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Bilophila wadsworthia, an under-appreciated anaerobic organism, was originally described in 1989. Ninety-nine Bilophila wadsworthia isolates, recovered form environmental and clinical specimens in Germany and in Southern California, were examined in this study. Many isolates were recovered in mixed culture with facultative aerobic and other anaerobic bacteria. All isolates were identified by standard laboratory procedures, including gas–liquid chromatography (GLC). A PCR fingerprint assay was established to compare the profiles of clinical and environmental isolates to the type strain (ATCC 49260) and to an environmental (sewage) reference strain (DSM 11045, RZATAU) for intra-species differences. Two primers, one universal primer, M13 core, and one tDNA primer, T3B, were used individually to analyse the strains. Homogeneous PCR fingerprint profiles were found for the majority of strains using the M13 core primer; two PCR groups were determined with T3B, one matching the type strain and one matching the environmental reference strain (DSM 11045, RZATAU). Two urease negative strains, WAL 11470 (blood isolate from California) and TUB 754 (intra-abdominal isolate from Germany) formed unique PCR fingerprint profiles with each of these primers. These results were confirmed by PCR fingerprinting using the T3A primer. These latter results suggest a possible genetic diversity in B. wadsworthia.

The susceptibility of eighty-seven strain of Bilophila wadsworthia to five beta-lactams, two beta-lactamase inhibitors, meropenem, metronidazole, clindamycin and two quinolones was determined. Tests were performed by the modified reference agar dilution technique using triphenyltetrazolium chloride for endpoint reading. The test strains showed a reduced susceptibility to the beta-lactams, penicillin G (MIC90 4 micrograms/ml), ampicillin (MIC90 32 micrograms/ml), piperacillin (MIC90 64 micrograms/ml), cephalothin (MIC90 2 micrograms/ml and cefotaxim (MIC90 4 micrograms/ml). The activity of ampicillin was increased by addition of the beta-lactamase inhibitor, sulbactam (MIC90 2 micrograms/ml), as was the activity of piperacillin by the addition of tazobactam (MIC90 4 micrograms/ml) 90.8% of the strains were found to produce beta-lactamase by the nitrocefin tube method. All strains were shown to be highly susceptible to meropenem, metronidazole and clindamycin (MICs < or = 1 microgram/ml). Sparfloxacin (MIC90 1 microgram/ml) and ciprofloxacin (MIC90 0.5 microgram/ml) were found to be active against most of the strains tested.

Bilophila wadsworthia isolates recovered from a right-ear cholesteatoma and brain abscess of the same patient were analyzed by means of polymerase chain reaction (PCR) fingerprinting with single primers (T3B and M13 core) to ascertain if they originated from the same clone. Their PCR fingerprint profiles were compared with those of three additional B. wadsworthia clinical isolates and the type strain (ATCC 49260). The two isolates from the same patient produced PCR fingerprint profiles identical to each other, regardless of which primer was used. All isolates' PCR fingerprint profiles, with use of either the T3B or M13 core primer, shared some major and minor bands. However, differences in additional major and minor bands distinguished each of the additional isolates, suggesting that there are different subgroups of B. wadsworthia.

There is still inadequate information on the role of certain newly described or reclassified anaerobes in disease processes, on their normal sites of carriage, and on their antimicrobial susceptibilities. Herein, we summarize this information (most of the literature reviewed is from the past 5 years, but a few of the articles are approximately 10 years old). Porphyromonas species had seemed to be relatively nonpathogenic, but recent work indicates that this belief is incorrect. P. gingivalis, P levii-like organisms, and P. endodontalis-like organisms have been recovered from a variety of oral and extraoral infections. P. macacae has been recovered from infected cat bite wounds. Sutterella wadsworthensis, recently differentiated from Campylobacter gracilis, has been found in a variety of infections. Bilophila wadsworthia has also been recovered from a wide variety of infections. Newly described anaerobic cocci, gram-positive nonsporeforming rods, and clostridia have also been isolated from various infections.

