Bilophila wadsworthia: a Unique Gram-negative Anaerobic Rod

Anaerobe (1997) 3, 83–86

Bilophila wadsworthia: a Unique Gram-negative Anaerobic Rod Ellen Jo Baron Department of Medicine, University of California, Los Angeles and Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, U.S.A. (Received 3 July 1996, accepted in revised form 21 February 1997) Key Words: Bilophila, Bilophila wadsworthia, appendicitis

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 asaccharolytic, 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 β-lactamase production. Ribosomal RNAbased 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 β-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 Bilophila wadsworthia was first recognized in 1988 during the course of a study designed to describe the microbiota of gangrenous and perforated appendicitis Address correspondence to Ellen Jo Baron at: 756 Haverford Avenue, Pacific Palisades, CA 90272, U.S.A. E-mail: [email protected]

1075-9964/97/020083 + 04 $25.00/0/an970075

and the effects of various antibiotic regimens on patient recovery. The organism was recovered from approximately one-half of all specimens of appendiceal tissue or peritoneal fluid collected from patients with appendicitis [1], although it had not been recognized previously [2,3]. Since its description, it has been the subject of or has been mentioned in approximately 20 publications. This review attempts to summarize the current information available with regard to this unique anaerobic organism. © 1997 Academic Press

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Characteristics of the Organism Strains of B. wadsworthia are asaccharolytic and able to reduce nitrate to nitrite and occasionally to N2; approximately 75% of strains are urease positive ([2], M. McTeague, personal communication). There appears to be no genetic difference among ureasepositive and urease-negative strains [4]. Growth is stimulated by 20% bile (oxgall) and 1% pyruvate [2]. In fact, demonstration of β-lactamase production requires growth on a pyruvate-containing medium [5]. Members of the genus produce a major acetic acid peak and minor to trace amounts of succinic acid as detected by gas-liquid chromatography performed on growth in pyruvate-supplemented pre-reduced, anaerobically sterilized (PRAS) peptone-yeast and peptone-yeast-glucose broths [2]. Two key distinguishing characteristics are its very rapid and strong catalase reaction with 15% H2O2, the standard catalase reagent used for anaerobic bacteria, and production of hydrogen sulfide from sulfurcontaining amino acids. The production of H2S results in the presence of a small dark central spot on colonies grown for at least 48–72 h on Bacteroides bile esculin (BBE) agar. This characteristic feature on the otherwise small and translucent colonies coupled with a strong positive catalase reaction are virtually definitive for identification of the species. Unlike many other sulfide producing genera, Bilophila does not contain desulfoviridin pigment or reduce sulfate. The cellular fatty acids are unique among bacterial species, consisting of iso-15:0, 16:0, 17:0cyc9,10 and 19:0cyc9,10 [2]. Freeze-fracture electron micrographs show a relatively smooth cell membrane with few protruding structures [6]. Transmission electron micrographs display a Gram-negative cell wall with no obvious peptidoglycan layer [6,7]. By thermal melting point determination, Bilophila species are 39–40 mol% G + C [2]. The cellular enzymes malate dehydrogenase and glutamate dehydrogenase were detected in some strains in small amounts and only under anaerobic conditions (Paula Summanen, personal communication). Their relative electrophoretic mobilities differed and may prove useful for taxonomic differentiation. 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 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 homology only with a strain of Bacteroides vulgatus [2]. Several natural habitats of the organism have been discovered. Using dilution plating, Bilophila species

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 three of 16 (19%) dogs with periodontitis that were sampled [8]. The rate of recovery from saliva and human vaginal secretions from 100 normal volunteers was only 4% and 3%, respectively [8]. At least five possible virulence mechanisms have been associated with Bilophila species. Similar to Bacteroides fragilis and unlike most other anaerobic species, Bilophila strains were shown to induce intraabdominal abscess formation when injected as pure cultures into the peritoneal cavities of mice (Andrew Onderdonk, personal communication). Although abscess formation is usually associated with presence of capsular polysaccharide, Bilophila does not appear to have a capsule; thus the specific virulence factor responsible for abscess formation is not yet known. As do most Gram-negative bacteria, Bilophila species induces clotting of Limulus amoebocyte lysate and promotes procoagulant activity by human mononuclear cells, both characteristics associated with the presence of endotoxin [9]. However, the magnitude of the endotoxin-like activity of the Bilophila species tested was less than that observed for typical Gramnegative organisms [9]. Tissue culture assays for cytotoxicity demonstrated some cytotoxic activity in two cell lines, but this factor has not been further studied. Additional in vitro studies demonstrated great variability in the ability of strains of Bilophila to adhere to a human embryonic intestinal cell line (Sharon Gerardo, personal communication), although the adherence characteristics of individual strains were stable during multiple passages. No structural features were detected by electron microscopy to account for the adherence capabilities of some strains, although differences in outer membrane proteins between adherent and non-adherent strains were found (Gerardo). Finally, the presence of iron-regulated outer membrane proteins, which may have a role in virulence, has been demonstrated [10]. Under irondepleted conditions, four new outer membrane proteins were expressed by all four separate strains of Bilophila wadsworthia tested [10]. The contribution of these proteins, if any, to the virulence of these organisms is unknown.

