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International 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
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-
e. agglomerans and related strains isolated from buckwheat seeds. agglomerans (i. including the two type strains. Naito and Shimizu. Erwinia uredo-vora and Erwinia stewartii (Dye. 1987. E. E. 2. 1969a. Buckwheat seeds Buckwheat seeds harvested in Nagano (4 samples) and Hokkaido (2 samples). 1976. agglomerans strains.1. Referring to Ewing and Fife. 1989.244 K. Materials and methods 2. Miyoshi et al.1965). 90 ml of sterile physiological saline was added to 10 g of ground buckwheat sample. 1988)) is a heterogeneous species. 1981) have been suggested for the ‘Erwinia herbicolaEnterobacter agglomerans complex’.. and has traditionally been used in Japan to produce flour mainly for making buckwheat noodles. Dye. a consensus has not been reached (Beji et al. agglomerans. herbicola. Introduction Buckwheat (Fagopyrum esculentum). The seed samples were kept in a refrigerator (4°C) and used for the isolation within one month after harvesting. whereas clinical bacteriologists use the nomenclature of Ewing and Fife. some strains of E. 1393. Er. Sakazaki et al. J.e. A.. a native of central Asia. 1986). They were treated in a blender (Stomacher Lab-Blender 400. Seward .. Substantial research on the bacterial flora on buckwheat seeds was performed by several investigators (Obinata et al.. Hosono / ht.. were used for the isolation of E. agglomerans and classified it into 11 biogroups. synonymous with Erwinia herbicola. agglomerans and E. We have reported that bacteria on buckwheat seeds harvested in Nagano and Hokkaido. Although many rearrangements (Sakazaki et al. 1969a. 1972 and Gavini et al. 1989). Different taxonomic views have resulted in an ambiguous situation: phytopathologists prefer to use the nomenclature of Dye. i.2. were in the range of 105-10*/g. Isolation of E. 1972. Muraschi et al.. 1972 investigated the biochemical characteristics of E. 1994). Recently.. Iimura and Hosono. and with animal as well as human reservoirs (Dye. Japan. 1993). Japan.. based on total DNA homology and electrophoretic protein pattern similarities. is a minor grain crols.. .. agglomerans and related glucose fermentative bacteria Gram-negative Methods for the isolation of E. Ewing and Fife.. 1976. Iimura. agglomerans and related strains were essentially the same as those described in our previous paper (Iimura and Hosono.. we investigated the biochemical characteristics of E. Food Microbiology 30 (1996) 243-253 1. 1988). and have identified Enterobacter agglomerans as one of the dominant species in these samples (Iimura and Hosono. were proposed to form a new genus called Pantoea (Gavini et al. i. 1988. In brief.e. 2. 1969b. and is considered to be associated with plant materials such as saprophytes or pathogens. herbicola. Beji et al.
France). Incubation was carried out at 30°C for 4 days.3.3. D-ribose. Japan) and incubated at 30°C for 48 h.3.1. Ltd. Dfructose. followed by filtration with sterilized gauze.. D-xylose. The oxidase test was carried out by use of cytochrome oxidase test paper (Nissui Pharmaceutical Co... pH 7. . D-mannose. Pigmentation The organisms were grown on ‘Nissui’ nutrient agar (nutrient broth solidified with 1.5% agar.3. London. maltose.2. 2. Marcy-I’Etoile. Growth and pigment production were observed after incubation at 30°C for 4 days. lactose. Ltd.2). These cultures were purified.3. growing on the same medium at 30°C for 48 h. L-arabinose. Ltd.. 2. Mich. Biochemical properties of the strains identified as Enterobacter agglomerante were further examined. Japan) encompassing Pserudomonas aeruginosa IF0 12689 as a positive control and Escherichia coli JCM 1649 as a negative control. The organisms were incubated at 30°C in prepared tubes of LIM medium by stabbing. The isolates were examined for Gram reaction and glucose fermentation. Gas formation Gas formation from glucose was detected with Durham tubes in Bacto OF basal medium (Difco Laboratories. Hosono / ht. 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. Tokyo. 2. L-rhamnose. The presence of indole was detected with Kovacs’ P-dimethyl-amino-benzalaldehyde reagent. Tokyo.5. 41°C and 44°C for 2 to 7 days.. D-cellobiose.3.A. Detroit. About ten colonies per sample were randomly isolated..) incorporated at 1. 2.0% in ‘Eiken’ brom cresol purple (BCP) semisolid medium: D-glucose. Motility and indole production This was determined using ‘Nissui’ lysine indole motility medium (LIM medium). Growth at 4”C. UK) for 1 min. Food Microbiology 30 (1996) 243-253 245 Medical Co. J.O% D-glucose. Biochemical tests for E.6. Acid production of sugars and related compounds The following compounds were incorporated at 1. 2. D-galactose.K.3. Catalase and oxidase tests Catalase production was detected by the production of bubbles in a 3% hydrogen peroxide solution at 30°C. 2. Iimura. Motility was checked by observing a clouding of the medium or growth extension from the inoculation line following incubation for 48 h.3.. A. Gram-negative glucose fermentative bacteria were identified by use of API 20E (Bio Merieux S. agglomerans and related strains isolated 2. A decimal dilution of the filtrate was plated on ‘Eiken’ trypto-soy agar (TSA) (Eiken Chemical Co.4.
