Rapid presumptive identification of human black pigmented Bacteroides species.

A simple and reliable technique is described for the rapid presumptive identification of black pigmented Bacteroides species of human origin. This method involved a microtitration technique that detected the hydrolysis of specific chromogenic enzyme substrates and haemagglutination of sheep erythrocytes. Pure cultures of black pigmented Bacteroides strains, representing the eight human species, were successfully differentiated and identified within 4 h by the identification scheme developed with this method. This is a highly reproducible method and the scheme should be useful in laboratories lacking the sophisticated equipment often needed for the identification of black pigmented Bacteroides.

The prevalence of beta-lactamase producing bacteria in subgingival plaque and their sensitivity to Augmentin.

Subgingival plaque samples from 20 patients with chronic periodontitis who had received no antibiotics for at least 3 months were screened for the presence of beta-lactamase-producing bacteria. Thirteen of the patients harboured beta-lactamase producing bacteria, most of which were members of the genus Bacteroides. The most frequently isolated species were Bacteroides melaninogenicus and Bacteroides capillosus which are often implicated in acute oral infections. All of the beta-lactamase-producing bacteroides strains were sensitive to a combination of amoxycillin with clavulanic acid (Augmentin).

Proteolytic activity in black-pigmented bacteroides species.

Black-pigmented Bacteroides species are frequently found in dentoalveolar abscesses. One general mechanism of bacterial virulence is the production of extracellular enzymes which degrade connective tissue or molecules associated with host defense. In this study the proteolytic activity of 18 bacterial strains from 9 black-pigmented Bacteroides species was examined. Bacteroides gingivalis degraded the greatest number of substrates studied and produced the highest levels of enzymatic activity. B. gingivalis was the only species that degraded collagen and produced high levels of enzymes that degraded N-benzoyl-DL-arginine (BANA) and N-CBz-glycyl-glycyl-arginine. Bacteroides intermedius degraded several substrates including PZ peptide. Bacteroides endodontalis produced enzymes that degraded beta-naphthylamide derivatives of glycylproline and glycylphenylalanine. There were considerable differences in enzyme production between strains of the same species. Such heterogeneity between strains in the production of proteolytic enzymes may be relevant to the in vivo infections produced in the host.

Use of the RapID-ANA System to screen for enzyme activities that differ among species of bile-inhibited Bacteroides.

The RapID-ANA System (Innovative Diagnostics Systems, Inc., Atlanta, Ga.) was used to test 102 strains of 14 species of phenotypically similar bile-inhibited Bacteroides from humans. Bacteroides oris, Bacteroides veroralis, Bacteroides buccalis, Bacteroides melaninogenicus, Bacteroides loescheii, and Bacteroides denticola had very similar enzyme activity profiles. Clear differentiation of these six species by the RapID-ANA System was not possible, but tests for arginine aminopeptidase and beta-glucosidase were helpful. Bacteroides oralis, Bacteroides intermedius, Bacteroides corporis, Bacteroides disiens, Bacteroides bivius, Bacteroides gingivalis, Bacteroides asaccharolyticus, and Bacteroides buccae each had unique enzyme activity profiles. No consistent differences in enzyme activities were found between the two DNA homology groups within Bacteroides melaninogenicus, Bacteroides loescheii, or Bacteroides intermedius. Tests for glycine aminopeptidase, alpha-galactosidase, arginine aminopeptidase, alpha-fucosidase, N-acetylglucosaminidase, reduction of triphenyltetrazolium, and production of indole were helpful in the differentiation of the species studied.

Purification and characterization of an immunoglobulin A1 protease from Bacteroides melaninogenicus.