Clinical Associations Several publications have catalogued the clinical syndromes and specimen types from which Bilophila wadsworthia has been isolated. In addition to isolates listed in previous reviews [8,11,12], the organism has been isolated from soft tissue infection in an intra-

Bilophila wadsworthia Table 1. Summary of sources of clinical isolates of Bilophila wadsworthia other than appendicitis tissue and peritoneal fluid Site Bartholin cyst abscess Bile Blood Brain abscess Breast abscess Empyema Fournier’s gangrene Gingivitis Hidradenitis suppurativa Liver abscess Osteomyelitis or bone site Pericardial fluid Soft tissue abscess, necrotizing fasciitis, or decubitus Vaginal discharge Total

Number of isolates reported

References

1 3 6 1 1 1 2 1 1 1 2 1

12 12 *8, 12, 17, 18 14 *11, 19 *8, 11, 20 8, 12 8 8 12 8 8

8 1

8, 12, 13 8

30

*The same isolate is mentioned in more than one publication.

venous drug user [13], and both cholesteatoma and brain abscess material from a patient with chronic otitis media [14]. Table 1 summarizes published data on the number and types of specimens from which Bilophila has been isolated. Although it is most commonly seen in specimens from patients with appendicitis, this species can clearly be expected as a component of anaerobic infections in any site. Interestingly, its association with appendicitis varies with the stage of disease, as determined by histopathology [15]. Investigation of 59 isolates from patients with appendicitis revealed that Bilophila was present in 25% of specimens from patients in the acute stage of disease, in 37% of specimens from patients with gangrenous appendicitis, and in 55% of specimens obtained from patients with perforated appendicitis [15]. On average, half of all properly collected tissues or peritoneal fluids from patients with appendicitis are found to harbor Bilophila wadsworthia, the third most common anaerobe isolated from such specimens [1].

Antimicrobial Susceptibilities Similar to several other genera of anaerobes, such as Fusobacterium, agar dilution susceptibilities of Bilophila spp. occasionally exhibited a trailing ‘haze’ of growth that made endopoint determination difficult [5,7,16]. The organisms in the haze were found to be spheroplasts, often as large as 30 µm across, or eight times the diameter of the unaffected cell [5,7]. Triphenylte-

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trazolium chloride, a dye that is reduced to a red color by enzymes of viable bacteria, was used to clarify the endpoint determination [5]. During the course of this study, it was determined that many enzymes of Bilophila wadsworthia, and presumably other anaerobic bacteria, are inhibited in the presence of oxygen ([5], Summanen, personal communication). It was also discovered that reliable susceptibility and β-lactamase results were dependent on the addition of 1% pyruvate to the testing growth medium [5]. Further investigation of the action of β-lactam antibiotics on cell wall structure was undertaken using imipenem. Inhibition of penicillin-binding proteins was found to be associated with spheroplast formation and ‘hazy’ growth on agar plates [7]. These distorted cells, however, were viable and able to return to normal morphology upon subculture. Time-kill kinetic studies of eight strains of Bilophila in pyruvate-containing medium illustrated the difficulty of interpreting susceptibility testing results. In these studies, breakpoint concentrations of ticarcillin/clavulanate, ampicillin/ sulbactam, imipenem, and cefoxitin effected two-tofour-log decreases in CFU over 30 h of incubation, but only two of the strains were killed completely (by ticarcillin/clavulanate, ampicillin/sulbactam, or imipenem) in these trials [16]. A proportion of the initial inoculum of most strains was still viable at the end of the 30 h time period of the experiment, and CFUs did not decline at all for several strains growing in clindamycin and chloramphenicol [16]. Perhaps these remaining viable cells were the distorted forms and spheroplasts seen in the triphenyltetrazolium chloride studies cited. Only metronidazole displayed consistent bactericidal activity against all eight strains reported [16]. Clinical implications of these results, however, are not known. With rare exceptions, Bilophila species are recovered in mixed cultures with other aerobic and anaerobic bacteria and are easy to overlook. Laboratory scientists should be encouraged to further characterize small, translucent colonies of Gram-negative bacilli on primary anaerobic blood agar plates or small, clear colonies with black centers on Bacteroides bile esculin agar. With just a rapid catalase test, many of these can be identified as Bilophila wadsworthia. By increasing the numbers of such strains recognized, we will increase our knowledge of the role of this organism in disease.

Acknowledgments I am grateful to Paula Summanen, Sharon Gerardo, and Andrew Onderdonk for sharing their unpublished observations. I also wish to thank Sydney Finegold for encouraging our pursuit of this unique organism.

