ELSEVIERInternational Journal of Food Microbiology 30 (1996) 243-253 Biochemical characteristics of Enterobacter agglomerans and related strains found in buckwheat seeds Kazuo Iimuraa, Akiyoshi Hosonob,* “MotojiyaCo., Ltd., Matsuntoto390, Japan bFaculty of Agriculture, Shinshu University,Nagano-Minamiminowa 399-45, Japan Received 9 March 1995; revised 19 June 1995; accepted 25 September 1995 Abstract Thirty strains of bacteria were randomly isolated and identified from buckwheat seeds. The phenotypic characteristics of these strains agree well with those of the Enterobacter agglomerans-Erwinia herbicola complex. On the basis of the difference in indole production and gas production from D-glucose, the isolates were divided into 3 phenotypic groups, viz. I, II and III. Twenty two strains were in phenotypic group I, which is negative for indole production and gas production from D-glucose, and resembles Pantoea agglomerans. All six strains in phenotypic group II, which is positive for indole production and negative for gas production from D-glucose, were identified as Erwinia ananas. Two strains in phenotypic group III, which is negative for indole production and positive for gas production from D-glucose, were identified as Rahnella aquatilis. Keywords: Enterobacter agglomerans; aquatilis; Buckwheat seed Erwinia ananas; Pantoea agglomerans; Rahnella * Corresponding author. 0168-1605/96/$15.000 1996 Elsevier Science B.V. All rights reserved PII SO1 1605(96)00949-X 68- is a minor grain crols. Introduction Buckwheat (Fagopyrum esculentum)... agglomerans and E. Iimura. 1393. Naito and Shimizu. agglomerans. 90 ml of sterile physiological saline was added to 10 g of ground buckwheat sample.. 1994). and with animal as well as human reservoirs (Dye... Dye. and is considered to be associated with plant materials such as saprophytes or pathogens. 1987.2. 1969b. J.. 2.. agglomerans strains. E. Japan. 1989. agglomerans and classified it into 11 biogroups. Referring to Ewing and Fife. some strains of E.1... 1972 and Gavini et al.1965). Ewing and Fife. In brief. Er. A. E. including the two type strains. Materials and methods 2. and have identified Enterobacter agglomerans as one of the dominant species in these samples (Iimura and Hosono. 1972 investigated the biochemical characteristics of E. Beji et al.244 K. Miyoshi et al.. The seed samples were kept in a refrigerator (4°C) and used for the isolation within one month after harvesting. . They were treated in a blender (Stomacher Lab-Blender 400.. 1969a. a native of central Asia. Recently. i.e. Buckwheat seeds Buckwheat seeds harvested in Nagano (4 samples) and Hokkaido (2 samples). herbicola. were proposed to form a new genus called Pantoea (Gavini et al. were used for the isolation of E. we investigated the biochemical characteristics of E. agglomerans (i. agglomerans and related strains isolated from buckwheat seeds. Sakazaki et al.. 1993). whereas clinical bacteriologists use the nomenclature of Ewing and Fife. synonymous with Erwinia herbicola.e. Seward . based on total DNA homology and electrophoretic protein pattern similarities. 1981) have been suggested for the ‘Erwinia herbicolaEnterobacter agglomerans complex’. Food Microbiology 30 (1996) 243-253 1. Hosono / ht. Isolation of E. Japan. 1988. Different taxonomic views have resulted in an ambiguous situation: phytopathologists prefer to use the nomenclature of Dye. 1976.e. Erwinia uredo-vora and Erwinia stewartii (Dye. We have reported that bacteria on buckwheat seeds harvested in Nagano and Hokkaido. 1989). herbicola. 