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Taurine-reduction Powers Rapid Growth of Bilophila wadsworthia: A Liquid Minimal-salts Medium for Clinical Research?
Anaerobe (1999) 5, 115±117 Article No. anae.1999.0210
LABORATORY METHODOLOGY (ORAL PRESENTATION)
Taurine-reduction Powers Rapid Growth of Bilophila wadsworthia: A Liquid Minimal-salts Medium for Clinical Research? Heike Laue1*,Ulrike K. Schumacher2, and Alasdair M. Cook1 1
Department of Biology, University of Konstanz, Konstanz 2 Department of Medical Microbiology, Eberhard-Karls-University, TuÈbingen, Germany
Key Words: Bilophila wadsworthia, taurine, minimal medium, bile acids
Clinical isolates of Bilophila wadsworthia grew rapidly (1±3 days) in the liquid taurine-minimal-salts medium developed for B. wadsworthia RZATAU, whereas growth on Bacteroides Bile Esculin Agar takes up to 1 week. Though rapid growth of B. wadsworthia was achieved, and no other pure cultures grew, the medium was not selective for the organism in human faeces or in intra-abdominal specimens. We hope, however, that our understanding of the physiological and biochemical characteristics of the organism supplies a tool for further research.
Introduction Bilophila wadsworthia, an obligately anaerobic, asaccharolytic Gram-negative rod, was first isolated from perforated and gangrenous appendices [1]. The bacterium can also be recovered from other intraabdominal and extra-abdominal infections [2,3], including infection of the brain [4,5]. Routine isolation of B. wadsworthia involves Bacteroides Bile Esculin Agar. Growth is slow, requiring 4 to 7 days, and yields small, black-centred colonies. The requirement for bile [1] was narrowed to taurine-conjugated bile acids or
* Corresponding author. Heike Laue, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany. Tel.: 49 7531 88 4385; Fax: 49 7531 88 2966; E-mail: [email protected]
1075±9964/99/030115 + 03 $30.00/0
# 1999 Academic Press
taurine [6], one of the most abundant of the low molecular weight organic molecules in mammals [7]. The role of taurine in the nutrition of a bacterium, strain RZATAU, isolated from a sewage works, and later identified as a strain of B. wadsworthia, was elucidated by Laue et al. [8] (Figure 1). The compound functions as a source of sulphite in a sulphite respiration, i.e. energy conservation. The sulphite accepts electrons from formate, lactate, pyruvate or other compounds (cf. [8]). The respiration must involve an electron transport system, one of whose components requires the precursor naphthoquinone which is a growth requirement of this organism. We now find growth of clinical isolates of B. wadsworthia to be identical with that of strain RZATAU in minimal-medium, so we are exploring the application of the liquid taurine-minimal-salts medium for selective isolation of B. wadsworthia in a clinical laboratory. # 1999 Academic Press
116
H. Laue et al.
Figure 1. The degradative pathway of taurine in Bilophila wadsworthia. Four enzymes are involved in the reduction of taurine: a taurine: pyruvate aminotransferase (I), an alanine dehydrogenase (II), a sulphoacetaldehyde sulpholyase (III) and a sulphite reductase (IV). The electrons ([H]) utilized for the reduction of sulfite derive mainly from the oxidation of an external electron donor, e.g. formate.
Materials and Methods Cultures and growth medium Bilophila wadsworthia RZATAU [8] was used with nine clinical isolates: two strains (App 772 and App 796) were isolated from appendicitis; two (ST 749 and ST 767) from human faeces; two (PT 6523 and PT 6540) from peritonitis; one (TS 36223) from the trachea; one (Knoch 35420) from a wound; and one (Vag 37954) from a vaginal specimen. Bacteroides bile esculin (BBE) Agar was supplemented with 1% taurine. The liquid taurine-minimalsalts medium consisted of a basal salts medium representing freshwater which was prepared anoxically as described by Widdel and Pfenning [9]. The medium was buffered with 50 mM NaHCO3 (pH 7.0± 7.1), supplemented with 200 mg 1,4-naphthoquinone per litre, a selenite±tungsten solution, trace elements, vitamins (cf. [8]) and reduced with about 1 mM titanium(III) nitrilotriacetate. The sterile stock solution (pH 7.2) of the reductant contained about 100 mM titanium(III) chelated in about 150 mM nitrilotriacetate and about 600 mM NaCl, prepared by the method of Moench and Zeikus [10]. The medium was further supplemented with 200 mM sulphide as a sulphur source, 10 mM taurine as an electron acceptor and 60 mM formate as an electron donor. B. wadsworthia RZATAU was incubated under an atmosphere of N2 plus CO2 (80 : 20) at 308C whereas the clinical isolates were incubated at 378C.
