biochemical characteristics of E. sertolicida and reported similar results except in the fementation of several sugars, the VP test, and citrate utilization. Using API Strep, Hashimoto (1982) and Iida et al. (1991) also reported that strains of this bacterium showed similar biochemical characteristics. @-Hemolytic streptococcal infection occurs in yellowtail, rainbow trout (OncorBzg..nchus mykiss, formerly Sakmo guirdrseri), tilapia (Orsochrornks sp.) (Kitao et al. 198I), ayu (PlecogBsssus aktivelis) (Ohnishi and Jo 1981 ), and Japanese flounder (Nakatsugawa 1983). The biochemical characterization of P-hemolytic streptococcus strains isolated from each of the fish species shows close similarities, suggesting that the infection is caused by one bacterial strain, identified as a biotype of Srreptococcus iniae (T. Kitao, personal communication). Iida et a%.(199 I), using API Strep, reported that strains of P-hemolytic Streptococcus sp. isolated from ayu and tilapia had similar characteristics, although this bacterium was not classified like any Strspgrtococcus species previously reported. In this study, Can. J. Fish. Aqraat. Sci., Vob. 50, I993

3 1 @-hemolytic Streptococcus sp. isolated from yellowtail showed several patterns. The 31 strains of P-hemolytic Strep~QCOCCUSsp. were classified into 14 types by the API ZYM test (Tables 3 and 4). Prior to this study, these bacteria had only been examined only by Gram stain, cytochrome oxidase, catalase, hemolysin, and an agglutination test using antiserum to P-hemolytic Streptococcus sp. isolated from ayu. The results of the AP%ZYM test suggest that these infections are caused by several types of P-hemolytic Streptococcus sp. Renibacterium sakrnosetnarurn is the causative agent of bacterial kidney disease in salmonid fish. The biological characteristics of isolates of this bacterium from several regions are similar (Bruno and Monro 1986). In the APH ZYM test dewere scribed here, only three types of R. sa&~nonknararsr&m detected (1 27223440, 1273214463, and 12732340). Austin and Austin (1987) reported that 448 R . salmonknwrum isolates had alkaline phosphatase, caprylate esterase (C8), leucine arylamidase, trypsin, acid phosphatase, phosphoamidase, P-glucosidase, and ax-mannosidase. Our R. salmsninamm strains isolated in Japan showed a positive reaction for butyrate esterase (C4), in addition to those cited by Austin and Austin (198'7). This difference in butyrate esterase QC4)activity may reflect a geographic difference in types of bacterial enzymes present. Aeromonas salmonkcida is one of the most studied fish pathogens. It can be separated into three subspecies, sa/naopzkcs'da, ackeromogenes, and masoucida, that differ in their biochemical characteristics such as brown water-soluble pigment formation and sucrose and mannitol fementation (Popoff 1984). In this study, we used six strains of A. sakmonicida ssp. salrnsnicida and no differences were detected using the API ZYM test (Table 5, 037723036)). Thus, this method is very useful for identification of this species, although other subspecies must be examined. Aeromonas I~ydrophilk~ has keen described as an etiological agent, not only in diseases of fish (Austin and Austin 1987) but also in those of amphibians, reptiles (Shotts et al. 1972), and humans (Von Graevenitz and Mensch 1968). This bacterium is 1139

++ ++

I I I

I I I

I I I

I I I

I I I

+ I 1 I

I

I

l

I

l


++


V. ordalii, V. salrnonickda, and V. vcrlr;eificus, have been described as causative agents of vibriosis (Egidius et al. 1986; Austin and Austin 1987). Grisez et al. (1991) reported that V. anpillarurn could be classified into six phenotypes using API 20E. Vibrio angui8larurn strains isolated from coho salmon (Oracorhypzchus kisutch) were examined by API ZYM and seven profiles resulted (Table 4). These strains showed different patterns of carbohydrate fermentation (see Kurose et ale 1989). In this study, we studied only the API ZYM profiles for V. anguk'llarurn, but other important vibrio species (V. ordalii and V. salmoniciikn) should be examined. Posteurella piscicida, the causative agent of pasteurellosis, and E. twrda, which causes edwardsiellosis, were shown to have similar enzymatic activities as assessed by API ZYM (Table 5). The biochemical characteristics sf P. piscicida or E. tar& are very stable, and no biotypes have been reported. The enzymatic activities of other fish pathogenic bacteria such as P. fluore-escens,6". colermnaris, and F. maritimus' were also investigated by API ZYM in this study. The specific API ZYM profiles for each bacterial species are shown in Table 5. Only one strain of each sf these species was examined, so results should be considered preliminary. En this study, the enzymatic activities of 11 pathogenic fish bacteria were examined with the APH ZYM kit in conjunction with identification using Gram stain and cytochrome oxidase and catalase assays. Moreover, the enzymatic activities and the additional tests of each bacteria were number coded for easy analysis. These code numbers identify individual bacterial species. Although other kits, including API 20NE, API 20E, and API strep, can also be used to identify fish pathogens (Hashimoto 1982; Kent 1982: Grisez et al. 1991; Iida et a%. 1991 ;Romalde and Toranzo 199 I), the application sf these kits is limited to closely related species (e.g., Enterobacteriaceae, Streptococcus); it cannot be used for all bacterial species. We have demonstrated that the API ZYM kit can be used for almost all bacterial fish pathogens. Thus, the API ZYM test may be one of the most valuable diagnostic methods for identifying bacterial fish disease. Further studies will need to test other bacterial species such as V. ordalii and H4 ruckeri.

Acknowledgements This study was funded in part by a Grant-in-Aid from the Ministry sf Education, Science and Culture, Japan (84556027).

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