1988)) is a heterogeneous species. i. 1969a. Muraschi et al. a consensus has not been reached (Beji et al. 1972. agglomerans and related strains were essentially the same as those described in our previous paper (Iimura and Hosono. Iimura and Hosono. agglomerans and related glucose fermentative bacteria Gram-negative Methods for the isolation of E. 2. and has traditionally been used in Japan to produce flour mainly for making buckwheat noodles. Substantial research on the bacterial flora on buckwheat seeds was performed by several investigators (Obinata et al. 1986). 1988). were in the range of 105-10*/g. Although many rearrangements (Sakazaki et al. 1976. followed by filtration with sterilized gauze. Biochemical properties of the strains identified as Enterobacter agglomerante were further examined.1. pH 7. About ten colonies per sample were randomly isolated.2. A.. London. A decimal dilution of the filtrate was plated on ‘Eiken’ trypto-soy agar (TSA) (Eiken Chemical Co. Growth and pigment production were observed after incubation at 30°C for 4 days. France). 41°C and 44°C for 2 to 7 days. D-mannose. The oxidase test was carried out by use of cytochrome oxidase test paper (Nissui Pharmaceutical Co. Ltd. Ltd. Japan) encompassing Pserudomonas aeruginosa IF0 12689 as a positive control and Escherichia coli JCM 1649 as a negative control.4. Biochemical tests for E. maltose.5% agar. Japan) and incubated at 30°C for 48 h. Incubation was carried out at 30°C for 4 days.5. lactose. Gas formation Gas formation from glucose was detected with Durham tubes in Bacto OF basal medium (Difco Laboratories. Catalase and oxidase tests Catalase production was detected by the production of bubbles in a 3% hydrogen peroxide solution at 30°C..3. 41°C and 44°C One drop of a light suspension of organisms in distilled water was added to ‘Nissui’ nutrient broth filled to a depth of about 5 cm in test tubes and incubated in a water bath at 4”C. Hosono / ht. Motility was checked by observing a clouding of the medium or growth extension from the inoculation line following incubation for 48 h. The organisms were incubated at 30°C in prepared tubes of LIM medium by stabbing.. Pigmentation The organisms were grown on ‘Nissui’ nutrient agar (nutrient broth solidified with 1. Detroit. Food Microbiology 30 (1996) 243-253 245 Medical Co.. Iimura.3. The presence of indole was detected with Kovacs’ P-dimethyl-amino-benzalaldehyde reagent. The isolates were examined for Gram reaction and glucose fermentation. Motility and indole production This was determined using ‘Nissui’ lysine indole motility medium (LIM medium). UK) for 1 min. J. D-galactose. These cultures were purified.0% in ‘Eiken’ brom cresol purple (BCP) semisolid medium: D-glucose. D-cellobiose. D-ribose. Ltd. Growth at 4”C.3.3. agglomerans and related strains isolated 2.A.3. Acid production of sugars and related compounds The following compounds were incorporated at 1.K. growing on the same medium at 30°C for 48 h.O% D-glucose. 2. Dfructose. 2. 2. L-rhamnose. 2.. 2. Marcy-I’Etoile. Gram-negative glucose fermentative bacteria were identified by use of API 20E (Bio Merieux S. D-xylose. .2).) incorporated at 1.3.. Mich. 2. L-arabinose. Tokyo.6.3. Tokyo.3.. .14..3. Tokyo.3. pH 7.13.of ‘Eiken’ DNA agar were heavily spotted with loopfuls of organisms from nutrient agar and incubated at 30°C for 48 h.246 K.7. 1988). meso-erythritol.0. .A. with bromothymol blue replacing bromocresol purple indicator. 2. 1955) containing 0. Methyl red (MR) and Voges-Proskauer (V-P) tests Cultures were grown at 30°C in Bacto MR-VP medium (Difco) containing glucose. 2. H2S was detected by blackening of the medium. 1%. melezitose.3. sucrose. Hosono / ht.8. DNase production was detected by flooding plates with n-HCl and observing zones of clearing around growth. D-sorbitol. 