Failure of the fluorescent antibody reaction to identify penicillinase-producing gonococci.

The fluorescent antibody test is now widely used to confirm the identity of Neisseria gonorrhoeae but may fail to identify penicillinase-producing strains (PPNG). This problem arises when conjugates are used that incorporate only gonococci that are not penicillinase-producers. We have shown that conjugates prepared from mixtures of PPNG and non-penicillinase producing gonococci give good fluorescent reactions. This difference in the reactions of PPNG strains is clearly related to their penicillinase-producing abilities, further study of the antigenic relation between penicillinase production and the antigenic structure of N gonorrhoeae is evidently required.     

Sensitivity of Neisseria gonorrhoeae to partially purified R-type pyocines and a possible approach to epidemiological typing.

Strains of Neisseria gonorrhoeae from a variety of sources were examined for sensitivity to 11 partially purified R-type pyocines from Pseudomonas aeruginosa. Selective inhibition of gonococci by pyocines of Kageyama groups R1 and R5 was observed. "Matched isolates", those from consorts or different body sites of individual patients, usually had very similar pyocine-sensitivity patterns and identical sensitivities to five antibiotics tested. This study included local isolates, strains from diverse geographic regions, and strains from disseminated gonococcal infections. It also proposed a relationship between pyocine-receptor sites in the lipopolysaccharide of Ps. aeruginosa and N. gonorrhoeae. Topics needing further evaluation are discussed.     

Evaluation of a fluorescent monoclonal antibody reagent for identification of cultured Neisseria gonorrhoeae

We evaluated a new fluorescent monoclonal antibody reagent for confirmation of identity of Neisseria gonorrhoeae. The reagent correctly identified all 161 fresh clinical isolates of N. gonorrhoeae, which included 11 penicillinase producing strains (PPNG). The reagent also correctly identified 21 stored PPNG strains. No cross reactions were seen with 58 fresh clinical isolates of N. meningitidis, 12 stored strains of N. lactamica, or with strains of Gardnerella vaginalis, lactobacilli, Candida spp., Staphylococcus epidermidis or Enterobacteriaceae. Some cross reaction was noted with strains of S. aureus, probably related to cell-wall protein. A. However, this reagent was highly sensitive and specific for use against oxidase positive, gram-negative cocci isolated in London.     

Differential Attachment by Piliated and Nonpiliated Neisseria gonorrhoeae to Human Sperm

Piliated type 1 Neisseria gonorrhoeae attached to 50% of human sperm after incubation of mixtures in vitro for 15 min at 35 C. In contrast, nonpiliated type 4 N. gonorrhoeae attached to only 23% of sperm. Similar results were obtained with three different strains of N. gonorrhoeae. Treatment with heat or formaldehyde to kill bacteria did not affect the amount of attachment by piliated or nonpiliated types. Escherichia coli and N. subflava, other species of piliated bacteria, attached to about 40% of sperm, and the nonpiliated species N. meningitidis and N. catarrhalis attached to a comparable number of sperm, as did type 4 N. gonorrhoeae. Prior incubation of type 1 N. gonorrhoeae with purified antibody prepared against gonococcal pili reduced the percentage of sperm with attached bacteria to the same level as that for nonpiliated type 4 gonococci. Similar treatment of other piliated organisms or of nonpiliated Neisseria did not affect the attachment of the bacteria to sperm.     

Superoxol and amylase inhibition tests for distinguishing gonococcal and nongonococcal cultures growing on selective media.