formate and taurine. No significant differences among the clinical isolates and strain RZATAU were observed (Figure 2). The molar growth yields were in the same range as described for B. wadsworthia RZATAU [8]. In a growth experiment with strain App 796, the specific growth rate m (0.12 h71) was similar to that for strain RZATAU (m = 0.15 h71) [8]. There was no significant difference between growth at 308C or 378C. Pure cultures of Staphylococcus epidermidis, S. aureus, Streptococcus intermedius, Enterococcus faecalis, E. casseliflavus, Escherichia coli, Pantoea agglomerans, Citrobacter diversus, Klebsiella oxytoca, Serratia marcescens, Bacteroides thetaiotaomicron, B. vulgatus, B. distasonis, Propionibacterium acnes, Veillonella parvula, Actinomyces neuii, Lactobacillus sp., Proteus mirabilis, Shigella flexneri, Agrobacter radiobacter and Acinetobacter baumanii did not grow in the liquid taurine-minimal-salts medium. We compared growth of clinical specimens on rich (BBE agar) and in minimal, liquid medium
Analytical methods Determination of taurine, formate, acetate, ammonia, sulphide and protein has been described previously [8].
Results All nine clinical isolates grew in 1 to 3 days in the liquid taurine-minimal-salts medium and utilized
Figure 2. Utilization of taurine as an electron acceptor in the presence of formate as an electron donor, and formation of the metabolic products acetate, ammonia and sulphide by nine clinical isolates of Bilophila wadsworthia and by B. wadsworthia RZATAU.
Bilophila wadsworthia in a taurine-minimal-salts medium
117
Table 1. Comparison of growth of cultures obtained from human faeces and intra-abdominal specimens (mainly wounds and abscesses) on taurine-supplemented BBE agar and in liquid taurine-minimal-salts medium Growth observed/Bilophila wadsworthia Source of inoculum faeces Wounds and abscesses
No samples
Taurine-minimalsalts medium
BBE agar
20 69
20/8 4/1
7 1
(taurine-minimal-salts), in order to test whether the taurine-minimal-salts medium is suitable for selective isolation of B. wadsworthia for clinical investigations (Table 1). After growth in the liquid minimal medium the cultures were subcultured on taurine-supplemented BBE agar for identification of B. wadsworthia. All 20 samples from faeces grew in the minimal medium, but just eight of them contained B. wadsworthia. The other bacteria were not identified. In contrast, direct plating on BBE agar yielded only seven isolates of B. wadsworthia. Four of 69 cultures from the intraabdominal specimens grew in the liquid taurineminimal-salts medium. In addition to B. wadsworthia, one culture contained Bacteroides fragilis, Porphyromonas gingivalis, Eubacterium lentum, E. limosum, Peptostreptococcus anaerobius, Fusobacterium sp. and Clostridium clostridioforme. Two cultures comprised Proteus mirabilis and other unidentified bacteria. The fourth culture consisted of Escherichia coli. Direct cultivation of the specimens on BBE agar also yielded only one B. wadsworthia isolate.
Discussion The liquid taurine-minimal-salts medium is optimal for rapid growth of pure cultures of B. wadsworthia (Figure 2). However, although many pure cultures do not grow in this medium, it is not selective for isolation of B. wadsworthia from clinical specimens, especially not from faeces (Table 1). If the liquid taurine-minimal-salts medium can be modified to allow only B. wadsworthia to grow, this would be a powerful tool for selective isolation. We suspect that the growth in faecal samples is due to faecal nutrients, but we have not overcome the problem. Statistically, the intra-abdominal samples were less subject to error (Table 1), but here also we have not eliminated possible cross-feeding or the mere survival of other bacteria, rather than their growth.
