Experimental Bacteroides fragilis endocarditis in rabbits

A serum-resistant strain of Bacterioides fragilis that did not produce heparinase was used to study the characteristics of B. fragilis endocarditis in the rabbit experimental model. The infective dose required to produce endocarditis in 50% of rabbits was significantly lower for rabbits with left-sided intracardiac catheters (log10 6.3 colony-forming units +/- 0.6/ml) as compared with right-sided intracardiac catheters (log10 7.7 colony-forming units +/- 0.8/ml). After 3 days of infection, bacterial titers of the tricuspid vegetations were significantly lower than titers of aortic vegetations (P less than 0.01), although at 5 days the titers were similar (P greater than 0.05). The weights of tricuspid vegetations, although similar at 3 days (P less than 0.05). There were no spontaneous deaths during 12 days of infection. In rabbits with the catheter removed before infection, bacterial titers were similar to those titers in rabbits with the catheter continuously in place. This model will permit study of various drug regimens for treatment of this disease.     

Serogrouping of Bacteroides fragilis subsp. fragilis by the agglutination test

The agglutination technique was used to establish a serological classification scheme for 98 strains of Bacteroides fragilis subsp. fragilis isolated from clinical specimens and normal human feces. Absorbed antisera were prepared to seven strains of B. fragilis subsp. fragilis. These seven absorbed antisera were species as well as subspecies specific and provided the basis of the serological classification scheme. This scheme was composed of 21 serogroups; seven of these serogroups contained only one group component. There was a total of 45 serological patterns. This serological scheme may be used for the serological classification of strains of B. fragilis subsp. fragilis and to study the epidemiology of this organism.     

Diagnosis of Bacteroides fragilis infection with counter-immunoelectrophoresis

In a study of 188 patients and 109 controls, the detection of antibody by counterimmunoelectrophoresis was used as a diagnostic aid in human infections with Bacteroides fragilis. It was found that positive results indicated current infection and negative results were not conclusive. The method used was simple, rapid, and easily performed in a routine laboratory, but further work is needed to enhance antigen potency.     

Molecular characterization of a heme-binding protein of Bacteroides fragilis BE1

An iron-repressible 44-kDa outer membrane protein plays a crucial role in the acquisition of heme by the anaerobic bacterium Bacteroides fragilis. The DNA sequence of the gene encoding the 44-kDa protein (hupA) was determined. The hupA gene encodes a protein of 431 amino acid residues with a calculated molecular mass of 48,189 Da. The hupA gene is preceded by an open reading frame of 480 bp that probably encodes a protein with a calculated molecular mass of 18,073 Da. hupA and this open reading frame are likely organized in an operon, and a sequence homologous to the Escherichia coli consensus Fur box was present in the putative promoter region of the operon. Heme-binding studies showed that HupA binds heme. Analysis of the deduced amino acid sequence revealed signature heme-binding consensus motifs, characteristic of heme lyases. Subcellular localization studies in E. coli revealed that HupA was mainly found in the cytoplasmic membrane but not in the outer membrane of E. coli. This suggested that B. fragilis uses another strategy for the translocation of this outer membrane protein across its cell envelope than E. coli does. HupA did not have significant homology with other putative bacterial heme receptors.     

Effect of chilling on the survival of Bacteroides fragilis

Factors affecting the susceptibility of Bacteroides fragilis subsp. fragilis to low temperature were examined. Predetermined numbers of cells were spread on agar media or suspended in enriched Trypticase soy broth and exposed to low temperature under both aerobic and anaerobic conditions. Exposure of 18-h growth of a freshly isolated B. fragilis strain to 4 degrees C aerobically or anaerobically resulted in a loss of at least 50% viability after 12 h. B. fragilis cells in early growth (6 h) were more tolerant to exposure at 4 degrees C than older cells (18 h). When the freshly isolated strain was repeatedly subcultured in the laboratory it was uniformly more cold tolerant than fresh clinical isolates. The incorporation of 1.0 M sucrose and 5 mM magnesium chloride into liquid media partially alleviated the lethal effects of cold temperature on B. fragilis subsp. fragilis.     