. duJcito1. 0. with bromothymol blue replacing bromocresol purple indicator. NaCl.5% for 48 h.of ‘Eiken’ DNA agar were heavily spotted with loopfuls of organisms from nutrient agar and incubated at 30°C for 48 h. 2. 12%. 2.1 I.5%. Urease test Christensen’s urea agar (Christensen. gelatin (Difco).7. 1%.7%. H. 2. 1988). was dispensed to a depth of 5 cm in test tubes and bacterial strains were inoculated by stabbing. melezitose. 0. 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. adonitol. Results were taken after 4 days of incubation at 30°C.0. 1%.3.3. Tokyo. 2. buffered peptone.8. 0. Liquefaction was observed after chilling tubes at 4°C after incubation.3. J.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.13.3. Deoxyribonuclease (DNase) test Plates .12.3. Decarboxylase test Moeller’s medium (Moeller. glycerol.3. sterilized at 121°C for 15 min. 2.9.. .3. trehalose. Liquefaction of gelatin A medium containing beef extract (Kyokuto Pharmaceutical Co.246 K.1% of KNO.A.10. meso-erythritol. Iimura. Methyl red (MR) and Voges-Proskauer (V-P) tests Cultures were grown at 30°C in Bacto MR-VP medium (Difco) containing glucose. sucrose.3% agar was employed. K. Gas formation at 30°C was detected with Durham tubes over a period of 30 days.14. peptone (Nissui).HPO. Food Microbiology 30 (1996) 243-253 melibiose. salicin. Acetylmethylcarbinol was detected using the Barritt modified method (Sakazaki et al. H2S was detected by blackening of the medium. 2. inositol. 2.. pH 7. Reduction of nitrate This was determined by growing the organism in ‘Nissui’ nutrient broth containing 0.3. at 30°C for 2. D-sorbitol. Cultures were incubated at 30°C for 30 days.5%. D-mannitol. 1955) containing 0. Ltd. inulin and starch. raffinose. 3 or 7 days and testing for the presence of nitrite with dimethyl a-naphtylamine-sulphanilic acid reagent.. Hosono / ht. 1946) was used with incubation for 48 h.. 2. DNase production was detected by flooding plates with n-HCl and observing zones of clearing around growth. Phenylalanine deaminase test Phenylalanine agar (Ewing et al. Japan). 0. The same medium without urea was used as a control for avoiding misjudgment. or-methyl-D-glucoside.
motility at 30°C gelatin liquefaction. T-8. 2. agglomerans. 1988).. The negative results were for cytochrome oxidase. 2. 1988). D-galactose.17.K. 2.3. L-arabinose. urease. The API 20E system codes 1005132 (strain T-33). T-15 and T-19) and 1045573 (strain T-9) were assigned to E.. Digestion of es&in This was examined by the methods described by Sakazaki (Sakazaki et al.. Observations were made at 30°C over 48 h. However. 2. T-17. Citrate utilization test Citrate utilization was determined using Bacto Simmons citrate agar (Difco). T-13. agglomerans-E.S. 1984). filled to a depth of 5 cm with ‘Eiken’ KCN broth base containing the same volume of 0. J. 1005333 (T-l. trehalose and D-mannitol. Kauffmann-Petersen’s organic acid utilization test Kauffmann-Petersen’s test was examined by the methods described by Sakazaki (Sakazaki et al. Iimura. (Brenner et al.3. These strains showed positive reactions for the following productions: catalase.18. and acids from D-glucose. tightly closed with rubber stoppers and observed for growth at 30°C over 48 h. T-16. Codes 1045773 (strain T-14) and 1245773 (strain T-4. and H. agglomerans. arginine dihydrolase. T-10. . T-18. T-20 and T-31) was not in this data base.3. 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. lysine and ornithine decarboxylase. the API 20E system code 1205133 (strains T-7.015% KCN solution were each inoculated with a loopful of a 24 h nutrient broth culture. Malonate utilization test Malonate utilization was determined using ‘Eiken’ malonate broth. anaerobic growth. D-mannose. Growth was indicated by a clouding of the medium. agglomerans or to Erwinia sp. T-12. Table 2 shows phenotypic characteristics of the 30 strains isolated. These results agree well with those of the E. D-xylose. 1005133 (strains T-3. herbicola complex. T-21.3. T-2. 3. T-6.15. acetoin from D-glucose and a-galactosidase. p-Galactosidase was determined using an ONPG (o-nitrophenyl-l)-galactosidase) Disc (Nissui). sucrose.19. Growth in KCN Test tubes (16-150 mm). Results and discussion We used the API 20E system to clarify the taxonomic position of the strains isolated from buckwheat seed samples. T-36) was assigned to Rahnella aquatilis. Observations were made at 30°C over 4 days. Food Microbiology 30 (1996) 243-253 241 2. Codes 1005332 (strain T-22). whereas code 1205573 (strains T-5. L-rhamnose.16. Erwinia sp.3. Klebsiella oxytoca or Klebsiella planticola. D-fructose.. maltose. Table 1 shows the API 20E system codes of 30 strains isolated. A. D-ribose. T-23 and T-32) were assigned to E. Hosono / Int.