2. buffered peptone. Japan). Results were taken after 4 days of incubation at 30°C.10.5%.3.. was dispensed to a depth of 5 cm in test tubes and bacterial strains were inoculated by stabbing. D-mannitol. Cultures were incubated at 30°C for 30 days. inositol.7%.. 2. Acetylmethylcarbinol was detected using the Barritt modified method (Sakazaki et al. 1%.1 I. or-methyl-D-glucoside. Deoxyribonuclease (DNase) test Plates . 2. peptone (Nissui). inulin and starch. at 30°C for 2. adonitol. 12%. Phenylalanine deaminase test Phenylalanine agar (Ewing et al. 3 or 7 days and testing for the presence of nitrite with dimethyl a-naphtylamine-sulphanilic acid reagent.5%. 2.3. trehalose.9. 0. gelatin (Difco). Ltd.5% for 48 h. raffinose.3% agar was employed. duJcito1. Liquefaction was observed after chilling tubes at 4°C after incubation. J. 0. 0. Urease test Christensen’s urea agar (Christensen.12. Food Microbiology 30 (1996) 243-253 melibiose. The same medium without urea was used as a control for avoiding misjudgment.HPO. Gas formation at 30°C was detected with Durham tubes over a period of 30 days. 1946) was used with incubation for 48 h. Iimura.. Liquefaction of gelatin A medium containing beef extract (Kyokuto Pharmaceutical Co. H.S production Cultures were grown in ‘Nissui’ Kligler iron agar at 30°C for 48 h. 1957) was used with incubation at 30°C for 48 h. 2.3.3. Decarboxylase test Moeller’s medium (Moeller. NaCl. K. Reduction of nitrate This was determined by growing the organism in ‘Nissui’ nutrient broth containing 0. 2. glycerol. The production of phenylpyruvic acid (by deaminase) was indicated by the formation of the characteristic green color upon the addition of an acidified ferric chloride solution to the culture.3. 0. sterilized at 121°C for 15 min. salicin.1% of KNO. 3. agglomerans. T-13. arginine dihydrolase. Growth was indicated by a clouding of the medium. L-rhamnose. urease. T-8. 1988). These strains showed positive reactions for the following productions: catalase. 2. T-2. These results agree well with those of the E.19. . D-xylose. Observations were made at 30°C over 48 h. T-16. T-21. T-23 and T-32) were assigned to E. 2. anaerobic growth. tightly closed with rubber stoppers and observed for growth at 30°C over 48 h. The API 20E system codes 1005132 (strain T-33). Codes 1005332 (strain T-22). sucrose. D-galactose.K. T-20 and T-31) was not in this data base. Growth in KCN Test tubes (16-150 mm). filled to a depth of 5 cm with ‘Eiken’ KCN broth base containing the same volume of 0. agglomerans. p-Galactosidase was determined using an ONPG (o-nitrophenyl-l)-galactosidase) Disc (Nissui). Malonate utilization test Malonate utilization was determined using ‘Eiken’ malonate broth. D-mannose.15. motility at 30°C gelatin liquefaction.3. 3. J. T-15 and T-19) and 1045573 (strain T-9) were assigned to E. whereas code 1205573 (strains T-5. the API 20E system code 1205133 (strains T-7. Hosono / Int. T-12. D-ribose. Citrate utilization test Citrate utilization was determined using Bacto Simmons citrate agar (Difco). T-17. and H. and acids from D-glucose. Food Microbiology 30 (1996) 243-253 241 2.. 2.18. trehalose and D-mannitol. Results and discussion We used the API 20E system to clarify the taxonomic position of the strains isolated from buckwheat seed samples. However. 1005333 (T-l.16. Digestion of es&in This was examined by the methods described by Sakazaki (Sakazaki et al. Erwinia sp. Table 2 shows phenotypic characteristics of the 30 strains isolated. herbicola complex.. T-36) was assigned to Rahnella aquatilis.S. T-18. (Brenner et al.17..3. A. D-fructose.015% KCN solution were each inoculated with a loopful of a 24 h nutrient broth culture. T-6. Codes 1045773 (strain T-14) and 1245773 (strain T-4. T-10. L-arabinose. acetoin from D-glucose and a-galactosidase. The negative results were for cytochrome oxidase..3. agglomerans or to Erwinia sp. 1988).3. Table 1 shows the API 20E system codes of 30 strains isolated. 