Bacteroides fragilis enterotoxin cleaves the zonula adherens protein, E-cadherin

Strains of Bacteroides fragilis associated with diarrheal disease (enterotoxigenic B. fragilis) produce a 20-kDa zinc-dependent metalloprotease toxin (B. fragilis enterotoxin; BFT) that reversibly stimulates chloride secretion and alters tight junctional function in polarized intestinal epithelial cells. BFT alters cellular morphology and physiology most potently and rapidly when placed on the basolateral membrane of epithelial cells, suggesting that the cellular substrate for BFT may be present on this membrane. Herein, we demonstrate that BFT specifically cleaves within 1 min the extracellular domain of the zonula adherens protein, E-cadherin. Cleavage of E-cadherin by BFT is ATP-independent and essential to the morphologic and physiologic activity of BFT. However, the morphologic changes occurring in response to BFT are dependent on target-cell ATP. E-cadherin is shown here to be a cellular substrate for a bacterial toxin and represents the identification of a mechanism of action, cell-surface proteolytic activity, for a bacterial toxin.     

Extrachromosomal elements in a variety of strains representing the Bacteroides fragilis group of organisms

Previous nucleic acid association studies have identified at least nine deoxyribonucleic acid (DNA) homology classes of the Bacteroides fragilis group of organisms. Using these classes as a taxonomic framework, we have screened representative strains of the B. fragilis group for the presence of extrachromosomal (plasmid) DNA. [3H]thymidine-labeled cell lysates were subjected to sodium dodecyl sulfate-salt precipitation, and supernatant fractions from such preparations were analyzed using cesium chloride-ethidium bromide equilibrium centrifugation. One strain from each group was examined in this fashion. Five of the strains were judged to contain no detectable plasmid DNA; however, four strains were observed to yield satellite bands corresponding to covalently closed circular plasmid DNA. Plasmid DNA from such gradients was subjected to velocity sedimentation through both neutral and alkaline sucrose gradients to determine molecular size. A 23 X 10(6)-molecular-weight plasmid was found in a B. fragilis strain representing one DNA homology group of this species, whereas a 3 X 10(6)-molecular-weight plasmid was found in a B. fragilis strain representing a second homology group. Similarly, a 31 X 10(6)-molecular-weight plasmid was found in a Bacteroides thetaiotaomicron strain representing one DNA homology group of this species, whereas a 3 X 10(6)-molecular-weight plasmid was found in a B. thetaiotaomicron strain representing a second homology group. In all instances, the small-molecular weight plasmids were present to the extent of about 15 copies per chromosomal equivalent, whereas the large plasmids were present to the extent of approximately 1 copy per chromosomal equivalent. The biological function of these plasmids is unknown.     

Molecular characterization of the fragilysin pathogenicity islet of enterotoxigenic Bacteroides fragilis

Enterotoxigenic strains of Bacteroides fragilis produce an extracellular metalloprotease toxin (termed fragilysin) which is cytopathic to intestinal epithelial cells and induces fluid secretion and tissue damage in ligated intestinal loops. We report here that the fragilysin gene is contained within a small genetic element termed the fragilysin pathogenicity islet. The pathogenicity islet of B. fragilis VPI 13784 was defined as 6,033 bp in length and contained nearly perfect 12-bp direct repeats near its ends. Sequencing across the ends of the pathogenicity islet from two additional enterotoxigenic strains, along with PCR analysis of 20 additional enterotoxigenic strains, revealed that the islet is inserted at a specific site on the B. fragilis chromosome. The site of integration in three nontoxigenic strains contained a 17-bp GC-rich sequence which was not present in toxigenic strains and may represent a target sequence for chromosomal integration. In addition to the fragilysin gene, we identified an open reading frame encoding a predicted protein with a size and structural features similar to those of fragilysin. The deduced amino acid sequence was 28.5% identical and 56.3% similar to fragilysin and contained a nearly identical zinc-binding motif and methionine-turn region.     

Prevalence of enterotoxigenic Bacteroides fragilis in children with diarrhea in Japan

In age-matched controlled studies performed in Japan, enterotoxigenic Bacteroides fragilis was isolated from 14.9% of 114 children aged 1 to 14 years with antibiotic-unassociated diarrhea (AUD) and 6.5% of 108 children aged 1 to 6 years with antibiotic-associated diarrhea (AAD). The difference in comparison with control children, was significant for AUD children but not AAD children.     