Attention has recently been focused on bacterial proteases with the capacity to cleave immunoglobulin A (IgA proteases) as possible pathogenic factors in bacterial meningitis, gonorrhoea, and destructive periodontal disease. Here, we describe a method for the rapid purification of a specific IgA1 protease from Bacteroides melaninogenicus. The IgA1 protease was purified 6,172-fold with a yield of 9% by ammonium sulfate precipitation, DEAE-ion exchange chromatography, and separation on a preparative TSK-G 3000SWG high-pressure gel permeation chromatography column. The enzyme was specific for human IgA1 and cleaved a prolyl-seryl peptide bond in the hinge region of the alpha 1 chain between residues 223 and 224. The molecular weight of the enzyme was 62,000, the isoelectric point was 5.0, and the Km was 3.4 X 10(-6). The enzyme was active over a broad pH range and had maximal activity at pH 5.0. B. melaninogenicus IgA1 protease was classified as a thiol protease on the basis of its inhibition by traditional protease inhibitors and the fact that it was active only under reducing conditions.

An improved method for assessing the incorporation of labeled precursors into DNA by human mononuclear cells.

The blastogenic responsiveness of activated lymphoid cells is usually assessed in vitro by measuring the incorporation of radioactive thymidine or iododeoxyuridine, a thymidine analog, into DNA. The accuracy of this method is compromised by the presence in activated and unactivated lymphocytes and in some of the substances used to activate them, of degradative enzymes which compete with DNA synthetase, the incorporation efficiency of exogenous precursor is inherently low. We have done studies aimed at improving both the efficiency and the accuracy of the assay system by selectively inhibiting the enzymes responsible for thymidylate synthesis de novo and DNA precursor degradation. Culture conditions were investigated and potential inhibitors were tested using human peripheral blood mononuclear cells activated with phytohemagglutinin. Nucleoside-degrading activity of mammalian and bacterial cells is due largely to nucleoside phosphorylases, enzymes that require orthophosphate for activity. We partly inhibited DNA precursor degradation by lowering the phosphate concentration in the culture medium and lowering the pH, thereby reducing the orthophosphate concentration. To reduce precursor degradation further, we tested several potential nucleoside phosphorylase and thymidylate synthetase inhibitors at various concentrations. Our data show that the addition of 1 mM fluorouracil and 1 mM deoxyuridine to the culture medium largely prevents degradation of radioactive thymidine and iododeoxyuridine without unduly compromising the DNA-labeling efficiency of cells activated with mitogens or bacterial homogenates. Under these conditions, label incorporation increases linearly as the number of blast cells or the labeling time increases.

Beta-lactamase producing bacteria in the human oral cavity.

Twenty-six individuals who had no history of long-term antibiotic therapy were examined for the prevalence of beta-lactamase producing bacteria in the oral cavity. Samples from a total of 159 normal and diseased periodontal sites, 44 cheek mucosae, 22 tongue dorsa and 22 salivas were studied. Penicillin resistant organisms were recovered from Trypticase soy blood agar plates containing 1.3 microgram/ml or 2 microgram/ml Benzylpenicillin. Beta-lactamase formation by these isolates was determined using a micro-iodometric assay. Low levels of penicillin resistant organisms were found in all samples. Approximately 10% of the samples yielded Beta-lactamase producing strains. Except for a few Veillonella parvula strains, all Beta-lactamase forming isolates were members of the Bacteroides melaninogenicus subspecies melaninogenicus - Bacteroides oralis group of organisms. These species can produce severe infections and, therefore, the present findings may be important in the clinical management of oral and nonoral infection.

Isolation and characterization of proteases from Bacteroides melaninogenicus.

We isolated two types of intracellular proteases from a strain of Bacteroides melaninogenicus. These enzymes were extracted from cells by ultrasonic treatment and were partially purified. These two enzymes (proteases I and II) differed in molecular weight, heat stability, sensitivity to reducing agents, Km value, and optimum pH for activity.

Isoelectric focusing of Bacteroides melaninogenicus group beta-lactamases.

beta-Lactamases extracted by sonication from the Bacteroides melaninogenicus group organisms (B. asaccharolyticus, B. melaninogenicus, B. bivius, and B. oralis) were found to be in the form of complexes with molecular weights of greater than or equal to 40 x 10(6), and this resulted in failure to characterize them by isoelectric focusing. Purification by el filtration in the presence of deoxycholate resulted in beta-lactamase preparations from B. bivius with pI's of 5.7. A beta-lactamase preparation extracted by osmotic shock from B. bivius also had a pI of 5.7. Osmotic shock preparations from B. asaccharolyticus, B. melaninogenicus, and B. oralis had two bands of equal intensity with pI's of 4.2 and 4.35.

