RESUMO
In tests in vitro with the phagocytes of human buffy coat, a recent isolate of Neisseria gonorrhoeae, which was pilated, formed small colonies and resembled the virulent Kellogg type 2 (strain BS), resisted ingestion more than did another isolate (strain AL), which was non-pilated, formed large colonies and resembled the avirulent Kellogg type 4. Some members of both strains survived for significant periods within the phagocytes in test conditions that tended to minimise rather than maximise such survival; and strain BS had a greater capacity for intracellular survival than strain AL, with some of its members surviving for at least 8 h. Resistance to phagocytic ingestion is one important invasive mechanism of gonococci, and resistance to phagocytic digestion may also play a role in pathogenicity.
Assuntos
Monócitos/imunologia , Neisseria gonorrhoeae/imunologia , Neutrófilos/imunologia , Fagocitose , Contagem de Células , Humanos , Monócitos/microbiologia , Neisseria gonorrhoeae/patogenicidade , Neutrófilos/microbiologia , Especificidade da Espécie , VirulênciaRESUMO
Strains of Neisseria gonorrhoeae adhered to pieces of human endocervix and appeared to be embedded in the surface mucus. Although a pilate strain adhered better than a non-pilate strain, the difference was small and pilation did not appear to be exclusively responsible for adherence. The pilate strain showed better adherence to pieces of human ectocervix and fallopian tube, but both strains were similarly adsorbed to human bronchus and guinea-pig uterus, cervix, male urethra and bladder, although to different degrees for different tissues. Since gonococci adhered to all tissues examined, their ability to infect human endocervix and fallopian tube and their failure to infect human ectocervix or guinea-pig urogenital tract mucosae are determined by factors other than a capacity for primary adherence to the tissue.
Assuntos
Brônquios/microbiologia , Colo do Útero/microbiologia , Tubas Uterinas/microbiologia , Neisseria gonorrhoeae/fisiologia , Adesividade , Animais , Brônquios/citologia , Colo do Útero/citologia , Epitélio/microbiologia , Tubas Uterinas/citologia , Feminino , Cobaias , Humanos , Masculino , Neisseria gonorrhoeae/ultraestrutura , Organoides , Especificidade da Espécie , Doenças do Colo do Útero/microbiologiaRESUMO
Perforated plastic chambers implanted subcutaneously in guinea-pigs and rabbits became encapsulated and filled with sterile transudate. When these chambers in guinea-pigs were inoculated with various strans of Neisseria gonorrhoeae, persistent infections were achieved without the use of anti-inflammation agents and in the presence of a substantial predominantly polymorphonuclear inflammatory response. Two strains with small colonies similar to types 1 and 2, and one strain with large colonies similar to type 4 of Kellogg et al. (1963 and 1968), showed differences in infectivity comparable with those that might be expected in man, and passage through guinea-pig chambers increased this infectivity. Rabbit chambers could not be infected without the use of an anti-inflammation drug (betamethasone), and differences in infectivity between strains were not as clear cut. The growth of N. gonorrhoeae in chambers in the guinea-pig provides a convenient model system for studying some aspects of the pathogenicity of this organism.
Assuntos
Técnicas Bacteriológicas , Exsudatos e Transudatos/microbiologia , Inflamação , Neisseria gonorrhoeae/patogenicidade , Animais , Betametasona/farmacologia , Eritrócitos/citologia , Exsudatos e Transudatos/citologia , Gonorreia/microbiologia , Cobaias , Leucócitos/citologia , Linfócitos/citologia , Modelos Biológicos , Neisseria gonorrhoeae/crescimento & desenvolvimento , Plásticos , Coelhos , Especificidade da EspécieRESUMO
The use of spectinomycin to kill extracellular bacterial in phagocytosis tests with gonococci and human polymorphonuclear phagocytes allowed the demonstration of a greater degree of intracellular survival and growth than in previous tests using penicillin.