Our better understanding of taurine metabolism in B. wadsworthia may allow studies of the pathogenic mechanism and importance of this organism in infections. We would also like to find out whether the presence of B. wadsworthia in infections correlates to the taurine concentration in the affected tissue.
Acknowledgements This research was supported in part by funds from the DFG.
References 1. Baron E.J., Summanen P., Downes J., Roberts M.C., Wexler H. and Finegold S.M. (1989) Bilophila wadsworthia, gen. nov. and sp. nov., a unique gram-negative anaerobic rod recovered from appendicitis specimens and human faeces. J Gen Microbiol 135: 3405±3411 2. Finegold S., Summanen P., Hunt Gerardo S. and Baron E. (1992) Clinical importance of Bilophila wadsworthia. Eur J Clin Microbiol Inf Dis 11: 1058±1063 3. Schumacher U. and BuÈcheler M. (1997) First isolation of Bilophila wadsworthia in otitis externa. HNO 45: 567±569 4. Marina M., Ivanova K., Ficheva M. and Fichev G. (1997) Bilophila wadsworthia in brain abscess: case report. Anaerobe 3: 107±109 5. Schumacher U.K., Bless D., Plinker P.K., Laue H. and Werner H. (1999) Bilophila wadsworthia in ear infections: report of three cases 6. Schumacher U.K., Lutz F. and Werner H. (1996) Taurine and taurine conjugated bile acids enhance growth of Bilophila wadsworthia. In 21st International Congress on Microbial Ecology and Disease, Paris 7. Huxtable R.J. (1992) Physiological actions of taurine. Physiol Rev 72: 101±163 8. Laue H., Denger K. and Cook A.M. (1997) Taurine reduction in anaerobic respiration of Bilophila wadsworthia RZATAU. Appl Environ Microbiol 63: 2016±2021 9. Widdel F. and Pfenning N. (1981) Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids. I. Isolation of new sulfate-reducing bacteria enriched with acetate from saline environments. Description of Desulfobacter postgatei gen. nov., sp. nov. Arch Microbiol 129: 395±400 10. Moench T.T. and Zeikus J.G. (1983) An improved preparation method for a titanium (III) media reductant. J Microbiol Meth 1: 199±202
LABORATORY METHODOLOGY (ORAL PRESENTATION)
Taurine-reduction Powers Rapid Growth of Bilophila wadsworthia: A Liquid Minimal-salts Medium for Clinical Research? Heike Laue1*,Ulrike K. Schumacher2, and Alasdair M. Cook1 1
Department of Biology, University of Konstanz, Konstanz 2 Department of Medical Microbiology, Eberhard-Karls-University, TuÈbingen, Germany
Key Words: Bilophila wadsworthia, taurine, minimal medium, bile acids
Clinical isolates of Bilophila wadsworthia grew rapidly (1±3 days) in the liquid taurine-minimal-salts medium developed for B. wadsworthia RZATAU, whereas growth on Bacteroides Bile Esculin Agar takes up to 1 week. Though rapid growth of B. wadsworthia was achieved, and no other pure cultures grew, the medium was not selective for the organism in human faeces or in intra-abdominal specimens. We hope, however, that our understanding of the physiological and biochemical characteristics of the organism supplies a tool for further research.
Introduction Bilophila wadsworthia, an obligately anaerobic, asaccharolytic Gram-negative rod, was first isolated from perforated and gangrenous appendices [1]. The bacterium can also be recovered from other intraabdominal and extra-abdominal infections [2,3], including infection of the brain [4,5]. Routine isolation of B. wadsworthia involves Bacteroides Bile Esculin Agar. Growth is slow, requiring 4 to 7 days, and yields small, black-centred colonies. The requirement for bile [1] was narrowed to taurine-conjugated bile acids or
* Corresponding author. Heike Laue, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany. Tel.: 49 7531 88 4385; Fax: 49 7531 88 2966; E-mail: [email protected]
1075±9964/99/030115 + 03 $30.00/0
# 1999 Academic Press
taurine [6], one of the most abundant of the low molecular weight organic molecules in mammals [7]. The role of taurine in the nutrition of a bacterium, strain RZATAU, isolated from a sewage works, and later identified as a strain of B. wadsworthia, was elucidated by Laue et al. [8] (Figure 1). The compound functions as a source of sulphite in a sulphite respiration, i.e. energy conservation. The sulphite accepts electrons from formate, lactate, pyruvate or other compounds (cf. [8]). The respiration must involve an electron transport system, one of whose components requires the precursor naphthoquinone which is a growth requirement of this organism. We now find growth of clinical isolates of B. wadsworthia to be identical with that of strain RZATAU in minimal-medium, so we are exploring the application of the liquid taurine-minimal-salts medium for selective isolation of B. wadsworthia in a clinical laboratory. # 1999 Academic Press