1984). 1005133 (strains T-3. 2. Iimura. T-24 and T-34) and 1205132 (strains T-11 and T-35) were interpreted with the API data base (API system 3rd edition) as E. Kauffmann-Petersen’s organic acid utilization test Kauffmann-Petersen’s test was examined by the methods described by Sakazaki (Sakazaki et al. agglomerans-E. Observations were made at 30°C over 4 days. lysine and ornithine decarboxylase. Klebsiella oxytoca or Klebsiella planticola. maltose. 1987. 22 strains (73. Nagano 1005132 1005133 1005133 1005133 1005133 1005133 1005133 1005133 1005133 1005133 1005332 1005333 1005333 1005333 1005333 1205132 1205132 1205133 1205133 1205133 1205133 1205133 1045573 1045773 1245773 1245773 1245773 1245773 1205573 1205573 . Gavini et al. Hokkaido Uryu.. Hosono / ht.. 1984. Hokkaido Hata. A. Hokkaido Uryu. 1. Table 1 Enterobucter ugglomerans isolated from buckwheat seeds and their API 20E profile numbers Strain number Buckwheat harvested Place Year 1991 1991 1991 1991 1991 1991 1991 1991 1991 1991 1991 1991 1991 1991 1991 1991 1990 1991 1991 1991 1991 1990 1991 1991 1991 1991 1991 1990 1991 1991 API 20E profile T-33 T-3 T-6 T-8 T-12 T-17 T-18 T-21 T-24 T-34 T-22 T-l T-2 T-15 T-19 T-11 T-35 T-7 T-13 T-16 T-20 T-31 T-9 T-14 T-4 T-10 T-23 T-32 T-5 T-36 Hata. However. Many taxonomical data have been published on the E. Nagano Matsumoto. Nagano Matsumoto. Nagano Azumi. the isolates were divided into 3 phenotypic groups.248 K.. Beji et al. Nagano Kawahigasi. Hokkaido Matsumoto. Brenner et al. I. 1005332. Nagano Saku. Hokkaido Kawahigasi. Nagano Kawahigasi. Nagano Uryu. herbicola complex (Gavini et al. which were grouped in phenotype I. Hokkaido Uryu. 1005333. all the strains involved in this group were negative for acid production on the BCP semisolid medium and positive for citrate utilization on the Bacto Simmons citrate agar (Table 2). and of acid production from amygdalin and inositol as illustrated in Fig. Food Microbiology 30 (1996) 243-253 On the basis of the differences in indole production and gas production from D-glucose. Hokkaido Uryu. 1988). Hokkaido Saku. J. Nagano Kawahigasi. Nagano Hata. Nagano Hata. Nagano Kawahigasi. viz.. Iimura. Nagano Azumi. II and III. Hokkaido Kawahigasi. Recently. Nagano Azumi..3% of the total isolates) were negative for indole production and gas production from D-glucose. Hokkaido Azumi. Nagano Azumi. Hokkaido Uryu. agglomerans-E. The API 20E system codes of these strains were 1005132. These codes were characterized by the modes of citrate utilization. 1005133. Out of the 30 strains isolated. Hokkaido Matsumoto. 1205132 and 1205133.. Hokkaido Uryu. Verdonck et al. Hokkaido Uryu. 1983. Hokkaido Uryu. Table 2 Penotypic characteristics of Enterohacter ugglomeruns (22 aquatilis and related strains isolated from buckwheat seeds Phenotype II Erwinia ananas (6 strains) Reaction Phenotype III Rahnella (2 strains) Reaction h Characteristics Phenotype I Pontoea agglomerans strains) Reaction (+) _ _ + + + _ 95 + _ + 95 _ 91 _ _ _ + + _ + 91 5 _ + + + + 83 + + + _ 61 _ _ _ _ 17 + _ + _ _ _ + Growth at 4°C Growth at 41°C Growth at 44°C Production of yellow pigment Motility Catalase Cytochroomoxidase KCN (growth) Gelatin liquefaction Indole production Voges-Proskauer reaction Nitrate reduced to nitrite H.S production Hydrolysis of esculin Gas production from D-glucose Arginine dihydrolase (Moeller) Lysine decarboxylase Omithine .decarboxylase Phenylalanine deaminase Deoxyribonuclease p-Galactosidase Urease Utilization of: Citrate (Simmons) Malonate D-Tartrate (K-P) Mutate (K-P) Acid produced from: L-Arabinose (33) _ _ . + + + + 5 18 83 + + 83 67 f - $ 4 3 B + + + + + + + f t + 83 + + + + + + + + s’o -t + + + - 3 3 & -.!z D-Ribose D-Xylose D-Fructose D-Glucose D-tiannose L-Rhamnose D-Cellobiose D-Galactose Lactose Maltose Melibiose Sucrose Trehalose Melezitose Rafkose Glyserol meso-Erytheritol Adonitol Dulcitol Inositole D-Mannitol D-sd’rbitol a-Methyl-D-glucoside Salicin Inulin Starch + 86 - +.Table 2 (continued) Phenotype I Puntoea agglomerans (22 strains) Reaction tion + + + + + + + Characteristics Phenotype II Erwinia ananus (6 strains) Reaction Phenotype III Rahnella aquatilis (2 strains) Reac3 k? 2 “3 . number indicated percentage of positive strains. all strains negative. . a ijP ti T= 8 k ‘.. delayed reaction. all strains positive.k + + + + + + 9 P 0 . -. 0. ananas or E. which is positive for indole production and negative for gas production from D-glucose. A. uredovora. Gavini et al. and results were coded by seven-digit profile numbers as described by the manufactuer. agglomerans (Gavini et al. we . viz. dulcitol and a-methyl-D-glucoside.. we recognized that most of the phenotypic characteristics of the strains of phenotypic group I were very similar to P. but not for adonitol. melibiose. phenotypic group II phenotypic group I T-34 c_______________________________________-_____. AMY. acid production from inositol. taking into account that the strain was positive for indole production and negative for gas production from D-glucose. sucrose. T-14. 1989). 1989 analyzed the E. 1989 reported that E. ananas showed positive reactions for the following: yellow pigment and indole. agglomerans or as Erwinia sp. as Pantoea agglomerans comb. API 20E profile and phenotype of Enterobacter agglomerans isolated from buckwheat seeds. 1. Referring to the phenotypic characteristics of P. Beji et al.. T-10. utilization of citrate. API 20E reaction: CIT. sorbitol and salicin. agglomerans. T-9. 1988 and Gavini et al. ____. nov. Furthermore. but with a few exceptions. Iimura. lactose. As shown in Table 1 and Fig. Fig._. 1988. agglomerans-E. Food Microbiology 30 (1996) 243-253 Entervtmcter zggkmem idated from buckvdheat Seeds 251 zkT-5 indole production 756. herbicola complex on the basis of phenotypic characteristics and DNA-DNA hybridization data and proposed the transfer of E. ananas (Beji et al. nov. 1.. T-23 and T-32) in phenotypic group II. phenwpic group + III CIT + IN0 + 1245773 CIT IN0 + 1045773 T-14 CIT IN0 1045573 T-9 1205133 T-7 T-l 3 T-l 6 T:E 1205132 T-l 1 T-35 1005333 T-l T-2 T-l 5 T-19 1005332 T-22 1005133 T-3 T-6 T-8 T-12 T-1’:. Hosono / ht. J. acid production from amygdalin.. Mergaert et al. raffinose. In reference to the phenotypic characteristics of E.. 1984 reported that a strain having the code 1245773 could be interpreted with the API data base as E. API 20E was performed at 30°C for 24 h. maltose. in production of ornithine decarboxylase and in acid production from lactose and D-cellobiose.K. 1989).. were identified either as E. T-21 T-24 1005132 T-33 T:?0 T-23 T-32 _----~--~~~_______. INO. agglomerans to Pantoea gen. tests in the API 20E system gave the results that the strains (T-4. and acids from D-cellobiose. Food Microbiology 30 (19%) 243-253 have recognized that most of the phenotypic characteristics of the strains of phenotypic group II closely resembled E. inositol. aquatilis is a rhizosphere-associated bacteria and also a nitrogen fixer. inulin and esculin (Table 2). maltose. Hamze. D-fructose. raffinose. Sci. 39. Syst.F. L-arabinose. New Zeal.. Ewing. 22. (1969b) A taxonomic study of the genus Erwinia. as Pantoeu agglomerans comb. Int. a potential contaminant in lager beer breweries. 12. a nitrogen fixing enteric bacterium associated with the rizosphere of wheat and maize. F. 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