J. Gen. App!. Microbiol., 37, 479-494 (1991)

IDENTIFICATION OF CARNOSUS STRAINS FROM FERMENTED FISH AND SOY SAUCE MASH

SOMBOON TANASUPAWAT, YASUHIRO HASHIMOTO,' TAKAYUKI EZAKI,' MICHIO KOZAKI,* AND KAZUO KOMAGATAZ>t

Department of Agricultural Chemistry, Tokyo University of Agriculture, Sakuragaoka 1-1-1, Setagaya-ku, Tokyo 156, Japan 'Gifu University School of Medicine, Tsukasa-machi 40, Gifu 500, Japan Institute of Applied Microbiology, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan

(Received September 30, 1991)

Gram-positive and -positive cocci isolated from fermented fish and soy sauce mash were systematically studied based on phenotypic and chemotaxonomic characteristics. They were separated into 2 groups (A and B) by photobiotin labeling DNA-DNA hybridization. Of 18 isolates studied, 10 (Group A) showed DNA homologies of more than 69% compared to the type strain of Staphylococcus carnosus and were identified as S carnosus. Eight isolates (Group B) showed DNA homologies of less than 50% compared to the type strain of S carnosus and no relatedness to the reference strains of Staphylococcus . The DNA base compo- sition of these two groups ranged from 34.4 to 37.3 mol% of guanine plus cytosine. They contained MK-7 as a major menaquinone and MK-6 or MK-6 and MK-8 as minor menaquinones, and the tested strains had anteiso-C15.0as a major fatty acid. Detailed characteristics of S. carnosus were described.

In the previous studies of lactic acid producing found in the fermented food in Thailand (38, 39), gram-positive and catalase-negative tetracocci were identified as Pediococcus pen tosaceus, Pediococcus acidilactici, Pediococcus halophi- lus, and Pediococcus sp. Further, other gram-positive and catalase-positive cocci were also isolated from the fish fermented with salt, such as ka pi (fish or shrimp

* Address reprint requests to: Dr . Michio Kozaki, Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Tokyo University of Agriculture, Sakuragaoka 1-1-1, Setagaya-ku, Tokyo 156, Japan. t Present address: NODAI Research Institute , Tokyo University of Agriculture, Sakuragaoka 1-1-1, Setagaya-ku, Tokyo 156, Japan.

479 480 TANASUPAWAT, HASHIMOTO, EZAKI, KOzAKI, and KOMAGATA VOL. 37 paste), kung-chom (fermented shrimp), pla-chom (fermented small fish), pla-ra (fermented fish), and bu-du (fermented fish in liquid form) in Thailand. The role of the cocci in the fermentation was discussed (38). Such cocci were included in the Staphylococcus on the basis of phenotypic characteristics, quinone systems, cellular fatty acid compositions, and DNA base compositions, but were rarely identified as any species based on the above-mentioned characteristics. Recently, the usefulness of fluorometric DNA-DNA hybridization has been reported for the identification of the strains in the genus Campylobacter (12), the genus Legionella (13), and the viridans group of streptococci (14). This paper deals with the identification of isolates from the fermented fish and "Tetracoccus soyae" isolated from soy sauce mash (41) , based on phenotypic and chemotaxonomic characteristics including fluorometric DNA-DNA hybridization. Detailed characteristics of Staphylococcus carnosus are described because some information is available for this species.

