Selective medium for Branhamella catarrhalis with acetazolamide as a specific inhibitor of Neisseria spp.

Several semiselective media for Branhamella catarrhalis have been proposed. These media allow growth of all members of the family Neisseriaceae, and further differentiation is necessary. By addition of 10 micrograms of acetazolamide, a carbonic anhydrase inhibitor, per ml and incubation in air, a medium was created which reduced growth of Neisseria spp. When saliva samples from 178 healthy schoolchildren were screened for the presence of B. catarrhalis, the carrier rate for this organism was estimated to be 48.9% with the selective medium compared with 12.4% when a semiselective medium, which contains only 10 micrograms of vancomycin, 5 micrograms of trimethoprim, and 2 micrograms of amphotericin B per ml, was used and 6.2% when a nonselective blood agar plate was used. The number of Neisseria spp. isolated dropped from 297 on the semiselective agar to 55 on the selective agar.     

API QuadFERM+ with rapid DNase for identification of Neisseria spp. and Branhamella catarrhalis.

The QuadFERM+ system (Analytab Products, Plainview, N.Y.), a 2-h carbohydrate degradation method for the identification of Neisseria spp., was evaluated along with a rapid DNase test for confirmation of Branhamella catarrhalis. QuadFERM+ identified 100% of 82 N. gonorrhoeae and 96% of 54 N. meningitidis strains. The two misidentified meningococcal strains were biochemically atypical and were also misidentified by the conventional method. Of 26 N. lactamica strains, 25 (96%) were correctly identified. Of 21 Neisseria spp., 14 (67%) produced carbohydrate reactions in agreement with the conventional procedure, and 7 strains produced detectable acid in the QuadFERM+ from maltose and sucrose but not glucose. All 9 N. cinerea and 30 B. catarrhalis strains were asaccharolytic by QuadFERM+. The rapid DNase test was positive for all B. catarrhalis strains and negative for all other organisms. Two beta-lactamase-positive N. gonorrhoeae strains and 25 (93%) of 27 beta-lactamase-positive B. catarrhalis strains were detected by the 2-h acidometric beta-lactamase test on the strip. QuadFERM+ with rapid DNase is a simple and easily interpretable method for identification of these organisms in the clinical laboratory.     

Use of the API NeIdent system for identification of pathogenic Neisseria spp. and Branhamella catarrhalis.

The API NeIdent system (Analytab Products, Plainview, N.Y.) was evaluated for identifying Neisseria spp. and Branhamella catarrhalis commonly isolated from clinical specimens. The system identified 90% of 303 Neisseria gonorrhoeae isolates, 71% of 113 Neisseria meningitidis isolates, and 63% of 16 Neisseria lactamica isolates but failed to identify any of 22 B. catarrhalis isolates. Testing of gonococcal strains of various auxotypes revealed no relationship between nutritional requirements and NeIdent profile numbers. With the Neisseria species, interpretation of the cinnamaldehyde-coupled beta-naphthylamine reactions was difficult and resulted in profile numbers not listed in the Profile Register. Positive resazurin-glucose reactions resulted in unlisted numbers for all B. catarrhalis strains. Inconsistent results were also obtained when 62 N. gonorrhoeae isolates were tested more than once on the strip. In all cases, profile variability and failure to identify these organisms were related to the beta-naphthylamide substrate tests. Expansion of the data base and modification of the substrate formulations or their interpretive criteria may increase the reliability of the NeIdent system for identifying Neisseria spp. and B. catarrhalis.     

Otitis media caused by beta-lactamase-producing Branhamella (Neisseria) catarrhalis.

A beta-lactamase-producing Branhamella catarrhalis was isolated in pure culture from the right middle ear aspiration of an otitis media in a 3-month-old girl. The patient responded well to cefamandole treatment.     

Rapid identification of pathogenic Neisseria species and Branhamella catarrhalis.

