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Background and Objective: Bilophlia spp. are gram-negative, pleomorphic rod, obligate anaerobe, oxidase-negative, catalase-positive and non-motile bacteria. B. wadsworthia is type species of genus Bilophila with the additional characteristic of urea hydrolysis. B. wadsworthia can be found in a variety of anaerobe infections, particularly appendicitis and intra-abdominal infection that are considered as important opportunistic pathogens. Methods: This study was designed to identify Bilophila spp. in clinical specimens by culture and PCR. We examined 91 DNA samples extracted from infected appendix tissues with specific primers. Results: Data showed that Bilophila spp. DNA existence in 53.85% (n=49) provided appendiceal tissue. Conclusion: The pathological and molecular examination of infected appendiceal tissues revealed that B. wadsworthia is able to act as the primary cause of significant lesions in the appendicle tissues. Key words: Bilophila spp., Appendectomy, Appendicle specimens, PCR, Nucleotide sequencing

A recently introduced quantitative framework for gut microbiota analysis indicated that microbial load alterations can be linked to various diseases, making it essential to pinpoint its determinants. We identified nutrient load as a main driver of the quantitative microbial community composition and functionality in vitro by stepwise decreasing standardized feed concentrations from 100% to 33%, 20% and 10% in 5-day intervals. While the proportional composition and metabolic profile were mainly determined by the inter-individual variability (35% and 41%), nutrient load accounted for 58%, 23% and 65% of the observed variation in the microbial load, quantitative composition and net daily metabolite production, respectively. After the 10-fold nutrient reduction, the microbial load decreased by 79.72 ± 9% and 82.96 ± 1.66% in the proximal and distal colon, respectively, while the net total short-chain fatty acid production dropped by 79.42 ± 4.42% and 84.58 ± 2.42%, respectively. The majority of microbial taxa quantitatively decreased, whereas a select group of nutritional specialists, such as Akkermansia muciniphila and Bilophila wadsworthia, and a number of opportunistic pathogens remained unaffected. This shows that nutrient load is an important driver of the human gut microbiome and should be considered in future in vitro and in vivo dietary research. ispartof: FEMS MICROBIOLOGY ECOLOGY vol:97 issue:9 ispartof: location:England status: published

Purpose Controversy exists over whether bacterial flora within the appendix differs between patients with and without appendicitis. To examine these potential differences, we cultured the appendiceal luminal microbiota of patients with and without acute appendicitis, and identified the bacterial species therein. Methods Fifty-seven patients with acute appendicitis and 37 patients without acute appendicitis who underwent curative resection of colorectal cancer and prophylactic appendectomies (control group) were included. Appendicitis patients were classified into the phlegmonous group or the gangrenous appendicitis group histopathologically. There was no patient with perforated appendicitis. Aerobic isolates were identified using standard identification schemata, and anaerobic isolates were identified according to the Japanese guidelines. Results There were no significant differences among the three groups in the median number aerobe species present per patient. However, the median number anaerobe species in the gangrenous appendicitis group was significantly higher than that of the control group and the phlegmonous appendicitis group. In addition, the incidence of patients with Bacillus species, Fusobacterium nucleatum, and Bilophila wadsworthia increased as the disease progressed from phlegmonous to gangrenous appendicitis. Conclusion The present results suggest that increased diversity of anaerobes and the translocation of Bacillus species, F. nucleatum, and B. wadsworthia are associated with the progression of acute appendicitis.

Responses of the microbiota to diet are highly personalized but mechanistically not well understood because many metabolic capabilities and interactions of human gut microorganisms are unknown. Here we show that sulfoquinovose (SQ), a sulfonated monosaccharide omnipresent in green vegetables, is a selective yet relevant substrate for few but ubiquitous bacteria in the human gut. In human feces and in defined co-culture, Eubacterium rectale and Bilophila wadsworthia used recently identified pathways to cooperatively catabolize SQ with 2,3-dihydroxypropane-1-sulfonate as a transient intermediate to hydrogen sulfide (H2S), a key intestinal metabolite with disparate effects on host health. SQ-degradation capability is encoded in almost half of E. rectale genomes but otherwise sparsely distributed among microbial species in the human intestine. However, re-analysis of fecal metatranscriptome datasets of four human cohorts showed that SQ degradation (mostly from E. rectale and Faecalibacterium prausnitzii) and H2S production (mostly from B. wadsworthia) pathways were expressed abundantly across various health states, demonstrating that these microbial functions are core attributes of the human gut. The discovery of green-diet-derived SQ as an exclusive microbial nutrient and an additional source of H2S in the human gut highlights the role of individual dietary compounds and organosulfur metabolism on microbial activity and has implications for precision editing of the gut microbiota by dietary and prebiotic interventions.

