APPLiED
AND
ENVIRONMEZNTAL MICROBIOLOGY, Oct. 1977, p. 419-423
Copyright X) 1977 American Society for Microbiology
Vol. 34, No. 4 Printed in U.S.A.
Bile Salt Degradation by Nonfermentative Clostridia DAVID.E.
MAHONY,1 C. ELIZABETH MEIER,' IAN A. MACDONALD,l*
AND
LILLIAN V. HOLDEMAN2
Departments of Microbiology and Medicine, Dalhousie University, Halifax, Nova Scotia, Canada,' and The Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 240602
Received for publication 19 April 1977
Eight strains of nonfermentative clostridia were characterized on the basis of their intracellular nicotine adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-dependent hydroxysteroid dehydrogenase (HSDH) content, ability to deconjugate taurocholate, growth characteristics, and metabolic products, including utilization of lactate and pyruvate. Two cultures of Clostridium sporosphaeroides (representing one strain obtained from two different sources), one strain of Clostridium irregularis, four strains of an unnamed species (Clostridium group SPH-1), and one strain of an unnamed species (Clostridium group P) were studied. Both cultures of C. sporosphaeroides contained low amounts of 7a-HSDH; C. irregularis contained only a low amount of 3a-HSDH. All four strains of Clostridium SPH-1 contained both 12a- and 7a-HSDH in the ratio of approximately 10:1. The strain of Clostridium group P contained only 12a-HSDH and was devoid of any other bile salt oxidoreductases. The enzyme preparation from Clostridium group P was useful in spectrophotometric quantitative studies of 12a-OH groups. Correlation of bile salt degradative activities with other phenotypic tests for characterization of and differentiation among such organisms is discussed. A variety of common intestinal organisms is known to degrade bile salts (1, 2, 5). Two common types of degradative reactions are deconjugation [by bile salt hydrolase (EC 3.5-) as found in Clostridium perfringens (17) and Bacteroides fragilis (20)] and dehydrogenation of the 3a-, 7a-, and 12a-OH groups (2). Of the three oxidoreductases, 7a-hydroxysteroid dehydrogenase (7a-HSDH) (EC 1.1.1.159) appears to be the most common and is found in Escherichia coli (4, 12, 13) and B. fragilis (8, 15), whereas 3a-HSDH (EC 1.1.1.50) is found in C.) perfringens (11). Very few intestinal organisms, including selected strains of C. perfringens, have been reported to contain 12a-HSDH (2, 11) (EC 1.1.1._). In this communication we report the bile salt degradation patterns and other characteristics that appear useful in distinguishing four different species of nonfermentative clostridia. We document the presence of nicotinamide adenine dinucleotide phosphate (NADP)-dependent 12a-HSDH in an unnamed species of Clostridium (group P) and the absence of other group-specific dehydrogenases, as well as the use of this enzyme in the spectrophotometric quantitative measurements of 12a-OH groups.
2392, Novotny SZU 277/51). Strain 4527 was deposited with the American Type Culture Collection (ATCC 25781) as the reference strain for this species (Smith and Hobbs, 19). The culture received from ATCC was also used in this study. Clostridium group SPH-1 strains Cl-11, S7A-36, and 10903 were isolated by us from feces of clinically healthy humans. Strain 9869 was received by one of us (L.V.H.) from D. W. Lambe, Jr., and was one of several kinds of bacteria isolated from ascitic fluid from a patient with esophageal varices. Clostridium irregularis 4428 was a labeled reference strain obtained from A. R. Prevot, Paris; Pr& vot number 6 VI. The Clostridium group P strain, C48-50, was isolated from human feces at the Anaerobe Laboratory, Virginia Polytechnic Institute and State University (VPI). All strain numbers, except ATCC 25781, are those of the VPI Anaerobe Laboratory. Cultural procedures. Stock cultures were maintained in chopped meat medium as described by Holdeman and Moore (6). Cultures were characterized by using methods previously described (6). For enzyme studies, cultures were grown for 48 h at 37°C in 200 ml of freshly boiled and cooled brain heart infusion (BHI) broth (Difco) from a 10-ml starter inoculum. For determination of characteristic colonial morphology, the strains were grown on BHI agar with 10% human blood for 7 days in an anaerobe jar under an atmosphere of hydrogen and carbon dioxide provided by a GasPak (BBL) generator. Colonies were photographed for comparison.
MATERIALS AND METHODS Strains of clostridia. Clostridium sporosphaeroides 4527 was received from G. Hobbs (McClung 419
420
MAHONY ET AL.
