D. R. Veale

Morphological, biological and antigenic properties of Neisseria gonorrhoeae adapted to growth in guinea-pig subcutaneous chambers.

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.

The intracellular survival and growth of gonococci in human phagocytes.

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.

Penetration of Penicillin into Human Phagocytes Containing Neisseria gonorrhoeae : Intracellular Survival and Growth at Optimum Concentrations of Antibiotic

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.

Demonstration of intracellular growth of gonococci in human phagocytes using spectinomycin to kill extracellular organisms.

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.

Factors Affecting the Induction of Phenotypically Determined Serum Resistance of Neisseria gonorrhoeae Grown in Media Containing Serum or its Diffusible Components

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.

Resistance of Neisseria gonorrhoeae Grown in vivo to Ingestion and Digestion by Phagocytes of Human Blood

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.

Phenotypically Determined Resistance of Neisseria gonorrhoeae to Normal Human Serum: Environmental Factors in Subcutaneous Chambers in Guinea Pigs

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.

Fractionation of Guinea Pig Serum for an Inducer of Gonococcal Resistance to Killing by Human Serum: Active Fractions Containing Glucopeptides Similar to Those from Human Red Blood Cells

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.

Investigation of the Determinants of the Survival of Neisseria gonorrhoeae within Human Polymorphonuclear Phagocytes

In studies aimed at identifying the determinants responsible for the ability of gonococci to survive and grow within human phagocytes, the reduction of intracellular survival of phagocyte-resistant gonococci by prior treatment of the bacteria with homologous antiserum provided two indirect means of testing for possible determinants. First, surface washes of the resistant gonococci and fractions of these extracts were examined for ability to neutralize the above effect of antisera. Second, antisera raised to purified surface components of the resistant organisms were examined for ability to promote intracellular killing. A combination of these methods indicate that the aggressins were not pili but components of the outer membrane. A surface wash of a phagocyte-resistant, pilated strain, BS4 (agar), neutralized the activity of homologous antisera before and after centrifuging at 20 000 g, but pili separated from the supernatant did not. A corresponding supernatant from the surface wash of a phagocyte susceptible strain, BSSH, had little neutralizing ability. Antisera to pili failed to reduce intracellular survival of resistant gonococci, whereas antisera against outer membrane vesicles did so. Finally, the neutralizing activity of the surface wash supernatant was removed by centrifugation after treatment with wheat germ agglutinin, which precipitates outer membrane vesicles.

Phenotypic changes in the resistance of Neisseria gonorrhoeae to killing by normal human serum.

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.