The interplay between diet, intestinal microbiota and host is a major factor impacting health. A diet rich in unsaturated fatty acids has been reported to stimulate the growth of Bilophila wadsworthia by increasing the proportion of the sulfonated bile acid taurocholate (TC). The taurine-induced overgrowth of B. wadsworthia promoted the development of colitis in interleukin-10-deficient (IL-10-/-) mice. This study aimed to investigate whether intake of the sulfonates sulfoquinovosyl diacylglycerols (SQDG) with a dietary supplement or their degradation product sulfoquinovose (SQ), stimulate the growth of B. wadsworthia in a similar manner and, thereby, cause intestinal inflammation. Conventional IL-10-/- mice were fed a diet supplemented with the SQDG-rich cyanobacterium Arthrospira platensis (Spirulina). SQ or TC were orally applied to conventional IL-10-/- mice and gnotobiotic IL-10-/- mice harboring a simplified human intestinal microbiota with or without B. wadsworthia. Analyses of inflammatory parameters revealed that none of the sulfonates induced severe colitis, but both, Spirulina and TC, induced expression of pro-inflammatory cytokines in cecal mucosa. Cell numbers of B. wadsworthia decreased almost two orders of magnitude by Spirulina feeding but slightly increased in gnotobiotic SQ and conventional TC mice. Changes in microbiota composition were observed in feces as a result of Spirulina or TC feeding in conventional mice. In conclusion, the dietary sulfonates SQDG and their metabolite SQ did not elicit bacteria-induced intestinal inflammation in IL-10-/- mice and, thus, do not promote colitis.

Tedizolid’s anaerobic activity is unappreciated. In this study, it was active against all 332 anaerobic isolates tested at ≤2 μg/ml exceptBilophila wadsworthiaand was more active than linezolid againstBacteroides fragilisgroup species (MIC90, 1 μg/ml versus 2 to 4 μg/ml). Tedizolid was active against Gram-positive anaerobes (MIC90for clostridia, 0.25 to 1 μg/ml; MIC90for anaerobic cocci, ≤0.

The influence of dairy on the gut microbiome has not been studied extensively. We performed a randomized cross-over study to analyze the effect of high dairy intake on the gut microbiome. Subjects were randomly assigned to a high-dairy diet (HDD) (5&ndash, 6 dairy portions per day) and a low-dairy diet (LDD) (&le, 1 dairy portion per day) for 6 weeks with a washout period of 4 weeks in between both diets. The gut microbiome was assessed using 16S rRNA gene sequencing. Compositionality and functionality of the gut microbiome was assessed using Quantitative Insights Into Microbial Ecology (QIIME) and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). Stool consistency was evaluated using the Bristol stool chart. In total, 46 healthy overweight subjects (BMI range 25&ndash, 30 kg/m2) completed both intervention periods. During the HDD, there was a significantly higher abundance of the genera Streptococcus, Leuconostoc, and Lactococcus, and the species Streptococcus thermophilus, Erysipelatoclostridium ramosum and Leuconostoc mesenteroides (pFDR &lt, 0.10). Furthermore, during the HDD, there was a significantly lower abundance of the genera Faecalibacterium and Bilophila, and the species Faecalibacterium prausnitzii, Clostridium aldenense, Acetivibrio ethanolgignens, Bilophila wadsworthia and Lactococcus lactis (pFDR &lt, 0.10). There were eight subjects who became constipated during the HDD and these subjects all had a lower abundance of F. prausnitzii. This is the first cross-over study in which the effect of an HDD compared to an LDD on the gut microbiome has been studied. An HDD led to a significantly different composition of the gut microbiome, with a particularly lower abundance of F. prausnitzii and a higher abundance of S. thermophilus. Constipation was observed in several subjects during the HDD. Predicted metabolic pathways were not significantly altered due to an HDD.