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. 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. 22 strains (73. herbicola complex (Gavini et al. Brenner et al. Nagano Matsumoto. Hokkaido Uryu. 1005332. These codes were characterized by the modes of citrate utilization.. 1983. 1205132 and 1205133. Nagano Saku. Nagano Kawahigasi. II and III. Nagano Uryu. Nagano Matsumoto. Nagano Azumi. Hokkaido Saku.3% of the total isolates) were negative for indole production and gas production from D-glucose. Beji et al. 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). Nagano Kawahigasi. 1988). Iimura. Nagano Azumi. Hokkaido Azumi. Nagano Hata. Hokkaido Kawahigasi.. Hokkaido Matsumoto. I. Hosono / ht. Hokkaido Kawahigasi. Verdonck et al. Hokkaido Uryu. 1. Hokkaido Uryu. Nagano Azumi. The API 20E system codes of these strains were 1005132.248 K. which were grouped in phenotype I. Nagano Kawahigasi. Recently. A. Hokkaido Matsumoto. 1005133. 1987. Gavini et al. the isolates were divided into 3 phenotypic groups. Nagano Kawahigasi... Hokkaido Uryu. Nagano Hata. Hokkaido Uryu. Out of the 30 strains isolated. Hokkaido Uryu. 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 . 1984. J. agglomerans-E.. viz. Hokkaido Uryu.. Nagano Azumi. Hokkaido Uryu. Hokkaido Hata. However. Many taxonomical data have been published on the E.
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 & -. all strains negative. all strains positive.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. 0.k + + + + + + 9 P 0 . delayed reaction. -. . a ijP ti T= 8 k ‘.!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 - +.
T-14. 1. acid production from amygdalin. 1989 reported that E. INO. 1989).. nov. 1988 and Gavini et al. and acids from D-cellobiose. Beji et al. Iimura. sucrose. T-10. 1984 reported that a strain having the code 1245773 could be interpreted with the API data base as E. maltose. Furthermore.. agglomerans or as Erwinia sp. herbicola complex on the basis of phenotypic characteristics and DNA-DNA hybridization data and proposed the transfer of E. 1. nov. viz. Mergaert et al. API 20E was performed at 30°C for 24 h. AMY. J.. in production of ornithine decarboxylase and in acid production from lactose and D-cellobiose. we .. utilization of citrate. agglomerans (Gavini et al. agglomerans to Pantoea gen. and results were coded by seven-digit profile numbers as described by the manufactuer. T-23 and T-32) in phenotypic group II. agglomerans-E. melibiose.. T-21 T-24 1005132 T-33 T:?0 T-23 T-32 _----~--~~~_______. 1989 analyzed the E. Fig. uredovora. 1988. ____. acid production from inositol. which is positive for indole production and negative for gas production from D-glucose. lactose. 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. Food Microbiology 30 (1996) 243-253 Entervtmcter zggkmem idated from buckvdheat Seeds 251 zkT-5 indole production 756. taking into account that the strain was positive for indole production and negative for gas production from D-glucose. ananas or E. tests in the API 20E system gave the results that the strains (T-4. ananas showed positive reactions for the following: yellow pigment and indole. Referring to the phenotypic characteristics of P.K. we recognized that most of the phenotypic characteristics of the strains of phenotypic group I were very similar to P. A. Gavini et al. As shown in Table 1 and Fig. sorbitol and salicin. dulcitol and a-methyl-D-glucoside. 1989). phenotypic group II phenotypic group I T-34 c_______________________________________-_____. API 20E reaction: CIT. API 20E profile and phenotype of Enterobacter agglomerans isolated from buckwheat seeds. agglomerans. were identified either as E._. but not for adonitol. In reference to the phenotypic characteristics of E. but with a few exceptions. as Pantoea agglomerans comb. raffinose. T-9. ananas (Beji et al..