Assuntos
Neisseria gonorrhoeae/crescimento & desenvolvimento , Neutrófilos/microbiologia , Espectinomicina/farmacologia , Humanos , Neisseria gonorrhoeae/efeitos dos fármacos , FagocitoseRESUMO
Gonococci from subcutaneously implanted chambers in guinea pigs produced, on agar, more than 95% small colonies showing a "double highlight" (DH) effect in oblique reflected light combined with transmitted light. Laboratory strains of gonococci produced some DH colonies, but other showed a single highlight (SH) or no highlight (NH). Selection of DH colonies and comparison of their organisms with gonococci grown in vivo and with those from SH colonies, showed that the DH character was associated with high infectivity for guinea-pig chambers, resistance to killing by human phagocytes and heavy pilation. Furthermore, DH colonies were found in the first culture of three fresh samples of urethral pus. Thus, the DH colony characteristic may be a more reliable criterion of pathogenicity of gonococcal isolates than systems used previously. There were, however, some differences between the gonococci grown in vivo and the DH colony types. The gonococci grown in vivo and cultured once on solid medium possessed one or two antigens which differed from those of DH (or SH) colonies. They also formed smooth suspensions (which separated slowly) in saline, compared with the rough suspensions (which separated quickly) formed by gonococci from DH (or SH) colonies. Finally, the organisms grown in vivo were resistant to killing by human serum whereas the DH (and SH) colony types were susceptible; the resistance of the organisms grown in vivo was lost during one subculture on agar suggesting that the property is a phenotypic characteristic. Hence, in addition to selecting DH colony types the conditions in vivo produce organisms which differ, probably phenotypically, from cultured organisms.
Assuntos
Neisseria gonorrhoeae/crescimento & desenvolvimento , Adaptação Fisiológica , Animais , Antígenos de Bactérias/análise , Membrana Celular , Cobaias , Humanos , Neisseria gonorrhoeae/imunologia , Fagócitos/fisiologia , Fenótipo , Seleção Genética , VirulênciaRESUMO
Phagocytes obtained from fresh human buffy coat (predominantly polymorphonuclear phagocytes) or from human buffy coat which had been incubated on a glass surface for 1 to 3 days (predominantly mononuclear phagocytes) were allowed to ingest gonococci, and then incubated with penicillin. More intracellular gonococci were killed at high than at low penicillin concentrations, indicating that penicillin penetrated the phagocytes. This was supported by autoradiography experiments with radiolabelled penicillin. A pilated, small-colony-forming gonococcal strain survived and multiplied for at least 15 h in polymorphonuclear phagocytes which were incubated with penicillin at the optimum concentration for killing the extracellular bacteria but not the intracellular ones; whereas a non-pilated, large-colony-forming strain survived for only 10 h. The former strain survived for at least 6 h in similar experiments with mononuclear phagocytes. Intracellular survival and in growth may be an important facet of the pathogenicity of gonococci.
Assuntos
Monócitos/microbiologia , Neisseria gonorrhoeae/crescimento & desenvolvimento , Neutrófilos/microbiologia , Penicilinas/farmacologia , Permeabilidade da Membrana Celular , Humanos , Monócitos/metabolismo , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/patogenicidade , Neutrófilos/metabolismo , Penicilinas/metabolismo , FagocitoseRESUMO
Human phagocytes that had ingested gonococci were incubated with increasing concentrations of penicillin in fresh human serum. Viable counts on phagocyte deposits before and after incubation indicated penicillin penetration at high concentrations but not at lower concentrations which were sufficient to kill extracellular organisms. With lower concentrations significant intracellular survival of gonococci occurred.