Summary Desulfonation of isethionate by the bacterial glycyl radical enzyme (GRE) isethionate sulfite-lyase (IslA) generates sulfite, a substrate for respiration that in turn produces the disease-associated metabolite hydrogen sulfide. Here, we present a 2.7 Å resolution X-ray structure of wild-type IslA from Bilophila wadsworthia with isethionate bound. In comparison with other GREs, alternate positioning of the active site β strands allows for distinct residue positions to contribute to substrate binding. These structural differences, combined with sequence variations, create a highly tailored active site for the binding of the negatively charged isethionate substrate. Through the kinetic analysis of 14 IslA variants and computational analyses, we probe the mechanism by which radical chemistry is used for C-S bond cleavage. This work further elucidates the structural basis of chemistry within the GRE superfamily and will inform structure-based inhibitor design of IsIA and thus of microbial hydrogen sulfide production., Graphical abstract, Highlights • Molecular basis for sulfite production in the human gut • Crystallographic snapshots of a glycyl radical enzyme found in the human gut • Molecular mechanism of radical-based C-S bond cleavage investigated • Canonical glycyl radical enzyme β barrel is tailored to enable sulfonate binding, Dawson, Irwin et al. provide a molecular depiction of how sulfite, a precursor to the cancer- and Crohn's disease-associated metabolite hydrogen sulfide, is generated from isethionate by the enzyme isethionate sulfite-lyase in the human gut microbiota.

Diets rich in animal source foods vs plant-based diets have different macronutrient composition, and they have been shown to have differential effects on the gut microbiome. In this study, we hypothesized that diets with very different nutrient composition are able to change gut microbiome composition and metabolites in a very short period. We compared a fast food (FF) diet (ie, burgers and fries) with a Mediterranean (Med) diet, which is rich in vegetables, whole grains, olive oil, nuts, and fish. Ten healthy subjects participated in a controlled crossover study in which they consumed a Med diet and FF diet in randomized order for 4 days each, with a 4-day washout between treatments. Fecal DNA was extracted and the 16S V4 region amplified using polymerase chain reaction followed by sequencing on an Illumina MiSeq. Plasma metabolites and bile acids were analyzed using liquid chromatography-mass spectrometry. Certain bile-tolerant microbial genera and species including Collinsella, Parabacteroides, and Bilophila wadsworthia significantly increased after the FF diet. Some fiber-fermenting bacteria, including Lachnospiraceae and Butyricicoccus, increased significantly after the Med diet and decreased after the FF diet. Bacterially produced metabolites indole-3-lactic acid and indole-3-propionic acid, which have been shown to confer beneficial effects on neuronal cells, increased after the Med diet and decreased after the FF diet. Interindividual variability in response to the treatments may be related to differences in background diet, for example as shown by differences in Bilophila response in relationship to the saturated fat content of the baseline diet. In conclusion, an animal fat-rich, low-fiber FF diet v. a high-fiber Med diet altered human gut microbiome composition and its metabolites after just 4 days.

Within the ENRIA project, several 'expertise laboratories' collaborated in order to optimize the identification of clinical anaerobic isolates by using a widely available platform, the Biotyper Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Main Spectral Profiles (MSPs) of well characterized anaerobic strains were added to one of the latest updates of the Biotyper database db6903; (V6 database) for common use. MSPs of anaerobic strains nominated for addition to the Biotyper database are included in this validation. In this study, we validated the optimized database (db5989 [V5 database] + ENRIA MSPs) using 6309 anaerobic isolates.Using the V5 database 71.1% of the isolates could be identified with high confidence, 16.9% with low confidence and 12.0% could not be identified. Including the MSPs added to the V6 database and all MSPs created within the ENRIA project, the amount of strains identified with high confidence increased to 74.8% and 79.2%, respectively. Strains that could not be identified using MALDI-TOF MS decreased to 10.4% and 7.3%, respectively. The observed increase in high confidence identifications differed per genus. For Bilophila wadsworthia, Prevotella spp., gram-positive anaerobic cocci and other less commonly encountered species more strains were identified with higher confidence. A subset of the non-identified strains (42.1%) were identified using 16S rDNA gene sequencing. The obtained identities demonstrated that strains could not be identified either due to the generation of spectra of insufficient quality or due to the fact that no MSP of the encountered species was present in the database. Undoubtedly, the ENRIA project has successfully increased the number of anaerobic isolates that can be identified with high confidence. We therefore recommend further expansion of the database to include less frequently isolated species as this would also allow us to gain valuable insight into the clinical relevance of these less common anaerobic bacteria. (C) 2018 Elsevier Ltd.

