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.     

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.     

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.     

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.     

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.     

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]     

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.     

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.     

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.     

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.     

The junction-associated protein, zonula occludens-1, localizes to the nucleus before the maturation and during the remodeling of cell-cell contacts

The junction-associated protein zonula occludens-1 (ZO-1) is a member of a family of membrane-associated guanylate kinase homologues thought to be important in signal transduction at sites of cell-cell contact. We present evidence that under certain conditions of cell growth, ZO-1 can be detected in the nucleus. Two different antibodies against distinct portions of the ZO-1 polypeptide reveal nuclear staining in subconfluent, but not confluent, cell cultures. An exogenously expressed, epitope-tagged ZO-1 can also be detected in the nuclei of transfected cells. Nuclear accumulation can be stimulated at sites of wounding in cultured epithelial cells, and immunoperoxidase detection of ZO-1 in tissue sections of intestinal epithelial cells reveals nuclear labeling only along the outer tip of the villus. These results suggest that the nuclear localization of ZO-1 is inversely related to the extent and/or maturity of cell contact. Since cell-cell contacts are specialized sites for signaling pathways implicated in growth and differentiation, we suggest that the nuclear accumulation of ZO-1 may be relevant for its suggested role in membrane-associated guanylate kinase homologue signal transduction.     

Longitudinal study of susceptibilities of species of the Bacteroides fragilis group to five antimicrobial agents in three medical centers

A total of 579 clinical isolates of the Bacteroides fragilis group collected from three Canadian hospitals were tested for susceptibility to five antimicrobial agents by using an agar dilution method. During the 4-year survey, isolates from intra-abdominal infections were collected from the following sites: abdominal abscesses (48%), peritoneal fluid (39%), blood (10%), and bile (3%). B. fragilis was the most prevalent species (35.4%), followed by B. thetaiotaomicron (19.2%), B. ovatus (15.9%), and B. vulgatus (11%). No metronidazole- or imipenem-resistant strains were found during the survey. Resistance profiles varied among the different species tested: 7.8, 2.9, and 7.3% of B. fragilis strains (n = 205) and 68.1, 17.2, and 9.4% of non-B. fragilis strains (n = 373) were resistant to cefotetan, cefoxitin, and clindamycin, respectively. B. fragilis and B. vulgatus demonstrated lower resistance rates than B. thetaiotaomicron, B. ovatus, B. distasonis, and B. caccae. During the study, rates of resistance to cefotetan and clindamycin fluctuated but rates of resistance to cefoxitin increased, particularly at one center. These data indicate a need to determine the susceptibility patterns of the B. fragilis group periodically at each hospital.     

Phosphorylation of the Drosophila adherens junction protein Armadillo: roles for wingless signal and zeste-white 3 kinase

The Drosophila segment polarity gene product Armadillo provides a link between two seemingly separate processes, regulation of segmental pattern by the Wingless intercellular signal and the function of cell-cell adherens junctions. armadillo was originally identified because of its segment polarity phenotype but subsequently was found to be the homolog of the vertebrate adherens junction protein beta-catenin. We examined the nature of the post-translational modification of Armadillo and its possible role in regulating Armadillo function. Armadillo is a phosphoprotein. Its level of phosphorylation varies both during embryonic development and from tissue to tissue. Phosphorylation occurs on both serine or threonine and tyrosine residues. Finally, Wingless signal negatively regulates Armadillo phosphorylation, while the segment polarity gene product Zeste-white 3, a serine/threonine protein kinase, promotes Armadillo phosphorylation. We discuss the implications of these results for regulation of Wingless/Wnt-1 signaling and adherens junction function.     

