- Email: [email protected]
Bacteriology and nutrition of environmental strains of Klebsiella pneumoniae involved in wood and bark decay
Ann. Microbiol. (Inst. Pasteur) 1983, 134 A, 189-196
BACTERIOLOGY OF OF
NUTRITION
ENVIRONMENTAL
KLEBSIELLA IN
AND
STRAINS
PNEUMONIAE
WOOD
AND
BARK
INVOLVED
DECAY
by A. M. Deschamps (1), C. Richard (3) and J.-M. Lebeault (1) O) Division Procddds biotechnologiques, Universitd de Technologic, BP 253, 60206 Compi~gne Cedex (France), and (0 Inslilut Pasteur, Service des Enldrobactdries, 75724 Paris Cedex 15
SUMMARY Strains of Klebsiella pneumoniae were isolated from screening procedures for wood- and bark-decaying bacteria. Their bacteriological and biochemical properties, their sensitivity to antibiotics and assimilation of wood components were analysed. Most were pectlnolytic, degraded xylan and different phenolic compounds. Unusual associations of capsular polysaccharides and biotypes were observed. The nitrogen fixation ability of these strains was also tested. KEY-WORDS: Wood, Klebsiella pneumoniae; Environmental strains, Nutrition, Decay, Ecology.
INTRODUCTION Many strains of Enterobacteriaceae have been isolated during screenings for bacteria which degrade kraft-lignin [5], tannic acid [6], pine bark [7] and condensed tannins [9]. Most were identified as isolates of Klebsiella pneumoniae. The samples screened were from decaying or living wood and bark or composted wood [4]. Isolates of K. pneumoniae have been described several times in forest environments [2, 14], but the screening media we used provided important information on the nutritional capacities of these bacteria. As the strains we isolated were good degraders of lignin [5] and tannic acid [6], a survey of the biochemical, nutritional and bacteriological proM a n u s c r i t re~u le 8 juillet 1982, accept6 le 22 n o v e m b r e 1982.
190
Ao M. DESCHAMPS, C. RICHARD AND J.-M. LEBEAULT
perties of these strains was made, especially as regards their ability to grow on pectin, xylan, phenolic compounds, kraft-lignin, carboxymethylcellulose (CMC) and tannic acid. Capsular polysaccharides and nitrogen fixation were also investigated. P a r t of these results have previously been described for some strains in a different way [10].
M A T E R I A L S AND M E T H O D S
Strains. Sixteen strains of K. pneumoniae were isolated and identified. These strains were grouped into 9 different types (serotype and biotype) as some strains were probably the same. Representative strains of the 9 types were deposited in the ~ Collection de l'Institut Pasteur ~ (CIP), Paris, with references 80.41 to 80.49. The origin of each strain is listed in table I. A primary identification of these strains was realized with API-20E strips (API System, La Balme-les-Grottes, France) and was confirmed with conventional and particular methods, according to the recommendation of Edwards and Ewing [11], Le Minor [15] and Richard [18, 19, 20].
Bacleriological tests. Routine bacteriological tests were used for acetoin (VP), methyl-red, oxidase, KCN, citrate (Simmons), indole and motility [15]. Acid production from carbohydrates was tested in 1 % peptone water (w/v) at pH 7.4 with 12 ml per litre of bromothymol-blue solution (bromothymol-blue 1 g and NaOH 0.1 N, 25 ml added to 475 ml of water). The concentration of each carbohydrate tested was 1 % (w/v) [15]. The enzymes urease, lysine and ornithinedecarboxylase (ODC), arginine-dihydrolase, thiosulphate-reductase, gelatinase, ~-xylosidase and nitrate-reductase were detected according to previously detailed methods [20]. The degradation of d-tartrate and the fermentation of mucate were tested according to Le Minor [15].
Sensitivity to antibiotics. Routine antibiograms were performed with antibiotic discs (Institut Pasteur Production, Paris) including: penicillin, ampicillin, cephalotin, streptomycin, kanamycin, gentamicin, chloramphenicol, novobiocin, carbenicillin, colimycin, polymyxin B, nalidixic acid and trimethoprime-sulphamethoxazole.
Assimilation o] wood and vegetable components. A basal mineral medium previously described [5] was complemented with a sole carbon source including citrus pectin, xylan and carboxymethyl-cellulose (Sigma), kraft-lignin indulin AT (Westvaco Polychemicals, USA), protoeatechuic and tannic acids (Sigma) and gallic acid (Fluka ACT). Each compound was added to obtain a final concentration of 1 ~ (w/v). The degradation of cellulose was confirmed with the cellulose-azure test [7]. Abundant growth, as compared to the
CIP CMC ODC VP
= = : =
Collection Institut Pasteur, Paris. carboxymethyl-cellulose. ornithine-deearboxylase. Voges-Proskauer.
K. PNEUMONIAE ON WOOD AND BARK COMPONENTS
191
null or only slight growth of the control (lacking the carbon source) on the minimal medium was considered as positive assimilation. The degradation of metahydroxybenzoate was included in the bacteriological tests [16, 19].
