‹›•aŒ¤‹† Fish Pathology 14 (2) 71-78, 1979. 9

A further Characterization of ruckeri (Enteric Redmouth Bacterium)

P. J. O'LEARY, J. S. ROHOVEC and J. L. FRYER

Department of Microbiology, Oregon State University, Corvallis, OR 97331

(Received May 22, 1979)

Seventeen cultures of Enteric Redmouth Bacterium were examined biochemically at selected

temperatures. Incubation temperature altered the motility of the bacterium. At 9•Ž non functional peritrichous flagella were produced, while at 18, 22, and 27•Ž the bacterium was

motile. The cells were nonmotile at 37•Ž due to a lack of flagellar production. The percent

guanine plus cytosine was determined to be 47.95•}0.45 (P=0.05). This work supports the proposal of Yersinia ruckeri as the genus and species designation of the Entric Redmouth Bacterium.

hynchus tshawytscha) and coho (O. kisutch) Introduction salmon, and (Salmo gairdneri). In the early 1950's, Rucker isolated a gram Each isolate was stored on Brain Heart In- negative, oxidase negative, motile bacterium fusion (BHI: Difco) slants under sterile mineral from moribund rainbow trout (Salmo gaird oil. neri). The first publication concerning this Biochemical tests bacterium did not appear until 1966 (Ross et The methods used to study most of the al., 1966) in which the definitive taxonomic biochemical properties of this organism have position of the bacterium could not be defined, been described previously (EDWARDSand EWING although it was placed in the family Entero 1972; LENETTE et al., 1974). For carbohydrate bacteriaceae. Hence the name Enteric Red utilization tests, sugars were incorporated into mouth Bacterium (ERMB) arose. Various in purple broth base (Difco) at a concentration vestigators have tried unsucessfully to classify of 0.5% (w/v). The NaCl concentration to the bacterium (BUSCH,1973; ROSS et al., 1966; determine salt tolerance was measured as WOBESER, 1973). Not until recently, with the percent salt (w/v) added to BHI broth. The acceptance of the genus Yersinia (BUCHANAN method of JEFFRIES et al. (1956) was used for et al., 1974) and refinement of the techniques the detection of deoxyribonuclease (DNAase) of DNA homology studies, has the character and ribonuclease (RNAase). DNA (Sigma; ization of ERMB been possible. type II-S, sodium salt, highly polymerized, EWING et al. (1978) performed by the neces from calf thymus) and RNA (Sigma ; type II sary DNA homology studied and biochemical -S , sodium salt from Torula Yeast) were in tests to place ERMB in the genus Yersinia. corporated into BHI broth with 1.5% Noble The bacterium was sufficiently different from agar (Difco) at a final concentration of 2 mg/ the existing species to ascribe a new species, ml. Zones of clearing after flooding the plate Yersinia ruckeri. This paper offers additional with 1 N HCl indicated hydrolysis of the substrate support for the new species designation indi . Lipase was assayed by the method of cated by EWING et al (1978). HOLDING and COLLE (1971). Tween 20, 40, 60 or 80 (Sigma) was added to give a final Materials and Methods concentration of 1% (v/v) with 1.5% (w/v) Isolation of Noble agar. Lipolytic activity was indicated Seventeen cultures of Y. ruckeri were by the presence of a precipitate in the agar isolated from kidney tissue in chinook (Oncor around the colony. Elastase and fibrinolysin 72 P.J.0’LEARY, J.S. RＯＨＯＶＥＣE and J.L. FRYER

Table 1. Biochemical comparison of 17 Yersinia ruckeri isolates at different incubation

temperatures. Characterization of Yersinia ruckeri 73

were determined by the method of SBARRA et (80: 20) was used to shadow cast at approxi al. (1963). Each substrate, elastin and fibrino mately 35•Ž angle with a specimen to source gen, was incorporated into a minimal salts distance of 6 cm. Images were observed on preparation and served as the sole carbon a Philips EM-300 transmission electron micro source. Zones of clearing around the colony scope and recorded on Kodak electron image

evidenced the enzymatic activity. plates. These biochemical reactions and API 50 R Results test strips (Analytab Products, Inc., Plainview, N.Y.) were incubated at 9, 18, 22, 27, and Homogeneity with regard to carbohydrate 37•Ž. fermentation was demonstrated among the Determination of percent guanine plus isolates and showed no variation at the dif cytosine1 (GC%) ferent temperatures tested (Table 1). Sucrose, The DNA from 3 isolates was extracted and lactose, xylose, arabinose, cellobiose, dulcitol, purified by conventional techniques (MARMUR, salicin, inositol, raffinose, rhamnose, melibiose, 1961; SEIDLER et al., 1969). The GC% deter and esculin could not be degraded. Only four minations were performed in duplicate in a out of 17 isolates were positive for sorbitol. Gilford Model 2000 Recording Spectrophoto The IMViC reactions for ERMB were meter using E. coli WP 2 as a standard. The +-+ at all temperatures tested. Tartrate- Tm was determined and the GC% was calcu could also be utilized as a sole carbon source, lated by the method of MANDEL et al. (1970). but malonate could not. The isolates did not

