An unusual Neisseria isolated from conjunctival cultures in rural Egypt

Seven isolates of an unusual Neisseria sp. were obtained from eye cultures of children in two rural Egyptian villages. These Neisseria utilized only glucose, they exhibited a positive reaction when tested with antisera to crude antigen from Neisseria meningitidis and N. gonorrhoeae, and they did not react with the fluorescent antibody tests for N. gonorrhoeae or with the monoclonal antibodies used to serotype gonococci. The Egyptian isolates had colony morphology more typical of meningococci than gonococci and showed opaque and transparent colony variants. On SDS-PAGE, the major outer-membrane proteins had different patterns than those noted for comparable proteins of meningococci and gonococci; heat-modifiable outer-membrane proteins were present. Four of the six isolates examined had cryptic plasmids of 2.8 megadaltons, which were slightly larger than the cryptic plasmid of N. gonorrhoeae. These plasmids were homologous to the gonococcal cryptic plasmid, but had different restriction enzyme fragment patterns. The DNA from the Egyptian isolates, like DNA from N. meningitidis but unlike DNA from N. gonorrhoeae, could be cut with the restriction enzyme HaeIII. The frequency of transformation into a temperature-sensitive mutant of N. gonorrhoeae was 0.2 for the Egyptian isolates and 0.1 for N. meningitidis, a frequency that was 5-10-fold lower than that for the N. gonorrhoeae control isolates. Whole-cell DNA from the Egyptian isolates showed 68%-73% homology with N. gonorrhoeae and 57%-63% with N. meningitidis. On the basis of our observations, the Egyptian isolates are distinct from N. meningitidis and may represent a variant of N. gonorrhoeae. We suggest that the isolates be called Neisseria gonorrhoeae ssp. kochii.     

Definition of a virulence-related antigen of Neisseria gonorrhoeae with monoclonal antibodies and lectins

Variants of one strain of Neisseria gonorrhoeae, grown in vivo or in vitro, that have been previously shown to differ in infectivity, serum resistance, and capsule production were compared with use of monoclonal antibodies and lectins. Monoclonal antibodies to virulent gonococci recognized an antigenic site of the lipopolysaccharide (LPS) produced in large amounts by gonococci grown in vivo but present only in a small proportion of in vitro-grown gonococci. This antigen (C-LPS) was found in all 85 different gonococcal isolates studied but not among nonpathogenic neisseriae. It was shared by group B and C meningococci but not by groups A and D. Enzyme-linked immunosorbent assay and Western blot analysis showed that N-acetylglucosamine and N-acetylgalactosamine form part of the epitope. The C-LPS antigen was shown by immunofluorescence to be present on the surface of the gonococci and also free as slime. This antigen appears to confer resistance to killing by normal sera.     

Attachment of pathogenic Neisseria to human mucosal surfaces: Role in pathogenesis

Studies of the interaction between Neisseria gonorrhoeae and human fallopian tube mucosa in organ culture suggest that attachment of gonococci is important, not only to secure th organism in the host, but also to initiate the disease process. The steps observed in gonococcal infection of fallopian tube organ cultures are: 1) attachment of gonococci to microvilli of nonciliated cells; 2) release from gonococci of lipopolysaccharide and possibly other toxic moities to cause mucosa damage; 3) engulfment or phagocytosis of gonococci by nonciliated cells; 4) transport of phagocytic vacuoles containing gonococci to the base of the nonciliated cells; and 5) exocytosis of gonococci within phagocytic vacuoles into the subepithelial tissues. In vivo, these steps might result in extensive local disease (e.g. salpingitis) or in the invasion of blood vessels to cause disseminated disease. Preliminary studies of human nasopharyngeal tissue in organ culture infected with Neisseria meningitidis indicate that meningococci attach to microvilli of nonciliated cells and are phagocytized by these cells. Meningococci subsequently appear in subepithelial tissues, though the route they take is not yet certain. These observations suggest at least some of the ways in which attachment may play a role in disease caused by N. gonorrhoeae and N. meningitidis. Mechanisms to block this attachment may provide new approaches to the prevention of infections caused by the pathogenic Neisseria.     

Aseptic arthritis after gonorrhoea.