Typing o[ capsular polgsaccharides. The standard procedure of Edwards and Ewing [11] was followed for preparing tile 72 anticapsular sera. The strains were cultivated in the Worfel-Ferguson medium [11] before typing in order to obtain satisfying capsules. The complete procedure has previously been described by Richard [18]. A suggested scheme of biotypes based on the dulcitol, sorbose and d-tartratc tests was also described. This classification of K. pneumoniae biotypes was used and detailed.
Assag /or nitrogen fixation. Nitrogenase activity of whole cells was assayed by the acetylene reduction test [17] under anaerobic conditions [13]. The procedure followed was described by Elmerich el at. [12]. Cultures in nitrogen-free medium were incubated for 16 h, and 1 ml acetylene was added to each 10-ml bottle. Ethylene produced was detected 1 h later with a 204 (( Pye Unicam)) gas chromatograph by injecting 0.25 ml of gas sample. RESULTS
Common bacteriological properties of the strains. The strains had the following common properties: glucose fermentation positive, non-motility, production of H~S negative, KCN test positive and oxidase negative. Acid from sugars was positive for every glucide tested: arabinose, cellobiose, glucose, glycerol, inositol, lactose, maltose, mannitol, melibiose, ~-methyl glucoside, raffinose, rhamnose, saccharose, salicine, sorbitol, sorbose, trehalose and xylose. The strains gave different results with adonitol and dulcitol. Metabolism of nitrogen compounds: each strain was positive for lysinedecarboxylase, but negative for arginine-dihydrolase, tryptophane-deaminase, indole and gelatinase. Assimilation of organic acids: each strain was positive for citrate (Simmons) and malonate. Enzymes: each strain was positive for nitratase and ~-xylosidase. All of the strains were highly sensitive to m a n y groups of the antibiotics tested except for carbenicillin and ampicillin.
Differential properties of strains. The 9 strains possessed 9 different capsular antigens (table I) but these types are not common and usually correspond to non-pathogenic strains. Four biotypes (Dd, d, Dc and c) based on the dulcitol, sorbose and tartrate tests [18] were observed (table I). The strains differed from each other when tested for the following bacteriological properties: urease, acetoin and methyl red tests, acid from dulcitol and adonitol, ODC, assimilation of metahydroxybenzoate, degraAnn. Microbiol. (lust. Pasteur), 134 A,
n ~ 2, 1983.
13
192
A . M . DESCHAMPS, C. RICHARD AND J.-M. LEBEAULT TABLE I. - - Origins and biotyping the <( K. p n e u m o n i a e >> strains.
Strain CIP
Capsular antigens
Biot y p e (*)
80.41 80.42 80.43 80.44 80.45 80.46 80.47 80.48 80.49
17 6 14 52 36 47 64 8 61
Dd d d d Dd Dd Dc d c
Origin
Reference
Fresh bark Decaying wood Decaying wood Soil Forest litter Decaying bark Decaying bark Decaying bark Decaying bark
[8, [5, [5, [6] [6] [91 [9] [9] [9]
10] s t r a i n B 1 2 10] s t r a i n 1.5 10] s t r a i n L 1 3 s t r a i n M21 s t r a i n M24
(*) A c c o r d i n g t o t h e c l a s s i f i c a t i o n o f R i c h a r d [18].
D i f f e r e n t i a l properties of the <( K. pneumoniae )> strains.
T A B L E l I. - -
Test/ClP
No.
Acetoin (VP) Adonitol Methyl red Duleitol MHB (m-hydroxy-benzoate) ODC D-tartrate Urease (Ferguson) Mueate N i t r o g e n a s e ( n i f +)
80.41
80.42
80.43
80.44
80.45
80.46
80.47
80.48
80.49
_ + § + -c -. -§ -
+ 4-, . --+ . -+ q-
+ ~-
+ --
+ --
+ +
+ -. + +
+ -}-
_L -
~-
4-
+ -
_a 7+ +
.
.
.
.
-+ . .
. + + .
.
--c .
§ -.
+ ~-
4+
. T -.
.
. .
.
.
4+
+ + § +
dation of d-tartrate and fermentation of mucate. These properties are shown in table II. All strains were positive for aeetoin and negative for m e t h y l red, except strain 80.41. Two strains were negative for adonitol and five were negative for duleitol. One strain (80.42) failed to grow on m e t a h y d r o x y b e n z o a t e b u t was positive for ODC. Only one strain was negative on mueate and two were d-tartrate-positive. Two strains were urease-negative. This variability for i m p o r t a n t bacteriological tests is discussed below.
Growth on wood and vegetable compounds. Some new and i m p o r t a n t data were obtained when screening for the nutritional properties of these strains of K. pneumoniae (table III). Each strain tested was able to grow on 6 9/o m e t h y l a t e d pectin as the sole carbon source, and 5 strains were able to grow with x y l a n as t h e sole carbon source. Only one strain produced a slight growth on CMC (strain
K. PNEUMONIAE ON WOOD AND BARK COMPONENTS TABLE III.
Strain
80.4l 80.42 80.43 80.44 80.45 80.46 80.47 80.48 80.49
Xylan
-~ + d+ + ---
193
Growth on wood and bark components.