Although comprising less than 0.01% of the normal human gastrointestinal microbiota, Bilophila wadsworthia is the third most common anaerobe recovered from clinical material obtained from patients with perforated and gangrenous appendicitis. Since its discovery in 1988, B. wadsworthia has been recovered from clinical specimens associated with a variety of infections, including sepsis, liver abscesses, cholecystitis, Fournier's gangrene, soft tissue abscesses, empyema, osteomyelitis, Bartholinitis, and hidradenitis suppurativa. In addition, it has been found in the saliva and vaginal fluids of asymptomatic adults and even in the periodontal pockets of dogs. The organism is a saccharolytic, fastidious, and is easily recognized by its strong catalase reaction with 15% H2O2, production of hydrogen sulfide, and growth stimulation by bile (oxgall) and pyruvate. Approximately 75% of strains are urease positive. When grown on pyruvate-containing media, > 85% of strains demonstrate beta-lactamase production. Ribosomal RNA-based phylogenetic studies show Bilophila to be a homogeneous species, most closely related to Desulfovibrio species. Both adherence to human cells and endotoxin have been observed, and preliminary work suggests that environmental iron has a role in expression of outer membrane proteins. Penicillin-binding proteins appear to mediate the organism's susceptibility to at least some beta-lactam agents, which induce spheroplast formation that results in a haze of growth on agar dilution susceptibility test plates which is difficult to interpret. Bilophilastrains are inhibited in vitro by most antibiotics.

Bilophila wadsworthia, an obligately anaerobic Gram-negative bacillus, was first isolated from appendicitis specimens and human faeces. The two cases described here are the first report of the isolation of B. wadsworthia in Hungary, and include the first isolation from chronic mastoiditis complicated with a brain abscess.

The composite human microbiome of Western populations has probably changed over the past century, brought on by new environmental triggers that often have a negative impact on human health. Here we show that consumption of a diet high in saturated (milk-derived) fat, but not polyunsaturated (safflower oil) fat, changes the conditions for microbial assemblage and promotes the expansion of a low-abundance, sulphite-reducing pathobiont, Bilophila wadsworthia. This was associated with a pro-inflammatory T helper type 1 (T(H)1) immune response and increased incidence of colitis in genetically susceptible Il10(−/−), but not wild-type mice. These effects are mediated by milk-derived-fat-promoted taurine conjugation of hepatic bile acids, which increases the availability of organic sulphur used by sulphite-reducing microorganisms like B. wadsworthia. When mice were fed a low-fat diet supplemented with taurocholic acid, but not with glycocholic acid, for example, a bloom of B. wadsworthia and development of colitis were observed in Il10(−/−) mice. Together these data show that dietary fats, by promoting changes in host bile acid composition, can markedly alter conditions for gut microbial assemblage, resulting in dysbiosis that can perturb immune homeostasis. The data provide a plausible mechanistic basis by which Western-type diets high in certain saturated fats might increase the prevalence of complex immune-mediated diseases like inflammatory bowel disease in genetically susceptible hosts.

The relative abundance of different groups of sulphate-reducing bacteria (SRB) in faecal DNA collected before and after therapy from patients suffering from Crohn's disease (CD), irritable bowel syndrome (IBS) or ulcerative colitis (UC) has been compared with that from healthy controls. Growth tests revealed that SRB were not more abundant in samples from patients with CD before treatment than in the healthy control group. For most of the 128 samples available, these preliminary results were confirmed using degenerate PCR primers that amplify the dsrAB gene. However, some samples from patients with CD before treatment contained a growth inhibitor that was absent from IBS or UC samples. In-depth sequencing of PCR-generated dsrB fragments revealed that the diversity detected was surprisingly low, with only eight strains of SRB and the sulphite-reducing bacterium, Bilophila wadsworthia, detected above the 0.1% threshold. The proportion of the two major species detected, B. wadsworthia and Desulfovibrio piger, was as high as 93.5% of the total SRB population in the healthy control group and lower in all patient groups. Four previously undescribed species were found: it is impossible to predict whether they are sulphate or sulphite-reducing bacteria.

Hydrogenotrophic microbiota have a significant impact on colonic health; however, little is known about their diversity and ecology in situ. Here, molecular-based methods and multivariate analyses were used to examine the abundance and diversity of mucosa-associated hydrogenotrophic microbes in 90 biopsies collected from right colon, left colon and rectum of 25 healthy subjects. Functional genes of all three hydrogenotrophic groups were detected in at least one colonic region of all subjects. Methanogenic archaea (MA) constituted approximately one half of the hydrogenotrophic microbiota in each colonic region. Sulfate-reducing bacteria (SRB) were more abundant than acetogens in right colon, while acetogens were more abundant than SRB in left colon and rectum. MA genotypes exhibited low diversity, whereas SRB genotypes were diverse and generally similar across the three regions within subject but significantly variable among subjects. Multivariate cluster analysis defined subject-specific patterns for the diversity of SRB genotypes; however, neither subject- nor region-specific clusters were observed for the abundance of hydrogenotrophic functional genes. Sequence analyses of functional gene clones revealed that mucosa-associated SRB were phylogenetically related to Desulfovibrio piger, Desulfovibrio desulfuricans and Bilophila wadsworthia; whereas MA were related to Methanobrevibacter spp., Mb. smithii and the order Methanomicrobiales. Together these data demonstrate for the first time that the human colonic mucosa is persistently colonized by all three groups of hydrogenotrophic microbes, which exhibit segmental and interindividual variation in abundance and diversity.