Sixteen patients with aseptic arthritis developing after gonorrhoea and 14 patients with arthritis after nongonococcal urogenital infection have been analysed with respect to clinical course, roentgenological signs, and humoral as well as cellular immune responses to Neisseria gonorrhoeae antigen. Fifty-eight healthy blood donors were used as controls. The clinical pattern did not differ significantly between the 2 groups. Eye or skin lesions indicative of Reiter's syndrome were found in 5 patients of both groups. Signs of sacroiliac arthritis were found in 8 and 6 patients respectively. Gonococcal complement fixation was positive in 9 of 16 patients in the postgonorrhoeal arthritis group and in 0 of 14 patients in the arthritis group with nongonococcal urogenital infection. The lymphocyte stimulation induced by gonococcal antigen was significantly greater in patients with postgonorrhoeal arthritis than in healthy controls. When reference was made to the results of stimulation of the lymphocytes with PPD, there was also a significant difference in the lymphocyte reactivity to gonococcal antigen between the group of patients with postgonorrhoeal arthritis and that of patients with arthritis after non-gonococcal urogenital infection. No such difference was noted between the latter group and the healthy controls. The clinical and immunologic data argue in favour of the hypothesis that Neisseria gonorrhoeae may induce an aseptic arthritis which sometimes presents as a complete Reiter's syndrome.     

The acute arthritis-dermatitis syndrome. The changing importance of Neisseria gonorrhoeae and Neisseria meningitidis

Sexually active young adults with an acute arthralgia or arthritis, with or without associated skin lesions, often have disseminated gonococcal infection (DGI). In recent years, an increasing proportion of patients seen with such complaints at the University of Washington Hospitals, Seattle, have had systemic meningococcal infection rather than DGI. Among 151 patients with acute arthritis studied prospectively from 1970 to 1972, blood or synovial fluid cultures yielded Neisseria gonorrhoeae in 30 patients and Neisseria meningitidis in two. Among 62 patients meeting the same criteria who were studied prospectively from 1980 to 1983, blood or synovial fluid cultures yielded gonococci in nine and meningococci in five. Separate analysis of blood culture results from two University of Washington Hospitals also revealed a decline in the number of cases of gonococcemia from 1970 through 1984 and a shift in the relative numbers of patients with bacteremia due to N gonorrhoeae and N meningitidis. The observed decline in gonococcemia coincides with a decline in the proportion of gonorrhea in Seattle caused by gonococcal strains that have been associated with DGI.     

Gonococcal cervicitis: a role for biofilm in pathogenesis

Neisseria gonorrhoeae forms a biofilm in flow cells on glass coverslips as well as on primary cervical epithelial cells. Electron microscopic studies of cervical biopsy specimens from 10 patients with culture-proven N. gonorrhoeae infection revealed evidence of biofilm formation in 3 of the biopsy specimens. These biofilms showed gonococci in networks of bacterial membrane within the biofilm structure. This finding was also observed in biofilms formed over glass cover slips and after infection of primary cervical tissue in vitro. The importance of membranous networks in Neisseria biofilm formation was demonstrated with N. gonorrhoeae strain 1291-msbB, which shows a markedly decreased ability to bleb. This mutant formed significantly less biofilm over glass surfaces and cervical epithelial cells, and complementation showed reversion to wild-type biofilms. Gonoccal biofilms, as part of the cervical infection, may be involved in the mechanisms by which asymptomatic infections, persistence, and increased antibiotic resistance occur.     

Phagocytosis and killing of Neisseria gonorrhoeae by Trichomonas vaginalis

Type 2 and 4 transparent and opaque Neisseria gonorrhoeae demonstrated a logarithmic loss of viability with a half life of approximately 10-30 min when incubated in the presence of Trichomonas vaginalis. Although this effect was observed in the absence of serum for most types of gonococci tested, it was consistently enhanced by the addition of human serum. Only for type 4 transparent gonococci did this process show an absolute serum requirement. Cytochalasin B inhibited the loss of viability. The nonphagocytic cattle parasite Tritrichomonas foetus did not ingest or kill N. gonorrhoeae. Electron microscopy revealed phagocytic uptake and degradation of N. gonorrhoeae in T. vaginalis, indicating that the loss of viability of N. gonorrhoeae was the result of phagocytosis followed by intracellular killing of gonococci by T. vaginalis.     

Studies on gonococcus infection. XVII. IgA1-cleaving protease in vaginal washings from women with gonorrhea.

Vaginal washings from women attending a veneral disease clinic were examined for the presence of protease that cleaved IgA subclass 1 (IgA1). In a crude assay, vaginal washings cleaved [125I]IgA1 in 19 of 25 specimens from individuals from whom Neisseria gonorrhoeae were cultivated. Forty-six specimens from 104 women whose cultures were negative for N. gonorrhoeae also cleaved [125I]IgA1. Vaginal washings from six of six women with culture-proven gonorrhea cleaved [125I]IgA1 into low-molecular-weight components identical to those produced by partially purified IgA1-specific protease from gonococci. The hydrolysis of [125I]IgA1 by vaginal washings from women whose cultures were negative for N. gonorrhoeae yielded cleavage products that resembled those of trypsin or alpha-chymotrypsin. These findings indicate that gonococci residing in the female genital tract produce IgA1-specific protease that can be detected in the vaginal washings of infected women.     