Two systems, the Identicult-Neisseria (IDN; Scott Laboratories, Inc., Fiskeville, R.I.) strip and the Neisseria/Haemophilus Identification Test Kit (NHI; Vitek Systems, Inc., Hazelwood, Mo.) card, were compared with the 4-h Minitek system (BBL Microbiology Systems, Cockeysville, Md.) for their ability to rapidly identify 157 pathogenic Neisseria and Branhamella catarrhalis isolates. IDN, limited in its identification to four species, when incubated at 35 degrees C for 10 min identified 99% of the isolates. However, when IDN was incubated at 22 degrees C for 20 min, it identified only 92% of the isolates. The NHI card, a rapid semiautomated system with the ability to identify 25 organisms to the species level, correctly identified all of the isolates. A test for beta-lactamase production included in the NHI card identified the 12 Neisseria gonorrhoeae and 10 B. catarrhalis beta-lactamase-positive isolates included in the study. The IDN strip (35 degrees C) and the NHI card compared favorably with the Minitek system.     

Branhamella (Neisseria) catarrhalis: criteria for laboratory identification.

Eleven clinically significant isolates of Branhamella catarrhalis grew well on modified Thayer-Martin medium and produced beta-lactamase, but did not grow on nutrient agar at 22 degrees C. Minimum inhibitory concentrations of vancomycin, colistin, and trimethoprim were found to be higher than the concentrations of these antibiotics in modified Thayer-Martin medium. The criteria necessary for laboratory identification of B. catarrhalis are discussed.     

Characterisation of Branhamella catarrhalis and differentiation from Neisseria species in a diagnostic laboratory.

To distinguish Branhamella catarrhalis from Neisseria species a study of 140 strains was made on simple laboratory media, with particular reference to deoxyribonuclease (DNase) production, superoxol reaction, and growth characteristics. All 97 clinical isolates of B catarrhalis (58 of which were beta-lactamase positive) and eight strains of B catarrhalis from the National Collection of Type Cultures were DNase positive and superoxol positive. None grew on modified New York City medium, modified Thayer Martin medium, MacConkey agar, crystal violet blood agar, nor under anaerobic conditions. Of the 16 different non-pathogenic Neisseria species tested, all were DNase negative, eight (50%) were superoxol reaction negative, and 13 (81%) grew on crystal violet blood agar. Using simple laboratory media, DNase, and superoxol tests, it was possible to identify B catarrhalis and to distingish it from pathogenic and non-pathogenic Neisseria species.     

Pseudogonococcal ophthalmia neonatorum. Branhamella (Neisseria) catarrhalis conjunctivitis.

A culture from conjunctivitis occurring in a neonate in association with a recurrent fever yielded a nearly pure growth of Branhamella (Neisseria) catarrhalis. The conjunctivitis was not appreciated and effectively treated until a second hospitalization in the fourth week of life. Partial suppression of symptoms had followed short-term parenteral antibiotic therapy during the first admission. Resolution quickly occurred in response to instillation of sodium sulfacetamide ophthalmic solution. Although B. catarrhalis is considered a non-pathogen, the literature reviewed included a number of diverse infections, but no previous instance of conjunctivitis. The organism's close similarities to Neisseria gonorrhoeae necessitate isolation and correct biochemical differentiation. Misdiagnosis of gonococcal conjunctivitis carries obvious social, psychological and medical impact.     

Lack of immunoglobulin A1 protease production by Branhamella catarrhalis.

Clinical isolates of Branhamella catarrhalis from the sputum of 20 patients with acute bronchopulmonary infection were examined for synthesis of immunoglobulin A1 protease by immunoelectrophoresis. Ten strains produced beta-lactamase, and 10 were beta-lactamase negative. None of the strains demonstrated immunoglobulin A1 protease activity despite the fact that three different culture media were used.     

Evaluation of the RIM-N, Gonochek II, and Phadebact systems for the identification of pathogenic Neisseria spp. and Branhamella catarrhalis.

Methods for identifying Neisseria spp. include conventional and modified carbohydrate degradation procedures, chromogenic enzyme substrate tests, and immunologic coagglutination tests for Neisseria gonorrhoeae. In this study, we evaluated the abilities of the RIM-N carbohydrate degradation system (American MicroScan, Campbell, Calif.), the Gonochek II enzymatic identification system (Du Pont Co., Wilmington, Del.), and the Phadebact Gonococcus coagglutination test (Pharmacia Diagnostics, Piscataway, N.J.) to identify pathogenic Neisseria spp. and Branhamella catarrhalis. Both stock strains and clinical isolates, including 176 N. gonorrhoeae, 173 Neisseria meningitidis, 48 Neisseria lactamica, and 12 B. catarrhalis strains, were tested. The RIM-N identified 98% of the gonococci, 99% of the meningococci, 94% of the N. lactamica strains, and 100% of the B. catarrhalis strains within 1 h. The Gonochek II system identified 99% of the gonococci, 97% of the meningococci, 100% of the N. lactamica strains, and 100% of the B. catarrhalis strains within 30 min. Phadebact coagglutination provided clearly positive results for only 77% of the N. gonorrhoeae strains, producing negative or equivocal results with 23% of the strains. The RIM-N and Gonocheck II tests generally produced clear-cut reactions. An additional advantage of the Gonocheck II system was the small inoculum required for the performance of the test compared with the other systems, thus allowing the identification of N. gonorrhoeae directly from the primary isolation medium.     