Initial susceptibility studies of Bilophila wadsworthia indicated significant resistance to several beta-lactam antibiotics, including imipenem and cefoxitin. NO beta-lactamase production was detected. However, some B. wadsworthia strains may grow as a heavy "haze" at up to the highest concentration of an antibiotic on standard antimicrobial agent-containing plates, and it is often difficult to determine the point at which conventional growth stops and haze growth begins. We investigated the nature of the haze growth of B. wadsworthia by using triphenyltetrazolium chloride (TTC) as an indicator of viability during antimicrobial susceptibility testing, by determining viability counts on antimicrobial agent-containing plates at various times, and by microscopically inspecting stained preparations of the growth on the control plate and the haze area. TTC MICs were determined by applying a TTC solution over the growth on plates inside the anaerobic chamber or within 5 min after exposure to air (aerobic TTC MICs). The haze growth reduced TTC in the chamber but not under aerobic conditions, whereas TTC was reduced by the conventional growth in both atmospheres. The aerobic TTC MICs correlated with the viability counts. Separated proteins resolved by polyacrylamide gel electrophoresis and isoelectric focusing showed TTC-reactive bands only when stained under anaerobic conditions, further demonstrating the sensitivity of TTC reduction to aerobic conditions. Microscopic examination of the haze growth indicated spheroplast formation. A new antibiogram for B. wadsworthia has been established by use of aerobic TTC endpoints; we believe that the lower MICs obtained with the TTC method are likely the ones that are clinically relevant and should be used in tests of B. wadsworthia. Also, we found that when the organisms were grown on pyruvate-containing medium, 87% of 56 strains tested were Beta-lactamase positive.

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.

Bilophila wadsworthia ist ein 1989 erstmals beschriebenes gramnegatives, obligat anaerobes Stabchenbakterium. Der naturliche Standort von Bilophila wadsworthia scheint in erster Linie der humane Intestinaltrakt zu sein, wo die Spezies bei etwa 60% der untersuchten Personen nachweisbar war. Klinische Isolate von Bilophila wadsworthia stammen uberwiegend aus intraabdominalen Untersuchungsmaterialien. Als Bestandsteil mischinfizierter Prozesse wurde Bilophila wadsworthia auch bei extraabdominalen Infektionen bereits mehrfach isoliert, jedoch sind keine Nachweise im Zusammenhang mit otologischen Erkrankungen beschrieben worden. Wir stellen den Fall einer Patientin mit unmittelbar postoperativer Otitis externa nach Stapesplastik vor, bei der die Anaerobier Bacteroides fragilis und Bilophila wadsworthia als wahrscheinliche Erreger isoliert wurden.

A case of a patient with a 20-year history of chronic otitis media complicated by cholesteatoma and brain abscess is described. A CT scan with contrast material showed three abscess cavities in the right cerebellar hemisphere. A culture from a specimen of the cholesteatoma yielded a significant amount of growth of Bilophila wadsworthia, Bacteroides fragilis and Prevotella oris and a moderate growth of alpha-streptococci and Staphylococcus simulans. From the pus of the brain abscess we also isolated numerous Bilophila wadsworthia, Bacteroides fragilis and Prevotella oris and some Prevotella buccae and Peptostreptococcus anaerobius. No aerobes were present. The patient underwent a craniotomy and the biggest abscess was removed together with the capsule. The antimicrobial therapy included penicillin plus metronidazole and later augmentin. The result of the treatment was a complete cure and total recovery of the patient. This is the first documentation of isolation of B. wadsworthia in chronic otitis media and in brain abscess.