The role of natural IgG and complement in the phagocytosis of type 4 Neisseria gonorrhoeae by human polymorphonuclear leukocytes.

The role of human serum components in the phagocytosis of logarithmic-phase type 4 Neisseria gonorrhoeae by human polymorphononuclear leukocytes was investigated. The requirement of fresh normal human serum (FHS) for optimal phagocytosis and the fixation of human immunoglobulin (IgG) and complenet (C3) to the gonococcal cell surface suggested that both serum factors participate in the phagocytosis of these organisms. The percentage of neutrophils containing ingested organisms was directly proportional to the concentration of IgG purified from FHS. Absorption studies suggested that this natural IgG binds to a trypsin-sensitive surface protein on type 4 gonococci and cross-reacts with stationary-phase type 2 N. gonorrhoeae, group C Neisseria meningitidis, and Branhamella catarrhalis, but not with logarithmic-phase type 2 gonococci or other Neisseria species. Although complement alone did not promote phagocytosis, it enhanced IgG-mediated ingestion. Studies using C2-deficient serum or serum chelators indicated that the alternative complement pathway participates in this interaction.     

Immunologic profile of patients with cured Hodgkin's disease.

Blood lymphocytes from 9 patients with Hodgkin's disease (HD) were studied. The results were compared with those of 6 seminoma testis patients and 9 healthy unrelated controls. All patients were in complete and unmaintained remission more than 10 years after termination of radiotherapy. The mean T-lymphocyte count of HD patients was lower than that of controls and seminoma testis patients. Lymphocyte DNA synthesis induced by pokeweed mitogen and phytohaemagglutinin was normal in both patient groups. Concanavalin A-induced DNA synthesis was low in 4 patients with HD although the mean stimulation of the group did not differ from controls or seminoma testis patients. Lymphocyte activation by PPD was slightly decreased in the 2 patient groups. No increase in spontaneous lymphocyte DNA synthesis was observed. The responding and stimulatory capacity in mixed lymphocyte culture was decreased in 3 and 2 HD patients respectively. 4 out of the 9 patients with HD but none with seminoma testis displayed severe impairment in T-lymphocyte functions. As 1 of the 4 had been treated solely by surgery, late effects of irradiation can only partly explain the results. The results may favour a hypothesis postulating a constitutional defect contributing to the immunoincompetence in HD.     

Evaluation of a DNA-hybridization method for detection of African and Asian strains of Neisseria gonorrhoeae in men with urethritis

The 2.6-megadalton (MDa) cryptic plasmid and the 4.4-MDa beta-lactamase plasmid of Neisseria gonorrhoeae were radiolabeled with [32P] nucleotides and used as probes for direct detection of gonococci and beta-lactamase plasmids in urethral exudates from men with urethritis. The sensitivity and specificity of the DNA probes were compared with culture isolation of N. gonorrhoeae and biochemical tests of gonococcal isolates for beta-lactamase production. Of 216 urethral specimens, 180 were positive for N. gonorrhoeae by DNA probe and culture, 27 were negative by both tests, and 9 gave discordant results. Compared with culture and with the chromogenic cephalosporin assay, the sensitivity and the specificity of the DNA probe was 99% and 93% and that of the beta-lactamase probe assay was 91% and 96%, respectively. Electrophoresis of plasmids isolated from 90 gonococcal cultures showed that all contained the 2.6-MDa plasmid, 29 possessed a 3.2-MDa plasmid, 18 a 4.4-MDa beta-lactamase plasmid, and 11 had a 24.5-MDa conjugal plasmid. We conclude that the sensitivity of our DNA probes was comparable to that of culture for diagnosis of gonorrhea and to conventional tests for detection of beta-lactamase.     

Analysis of the genetic differences between Neisseria meningitidis and Neisseria gonorrhoeae: two closely related bacteria expressing two different pathogenicities.