Colorectal cancer (CRC) is the third most common newly diagnosed cancer in both men and women in the Unites States. Colonoscopy has become increasingly popular in CRC screening and represents the gold standard for detecting and removing pre-cancerous lesions. Although colonoscopy is considered a relatively safe procedure, it is invasive and bowel preparation can be challenging for patients. As interest in the gut microbiome has expanded, there have been new links established between bacteria and the development of CRC. These developing associations could prove to be a useful adjunct to colonoscopy for CRC screening in the future. This review examines current research evaluating multiple proposed pathogenic microorganisms including sulfidogenic bacteria such as Bilophila wadsworthia, as well as Streptococcus bovis, Helicobacter pylori, Bacteroides fragilis, and Clostridium septicum. This discussion primarily focuses on bacterial pathogenesis, evidence of association with CRC, and the proposed mechanisms of carcinogenesis.

Dietary lipids favor the growth of the pathobiont Bilophila wadsworthia, but the relevance of this expansion in metabolic syndrome pathogenesis is poorly understood. Here, we showed that B. wadsworthia synergizes with high fat diet (HFD) to promote higher inflammation, intestinal barrier dysfunction and bile acid dysmetabolism, leading to higher glucose dysmetabolism and hepatic steatosis. Host-microbiota transcriptomics analysis reveal pathways, particularly butanoate metabolism, which may underlie the metabolic effects mediated by B. wadsworthia. Pharmacological suppression of B. wadsworthia-associated inflammation demonstrate the bacterium’s intrinsic capacity to induce a negative impact on glycemic control and hepatic function. Administration of the probiotic Lactobacillus rhamnosus CNCM I-3690 limits B. wadsworthia-induced immune and metabolic impairment by limiting its expansion, reducing inflammation and reinforcing intestinal barrier. Our results suggest a new avenue for interventions against western diet-driven inflammatory and metabolic diseases., Lipid intake is known to promote Bilophila wadsworthia growth. Here the authors show that B. wadsworthia aggravates high fat diet induced metabolic dysfunctions and its suppression, both pharmacologically or mediated by Lactobacillus rhamnosus, limits the severity of metabolic impairment.

Colorectal cancer is the fourth most common neoplasia in Europe, where it accounts for 28.2 new cases per 100,000 inhabitants. In an effort to decrease the incidence of this disease, various prevention measures are being studied, one of which are anthocyanin-rich foods. Anthocyanins are potent antioxidant flavonoids mainly found in flowers and colorful fruits and vegetables. These nutraceuticals have diverse biological functions once ingested, including immunomodulatory, anti-inflammatory and antitumor functions. In order to test the preventive effect of these flavonoids against colorectal cancer, an animal model (Rattus norvegicus F344) was developed. In this model two doses of azoxymethane (10 mg/kg) and two treatments with dextran sodium sulfate (DSS) were administered to the animals. For 20 weeks they were fed either control rat feed, control sausages, or functional sausages containing 0.1% (w/w) of anthocyanins from a mixture of dehydrated blackberries and strawberries. At the end of that period, the animals were sacrificed and their antioxidant plasma levels and digestive tract tissues were analyzed. The results revealed a statistically significant reduction in the number of colon tumors in the functional sausages cohort with respect to the control animals and an increase in the FRAP (ferric reducing ability of plasma) total antioxidant activity in that same cohort. Colon microbiota differences were also examined via metagenomics 16S ribosomal RNA (rRNA) sequencing, revealing a significant reduction in populations of the pro-inflammatory Bilophila wadsworthia. Therefore, the design of functional processed meat products, such as ones enriched with anthocyanins, may be an effective strategy for preventing inflammatory digestive diseases and colorectal cancer in human populations.