Objective To investigate the in vitro activity of a range of anti-anaerobe antimicrobials against non- Bacteroides fragilis group anaerobic Gram-negative bacilli isolated in Europe. Methods Isolates from 15 laboratories in 13 countries were identified by conventional methods. The MICs of 20 antibiotics were determined by an agar dilution method. Results There were 488 Prevotella spp., 174 fusobacteria, 69 Porphyromonas spp., 33 Bacteroides spp., 28 Bilophila wadsworthia and 16 Campylobacter spp. isolates, one Sutterella wadsworthensis isolate and four unidentified isolates. Penicillin resistance (and diminished susceptibility to piperacillin) was most common in Prevotella spp. and Bilophila wadsworthia but was also seen in many other species. All isolates, except three of Bilophila wadsworthia, were susceptible to amoxycillin/clavulanate. Most isolates were susceptible to cefoxitin (except Bilophila wadsworthia ) and all were susceptible to the carbapenems. Clinafloxacin was the most active quinolone, followed by trovafioxacin and then sparfloxacin. Most fusobacteria were inherently resistant to the macrolides, as expected, but resistance to macrolides and a ketolide in other species was uncommon. Most Fusobacterium varium isolates were resistant to clindamycin, but resistance to clindamycin in all other species was rare. Tetracycline resistance was common but this did not affect the glycylcyclines. There was one isolate of Bacteroides putredinis resistant to chloramphenicol, and three isolates, a Bacteroides ureolyticus isolate, the Sutterella wadsworthensis isolate and one of the unnamed isolates, were metronidazole resistant. Rifampicin was active against most Prevotella and Porphyromonas spp., but not against many other genera. Conclusions Penicillin resistance has increased in Europe among non -Bacteroides fragilis anaerobic Gram-negative bacilli, much of it due to β-lactamase. Acquired resistance to other β-lactams, macrolides and rifampicin has not significantly increased, and chloramphenicol and metronidazole are unaffected. However, resistance to tetracycline is common. The new compounds, a ketolide (HMR 3647), the glycylcyclines and clinafloxacin, are highly active.

Bilophila wadsworthia is a common inhabitant of the human colon and has been associated with appendicitis and other local sites of inflammation in humans. Challenge-exposure or prevalence studies in laboratory and other animals have not been reported. B. wadsworthia is closely related phylogenetically to Desulfovibrio sp. and Lawsonia intracellularis , which are considered colon pathogens. We developed a PCR specific for B. wadsworthia DNA. Samples of bacterial DNA extracted from the feces of pigs on six farms in Australia and four farms in Venezuela were examined. Specific DNA of B. wadsworthia was detected in the feces of 58 of 161 Australian and 2 of 45 Venezuelan pigs, results comprising 100% of the neonatal pigs, 15% of the weaned grower pigs, and 27% of the adult sows tested. Single-stranded conformational polymorphism analysis of PCR product DNA derived from pigs or from known human strains showed an identical pattern. Histologic examination of the intestines of weaned B. wadsworthia -positive pigs found no or minor specific lesions in the small and large intestines, respectively. B. wadsworthia is apparently a common infection in neonatal pigs, but its prevalence decreases after weaning. The possible role of B. wadsworthia as an infection in animals and in human colons requires further study.

A dissimilatory sulfite reductase (DSR) was purified from the anaerobic, taurine-degrading bacterium Bilophila wadsworthia RZATAU to apparent homogeneity. The enzyme is involved in energy conservation by reducing sulfite, which is formed during the degradation of taurine as an electron acceptor, to sulfide. According to its UV-visible absorption spectrum with maxima at 392, 410, 583, and 630 nm, the enzyme belongs to the desulfoviridin type of DSRs. The sulfite reductase was isolated as an α 2 β 2 γ n ( n ≥ 2) multimer with a native size of 285 kDa as determined by gel filtration. We have sequenced the genes encoding the α and β subunits ( dsrA and dsrB , respectively), which probably constitute one operon. dsrA and dsrB encode polypeptides of 49 (α) and 54 kDa (β) which show significant similarities to the homologous subunits of other DSRs. The dsrB gene product of B. wadsworthia is apparently a fusion protein of dsrB and dsrD . This indicates a possible functional role of DsrD in DSR function because of its presence as a fusion protein as an integral part of the DSR holoenzyme in B. wadsworthia . A phylogenetic analysis using the available Dsr sequences revealed that B. wadsworthia grouped with its closest 16S rDNA relative Desulfovibrio desulfuricans Essex 6.

Alanine dehydrogenase [l-alanine:NAD+ oxidoreductase (deaminating), EC 1.4.1.4.] catalyses the reversible oxidative deamination of l-alanine to pyruvate and, in the anaerobic bacterium Bilophila wadsworthia RZATAU, it is involved in the degradation of taurine (2-aminoethanesulfonate). The enzyme regenerates the amino-group acceptor pyruvate, which is consumed during the transamination of taurine and liberates ammonia, which is one of the degradation end products. Alanine dehydrogenase seems to be induced during growth with taurine. The enzyme was purified about 24-fold to apparent homogeneity in a three-step purification. SDS-PAGE revealed a single protein band with a molecular mass of 42 kDa. The apparent molecular mass of the native enzyme was 273 kDa, as determined by gel filtration chromatography, suggesting a homo-hexameric structure. The N-terminal amino acid sequence was determined. The pH optimum was pH 9.0 for reductive amination of pyruvate and pH 9.0–11.5 for oxidative deamination of alanine. The apparent K m values for alanine, NAD+, pyruvate, ammonia and NADH were 1.6, 0.15, 1.1, 31 and 0.04 mM, respectively. The alanine dehydrogenase gene was sequenced. The deduced amino acid sequence corresponded to a size of 39.9 kDa and was very similar to that of the alanine dehydrogenase from Bacillus subtilis.

