Nosocomial pneumonia caused by a glucose-metabolizing strain of Neisseria cinerea.

We describe what appears to be the first reported case of nosocomial pneumonia caused by Neisseria cinerea. The isolate metabolized glucose when tested in BACTEC Neisseria Differentiation Kits (Johnston Laboratories), but did not produce detectable acid in cystine-Trypticase (BBL Microbiology Systems) agar medium or in modified oxidation-fermentation medium. Clinical laboratories that rely on the BACTEC method for differentiation of pathogenic neisseriae should be aware of the fact that N. cinerea may mimic N. gonorrhoeae when tested in BACTEC Neisseria Differentiation kits. The ability of N. cinerea to grow well on tryptic soy and Mueller-Hinton agars and its inability to grow on modified Thayer-Martin medium are characteristics which help to distinguish N. cinerea from N. gonorrhoeae.     

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.     

Difficulties in differentiating Neisseria cinerea from Neisseria gonorrhoeae in rapid systems used for identifying pathogenic Neisseria species.

Neisseria cinerea and Neisseria gonorrhoeae may occur at the same body sites and may have similar colony morphologies. Ideally, systems used for rapid identification of N. gonorrhoeae should be able to differentiate N. cinerea from gonococci. We tested seven N. cinerea strains using the Gonochek II (Du Pont Diagnostics), Minitek (BBL Microbiology Systems), RapID-NH (Innovative Diagnostics, Inc.), RIM-N (American Microscan), and Phadebact (Pharmacia Diagnostics) systems. We found that the reactions produced by N. cinerea in Gonochek II, Minitek, and RapID-NH kits could be confused with the results produced by some strains of N. gonorrhoeae. The susceptibility of N. cinerea to colistin, its ability to grow on tryptic soy or Mueller-Hinton agar, and its inability to grow on modified Thayer-Martin medium help differentiate it from gonococci.     

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.     

Prevalence and persistence of Neisseria cinerea and other Neisseria spp. in adults

Neisseria cinerea is a commensal Neisseria sp. which was first described in 1906 but was subsequently misclassified as a subtype of Branhamella catarrhalis. N. cinerea resembles Neisseria gonorrhoeae in both cultural and biochemical characteristics and, thus, may also have been misidentified as N. gonorrhoeae. Of 202 patients whose oropharynges were colonized by Neisseria spp., N. cinerea was isolated in 57 (28.2%) patients, including 25 (30.1%) of 83 women, 22 (23.9%) of 92 heterosexual men, and 10 (37.0%) of 27 homosexual men in Seattle, Wash., in 1983 to 1984. N. cinerea was isolated from the urethra of only one (1.1%) patient. The oropharynges of many individuals were colonized persistently by strains of N. cinerea and other Neisseria spp.     

Identification of Neisseria spp., Haemophilus spp., and other fastidious gram-negative bacteria with the MicroScan Haemophilus-Neisseria identification panel.

The Haemophilus-Neisseria identification (HNID) panel (American MicroScan, Sacramento, Calif.) is a 4-h microdilution format system for identification of Haemophilus and Neisseria spp., Branhamella (Moraxella) catarrhalis, and Gardnerella vaginalis. The HNID panel was evaluated by using 423 clinical isolates and stock strains of these organisms, and HNID identifications were compared with those obtained by conventional methods. In addition, 32 isolates representing six genera not included in the HNID data base were tested to determine whether these organisms would produce unique biotype numbers for possible inclusion in the data base. The HNID panel correctly identified 95.3% of 86 Neisseria gonorrhoeae strains, 96% of 25 G. vaginalis strains, and 100% of 28 Neisseria lactamica strains and 48 B. catarrhalis strains. Only 64.7% of 68 Neisseria meningitidis isolates were identified correctly owing to false-negative or equivocal carbohydrate and/or aminopeptidase reactions. Among the Haemophilus spp., 98.8% of 83 H. influenzae strains, 97.1% of 34 H. parainfluenzae strains, and 80% of 15 H. aphrophilus and H. paraphrophilus strains were correctly identified. Eight strains of Neisseria cinerea, a species not included in the data base, produced profiles identical with those for B. catarrhalis and N. gonorrhoeae. Isolates of other species not included in the data base, including Eikenella corrodens, Kingella spp., and Cardiobacterium hominis, produced unique biochemical reaction patterns on the panel. Modification of interpretative criteria for certain tests, expansion of the data base to include other species, and suggestions for additional confirmatory tests will increase the accuracy and utility of the HNID panel.     