Mouse inoculation produce urease, nor reduce nitrite; however, Three groups of 5 white Swiss Webster nitrate reduction was observed. The Triple mice were injected with one of three ERMB Sugar Iron agar slants (TSI) were alkaline isolates. These strains were grown to late over acid with no gas or hydrogen sulfide

log phase in BHI broth at 22•Ž. Each mouse production as would be expected from the was injected intraperitonally (IP) with 0.2 ml glucose, sucrose, and lactose reactions given of the straight broth culture (•`2•~108 cells) above. The bacterium could tolerate NaCl and observed for 14 days. concentrations of •…3% but not •†7%.

Determination of flagellation . Metabolic reactions of the bacterium were Isolate HI-70 of Y. ruckeri was grown in influenced by temperature as exhibited by the BHI broth at 9, 22 or 37•Ž for three passages decarboxylation of amino acids. Lysine and to acclimate the bacteria to the respective arginine were decarboxylated only at 18 and temperature. The cells were then inoculated 22•Ž while temperature showed no effect on into 15 ml BHI broth and grown at the selected ornithine decarboxylase (positive) and phenyal temperatures to middle log phase. Glutaral anine deaminase (negative).The cells were dehyde was added to a final concentration of nonmotile at 9 and 37•Ž, but were motile at 1.5% (v/v) and the culture was allowed to 18, 22, and 27•Ž exhibiting luxurious growth incubate for an additional 2 hr. The cultures throughout the culture medium. from each of the three temperatures were The assays for various extracellular enzymes then acclimated to 22•Ž and dialyzed overnight displayed some temperature dependency. against distilled water. The cells were centri DNAase was produced only at 37•Ž and fuged at 1085•~g. The supernatant was dis RNAase was not produced at any of the tem carded and the pellet was resuspended in 2-4 peratures tested. Fibrinolysin and elastase drops of distilled water. One drop of the cell were not produced by any of the isolates. suspension was placed on a 300 mesh Formvar Chondroitin sulfatase was produced at 27 and copper coated screen. The excess liquid was 37•Ž. Hyaluronidase was produced by certain isolates blotted off with filter paper and the screen at 22 and 27•Ž. The lipases as assayed was allowed to air dry. The sample was by Tween 20, 40, 60 and 80 also showed some shadow cast in a Varian Model VE-10 vacuum temperature dependency. All of the isolates evaporator at 1•~10-5 Torr. Platinum-palladium could utilize Tween 20 and none could utilize P.J. O'LEARY, J.S. ROHOVEC and J.L. FRYER

Table 2. Individual and average guanine plus hydrolase. Incubation temperature has been

cytosine values for Ｙｅｒｓｉｎｉａ rucheri offered as a partial explanation for this vari as determined by thermal melting ability. Therefore, individual biochemical tests (Tm). were conducted at 9,18,22,27, and 37℃. It was found that many of these biochemical reactions have a temperature optimum. Dif ferences observed by other investigators con cerning lysine deearboxylase and arginine dihydrolase can be resolved on the basis of temperature. The variahiiity in the production of hydrogen sulfide may rely on the sensitivity of the assays used. BUSCH (1973) obtained a positive reac Tween 80. Tween 40 and 60 could be degrarded tion with all of his isolates using lead acetate at 9,18,22, and 27℃ but not at 37℃. Gelatin medium. Others, including work reported was not liquefied at 9,18,22,or 37℃. At here, relied on gross production of H2S in 27℃,only the top one-third of the tube was TSI slants. The sensitivity of lead acetate iquefied even when allowed to incubate three l medium exceeds TSI slants;therefore, this additional weeks. Gamma hemolysis was ob difference in sensitivity could explain the dis served on 5%sheep blood agar. crepancies observed. only the fermentation The percent guanine plus cytosine for three of sorbitol differs from that which has been isolates was found to be 48.45,47.77, and reported. Previously, ERMB was thought to 47.4,respectively (Table 2). A One-Way be sorbitol negative;however, in this study ANOVA table was constructed(SNEDECOR and four of seventeen isolates were found to be COCHRAN 1973)and no significant differences positive. between the GC% of the isolates could be shown The bacterium has been shown to be related at the 95% confidence level. The average GC% biochemically (EWlNG et al.,1978) and by DNA for ERMB from all six determinations was homologies(STEIGERWALT et al.,1975)to the 47.95±9.45(95% canfidence interval). two genera, Serratia and Yersinia(BRENNER Electron micrographs were taken of ERMB et al.,1976;EWlNG at.,1978). The GC% grown at 9,22 and 37℃. It can be seen at for Serratia falls in the range of 50-53%, and 22℃(Figs.1A-1B)the bacterium is peri 9 whereas Yersinia is 45-47% (BUCHANAN and trichously flagellated while at 37℃ (Figs.1C GIBBONS 1974). The value obtained here of -1D)no flagella are noted . 47.95% GC for ERMB would preclude its in No reproducible disease or mortality was clusion in the genus Serratia in favor of