116
H. Laue et al.
Figure 1. The degradative pathway of taurine in Bilophila wadsworthia. Four enzymes are involved in the reduction of taurine: a taurine: pyruvate aminotransferase (I), an alanine dehydrogenase (II), a sulphoacetaldehyde sulpholyase (III) and a sulphite reductase (IV). The electrons ([H]) utilized for the reduction of sulfite derive mainly from the oxidation of an external electron donor, e.g. formate.
Materials and Methods Cultures and growth medium Bilophila wadsworthia RZATAU [8] was used with nine clinical isolates: two strains (App 772 and App 796) were isolated from appendicitis; two (ST 749 and ST 767) from human faeces; two (PT 6523 and PT 6540) from peritonitis; one (TS 36223) from the trachea; one (Knoch 35420) from a wound; and one (Vag 37954) from a vaginal specimen. Bacteroides bile esculin (BBE) Agar was supplemented with 1% taurine. The liquid taurine-minimalsalts medium consisted of a basal salts medium representing freshwater which was prepared anoxically as described by Widdel and Pfenning [9]. The medium was buffered with 50 mM NaHCO3 (pH 7.0± 7.1), supplemented with 200 mg 1,4-naphthoquinone per litre, a selenite±tungsten solution, trace elements, vitamins (cf. [8]) and reduced with about 1 mM titanium(III) nitrilotriacetate. The sterile stock solution (pH 7.2) of the reductant contained about 100 mM titanium(III) chelated in about 150 mM nitrilotriacetate and about 600 mM NaCl, prepared by the method of Moench and Zeikus [10]. The medium was further supplemented with 200 mM sulphide as a sulphur source, 10 mM taurine as an electron acceptor and 60 mM formate as an electron donor. B. wadsworthia RZATAU was incubated under an atmosphere of N2 plus CO2 (80 : 20) at 308C whereas the clinical isolates were incubated at 378C.
formate and taurine. No significant differences among the clinical isolates and strain RZATAU were observed (Figure 2). The molar growth yields were in the same range as described for B. wadsworthia RZATAU [8]. In a growth experiment with strain App 796, the specific growth rate m (0.12 h71) was similar to that for strain RZATAU (m = 0.15 h71) [8]. There was no significant difference between growth at 308C or 378C. Pure cultures of Staphylococcus epidermidis, S. aureus, Streptococcus intermedius, Enterococcus faecalis, E. casseliflavus, Escherichia coli, Pantoea agglomerans, Citrobacter diversus, Klebsiella oxytoca, Serratia marcescens, Bacteroides thetaiotaomicron, B. vulgatus, B. distasonis, Propionibacterium acnes, Veillonella parvula, Actinomyces neuii, Lactobacillus sp., Proteus mirabilis, Shigella flexneri, Agrobacter radiobacter and Acinetobacter baumanii did not grow in the liquid taurine-minimal-salts medium. We compared growth of clinical specimens on rich (BBE agar) and in minimal, liquid medium
Analytical methods Determination of taurine, formate, acetate, ammonia, sulphide and protein has been described previously [8].
Results All nine clinical isolates grew in 1 to 3 days in the liquid taurine-minimal-salts medium and utilized
Figure 2. Utilization of taurine as an electron acceptor in the presence of formate as an electron donor, and formation of the metabolic products acetate, ammonia and sulphide by nine clinical isolates of Bilophila wadsworthia and by B. wadsworthia RZATAU.