Relebactam is an important beta-lactamase inhibitor for certain aerobic organisms, but alone it has no antianaerobic activity, with most anaerobes having MICs of ≥32 μg/ml with the exception of a very few strains. There was no enhancement or antagonism of imipenem activity with the addition of relebactam, including activity against imipenem-resistant strains. The relebactam-imipenem combination had excellent overall activity against the anaerobes tested.

The antibiotic susceptibility profile of the Bacteroides fragilis group, Gram-positive anaerobic cocci (GPAC), Fusobacterium spp., Prevotella spp., Veillonella spp. and Bilophila wadsworthia for amoxicillin, amoxicillin-clavulanic acid, clindamycin and metronidazole was determined. Human clinical isolates were isolated between 2011 and 2013 at the Microbiological Diagnostic Laboratory of the University Medical Center Groningen, The Netherlands and subjected to MALDI-TOF MS identification and susceptibility testing using E-test for MIC determination.Differences in clindamycin susceptibility between species of the B. fragilis group and GPAC were observed, with Bacteroides ovatus and Peptoniphilus harei having the highest resistance rates. Compared to other European countries, in The Netherlands the MIC90 for clindamycin of fusobacteria is low. Metronidazole resistance was first encountered in the genus Prevotella in 2013, but not in species of GPAC as reported in Belgium and Bulgaria. The differences in clindamycin resistance between the different European countries and reports of metronidazole resistance within the genera Prevotella and GPAC warrant more extensive susceptibility studies on anaerobic pathogens. (C) 2014 Elsevier Ltd. All rights reserved.

The composite human gut microbiomes of Western populations have changed over the past century, brought on by new environmental triggers that often have a negative impact on human health. Diets high in saturated fats and refined sugars and low in fiber are leading candidates for these events and for triggering the increased prevalence of immune-mediated diseases like inflammatory bowel disease (IBD). Our studies have shown that consumption of a ‘Western' diet high in saturated (milk-derived) fat (MF) or n-6 polyunsaturated (safflower oil) fat have similar effects on the structure of the colonic microbiome of wild-type and IL- 10-/- mice, characterized by increased Bacteroidetes and decreased Firmicutes. However, the MF diet uniquely promotes the expansion of an immunogenic sulfite-reducing pathobiont, Bilophila wadsworthia, a member of the Deltaproteobacteria and minor component of the gut microbiome. This bacterial bloom results from a MF diet-induced shift in hepatic conjugation of bile acids, from glycocholic to taurocholic (TC) acid, which is important for solubilizing the more hydrophobic MF diet. However, it is also responsible for delivery of taurine-derived sulfur to the distal bowel, promoting the assemblage of bile-tolerant microbes such as B. wadsworthia. The bloom of this species promotes a Th1-mediated immune response and the development of colitis in IL-10-/- mice. A similar bloom of B. wadsworthia is seen when IL-10-/- mice are fed a low-fat diet supplemented with TC. B. wadsworthia colonization of monoassociated germ-free IL-10-/- mice was dependent on the host consuming either a high-saturated MF diet or the gavage with TC. Together, these data provide a plausible explanation for the link between diseases such as IBD and dietary-mediated selection of gut microbial pathobionts in genetically susceptible hosts. With this knowledge, it may be possible to mitigate the bloom of these types of pathobionts by modifying the conjugation states of bile acids.

Background Bilophila wadsworthia is a major member of sulfidogenic bacteria in human gut, it was originally recovered from different clinical specimens of intra-abdominal infections and recently was reported potentially linked to different chronic metabolic disorders. However, there is still insufficient understanding on its detailed function and mechanism to date. Methods A B. wadsworthia strain was isolated from fresh feces of a latent autoimmune diabetes in adults patient and we investigated its pathogenicity by oral administration to specific-pathogen-free mice. Tissue samples and serum were collected after sacrifice. Stool samples were collected at different time points to profile the gut microbiota. Results Bilophila wadsworthia infection resulted in the reduction of body weight and fat mass, apparent hepatosplenomegaly and elevated serum inflammatory factors, including serum amyloid A and interleukin-6, while without significant change of the overall gut microbiota structure. Conclusions These results demonstrated that higher amount of B. wadsworthia caused systemic inflammatory response in SPF mice, which adds new evidence to the pathogenicity of this bacterium and implied its potential role to the chronic inflammation related metabolic diseases like diabetes.

