Bilophila wadsworthia Clinical Isolates Compared by Polymerase Chain Reaction Fingerprinting

S. Hunt Gerardo 0 M. Marina 0 D. M. Citron 0 M. C. Claros 0 M. K. Hudspeth 0 E. J. C. Goldstein 0 0 From the R. M. Alden Research Laboratory, Santa Monica/UCLA Medical Center , Santa Monica, California, USA ; National Center of Infectious and Parasitic Diseases , Sofia, Bulgaria ; Institute of Medical Microbiology, University of Leipzig , Leipzig, Germany ; and UCLA School of Medicine, Department of Medicine , Los Angeles, California, USA 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. - Bilophila wadsworthia, a recently described obligately anaerobic, gram-negative rod originally isolated from perforated and gangrenous appendicitis [1], has also been isolated from a variety of other clinical specimens [2 4]. B. wadsworthia colonies may be overlooked on routine blood agar plates, as they are small and nonpigmented and grow slowly (4 7 days). Because the organism is asaccharolytic, other biochemical tests are necessary for identification. Recently, a species-specific oligonucleotide probe for B. wadsworthia rRNA was used in hybridization experiments with 12 strains of B. wadsworthia and 16 other aerobic and anaerobic isolates (13 species), as well as with mixed aerobic and anaerobic cultures [5]. That preliminary study indicated that molecular techniques can provide a rapid means for identification of this organism. In this study, a PCR fingerprinting technique was used to ascertain if two B. wadsworthia isolates recovered from a rightear cholesteatoma and brain abscess of the same patient [6] originated from the same clone. The results were compared with the PCR fingerprint profiles of the American Type Culture Collection (ATCC) type strain and three additional clinical isolates of B. wadsworthia. Materials and Methods Specimens. Five clinical isolates of B. wadsworthia were provided by one of the authors (M. M.). Two strains were isolated in mixed cultures of (1) the tissue of a right-ear cholesteatoma (R. M. Alden isolate [RMA] 8070) and (2) pus from a cerebellar brain abscess (RMA 8068) in the same patient, who had a history of chronic otitis media [6]. Bacteroides fragilis and Prevotella oris were abundantly present in both of these specimens. Prevotella buccae and Peptostreptococcus anaerobius were present in moderate numbers only in the brain abscess specimen, whereas a-streptococci and Staphylococcus simulans were present in moderate numbers only in the cholesteatoma specimen. Additional clinical strains, obtained from three different patients, were isolated in mixed cultures of (1) a specimen from the retroperitoneal space of a patient with paraproctitis, a gangrenous scrotum, and necrotizing fasciitis (RMA 7355), (2) the necrotic tissue of a patient with gangrenous appendicitis and phlegmon in the retroperitoneum and femoral area (RMA 8066), and (3) the necrotic tissue of a patient with perforation of the sigma by a foreign body, complicated by phlegmon and necrotic fasciitis of the abdominal wall (RMA 8069). All isolates were stored in 20% skim milk at 0707C until use. Isolates were subcultured for purity and good growth on brucella agar supplemented with sheep blood, vitamin K1, hemin, and 1% pyruvate prior to testing. Control strains included the B. wadsworthia type strain ATCC 49260 (Wadsworth Anaerobe Laboratory isolate [WAL] 7959); Bacteroides gracilis ATCC 33236 (WAL 6989); and Sutterella wadsworthensis ATCC 51579. Biochemical analyses. All isolates were tested for the following: catalase production (15% v/v H2O2); nitrate reduction and indole formation (prereduced anaerobically sterilized [PRAS] biochemicals; Carr Scarborough Microbiologicals, Decatur, GA); growth on bacteroides bile esculin (BBE) agar (Anaerobe Systems, San Jose, CA); and the preformed enzymes urease and arginine aminopeptidase (WEE-TABS, Key Scientific, Round Rock, TX). Analysis of a variety of other preformed enzymes was performed with the RapID ANA II test system (Innovative Diagnostics Systems, L. P., Norcross, GA). PCR analyses. Several bacterial colonies of each isolate were suspended in 1 mL of sterile distilled water to a turbidity approximately equal to a number 3 McFarland standard. Two hundred microliters of the cell suspension were removed and pelleted, resuspended in 100 200 mL of Insta-Gene Matrix (BioRad, Hercules, CA), and then incubated at 507C for 15 30 minutes. Cell solutions were vortexed and then heated for 8 10 minutes at 1007C. After the vortexing, cell lysates containing the DNA extract were centrifuged to remove cellular debris. Cell lysates were stored at 0207C until use. Ten to 30 mL of each cell lysate supernatant was subjected to PCR amplification with use of the following as single primers: the core sequence of phage M13 (M13 core; 5*-GAGG GTGGCGGTTCT-3*) and a nonspecific primer derived from t-DNA intergenic spacer (T3B 5*-AGGTCGCGGGTTCGA ATCC-3*) [7, 8]. An ultrapure preparation of Escherichia coli strain B DNA (Sigma, St. Louis) was used as a positive control for PCR amplification. Amplification reactions and absorbance profiles were analyzed as previously described [9]. Results Biochemical reactions for all the clinical isolates matched the profile previously described [2]. All strains grew on BBE, reduced nitrate, and were positive for catalase and arginine aminopeptidase. Urease activity was variable among the strains: RMA 7355 and RMA 8069 were urease-positive; RMA 8070 and RMA 8068 were weakly urease-positive; and RMA 8066 was urease-negative. PCR fingerprint analysis of the two B. wadsworthia isolates, RMA 8070 and RMA 8068, produced identical fingerprint profiles with use of the T3B single primer (figure 1B). Analysis of three additional B. wadsworthia clinical isolates demonstrated that these strains exhibited a major band and two minor bands in common with B. wadsworthia ATCC 49260 and the two strains RMA 8070 and RMA 8068 (figures 1A and 1B). Nevertheless, on the basis of the presence or absence and size of three additional major bands, these three additional isolates demonstrated unique PCR fingerprint profiles, distinct from those of RMA 8070 and RMA 8068 as well as each other. The ATCC type strain, although not identical to any of the clin (...truncated)