MATERIALS AND METHODS

Bacterial strains and cultivation. Isolated strains used in this study, their sources, and reference strains (8-10,17, 22, 23, 26, 29-32, 38, 41) are listed in Table 1. Names in quotation marks refer to the species not listed on the Approved Lists of Bacterial Names (34) or not yet validly published. All cultures were cultivated on -yeast extract-peptone (GYP) agar medium at 30°C for 2 days. The medium was composed of 5 g glucose, 5 g yeast extract (Difco Laboratories, Detroit, Mich.), 5 g polypeptone (Daigo Eiyo, Tokyo), 15 g agar, and 1,000 ml distilled water, pH 6.8. In chemotaxonomic studies, cells were grown in a 5,000 ml-flask containing 1,000 ml of GYP-sodium acetate-mineral salt broth composed of 5 g glucose, 5 g yeast extract (Difco), 5 g polypeptone (Daigo Eiyo), 5 g sodium acetate, 200 mg MgSO4. 7H2O, 10 mg Mn2SO4.4H2O, 10 mg Fe504 7H2O, 10 mg NaCI, and 1,000 ml distilled water, pH 6.8. The cultures were incubated at 30°C for 2 days with a rotary shaker (250 rpm). Identification methods. Phenotypic characteristics: Cell forms, cell arrange- ments, and gram stain were examined on the cells grown on GYP at 30°C for 2 days. Gram staining was carried out by the modified method of Hucker and Conn (18). Motility was tested by appearance of stab cultures in soft agar medium (45). Spore formation was examined in gram-stained specimens. Colonial appearance was examined by the growth of cultures on GYP agar plate incubated at 30°C for 5 days. Catalase activity was detected by production of bubbles in a 3% hydrogen peroxide solution and determined by the iron porphyrin benzidine test (7). activity was tested by using rabbit plasma (Eiken Chemical Co., Ltd., Tokyo). of blood was performed on a blood agar plate (Eiken). Oxidase was detected with cytochrome oxidase test paper (Nissui Pharmaceutical Co., Ltd., Tokyo). Urease was determined on urea medium (3). Phosphatase was tested by the method of Barber and Kuper (2). Voges-Proskauer (VP) reaction in 1991 Staphylococcus carnosus from Fermented Food 481 glucose-peptone broth, lipolytic activity for Tween 80, hydrolysis of esculin and gelatin, lecithovitellin (LV) reaction, reduction of nitrate, and utilization of citrate were determined by the methods described by Cowan and Steel (5). Hydrolysis of arginine was performed by the method of Thornley (40). Lactic acid production was determined chemically (6). Oxidation and fermentation test (OF test) was examined in peptone-yeast extract-phenol red (PYP) medium (Nissui) (46). Uti- lization of mannitol in anaerobic conditions was tested in a modified Baird-Parker's medium (1) in which glucose was replaced with mannitol. Gaspak anaerobic system (BBL Microbiology Systems, Cockeysville, Md.) was used for anaerobic cultures. Acid production from 19 kinds of carbohydrate was tested as described in the previous paper (38). Aerobiosis was detected in soft agar (45). Relation to temperature was tested by growth in GYP-sodium acetate-mineral salt broth incubated at 18°C and 42°C for 5 days. The effect of initial pH on growth was tested by incubating in GYP-sodium acetate-mineral salt broth adjusted to pH 4.5, 5.0, 6.8, 8.2, or 9.0 at 30°C for 5 days. Halotolerant characteristic was examined by incubating in GYP-sodium acetate-mineral salt broth containing 6%, 8%, 10% or 15% NaCI at 30°C for 5 days. Susceptibility to lysozyme (400 cg/ml and 1,600 , tg/ml), lysostaphin (2001ug/ml), and (1.6 iig/ml and 5.0 icg/ml) was determined by the method of Kloos et al. (21). DNA base composition. DNA was isolated from cells grown in GYP-sodium acetate mineral salt broth and purified by the method of Saito and Miura (28). For the strains showing difficulty in isolation of DNA, the medium was supplemented with 1 to 2% of glycine (47). The purified DNA was submitted to determination of DNA base compositions by the method of Tamaoka and Komagata (37). DNA-DNA hybridization. Photobiotin-binding DNA-DNA hybridization was carried out in 2 X SSC (saline-trisodium citrate) and 50% formamide solution at 37°C as previously reported (11-15). Cellular fatty acid composition. Fatty acids were analyzed by gas chroma- tography according to the method described by Ikemoto et al. (20). Quinone systems. Quinones were extracted from freeze-dried cells (200 mg) and purified as described by Collins et al. (4). The purified quinones were examined by reversed-phase high performance liquid chromatography (36). The abbreviation (e.g. MK-6, MK-7, MK-8, etc.) used for menaquinones indicated the number of isoprene units in a side chain.