Pectin
§ ~H-= § -§ +
CMC
--------
Tannic acid
+ + -4+ + + + + +
Gallic acid
Indulin (AT)
+ + -~ § + § + + +
+ + -P ------
Protocatechuic acid
§ A§ q"§ d§ +
80.48), b u t unfortunately this strain was negative using the more precise cellulose-azure test [7]. When tested for phenolic compounds, each strain was positive with gallic and protocatechuic acids, and only one strain failed to grow on tannic acid. Three strains gave a positive result with kraft-lignin as the sole carbon source [5].
Nilrogenase activity. Among these 9 strains, only 3 were positive for nitrogenase activity (strains 80.42, 80.47 and 80.49). These strains were from decaying bark and wood (tables I and II). Strain 80.42 was definitely superior in nitrogen fixation, and compared favorably to the K. pneumoniae strain M5al used for genetic experiments on nif genes [12].
DISCUSSION The environmental strains of K. pneumoniae studied here showed unusual associations of capsular antigens and biotypes. Among the collection of 900 strains of K. pneumoniae of the (( Institut Pasteur )) (Service des Ent6robact6ries), the associations 17Dd, 6d-ODC +, 14d and 8d-mucatehave never been described before, b u t the associations 52d and 36Dd were described for eight strains of clinical and environmental origin. The capsular polysaccharides of these strains correspond to non-pathogenic isolates, b u t environmental strains have sometimes been proved to be as potentially pathogenic as clinical strains [1]. The bacteriological properties of these strains were variable for different i m p o r t a n t characteristics (ODC, adonitol, dulcitol, mucate, acetoin and tartrate) as described before. This variability is also i m p o r t a n t in clinical isolates, as shown by Naemura el al. [16]. These authors classified the K. pneumoniae isolates into 5 groups, including an indole-positive group 1 considered to be the species K. oxgtoca. These authors included the degradation of pectin (sodium polypectate) in the
194
A . M . DESCHAMPS, C. RICHARD AND J.-M. LEBEAULT
bacteriologically i m p o r t a n t properties such as those described by Von Riesen [22]. In a previous paper [10] we demonstrated t h a t strains 80.42, 80.43, 80.44 and 80.45 were able to grow with polygalacturonic acid (sodium salt, Sigma), i. e. the same acid as t h a t used by Von 1Riesen. These strains were also able to grow on m e t h y l a t e d pectin [10]. In this paper we report the positive growth on citrus pectin for every strain. So we m a y conclude t h a t pectinolysis is not restricted to indole-positive strains, as was suggested by Von Riesen [22], but may also be described for indole-negative strains. This pectinolysis m a y also be generalized to highly m e t h y l a t e d pectins, polygalaeturonic acid [3] and crude citrus pectin. According to the classification of Naemura, strain 80.41 m a y be included as a true m e m b e r of group 5 whereas the other strains m a y be considered as members of group 4, but t h e latter strains are peetinolytic. The comprehensive results described here for the assimilation of wood and vegetable components show once again t h a t K. pneumoniae is very well adapted to botanical environments. As previously shown [10], strain 80.43 is able to grow on any component tested except cellulose. The fact t h a t these strains were mostly isolated from decaying bark m a y explain t h a t t h e y assimilate tannic, gallic and protocatechuic acids where these phenolic compounds are a b u n d a n t [5, 6, 8]. The ability of these strains to fix nitrogen is one more advantage not only for growth in nitrogen-deficient media such as inside living wood [2, 21] but also for growth as epiphytic bacteria. The nitrogenase activity of some strains tested is significant. The results reported in this paper give a deeper understanding of these ubiquitous bacteria which are able to assimilate several wood or vegetable components, sometimes in a better w a y t h a n purely saprophytic strains. One i m p o r t a n t fact remains, t h a t is the possibility for clinical strains to colonize botanical environments [14] and reciprocally, botanical strains m a y be pathogenic for mice [2] as well as for humans.
RESUME BACTI~RIOLOGIE ET NUTRITION DE DE
(( K L E B S I E L L A
SOUCHES ENVIRONNEMENTALES
PNEUMONIAE
ET D'ECORCE
EN
)) ISOLI~ES D E
BOIS
Dt~COMPOSITION
Des souches de Klebsiella pneumoniae ont ~t~ isol&s ~ diverses reprises, de bois et d'&orce en d&omposition, parmi divers types de bact~ries. Leurs caraet~res baet~riologiques et bioehimiques, leur sensibilit6 aux antibiotiques et l'assimilation de divers eomposants du bois et des 6corees comme seule source de carbone ont ~t~ analyses. La plupart des souches sont pectinolytiques et d~gradent le xylane et diff~rents compos~s ph6noliques. Des associations inhabituelles de groupes capsulaires et de biotypes ont ~t~ observ&s. Certaines de ces souches sont ~galement capables de fixer
K. P N E U M O N I A E ON WOOD AND BARK COMPONENTS
195
l'azote libre. Ces r6sultats eonfirment l'aptitude de K. pneumoniae i~ se d6velopper en milieu v6g~tal. MOTS-CLI~S : Bois, Klebsiella pneumoniae; Souehes environnementales, Nutrition, D6gradations, I~cologie.
ACKNOWLEDGEMENTS The authors acknowledge C. Elmerieh (Ddpartement de Biochimie et Gdndtique Mierobienne, Institut Pasteur de Paris) for the nitrogen reduction tests.