. Agric. 24. D. The ‘carotovora’ group. Leclerc. J. were identified as R. Gavini. Erwinia milletiae and Enterobacter agglomerans and redefinition of the taxon by genotypic and phenotypic data. inositol. D-cellobiose. as Pantoeu agglomerans comb.. Beji.L.. 1979 E. Bacterial. F. D. Syst. M. cr-methyl-D-glucoside. D-mannitol. 77-88. (1991) Rahnella aquatilis.W. J.. Dye. Gavini.. Lefebvre. Can. F. Hosono / Int. adonitol. J. Kersters.. W. and De Ley. D-galactose. translucent.I.J. 223-229. limura. A. J. A. melibiose. 45-55. Sci. soil. which are negative for indole production and positive for gas production from D-glucose. J. Knutson. . aquatilis with the API 20E system. from human clinical specimens. M. Acid production was observed from D-glucose. 34. and Achouak. Izard. J. C. H. Christensen. K. 81-97. trehalose. 337-345. J. Davis. Izard. A. Int.J.K. Kersters. 1955203. aquatilis has also been isolated mostly from water. Microbial. W. 12.. and De Ley.F. Res. nov. Gavini.. a nitrogen fixing enteric bacterium associated with the rizosphere of wheat and maize. et a I’espece Enterobacter ugglomeruns. D-fructose. 37. J. and Reavis. Steigerwalt. 1991 reported that R. Syst. (1969a) A taxonomic study of the genus Erwiniu. W. D-sorbitol and salicin but not from melezitose. Berge. Bacterial. Van Vuuren. Bacterial.. J. lager beer breweries and in a few cases. (1988) Subjective synonymy of Erwinia herbicola. H. (1957) Phenylalanine and malonate media and their use in enteric bacteriology. Ewing. Ewing. Mielcarek. 63-68. Food Microbial.. 38. O. (1969b) A taxonomic study of the genus Erwinia. (the herbicolalathry bacteria). Lab. B. a potential contaminant in lager beer breweries. J. Syst. New Zeal. erythritol. H. Bacterial. Int. 15. Beji. Acetoin was positive but lysine and ornithine decarboxylase. 4-l 1. Food Microbiology 30 (19%) 243-253 have recognized that most of the phenotypic characteristics of the strains of phenotypic group II closely resembled E. Hlth. D. aquatilis strains described by Izard et al. nov. 39. D. and Fife. Mergaert. ananas. lactose. Brenner. J. Gavini. Syst. arginine dihydrolase and DNase were negative. References Beji.. 461-466. J. These characteristics fit well with those of R. (1981) A numerical taxonomic study of the genus Erwiniu. J. Hamze.B.. Mergaert. Int... K. 833-839. Syst. (1946) Urea decomposition as a means of differentiating Proteus and paracolon cultures from each other. 4. agglomerans. R.R. 1972 to Puntoeu gen. Microbial. and convex with an entire margin (data not shown).G. and Leclerc. Appl.H.. inulin and esculin (Table 2).. J. nov. A. K. Bacterial. nov. D. 1991 reported that R. Izard. 12. D. (1984) Attempts to classify herbicola group Enterobacter agglomerans strains by deoxyribonucleic acid hybridization and phenotypic tests.252 K. sucrose. and description of Puntoeu dispersu sp..R. Publ.. (1989) Transfer of Enterobucter ugglomerans (Beijerinck 1988) Ewing and Fife. 13. (1983) Etude taxonomiqu de souches appartenant ou apparentees au genre Erwinia group Herbicola. F.. D.H. (1972) Enterobacter aggfomerans (Beiierinck) comb. maltose. M.W. J. D-ribose. T. 22.. Mergaert.. D-mannose. 218-235. Int. II.W..J. L-rhamnose. J. W.W. B. New Zeal. 153-161.. Hamze et al.. 52. Berge et al. L-arabinose. Heuhn. IV. Sci. and Kersters. G. Fanning. and Krichevsky. F. dulcitol. A. Colonies were smooth. Int. Dye. D-xylose. Dye.. New Zeal. All the strains (T-5 and T-36) in phenotypic group III. J. (1991) RuhneNu aquatilis. aquatilis is a rhizosphere-associated bacteria and also a nitrogen fixer. raffinose. ‘A typical Erwinias’.
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