Antonie van Leeuwenhoek 47 (1981) 147-157
147
Gluconobacters from honey bees B. LAMBERT,K. KERSTERS,F. GOSSELI~, J. SWINGS AND J. DE LEY Laboratorium voor Microbiotogie en mierobiYte Genetica, Rijksuniversiteit, Ledeganckstraat 35, B-9000 Gent, Belgium
LAMBERT, B., KERSTERS, K., GOSSELI~,F., SWINGS, J. and DE LEY, J, 1980. Gluconobacters from honey bees. Antonie van Leeuwenhoek 47:147-157. Fifty-six Gluconobacter strains and one Acetobacter strain were isolated from honey bees and their environment in three different regions in Belgium and identified phenotypically. Polyacrylamide gel electrophoresis of the soluble cell proteins showed that two different types exist within the Gluconobacter isolates: strains from type A were found in samples of the three regions, whereas strains from type B were only isolated in two of the three regions. Both types could occur in bees from the same region, from several hives of one bee keeper and from one hive. Strains from type A were almost identical with collection strain G. oxydans subsp, suboxydans NCIB 9018, whereas strains from type B consituted a new protein electrophoretic type within the genus Gluconobacter. Although Gluconobacter is apparently associated with honey bees, it is not known whether it is important or required for the bees or any hive product.
INTRODUCTION The bacteria isolated from honey bees have been assigned to one of the following genera: Bacillus, Bifidobacterium, Brevibacterium, Enterobacter, Eseh-
erichia, Hafnia, Klebsiella, Lactobacillus, Proteus, Pseudomonas, Salmonella, Serratia, Shigella, Staphylococcus and Streptococcus" (Borchert, 1966; Baily, 1968; Gilliam and Morton, 1974; Scardovi and Trovatelli, 1969). It is also known that bacteria are associated with important diseases of the honey bee e.g. American and European foulbrood, septicaemia and melanosis of reproductive organs. Although several antimicrobial barriers exist (Lavie, 1968) both in the hive and in the honey bee, a complex bacterial flora develops gradually in the bee. Its composition is variable, depending on the food and the hive to which the bee belongs.
148
a. LAMBERTET AL.
It remains questionable whether the hive and the honey bee harbour a specific and well-adapted bacterial flora, or a completely accidental one, acquired by each individual bee from the multitude of microorganisms in the environment. Bacteria belonging to the sugar-loving, acid- and ethanol-tolerating genus Gluconobacter were found to be associated with nectar and bees in Spain. Each bee appeared to harbour 103 to 106 of these bacteria (Ruiz-Argfieso and Rodriguez-Navarro, 1975). We wanted to verify whether Gluconobacter is associated with honey bees in Belgium. In the present paper we report on the enrichment, the isolation and the phenotypic identification of Gluconobacter from bees from three different regions in Northern Belgium. Polyacrylamide gel electrophoresis of soluble cell proteins enables to detect genetically highly related strains (Kersters and De Ley, 1975, 1980). We used this technique to answer some questions related to the microecology of the Gluconobacter strains isolated from bees. (i) How many different electrophoretic types occur and how are they distributed geographically? (ii) Which electrophoretic types can be found respectively in one hive, in different hives from one bee keeper and in hives of neighbouring bee keepers? (iii) Is it possible to identify the isolates from bees with any of the known collection strains of Gluconobacter?
MATERIALS AND METHODS
Samples Four hundred and eleven samples, mostly bees, but also flowers visited by bees, honey and other materials from the hive, and some wasps were collected during August and September 1979 in Aalter, Neeroeteren, Zottegem and Gent (Belgium). The bees were killed by keeping them at 4~ during 15 minutes. Tests, media and conditions The basal medium for growth of Gluconobacter contained 0.5 ~o yeast extract (Oxoid) and 5 ~o D-glucose in water. Enrichment of Gluconobacter was carried out in the basal medium to which 0.0016 ~ bromophenol blue and 0.01 ~ actidione (Upjohn) was added. The temperature for growth was 28~ The tubes showing acidification after 2 to 4 days were plated out on a medium containing 5 ~ D-glucose, 1 ~ yeast extract (Oxoid), 3 ~ C a C O 3 and 2 ~ agar. Colonies that were surrounded by a zone of dissolved C a C O 3 w e r e purified further. Pure cultures were maintained on the same medium. Growth in 0.5 ~o yeast extract was observed after 7 days. Growth on and oxidation of D, L-lactate, ketogenesis on glycerol, D-mannitol and sorbitol, the presence of catalase and growth in the Hoyer-ethanol medium were tested according to Frateur (1950). The oxidation and overoxidation of ethanol was investigated on the media described by Frateur (1950) and Carr (1968). Growth at pH 3.55 was tested in the basal medium made
149
G L U C O N O B A C T E R S F R O M H O N E Y BEES
up in 0.1 M citric acid-0.2 M phosphate buffer at pH 3.55. Resistance towards ethanol was studied in the basal medium to which absolute ethanol was added to the final concentrations of 1 and 5 ~ . The medium of Asai et al. (1964) was used to detect acid formation from D-glucose and D-xylose. The pH was measured after 7 days of incubation. The formation of 2-keto-, 5-keto- and 2,5-diketogluconic acids from glucose was tested as described by Gossel6 et al. (1980). H/S formation was detected by lead acetate paper strips in the basal medium supplemented with 0.5~o peptone (Oxoid), 0 . 0 5 ~ NazSO 4 and 0.1 ~o L-cystein. Nitrate reduction was tested in the basal medium to which 0.1 ~o KNO3 was added. Hydrolysis of gelatin was determined in 0.5 ~ yeast extract (Oxoid), 2~o D-mannitol, 0.1 ~ D-glucose, 0.3 ~o peptone and 12 ~o gelatin. Oxidase was determined by the Kovacs' method (1956).