By using an agar dilution method, the comparative in vitro activities of ertapenem (MK-0826) were studied against 1,001 anaerobes isolated from human intra-abdominal infections in 17 countries worldwide. MK-0826 was uniformly active against all isolates, including all Bacteroides fragilis group species isolates, with the exception of 12 of 61 (20%) strains of Bilophila wadsworthia , 3 strains of lactobacilli, and 1 isolate of Acidaminococcus fermentans . Geographical variation in activity was not observed.

The influence of growth medium and incubation conditions on the detection of Bilophila wadsworthia β-lactamase was tested with Cefinase and Cefinase Plus disks. The tests involved aerobic and anaerobic incubation with conventional disk and quantitative tube assays. The production of β-lactamase was correlated with penicillin G, ampicillin, and ampicillin-sulbactam MICs and inhibition zones on penicillin (2-U) disks. The strains were grown on (i) brucella agar (brucella), (ii) brucella agar supplemented with 1% pyruvate (brucella-pyruvate), and (iii) brucella agar supplemented with 1% taurine (brucella-taurine). With the aerobic disk assay, 100, 100, and 7% of strains were positive after 30 min from growth on brucella-pyruvate, brucella, and brucella-taurine plates, respectively; of strains grown on brucella-taurine, 54% remained negative by the Cefinase assay, and 23% remained negative by the Cefinase Plus assay at 2 h. In quantitative assays, the strains became positive after 30 min from brucella-pyruvate plates and after 1 h from brucella plates. The intensities of the reactions were strongest with brucella-pyruvate plates under anaerobic test conditions. Anaerobic incubation enhanced β-lactamase detection of growth on brucella-taurine: at 3 h, 85% of strains were positive in comparison to 38% with aerobic incubation. All β-lactamase-negative strains were susceptible to penicillin G and ampicillin; all β-lactamase-positive strains were resistant to ampicillin and, with the exception of two strains, penicillin G. In conclusion, β-lactamase production correlated with susceptibility to penicillin G and ampicillin. Brucella agar supplemented with 1% pyruvate was the most reliable medium for testing B. wadsworthia β-lactamase, and anaerobic incubation expedited positive results. Brucella agar supplemented with taurine was unsuitable for B. wadsworthia β-lactamase testing. Cefinase and Cefinase Plus results were in agreement, but Cefinase Plus yielded faster reactions.

Bilophila wadsworthia RZATAU is a Gram-negative bacterium which converts the sulfonate taurine (2-aminoethanesulfonate) to ammonia, acetate and sulfide in an anaerobic respiration. Taurine:pyruvate aminotransferase (Tpa) catalyses the initial metabolic reaction yielding alanine and sulfoacetaldehyde. We purified Tpa 72-fold to apparent homogeneity with an overall yield of 89%. The purified enzyme did not require addition of pyridoxal 5'-phosphate, but highly active enzyme was only obtained by addition of pyridoxal 5'-phosphate to all buffers during purification. SDS/PAGE revealed a single protein band with a molecular mass of 51 kDa. The apparent molecular mass of the native enzyme was 197 kDa as determined by gel filtration, which indicates a homotetrameric structure. The kinetic constants for taurine were: Km = 7.1 mm, Vmax = 1.20 nmol·s-1, and for pyruvate: Km = 0.82 mm, Vmax = 0.17 nmol·s-1. The purified enzyme was able to transaminate hypotaurine (2-aminosulfinate), taurine, beta-alanine and with low activity cysteine and 3-aminopropanesulfonate. In addition to pyruvate, 2-ketobutyrate and oxaloacetate were utilized as amino group acceptors. We have sequenced the encoding gene (tpa). It encoded a 50-kDa peptide, which revealed 33% identity to diaminopelargonate aminotransferase from Bacillus subtilis.

MICs of antibiotics against Bilophila wadsworthia isolates were measured by agar and broth microdilution with pyruvic acid and by Etest. The inoculum size influenced greatly agar dilution. Despite discrepancies in MICs depending on the measurement method used, clindamycin consistently showed potent activity. Broth microdilution and Etest appear to be candidates for laboratory susceptibility testing.

