Identi¢cation of Bilophila wadsworthia by speci¢c PCR which targets the taurine:pyruvate aminotransferase gene
Heike Laue1, Theo H. M. Smits1, Ulrike K. Schumacher2, Marina C. Claros3, Ralf Hartemink4&
Alasdair M. Cook1
1Department of Biological Sciences, University of Konstanz, Konstanz, Germany;2Institute for Medical Microbiology & Hygiene, Eberhard- Karls-University, T ¨ubingen, Germany;3Institute of Medical Microbiology and Infectious Epidemiology, University of Leipzig, Leipzig, Germany;
and4Department of Food Technology and Nutritional Sciences, Wageningen University and Research Centre, Wageningen, The Netherlands
Correspondence:Theo H. M. Smits, Department of Biological Sciences, University of Konstanz, D-78457 Konstanz, Germany.
Tel.:149 7531 883270; fax:149 7531 882966; e-mail: theo.smits@uni-konstanz.de
Present address:Heike Laue, Arpida Ltd., Dammstrasse 36, CH-4142 M ¨unchenstein, Switzerland.
Present address:Marina C. Claros, Viollier AG, Spalenring 145/147, Postfach, CH-4002, Basel, Switzerland.
Editor: William Wade
Keywords
Bilophila wadsworthia isolation; taurine dissimilation; taurine:pyruvate aminotransferase.
Abstract
The bile-resistant, strictly anaerobic bacteriumBilophila wadsworthiais found in human faecal flora, in human infections and in environmental samples. A specific PCR primer set for the gene encoding the first metabolic enzyme in the degradative pathway for taurine inB. wadsworthia, taurine:pyruvate aminotransferase (tpa), was developed and tested. In addition, enrichment cultures were started from faecal samples of primates and felines and shown to containB. wadsworthia. These were subcultured on agar media and then identified by PCR fingerprinting. PCR for tpa was successful in all positive enrichment cultures and showed no amplification signal in a variety of other bacterial species. Therefore, this PCR method could be a promising tool for rapid detection of B. wadsworthia in biological samples.
Introduction
The strictly anaerobic bacterium Bilophila wadsworthiahas been recovered from a variety of intra-abdominal infections, especially appendicitis, and extraabdominal infections (Baron et al., 1992; Finegoldet al., 1992; Rautioet al., 2000), as well as human and pig faeces (Baronet al., 1989; Schumacheret al., 1997; McOristet al., 2001), anaerobic digestors of communal wastewater treatment plants (Laueet al., 1997) and pristine lake sediments (K. Denger and A.M. Cook, unpublished).
Bilophila wadsworthiawas named for its apparent requirement for 20% bile in the growth medium (Baronet al., 1989). Bile could be replaced by taurine (2-aminoethanesulphonate)- conjugated bile acids (Schumacheret al., 1996), and growth with taurine was elucidated as an anaerobic respiration (Laue et al., 1997). Bilophila wadsworthia conserves energy by
sulphite reduction, with taurine as a source of sulphite, in combination with organic (e.g. formate) or inorganic elec- tron donors (Laueet al., 2001). Taurine is usually the most abundant low-molecular-weight organic compound in mammals (Huxtable, 1992), and one of its many functions is conjugation with bile acids to form bile salts.
At least six enzymes are involved in the formation of the metabolic end products (ammonium, acetate and sulphide ions) during the degradation of taurine byB. wadsworthia (Laueet al., 1997, 1999). Taurine-pyruvate aminotransferase (Tpa) [EC 2.6.1.77] catalyzes transamination of taurine to sulphoacetaldehyde and alanine (Laue & Cook, 2000b).
Alanine dehydrogenase [EC 1.4.1.4] regenerates pyruvate and releases the ammonium ion (Laue & Cook, 2000a). A sulphoacetaldehyde acetyltransferase [EC 2.3.3.15] is pre- sumed to generate acetyl phosphate and release sulphite
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(Ruffet al., 2003). Dissimilatory sulphite reductase (DSR, encoded bydsrABC) [EC 1.8.99.3] generates sulphide from sulphite (Laueet al., 2001). A phosphate acetyltransferase [EC 2.3.1.8], to yield acetyl-CoA for biosynthesis, and an acetate kinase [EC 2.7.2.1] to yield ATP (and acetate) (Cook & Denger, 2002) have been detected at high levels (K. Denger & A.M. Cook, unpublished data). Just as the presence of taurine is widespread, its utilization by bacteria is also widespread, and many organisms initiate dissimila- tion via Tpa (Cook & Denger, 2002; Dengeret al., 2004;
Gorzynskaet al., 2006). Further, anaerobic bacteria seem to initiate the assimilation of sulphur from taurine via Tpa (Chienet al., 1997; Masepohlet al., 2001).