Phospholipase A activity in supernatants from cultures of Bacteroides melaninogenicus.

The phospholipase A activity in culture supernatants of two strains of Bacteroides melaninogenicus is described. The enzyme utilize phosphatidylcholine as substrate and produce mainly lysophosphatidylcholine and free fatty acids. The activities are Ca2+-independent, are not affected by the presence of a chelating agent, have a broad pH range (5-9) and an optimum temperature for activity of approx. 50 degrees C. The activity in a growing bacterial culture increases from the end of the lag phase to the late exponential phase of growth. Analysis of the products resulting from the actions of the enzymes on L-alpha-palmitoyl-beta-oleoyl[1-14C]phosphatidylcholine indicates that the enzymes are phospholipase A1 (EC 3.1.1.32).

Characterization of anaerobic gram-negative bacilli by using rapid slide tests for beta-lactamase production.

A total of 175 isolates of anaerobic gram-negative bacilli were tested for beta-lactamase production by using a slide test modification of the chromogenic cephalosporin (Nitrocefin, Glaxo, Middlesex, England) assay and the iodometric slide test. Included isolates were Bacteroides melaninogenicus (46), B. fragilis (78), other Bacteroides isolates (21), Fusobacterium (25), and other gram-negative bacilli (5). Both slide tests detected 25 B. melaninogenicus isolates that were beta-lactamase producers (minimal inhibitory concentration of penicillin was greater than 0.78 micrograms/ml). beta-Lactamase produced by the other gram-negative anaerobes could only be detected by the Nitrocefin assay. This assay was positive in 70 or 77 B. fragilis against which the minimal inhibitory concentration of penicillin was greater than 0.78 micrograms/ml. Ten of 11 other species of Bacteroides against which the minimal inhibitory concentration of penicillin was greater than 0.78 micrograms/ml were also Nitrocefin test positive. Minimal inhibitory concentrations of penicillin against all isolates of Fusobacterium and unidentifified gram-negative bacilli were less than or equal to 0.78 micrograms/ml and were Nitrocefin assay negative. beta-Lactamase-producing strains of B. melaninogenicus can be differentiated because both the slide iodometric and Nitrocefin assays will be positive, whereas beta-lactamase produced by other Bacteroides will only be detected by the Nitrocefin assay. Such penicillin-resistant isolates could be detected and reported to clinicians before final identification.

Chromogenic cephalosporin spot test to detect beta-lactamase in clinically significant bacteria.

lactamase production. Reacteroides melaniongenicus, 14

Antibiotic susceptibility of anaerobic bacteria with special reference to Bacteroides fragilis.

It was shown that recent Swedish clinical isolates of anaerobic bacteria are susceptible to many antibiotics by the agar dilution method with the exception of the Bacteroides group versus beta-lactam antibiotics or tetracyclines. Strains of B. fragilis were inhibited by 4--greater than 128 micrograms benzylpenicillin or cephalothin/ml, 1.0--64 micrograms cefoxitin/ml, 0.064--2 micrograms clindamycin or metronidazole/ml, 2--8 micrograms chloramphenicol/ml, 2--16 micrograms fusidic acid/ml and 0.032--32 micrograms doxycycline/ml. Resistance to beta-lactam antibiotics was partly due to the production of beta-lactamase. Growth of beta-lactamase producing strains in the presence of enzyme inhibitors such as clavulanic acid or CP-45899 together with cephaloridine lowered the MIC's manyfold. Cefoxitin with relative resistance to beta-lactamases inhibited the majority of the strains at 8 micrograms/ml. Cefoxitin-resistant strains (MIC greater than or equal to 16 micrograms/ml) were also resistant to the new cephalosporins BL-S786 and HR-756 as well as to the new cefamycins A, B, CL619-183, CS-1170 and Sq-14359 and to thienamycin. Cefamycin CL619-183, only showed a slightly higher in vitro activity than cefoxitin. Resistance to the cefamycins could not be correlated to the production of beta-lactamases.