REFERENCES [1] BAGLEY,S. T. & SEIDLEB, R. J., Comparative pathogenicity of environmental and clinical Klebsiella. Hlth Lab. Sci., 1978, 15, 104-111. [2] BAGLEY, S. T., SEIDLER, R. J., TALBOT, H. W. & MORROW,J. E., Isolation of Klebsiellae within living wood. Appl. Environ. Microbiol., 1978, 36, 178-185. [3] CHATTERJEE, A. K., BUCHANAN, G. E., BEHRENS, M.K. & STARH, M. P., Synthesis and excretion of polygalacturonic acid trans-eliminase in Erwinia, Yersinia and Klebsiella species. Canad. J. Microbiol., 1979, 25, 94-102. [4] DESCHAMPS,A. M., HENNO, P., PERNELLE, C., CAIGNAULT,L. & LEBEAULT, J. M., Bench scale reactors for composting research. Biotech. Lelters, 1979, 1,239-244. [5] DESCRAMPS, A. M., MAHOUDEAU, G. & LEBEAULT, J. M., Fast degradation of kraft-lignin by bacteria. Europ. J. appl. Microbiol. Biolechnol., 1980, 9, 45-51. [6] DESCHAMPS,A. M., ~IAHOUDEAU,G., CONTI, M. & LEBEAULT, J. M., Bacteria degrading tannic acid and related compounds. J. Ferment. Technol., 1980, 58, 93-97. [7] DESCHAMPS,A. M. & LEBEAULT,J. M., Recherche de bactgries cellnlolytiques par la mfithode fi la cellulose-azure. Ann. Microbiol. (lust. Pasteur), 1980, 131 A, 77-81. [8] DESCHAMPS, A. M., MAHOUDEAU, G., LEULLIETTE, L. & LEBEAULT, J. M., Bark degradation and utilization by bacteria of various origins. Rev. Ecol. Biol. Sol, 1980, 17, 577-581. [9] DESCm~MPS, A.M. & LEBEAULT, J. M., Bacterial degradation of tannins, in ,< Advances in biotechnology )), (M. Moo-Young & C. W. Robinson), Vol. 2, (639-644). Pergamon Press, New York, 1981. [10] DrSCUAMPS, A. M. & LEBEAULT,J. M., Klebsiella pneumoniae as a potential pathogen proliferating in wood waste and lignin wastewater, in ((Advances in bioteehnology )), (M. Moo-Young & C. W. Robinson), Vol. 2, (659-662). Pergamon Press, New York, 1981. [11] EDWARDS,P. R. & EWING,N. H., Identification of Enterobacleriaceae. 3rd edition, (290-300). Burgess, Minneapolis, 1972. [12] ELMERICH, C., I-~OUMARD,J., SIBOLD, L., MANHEIMEH, 1. & CHARPIN, N., Genetic and biochemical analysis of mutants induced by bacteriophage Mu DNA integration into Klebsiella pneumoniae nitrogen fixation genes. Mol. gen. Genetics, 1978, 165, 181-189. [13] HILL, S., Influence of atmospheric oxygen concentration on acetylene reduction and efficiency of nitrogen fixation in intact Klebsiella pneumoniae. d. gen. Microbiol., 1976, 93, 335-345.
196
A.M.
DESCHAMPS, C. RICHARD AND J.-M. L E B E A U L T
[14] KNITTEL, M. D., SEIDLER, R. J., EBY, C. • CABE, L. M., Colonization of the botanical environment by Klebsiella isolated of pathogenic origin. Appl. Environ. Mierobiol., 1977, 34, 557-563. [15] LE MINOH, L., Le diagnostic de laboratoire de bacilles h Gram negatif. - - I. Ent@obactdries. I~ditions de la Tourelle, Saint-Mand6, 1972. [16] NAEMURA, L. G., BAGLEY, S. T., SEIDLER, R. J., KAPER, J. B. ~ COLWELL, R. R., Numerical taxonomy of Klebsiella pneumoniae strains isolated from clinical and non clinical sources. Curr. Mierobiol., 1979, 2, 175-180. [17] POSTGATE, J . R . , The acetylene reduction test for nitrogen fixation, in (( Methods in microbiology )), (J. R. Norris & D. W. Ribbons), Vol. 6 B, (343-356). Academic Press, London, New York, 1972. [18] I~ICHARD,C., Etude antig6nique et biochimique de 500 souches de Klebsiella. Ann. Biol. clin., 1973, 31, 295-303. [19] I-{ICHARD, C., Prdsence chez Enlerobacler aerogenes d'antig6nes capsulaires apparentds h ceux de Klebsiella : intdr~t de l'utilisation du metahydroxybenzoate dans le diagnostic diffdrentiel E. aerogenes-K, pneumoniae. Ann. Mierobiol. (Inst. Pasteur), 1977, 128 A, 289-295. [20] RICHAI~D, C., Techniques de recherche d'enzymes utiles au diagnostic de bacteries ~ Gram n6gatif. Ann. Biol. clin., 1978, 36, 407-424. [21] SEIDLEn, R. J., AHO, P. E., RAJU, P. N. & EVANS, H. J., Nitrogen fixation by bacterial isolates from decay in living white fir trees. J. gen. Microbioh, 1972, 73, 413-416. [22] Von I~IESEN, V. L., Pectinolytic, indole-positive strains of Klebsiella pneumoniae. Int. J. syst. Baet., 1976, 26, 143-145.