There is very little gene-specific sequence information available to identifyB. wadsworthia. The DSR shares more than 80% amino acid identity with other DSRs (Laueet al., 2001), and the alanine dehydrogenase shares more than 60%
identity with other alanine dehydrogenases (Laue & Cook, 2000a). In contrast, Tpa usually has low similarity to other aminotransferases with known function (Laue & Cook, 2000b; Supplementary Fig. S1). Genome sequence data reveal that the Tpa from Rhodobacter capsulatus SB1003, involved in the assimilation of sulphonate sulphur (Mase- pohlet al., 2001), shares 58% identity with the Tpa of B.
wadsworthia. The highest level of amino acid sequence identity (around 60%) is to hypothetical proteins from the (unfinished) genome sequences of three strains ofR. sphaer- oides(strains 2.4.1, ATCC 17025 and ATCC 17029). At least one of these does not have an active Tpa when the strain is growing on taurine as sole carbon or nitrogen source (T.H.M. Smits, K. Denger & A.M. Cook, unpublished data).
Thetpagene thus seems to be a suitable candidate for the specific identification ofB. wadsworthia. To test this possi- bility, and to investigate the general abundance of both B. wadsworthiaand itstpagene, a specific PCR primer set for the tpa gene of B. wadsworthia was evaluated for its specificity. Enrichments with taurine started from faecal samples of several felines and primates containedB. wads- worthia, and their presence was confirmed by specific PCR for thetpagene anddsrABgenes.
Materials and methods
Growth media and enrichment cultures
The freshwater mineral salts medium of Widdel & Pfennig (1981) was used as the basis for the anoxic media. Enrich- ment cultures for, and pure cultures ofB. wadsworthiawere incubated in medium with 10 mM taurine and 60 mM sodium formate. All liquid cultures were incubated at 301C under an atmosphere of N2 plus CO2 (80 : 20) in serum bottles sealed with butyl rubber septa.
Faecal samples for enrichment cultures were collected from eight species of primates and five species of felines (Table 1) at several zoos in The Netherlands, and two samples of sheep faeces were collected near Konstanz. Faeces (10–25 g) were suspended in 90 mL buffered peptone water (Oxoid) supplemented with cysteine HCl (0.5 g L1) and resazurine, and homogenized in an anoxic chamber. The flask was filled with sterile glycerol (final concentration about 50%) and stored at 801C before use.
Table 1.Enrichment ofBilophila wadsworthiafrom animal faeces, and detection oftpaanddsrgenes in the enrichment cultures
Source of inoculum Growth Sulphidew tpaz dsr‰
Celebes Macaque Macaca nigra 1 1 1 1
Chimpanzee Pan troglodytes 1 11 1 1
Galago Galagosp. 11 11 1 1
Gorilla Gorilla gorilla 11 11 1 1
Lesser Mouse Lemur Microcebus murinus ND ND
Mangabey Cercocebus totquates 11 11 1 1
Siamang Hylobates syndactulus 11 11 1 1
Orang-utan Pongo pygmaeus 11 11 1 1
Bobcat Lynx lynx 11 11 1 1
Cheetah Acinonyx jubatus 11 11 1 1
Panther Panthera pardus 11 11 1 1
Tiger Panthera tigrus 11 11 1 1
Lion Panthera leo ND ND
Sheep Ovis aries ND ND
Growth was monitored as turbidity:11, strong growth;1, significant growth;, no growth.
wSulphide formation:11, large amounts;1, significant amounts;, none detected.
zAmplification bytpa-specific PCR of a 1.0 kb DNA fragment.
‰Partial gene fragments of the dissimilatory sulphite reductase genesdsrABwere detected by the 1.9 kb amplification product obtained with the consensus PCR primers DSR1F and DSR4R (Wagneret al., 1998).
ND, not determined.