The systematic serology of Neisseria gonorrhoeae: antigens associated with pathogenesis in Neisseria spp. from man

Sonicates of eight Neisseria species from man were analysed in a micro-Ouchterlony double-diffusion absorption assay in comparison with a gonococcal reference antiserum-antigen system. Five major gonococcal precipitin zones were identified which comprised genus-, species- and type-specific components. One antigen was found in all strains of three species with pathogenic capability--N. gonorrhoeae, N. meningitidis and N. flavescens. It was not detected in N. lactamica, N. pharyngis, N. elongata, N. cinerea or N. catarrhalis.     

Comparison of three methods for identification of pathogenic Neisseria species.

A radiometric procedure was compared with the Minitek and Cystine Trypticase Agar sugar degradation methods for identification of 113 Neisseria species (58 Neisseria meningitidis, 51 Neisseria gonorrhoeae, 2 Neisseria lactamica, 2 Neisseria sicca). Identification of meningococci and gonococci was confirmed by agglutination and fluorescent antibody techniques, respectively. The Minitek method identified 97% of meningococci, 92% of gonococci, and 100% of other Neisseria after 4 h of incubation. The radiometric (Bactec) procedure identified 100% of gonococci and 100% of miscellaneous Neisseria after 3 h, but problems were encountered with meningococci: 45% of the later strains yielded index values for fructose between 20 and 28 (recommended negative cut-off point, less than 20), with strongly positive (greater than 100) glucose and maltose and negative o-nitrophenyl-beta-D-galactopyranoside reactions in all 58 strains. The Cystine Trypticase Agar method identified 91% of meningococci, 90% of gonococci, and 100% of other Neisseria after 24 to 48 h. Prolongation of the Cystine Trypticase Agar incubation period led to abnormal lactose/sucrose reactions in some meningococci and gonococci. Radiometric and Minitek systems are more accurate and convenient than Cystine Trypticase Agar techniques, but, on the basis of these results, radiometric fructose sensitivity levels for meningococci need reevaluation.     

Distribution of an antigenically related iron-regulated protein among the Neisseria spp.

Several iron-regulated proteins of Neisseria gonorrhoeae have been reported. One of these, a 37,000-molecular-weight protein (37K protein), appears to be common to all gonococcal isolates. Recently, the occurrence of a similar protein has also been noted in N. meningitidis. The gonococcal 37K protein has been purified and used to produce both rabbit monospecific antiserum and murine monoclonal antibodies. Using these antibody reagents, we analyzed 57 strains from nine species of Neisseria and the closely related organism Branhamella catarrhalis for the presence of proteins antigenically related to the gonococcal 37K protein. Strains grown on medium with low iron content were probed for antigenic reactivity by Western blot techniques and an enzyme-linked immunosorbent assay. Proteins which cross-reacted with the rabbit monospecific antiserum were designated as AgR-37K proteins. The data indicated that the AgR-37K proteins were conserved among the 40 strains of N. gonorrhoeae, N. meningitidis, N. lactamica, and N. cinerea tested. Seventeen strains from other species of Neisseria and Branhamella did not express AgR-37K proteins with the exception of one N. subflava isolate. All AgR-37K proteins appeared to be regulated by the amount of available iron in the growth medium. Murine monoclonal antibodies were used to probe the antigenic heterogeneity of the AgR-37K proteins from different Neisseria spp. Two of seven monoclonal antibodies were broadly cross-reactive, recognizing the AgR-37K proteins from all species examined. The remaining five monoclonal antibodies were more discriminating, recognizing the AgR-37K proteins from certain species. The antigenic conservation of these AgR-37K proteins, particularly among the pathogenic members of the genus Neisseria, may imply that these proteins serve a common function in pathogenicity.     