1,25-dihydroxyvitamin D3 stimulates the assembly of adherens junctions in keratinocytes: involvement of protein kinase C

Signaling via intercellular junctions plays an important role in the regulation of growth and differentiation of epithelial cells. Loss of cell-cell contacts has been implicated in carcinogenesis, tumor progression, and metastasis. Here, we investigated whether 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] was able to stimulate the assembly of adherens junctions and/or desmosomes in cultured human keratinocytes. After 4-day incubation, 1,25-(OH)2D3 caused assembly of adherens junctions, but not desmosomes. The adherens junctions were identified upon known ultrastructural criteria and evidence of the translocation of specific junctional proteins (E-cadherin, P-cadherin, alpha-catenin, and vinculin) to the cell-cell borders. The presence of alpha-catenin and vinculin at cell-cell borders indicated that the adherens junctions were functional. This was further supported by showing that anti E-cadherin antibody inhibited the 1,25-(OH)2D3-induced keratinocyte stratification. A relation between protein kinase C and adherens junction regulation was noticed. 1,25-(OH)2D3-dependent formation of junctions was blocked by the inhibitors of protein kinase C, bisindolylmaleimide and 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), and treatment of keratinocytes with 1,25-(OH)2D3 caused a rapid activation of protein kinase C and its translocation to the membranes. Formation of intercellular contacts may be an important mechanism of 1,25-(OH)2D3 action in hyperproliferative and neoplastic diseases.     

Crystallization and preliminary crystallographic analysis of RepA1, a replication control protein of the RepFIC replicon of enterotoxin plasmid EntP307

RepA1 protein is essential for replication of the RepFIC replicon of enterotoxin plasmid EntP307 and is thought to interact directly with the origin of replication. We have purified RepA1 from an over-producing expression system and have prepared single crystals using a macroseeding technique. The crystals belong to space group P2(1)2(1)2(1) or P2(1)2(1)2, with cell dimensions a = 61 A, b = 67 A, and c = 243 A. They diffract X-rays to 3.3 A resolution and probably contain two 40,000 molecular weight RepA1 molecules per asymmetric unit.     

Expression of the adherens junction protein vinculin in human basal and squamous cell tumors: relationship to invasiveness and metastatic potential

The acquisition of an invasive or metastatic phenotype in malignant neoplasms is often correlated with reduced cellular adhesiveness. We investigated the expression of the adhesion-associated cytoplasmic protein, vinculin, in normal and neoplastic human squamous epithelia, as well as in metastases of squamous cell carcinomas, and correlated the results with invasiveness and metastatic potential. Tissue samples from various tumors were examined, including basal cell carcinomas (BCC), keratoacanthomas, and squamous cell carcinomas (SCC). In addition, lymph node metastases from nine of the SCC were tested in this study. Our results indicate that most BCC, keratoacanthomas, and in situ SCC display strong positive staining for vinculin. The level of immunolabeling for vinculin and its pattern of distribution in the low malignant, nonmetastasizing lesions was similar to those observed in normal squamous epithelia. In contrast, in SCC, which are invasive and possess metastatic potential, as well as in their metastases, vinculin labeling was negative or poor, irrespective of their degree of differentiation. In conclusion, poor vinculin labeling in tumors of squamous epithelial origin examined here appears to be related to the metastatic potential of the tumor. Vinculin immunostaining of primary tumors originating in stratified squamous epithelia may thus be of value in helping to determine the metastatic potential of these neoplasms.     

Superantigen-based immunotherapy: a phase I trial of PNU-214565, a monoclonal antibody-staphylococcal enterotoxin A recombinant fusion protein, in advanced pancreatic and colorectal cancer