Antibiotic sensitization using biphenyl tetrazoles as potent inhibitors of Bacteroides fragilis metallo-beta-lactamase

BACKGROUND: High level resistance to carbapenem antibiotics in gram negative bacteria such as Bacteroides fragilis is caused, in part, by expression of a wide-spectrum metallo-beta-lactamase that hydrolyzes the drug to an inactive form. Co-administration of metallo-beta-lactamase inhibitors to resistant bacteria is expected to restore the antibacterial activity of carbapenems. RESULTS: Biphenyl tetrazoles (BPTs) are a structural class of potent competitive inhibitors of metallo-beta-lactamase identified through screening and predicted using molecular modeling of the enzyme structure. The X-ray crystal structure of the enzyme bound to the BPT L-159,061 shows that the tetrazole moiety of the inhibitor interacts directly with one of the two zinc atoms in the active site, replacing a metal-bound water molecule. Inhibition of metallo-beta-lactamase by BPTs in vitro correlates well with antibiotic sensitization of resistant B. fragilis. CONCLUSIONS: BPT inhibitors can sensitize a resistant B. fragilis clinical isolate expressing metallo-beta-lactamase to the antibiotics imipenem or penicillin G but not to rifampicin.     

Bacteremia caused by the Bacteroides fragilis group in patients with hematologic diseases

During a 22-year period, 13 patients with hematologic diseases developed bacteremia caused by the Bacteroides fragilis group, with a frequency which remained almost unchanged. Nine patients (69%) had polymicrobial infections. Acute leukemia was the most common underlying disease. The lower intestinal tract (necrotizing enterocolitis and anorectal abscesses) was the most common source of infection. Prior antibiotic therapy was the most frequent host condition before bacteremia, followed by cancer chemotherapy, neutropenia, thrombocytopenia and hypoproteinemia. Septic shock occurred only in seven patients with polymicrobial infections. Six patients, including five with shock, died within a week of onset, while the other seven survived for at least three weeks. Despite its clinical similarity to aerobic gram-negative infection, bacteremia due to the B. fragilis group may well, therefore, be suspected particularly when neutropenic patients who present with lower intestinal symptomatology develop a persistent fever unresponsive to the initial empiric antibiotic therapy.     

Escherichia coli meningitis in a 16-month old infant revealing a posterior fossa epidermoid cyst

Surgical management of patients with simultaneous coexisting malignancy of the digestive organs and an abdominal aortic aneurysm (AAA) remains controversial. In the five patients who underwent the aneurysmectomy first, no complications developed after an aneurysmectomy and a resection of malignancy could be performed within 4 weeks, whereas postoperative complications after the resection of malignancy developed in two of them. Two patients underwent a one-stage operation, in which one was unable to tolerate the two procedures, and no postoperative complications were seen; however, one patient with cardiac dysfunction who first underwent an aneurysmectomy died 3 months after operation due to cardiac and renal failure. These results indicate that the aneurysmectomy first is preferred, when such patients do not have absolute indications of malignancy or AAA; however, a one-stage operation should be chosen when the patients show a disturbance of key organs.     

Structural elucidation of the capsular polysaccharide of Bacteroides fragilis strain 23745M1

The capsule of Bacteroides fragilis (ATCC23745) consists of two distinct polysaccharides, the separation of which could not be accomplished. The mouse-passaged strain (23745M1), however, yielded a preponderant polysaccharide which was isolated and purified. Using mainly high resolution NMR spectroscopy, the structure of the polysaccharide was elucidated and it is composed of the following repeating unit: [formula see text]     

Purification, characterization, and kinetic studies of a soluble Bacteroides fragilis metallo-beta-lactamase that provides multiple antibiotic resistance

Resistance to multiple beta-lactam antibiotics traced to the expression of Zn(II) requiring metallo-beta-lactamases has emerged in clinical isolates of several bacterial strains including Bacteroides fragilis, a pathogen commonly found in suppurative/surgical infections. A soluble B. fragilis metallo-beta-lactamase has been purified to homogeneity from the cell growth medium after expression as a secretory protein in Escherichia coli. The enzyme requires two tightly bound Zn(II) ions for full activity, and the Zn(II) ions can be removed by EDTA from the enzyme. The apoenzyme is reactivated by stoichiometric amounts of Zn(II) and Co(II) ions. The Co(II)-substituted enzyme exhibits a UV-visible spectrum characterized by strong Co(II) d-d transitions at 510, 548, 615, and 635 nm and an EPR spectrum with g values of 5. 52, 4.25, and 2.01: features that serve as useful spectroscopic handles for the mechanistic studies of the enzyme. Although steady-state and transient-state kinetic studies of the soluble Zn(II) enzyme with nitrocefin as substrate found no ionizable groups with pKa values between 5.25 and 10.0 involved in catalysis, a kinetically significant proton transfer step in turnover was implicated by studies in deuterium oxide. These studies also detected the accumulation of an enzyme-bound intermediate and provide the basis for a minimal kinetic scheme describing metallo-beta-lactamase-catalyzed nitrocefin hydrolysis.     