Two inexpensive screening tests were evaluated singly and in tandem for distinguishing Neisseria gonorrhoeae from other oxidase-positive microorganisms growing on selective gonococcal media. In tests of 728 cultures, including 460 N. gonorrhoeae, 4 Neisseria lactamica, 257 Neisseria meningitidis, and 7 Branhamella catarrhalis, both Superoxol (30% H2O2; J. T. Baker Chemical Co., Phillipsburg, N.J.) and amylase inhibition tests were 100% sensitive (positive) for 20-h cultures of N. gonorrhoeae. Singly, the Superoxol test was 92.7% specific for N. gonorrhoeae, compared with a specificity of 82.3% for the amylase inhibition test. By using tandem screening tests to distinguish gonococci, we achieved an overall specificity of 98.6%. Group A meningococci were the primary source of error in the Superoxol test, with 97% (37 of 38) strains producing gonococcal like reactions for catalase. From 5 to 20% of N. meningitidis serogroups X, Y, Z, and Z' and nontypable strains, as well as about 50% of B. catarrhalis and N. lactamica strains, were also strong catalase producers.     

Strain differentiation of Neisseria gonorrhoeae by reverse passive hemagglutination.

A reverse passive hemagglutination test that utilizes human erythrocytes coated with antibody to gonococci was developed to distinguish differences among 11 strains of Neisseria gonorrhoeae. Different rabbits were immunized with each strain of gonococcus. Antibody was purified by passing antiserum over an immunoadsorbent column containing homologous cell walls trapped in a cross-linked polyacrylamide gel. Antibody, after absorption with N. meningitidis, was used for coating 11 individual suspensions of erythrocytes, each with antibody to one gonococcal strain. The panel of coated erythrocytes was added to microtiter trays containing dilutions of homologous bacterial lysate and lysates from 10 heterologous strains. Agglutination titers were highest with homologous lysates, although cross-reactions occurred among some heterologous lysates. Lysates of nongonococcal Neisseria species and of other genera did not agglutinate coated erythrocytes. The reverse passive hemagglutination test can be a useful procedure to distinguish differences among strains of N. gonorrhoeae.     

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.     

Prevalence of fluorescent monoclonal antibody-nonreactive Neisseria gonorrhoeae in five North American sexually transmitted disease clinics.

We compared a direct fluorescent monoclonal antibody (DFA) test with alternative enzymatic and fermentation tests for identifying presumptive gonococcal isolates in a systematic sample from patients attending five sexually transmitted disease clinics in five cities. Fourteen (2.5%) of 556 isolates from three clinics were nonreactive with DFA confirmatory reagent and reactive by both the Quad-Ferm (BioMerieux Vitek Inc.) and the Rapid NH (Innovative Diagnostic Systems, Inc.) tests. The prevalence of DFA-nonreactive Neisseria gonorrhoeae isolates varies geographically and is independent of local methods for the identification of possible gonococci.     

Distribution of gonococcal lipopolysaccharide biosynthesis genes among strains of Neisseria gonorrhoeae and other neisserial species

A plasmid, pTME6, containing Neisseria gonorrhoeae lipopolysaccharide biosynthesis genes was used as a probe to analyze DNA from strains of N. gonorrhoeae, N. meningitidis and various commensal Neisseria by Southern blotting. Chromosomal DNA from 26 gonococcal strains probed with 32P-labeled pTME6 produced five different hybridization patterns. No correlation between hybridization pattern and auxotype, serotype, serum sensitivity or SDS-urea-PAGE migration of LPS was observed. DNA from strains of N. meningitidis, N. lactamica and N. cinerea, but not other commensal Neisseria species, hybridized strongly to pTME6.     

Imferon agar: improved medium for isolation of pathogenic Neisseria.

Imferon, an iron-dextran complex, enhances the growth of Neisseria gonorrhoeae and N. meningitidis. The use of Imferon as a replacement for ferric nitrate, in a defined supplement for GC agar significantly increased the average colony sizes of both gonococci and meningococci. In comparison with Thayer-Martin medium, Imferon agar increased the speed and rate of isolation of gonococci from clinical specimens.     

Reevaluation of bacteriocinogeny in Neisseria gonorrhoeae.