Polyacrylamide gel electrophoresis of soluble proteins Bacteria were grown in Roux flasks at 28~ for 40 h on the glucose-yeast extract-CaCO3-agar medium. The preparation of cell-free extracts, dialysis (Swings et al., 1980), polyacrylamide gel electrophoresis of soluble proteins, densitometry, normalization of densitometric tracings and photography were carried out as described previously (Kersters and De Ley, 1975). Each protein extract was examined in at least two independent electrophoretic runs. The normalized densitometric tracings were converted into a sequence of 120 numbers, representing the optical densities (expressed in mm height) of each position on a scan. The Pearson product-moment correlation coefficient (r) between any pair of densitometric tracings was calculated and the electrophoregrams were grouped by the unweighted average-pair group method using programs previously developed (Kersters and De Ley, 1975) and the Clustan program (Version IC) of Wishart (1978) on a Siemens 7755 (BS2000) computer of the Centraal Digitaal Rekencentrum, Rijksuniversiteit, Gent.
RESULTS
Isolation and phenotypic characterization of Gluconobacter strains from bees Fourty percent of all samples acidified the enrichment medium and might contain Gluconobacter. After enrichment and isolation, we used the following features to identify Gluconobacter: gram-negative cells; no growth in 0.5 ~o yeast extract, in Hoyer-ethanol medium, in the presence of 5 ~ ethanol or on D, Llactate; growth at pH 3.55, in the presence of 1 ~o ethanol; oxidation of ethanol to acetic acid; acid from D-glucose and D-xylose (pH < 4); oxidase negative; catalase positive; no HzS formation; no gelatin liquefaction; no nitrate reduction; ketogenesis on sorbitol, glycerol and D-mannitol; formation of 2-ketoand 5-ketogluconic acids from D-glucose (Gossel6 et al., 1980; De Ley and Frateur, 1974; Swings and De Ley, 1981).
Colony colour on GYC c : light-yellow brown dark coffee-brown pink C r y s t a l s visible o n G Y C c Gram reaction Rods Ellipsoidal cells F i l a m e n t o u s cells C h a i n s o f cells G r o w t h a t p H 3.55 G r o w t h in the presence o f 5 ~o e t h a n o l G r o w t h o n D, L-lactate O x i d a t i o n o f D, L-lactate Oxidation of ethanol Overoxidation of ethanol H2S K e t o g e n e s i s o n sorbitol K e t o g e n e s i s o n glycerol Ketogenesis on D-mannitol F o r m a t i o n of: 2 - k e t o g l u c o n i c acid 5-ketogluconic acid 2 , 5 - d i k e t o g l u c o n i c acid
Features b
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95
" T h e f o l l o w i n g strains s h o w e d identical f e a t u r e s : 30, 48 a n d 50; 36 a n d 41 ; 39 a n d 47; 42 a n d 45; 43, 44 a n d 46; 54 a n d 57; 66, 68, 74, 85, 87, 90 a n d 93; 81, 84, 86, 92 a n d 94. S t r a i n s n ~ 29 to 62 were isolated f r o m m a t e r i a l collected in A a l t e r , n ~ 65 to 73 in N e e r o e t e r e n , n ~ 74 to 94 in Z o t t e g e m a n d n ~ 95 in Gent. b S y m b o l s u s e d : ( + ) w e a k r e a c t i o n , - f e a t u r e a b s e n t o r negative, + f e a t u r e p r e s e n t o r positive, V G r a m variable. c G Y C : 5 ~ glucose + 1 ~ y e a s t e x t r a c t ( O x o i d ) + 3 ~ C a C O 3 + 2 ~ a g a r .