For the initial enrichment cultures, about 10% (v/v) inoculum was used. Enrichment cultures were transferred to fresh medium after 5 days for the initial cultures, and after 2–4 days for later cultures. Pure cultures were isolated from the fourth transfer of the enrichment cultures by serial streaking the cultures on Bacteroides Bile Esculin (BBE) agar plates, which were supplemented with 1% taurine and incubated under anoxic conditions at 371C.
Bacteria
The following pure cultures ofB. wadsworthiawere used in addition to the new isolates: the type strain (ATCC 49260) (Baronet al., 1989), two environmental isolates, RZATAU (DSM 11045) and KNATAU (Laue et al., 1997), and 11 clinical isolates (Claroset al., 1999) recovered from blood (one), appendicitis (two), abdominal specimens (two), hu- man faeces (one), the trachea (one), ulcus cruris (one), a vaginal specimen (one), peritonitis (one), and an ear abscess (one). A further 14 characterized pure cultures and their growth characteristics are cited in the supplementary data (Supplementary Table S1). A set of 19 clinical isolates was used as a suite of controls: Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Bifidobacterium breve, Clostridium perfringens, Enterococcus faecalis, Escherichia coli, Fusobacterium nucleatum, Klebsiella pneumoniae, Lactobacil- lus acidophilus, Peptostreptococcus anaerobius, Finegoldia mag- na(formerlyPeptostreptococcus magnus), Micromonas micros (formerlyPeptostreptococcus micros), Prevotella bivia, Prevo- tella intermedia, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureusandStreptococcus pyogenes.
Analytical methods
Taurine was quantified by high-performance liquid chroma- tography after derivatization with 2,4-dinitrofluorobenzene (Denger et al., 1997). Sulphide was detected by the formation of a brown precipitate with copper salts (Widdel
& Bak, 1992).
Molecular methods
Total DNA was prepared from pure and enrichment cultures according to the cetyltrimethylammonium bromide preci- pitation method (Ausubelet al., 1987). For the 19 clinical strains, genomic DNA was isolated from the Gram-negative bacteria by alkaline lysis, and from the Gram-positive bacteria as described elsewhere (Bolletet al., 1991).
Specific PCR primers were derived from thetpagene of B. wadsworthiaRZATAU (Laue & Cook, 2000b) (accession no. AF269146) to amplify a large portion (1003 bp) of the gene (1371 bp): TPA-F, 50-caacgtccccaccatcaagttctctg-30, and TPA-R, 50-tgaattcgcggaaggagcgagaggtc-30. PCR amplifica- tion was carried out in a Master Cycler gradient thermo-
cycler (Eppendorf). The reaction mixture (20mL) contained 2.25 mM MgCl2, 50 mM Tris-HCl, pH 9.2, 14 mM (NH4)2SO4, 10% dimethyl sulphoxide, 0.5 mM dNTPs, 0.3mM of each primer, 1 U Taq polymerase (MBI Fermen- tas), and 2mL template. Washed and concentrated cells as template were added directly to PCR reaction mixtures instead of purified DNA, where the simplification was effective.
The PCR primer pair DSR1F and DSR4R (Wagneret al., 1998) was used to amplify a 1.9 kb DNA fragment encoding most of the a and b subunits of dissimilatory sulphite reductase as described previously (Laueet al., 2001). PCR products and a 1 kb DNA ladder molecular size marker (MBI Fermentas) were loaded on to a 1.5% agarose gel, which was subsequently stained with ethidium bromide (Sambrooket al., 1989), to evaluate the PCR. To verify the identity of candidateB. wadsworthiaisolates obtained from the enrichment cultures, the PCR fingerprinting technique was used (Hunt Gerardo et al., 1997). Single primer amplifications were carried out with the tDNA primer T3B, and PCR products were visualized on agarose gels. Bands were compared with the pattern for the type strain.
Results
Development of PCR assays forB. wadsworthia Few genes encoding metabolic enzymes have been se- quenced from B. wadsworthia RZATAU, and they are all involved in the anaerobic dissimilation of taurine (Laue &
Cook, 2000a, b; Laueet al., 2001). We designed a primer set, which is highly specific for thetpagene, and which should yield a 1003 bp PCR fragment. The primer set was tested on the B. wadsworthia environmental isolates RZATAU and KNATAU, on the type strain ATCC 49260Tand on 11 clinical isolates ofB. wadsworthia. All yielded the 1 kb fragment (not shown). This suggests that little genetic diversity is present among isolates of the genus Bilophila, which is currently represented by only one species (B. wadsworthia).