Evaluation of substrates for radiometric detection of bacteria in blood cultures.

Various 14C-labeled substrates were evaluated for their potential use in blood culture media. These uniformly labeled compounds were added to hypertonic and anaerobic formulations of modified Columbia broth and compared with analogous BACTEC media with the BACTEC 460. Different bacterial species gave significant growth indices when 2.0 microCi of labeled glucose, glutamic acid, aspartic acid, arginine, or formate was used alone or in combinations in the experimental media. The combination of glucose, glutamic acid, and sodium formate was selected, and simulated blood cultures with representative aerobic, facultative, and anaerobic bacteria and a yeast were compared with BACTEC vials. Under these conditions, the experimental media often became positive several hours earlier than the BACTEC vials and usually produced higher growth indices.     

Electron capture gas chromatographic detection of acethylmethylcarbinol produced by neisseria gonorrhoeae.

Acetylmethylcarbinol (acetoin) production by Neisseria gonorrhoeae and other Neisseria species was established by gas-liquid chromatography and by mass spectrometric data. Sixty-nine isolates of Neisseria were tested by incubating them in a chemically defined fluid medium. The medium was extracted with organic solvents and derivatized with heptafluorobutryic anhydride for gas chromatography and mass spectrometry. Cultures of 58 of the same strains were tested with the conventional Voges-Proskauer reagents, and results were compared with those of gas-liquid chromatography. When glucose was used as an energy source, N. gonorrhoeae, some N. meningitidis, and N. lactamica produced enough acetoin in 16 h to be detectable by either method, whereas other Neisseria species produce amounts detectable only by gas chromatography. The conventional acetylmethylcarbinol test with the chemically defined medium and maltose as an energy source might be used to develop methods that would differentiate certain members of the genus, including the pathogenic species.     

Evaluation of eight methods for identification of pathogenic Neisseria species: Neisseria-Kwik, RIM-N, Gonobio-Test, Minitek, Gonochek II, GonoGen, Phadebact Monoclonal GC OMNI Test, and Syva MicroTrak Test.

The performance of eight methods in identifying Neisseria species, particularly N. gonorrhoeae, was evaluated. These methods included four rapid carbohydrate utilization tests (Gonobio-Test, Neisseria-Kwik, RIM-N, and Minitek); the Gonochek II, a test which is based on the utilization of chromogenic substrates; and three monoclonal antibody tests (Syva MicroTrak, GonoGen, and Phadebact Monoclonal GC OMNI Test). In all, 182 isolates comprised in six species of Neisseria as well as Branhamella catarrhalis and Moraxella sp. were tested. Cystine-tryptic digest agar supplemented with sugars was included for reference purposes. In the carbohydrate utilization tests, the sensitivity and specificity of the Neisseria-Kwik and Minitek tests for the identification of N. gonorrhoeae were 100%. This compared with sensitivities and specificities, respectively, of 100 and 99.1% for the Gonobio-Test and 99.1 and 100% for cystine-tryptic digest agar sugars and the RIM-N test. The sensitivity and specificity of the Gonochek II test were 99.0 and 86.7%, respectively. Although most test kits did not claim to identify all Neisseria species, in several cases isolates of N. subflava were misidentified or could be misinterpreted as N. gonorrhoeae or N. meningitidis. With the monoclonal reagents, the Syva MicroTrak system was 100% sensitive and 100% specific. The GonoGen test was both 99.1% sensitive and specific, while the Phadebact Monoclonal GC OMNI Test was 99.1% sensitive but 91.2% specific. With this latter test, cross-reactions were observed with strains of B. catarrhalis, N. cinerea, and N. lactamica.     

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.     