Assuntos
Neisseria gonorrhoeae/efeitos dos fármacos , Penicilinas/farmacologia , Fagócitos/efeitos dos fármacos , Gonorreia/tratamento farmacológico , Humanos , Penicilinas/uso terapêutico , Fagocitose/efeitos dos fármacosRESUMO
Phenotypically determined resistance of gonococci to killing by normal human serum can be generated by growth of susceptible organisms in media containing guinea pig, calf or human serum. However, even in the best medium tested, i.e. defined medium containing 50% (v/v) guinea pig serum, resistance was greatly reduced after 24 h incubation and the maximum number of colony-forming units generated was 10(7) to 10(8) ml-1. Resistance was not acquired after incubation in guinea pig serum at low temperature (8 degrees C), supporting previous indications that metabolic activity was necessary for the generation of resistance. Alteration of the concentration of glutamine, proline, lactate or iron had little or no effect on the generation of serum resistance under the conditions used. Optimum conversion to resistance occurred at pH 6.0 to 6.5 and both non-diffusible and diffusate fractions of dialysed guinea pig serum promoted resistance. Furthermore, resistant organisms could be produced by incubation in defined medium containing the diffusate from guinea pig serum and 0.1% bovine serum albumin, a step which should facilitate identification of the resistance-promoting factor.
Assuntos
Atividade Bactericida do Sangue , Neisseria gonorrhoeae/imunologia , Atividade Bactericida do Sangue/efeitos dos fármacos , Meios de Cultura , Desoxirribonucleases/farmacologia , Diálise , Glutamina/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Ferro/farmacologia , Lactatos/farmacologia , Ácido Láctico , Neisseria gonorrhoeae/efeitos dos fármacos , Fenótipo , Prolina/farmacologia , TemperaturaRESUMO
The toxicity of gonococci [strain BS4 (agar)] for human peripheral blood polymorphonuclear phagocytes, infected in vitro, was assessed by light microscopic examination of Giemsa stained cell deposits of polymorphonuclear phagocytes which had ingested these bacteria. The cytotoxicity elicited by viable gonococci, assessed by percentage lysis and concomitant reduction in the number of polymorphonuclear phagocytes increased as the ratio of gonococci to phagocytes in the original suspension mixture was raised. Pretreatment of viable gonococci with antiserum raised to whole organisms increased the cytotoxic effect produced by the organisms. Killed (heat or UV irradiation) gonococci caused little or no cytotoxicity, even when the organisms were pretreated with specific antiserum. Hence, the lysis of polymorphonuclear phagocytes appears to be caused by a factor or factors produced by viable gonococci and not by LPS per se.
Assuntos
Citotoxinas/imunologia , Neisseria gonorrhoeae/imunologia , Neutrófilos/imunologia , Citotoxinas/efeitos da radiação , Temperatura Alta , Humanos , Soros Imunes/farmacologia , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/efeitos da radiação , Raios UltravioletaRESUMO
Attempts to study quantitatively the phagocytosis of gonococci from urethral pus failed because of the small numbers of organisms and technical difficulties. However, gonococci from chambers implanted subcutaneously in guinea pigs, which were similar to gonococci from urethral pus in their resistance to killing by human serum, were obtained in sufficient quantities for comparison in phagocytosis tests with the in vitro grown strains from which they were derived. Microscopic and viable counts of gonococci in phagocytes showed that in vivo grown organisms (strain BSV) were readily phagocytosed by human polymorphonuclear phagocytes. There was little difference betweee to ingestion. There was, however, a marked difference in the intracellular survival of strains BSV and BS during the first hour of phagocytosis. Whereas BSV organisms survived well, many BS organisms were killed. Subsequently, strain BSV and the survivors of the strain BS inoculum responded similarly to the intracellular bactericidins. These results were supported by electron microscopy of infected phagocytes. Resistance of gonococci in vivo to ingestion and digestion by human phagocytes seem to be important facets of the pathogenesis of gonorrhoea.