BACTERIOLOGY OF OF
NUTRITION
ENVIRONMENTAL
KLEBSIELLA IN
AND
STRAINS
PNEUMONIAE
WOOD
AND
BARK
INVOLVED
DECAY
by A. M. Deschamps (1), C. Richard (3) and J.-M. Lebeault (1) O) Division Procddds biotechnologiques, Universitd de Technologic, BP 253, 60206 Compi~gne Cedex (France), and (0 Inslilut Pasteur, Service des Enldrobactdries, 75724 Paris Cedex 15
SUMMARY Strains of Klebsiella pneumoniae were isolated from screening procedures for wood- and bark-decaying bacteria. Their bacteriological and biochemical properties, their sensitivity to antibiotics and assimilation of wood components were analysed. Most were pectlnolytic, degraded xylan and different phenolic compounds. Unusual associations of capsular polysaccharides and biotypes were observed. The nitrogen fixation ability of these strains was also tested. KEY-WORDS: Wood, Klebsiella pneumoniae; Environmental strains, Nutrition, Decay, Ecology.
INTRODUCTION Many strains of Enterobacteriaceae have been isolated during screenings for bacteria which degrade kraft-lignin [5], tannic acid [6], pine bark [7] and condensed tannins [9]. Most were identified as isolates of Klebsiella pneumoniae. The samples screened were from decaying or living wood and bark or composted wood [4]. Isolates of K. pneumoniae have been described several times in forest environments [2, 14], but the screening media we used provided important information on the nutritional capacities of these bacteria. As the strains we isolated were good degraders of lignin [5] and tannic acid [6], a survey of the biochemical, nutritional and bacteriological proM a n u s c r i t re~u le 8 juillet 1982, accept6 le 22 n o v e m b r e 1982.
190
Ao M. DESCHAMPS, C. RICHARD AND J.-M. LEBEAULT
perties of these strains was made, especially as regards their ability to grow on pectin, xylan, phenolic compounds, kraft-lignin, carboxymethylcellulose (CMC) and tannic acid. Capsular polysaccharides and nitrogen fixation were also investigated. P a r t of these results have previously been described for some strains in a different way [10].
M A T E R I A L S AND M E T H O D S
Strains. Sixteen strains of K. pneumoniae were isolated and identified. These strains were grouped into 9 different types (serotype and biotype) as some strains were probably the same. Representative strains of the 9 types were deposited in the ~ Collection de l'Institut Pasteur ~ (CIP), Paris, with references 80.41 to 80.49. The origin of each strain is listed in table I. A primary identification of these strains was realized with API-20E strips (API System, La Balme-les-Grottes, France) and was confirmed with conventional and particular methods, according to the recommendation of Edwards and Ewing [11], Le Minor [15] and Richard [18, 19, 20].
Bacleriological tests. Routine bacteriological tests were used for acetoin (VP), methyl-red, oxidase, KCN, citrate (Simmons), indole and motility [15]. Acid production from carbohydrates was tested in 1 % peptone water (w/v) at pH 7.4 with 12 ml per litre of bromothymol-blue solution (bromothymol-blue 1 g and NaOH 0.1 N, 25 ml added to 475 ml of water). The concentration of each carbohydrate tested was 1 % (w/v) [15]. The enzymes urease, lysine and ornithinedecarboxylase (ODC), arginine-dihydrolase, thiosulphate-reductase, gelatinase, ~-xylosidase and nitrate-reductase were detected according to previously detailed methods [20]. The degradation of d-tartrate and the fermentation of mucate were tested according to Le Minor [15].
Sensitivity to antibiotics. Routine antibiograms were performed with antibiotic discs (Institut Pasteur Production, Paris) including: penicillin, ampicillin, cephalotin, streptomycin, kanamycin, gentamicin, chloramphenicol, novobiocin, carbenicillin, colimycin, polymyxin B, nalidixic acid and trimethoprime-sulphamethoxazole.
Assimilation o] wood and vegetable components. A basal mineral medium previously described [5] was complemented with a sole carbon source including citrus pectin, xylan and carboxymethyl-cellulose (Sigma), kraft-lignin indulin AT (Westvaco Polychemicals, USA), protoeatechuic and tannic acids (Sigma) and gallic acid (Fluka ACT). Each compound was added to obtain a final concentration of 1 ~ (w/v). The degradation of cellulose was confirmed with the cellulose-azure test [7]. Abundant growth, as compared to the
CIP CMC ODC VP
= = : =
Collection Institut Pasteur, Paris. carboxymethyl-cellulose. ornithine-deearboxylase. Voges-Proskauer.
K. PNEUMONIAE ON WOOD AND BARK COMPONENTS
191
null or only slight growth of the control (lacking the carbon source) on the minimal medium was considered as positive assimilation. The degradation of metahydroxybenzoate was included in the bacteriological tests [16, 19].
Typing o[ capsular polgsaccharides. The standard procedure of Edwards and Ewing [11] was followed for preparing tile 72 anticapsular sera. The strains were cultivated in the Worfel-Ferguson medium [11] before typing in order to obtain satisfying capsules. The complete procedure has previously been described by Richard [18]. A suggested scheme of biotypes based on the dulcitol, sorbose and d-tartratc tests was also described. This classification of K. pneumoniae biotypes was used and detailed.