PURPOSE: To establish the maximum-tolerated dose (MTD) and define the toxicities of a single-dose infusion of PNU-214565, a recombinant Escherichia coli-derived fusion protein of Staphylococcal enterotoxin A (SEA) and the Fab-fragment of the C242 monoclonal antibody in patients with advanced colorectal and pancreatic carcinomas. To investigate the capability of PNU-214565 to induce a superantigen (SAg) response resulting in cytokine production and tumor regression. PATIENTS AND METHODS: Twenty-one patients (age range, 39 to 76 years; median, 64; 12 men, nine women; 18 colorectal, three pancreatic cancers) were treated with a single 3-hour infusion of PNU-214565, with doses ranging from 0.01 to 1.5 ng/kg. All patients had prior chemotherapy and a good performance status Eastern Cooperative Oncology Group [ECOG] performance status [PS] = 0 [n = 10]; PS = 1 [n = 11]), 10 had prior radiation, and 18 had prior surgery. RESULTS: Fever and hypotension were the most common toxicities. Fever of any grade occurred in 16 of 21 patients (76%): four of 21 (19%) with grade 2 and two of 21 (9.5%) with grade 3. Hypotension of any grade occurred in 13 of 21 (62%): four of 21 with grade 2 and one of 21 (5%) with grade 3. Interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF alpha) induction correlated with toxicity. In the two patients with grade 3 fever, peak IL-2 and TNF alpha levels were 2.9 IU/mL and 165 pg/mL, and 8.3 IU/mL and 245 pg/mL, respectively. Transient, > or = 50% decreases in circulating monocytes were observed in 17 of 21 patients as early as 0.5 hours (median time, 2 hours) from the start of infusion. Decreases (mean 33%) in circulating lymphocytes were observed in seven of 21 patients. All three patients with grade 3 toxicity were treated at the 0.5-ng/kg dose. The significance of baseline anti-SEA, human antimouse antibody (HAMA), CA242-soluble antigen levels, and T-cell receptor variable beta region (TCR V beta) subsets and histocompatibility leukocyte antigen-DR (HLA-DR) genotypes was assessed as possible predictors of toxicity. All toxicities were transient and easily managed. No grade 3 toxicity occurred at the higher dose levels. CONCLUSION: PNU-214565, a SAg-based tumor targeted therapy, is safe when given as a single 3-hour infusion at doses up to 1.5 ng/kg. The MTD for a single dose was not determined. The safety of a repeated dose schedule is currently under investigation, beginning with doses determined to be safe in this trial.     

Mouse hepatitis virus 3C-like protease cleaves a 22-kilodalton protein from the open reading frame 1a polyprotein in virus-infected cells and in vitro

The 3C-like proteinase (3CLpro) of mouse hepatitis virus (MHV) is predicted to cleave at least 11 sites in the 803-kDa gene 1 polyprotein, resulting in maturation of proteinase, polymerase, and helicase proteins. However, most of these cleavage sites have not been experimentally confirmed and the proteins have not been identified in vitro or in virus-infected cells. We used specific antibodies to identify and characterize a 22-kDa protein (p1a-22) expressed from gene 1 in MHV A59-infected DBT cells. Processing of p1a-22 from the polyprotein began immediately after translation, but some processing continued for several hours. Amino-terminal sequencing of p1a-22 purified from MHV-infected cells showed that it was cleaved at a putative 3CLpro cleavage site, Gln_Ser4014 (where the underscore indicates the site of cleavage), that is located between the 3CLpro domain and the end of open reading frame (ORF) 1a. Subclones of this region of gene 1 were used to express polypeptides in vitro that contained one or more 3CLpro cleavage sites, and cleavage of these substrates by recombinant 3CLpro in vitro confirmed that amino-terminal cleavage of p1a-22 occurred at Gln_Ser4014. We demonstrated that the carboxy-terminal cleavage of the p1a-22 protein occurred at Gln_Asn4208, a sequence that had not been predicted as a site for cleavage by MHV 3CLpro. Our results demonstrate the usefulness of recombinant MHV 3CLpro in identifying and confirming cleavage sites within the gene 1 polyprotein. Based on our results, we predict that at least seven mature proteins are processed from the ORF 1a polyprotein by 3CLpro and suggest that additional noncanonical cleavage sites may be used by 3CLpro during processing of the gene 1 polyprotein.