Assuntos
Neisseria gonorrhoeae , Neutrófilos/fisiologia , Fagocitose , Humanos , Neisseria gonorrhoeae/ultraestrutura , Neutrófilos/ultraestrutura , Especificidade da EspécieRESUMO
In reassessment of previous tests for intracellular survival, results have been confirmed and additional evidence obtained indicating that some gonococci can survive and multiply in human phagocytes. Use was made of the ability of penicillin to penetrate phagocytes and to kill only actively growing organisms. In microscopic counts on 33 urethral exudate smears, an average of 49% of gonococci were associated with polymorphonuclear phagocytes. The organisms were unevenly distributed amongst the phagocytes, with most cells uninfected and some containing large numbers. Many phagocytes also remained uninfected in tests in vitro with low gonococcal inocula although experiments with large inocula showed that most phagocytes could ingest gonococci. It is proposed that ingestion of one gonococcus may stimulate the phagocytes to take up more. Phagocytes were killed and disintegrated after ingesting large numbers of gonococci and similar effect in vivo may be responsible for the large clumps of organisms seen in urethral exudate. These results underline the probable importance in the pathogenesis of gonorrhoea of intracellular survival in phagocytes.
Assuntos
Neisseria gonorrhoeae/crescimento & desenvolvimento , Fagócitos/microbiologia , Atividade Bactericida do Sangue , Exsudatos e Transudatos/microbiologia , Gonorreia/microbiologia , Humanos , Técnicas In Vitro , Masculino , Neisseria gonorrhoeae/efeitos dos fármacos , Neutrófilos/microbiologia , Penicilinas/farmacologia , Fagocitose , Uretra/microbiologiaRESUMO
Gonococci adapted to growth in guinea pig chambers [strain BS4 (agar)] were predominantly smooth organisms and produced a type-specific antigen. A vaccine prepared by treating these gonococci with formalin, protected guinea pig chambers against homologous challenge in contrast to a similarly treated laboratory strain (BSDH) which had been selected in vitro from the same parent strain and which did not produce the type-specific antigen. Surface washes of BS4 (agar) contained the type-specific antigen but attempts to immunize guinea pigs with complexes of rabbit antibody with this antigen excised from gels failed. However, good immunity could be produced by combining such complexes with formalin-killed rough gonococci (strain BS4R), lacking the type-specific antigen, which were found in some chambers of challenged guinea pigs that had been immunized with the complexes. Hence, at least two antigens -- one the type-specific antigen and the other(s) possessed by both BS4 (agar) and BS4R -- are needed for immunogenicity. Surface washes of BS4 (agar) and BS4R contained three antigens, distinct from the type-specific antigen, which might complement it in producing immunity. Similar antigens were present in surface washes of five fresh isolates from human urethral pus, but only a few organisms from these isolates possessed antigens similar to the type-specific antigen. The variability of gonococci in antigenicity, immunogenicity and probably virulence is discussed.
Assuntos
Antígenos de Bactérias/imunologia , Neisseria gonorrhoeae/imunologia , Animais , Anticorpos Antibacterianos/biossíntese , Complexo Antígeno-Anticorpo , Vacinas Bacterianas , Epitopos , Gonorreia/prevenção & controle , Cobaias , Imunoeletroforese BidimensionalRESUMO
Some gonococci obtained from human urethral exudate or from subcutaneously implanted chambers in guinea pigs show a resistance to killing by human serum which is lost on sub-culture in vitro after a few generations. The environmental factors which may influence the phenotypic expression of resistance to serum killing were investigated in guinea pig chambers and in chamber fluid in vitro. The redox potential in chambers before and after infection was lower than that of heart blood but conditions were not anaerobic; H2O2 increased the redox potential but did not decrease gonococcal serum resistance. The chambers were slightly alkaline before and after infection. When the concentration of glucose (depleted in infected chambers by the abundant polymorphonuclear cells) was restored to excess, the serum resistance of the gonococci was unaffected. Concentrations of free amino acids in chambers changed little during infection. Gonococci adapted to growth in chambers and subsequently rendered serum-sensitive by growing once on agar reverted to serum-resistance after 0.5 to 1 h incubation in chamber fluid in vitro at 37 degrees C but not at 25 degrees C or 4 degrees C. After 16 to 24 h growth at 37 degrees C, resistance was again lost. The reversion to serum resistance did not occur in a complex laboratory medium. Examination of the chamber fluid after growth of gonococci in vitro showed depletion of lactate, glutamine and proline.