Assag /or nitrogen fixation. Nitrogenase activity of whole cells was assayed by the acetylene reduction test [17] under anaerobic conditions [13]. The procedure followed was described by Elmerich el at. [12]. Cultures in nitrogen-free medium were incubated for 16 h, and 1 ml acetylene was added to each 10-ml bottle. Ethylene produced was detected 1 h later with a 204 (( Pye Unicam)) gas chromatograph by injecting 0.25 ml of gas sample. RESULTS
Common bacteriological properties of the strains. The strains had the following common properties: glucose fermentation positive, non-motility, production of H~S negative, KCN test positive and oxidase negative. Acid from sugars was positive for every glucide tested: arabinose, cellobiose, glucose, glycerol, inositol, lactose, maltose, mannitol, melibiose, ~-methyl glucoside, raffinose, rhamnose, saccharose, salicine, sorbitol, sorbose, trehalose and xylose. The strains gave different results with adonitol and dulcitol. Metabolism of nitrogen compounds: each strain was positive for lysinedecarboxylase, but negative for arginine-dihydrolase, tryptophane-deaminase, indole and gelatinase. Assimilation of organic acids: each strain was positive for citrate (Simmons) and malonate. Enzymes: each strain was positive for nitratase and ~-xylosidase. All of the strains were highly sensitive to m a n y groups of the antibiotics tested except for carbenicillin and ampicillin.
Differential properties of strains. The 9 strains possessed 9 different capsular antigens (table I) but these types are not common and usually correspond to non-pathogenic strains. Four biotypes (Dd, d, Dc and c) based on the dulcitol, sorbose and tartrate tests [18] were observed (table I). The strains differed from each other when tested for the following bacteriological properties: urease, acetoin and methyl red tests, acid from dulcitol and adonitol, ODC, assimilation of metahydroxybenzoate, degraAnn. Microbiol. (lust. Pasteur), 134 A,
n ~ 2, 1983.
13
192
A . M . DESCHAMPS, C. RICHARD AND J.-M. LEBEAULT TABLE I. - - Origins and biotyping the <( K. p n e u m o n i a e >> strains.
Strain CIP
Capsular antigens
Biot y p e (*)
80.41 80.42 80.43 80.44 80.45 80.46 80.47 80.48 80.49
17 6 14 52 36 47 64 8 61
Dd d d d Dd Dd Dc d c
Origin
Reference
Fresh bark Decaying wood Decaying wood Soil Forest litter Decaying bark Decaying bark Decaying bark Decaying bark
[8, [5, [5, [6] [6] [91 [9] [9] [9]
10] s t r a i n B 1 2 10] s t r a i n 1.5 10] s t r a i n L 1 3 s t r a i n M21 s t r a i n M24
(*) A c c o r d i n g t o t h e c l a s s i f i c a t i o n o f R i c h a r d [18].
D i f f e r e n t i a l properties of the <( K. pneumoniae )> strains.
T A B L E l I. - -
Test/ClP
No.
Acetoin (VP) Adonitol Methyl red Duleitol MHB (m-hydroxy-benzoate) ODC D-tartrate Urease (Ferguson) Mueate N i t r o g e n a s e ( n i f +)
80.41
80.42
80.43
80.44
80.45
80.46
80.47
80.48
80.49
_ + § + -c -. -§ -
+ 4-, . --+ . -+ q-
+ ~-
+ --
+ --
+ +
+ -. + +
+ -}-
_L -
~-
4-
+ -
_a 7+ +
.
.
.
.
-+ . .
. + + .
.
--c .
§ -.
+ ~-
4+
. T -.
.
. .
.
.
4+
+ + § +
dation of d-tartrate and fermentation of mucate. These properties are shown in table II. All strains were positive for aeetoin and negative for m e t h y l red, except strain 80.41. Two strains were negative for adonitol and five were negative for duleitol. One strain (80.42) failed to grow on m e t a h y d r o x y b e n z o a t e b u t was positive for ODC. Only one strain was negative on mueate and two were d-tartrate-positive. Two strains were urease-negative. This variability for i m p o r t a n t bacteriological tests is discussed below.
Growth on wood and vegetable compounds. Some new and i m p o r t a n t data were obtained when screening for the nutritional properties of these strains of K. pneumoniae (table III). Each strain tested was able to grow on 6 9/o m e t h y l a t e d pectin as the sole carbon source, and 5 strains were able to grow with x y l a n as t h e sole carbon source. Only one strain produced a slight growth on CMC (strain
K. PNEUMONIAE ON WOOD AND BARK COMPONENTS TABLE III.
Strain
80.4l 80.42 80.43 80.44 80.45 80.46 80.47 80.48 80.49
Xylan
-~ + d+ + ---
193
Growth on wood and bark components.
Pectin
§ ~H-= § -§ +
CMC
--------
Tannic acid
+ + -4+ + + + + +
Gallic acid
Indulin (AT)
+ + -~ § + § + + +
+ + -P ------
Protocatechuic acid
§ A§ q"§ d§ +
80.48), b u t unfortunately this strain was negative using the more precise cellulose-azure test [7]. When tested for phenolic compounds, each strain was positive with gallic and protocatechuic acids, and only one strain failed to grow on tannic acid. Three strains gave a positive result with kraft-lignin as the sole carbon source [5].