Colony colour on GYC c : light-yellow brown dark coffee-brown pink C r y s t a l s visible o n G Y C " Gram reaction Rods Ellipsoidal cells F i l a m e n t o u s cells C h a i n s o f cells G r o w t h at p H 3.55 G r o w t h in the presence o f 5 ~o e t h a n o l G r o w t h o n D, L-lactate O x i d a t i o n o f D,L-lactate Oxidation of ethanol Overoxidation of ethanol H2 S Ketogenesis on sorbitol K e t o g e n e s i s o n glycerol Ketogenesis on D-mannitol F o r m a t i o n of: 2-ketogluconic acid 5-ketogluconic acid 2 , 5 - d i k e t o g l u c o n i c acid
Features b
T a b l e 1.
U,
152
B. LAMBERTET AL.
The following fifty-six isolates were identified as Gluconobacter: n ° 29 to 39, 41 to 51, 53 to 57, 60, 61,62, 65, 66, 68, 69, 70, 73, 74, 76 to 94. Strain n ° 29 was from Solidago canadensis, n ° 57 from HeIeniurn sp., n ° 92 from Campanulapatula, the remaining fifty-three isolates were all from bees. Nine strains remained unidentified, and will not be considered further. One isolate (n ° 95) from a wasp proved to be an Acetobacter as it overoxidized ethanol to CO 2 and H 2 0 ; grew on D, L-lactate, was only slightly ketogenic, formed only 2-ketogluconic acid from D-glucose and grew in the presence o f 5 ethanol. Table 1 summarizes the isolation sites and phenotypic characterization of the 57 isolates.
29 95 80 ~9 77 30 36 ?0 50 87 ql ~8 5~ 56 60 66 57 53 7~ 65 76 39 51 93 86 92 73 61 82 6~ ~2 ~3 NCI6 9108 LMO 5 3 , I 1 38 69 90 9q 68 85 31 32 68 3q 35 83 55 89 78 91 61 62 q6 37 q7 79 33
Fig. l. Sorted and differentially shaded matrix of correlation coefficients based on the numerical analysis of protein electrophoregrams of 56 acetic acid bacteria isolated from bees and two culture collection strains of Gluconobacter oxydans NCIB 9108 and L M D 53.11.
GLUCONOBACTERS FROM HONEY BEES
153
The following features were uniformly present in all strains and are not mentioned in the table: no growth in yeast extract broth, no growth in Hoyerethanol medium, growth in the presence of 1 ~o ethanol; acid formation from Dglucose and D-xylose (pH < 4); oxidase negative; catalase positive; gelatinase negative; no reduction of nitrates,
Comparison of protein electrophoregrams of the isolates Protein electrophoregrams were prepared from all the isolates except n ~ 45. The reproducibility of the gel electrophoretic technique was checked by comparing electrophoregrams of soluble proteins from two independently grown cultures of the following isolates: n ~ 29, 31, 32, 33, 34, 80, 89 and 91. The correlation coefficients of 60 electrophoregrams from these 8 strains were computed. The lower limits of reproducibility were above r -~ 0.95 for each of these strains. The reproducibility levels of the protein electrophoregrams were thus sufficiently high, provided that standardized experimental conditions were used (Kersters and De Ley, 1975). The most typical electrophoregram of each strain (Swings et al., 1976) was used for the final clustering represented in Fig. 1 as a sorted and differentially shaded matrix of correlation coefficients. Normalized photographs of stained gels of 12 representative isolates and two culture collection strains of Gluconobacter oxydans (NCIB 9108 and L M D 53.11) are shown in Fig. 2. Two electrophoretically homogeneous groups A and B could be distinguished (Fig. 1 and 2) representing respectively 66 ~o and 27 ~o of the isolates investigated electrophoretically. Both groups were phenotypically indistinguishable with the tests used. The protein patterns of the following strains were different from those of clusters A and B and different from each other: n ~ 29, 33, 80 and 95 (Fig. 1 and 2). Phenotypic analysis indicated that strain n ~ 95 belongs to the genus
Acetobacter. The geographic distribution of the bee-keepers and the strains belonging to both electrophoretic groups is represented in Fig. 3. Strains belonging to group A were distributed randomly in the three areas investigated in northern Belgium. Strains of group B were isolated in Aalter and Zottegem but not in Neeroeteren (Limburg). In a second step we compared the electrophoregrams of the isolates from bees with those of more than 150 Glueonobacter and Acetobacter strains, which were mostly obtained from culture collections. The genus Glueonobacter consists of at least six electrophoretic groups and a number of ungrouped strains (Kersters and De Ley, unpublished results). The protein patterns of one of these electrophoretic groups of Gluconobacter were almost indistinguishable from the patterns of the bee isolates from group A. Two of these collection strains (NCIB 9108 and L M D 53.11), both isolated in the Netherlands, were included in the grouping represented in Fig. 1 and the photographs of Fig. 2. The strains of our group B constitute a new protein electrophoretic type within the genus Gluconobaeter.