Assuntos
Atividade Bactericida do Sangue , Neisseria gonorrhoeae/genética , Acetatos/metabolismo , Aminoácidos/metabolismo , Animais , Meios de Cultura , Glucose/metabolismo , Cobaias , Humanos , Concentração de Íons de Hidrogênio , Lactatos/metabolismo , Neisseria gonorrhoeae/imunologia , Neisseria gonorrhoeae/metabolismo , Oxirredução , FenótipoRESUMO
The immunogenicity of Neisseria gonorrhoeae in the guinea pig subcutaneous chamber model, assessed by serum bactericidal tests and challenge experiments, is complicated by diversity of immunotypes which may or may not show partial cross-reactions, by the need for antibodies to more than one type-specific antigen for full homologous protection, and possibly by the limited accessibility of the relevant antigens on the cell surface.
Assuntos
Anticorpos Antibacterianos/biossíntese , Neisseria gonorrhoeae/imunologia , Animais , Antígenos de Bactérias/análise , Atividade Bactericida do Sangue , Reações Cruzadas , CobaiasRESUMO
A type-specific antigen of Neisseria gonorrhoeae was previously demonstrated by two-dimensional immunoelectrophoresis, and was produced by strains adapted to growth in subcutaneous chambers in guinea pigs. This antigen was also present in 'smooth' (non-autoagglutinating) variants selected directly from the first agar cultures of three clinical isolates, without chamber passage. The type-specificity of this antigen paralleled the reactions of pili in immune electron microscopy, suggesting that the type-specific antigens were pili. However, 'rough' (autoagglutinating) variants lacking this type-specific antigen were nevertheless pilated. Examination of one strain by immune electron microscopy showed that the pili of the rough variant differed antigenically from those of the smooth variant. Pili on the rough variant tended to form extensive parallel aggregates, whereas pili on the smooth variant radiated individually from the gonococci. This physical difference might relate to the behaviour of the gonococci in suspension. The significance of pilus variation in immunity to gonococcal infection is discussed.
Assuntos
Antígenos de Bactérias/análise , Fímbrias Bacterianas/imunologia , Neisseria gonorrhoeae/imunologia , Aglutinação , Reações Cruzadas , Epitopos , Fímbrias Bacterianas/ultraestrutura , Microscopia Eletrônica , Neisseria gonorrhoeae/ultraestruturaRESUMO
Gonococci adapted to growth in chambers implanted subcutaneously into guinea pigs are resistant to killing by human serum. This resistance is lost after a few generations in vitro both in culture medium and in fluid taken from guinea-pig chambers. The rate of loss is too rapid to occur by mutation and selection. Furthermore, the resistance is regained after a few generations when bacteria from the first in vitro culture are inoculated back into guinea-pig chambers in vivo. Hence the loss of serum resistance in vitro and the gain in vivo are probably due to phenotypically controlled events. Such events could be important in the pathogenicity of Neisseria gonorrhoeae.
Assuntos
Atividade Bactericida do Sangue , Neisseria gonorrhoeae/genética , Animais , Meios de Cultura , Cobaias , Humanos , Neisseria gonorrhoeae/patogenicidade , Fenótipo , Fatores de Tempo , VirulênciaRESUMO
Infection of subcutaneusly implanted chambers in guinea pigs conferred immunity against homologous infection of other chambers in the same animals. However, attempts to immunize guinea pigs by subcutaneous injection of filtered fluid from infected chambers, or with small doses of formalin-killed, chamber gonococci were not successful. Thus, neither organisms grown in vivo nor their extracellular products appeared to be exceptionally immunogenic. In immunizing tests with different isolates of gonococci adapted to growth in guinea-pig chambers, cross-immunity to chamber infection with low challenge doses was detected only between two of six isolates. The killing of gonococci in chambers of immunized animals, which occurred only after homologous challenge or with the heterologous strain showing cross-immunity, was not due primarily to humoral factors in the chamber fluid but probably to an enhanced effectiveness of phagocytosis. The serum of immunized animals was bactericidal for homologous strains and for the strain showing cross-immunity but not for strains showing no cross-immunity. Hence, serum bactericidal activity might be a useful indicator for investigating the specificity of immunity produced by different gonococcal strains.