observed when mice were injected IP with 2 Yersinia. ERMB cells. The bacterium was con ×108 The distinguishing characteristics between sidered to be nonpathogenic for mice. the species of Yersinia have been compared to ERMB(Table 3). Enteric redmouth bacter Discussion ium is most closelyrelated to Y.pseudotuber The biochemical reactions observed for cell culosis and Y,enterocolitica, but many cha isolates were homogeneous (Table 1)with very raeteristicsdiffer sufficientlyto preclude the few exceptions and carrespanded quite well inclusion of ERMB into existing species. with those previously reported (BUSCH,1973; BRENNER et al.(1976) conducted DNA homology EWlNG et al.,1978; Ross et al., 1966; studies and found that ERMB possessed a WOBESER,1973). Results obtained using API relative binding ratio(RBR) of 29-31% when 50 R tests agreed with those observed by other compared with Y. enterocolitica 501-70. The biochemical means. Tests which have provided RBR for Y. pestis was 43%. When tested variable results are hydrogen sulfide produc against Y. pseudotuberculosis P 105, the RBR tifln, lysine decarboxyiase, and arginine di for various isolateswere as follows:Y. pseu- Characterization of Yersinia ruckeri 75Tabl

e 3. Distinguishing characteristics between the recognized species of Yersiniaa and ruckeri. Yersinia

dotuberculosis, 81-100%; Y. entercolitica, bacteriaceae (BUCHANAN nd GIBBONS 1974; 42-53% and ERMB, 29-30%. STEIGERWALT et LENETTE et al., 1974) but has eluded further al. (1975) found an RBR value for ERMB when classification. It was proposed that ERMB be compared to 868-57, classified in the genus Yersinia because of Serratia liquefaciens 446-68, and Serratia homology in GC% content and the definitive radideae 937-72 of 24-28%, 16.9-17.5% and biochemical reactions. Enteric redmouth bac 25%, respectively. terium does not correlate well by DNA homo The variability in motility with regard to logies (BRENNER et al., 1976) or biochemical temperature is also a characteristic of the reactions to existing species of Yersinia; there genus Yersinia. This phenomenon has been fore, EWING et al. (1978) have recently proposed noted in Y. pseudotuberculosis and Y. entero that Yersinia ruckeri be adopted as the genus colitica (BUCHANAN and GIBBONS 1974; NILEHN, and species designation for ERMB. This is a,1969c; PRESTON and MAITLAND 19691952). in recognition of R. R. Rucker, the first person This work indicates that ERMB is nonmotile to isolate the causative agent of enteric red at lower temperatures (9•Ž) due to the presence mouth disease. of inactive flagella and at higher tempera We feel that due to the biochemical similari tures (37•Ž) because of an absence of flagella. ties between ERMB and Y. enterocolitica, the The bacterium was flagellated and motile when bacterium may be frequently overlooked. "Yersinia •@ incubated at 18 to 27•Ž. -like" organisms are often described Another similarity occurs between Y. enter in papers dealing with aquatic and environ colitica and ERMB. No mortalities were mental microbiology (BOTZLER et al., 1976; observed when massive amounts of ERMB KAPPERUD, 1977; MARTYNY and BOTZLER, were injected IP into white mice. It was 1976) . Bacteria are isolated which correspond concluded that ERMB is not pathogenic for with many of the biochemical characteristics these animals. The inability to inflict a re of Y. enterocolitica, but not all, and cannot producible disease in white mice has also been be placed into a serological group. If these observed with Y. enterocolitica, isolated from organisms are examined more closely, they man, pigs, dogs, chinchillas, guinea pigs, may in fact be ERMB and thus extend the rabbits, and hares (CARTER et al., 1973; known host range of ERMB beyond that of NILEHN,1969b). salmonid fishes. The enteric redmouth bacterium has been The work presented here further describes recognized as a member of the family Entero ERMB with respect to its biochemical and 76 P. J.O'LEARY, J. S.ROHOVEC and J. L.FRYER

physical characteristics and GC% content. We LENETTE, E., E. SPAULDING, and J. TRUANT (1974): have reached the same conclusions as EWING Manual of clinical microbiology, second edition. et al. (1978) and support Yersinia ruckeri as Am. Soc. Microbiol., Washington, D. C. 970 p. the genus and species designation for ERMB. MANDEL, M., L. IGAMBI, J. BERGENDAHL, M. DODSON, and E. SCHELTGER (1970): Correlation of melting Acknowledgements temperature and cesium chloride buoyant density of bacterial DNA. J. Bacteriol., 101,333-338. This research was supported by the Oregon MARMUR, J. (1961): A procedure for the isolation Department of Fish and Wildlife under PL of deoxyribonucleic acid from microorganisms. J. 89304 Anadromous Fish Act. The information Mol. Biol. 3,208-218.