154
B. LAMBERTET AL.
The p r o t e i n patterns o f isolate 95 displayed in its lower part a g r o u p of b a n d s characteristic for several strains o f A c e t o b a c t e r p a s t e u r i a n u s . The electrophoretic p r o t e i n p a t t e r n s of isolates n ~ 29, 33 a n d 80 occupied each a separate position.
Fig. 2. Normalized protein electrophoregrams of 11 Gluconobacter strains, isolated from bees, 2 cutture collection strains ofGluconobacter oxydans (NCIB 9108 and LMD 53.1I), and one Acetobacter strain (n~95) isolated from a wasp.
GLUCONOBACTERS FROM HONEY BEES
155
.
Fig. 3. Geographic distribution of the Gluconobacter isolates from bees in northern Belgium. The n u m b e r of strains belonging to the electrophoretic groups A and B are shown.
DISCUSSION
In the older literature Gluconobacter has never been mentioned to be associated with bees. Only Ruiz-Argfieso and Rodriguez-Navarro (1975) described it recently in Spain. Our data show that these bacteria can readily be isolated from honey bees in northern Belgium. We identified 8 5 ~ of our isolates as Gluconobacter sp. Our Gluconobacter strains, although phenotypically very similar, fell apart in two different electrophoretic groups A and B. The former group consisted of 37 strains and the latter of 15 strains. The application of gel electrophoresis of soluble proteins indicated that the same, or at least genetically closely related, Gluconobacter strains were present in different hives of three geographical regions in northern Belgium. Gluconobacter strains belonging to our major group A (Fig. 1) were also isolated from flowers (strains n ~ 57 and 92). The protein patterns of the strains of group A were almost identical to the electrophoregrams of several collection strains of G. oxydans (e.g. N C I B 9108 and L M D 53.11), which have been isolated over 20 years ago from beer in the Netherlands. The comparison of electrophoretic protein patterns indicated that genetically different Gluconobacter strains were present in some hives of the same region (Fig. 3), in several hives of the same bee keeper and sometimes in one hive. It is not clear whether the presence of Gluconobacter has any significance for the bee or any hive product. The bee acts as a vector in the dissemination of Gluconobacter on the hive, flowers and mummified or damaged apples. Passmore (1973) has shown that infections in cider manufactories, due to acetic
156
B. LAMBERT ET AL.
acid bacteria, originate from infected apples. She found no acetic acid bacteria o n h e a l t h y a p p l e s u n t i l t h e y w e r e h a r v e s t e d in S e p t e m b e r . T h e y w e r e p r e s e n t o n fallen fruit and flowers. Passmore (1973) suggested that acetic acid bacteria o v e r w i n t e r i n m u m m i f i e d a p p l e s . O u r d a t a s t r o n g l y s u g g e s t t h a t t h e h i v e is a suitable overwintering place for Gluconobacter. J . D . L . is i n d e b t e d t o t h e F o n d s v o o r K o l l e c t i e f F u n d a m e n t e e l O n d e r z o e k f o r a r e s e a r c h a n d p e r s o n n e l g r a n t . K . K . a n d J.S. a r e i n d e b t e d t o t h e N a t i o n a a l Fonds voor Wetenschappelijk Onderzoek for research grants and F.G. to the I n s t i t u u t t o t A a n m o e d i g i n g v a n h e t W e t e n s c h a p p e l i j k O n d e r z o e k in N i j v e r h e i d en Landbouw for a scholarship. R e c e i v e d 5 N o v e m b e r 1980.
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Zentralbl. Bakteriol. Abt. II. 123: 64-68. SWINGS, J. and DE LEy, J. 1981. The genera Gluconobacter and Acetobacter. In M. P. Starr, H. Stolp, H. G. Trfiper, A. Balows and H. G. Schlegel (eds), The prokaryotes. A handbook on habitats, isolation and identification of bacteria. - - Springer Verlag, Berlin. In press. SWINGS,J., GILLIS,M., KERSTERS,K., DE VOS, P., GOSSEL~, F. and DE LEY, J. 1980. Frateuria, a new genus for "'Acetobaeter aurantius". - - Int. J. Syst. Bacteriol. 30: 547-556. SWINGS, J., KERSTERS,K. and De LEY, J. 1976. Numerical analysis ofelectrophoretic protein patterns of Z y m o m o n a s strains. - - J. Gen. Microbiol. 93; 266-271. WISHARX, D. 1978. Clustan, User Manual (3rd ed.) - - Program Library Unit, Edinburgh University, Edinburgh.