Bilophila wadsworthia in a taurine-minimal-salts medium
117
Table 1. Comparison of growth of cultures obtained from human faeces and intra-abdominal specimens (mainly wounds and abscesses) on taurine-supplemented BBE agar and in liquid taurine-minimal-salts medium Growth observed/Bilophila wadsworthia Source of inoculum faeces Wounds and abscesses
No samples
Taurine-minimalsalts medium
BBE agar
20 69
20/8 4/1
7 1
(taurine-minimal-salts), in order to test whether the taurine-minimal-salts medium is suitable for selective isolation of B. wadsworthia for clinical investigations (Table 1). After growth in the liquid minimal medium the cultures were subcultured on taurine-supplemented BBE agar for identification of B. wadsworthia. All 20 samples from faeces grew in the minimal medium, but just eight of them contained B. wadsworthia. The other bacteria were not identified. In contrast, direct plating on BBE agar yielded only seven isolates of B. wadsworthia. Four of 69 cultures from the intraabdominal specimens grew in the liquid taurineminimal-salts medium. In addition to B. wadsworthia, one culture contained Bacteroides fragilis, Porphyromonas gingivalis, Eubacterium lentum, E. limosum, Peptostreptococcus anaerobius, Fusobacterium sp. and Clostridium clostridioforme. Two cultures comprised Proteus mirabilis and other unidentified bacteria. The fourth culture consisted of Escherichia coli. Direct cultivation of the specimens on BBE agar also yielded only one B. wadsworthia isolate.
Discussion The liquid taurine-minimal-salts medium is optimal for rapid growth of pure cultures of B. wadsworthia (Figure 2). However, although many pure cultures do not grow in this medium, it is not selective for isolation of B. wadsworthia from clinical specimens, especially not from faeces (Table 1). If the liquid taurine-minimal-salts medium can be modified to allow only B. wadsworthia to grow, this would be a powerful tool for selective isolation. We suspect that the growth in faecal samples is due to faecal nutrients, but we have not overcome the problem. Statistically, the intra-abdominal samples were less subject to error (Table 1), but here also we have not eliminated possible cross-feeding or the mere survival of other bacteria, rather than their growth.
Our better understanding of taurine metabolism in B. wadsworthia may allow studies of the pathogenic mechanism and importance of this organism in infections. We would also like to find out whether the presence of B. wadsworthia in infections correlates to the taurine concentration in the affected tissue.
Acknowledgements This research was supported in part by funds from the DFG.
References 1. Baron E.J., Summanen P., Downes J., Roberts M.C., Wexler H. and Finegold S.M. (1989) Bilophila wadsworthia, gen. nov. and sp. nov., a unique gram-negative anaerobic rod recovered from appendicitis specimens and human faeces. J Gen Microbiol 135: 3405±3411 2. Finegold S., Summanen P., Hunt Gerardo S. and Baron E. (1992) Clinical importance of Bilophila wadsworthia. Eur J Clin Microbiol Inf Dis 11: 1058±1063 3. Schumacher U. and BuÈcheler M. (1997) First isolation of Bilophila wadsworthia in otitis externa. HNO 45: 567±569 4. Marina M., Ivanova K., Ficheva M. and Fichev G. (1997) Bilophila wadsworthia in brain abscess: case report. Anaerobe 3: 107±109 5. Schumacher U.K., Bless D., Plinker P.K., Laue H. and Werner H. (1999) Bilophila wadsworthia in ear infections: report of three cases 6. Schumacher U.K., Lutz F. and Werner H. (1996) Taurine and taurine conjugated bile acids enhance growth of Bilophila wadsworthia. In 21st International Congress on Microbial Ecology and Disease, Paris 7. Huxtable R.J. (1992) Physiological actions of taurine. Physiol Rev 72: 101±163 8. Laue H., Denger K. and Cook A.M. (1997) Taurine reduction in anaerobic respiration of Bilophila wadsworthia RZATAU. Appl Environ Microbiol 63: 2016±2021 9. Widdel F. and Pfenning N. (1981) Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids. I. Isolation of new sulfate-reducing bacteria enriched with acetate from saline environments. Description of Desulfobacter postgatei gen. nov., sp. nov. Arch Microbiol 129: 395±400 10. Moench T.T. and Zeikus J.G. (1983) An improved preparation method for a titanium (III) media reductant. J Microbiol Meth 1: 199±202