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References 1. Bennion R.S., Baron E.J., Thompson J.E., Downes J., Summanen P., Talan D.A. and Finegold S.M. (1990) The bacteriology of gangrenous and perforated appendicitis — revisited. Ann Surg 211: 165–171 2. Baron E.J., Summanen P., Downes J., Roberts M.C., Wexler H.M. and Finegold S.M. (1989) Bilophila wadsworthia, gen. nov. and sp. nov., a unique Gram-negative anaerobic rod recovered from appendicitis specimens and human faeces. J Gen Microbiol 135: 3405–3411 3. Baron E.J., Summanen P., Downes J., Roberts M.C., Wexler H. and Finegold S.M. (1990) Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List No. 34. Int J Syst Bacteriol 40: 320–321 4. Sapico F.L., Reeves D., Wexler H.M., Duncan J., Wilson K.H. and Finegold S.M. (1994) Preliminary study using species-specific oligonucleotide probe for rRNA of Bilophila wadsworthia. J Clin Microbiol 32: 2510–2513 5. Summanen P., Wexler H.M. and Finegold S.M. (1992) Antimicrobial susceptibility testing of Bilophila wadsworthia by using triphenyltetrazolium chloride to facilitate the endpoint determination. Antimicrob Agents Chemother 36: 1658–1664 6. Summanen P., Downes J., Karl M., Lounaimaa K., Baron E., Jousimies-Somer H. and Finegold S. (1989) Characteristics and ultrastructure of an unusual Gram-negative bacillus isolated from inflamed and noninflamed appendices. Abstr Eur Soc Clin Microbiol abstr. PA 12; p.5 7. Summanen P., Wexler H.M., Lee K., Becker S.A.W.E., Garcia M.M. and Finegold S.M. (1993) Morphological response of Bilophila wadsworthia to imipenem: correlation with properties of penicillin-binding proteins. Antimicrob Agents Chemother 37: 2638–2644 8. Baron E.J., Curren M., Henderson G., Jousimies-Somer H., Lee K., Lechowitz K., Strong C.A., Summanen P., Tun´er K. and Finegold S.M. (1992) Bilophila wadsworthia isolates from clinical specimens. J Clin Microbiol 30: 1882–1884 9. Mosca A., D’Alagni M., Del Prete R., De Michele G.P., Summanen P.H., Finegold S.M. and Miragliotta G. (1995) Preliminary evidence of endotoxic activity of Bilophila wadsworthia. Anaerobe 1: 21–24

10. Daniel C., Baron E.J. and Courcol R.J. (1995) Effect of iron depletion on protein profiles of Bilophila wadsworthia. Clin Infect Dis 20 (Suppl 2): S158–S159 11. Finegold S., Summanen P., Gerardo S.H. and Baron E. (1992) Clinical importance of Bilophila wadsworthia. Eur J Clin Microbiol Infect Dis 11: 1058–1063 12. Summanen P.H., Jousimies-Somer H., Manley S., Bruckner D., Marina M., Goldstein E.J.C. and Finegold S.M. (1995) Bilophila wadsworthia isolates from clinical specimens. Clin Infect Dis 20(Suppl2): S210–S211 13. Summanen P.H., Talan D.A., Strong C., McTeague M., Bennion R., Thompson J.E., Vaisanen M.-L., Moran G., Winer M. and Finegold S.M. (1995) Bacteriology of skin and soft-tissue infections: comparison of infections in intravenous drug users and individuals with no history of intravenous drug use. Clin Infect Dis 20(Suppl 2): S279–S282 14. Marina M., Ivanova K., Ficheva M. and Fichev G. (1996) Bilophila wadsworthia in brain abscess: case report. Abstracts, International Congress and Workshop on Anaerobic Bacteria and Anaerobic Infections, Athens, Greece 15. Baron E.J., Bennion R., Thompson J., Strong C., Summanen P., McTeague M. and Finegold S.M. (1992) A microbiological comparison between acute and complicated appendicitis. Clin Infect Dis 14: 227–231 16. Baron E.J., Ropers G., Summanen P. and Courcol R.J. (1993) Bactericidal activity of selected antimicrobial agents against Bilophila wadsworthia and Bacteroides gracilis. Clin Infect Dis 16(Suppl 4): S339–S343 17. Bernard D., Verschraegen G., Claeys G., Lauwers S. and Rosseel P. (1994) Bilophila wadsworthia bacteremia in a patient with gangrenous appendicitis. Clin Infect Dis 18: 1024–1025 18. Kasten M.J., Rosenblatt J.E. and Gustafson D.R. (1992) Bilophila wadsworthia bacteremia in two patients with hepatic abscess. J Clin Microbiol 30: 2502–2503 19. Giamarellou H., Soulis M., Antoniadou A. and Gogas J (1994) Periareolar nonpuerperal breast infection: treatment of 38 cases. Clin Infect Dis 18: 73–76 20. Civen R., Jousimies-Somer H., Marina M., Borenstein L., Shah H. and Finegold S.M. (1995) A retrospective review of cases of anaerobic empyema and update of bacteriology. Clin Infect Dis 20(Suppl2): S224–S229