Extraction and preparation of 3a-, 7a-, and 12aHSDH. The 48-h, 200-ml culture described above was centrifuged at 6,000 x g for 20 min at 4°C. The supernatant fluid was discarded and the pellet was suspended in 10 ml of 0.1 M sodium phosphate buffer (pH 7.0) and recentrifuged. The final sediment was resuspended in 15 ml of 0.1 M sodium phosphate buffer (pH 7.0) containing 10-3 M ethylenediaminetetraacetic acid. This suspension was sonicated at 4°C with a Fisher ultrasonic probe at 100 W for 4 min and then recentrifuged at 6,000 x g for 10 min to sediment the cell debris. The supernatant fluid was collected and assayed for 3a-, 7a-, and 12a-oxidoreductase activities. Assay for 3a-, 7a-, and 12a-HSDH. Assay conditions have been described in detail in earlier communications (11, 15). Substrates used were: 3a-,7adihydroxy-5,8-cholanoyl glycine, 10-3 M; 3a-,12adihydroxy-5,8-cholanoyl glycine 10-3 M; 3a-hydroxy5,8-cholanoyl taurine, 10-4 M; and 5a-androstan-3aol-17-one, 10-4 M. Cofactors used were: NAD and NADP, both 1.7 x 10-3 M. One unit is defined as the amount of enzyme required to yield 1 umol of NAD(P)H per minute using an extinction coefficient of 6.2 x 103 M-' cm-l for reduced nucleotide at 340 nm (7). Thin-layer chromatographic verification of group specificities of 3a-, 7a-, and 12a-OH oxidoreductases. Cell-free preparations (0.1 ml) were tested in a 3.0-ml-reaction mixture containing both substrate (3a-, 7a-dihydroxy-5,3-cholanate [CDC], 3a-, 12a-dihydroxy-5,3-cholanoate [DC], or 5a-androstan-3a-ol-17-one) and cofactor (as above, giving six possible combinations). After monitoring NAD(P)H generation at 340 nm until the absorbance exceeded 2.0, the reaction mixture was acidified to pH 3.0 and twice extracted with equal volumes of diethyl ether. The extract was evaporated to dryness under nitrogen and reconstituted in 0.3 ml of methanol-water (5:1 [vol/vol]). Twenty-five microliters of extract was spotted on thin-layer chromatography (TLC) plates and subjected to chromatography in three solvent systems: (A) chloroform-methanol-acetic acid (40:2:1 [vol/vol/vol], (B) chloroform-methanol-acetic acid (40:1:1 [vol/vol/ vol]) and (C) benzene-dioxane-acetic acid (75:20:2).
3a-Hydroxy-7-keto-5,8-cholanoate, 3a-hydroxy-12-
keto-5,8-cholanoate (3) and 5a-androstan-3,17-dione
(Steraloids) were used as standards. Plates were sprayed with p-hydroxybenzaldehyde reagent (9, 21) or phosphomolybdate and developed at 80 or 140°C, respectively. Quantiflcation of 12a-OH groups. Reaction mixtures (3.0 ml) consisting of 0.17 M glycine-NaOH, pH 9.5, 1.7 x 10-3 M NADP, and a variable concentration of bile salt (DC or 3ca-, 7a-, 12a-trihydroxy5/3-cholanoate [C]) were incubated with 100 ,ul of cell-free preparation of Clostridium group P, strain C48-50. The absorbance at 340 nm was monitored before enzyme addition and at subsequent 10-min intervals until no further increase was observed. Blanks were run in parallel. The initial absorbance was substracted from the final reading. Fifty microliters of a 10-mg/ml solution of purified Pseudomonas testosteroni 3a-HSDH was used in the tandem
APPL. ENVIRON. MICROBIOL.
oxidation studies. Assay for deconjugation. The organisms were grown in the presence of 5 x 10-4 M taurocholate in 10 ml of BHI broth for 48 h. The cells were centrifuged at 6,000 x g and the spent bacterial medium (SBM) was saved. Five milliliters of SBM was extracted as described above, reconstituted into a 0.5-ml volume, and subjected to TLC (solvent A). Taurocholate may be differentiated from cholate and other unconjugated degradation products by its relative non-extractability into the ether phase as well as by its lack of mobility on TLC, using solvent A. Additionally, 200 1,u of unextracted SBM was subjected to the direct chemical assay for deconjugation described earlier (10).