The specificity of the primer set was tested on a range of clinically relevant Gram-positive and Gram-negative organ- isms (not shown), and on a set of sulphonate-degrading bacteria including sulphate reducers of the related genus Desulfovibrio(Table S1). No amplification product fortpa was observed, even with a lower annealing temperature (down to 401C). The primers can thus be regarded as specific for B. wadsworthia. The dissimilatory sulphite reductase genesdsrABofB. wadsworthiaRZATAU have been sequenced (Laueet al., 2001). A primer set that had been developed to detect a 1.9 kb fragment of the dsrABgenes common to all sulphate reducers (Wagneret al., 1998) was used with the same set of organisms mentioned above. In this case, all strains ofB. wadsworthia(not shown) and the
Desulfovibriospp. (Table S1) gave a positive signal at 1.9 kb, whereas no other tested strain yielded the PCR fragment.
This confirms that the primer set is unable to discriminate between sulphate reducers and the phylogenetically related B. wadsworthia.
Enrichment ofB. wadsworthia from animal faeces
Anoxic enrichment cultures forB. wadsworthiain taurine- plus-formate-minimal-salts medium were set up with faecal samples from primates, felines and an ungulate. All of these cultures, with the exceptions of inocula from the lesser mouse lemur, lion and sheep, grew in about 5 days, produced significant amounts of sulphide (Table 1) with simultaneous utilization of taurine (data not shown), and could be reproducibly subcultured. After about four sub- cultures to fresh liquid medium, 11 enrichments were considered stable and positive. These grew in 2–4 days and contained predominantly short, nonmotile rods, i.e. the morphotype ofB. wadsworthia (Baron et al., 1989). Pure cultures were obtained from these enrichments after plating on taurine-supplemented BBE agar. Convex colonies with black centres and translucent margins were observed in each culture, which indicated the presence of B. wadsworthia (Baronet al., 1989). These identifications were confirmed by PCR fingerprinting using the primer T3B (Hunt Gerardo et al., 1997), yielding in all cases the characteristic band for B. wadsworthia.
Application oftpa and dsrAB primers on the enrichment cultures
DNA isolated from the 11 positive enrichment cultures (Table 1) was used as the template for the tpa-specific primer pair, and amplification of the 1 kb DNA fragment was observed in each case (Fig. 1). In addition, the presence
of genes encoding DSR in all 11 enrichment cultures was indicated by the amplification of the 1.9 kb dsrAB PCR fragment (not shown). The presence of B. wadsworthia in the enrichment cultures was thus indicated by the positive PCR reactions fortpaanddsrABand corresponded well with the results obtained by microscopy, by colony morphology on BBE agar plates, and by PCR fingerprinting (see above).
Discussion
Bilophila wadsworthiahas been recovered from the faeces of 60% of all humans tested (Baron et al., 1992; Schumacher et al., 1997) which indicates that the human gastrointestinal tract is a natural habitat for this organism (Baron et al., 1989; Eckburget al., 2005); it has also been detected in the intestinal tract of pig, chicken and eland (Baronet al., 1992;
McOrist et al., 2001, 2003; Nelsonet al., 2003). The data in Table 1 indicate that B. wadsworthiais also widespread but not omnipresent in the intestinal tract of primates and felines.
We were unable to isolateB. wadsworthiafrom the faeces of sheep, lion and lesser mouse lemur. The sample of lion faeces, however, was old, andB. wadsworthiatends to be lost from samples, which were exposed for longer time to oxic conditions (K. Denger & A.M. Cook, unpublished data). An alternative possibility is that, as in the case of humans, pigs and chickens (Baronet al., 1992; McOristet al., 2001, 2003;
Eckburget al., 2005), not all individuals contain a detectable population of B. wadsworthia within their gastrointestinal tract: populations ofB. wadsworthiain human faeces vary between 103and 108g1(wet weight) of faeces, with a total of 1011anaerobes g1(wet weight) (Baronet al., 1992). The lower limit number might not allow successful isolation of B. wadsworthiafrom faecal samples.
