Bilophila Wadsworthia: a Unique Gram-Negative Anaerobic Rod
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Anaerobe (1997) 3, 83–86 Bilophila wadsworthia: a Unique Gram-negative Anaerobic Rod Ellen Jo Baron Department of Medicine, Although comprising less than 0.01% of the normal human gastrointestinal University of California, Los microbiota, Bilophila wadsworthia is the third most common anaerobe Angeles and recovered from clinical material obtained from patients with perforated and Department of Molecular gangrenous appendicitis. Since its discovery in 1988, B. wadsworthia has been Microbiology and Immunology, recovered from clinical specimens associated with a variety of infections, University of Southern including sepsis, liver abscesses, cholecystitis, Fournier’s gangrene, soft California, Los Angeles, U.S.A. tissue abscesses, empyema, osteomyelitis, Bartholinitis, and hidradenitis suppurativa. In addition, it has been found in the saliva and vaginal fluids (Received 3 July 1996, of asymptomatic adults and even in the periodontal pockets of dogs. accepted in revised form 21 The organism is asaccharolytic, fastidious, and is easily recognized by its February 1997) strong catalase reaction with 15% H2O2, production of hydrogen sulfide, and growth stimulation by bile (oxgall) and pyruvate. Approximately 75% of Key Words: Bilophila, Bilophila strains are urease positive. When grown on pyruvate-containing media, wadsworthia, appendicitis > 85% of strains demonstrate â-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 â-lactam agents, which induce spheroplast formation that results in a haze of growth on agar dilution susceptibility test plates which is difficult to interpret. Bilophila strains are inhibited in vitro by most antibiotics. © 1997 Academic Press Introduction and the effects of various antibiotic regimens on patient recovery. The organism was recovered from approximately one-half of all specimens of appendi- Bilophila wadsworthia was first recognized in 1988 ceal tissue or peritoneal fluid collected from patients during the course of a study designed to describe the with appendicitis [1], although it had not been microbiota of gangrenous and perforated appendicitis recognized previously [2,3]. Since its description, it has been the subject of or has been mentioned in approximately 20 publications. This review attempts Address correspondence to Ellen Jo Baron at: 756 Haverford Avenue, Pacific Palisades, CA 90272, U.S.A. to summarize the current information available with E-mail: [email protected] regard to this unique anaerobic organism. 1075-9964/97/020083 + 04 $25.00/0/an970075 © 1997 Academic Press 84 E. J. Baron Characteristics of the Organism were detected in fecal specimens from 34 of 57 (60%) normal human volunteers [2,8]. The same author was able to recover the species from periodontal pockets of Strains of B. wadsworthia are asaccharolytic and able to three of 16 (19%) dogs with periodontitis that were reduce nitrate to nitrite and occasionally to N2; sampled [8]. The rate of recovery from saliva and approximately 75% of strains are urease positive ([2], human vaginal secretions from 100 normal volunteers M. McTeague, personal communication). There was only 4% and 3%, respectively [8]. appears to be no genetic difference among urease- At least five possible virulence mechanisms have positive and urease-negative strains [4]. Growth is been associated with Bilophila species. Similar to stimulated by 20% bile (oxgall) and 1% pyruvate [2]. Bacteroides fragilis and unlike most other anaerobic In fact, demonstration of â-lactamase production species, Bilophila strains were shown to induce intra- requires growth on a pyruvate-containing medium [5]. abdominal abscess formation when injected as pure Members of the genus produce a major acetic acid cultures into the peritoneal cavities of mice (Andrew peak and minor to trace amounts of succinic acid as Onderdonk, personal communication). Although detected by gas-liquid chromatography performed on abscess formation is usually associated with presence growth in pyruvate-supplemented pre-reduced, anae- of capsular polysaccharide, Bilophila does not appear robically sterilized (PRAS) peptone-yeast and pep- to have a capsule; thus the specific virulence factor tone-yeast-glucose broths [2]. responsible for abscess formation is not yet known. As Two key distinguishing characteristics are its very do most Gram-negative bacteria, Bilophila species rapid and strong catalase reaction with 15% H2O2, the induces clotting of Limulus amoebocyte lysate and standard catalase reagent used for anaerobic bacteria, promotes procoagulant activity