We have investigated genetic differences between the closely related pathogenic Neisseria species, Neisseria meningitidis and Neisseria gonorrhoeae, as a novel approach to the elucidation of the genetic basis for their different pathogenicities. N. meningitidis is a major cause of cerebrospinal meningitis, whereas N. gonorrhoeae is the agent of gonorrhoea. The technique of representational difference analysis was adapted to the search for genes present in the meningococcus but absent from the gonococcus. The libraries achieved are comprehensive and specific in that they contain sequences corresponding to the presently identified meningococcus-specific genes (capsule, frp, rotamase, and opc) but lack genes more or less homologous between the two species, e.g., ppk and pilC1. Of 35 randomly chosen clones specific to N. meningitidis, DNA sequence analysis has confirmed that the large majority have no homology with published neisserial sequences. Mapping of the cloned DNA fragments onto the chromosome of N. meningitidis strain Z2491 has revealed a nonrandom distribution of meningococcus-specific sequences. Most of the genetic differences between the meningococcus and gonococcus appear to be clustered in three distinct regions, one of which (region 1) contains the capsule-related genes. Region 3 was found only in strains of serogroup A, whereas region 2 is present in a variety of meningococci belonging to different serogroups. At a time when bacterial genomes are being sequenced, we believe that this technique is a powerful tool for a rapid and directed analysis of the genetic basis of inter- or intraspecific phenotypic variations.     

Antigenic specificity of antibodies in vaginal secretions during infection with Neisseria gonorrhoeae

Antibodies in genital secretions of patients with gonorrhea have been shown to inhibit the attachment of gonococci to epithelial cells. The gonococcal antigens for which these antibodies are specific were studied by adsorption of the genital secretions from a patient infected with gonorrhea with purified lipopolysaccharide, outer membrane complex, or purified pili of homologous Neisseria gonorrhoeae and measurement of the reduction of inhibition of attachment of the gonococci to epithelial cells. The removal of antibodies was documented with the use of a solid-phase radioimmunoassay in which the amount of antibody in the adsorbed secretions that bound to a specific gonococcal antigen was shown to be reduced as compared with the amount of antibody in unadsorbed secretions. The antibody in the secretions that inhibited attachment was removed primarily by adsorption with the homologous pili, not with homologous lipopolysaccharide. A preparation of the homologous outer membrane complex that contained pili, cell-wall proteins, and lipopolysaccharide also blocked the inhibitory antibody.     

Immunologic Profile of Patients with Cured Hodgkin's Disease

Blood lymphocytes from 9 patients with Hodgkin's disease (HD) were studied. The results were compared with those of 6 seminoma testis patients and 9 healthy unrelated controls. All patients were in complete and unmaintained remission more than 10 years after termination of radiotherapy. The mean T-lymphocyte count of HD patients was lower than that of controls and seminoma testis patients. Lymphocyte DNA synthesis induced by pokeweed mitogen and phytohaemagglutinin was normal in both patient groups. Concanavalin A-induced DNA synthesis was low in 4 patients with HD although the mean stimulation of the group did not differ from controls or seminoma testis patients. Lymphocyte activation by PPD was slightly decreased in the 2 patient groups. No increase in spontaneous lymphocyte DNA synthesis was observed. The responding and stimulatory capacity in mixed lymphocyte culture was decreased in 3 and 2 HD patients respectively. 4 out of the 9 patients with HD but none with seminoma testis displayed severe impairment in T-lymphocyte functions. As 1 of the 4 had been treated solely by surgery, late effects of irradiation can only partly explain the results. The results may favour a hypothesis postulating a constitutional defect contributing to the immunoincompetence in HD.     

Epidemiology and molecular basis of penicillin-resistant Neisseria meningitidis in Spain: a 5-year history (1985-1989).

Penicillin-resistant (penr) clinical isolates of Neisseria meningitidis, which do not produce beta-lactamase, were first identified in Spain in 1985; the frequency of their recovery, which has been increasing in the past few years, reached 20% in 1989. Serogrouping, determination of serotypes and subtypes, and multilocus enzyme electrophoresis of the penr strains showed an extensive diversity. Resistance is due, at least in part, to a decreased affinity of penicillin-binding protein (PBP) 2 for penicillin. Similar low-affinity forms of PBP 2 are also found in penr isolates of Neisseria lactamica, Neisseria polysaccharea, and Neisseria gonorrhoeae. Genetic transformation of an N. meningitidis type strain to low-level penicillin resistance with DNA from resistant meningococci and other Neisseria species resulted in transformants that possessed low-affinity forms of PBP 2. These altered forms of PBP 2 have been shown to arise from recombinational events that replace parts of the PBP 2 gene with the corresponding regions from the PBP 2 genes of commensal Neisseria species.     