The outer membrane proteins of Desulfovibrio piger and Bilophila wadsworthia (Omp-DP and Omp-BW, respectively) and the genes encoding them (omp-DP and omp-BW) were isolated and characterized. Native Omp-DP and Omp-BW form a trimeric structure of approximately 120 kDa. These proteins disaggregated into monomers with a molecular weight of approximately 53 kDa after heating at 95 degrees C for 10 min. The pore-forming abilities of these oligomeric proteins demonstrated that they form small nonspecific channels with an exclusion limit of 260-300 Da. The omp-DP and omp-BW genes were cloned and sequenced. Sequence analyses revealed an open reading frame of 1,512 bp for omp-DP and 1,440 bp for omp-BW. The mature Omp-DP protein consisted of 480 amino acids and had a calculated MW of 53,290 Da. The mature Omp-BW protein consisted of 456 amino acids and had a calculated MW of 50.050 Da. Alignment of Omp-DP with Omp-BW revealed 54% homology, whereas alignment with other known porins showed a low level of homology. Analysis of the secondary structures indicated that both proteins span the outer membrane 18 times with amphipathic beta-strands. This research presents porins which were isolated and characterized for the first time from bacteria belonging to the Desulfovibrionaceae family.

The gram-negative anaerobic gut bacterium Bilophila wadsworthia is the third most common isolate in perforated and gangrenous appendicitis, being also found in a variety of other infections. This organism performs a unique kind of anaerobic respiration in which taurine, a major organic solute in mammals, is used as a source of sulphite that serves as terminal acceptor for the electron transport chain. We show here that molecular hydrogen, one of the major products of fermentative bacteria in the colon, is an excellent growth substrate for B. wadsworthia. We have quantified the enzymatic activities associated with the oxidation of H(2), formate and pyruvate for cells obtained in different growth conditions. The cell extracts present high levels of hydrogenase activity, and up to five different hydrogenases can be expressed by this organism. One of the hydrogenases appears to be constitutive, whereas the others show differential expression in different growth conditions. Two of the hydrogenases are soluble and are recognised by antibodies against a [FeFe] hydrogenase of a sulphate reducing bacterium. One of these hydrogenases is specifically induced during fermentative growth on pyruvate. Another two hydrogenases are membrane-bound and show increased expression in cells grown with hydrogen. Further work should be carried out to reveal whether oxidation of hydrogen contributes to the virulence of B. wadsworthia.

Correlations between mucosal sulfidogenic bacteria and diet were examined using sigmoid biopsies and the NIH Dietary History Questionnaire collected from 9 healthy subjects over 4 time points (0, 3, 6, and 9 months). Bacterial genera, namely Akkermansia muciniphilia, Bilophila wadsworthia, Desulfovibrio spp. (DSV), Desulfobulbus spp. (DBB), Desulfobacter spp. (DSB), and Desulfotomaculum spp. (DFM) were quantified by 16S rRNA gene-specific qPCR. Functional genes for pan-dissimilatory sulfite reductase (dsrA) and B. wadsworthia specific dsrA (dsrA-BW) were measured along with three genes utilized for cysteine fermentation by Fusobacterium nucleatum: L-cysteine desulfhydrase (Lcd), L-methionine-γ-lyase (FN1419), and cystathionine-β-synthase (FN1055). Significant positive correlations were observed between high saturated fat intake and increased abundance of DSB, dsrA-BW, and A. muciniphilia; specifically those present in milk fat, palm, and coconut oils. Similarly, DSB abundance was increased with total dair...

The bile-resistant, strictly anaerobic bacterium Bilophila wadsworthia is 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 in B. 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 contain B. 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.