Nilrogenase activity. Among these 9 strains, only 3 were positive for nitrogenase activity (strains 80.42, 80.47 and 80.49). These strains were from decaying bark and wood (tables I and II). Strain 80.42 was definitely superior in nitrogen fixation, and compared favorably to the K. pneumoniae strain M5al used for genetic experiments on nif genes [12].
DISCUSSION The environmental strains of K. pneumoniae studied here showed unusual associations of capsular antigens and biotypes. Among the collection of 900 strains of K. pneumoniae of the (( Institut Pasteur )) (Service des Ent6robact6ries), the associations 17Dd, 6d-ODC +, 14d and 8d-mucatehave never been described before, b u t the associations 52d and 36Dd were described for eight strains of clinical and environmental origin. The capsular polysaccharides of these strains correspond to non-pathogenic isolates, b u t environmental strains have sometimes been proved to be as potentially pathogenic as clinical strains [1]. The bacteriological properties of these strains were variable for different i m p o r t a n t characteristics (ODC, adonitol, dulcitol, mucate, acetoin and tartrate) as described before. This variability is also i m p o r t a n t in clinical isolates, as shown by Naemura el al. [16]. These authors classified the K. pneumoniae isolates into 5 groups, including an indole-positive group 1 considered to be the species K. oxgtoca. These authors included the degradation of pectin (sodium polypectate) in the
194
A . M . DESCHAMPS, C. RICHARD AND J.-M. LEBEAULT
bacteriologically i m p o r t a n t properties such as those described by Von Riesen [22]. In a previous paper [10] we demonstrated t h a t strains 80.42, 80.43, 80.44 and 80.45 were able to grow with polygalacturonic acid (sodium salt, Sigma), i. e. the same acid as t h a t used by Von 1Riesen. These strains were also able to grow on m e t h y l a t e d pectin [10]. In this paper we report the positive growth on citrus pectin for every strain. So we m a y conclude t h a t pectinolysis is not restricted to indole-positive strains, as was suggested by Von Riesen [22], but may also be described for indole-negative strains. This pectinolysis m a y also be generalized to highly m e t h y l a t e d pectins, polygalaeturonic acid [3] and crude citrus pectin. According to the classification of Naemura, strain 80.41 m a y be included as a true m e m b e r of group 5 whereas the other strains m a y be considered as members of group 4, but t h e latter strains are peetinolytic. The comprehensive results described here for the assimilation of wood and vegetable components show once again t h a t K. pneumoniae is very well adapted to botanical environments. As previously shown [10], strain 80.43 is able to grow on any component tested except cellulose. The fact t h a t these strains were mostly isolated from decaying bark m a y explain t h a t t h e y assimilate tannic, gallic and protocatechuic acids where these phenolic compounds are a b u n d a n t [5, 6, 8]. The ability of these strains to fix nitrogen is one more advantage not only for growth in nitrogen-deficient media such as inside living wood [2, 21] but also for growth as epiphytic bacteria. The nitrogenase activity of some strains tested is significant. The results reported in this paper give a deeper understanding of these ubiquitous bacteria which are able to assimilate several wood or vegetable components, sometimes in a better w a y t h a n purely saprophytic strains. One i m p o r t a n t fact remains, t h a t is the possibility for clinical strains to colonize botanical environments [14] and reciprocally, botanical strains m a y be pathogenic for mice [2] as well as for humans.
RESUME BACTI~RIOLOGIE ET NUTRITION DE DE
(( K L E B S I E L L A
SOUCHES ENVIRONNEMENTALES
PNEUMONIAE
ET D'ECORCE
EN
)) ISOLI~ES D E
BOIS
Dt~COMPOSITION
Des souches de Klebsiella pneumoniae ont ~t~ isol&s ~ diverses reprises, de bois et d'&orce en d&omposition, parmi divers types de bact~ries. Leurs caraet~res baet~riologiques et bioehimiques, leur sensibilit6 aux antibiotiques et l'assimilation de divers eomposants du bois et des 6corees comme seule source de carbone ont ~t~ analyses. La plupart des souches sont pectinolytiques et d~gradent le xylane et diff~rents compos~s ph6noliques. Des associations inhabituelles de groupes capsulaires et de biotypes ont ~t~ observ&s. Certaines de ces souches sont ~galement capables de fixer
K. P N E U M O N I A E ON WOOD AND BARK COMPONENTS
195
l'azote libre. Ces r6sultats eonfirment l'aptitude de K. pneumoniae i~ se d6velopper en milieu v6g~tal. MOTS-CLI~S : Bois, Klebsiella pneumoniae; Souehes environnementales, Nutrition, D6gradations, I~cologie.
ACKNOWLEDGEMENTS The authors acknowledge C. Elmerieh (Ddpartement de Biochimie et Gdndtique Mierobienne, Institut Pasteur de Paris) for the nitrogen reduction tests.