Auxotypes, penicillin susceptibility, and serogroups of Neisseria gonorrhoeae from disseminated and uncomplicated infections

We examined auxotypes, penicillin susceptibility, and outer membrane serogroups of 137 strains of Neisseria gonorrhoeae isolated from patients with disseminated gonococcal infection (DGI) and 137 control strains from patients with uncomplicated gonorrhea. We analyzed separately the data for strains isolated from systemic sites in patients with DGI and for strains from local sites in patients with the clinical syndrome of DGI (SDGI) who had negative systemic cultures. We found the nutritional requirement for arginine, hypoxanthine, and uracil (AHU auxotype) significantly more often among DGI strains than among SDGI strains. By using commercially available serogrouping reagents to detect outer membrane protein antigens, we found that regardless of strain auxotype, dissemination correlated best with the presence of protein IA antigens. We did not find that gonococci isolated from DGI are highly susceptible to penicillin. Susceptibility to low concentrations of penicillin correlated only with the AHU requirement, not with serogroup or isolation from a patient with DGI or SDGI.     

Evidence of serum antibodies to Neisseria gonorrhoeae before gonococcal infection

To characterize the serum antibody response to urethral infection with Neisseria gonorrhoeae, we examined pre- and postinfection sera from 13 men experiencing their first gonococcal infection. Using western blot analysis, we found that nine of 13 patients developed new serum IgG antibodies against one or more antigens, most commonly against lipooligosaccharide, followed in order by the H.8-antigen, pili, proteins I and II, and protein III. Twelve of 13 patients had preexisting IgG to gonococcal antigens, most commonly against the H.8 antigen, followed by pili, lipooligosaccharide, protein I, and protein III. Using serum obtained from other patients before and after nasopharyngeal carriage of Neisseria meningitidis, we demonstrated that carriage resulted in serum IgG cross-reactive to N. gonorrhoeae antigens. This is likely explanation for the presence of antigen-specific antibody in preinfection sera.     

Epidemiological and bacteriological study on gonococcal infections]

Epidemiological and bacteriological studies on Neisseria gonorrhoeae isolated in Sapporo, Japan, in 1980 and 1991 performed and the following results were obtained. 1. The range of age in the patients infected with Neisseria gonorrhoeae tended to be younger than those in the whole country. 2. Male patients in the early 20s or younger with gonococcal urethritis were often infected by bon-professional females but those in their late 20s or older were often infected from professional females, for example prostitutes and hostesses. 3. The rate of professional females who were positive to gonococci reached 17.4% and young females in their teens with cervicitis had the highest morbidity rate of gonococci than those in the older females. 4. The latent period in gonococcal infections tended to become longer gradually. 5. The isolation rate of penicillinase producing Neisseria gonorrhoeae (PPNG) showed a peak of 23.9% (61/255) in 1985, but gradually declined thereafter and it was 3.7% (1/27) in 1991. 6. An investigation on auxotype showed a decline of proto and Pro-strains and an increase of AHU-strains in non-PPNG. And most of the PPNG belonged to proto or Pro-strains. 7. With the relationship between auxotype and sensitivity to AMPC, AHU-strains were more sensitive than proto or Pro-strains.     

Studies on gonococcus infection. XI. Comparison of in vivo and vitro association of Neisseria gonorrhoeae with human neutrophils.

The levels of association between Neisseria gonorrhoeae and neutrophils as determined by microscopic study of smears of urethral exudate from men with acute gonorrhea and by in vitro assay of N. gonorrhoeae cultivated from those same individuals showed statistically significant correlation. The percentage of N. gonorrhoeae attached to or ingested by neutrophils varied from 30% to 90% in the clinical smears. Neither the apparent in vivo nor the in vitro association with neutrophils could be correlated with piliation of the N. gonorrhoeae studied.     

Detection of Neisseria gonorrhoeae in first-voided urine sediments from male urethritis patients by polymerase chain reaction]

Neisseria gonorrhoeae was detected from first-voided urine sediments of male patients with urethritis by polymerase chain reaction (PCR). Urine and urinary sediment were treated with proteinase K, and DNA was further purified by phenol extraction. Two oligonucleotides based on sequences within a ribosomal RNA gene from N. gonorrhoeae were used as primers for the PCR. A DNA fragment of 206 bp specific for N. gonorrhoeae was amplified by PCR and detected by agarose gel electrophoresis. In 19 specimens of urine sediments collected from 21 patients in whom N. gonorrhoeae was isolated from urethral swab by culture, 206 bp DNA fragment was amplified by PCR. In all specimens of urine sediments from 24 patients in whom cultures for N. gonorrhoeae were negative, no DNA was amplified by the PCR. The overall coincidence rate between the PCR for detecting N. gonorrhoeae in first-voided urine sediments and culture in urethral swab was 95.6% (43/45). PCR procedure for detection of pathogens from first-voided urine sediments would be noninvasive and would be applied for the diagnosis of gonococcal urethritis and chlamydial urethritis.