RESULTS AND DISCUSSION Colonial morphological characteristics of the strains studied suggested three groups: (i) C. sporosphaeroides, (ii) Clostridium group SPH1 and C. irregularis, and (iii) Clostridium group P (Fig. 1). Biochemical characterization data collected independently in three different laboratories and bile salt degradation data (Table 1) are in excellent agreement and appear to justify the grouping of these eight organisms into four distinct groups. On the basis of initial characterization studies, strains of Clostridium group SPH-1 were first identified as C. sporosphaeroides. Indeed, the Cl-11 strain was so designated previously (19). Like C. sporosphaeroides, it produced acetic, propionic, and butyric acids from peptone, stained gram negative, did not ferment any carbohydrates tested, was nonproteolytic, and was nonreactive in the other standard tests used. However, further study of these strains showed that the strains now designated Clostridium group SPH-1 did not produce hydrogen, converted pyruvate to butyrate, and three of four strains produced H2S in SIM (BBL) medium. C. sporosphaeroides produced abundant hydrogen, did not produce H2S, and did not convert pyruvate to butyrate. Rather, C. sporosphaeroides converted lactate to propionate, a relatively rare, but not unique, property. Strains of C. novyi, C. haemolyticum, C. botulinum types C and D, and Fusobacterium necrophorum also convert lactate to propionate (16). The differences in bile salt-degrading enzymes are additional indications that Cl-11, S7A-36, 9869, and 10903 are different from C. sporosphaeroides and should be assigned a new species name. It is interesting to note that all the organisms in the Clostridium group SPH-1 appear essentially identical, except for the 10903 strain, isolated in Nova Scotia, which appears different with respect to NAD-dependent 12a-HSDH and to deconjugation activity
FIG. 1. Colony morphology of eight strains of nonfermentative clostridia (x6.5). Morphological group I C. sporosphaeroides strains 4527 and ATCC 25781: irregular edge, flat colonies 1 mm in diameter; group II Clostridium group SPH-1 strains, C1-11,S7A-36, 9869, and 10903 and C. irregularis 4428: irregular edge, umbonate, 3 mm in diameter; group III Clostridium group P, strain C48-50 entire edge, convex, 0.5 mm in diameter. 421 =
=
422
MAHONY ET AL.
APPL. ENVIRON. MICROBIOL.
TABLE 1. Bile salt degradation and some other characteristics of strains of clostridia testeda Clostridium Characteristic
NADP/3a-HSDHb NADP/7a-HSDHb NADP/12a-HSDHb NAD/3a-HSDHb NAD/7a-HSDHb NAD/12a-HSDHb Deconjugation Growth in broth Butyric acid produced Propionate
AND
ENVIRONMEZNTAL MICROBIOLOGY, Oct. 1977, p. 419-423
Copyright X) 1977 American Society for Microbiology
Vol. 34, No. 4 Printed in U.S.A.
Bile Salt Degradation by Nonfermentative Clostridia DAVID.E.
MAHONY,1 C. ELIZABETH MEIER,' IAN A. MACDONALD,l*
AND
LILLIAN V. HOLDEMAN2
Departments of Microbiology and Medicine, Dalhousie University, Halifax, Nova Scotia, Canada,' and The Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 240602
Received for publication 19 April 1977
Eight strains of nonfermentative clostridia were characterized on the basis of their intracellular nicotine adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-dependent hydroxysteroid dehydrogenase (HSDH) content, ability to deconjugate taurocholate, growth characteristics, and metabolic products, including utilization of lactate and pyruvate. Two cultures of Clostridium sporosphaeroides (representing one strain obtained from two different sources), one strain of Clostridium irregularis, four strains of an unnamed species (Clostridium group SPH-1), and one strain of an unnamed species (Clostridium group P) were studied. Both cultures of C. sporosphaeroides contained low amounts of 7a-HSDH; C. irregularis contained only a low amount of 3a-HSDH. All four strains of Clostridium SPH-1 contained both 12a- and 7a-HSDH in the ratio of approximately 10:1. The strain of Clostridium group P contained only 12a-HSDH and was devoid of any other bile salt oxidoreductases. The enzyme preparation from Clostridium group P was useful in spectrophotometric quantitative studies of 12a-OH groups. Correlation of bile salt degradative activities with other phenotypic tests for characterization of and differentiation among such organisms is discussed. A variety of common intestinal organisms is known to degrade bile salts (1, 2, 5). Two common types of degradative reactions are deconjugation [by bile salt hydrolase (EC 3.5-) as found in Clostridium perfringens (17) and Bacteroides fragilis (20)] and dehydrogenation of the 3a-, 7a-, and 12a-OH groups (2). Of the three oxidoreductases, 7a-hydroxysteroid dehydrogenase (7a-HSDH) (EC 1.1.1.159) appears to be the most common and is found in Escherichia coli (4, 12, 13) and B. fragilis (8, 15), whereas 3a-HSDH (EC 1.1.1.50) is found in C.) perfringens (11). Very few intestinal organisms, including selected strains of C. perfringens, have been reported to contain 12a-HSDH (2, 11) (EC 1.1.1._). In this communication we report the bile salt degradation patterns and other characteristics that appear useful in distinguishing four different species of nonfermentative clostridia. We document the presence of nicotinamide adenine dinucleotide phosphate (NADP)-dependent 12a-HSDH in an unnamed species of Clostridium (group P) and the absence of other group-specific dehydrogenases, as well as the use of this enzyme in the spectrophotometric quantitative measurements of 12a-OH groups.