by human mono- and production of hydrogen sulfide from sulfur- nuclear cells, both characteristics associated with the containing amino acids. The production of H2S results presence of endotoxin [9]. However, the magnitude of in the presence of a small dark central spot on colonies the endotoxin-like activity of the Bilophila species grown for at least 48–72 h on Bacteroides bile esculin tested was less than that observed for typical Gram- (BBE) agar. This characteristic feature on the otherwise negative organisms [9]. Tissue culture assays for small and translucent colonies coupled with a strong cytotoxicity demonstrated some cytotoxic activity in positive catalase reaction are virtually definitive for two cell lines, but this factor has not been further identification of the species. Unlike many other sulfide studied. Additional in vitro studies demonstrated producing genera, Bilophila does not contain desulfo- great variability in the ability of strains of Bilophila to viridin pigment or reduce sulfate. adhere to a human embryonic intestinal cell line The cellular fatty acids are unique among bacterial (Sharon Gerardo, personal communication), although species, consisting of iso-15:0, 16:0, 17:0cyc9,10 and the adherence characteristics of individual strains 19:0cyc9,10 [2]. Freeze-fracture electron micrographs were stable during multiple passages. No structural show a relatively smooth cell membrane with few features were detected by electron microscopy to protruding structures [6]. Transmission electron account for the adherence capabilities of some strains, micrographs display a Gram-negative cell wall with although differences in outer membrane proteins no obvious peptidoglycan layer [6,7]. By thermal between adherent and non-adherent strains were melting point determination, Bilophila species are found (Gerardo). Finally, the presence of iron-regu- 39–40 mol% G + C [2]. The cellular enzymes malate lated outer membrane proteins, which may have a role dehydrogenase and glutamate dehydrogenase were in virulence, has been demonstrated [10]. Under iron- detected in some strains in small amounts and only depleted conditions, four new outer membrane pro- under anaerobic conditions (Paula Summanen, per- teins were expressed by all four separate strains of sonal communication). Their relative electrophoretic Bilophila wadsworthia tested [10]. The contribution of mobilities differed and may prove useful for taxo- these proteins, if any, to the virulence of these nomic differentiation. organisms is unknown. Phylogenetically, the genus Bilophila maps within the delta and epsilon subdivisions of the purple bacteria between two species of Desulfovibrio [4]. A probe constructed from Bilophila wadsworthia 16S Clinical Associations rRNA hybridized slightly to a strain of Bacteroides thetaiotaomicron and a strain of B. fragilis [4]. Crude whole cell chromosomal DNA dot-blots showed slight Several publications have catalogued the clinical homology only with a strain of Bacteroides vulgatus syndromes and specimen types from which Bilophila [2]. wadsworthia has been isolated. In addition to isolates Several natural habitats of the organism have been listed in previous reviews [8,11,12], the organism has discovered. Using dilution plating, Bilophila species been isolated from soft tissue infection in an intra- Bilophila wadsworthia 85 Table 1. Summary of sources of clinical isolates of Bilophila trazolium chloride, a dye that is reduced to a red color wadsworthia other than appendicitis tissue and peritoneal fluid by enzymes of viable bacteria, was used to clarify the Number of isolates endpoint determination [5]. During the course of this Site reported References study, it was determined that many enzymes of Bartholin cyst abscess 1 12 Bilophila wadsworthia, and presumably other anaerobic Bile 3 12 bacteria, are inhibited in the presence of oxygen ([5], Blood 6 *8, 12, 17, 18 Brain abscess 1 14 Summanen, personal communication). It was also Breast abscess 1 *11, 19 discovered that reliable susceptibility and â-lactamase Empyema 1 *8, 11, 20 results were dependent on the addition of 1% pyr- Fournier’s gangrene 2 8, 12 Gingivitis 1 8 uvate to the testing growth medium [5]. Further Hidradenitis suppurativa 1 8 investigation of the action of â-lactam antibiotics on Liver abscess 1 12 cell wall structure was undertaken using imipenem. Osteomyelitis or bone site 2 8 Pericardial fluid 1 8 Inhibition of penicillin-binding proteins was found to Soft tissue abscess, be associated with spheroplast formation and ‘hazy’ necrotizing fasciitis, or decubitus 8 8, 12, 13 growth on agar plates [7]. These distorted cells, Vaginal