In all vertebrates except for mammals, bile salts are the conjugates of taurine and cholesterol derivatives (Huxtable, 1992). Mammals, however, use glycine in addition to taurine, though some animals, including the carnivorous polar bear, are described as purely glycine-conjugators (Huxtable, 1992). As taurine-conjugated bile acids are a source of taurine for B. wadsworthia (Schumacher et al., 1996), this species presumably belongs to the normal flora of intestinal tracts, which contain taurine-conjugated bile acids.
Bilophila wadsworthia is one of the most important anaerobic pathogens especially in appendicitis and intra- abdominal infections (Schumacher et al., 1997). Culture- based detection and identification of B. wadsworthia on conventional agar media is time-consuming (4–7 days).
Therefore, a more rapid and specific detection method would improve the laboratory diagnosis of this important pathogen. The PCR test involving the tpa primer set is highly specific for theB. wadsworthia tpagene, with no false 1.5
1.2 1.0 0.5
1 2 3 4 5 7 8 9 10 11
0.8
6
Fig. 1.Amplification by PCR of a fragment (1003 bp) of thetpagene from representative enrichment cultures in taurine-and-formate-supple- mented minimal-salts medium. The enrichment cultures are detailed in Table 1. Lane 1, 1 kb ladder; lane 2, inoculum from a gorilla; 3, inoculum from a chimpanzee; 4, inoculum from a mangabey; 5, inoculum from a panther; 6, inoculum from a Celebes macaque; 7, inoculum from a siamang; 8, inoculum from a bobcat; 9, inoculum from a cheetah;
10, template from a pure culture ofBilophila wadsworthiaRZATAU;
11, control (water).
negatives in B. wadsworthia isolates from clinical and environmental sources and no false positives from closely related genera. Based on a recent database search at NCBI (February 2006), no bacterial sequences were obtained that would allow PCR with this primer set. The amino acid sequence most similar (60%) to theB. wadsworthiaTpa is a putative aminotransferase from the genome sequences of three strains of R. sphaeroidesrepresented by strain 2.4.1:
this gene product has negligible Tpa activity (T.H.M. Smits, K. Denger & A.M. Cook, unpublished data). Functional Tpa proteins (Masepohl et al., 2001; Denger et al., 2004;
Gorzynskaet al., 2006), whether involved in the assimilation of taurine sulphur, the assimilation of taurine nitrogen or the dissimilation of taurine carbon, do not cluster in phylogenetic trees, but are interspersed with proteins of different or unknown functions (Fig. S1).
ThedsrABprimer set (Wagneret al., 1998) could be used in a multiplex PCR approach. The amplification of the dsrAB genes using the current primer set in combination with the tpa gene by PCR is convincing proof for the presence of B. wadsworthia because the corresponding enzymes are both involved in the taurine degradative path- way. As this gene has been sequenced for B. wadsworthia RZATAU (Laueet al., 2001), it would be possible to design a gene-specific primer set that only detects thedsrABgenes of B. wadsworthia.
We have shown the presence ofB. wadsworthia tpagenes in clinical isolates and enrichments from a broad range of sources, demonstrating that the primer set is applicable to identifyB. wadsworthia, whether from clinical or environ- mental sources. The primer set fortpawe developed, with thedsrABprimers, would allow rapid screening of clinical samples using PCR technology, including quantitative PCR using real-time PCR technology (Bustin, 2000, 2002;
Mackay, 2004). Further studies are required to develop the applications for medical laboratories.
Acknowledgements
We are grateful to Ralf Rabus (Max Planck Institute for Marine Microbiology, Bremerhaven, Germany), who con- firmed our hypothesis thatDesulfotalea psychrophilaLSv54 would dissimilate taurine, to Lydia H¨ausermann, T¨ubingen, for preparing the DNA from clinical strains and to Daniela Adler, Leipzig, for doing the DNA fingerprinting. This research was supported by funds from the Deutsche Forschungsgemeinschaft, the European Union (SUITE:
ENV4-CT98-0723) and the University of Konstanz.
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Supplementary data
Supplementary data associated with this article can be found in the online version, at doi: 10.1016/j.femsle.
Fig. S1. Dendrogram of orthologues of taurine:pyru- vate aminotransferase (Tpa) within COG161.
Table S1. Some characteristics of pure cultures of sulfonate-degrading bacteria and the detection of the tpa gene and thedsrABgenes
This material is available as part of the online article from http://www.blackwell-synergy.com