2392, Novotny SZU 277/51). Strain 4527 was deposited with the American Type Culture Collection (ATCC 25781) as the reference strain for this species (Smith and Hobbs, 19). The culture received from ATCC was also used in this study. Clostridium group SPH-1 strains Cl-11, S7A-36, and 10903 were isolated by us from feces of clinically healthy humans. Strain 9869 was received by one of us (L.V.H.) from D. W. Lambe, Jr., and was one of several kinds of bacteria isolated from ascitic fluid from a patient with esophageal varices. Clostridium irregularis 4428 was a labeled reference strain obtained from A. R. Prevot, Paris; Pr& vot number 6 VI. The Clostridium group P strain, C48-50, was isolated from human feces at the Anaerobe Laboratory, Virginia Polytechnic Institute and State University (VPI). All strain numbers, except ATCC 25781, are those of the VPI Anaerobe Laboratory. Cultural procedures. Stock cultures were maintained in chopped meat medium as described by Holdeman and Moore (6). Cultures were characterized by using methods previously described (6). For enzyme studies, cultures were grown for 48 h at 37°C in 200 ml of freshly boiled and cooled brain heart infusion (BHI) broth (Difco) from a 10-ml starter inoculum. For determination of characteristic colonial morphology, the strains were grown on BHI agar with 10% human blood for 7 days in an anaerobe jar under an atmosphere of hydrogen and carbon dioxide provided by a GasPak (BBL) generator. Colonies were photographed for comparison.
MATERIALS AND METHODS Strains of clostridia. Clostridium sporosphaeroides 4527 was received from G. Hobbs (McClung 419
420
MAHONY ET AL.
Extraction and preparation of 3a-, 7a-, and 12aHSDH. The 48-h, 200-ml culture described above was centrifuged at 6,000 x g for 20 min at 4°C. The supernatant fluid was discarded and the pellet was suspended in 10 ml of 0.1 M sodium phosphate buffer (pH 7.0) and recentrifuged. The final sediment was resuspended in 15 ml of 0.1 M sodium phosphate buffer (pH 7.0) containing 10-3 M ethylenediaminetetraacetic acid. This suspension was sonicated at 4°C with a Fisher ultrasonic probe at 100 W for 4 min and then recentrifuged at 6,000 x g for 10 min to sediment the cell debris. The supernatant fluid was collected and assayed for 3a-, 7a-, and 12a-oxidoreductase activities. Assay for 3a-, 7a-, and 12a-HSDH. Assay conditions have been described in detail in earlier communications (11, 15). Substrates used were: 3a-,7adihydroxy-5,8-cholanoyl glycine, 10-3 M; 3a-,12adihydroxy-5,8-cholanoyl glycine 10-3 M; 3a-hydroxy5,8-cholanoyl taurine, 10-4 M; and 5a-androstan-3aol-17-one, 10-4 M. Cofactors used were: NAD and NADP, both 1.7 x 10-3 M. One unit is defined as the amount of enzyme required to yield 1 umol of NAD(P)H per minute using an extinction coefficient of 6.2 x 103 M-' cm-l for reduced nucleotide at 340 nm (7). Thin-layer chromatographic verification of group specificities of 3a-, 7a-, and 12a-OH oxidoreductases. Cell-free preparations (0.1 ml) were tested in a 3.0-ml-reaction mixture containing both substrate (3a-, 7a-dihydroxy-5,3-cholanate [CDC], 3a-, 12a-dihydroxy-5,3-cholanoate [DC], or 5a-androstan-3a-ol-17-one) and cofactor (as above, giving six possible combinations). After monitoring NAD(P)H generation at 340 nm until the absorbance exceeded 2.0, the reaction mixture was acidified to pH 3.0 and twice extracted with equal volumes of diethyl ether. The extract was evaporated to dryness under nitrogen and reconstituted in 0.3 ml of methanol-water (5:1 [vol/vol]). Twenty-five microliters of extract was spotted on thin-layer chromatography (TLC) plates and subjected to chromatography in three solvent systems: (A) chloroform-methanol-acetic acid (40:2:1 [vol/vol/vol], (B) chloroform-methanol-acetic acid (40:1:1 [vol/vol/ vol]) and (C) benzene-dioxane-acetic acid (75:20:2).