Comparative immunochemistry of lipopolysaccharides from Branhamella catarrhalis strains.

Lipopolysaccharides (LPS) were extracted and purified from the type strain and from a clinical isolate of Branhamella catarrhalis. Chemical analysis revealed the presence of glucose, galactose, and glucosamine in different molar proportions in the LPS from these two isolates, whereas there was no difference between the two isolates in the ratios of ketodeoxyoctonate, phosphate, and the fatty acids C12, 3-OH-C12, and 3-OH-C11 present. Heptose or 3-OH-C14 was not detectable in either preparation. LPS from both strains appeared semirough according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, presenting a core polysaccharide plus one repeating unit. Immunoblotting, passive hemolysis, and hemolysis inhibition assays using anti-LPS antibodies from immunized rabbits demonstrated cross-reactivity between the LPS preparations; however, antigenic dissimilarities were also found, suggesting that more than one serotype may exist. The lipid A isolated from the two LPS was serologically identical and exhibited cross-reactivity with lipid A of members of the family Enterobacteriaceae. The B. catarrhalis LPS were biologically active, causing lethality in D-galactosamine-sensitized C57/BL6 mice and inducing Limulus amoebocyte lysate gelation.     

Rapid method for differentiating strains of Branhamella catarrhalis.

The ability of Branhamella catarrhalis to cause nosocomial infections is a matter of some controversy. The API ZYM research kit for detecting 89 enzymes was used on 49 isolates of B catarrhalis to select enzymes of potential use in differentiating clinical isolates. Twenty nine enzymes were produced by all isolates (13 strongly positive) and many of these were esterases; 16 enzymes were not detected in any isolate (40 if a more stringent criterion was used). Twenty enzymes were selected to form a prototype biotyping panel which allowed 17 different patterns of reactivity to be recognised. Of the 49, 34 isolates were confined to the three commonest patterns. Only one isolate was untypable using this panel due to lack of reactivity. A kit with these 20 substrates may be sufficiently discriminatory to be useful in the rapid study of outbreaks of infection caused by B catarrhalis.     

Profiles of amino-acid utilisation and production amongst strains of Branhamella catarrhalis

Utilisation and production of amino acids by isolates of Branhamella catarrhalis was studied by ion exchange chromatography after cells had been grown in nutrient broth and Mueller-Hinton broth. The profiles of amino acids used and produced by each strain were compared by a single linkage cluster algorithm. The results of this study reflect the biochemical and physiological heterogeneity amongst strains of B. catarrhalis.     

Isolation of Branhamella catarrhalis from sputum and tracheal aspirate

Branhamella catarrhalis was suggested by the presence of gram-negative intracellular diplococci and confirmed by quantitative culture of the expectorated sputa of 11 patients with clinical evidence of lower respiratory tract infection. In quantitative culture, more than 20 colonies of B. catarrhalis were seen in each of the liquefied expectorated sputa at a dilution of 10(-7). Transtracheal aspirations were then performed on these 11 patients, and B. catarrhalis was isolated from the lower respiratory tract secretions of 10 of the patients. Of the 10 isolates, 5 produced beta-lactamase. None of the isolates grew on modified Thayer-Martin medium. The presence of gram-negative intracellular diplococci and the growth of B. catarrhalis on quantitative culture of expectorated sputum reliably indicates the presence of B. catarrhalis in the lower respiratory tract.     

Evaluation of a rapid method for identifying Branhamella catarrhalis.

The speed and precision of hydrolysis of tributyrin to butyric acid as a test to detect Branhamella catarrhalis were evaluated. The test proved consistently reliable in the identification of strains and correctly differentiated B catarrhalis from Neisseria sp. The combination of Gram stain, oxidase, and catalase tests to tributyrin hydrolysis provides a means of positive same day identification of B catarrhalis.     