High-yield expression, purification, and characterization of active, soluble Bacteroides fragilis metallo-beta-lactamase, CcrA

Two new areas of anchor development are biodegradable anchors and "mini" anchors. The group of biodegradable anchors tested include the Bio-Anchor, LactoSorb, Biofix, Bio-Statak, Mini Screw suture anchor, DePuy 4.5 molded, DePuy 4.5 machined, DePuy 3.5 machined, TAG Wedge 4, TAG Rod 2, TAG Wedge 3, TAG Wedge 2, and Stealth. "Mini anchors" have drill holes or minor diameters of < 2.2 mm. Those tested include the Mini Revo and Bio-Anchor, miniHarpoon, mini Mitek and Fast in 3, Statak 1.5 and 2.5, SB 2 and PeBA 3, Corkscrew 5, Corkscrew 3.5, and Fastak A2, Ogden 2.5, TAG Wedge 2, ROC 1.9, and Questus 2.5. Additional anchors tested that fit neither category include the Anspach, Questus 3.5 and 5.0, SB 3 and PeBA-C, Ogden 3.5, Fast in 4, Ultrafix, and the ROC 3.5, ROC 2.8, ROC 2.3, and ROC XS. An anchor comparison, using an established protocol in fresh porcine femurs, recorded failure strength, failure mode, eyelet size, minor and major diameters, and drill hole sizes. Except for the Bio-Anchor and TAG Wedge 2, biodegradable anchors tend to be larger to compensate for their lower strength relative to metal. Biodegradable screw anchors' predominant failure mode was eyelet cutout, whereas biodegradable nonscrew anchors failed to predominantly by anchor pullout. From an initial mechanical perspective, these biodegradable anchors perform acceptably. Both biodegradable and "mini" anchors include screw and nonscrew designs. As expected, screw designs perform well and generally fail at higher loads than nonscrew anchors. Although biodegradable anchors, as a group, are not as strong as metal anchors, they are stronger than the sutures for which they are designed. The move to smaller ("mini") and biodegradable anchors is supported by these data. Whether an anchor fails at twice the suture breaking strength or 10 times the suture breaking strength should make no difference.     

In vitro activity of josamycin and rosamicin against Bacteroides fragilis compared with clindamycin, erythromycin, and metronidazole

The inhibitory and bactericidal activities of josamycin and rosamicin against 29 clinical isolates of Bacteroides fragilis were compared with those of clindamycin, erythromycin, and metronidazole by a broth dilution technique. Josamycin and rosamicin had similar inhibitory activity to metronidazole and clindamycin. Rosamicin had similar bactericidal activity to clindamycin but was less bactericidal than metronidazole (the most bactericidal agent tested). Josamycin was slightly more bactericidal than erythromycin (the least bactericidal agent tested), but less so than rosamicin and clindamycin.     

Identification and DNA sequence of the mobilization region of the 5-nitroimidazole resistance plasmid pIP421 from Bacteroides fragilis

The nucleotide sequence of the DNA mobilization region of the 5-nitroimidazole resistance plasmid pIP421, from strain BF-F239 of Bacteroides fragilis, was determined. It contains a putative origin of transfer (oriT) including three sets of inverted repeats and two sequences reminiscent of specific integration host factor binding sites. The product of the mobilization gene mob421 (42.2 kDa) is a member of the Bacteroides mobilization protein family, which includes the MobA of pBI143, NBUs, and Tn4555. Sequence similarity suggests that it has both oriT binding and nicking activities. The transfer frequency of pIP421 in a B. fragilis donor strain possessing a Tc(r) or Tc(r) Em(r)-like conjugative transposon was significantly enhanced by tetracycline. Moreover, the mobilization region of pIP421 confers the ability to be mobilized from Escherichia coli by an IncP plasmid.