Bacteriocin typing has been described previously and proposed for typing gonococci. A survey has been made of 150 strains of N. gonorrhoeae from various places to determine the feasibility of a gonocin typing system. All strains were found to produce an inhibitory substance which inhibited all strains of gonococci tested, one strain of Neisseria flavescens, two strains of Neisseria meningitidis, as well as the producing strain itself. The inhibitory activity was enhanced by supplementary glucose, reduced by supplementary serum, and unaffected by the addition of HEPES buffer, by the temperature of incubation, or by the exposure of potential producer strains to sublethal concentrations of mitomycin C. This nonspecific inhibitory activity differed from that of a putative bacteriocin produced by a strain of N. meningitidis, in that the latter inhibited most other meningococci but not the producer strain itself. Bacteriocinogeny has not yet been convincingly demonstrated in N. gonorrhoeae, and gonocin typing has not yet been shown to be feasible. Production of the nonspecific inhibitor may have obscured past attempts to demonstrate type-specific gonococcal bacteriocin.     

Pathological features of experimental gonococcal infection in mice and guinea pigs.

The histopathological and immunofluorescent findings in tissues within and surrounding artificially created subcutaneous tissue cavities infected with Neisseria gonorrhoeae for 1 to 30 days were studied in mice and guinea pigs. Findings in the tissue cavities of the animal models were similar to the findings of disseminated gonococcal infection in humans. These similarities included an intense persistent polymorphonuclear leukocytic response with tissue necrosis, hemorrhage into the early lesion, a perivascular leukocytic response in adjacent tissue, difficulty in detecting large numbers of discrete morphologically typical gonococci by the tissue Gram stain and direct fluorescent antibody techniques, a decrease in the number of identifiable gonococci with duration of the infection, and moderate amounts of extracellular and intracellular immunofluorescent gonococcal debris. Studies into the pathogenesis of the animal infections may enhance our understanding of the pathogenic mechanism (s) associated with gonococcal infection in humans.     

Laboratory diagnosis of gonococcal infection by genetic transformation.

The transformation test for the detection of infection by Neisseria gonorrhoeae has been examined using pro gonococci as recipients and DNA preparations from 912 clinical isolates and from 240 direct swab specimens as donors. The reliability of the method was checked with DNA from clinical isolates; 82% of the N. meningitidis from throat swab specimens were capable of transforming the gonococcal recipients, but after identification of the meningococcus by the aminopeptidase profile, the transformation test was then 99.5% positive for the gonococcus with virtually no false-positives. The only other organism to give a positive reaction was N. lactamica, which occurred once in 912 specimens. When applied directly to swab specimens, the reliability of the test was reduced, but this may have been related to variability of the specimen itself. However, 7 of 15 specimens which were microscopically suspected to be gonococci but unculturable were positive; also, 9 out of 38 unculturable specimens that were not even suspected to be gonococci were positive. Hence the test was able to identify the presence of gonococci that were unculturable. The aminopeptidase activities were not sensitive enough to be detected in the direct swab specimens, and neither cys nor leu auxotrophs were suitable as recipients to give a differentiation between N. gonorrhoeae and N. meningitidis. Evidence was obtained which would support the proposition that the transfer of genetic material between N. gonorrhoeae and N. meningitidis may occur.     

Gono Gen coagglutination test for confirmation of Neisseria gonorrhoeae.

The Gono Gen (Micro-Media Systems, Inc., Potomac, Md.) coagglutination test was compared with the sugar utilization test and with a direct fluorescent antibody test for confirmation of Neisseria gonorrhoeae. Of 110 gonococcal clinical isolates, 109 were positive by the Gono Gen test. Of 57 nongonococcal gram-negative diplococci, all were negative by the Gono Gen test. We conclude that the Gono Gen test is sensitive and highly specific and provides a rapid method for the confirmation of N. gonorrhoeae.     

Antigenic similarities in lipopolysaccharides of Haemophilus and Neisseria and expression of a digalactoside structure also present on human cells.