Biophila wadsworthia is a recently recognized nonspore-forming anaerobic gram-negative rod and is reported to be associated with various infections such as gangrenous perforated appendicitis, peritonitis, osteomyelitis and bacteremia. Although the isolation of B. wadsworthia seems to be facilitated by using Bacteroides bile esculin (BBE) agar, the reliable scheme of identification of this species has been published. This study was conducted to find simple, rapid, and reliable measures for identification of B. wadsworthia. A total of 32 B. wadsworthia-suspected clinical isolates as well as B. wadsworthia WAL 7959 and Desulfomonas pigra DSM 749, which is the species that shows bacteriological characteristics similar to B. wadsworthia, were used. Through the study of various biochemical and enzymatic tests and culture on selective media, it is indicated that a nonspore-forming, gram-negative anaerobic rod that is nonmotile, forms "black-eyed" colonies on BBE agar, and demonstrates strongly positive catalase test, positive acid phosphatase test, and no-growth on Bacterides medium or to be susceptible to colistin (10 micrograms disk) can be identified as B. wadsworthia.

Adherence of Bilophila wadsworthia to the cultured human embryonic intestinal cell line, Intestine 407 (Int 407), varied among the strains tested from strongly adherent (76-100% cells positive for one or more adherent bacteria) to non- or weakly adherent (0-25% positive cells). Although negative staining revealed that infrequent cells of an adherent strain, WAL 9077, the adherent type-strain, WAL 7959, and a non-adherent strain, WAL 8448, expressed loosely associated fimbrial structures, a role for these structures in adhesion could not be confirmed with either scanning or thin-section electron micrography. Ruthenium red staining of thin-section preparations and subsequent electron microscopy failed to reveal an extensive extracellular polysaccharide layer. SDS-PAGE analysis of crude outer membrane fractions of WAL 9077 and WAL 8448 demonstrated clear differences in their major and minor outer membrane protein components. Thus, we postulate that the adherence of B. wadsworthia to Int 407 cells is mediated by an outer membrane or cell wall component.

Pathogenicity of microorganisms is a multifactorial phenomenon. Host environment plays a role in the development of the infectious process. The presence of key nutrients, such as iron, may be important for the expression of virulence factors. Iron is an essential component of a wide variety of biochemical processes in microorganisms, and its availability is of major importance in bacterial pathogenesis. In response to a low level of available iron in vivo, the creation of high-affinity iron-uptake systems and of a number of iron-regulated membrane proteins is induced. Although relationships between iron and virulence factors of aerobes have been better studied, a few such studies have been carried out with regard to anaerobes (mainly Bacteroides fragilis) [ 1, 2]. As discussed by Duerden [3], there is as yet no clear understanding of many of the virulence factors of anaerobes. Little is known about the mechanisms of iron uptake used by anaerobic bacteria, and nothing about those of Bilophila wadsworthia. The purpose of this work was to study the properties of this recently recognized anaerobe in relation to iron metabolism. B. wadsworthia is an anaerobic, gram-negative, asaccharolytic, bileresistant, and strongly catalase-positive bacillus [4]. This bacterium was first isolated from patients with gangrenous and perforative appendicitis. This study sought to determine whether B. wadsworthia expressed iron-regulated outer-membrane proteins (OMPs) in an iron-depleted environment and whether these proteins were immunogenic. Iron-free reagents and materials were used to prepare the ironfree medium. In this work, all glassware was deferrated. Chemicals used to prepare the medium were of highest purity. The medium was one previously described, to which oxgall (2%) and pyruvate (10 mM) were added [5]. The medium components were deferrated with Chelex-100 resin (Bio-Rad Laboratories, Hercules, CA), except for MgSO4, amino acids, and vitamins. Medium without iron contained

In this study we describe two properties of the Gram-negative bacterium Bilophila wadsworthia, namely the ability to clot Limulus lysate and the capacity to induce the production of tissue factor-like procoagulant activity by human mononuclear cells in vitro. Although exhibited at a lower degree when compared with those of typical Gram-negative bacteria or Gram-negative endotoxin those activities may account in part for Bilophila's pathogenicity. The capacity indeed to induce fibrin formation through the interaction with mononuclear cells suggests one mechanism by which the microorganism might cause abscess formation in the host. Moreover, since this activity is dependent on the number of Bilophila interacting with mononuclear cells, we hypothesize that this biological activity is closely influenced by growth environment.