contained in this report was part of the Na MARTYNY, J. and R. BOTZLER (1976): Yersinia isol tional Science Foundation U.S.-Japan Coopera ated from wapiti (Cerrus canadensis roosevelti).

tive Science Program. J. Wild. Dis., 12, 386-389.

NILEHN, B.(1969a): Electron microscope studies Bibliography on flagellation in different strains of Yersinia

BOTZLER, R., F. WETZLER, A. COWAN, and T. QUAN enterocolitica. Acta Path. Microbiol. Scand., 77,

(1976): Yersinia in pond water and snails. J. 527-541. Wildlife Dis., 12,492-496. (1969b): Studies on BRENNER, D., A. STEIGERWALT, D. FALCAO, R. with special reference to bacterial diagnosis and

WEAVER and G. FANNING (1976): Characteriza occurrence in human acute enteric disease. Acta tion of Yersinia enterocolitica and Yersinia Path. Microbiol. Scand. Supplementum 206,1-

pseudotuberculosis by dexoyribonucleic acid hy 39. bridization and by biochemical reactions. Inter. (1969c): Studies on Yersinia enterocolitica.

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manual of determinative bacteriology. Eighth PRESTON, N. and H. MAITLAND (1952): The in edition, The Williams and Wilkins Co., Baltimore, fluence of temperature on the motility of Pasteu

Maryland, 1246 p. rella pseudotuberculosis. J. Gen. Microbiol., 7, BUSCH, R. (1973): The serological surveillance of 117-128.

salmonid populations for presumptive evidence of Ross, A., R. RUCKER, and W. EWING (1966): De specific disease association. Ph. D. thesis, Univ. scription of a bacterium associated with red

of Idaho, Moscow, Idaho. 106 p. mouth disease of rainbow trout (Salmo gairdneri). CARTER, P., C. Vanga, and E. Keet (1973): New Can. J. Microbiol., 12, 763-770.

strain of Yersinia enterocolitica pathogenic for SEIDLER, R., M. STARR, and M. MANDEL (1969):

rodents. Appl. Microbiol., 26, 1016-1018. Deoxyribonucleic acid characterization of Bdel EDWARDS, P., and W. EWING (1972): Identification lovibrio. J. Bacteriol., 100,786-790.

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(1978): Yersinia ruckeri sp. nov., the redmouth SNEDECOR, G. and W. COCHRAN (1973): Statistical

(RM) bacterium. Int. J. Syst. Bacteriol., 28, 37- methods, sixth edition. The Iowa State Press, 44. Ames, Iowa. 593 p.

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water in Norway and Denmark. Acta Path. Saskatchewan. J. Fish. Res. Board Can., 30, Microbiol. Scand. Sec. B, 85,129-135. 571-575. Characterization of Yersinia ruckeri 77

Yersinia ruckeri(レッ ドマ ウ ス 菌)の 性 状 の 吟 味

レッ ドマ ウス菌17株 を もち い,そ の 性 状 を い くつ か の 異 な る温 度条 件下 に お い て比 較 検 討 した｡そ の結 果,培 養 温 度 は レ ッ ドマ ウス 菌 の 運 動 性 に 影 響 し,18,22お よ び27｡Cで は 運 動 す1るが･9｡Cで は 周 毛 が存 在 す る こ とに もか か わ らず 機 能 せ ず,ま た,37｡Cで は鞭 毛 が 生 じな い た め運 動 性 が失 な わ れ る こ とが 明 らか とな っ た｡･ま た,供 試 菌 株 の グ ア ニ ソ と シ トシ ソの含 有 率(GC%)は47.95±0.45%(P=0.05)で あ り,そ の 他 の 生 化 学 的 性 状 も レッ ドマ ウス菌 の 属 種 をyθＹｅｒsiniaruckeriと す る提 案 を 支 持 す る も ので あ っ た｡

Fig. Electron micrographs of Yersinia ruckeri cells incubated at selected temperatures. A. 9•Ž, cells

are flagellated but are not motile; B. 22•Ž, cells are flagellated and mottle; C. 37•Ž, cells are non flagellated and non motile; D. a higher magnification of a single cell incubated at 37•Ž showing

the absence of flagella. Bar =1 ƒÊm 78 P. J. O'LEARY. J.S. ROHOVEC and J.L. FRYER

A B

D C