RESULTS

Phenotypic characteristics All isolates were gram-positive, nonmotile cocci, and produced coccal forms appeared singly, in pairs, in tetrads, and in clusters. They did not produce spores. All cells measured from approximately 0.5 to 1.O um in diameter. Colonies on GYP agar plate were circular in shape low convex with entire margin, nonpig- mented and white with pale yellow-orange tint or yellow orange in color. Table 1. Strains stud ied, strain designations and their isolation sources. 1991 Staphylococcus carnosus from Fermented Food 483 Table 2. Phenotypic characteristics of staphylococcal isolates and related bacteria. 1991 Staphylococcus carnosus from Fermented Food 485 486 TANASIJPAWAT,HASHIMOTO, ELAKI, KOzAKI, and KOMAGATA VOL.37

The isolates showed positive responses for catalase, benzidine test, nitrate reduction, arginine hydrolysis, and lactic acid production, and negative responses for coagulase, hemolysis of blood, cytochrome oxidase, VP-reaction, gelatin hy- drolysis, lecithovitellin reaction, and citrate utilization. Variable responses were shown for urease, phosphatase, lipolytic activity, and esculin hydrolysis, as shown in Table 2. All isolates were facultatively anaerobic organisms and cleaved glucose fer- mentatively. They produced acid aerobically from D-glucose, D-, and D-, and moreover some isolates produced acid from D-galactose, D- , , maltose, sucrose, D-melezitose, glycerol, D-mannitol, and D- sorbitol. They did not produce acid from D-arabinose, D-cellobiose, L-fucose, gluconate, D-turanose, D-xylose, and xylitol, and failed to produce acid from D-mannitol in anaerobic conditions. All isolates grew at 18 and 42 ° C, and grew well between pH 6.8 and 9.0. Some isolates grew at pH 5.0, but none grew at pH 4.5. They grew in the medium containing 6%, 8%, or 10% NaCI, and some did in 15% NaCI. All isolates were susceptible to lysostaphin (200 beg/ml) and novobiocin (1.6 iig/ml and 5.0 beg/ml), but most isolates were not susceptible to lysozyme (400 iig/ ml and 1,600 ig/ml). As shown in Table 2, phenotypic characteristics of isolates and other Staphy- lococcus species were similar and they did not serve as distinguishing character- istics.

DNA base composition DNA base compositions of the isolates ranged from 34.4 to 37.3 mol°o and other Staphylococcus species contained 31.2 to 39.0 mol% of guanine plus cytosine (G + C content) (8,10,17, 22, 23, 29 33), as shown in Table 3.

DNA-DNA hybridization On the basis of DNA homologies, the isolates were separated into two groups as shown in Table 3. The DNA-DNA hybridization carried out under optimal (37°C) conditions indicated that Staphylococcus carnosus GIFU 10766T had ho- mology of more than 69% compared to the isolates of Group A and showed homology of less than 50% to the isolates of Group B and other Staphylococcus species. The isolate SK 03 (Group B) showed DNA homology of less than 60%, 43%, and 25% compared to the isolates of Group A, Staphylococcus carnosus GIFU 10766T, and the strains of Staphylococcus species, respectively.

Cellular fatty acid composition The isolates tested contained a branched-chain acid of anteiso (a)-C15.0 as a major component. The branched-chain acids of iso (i)-C14,0,i-C15.0, i-C1b.0, i-C17:0, i-C19., a-C17.0,and a-C19,0,and straight-chain saturated acids of 014:0,015:0, 016:0, C180, and 020:0 were also found. Most of the strains of Staphylococcus species 1991 Staphylococcus carnosus from Fermented Food 487

Table 3. DNA base composition and DNA homologies of staphylococcal isolates and related bacteria.

showed the same fatty acid profiles, and the amounts were variable as shown in Table 4. Staphylococcus caseolyticus GIFU 10760 contained a large amount of fatty acids of C14,0, C16.0 and C18.0, and S intermedius GIFU 8781T had a large amount of i-Cts:0. Table 4. Cellular fatty acid composition of staphylococcal isolates and related bacteria. 1991 Staphylococcus carnosus from Fermented Food 489

Table 5. Quinone systems of staphylococcal isolates and related bacteria.

