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.     

Laboratory issues in the detection and reporting of antibacterial resistance

The emergence of antimicrobial resistance among several common bacterial pathogens requires that clinical microbiology laboratories have the ability to promptly and accurately recognize resistance in patients' isolates. Laboratories have several options for performing routine susceptibility testing, including the broth microdilution procedure (with or without instrumentation for test reading), automated instrument systems that provide rapid results, antibiotic gradient diffusion, and disk diffusion procedures. In addition, there are definitive screening tests capable of recognizing resistance to drugs of choice among several common bacterial species based on single drug concentration tests or rapid spot tests. The likely emergence of still newer resistance mechanisms will provide a challenge to clinical microbiologists to devise accurate, yet cost-effective strategies for use in the future.     

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.     

Transport of 2-deoxy-D-galactose in Saccharomyces fragilis

2-Deoxy-D-galactose (dGal) transport in Saccharomyces fragilis is characterized by energy requirement and accumulation of the free sugar against a concentration gradient, indicating active transport. Besides free sugar dGal-1-phosphate, UDP-dGal and a trehalose-like derivative were found inside the cells. The accumulation of the phosphorylated derivatives was balanced by a concomitant decrease of ATP, orthophosphate and polyphosphates. With pulse labeling experiments it could be shown that the free sugar is transported into the cells. This conclusion was supported by several other experimental results, e.g. the lack of correlation between the sugar transport parameters and the dGal phosphorylation capacity, and the countertransport of free dGal evoked by galactose in the medium. The typical differences between this active transport mechanism and the transport-associated phosphorylation system, described previously, are discussed.     

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.     

Bacteroides fragilis meningitis revealing a meningorectal fistula

Cases of meningitis due to Bacteroides fragilis are rare; we report a case revealing a meningorectal fistula. CASE REPORT: A 2-month-old infant developed a severe sepsis syndrome following a rectosigmoidoscopy for rectal bleeding. Lumbar puncture diagnosed bacterial meningitis. Cerebrospinal fluid (CSF) culture evidenced B fragilis with betalactamase. The initial antibiotherapy was changed for imipenem-metronidazole, which is at present the recommended antibiotherapy. Malformation including pre-spinal tumor and meningorectal fistula was evoked on magnetic resonance imaging (MRI) and confirmed by surgery. The outcome was favorable after surgery and antibiotherapy. CONCLUSION: B fragilis meningitis are usually associated with sepsis, whose origin is obvious. In our case, meningitis was isolated, revealing a meningorectal fistula.     

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.     

Production of enterotoxins by Bacteroids fragilis strains--effect of clindamycin

Four B. fragilis strains were examined: one nonenterotoxigenic (NTBF) and three producing enterotoxin (ETBF). The growth of cultures was determined and enterotoxin, which is released to the culture medium during growth of strains, was detected. BHI broth and BHI broth with addition of subinhibitory doses (sub-MIC) of clindamycin were applied. Bacterial cultures were incubated at 37 degrees C for 48 hours. After 4, 8, 16, 24, 48 hours of cultivation, samples of bacterial cultures were collected and the optical density was measured. Then the samples were centrifuged, supernatants were filtered through 0.45 micron filters and concentrated three times with 5000 D ultrafilters. Prepared samples were kept frozen at -70 degrees C until used. The titre of enterotoxin in samples was determined on human colon adenocarcinoma cell line HT 29/C1. Neutralization assay was performed with culture filtrates, which were enterotoxin-positive and with rabbit anti-enterotoxin serum. The results of the experiments indicate that enterotoxin is detected after 16 hours of incubation of ETBF strains. Clindamycin at subinhibitory concentrations (sub-MIC) inhibits the growth of B. fragilis cultures. The antibiotic causes also delay and decrease in enterotoxin production by ETBF strains.     

New azacyclopropene derivatives from Dysidea fragilis collected in Pohnpei

The sponge Dysidea fragilis from Pohnpei contained four azacyclopropene derivatives, (4E)-S-dysidazirine [2], which is the optical enantiomer of the known compound dysidazirine [1], (4Z)-dysidazirine [3], (4E)-S-antazirine [4], and (4Z)-antazirine [5]. The structures of the new compounds were elucidated by interpretation of spectral data.     

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.     

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.     

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.     

Fluorescence properties of reconstituted forms of UDP-glucose 4-epimerase from Saccharomyces fragilis

UDP-glucose 4-epimerase from Saccharomyces fragilis exhibits a very characteristic intense fluorescence with an excitation maximum at 360 nm and an emission maximum at 433 nm. The fluorescence spectrum resembles the fluorescence of free NADH with an apparent blue shift and, although the exact nature of the fluorophore is not known, the protein-bound NAD, which is a coenzyme for this reaction, or its reduced form is obviously involved in the emission of the fluorescence. The fluorphore therefore constitutes part of the active site. The inactivation of epimerase with diazinedicarboxylic acid bis(N,N-diethylamide), a reaction shown in the previous paper to form a disulfide linkage across the subunits, results in a simultaneous and correlated loss of the characteristic fluorescence of the enzyme. Reaction with mercaptoethanol restores the native fluorescence with a 2 nm blue shift in emission maximum. These epxeriments provide additional evidence that the two conformationally vicinal sulfhydryl groups are located at the active site. Unlike the reconstituted enzyme obtained from the diamide-inactivated enzyme, the partialy active enzymes reconstituted from p-chloromercuribenzoate-inactivated and heat-inactivated enzymes fail to show the reappearance of the characteristic native fluorescence. Treatment with N-ethylmaleamide, on the other hand, leads to a form of the inactive enzyme that fully retains its fluorescent properties. A model depicting the minimal changes at the active site during the process of inactivation and reconstitution by these various treatments is presented.     

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.     

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.