REFERENCES [1] BAGLEY,S. T. & SEIDLEB, R. J., Comparative pathogenicity of environmental and clinical Klebsiella. Hlth Lab. Sci., 1978, 15, 104-111. [2] BAGLEY, S. T., SEIDLER, R. J., TALBOT, H. W. & MORROW,J. E., Isolation of Klebsiellae within living wood. Appl. Environ. Microbiol., 1978, 36, 178-185. [3] CHATTERJEE, A. K., BUCHANAN, G. E., BEHRENS, M.K. & STARH, M. P., Synthesis and excretion of polygalacturonic acid trans-eliminase in Erwinia, Yersinia and Klebsiella species. Canad. J. Microbiol., 1979, 25, 94-102. [4] DESCHAMPS,A. M., HENNO, P., PERNELLE, C., CAIGNAULT,L. & LEBEAULT, J. M., Bench scale reactors for composting research. Biotech. Lelters, 1979, 1,239-244. [5] DESCRAMPS, A. M., MAHOUDEAU, G. & LEBEAULT, J. M., Fast degradation of kraft-lignin by bacteria. Europ. J. appl. Microbiol. Biolechnol., 1980, 9, 45-51. [6] DESCHAMPS,A. M., ~IAHOUDEAU,G., CONTI, M. & LEBEAULT, J. M., Bacteria degrading tannic acid and related compounds. J. Ferment. Technol., 1980, 58, 93-97. [7] DESCHAMPS,A. M. & LEBEAULT,J. M., Recherche de bactgries cellnlolytiques par la mfithode fi la cellulose-azure. Ann. Microbiol. (lust. Pasteur), 1980, 131 A, 77-81. [8] DESCHAMPS, A. M., MAHOUDEAU, G., LEULLIETTE, L. & LEBEAULT, J. M., Bark degradation and utilization by bacteria of various origins. Rev. Ecol. Biol. Sol, 1980, 17, 577-581. [9] DESCm~MPS, A.M. & LEBEAULT, J. M., Bacterial degradation of tannins, in ,< Advances in biotechnology )), (M. Moo-Young & C. W. Robinson), Vol. 2, (639-644). Pergamon Press, New York, 1981. [10] DrSCUAMPS, A. M. & LEBEAULT,J. M., Klebsiella pneumoniae as a potential pathogen proliferating in wood waste and lignin wastewater, in ((Advances in bioteehnology )), (M. Moo-Young & C. W. Robinson), Vol. 2, (659-662). Pergamon Press, New York, 1981. [11] EDWARDS,P. R. & EWING,N. H., Identification of Enterobacleriaceae. 3rd edition, (290-300). Burgess, Minneapolis, 1972. [12] ELMERICH, C., I-~OUMARD,J., SIBOLD, L., MANHEIMEH, 1. & CHARPIN, N., Genetic and biochemical analysis of mutants induced by bacteriophage Mu DNA integration into Klebsiella pneumoniae nitrogen fixation genes. Mol. gen. Genetics, 1978, 165, 181-189. [13] HILL, S., Influence of atmospheric oxygen concentration on acetylene reduction and efficiency of nitrogen fixation in intact Klebsiella pneumoniae. d. gen. Microbiol., 1976, 93, 335-345.
196
A.M.
DESCHAMPS, C. RICHARD AND J.-M. L E B E A U L T
[14] KNITTEL, M. D., SEIDLER, R. J., EBY, C. • CABE, L. M., Colonization of the botanical environment by Klebsiella isolated of pathogenic origin. Appl. Environ. Mierobiol., 1977, 34, 557-563. [15] LE MINOH, L., Le diagnostic de laboratoire de bacilles h Gram negatif. - - I. Ent@obactdries. I~ditions de la Tourelle, Saint-Mand6, 1972. [16] NAEMURA, L. G., BAGLEY, S. T., SEIDLER, R. J., KAPER, J. B. ~ COLWELL, R. R., Numerical taxonomy of Klebsiella pneumoniae strains isolated from clinical and non clinical sources. Curr. Mierobiol., 1979, 2, 175-180. [17] POSTGATE, J . R . , The acetylene reduction test for nitrogen fixation, in (( Methods in microbiology )), (J. R. Norris & D. W. Ribbons), Vol. 6 B, (343-356). Academic Press, London, New York, 1972. [18] I~ICHARD,C., Etude antig6nique et biochimique de 500 souches de Klebsiella. Ann. Biol. clin., 1973, 31, 295-303. [19] I-{ICHARD, C., Prdsence chez Enlerobacler aerogenes d'antig6nes capsulaires apparentds h ceux de Klebsiella : intdr~t de l'utilisation du metahydroxybenzoate dans le diagnostic diffdrentiel E. aerogenes-K, pneumoniae. Ann. Mierobiol. (Inst. Pasteur), 1977, 128 A, 289-295. [20] RICHAI~D, C., Techniques de recherche d'enzymes utiles au diagnostic de bacteries ~ Gram n6gatif. Ann. Biol. clin., 1978, 36, 407-424. [21] SEIDLEn, R. J., AHO, P. E., RAJU, P. N. & EVANS, H. J., Nitrogen fixation by bacterial isolates from decay in living white fir trees. J. gen. Microbioh, 1972, 73, 413-416. [22] Von I~IESEN, V. L., Pectinolytic, indole-positive strains of Klebsiella pneumoniae. Int. J. syst. Baet., 1976, 26, 143-145.