Activation of B chronic lymphocytic leukaemia cells by Branhamella catarrhalis.

Cells from the blood of patients with chronic lymphocytic leukaemia were cultured in the presence of two polyclonal activators of human B cells, the bacteria Branhamella catarrhalis (Bc) and Staphylococcus aureus Cowan 1 (SAC). Although the magnitude of the responses varied, cells from seven of the eight patients studied were induced to proliferate in response to Bc. In contrast, the response to SAC was low or negligible in seven of the eight patients, and only one patient responded well to this mitogen. Bc was also effective in inducing secretion of IgM in cells from seven of the eight patients, and this was unaffected by removal of T cells. Fractionation of CLL cells on density gradients showed that the highest level of IgM production was induced in cells with a low buoyant density, whilst cells with a high buoyant density secreted little or no immunoglobulin in response to Bc. Together, these results demonstrate that Bc is an effective, T-independent activator of both DNA synthesis and immunoglobulin production in CLL cells.     

B.CAT CONFIRM, a rapid test for confirmation of Branhamella catarrhalis.

B.CAT CONFIRM (Scott Laboratories, Inc., Fiskeville, R.I.), a rapid test for detection of tributyrin hydrolysis, was evaluated for its ability to identify strains of Branhamella catarrhalis and to differentiate them from Neisseria species and related species. On initial testing, B.CAT CONFIRM was positive for 65 (96%) of 68 B. catarrhalis strains within 30 min after inoculation. Retesting of the remaining three strains resulted in their correct identification. B.CAT CONFIRM was negative for all Neisseria spp. (130 strains) and for Kingella spp. (3 strains). Two of the three Moraxella spp. were weakly positive in the B.CAT CONFIRM after 60 min, but these reactions were easily distinguishable from the strong reactions of B. catarrhalis strains. This test will be helpful in the clinical laboratory for the rapid identification of B. catarrhalis in clinical specimens.     

Characterization of Neisseria cinerea, a nonpathogenic species isolated on Martin-Lewis medium selective for pathogenic Neisseria spp.

An asaccharolytic, gram-negative, oxidase-positive diplococcus was isolated on Martin-Lewis medium from the cervix of a patient attending an arthritis clinic at Seattle Public Health Hospital, Seattle, Wash. This strain, NRL 32165, did not produce detectable acid from glucose, maltose, sucrose, fructose, mannitol, or lactose in either cystine Trypticase agar (BBL Microbiology Systems, Cockeysville, Md.) or modified oxidation-fermentation medium and was identified presumptively as a glucose-negative Neisseria gonorrhoeae strain, but was identified later as Neisseria cinerea on the basis of its biochemical reactions. Nitrate was not reduced, nitrite (0.001%, wt/vol) was reduced, and polysaccharide was not produced from sucrose. Proline, arginine, and cystine-cysteine were required for growth on defined medium. Strain NRL 32165 did not react with antigonococcal protein I monoclonal antibodies and did not produce immunoglobulin A protease. In DNA:DNA homology studies with N. gonorrhoeae NRL 8038 (F62) and N. cinerea type strain NRL 30003, strain NRL 32165 showed 95% homology relative to N. cinerea and 44% homology relative to N. gonorrhoeae. Thus, the identity of strain NRL 32165 was confirmed as N. cinerea (von Lingelsheim 1906) Murray 1939. Of all Neisseria spp., N. cinerea is most likely to be misidentified as a glucose-negative N. gonorrhoeae strain.     

Selective medium with DNase test agar and a modified toluidine blue O technique for primary isolation of Branhamella catarrhalis in sputum.

A selective medium with DNase test agar and incorporating vancomycin (10 micrograms/ml), trimethoprim (8 micrograms/ml), and amphotericin B (2 micrograms/ml) supported the growth of 305 Branhamella catarrhalis isolates. A modified toluidine blue O technique was used after 48 h of incubation in CO2 to overlay suspected B. catarrhalis colonies. A metachromatic color change was observed in 15 min, indicating DNase production. In 200 unselected sputum samples of hospitalized patients, this method was compared with routine microbiologic procedures; 31 B. catarrhalis isolates were recovered with the method, compared with 22 isolated from the clinical laboratory. This medium will be particularly useful for culture of sputum, which shows inflammatory cells and gram-negative diplococci on Gram-stained smears.