The antimicrobial activities of garenoxacin and eight other antibiotics against 641 anaerobic isolates were evaluated with the NCCLS agar dilution method. Overall, the MICs of garenoxacin for 50 and 90% of the strains tested (in micrograms per milliliter) were as follows: Bacteroides fragilis group, 0.5 and 2; Prevotella spp., 0.25 and 2; Fusobacterium spp., 0.25 and 0.5; Porphyromonas spp., 0.125 and 0.25; Bilophila wadsworthia , 0.5 and 1; Veillonella spp., 0.25 and 0.5; Clostridium spp., 0.25 and 1; Clostridium difficile , 2 and >64; Bifidobacterium spp., 1 and 2; Eggerthella lenta , 0.25 and 1; Propionibacterium spp., 0.5 and 0.5; gram-positive cocci, 0.125 and 0.25.

International audience; The gut microbiota (GM) consists of resident commensals and transient microbes conveyed by the diet but little is known about the role of the latter on GM homeostasis. Here we show, by a conjunction of quantitative metagenomics, in silico genome reconstruction and metabolic modeling, that consumption of a fermented milk product containing dairy starters and Bifidobacterium animalis potentiates colonic short chain fatty acids production and decreases abundance of a pathobiont Bilophila wadsworthia compared to a milk product in subjects with irritable bowel syndrome (IBS, n=28). The GM changes parallel improvement of IBS state, suggesting a role of the fermented milk bacteria in gut homeostasis. Our data challenge the view that microbes ingested with food have little impact on the human GM functioning and rather provide support for beneficial health effects.

Long-term dietary intake influences the structure and activity of the trillions of microorganisms residing in the human gut, but it remains unclear how rapidly and reproducibly the human gut microbiome responds to short-term macronutrient change. Here we show that the short-term consumption of diets composed entirely of animal or plant products alters microbial community structure and overwhelms inter-individual differences in microbial gene expression. The animal-based diet increased the abundance of bile-tolerant microorganisms (Alistipes, Bilophila and Bacteroides) and decreased the levels of Firmicutes that metabolize dietary plant polysaccharides (Roseburia, Eubacterium rectale and Ruminococcus bromii). Microbial activity mirrored differences between herbivorous and carnivorous mammals, reflecting trade-offs between carbohydrate and protein fermentation. Foodborne microbes from both diets transiently colonized the gut, including bacteria, fungi and even viruses. Finally, increases in the abundance and activity of Bilophila wadsworthia on the animal-based diet support a link between dietary fat, bile acids and the outgrowth of microorganisms capable of triggering inflammatory bowel disease. In concert, these results demonstrate that the gut microbiome can rapidly respond to altered diet, potentially facilitating the diversity of human dietary lifestyles.

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 0 -phosphate, but highly active enzyme was only obtained by addition of pyridoxal 5 0 -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: Kma 7.1 mm, Vmaxa 1.20 nmol·s 21 , and for pyruvate: Kma 0.82 mm, Vmaxa 0.17 nmol·s 21 . The purified enzyme was able to transaminate hypotaurine (2-aminosulfinate), taurine, b-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.

The phenotypic characteristics as well as the prevalence and quantity (colony forming units/mL/sample) of 39 bacterial isolates of agar pitting Gram-negative rods, from two diseased and one healthy subgingival site from each of 16 pet dogs with naturally occurring periodontitis, were investigated. Phenotypic features were determined with use of standard biochemical methods, by enzymatic profiling with the API ZYM system, and by cellular fatty acid profiling. The organisms detected were motile, catalase-negative Campylobacter sp., present in 69% of the dogs and in 29% of the subgingival samples (69/29), motile, catalase-positive Campylobacter sp. (63/29), Eikenella corrodens (25/10), organisms closely resembling E. corrodens but nitrate-negative and unable to grow in air, designated E. corrodens -like (19/8), Bilophila wadsworthia (6/2), and non-motile Campylobacter sp. (6/2). The most frequent organisms were the motile Campylobacter isolates constituting 72% of all isolates. No statistically significant differences were detected between the diseased and healthy subgingival sites, with regard to the prevalence of any of these groups of organisms. Furthermore, the bacterial isolates were detected in almost equal numbers in the diseased and healthy sample sites. Hence, no association between dog periodontitis and the agar pitting Gram-negative rods was established. The phenotypic data also suggest, that the organisms that were only presumptively identified in the present study (Campylobacter sp., E. corrodens -like), may not have been previously described.

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.