Monoclonal antibodies raised against Haemophilus influenzae and Neisseria gonorrhoeae were used to investigate similarities or differences in the lipopolysaccharide antigens of pathogenic and commensal strains of several Gram-negative bacteria indigenous to mucosal surfaces of humans. In immunoblotting experiments, 20 of 36 monoclonal antibodies showed cross-reactions between species of Neisseria and Haemophilus. The common epitopes were present on N. gonorrhoeae, N. meningitidis, N. lactamica, H. influenzae including biogroup aegyptius, and occasionally H. parainfluenzae. No other commensal Neisseria or Gram-negative organisms tested reacted with the monoclonal antibodies with one exception; a single strain of pathogenic Escherichia coli was recognised by a N. gonorrhoeae-specific monoclonal antibody. One monoclonal antibody, raised against N. gonorrhoeae lipopolysaccharide, reacted with N. gonorrhoeae (32 of 59 strains), N. meningitidis (9 of 26 strains), H. influenzae (6 of 16 strains). An epitope expressed by H. influenzae and implicated in its virulence was also present on 14 of 59 strains of N. gonorrhoeae and was shown to comprise a digalactoside structure, alpha-galactosyl-1,4-beta-galactose (Gal alpha 1,4Gal beta), also found on human cells.     

Identification of pathogenic neisseriae by genetic transformation

The detection of pathogenic neisseriae by genetic transformation of a naturally occurring proline auxotroph of Neisseria gonorrhoeae strain F62 is described. Of 169 clinical isolates of N. gonorrhoeae, approximately 90% gave a positive transformation assay. Twelve clinical isolates of N. meningitidis and stock cultures of the various meningococcal serogroups also gave a positive result. However, the sensitivity of the assay was found to be approximately 1000-fold lower with N. meningitidis as test organism. Eleven other members of the family Neisseriaceae failed to transform the recipient organism. Although proline requirement did not appear to limit the value of the assay greatly, it probably was the main reason for negative results. The sensitivity of the assay and its ability to detect non-viable gonococci suggests that this method merits further investigation as a possible aid to diagnosis of gonococcal infection in special circumstances.     

Antigenic specificity and heterogeneity of lipopolysaccharides from pyocin-sensitive and -resistant strains of Neisseria gonorrhoeae

Homologous antisera were raised against lipopolysaccharides (LPSs) isolated from pyocin 103-sensitive JW31 strain Neisseria gonorrhoeae and its isogenic, pyocin-resistant variant, JW31R. Changes in immunochemical reactivity of LPS antigen associated with pyocin-resistance were examined by enzyme-linked immunosorbent assay, employing homologous and heterologous anti-LPS immune sera. The acquisition of pyocin 103 resistance is accompanied by a loss in LPS antigen reactivity with homologous anti-LPS. The variant LPS of pyocin 103-resistant mutants is immunogenic and displays a new, distinct antigenic specificity shared with other pyocin 103-resistant variant gonococcal strains. The acquisition of pyocin 103 resistance by JW31 strain gonococci is also accompanied by a striking loss of LPS cross-reactivity with antistreptococcal polysaccharide reagents having an antibody combining site specificity directed against the chemically defined lactose polymer from Streptococcus faecalis cell wall and pneumococcal type 14 capsular polysaccharide. When examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis, JW31 and JW31R LPSs show banding patterns characteristic of microheterogeneous, rough-type LPS devoid of O-side chains. Immunoblot transfer analysis of gel-separated gonococcal LPS antigens shows a difference in the pattern of antibody binding by homologous versus cross-reactive anti-LPS, which suggests a heterogeneity in the distribution of cross-reactive determinants among LPS molecules.     