Quinone systems Quinone systems of the isolates were menaquinones consisting of MK-7 as a major component and MK-6 or MK-6 and MK-8 as minor quinones, as shown in Table 5. Staphylococcus caseolyticus GIFU 10760, Staphylococcus hyicus JCM 2423T(26), and Staphylococcus sciuri GIFU 9126T(26) contained MK-6 as a major component. 490 TANASUPAWAT, HASHIM0T0, EZAKI, KOZAKI, and KOMAGATA VOL. 37

DISCUSSION

The isolates were included in the members of the genus Staphylococcus (24) on the basis of phenotypic characteristics, DNA base compositions, cellular fatty acid compositions, and quinone systems, but were rarely separated into species. How- ever, on the basis of DNA homologies, the isolates were fell into two groups at the species level, as shown in Table 3. Group A contained 8 isolates (SK 07, SK 10, SK 06, SK 09, SK 11, SK 08, SK 13, SK 12), "TT soyae" F-2 (NRIC 1522; NRIC = NODAI Research Institute Culture Collection, Tokyo University of Agriculture, Tokyo), and "T soyae" F-8 (NRIC 1719). The strains in this group showed DNA homologies of more than 69% compared to the type strain of Staphylococcus carnosus. Thus, these isolates were identified as this species because the bacterial species is considered to be an assembly of strains with DNA homologies of approximately 70% (43). Group B contained 8 isolates (SK 03, SK 04, SK 05, SK 14, SK 15, SK 16, Ph 79S, and Ph 79L). Isolates in Group B showed DNA homologies of less than 50% compared to the type strain of S. carnosus and no relatedness to the valid species of the genus Staphylococcus. According to Bergey's Manual of Systematic Bacteriology vol 2. (24), recent studies (16, 19, 42), and the characteristics shown in Tables 2, 4, 5 of this study, these gram-positive and catalase-positive cocci were rarely identified as any valid species in the genus Staphylococcus. In addition, by the use of API STAPH (API System S. A., Montalieu-Vercieu, France) (25), SK 16 was identified as (API result number: 6716053), and SK 03, SK 14 and SK 15 as Staphylococcus hominis (API result number: 6712053), and the remaining isolates were not identified as any known species (unpublished). This identification did not coincide with the results of DNA-DNA hybridization. Referring to the data of DNA homologies, characteristics of ten strains in Group A identified as S carnosus are described as shown below because the phenotypic characteristics and DNA base composition have been reported only for a few strains of S carnosus (31), and quinone systems and cellular fatty acid compositions have not been reported so far. These data will be useful for a better understanding of this species.