Assuntos
Imunização , Neisseria gonorrhoeae/imunologia , Animais , Atividade Bactericida do Sangue , Reações Cruzadas , Gonorreia/prevenção & controle , Cobaias , Masculino , Especificidade da EspécieRESUMO
Gonococci (strain BS3) passaged three times and harvested directly from plastic chambers implanted subcutaneously in guinea pigs were compared with the parent strain (BS) grown in vitro. The strain grown in vivo produced smaller colonies than that grown in vitro and when examined directly in chamber fluid was sometimes not pilated. It was more resistant to the bactericidal action of human serum and more infective for guinea-pig chambers. In gel diffusion, extracts of the organisms adapted in vivo and cultured once on agar appeared to contain one or two antigens that were different from those in extracts of the in vitro grown organisms; and on polyacrylamide gels, electrophoresis of similar extracts showed one or more protein components for strain BS3 which were not seen for strain BS. Gonococci grown in guinea-pig subcutaneous chambers appear to be suitable for studies on the determinants of gonoccal pathogenicity.
Assuntos
Cobaias/microbiologia , Neisseria gonorrhoeae , Animais , Antígenos de Bactérias/análise , Atividade Bactericida do Sangue , Epitopos , Humanos , Neisseria gonorrhoeae/crescimento & desenvolvimento , Neisseria gonorrhoeae/imunologia , Neisseria gonorrhoeae/patogenicidade , Especificidade da Espécie , VirulênciaRESUMO
The high natural resistance of gonococci showing a characteristic 'double highlight' (DH) colonial morphology (Penn, Veale & Smith, 1977b) to intracellular killing by human phagocytes was markedly reduced by addition of rabbit antiserum to the phagocytosis medium or by preincubation of organisms with antiserum. Antisera raised to three different DH gonococcal strains showed a complex pattern of specificity in phagocytosis tests with the homologous organisms and three other DH strains. The effect of antiserum could be neutralized by adsorption with intact organisms or with extracts, prepared ultrasonically, of the homologous strain. Antiserum also promoted the intracellular killing of a strain which had a 'single highlight' colonial morphology (Penn et al., 1977b) and a low natural resistance to phagocytic killing, but adsorption with this strain neutralized the antiserum less consistently than the DH strain. The neutralization of antiserum-mediated promotion of intracellular killing by extracts of organisms naturally resistant to such killing may provide an assay for the aggressins responsible for this resistance.
Assuntos
Neisseria gonorrhoeae/imunologia , Fagócitos/imunologia , Adsorção , Especificidade de Anticorpos , Atividade Bactericida do Sangue , Humanos , Soros Imunes/farmacologia , FagocitoseRESUMO
The resistance of gonococci to complement-mediated killing by serum is important in the pathogenesis of gonorrhoea. Most urethal strains lose this resistance on subculture. The host product(s) which induces the resistance in vivo is therefore fundamental to pathogenesis. Human genital secretions and some sera induced gonococci to serum resistance in vitro. Guinea pig serum was more active than human serum and low molecular weight fractions from it conferred resistance to gonococci in 3 h at 37 degrees C. Similar active fractions were obtained from human sera. Now guinea pig serum has been further fractionated for the low molecular weight inducer by membrane filtration, gel filtration on Sephadex G25, high performance liquid chromatography (HPLC) with a Spherisorb ODS reverse phase column, chromatography on Sephadex LH20 and HPLC with a Partisil SCX cation exchange column. The small yield (less than 1 mg from 400 ml serum) of highly active material was contaminated with breakdown products from the Partisil SCX column and a mixture of compounds. However, analysis indicated the presence of one or more small glucopeptides containing cysteine, glutamic acid, aspartic acid, threonine, serine, glycine, alanine, valine and lysine. Similar glucopeptides are liberated from fresh human red blood cells in slightly hypertonic saline and samples of them induced gonococci to serum resistance.