Chlamydial infection increases gonococcal colonization in a novel murine coinfection model

Genital tract infections caused by Neisseria gonorrhoeae and Chlamydia trachomatis serovars D to K occur at high incidence in many areas of the world. Despite high rates of coinfection with these pathogens, investigations of host-parasite interactions have focused on each pathogen individually. We describe here a coinfection model in which female BALB/c mice were first infected with the mouse Chlamydia species C. muridarum and then inoculated with N. gonorrhoeae following treatment with water-soluble 17β-estradiol to promote long-term gonococcal infection. Viable gonococci and chlamydiae were recovered for an average of 8 to 10 days, and diplococci and chlamydial inclusions were observed in lower genital tract tissue by immunohistochemical staining. Estradiol treatment reduced proinflammatory cytokine and chemokine levels in chlamydia-infected mice; however, coinfected mice had a higher percentage of vaginal neutrophils compared to mice infected with either pathogen alone. We detected no difference in pathogen-specific antibody levels due to coinfection. Interestingly, significantly more gonococci were recovered from coinfected mice compared to mice infected with N. gonorrhoeae alone. We found no evidence that C. muridarum increases gonococcal adherence to, or invasion of, immortalized murine epithelial cells. However, increased vaginal concentrations of inflammatory mediators macrophage inflammatory protein 2 and tumor necrosis factor alpha were detected in C. muridarum-infected mice prior to inoculation with N. gonorrhoeae concurrently with the downregulation of cathelicidin-related antimicrobial peptide and secretory leukocyte peptidase inhibitor genes. We conclude that female mice can be successfully infected with both C. muridarum and N. gonorrhoeae and that chlamydia-induced alterations in host innate responses may enhance gonococcal infection.     

Nuclease enhancement of specific cell agglutination in a serodiagnostic test for Neisseria gonorrhoeae.

Antiserum to a purified type R lipopolysaccharide antigen isolated from Neisseria gonorrhoeae was used in a slide agglutination test and compared with conventional carbohydrate utilization and fluorescent antibody tests to confirm the identity of laboratory cultures classified as typical or "atypical" N. gonorrhoeae. Cultures of Corynebacterium vaginalis, N. meningitidis, N. catarrhalis, N. sicca, and N. lactamicus were also tested in the slide agglutination procedure. The addition of both deoxyribonuclease and ribonuclease (1 mg/ml) to the cell suspension medium of phosphate-buffered saline improved the sensitivity and specificity of the agglutination reaction for N. gonorrhoeae. Problems relating to the agglutination test as an aid in identification of N. gonorrhoeae are discussed.     

Incidence of Neisseria gonorrhoeae isolates negative by Syva direct fluorescent-antibody test but positive by Gen-Probe accuprobe test in a sexually transmitted disease clinic population.

To determine the accuracy of the Syva (Palo Alto, Calif.) direct fluorescent-antibody (DFA) test in comparison with the Gen-Probe (San Diego, Calif.) Accuprobe culture confirmation test, we tested 395 isolates of Neisseria gonorrhoeae from cultures obtained from patients attending a sexually transmitted disease clinic from 1 July 1991 through 30 June 1992. All isolates were tested for DFA reactivity with a polyclonal reagent (Difco Laboratories, Detroit, Mich.) and a monoclonal reagent (Syva, Inc., direct specimen test) and for specific molecular probe reactivity by the Gen-Probe Accuprobe culture confirmation test for N. gonorrhoeae. The 395 isolates gave positive results for the Gen-Probe culture confirmation test and the Difco polyclonal direct specimen test. However, 18 (4.6%) of the isolates were negative for N. gonorrhoeae by the Syva DFA test. With the exception of six beta-lactamase-positive isolates, all isolates that were negative by Syva DFA were sensitive to penicillin, tetracycline, spectinomycin, and ceftriaxone by disk-diffusion susceptibility testing. Auxotyping and serotyping studies indicated that strains negative by Syva DFA consisted of several variants. The frequency of N. gonorrhoeae isolates showing negative results by Syva DFA in this patient population ranged from 0 to 11.5%/month. Laboratories using only the Syva DFA test for confirmation of N. gonorrhoeae may incur a significant risk of misidentification.