Portions of the 16S RNA from a urease-positive Bilophila wadsworthia strain were sequenced, and a probe was constructed. The probe was end labeled with [32P]ATP and polynucleotide kinase and hybridized on a nylon filter (by dot blot hybridization) to the immobilized rRNA of 12 B. wadsworthia strains and eight other anaerobic isolates. The probe efficiently hybridized only to the Bilophila strains. Cross-reactivity at high RNA levels (2,000 ng) was observed with one strain of Bacteroides thetaiotamicron and one strain of Bacteroides fragilis (with 10x SET buffer [20x SET buffer is 0.5 M NaCl, 0.03 M Tris, and 2 mM EDTA]) but was not seen at lower RNA levels or with 5x SET buffer. When tested against mixed cultures of aerobic and anaerobic isolates representative of appendiceal abscess flora, the probe did not react with mixed cultures containing no Bilophila cells and could detect > or = 10(5) Bilophila CFU/ml when the mixture was seeded with Bilophila cells. This probe is of potential use in the rapid identification of pure isolates and in the direct identification of B. wadsworthia in clinical specimens.

The penicillin-binding protein (PBP) patterns of six strains of Bilophila wadsworthia were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and subsequent fluorography of membrane preparations labelled with [3H]benzylpenicillin. The PBP profiles among the strains were similar; generally, seven to nine PBP-reactive bands could be visualized; their molecular weights ranged from 31 to 137 kDa. The relative affinities of the PBPs of four strains of B. wadsworthia for imipenem were examined and correlated with the morphological responses of the cells to imipenem. Morphological changes were examined by light and electron microscopies. Light microscopy revealed that at low concentrations (less than the MIC), imipenem induced the formation of rounded and bulging cells; rarely, elongation without filamentation was observed. In the presence of imipenem at the MIC, spheroplast formation was observed. Scanning and transmission electron microscopies revealed round forms together with larger, multilobate cells in the presence of subinhibitory concentrations of imipenem, suggesting that new growth sites were initiated while cell division was inhibited. Peeling of the outer membrane was also seen. Spheroplasts were very large (up to 30 microns in diameter) and stable in aqueous solution. Inhibition of the PBPs could be seen in the presence of low imipenem concentrations.

Bactericidal assays of Bacteroides gracilis (six strains) and Bilophila wadsworthia (12 strains) in brucella broth with appropriate supplements were performed by the time-kill kinetic method. Antimicrobial agents tested were ampicillin/sulbactam (final concentrations, 16/8 micrograms/mL), ticarcillin/clavulanate (128/2 micrograms/mL), imipenem (8 micrograms/mL), cefoxitin (32 micrograms/mL), chloramphenicol (16 micrograms/mL), clindamycin (4 micrograms/mL), and metronidazole (16 micrograms/mL). Although all antimicrobial agents tested inhibited growth of all Bilophila strains during the first 24 hours, bactericidal activity was variable; only metronidazole was uniformly bactericidal. Most strains of Bilophila showed 1-2 log increases in growth at 6 hours with clindamycin and chloramphenicol. With chloramphenicol, some Bilophila strains tested showed regrowth starting at 30 hours. B. gracilis strains were generally more susceptible to all agents tested. Metronidazole, ticarcillin/clavulanate, chloramphenicol, and imipenem were most active. Several strains of B. gracilis were not killed by ampicillin/sulbactam, clindamycin, or cefoxitin. Activity was variable among strains and antimicrobial agents.

Bilophila wadsworthia is an anaerobic, gram-negative, asaccharolytic, urease-positive, bile-resistant, catalase-positive bacillus, originally recovered from infections in patients with gangrenous and perforated appendicitis. Additional isolations from clinical specimens, including pleural fluid, joint fluid, blood and pus from a scrotal abscess, mandibular osteomyelitis and axillary hidradenitis suppurativa are described here. Bilophila is found as normal flora in feces and, occasionally, in saliva and in the vagina. Isolates from humans are usually beta-lactamase positive and therefore resistant to certain beta-lactam antibiotics. Two percent of strains are also resistant to clindamycin.

Bilophila wadsworthia is an anaerobic, gram-negative, asaccharolytic, bile-resistant, catalase-positive bacillus that is usually urease positive and was originally recognized in specimens of peritoneal fluid and tissue from patients with appendicitis. Additional isolations from clinical specimens, including a scrotal abscess, mandibular osteomyelitis, axillary hidradenitis suppurativa, pleural fluid, joint fluid, and blood, are described here.