Serological classification of Neisseria gonorrhoeae with monoclonal antibodies

Hybrid cells producing monoclonal antibodies against antigens of Neisseria gonorrhoeae were obtained by the polyethylene glycol-mediated fusion of mouse myeloma cells and lymphocytes from mice immunized with gonococcal protein I or outer membrane proteins. From four fusions, 16 phenotypically stable, independently cloned hybrid cell lines were selected for continued study. Each of the cell lines produced a characteristically different monoclonal antibody which reacted in immunoprecipitation assays with a unique antigenic determinant on protein I of the outer membrane complex of the bacteria. In antibody binding, immunofluorescence, and coagglutination assays these antibodies each reacted with a restricted group of N. gonorrhoeae strains. None of the monoclonal antibodies reacted with 17 other different species of Neisseria or with Branhamella catarrhalis. When tested on 34 N. gonorrhoeae reference serotyping strains, the monoclonal antibodies demonstrated serological relationships between the strains which paralleled those observed with conventional polyvalent antisera. These antibodies now provide standardized reagents for the rapid and precise serological characterization of many strains of N. gonorrhoeae.     

Further antigenic similarities of Neisseria gonorrhoeae lipooligosaccharides and human glycosphingolipids.

Anticarbohydrate monoclonal antibodies were tested for their ability to bind to various strains of Neisseria. A monoclonal antibody that binds to the ganglio-series glycosphingolipid, ganglio-N-triaosylceramide, also bound to strains of Neisseria gonorrhoeae but not to other species of Neisseria. An antibody specific for the globo-series glycosphingolipid, globotriaosylceramide, also bound to strains of N. gonorrhoeae, Neisseria meningitidis, Neisseria lactamica, and Branhamella catarrhalis but not to any other strains of nonpathogenic Neisseria.     

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.     

Monoclonal antibody that recognizes an outer membrane antigen common to the pathogenic Neisseria species but not to most nonpathogenic Neisseria species.

A hybridoma derived from a mouse immunized with gonococcal outer membranes produced an antibody, designated H.8, that bound to all strains of Neisseria gonorrhoeae and N. meningitidis tested, and to N. lactamica and N. cinerea, but only rarely to other nonpathogenic Neisseria species. Studies with the gonococcal strain used in production of the antibody showed that the antibody bound to a surface-exposed, protease-sensitive, and heat-modifiable outer membrane antigen that we believe is distinct from previously described gonococcal outer membrane proteins.     

Isolation of Neisseria lactamicus from the nasopharynx

During 1971 and 1972, 71 cultures of neisseriae that attacked lactose were received by this laboratory. All strains except one from an eye swab were from the nasopharynx of healthy subjects. Nineteen similar strains from the nasopharynx were isolated in this laboratory.The characteristics of these strains were compared with those of Neisseria meningitidis, Neisseria pharyngis, Neisseria catarrhalis, and Neisseria lactamicus. The 90 strains under investigation closely resembled Neisseria meningitidis but could be differentiated by production of acid from lactose and beta-galactosidase activity and were therefore classified as Neisseria lactamicus.     

Evaluation of rapid carbohydrate degradation tests for identification of pathogenic Neisseria

A total of 156 clinical isolates were tested by the modified rapid fermentation test, the BACTEC Neisseria differentiation kit, and the cystine-Trypticase agar method. The modified rapid fermentation test and BACTEC methods accurately identified at least 95% of 101 strains of N. gonorrhoeae tested and at least 91% of 45 strains of N. meningitidis tested within 4 h. Overall, the cystine-Trypticase agar method was the most accurate (97%) but required as long as 48 h of incubation. The data presented appear to show that rapid carbohydrate degradation tests can provide reliable and specific identification results.     

Proctitis associated with Neisseria cinerea misidentified as Neisseria gonorrhoeae in a child.

An 8-year-old boy developed proctitis. Rectal swabs yielded a Neisseria sp. that was repeatedly identified by API (Analytab Products, Plainview, N.Y.), Minitek (BBL Microbiology Systems, Cockeysville, Md.), and Bactec (Johnston Laboratories, Towson, Md.) methods as Neisseria gonorrhoeae. Subsequent testing in a reference laboratory yielded an identification of Neisseria cinerea. It is suggested that identification of a Neisseria sp. isolated from genital or rectal sites in a child be confirmed by additional serological, growth, and antibiotic susceptibility tests and, if necessary, by a reference laboratory. The implications of such misidentifications are discussed.