3a-Hydroxy-7-keto-5,8-cholanoate, 3a-hydroxy-12-
keto-5,8-cholanoate (3) and 5a-androstan-3,17-dione
(Steraloids) were used as standards. Plates were sprayed with p-hydroxybenzaldehyde reagent (9, 21) or phosphomolybdate and developed at 80 or 140°C, respectively. Quantiflcation of 12a-OH groups. Reaction mixtures (3.0 ml) consisting of 0.17 M glycine-NaOH, pH 9.5, 1.7 x 10-3 M NADP, and a variable concentration of bile salt (DC or 3ca-, 7a-, 12a-trihydroxy5/3-cholanoate [C]) were incubated with 100 ,ul of cell-free preparation of Clostridium group P, strain C48-50. The absorbance at 340 nm was monitored before enzyme addition and at subsequent 10-min intervals until no further increase was observed. Blanks were run in parallel. The initial absorbance was substracted from the final reading. Fifty microliters of a 10-mg/ml solution of purified Pseudomonas testosteroni 3a-HSDH was used in the tandem
APPL. ENVIRON. MICROBIOL.
oxidation studies. Assay for deconjugation. The organisms were grown in the presence of 5 x 10-4 M taurocholate in 10 ml of BHI broth for 48 h. The cells were centrifuged at 6,000 x g and the spent bacterial medium (SBM) was saved. Five milliliters of SBM was extracted as described above, reconstituted into a 0.5-ml volume, and subjected to TLC (solvent A). Taurocholate may be differentiated from cholate and other unconjugated degradation products by its relative non-extractability into the ether phase as well as by its lack of mobility on TLC, using solvent A. Additionally, 200 1,u of unextracted SBM was subjected to the direct chemical assay for deconjugation described earlier (10).
RESULTS AND DISCUSSION Colonial morphological characteristics of the strains studied suggested three groups: (i) C. sporosphaeroides, (ii) Clostridium group SPH1 and C. irregularis, and (iii) Clostridium group P (Fig. 1). Biochemical characterization data collected independently in three different laboratories and bile salt degradation data (Table 1) are in excellent agreement and appear to justify the grouping of these eight organisms into four distinct groups. On the basis of initial characterization studies, strains of Clostridium group SPH-1 were first identified as C. sporosphaeroides. Indeed, the Cl-11 strain was so designated previously (19). Like C. sporosphaeroides, it produced acetic, propionic, and butyric acids from peptone, stained gram negative, did not ferment any carbohydrates tested, was nonproteolytic, and was nonreactive in the other standard tests used. However, further study of these strains showed that the strains now designated Clostridium group SPH-1 did not produce hydrogen, converted pyruvate to butyrate, and three of four strains produced H2S in SIM (BBL) medium. C. sporosphaeroides produced abundant hydrogen, did not produce H2S, and did not convert pyruvate to butyrate. Rather, C. sporosphaeroides converted lactate to propionate, a relatively rare, but not unique, property. Strains of C. novyi, C. haemolyticum, C. botulinum types C and D, and Fusobacterium necrophorum also convert lactate to propionate (16). The differences in bile salt-degrading enzymes are additional indications that Cl-11, S7A-36, 9869, and 10903 are different from C. sporosphaeroides and should be assigned a new species name. It is interesting to note that all the organisms in the Clostridium group SPH-1 appear essentially identical, except for the 10903 strain, isolated in Nova Scotia, which appears different with respect to NAD-dependent 12a-HSDH and to deconjugation activity
FIG. 1. Colony morphology of eight strains of nonfermentative clostridia (x6.5). Morphological group I C. sporosphaeroides strains 4527 and ATCC 25781: irregular edge, flat colonies 1 mm in diameter; group II Clostridium group SPH-1 strains, C1-11,S7A-36, 9869, and 10903 and C. irregularis 4428: irregular edge, umbonate, 3 mm in diameter; group III Clostridium group P, strain C48-50 entire edge, convex, 0.5 mm in diameter. 421 =
=
422
MAHONY ET AL.
APPL. ENVIRON. MICROBIOL.
TABLE 1. Bile salt degradation and some other characteristics of strains of clostridia testeda Clostridium Characteristic
NADP/3a-HSDHb NADP/7a-HSDHb NADP/12a-HSDHb NAD/3a-HSDHb NAD/7a-HSDHb NAD/12a-HSDHb Deconjugation Growth in broth Butyric acid produced Propionate