During a 3-year period routine anaerobic cultures were examined for the presence of Bilophila wadsworthia . Using taurine-supplemented Bacteroides–Bile–Esculin agar for isolation, we observed three cases of complicated polybacterial ear infections where B. wadsworthia was involved. The first case involved a 69-year-old female patient presenting an otitis externa following stapedectomy where B. wadsworthia was isolated. The second patient, 30-year-old woman, with a 2-decade history of otitis and otorrhoea presented with a cholesteatoma, complicated by brain abscess formation and B. wadsworthia was isolated from the purulent ear secretion as well as from the abscess material. The third case, a 39-year-old male patient suffering from cholesteatoma presented with otorrhoea and otalgia, B. wadsworthia was isolated from purulent ear secretion. In all cases, B. wadsworthia was part of mixed aerobic–anaerobic infections. Because this species was not found in 200 ear swabs from 100 healthy volunteers and was not detectable in throat swabs or saliva from of these patients, an exogenous origin of these outer and middle ear infections as well as an infection by fecal contamination seems more probable than ascending infections from the pharynx or the ear canal.

Bilophila wadsworthia, a recently described anaerobic gram-negative bacillus, was isolated from blood cultures of two patients with liver abscesses. These isolates exhibited the expected phenotypic characteristics, and identification was confirmed by cell wall fatty acid analysis by gas-liquid chromatography. This is the first documentation of bacteremia due to B. wadsworthia.

Objective To compare the Etest and a microbroth dilution system (Sceptor) to a reference agar dilution method for susceptibility testing of Bilophila wadsworthia. Methods The susceptibility of 15 clinical isolates of Bilophila wadsworthia was determined by the National Committee for Clinical Laboratory Standards (NCCLS) agar dilution method using triphenyltetrazolium chloride for endpoint determination. The results were compared with the results obtained by the E test and a commercial microbroth dilution system (Sceptor). Results Comparison of the MICs obtained by the reference method and the Etest revealed few discrepancies, with piperacillin and metronidazole being the only exceptions. The overall agreement was 70% within one dilution step. The discrepancies did not result in major interpretative errors. The overall essential agreement using susceptibility categories was 98% for the E test and 99% for the microdilution system. Conclusions Both methods may be considered as acceptable alternatives for testing individual isolates of B. wadsworthia.

Summary: Strongly catalase-positive Gram-negative anaerobic rods were isolated from approximately half of all intra-abdominal specimens received from patients with gangrenous and perforated appendicitis, and subsequently also from normal faecal specimens. The organism was originally detected on Bacteroides-bile-aesculin (BBE) agar, and grew slowly on non-selective anaerobic media containing blood. It was stimulated by bile and differed from other known genera by being urease- and catalase-positive, and by reducing nitrate. It did not reduce sulphate. Other anaerobic Gram-negative rods showed no homology by DNA dot-blot hybridization. The thermal melting profile of chromosomal DNA showed 39-40 mol% G + C. The whole-cell fatty acid methyl ester profile included cyclic and branched long-chain acids, and differed from those of all other anaerobes that have been tested. β-Lactamase was not detected. The name Bilophila wadsworthia gen. nov., sp. nov. is proposed for this organism.

Hydrogen sulfide (H2S) production in the intestinal microbiota has many contributions to human health and disease. An important source of H2S in the human gut is anaerobic respiration of sulfite released from the abundant dietary and host-derived organic sulfonate substrate in the gut, taurine (2-aminoethanesulfonate). However, the enzymes that allow intestinal bacteria to access sulfite from taurine have not yet been identified. Here we decipher the complete taurine desulfonation pathway in Bilophila wadsworthia 3.1.6 using differential proteomics, in vitro reconstruction with heterologously produced enzymes, and identification of critical intermediates. An initial deamination of taurine to sulfoacetaldehyde by a known taurine:pyruvate aminotransferase is followed, unexpectedly, by reduction of sulfoacetaldehyde to isethionate (2-hydroxyethanesulfonate) by an NADH-dependent reductase. Isethionate is then cleaved to sulfite and acetaldehyde by a previously uncharacterized glycyl radical enzyme (GRE), isethionate sulfite-lyase (IslA). The acetaldehyde produced is oxidized to acetyl-CoA by a dehydrogenase, and the sulfite is reduced to H2S by dissimilatory sulfite reductase. This unique GRE is also found in Desulfovibrio desulfuricans DSM642 and Desulfovibrio alaskensis G20, which use isethionate but not taurine; corresponding knockout mutants of D. alaskensis G20 did not grow with isethionate as the terminal electron acceptor. In conclusion, the novel radical-based C-S bond-cleavage reaction catalyzed by IslA diversifies the known repertoire of GRE superfamily enzymes and enables the energy metabolism of B. wadsworthia. This GRE is widely distributed in gut bacterial genomes and may represent a novel target for control of intestinal H2S production.