Characteristics of Staphylococcus carnosus strains examined in this study The above-mentioned ten strains of S. carnosus (Group A) shared almost the same characteristics as follows: Cells are gram-positive cocci, 0.5-1.0 im in di- ameter, and appear singly, in pairs, in tetrads, and in clusters. Cells are nonmotile and nonsporing. Colonies on glucose-yeast extract peptone agar plate are circular in shape, low convex with entire margin, white or white with pale yellow orange tint or yellow-orange. All strains are positive for catalase, benzidine test, nitrate reduction, arginine hydrolysis and lactic acid production, and are negative for coagulase, hemolysis of blood, cytochrome oxidase, VP-reaction, hydrolysis of 1991 Staphylococcus carnosus from Fermented Food 491 gelatin, lecithovitellin reaction, and citrate utilization. They are facultatively anaerobic and cleave glucose fermentatively. They produce acids from D-glucose, D-fructose, and D-trehalose aerobically. All fail to produce acids from D-arabinose, D-cellobiose, L-fucose, gluconate, D-turanose, D-xylose, and xylitol. The strains grow at 18°C and 42°C, and grow well between pH 6.8 and pH 9.0. They grow in medium containing 6%, 8%, or 10% of NaCI. All are susceptible to lysostaphin ( 200 ,ug/ml) and novobiocin (1.6 i g/ml and 5.0 a g/ml) . DNA base composition ranges from 34.4 to 37.1 mol% of guanine plus cytosine. Cellular fatty acids of the strains studied are branched fatty acid of a-C15.0as a major component and the straight-chain fatty acid of C20:0is found. The strains contain MK-7 as a major quinone and some strains contain MK-6 or MK-6 and MK-8 as minor quinones. The presence of C20:0acid is recognized in the species of the genus Staphylo- , but not in strains of the genera , Stomatococcus, Enterococcus, Lactococcus, Streptococcus, and Pediococcus (27). This acid seems to be a fatty acid marker for differentiation of Staphylococcus strains from the above-mentioned cocci. Staphylococcus caseolyticus GIFU 10760 has straight-chain fatty acids as major components (44, this study). This strain is also different from the valid Staphylococcus species in quinone systems (this study), and no relationship to other species on the basis of DNA homology (24, 30) was suggested. Most strains of Staphylococcus carnosus (Group A) are separated from those in Group B by crease, phosphatase, esculin hydrolysis, acid production from D- galactose, D-mannose, lactose, maltose, sucrose, melezitose, and growth at pH 5.0. However, these characteristics are not always consistent for the differentiation of the two groups. Fluorometric DNA-DNA hybridization is useful for identification of S carnosus. The isolates of Group B showed low DNA homologies compared to S. carnosus and other species in the genus Staphylococcus. The strains will not be designated as any known species in the genus Staphylococcus. Further studies are needed to make clear the taxonomic position of strains in Group B. The strains of Staphylococcus carnosus were originally found in dry sausage and in a starter for the production of dry sausage (31). The strains F-2 and F-8 of "Tetracoccus soyae" were isolated from soy sauce mash in Japan , and this species was described as a new species (41) but has not been validated so far. In this study, the "TT soyae" strains were identified as S. carnosus based on DNA-DNA hybrid- ization, but they differed a little in phenotypic characteristics from other SScarnosus strains. The strain of "Tetracoccus" sp. No. 100 was also isolated from soy sauce mash (35). This strain was reported to be gram-positive coccus and catalase positive, and to have phenotypic characteristics and DNA base composition similar to "Tetracoccus soyae" F-2 (35). Thus, this strain will be included in S, carnosus. S. carnosus shows coagulase negative and hemolysis negative (31), and has not been reported as causing infections of humans or animals (24). Strains studied were distributed in fermented fish containing NaCI of more than 500 and in soy sauce mash with NaCI of over 17%. The strains of "T. soyae" and "Tetracoccus" 492 TANASUPAWAT, HASHIMOTO, EZAKI, KOZAKI, and KOMAGATA VOL. 37 sp. were reported to play a role in the making of soy sauce (35), and the strains isolated from fermented fish in Thailand may also play some roles in fish fermen- tation (38). Therefore, it is interesting to note that such staphylococcal strains are widely distributed in the fermented foods. Further study will make clear the role of this group of bacteria in the fermentation. This paper is a part of the study by Somboon Tanasupawat presented in Annual Reports of ICBiotech, vol. 9, p. 239-252, 1986. The International Center of Cooperative Research in Biotechnology, Japan (ICBiotech), Faculty of Engi- neering, Osaka University, Osaka, Japan admits the scientific papers in the Annual Reports as personal communications and not as original publications. Thus this paper is the original publication by the authors.

We wish to thank Dr. J. Sugiyama, Dr. A. Hiraishi, and Dr. J. Tamaoka, Institute of Applied Microbiology, The University of Tokyo, for their suggestions and for helping with the determination of quinone systems and DNA base composition; Dr. K. Suzuki, Japan Collection of Microorganisms, RIKEN, Wako-shi, Saitama, for helping with the analysis of cellular fatty acids; and Dr. S. Okada, NODAI Research Institute, Tokyo University of Agriculture, for supplying cultures.

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