Competition between Neisseria gonorrhoeae and Staphylococcus epidermidis during iron-limited or replete continuous culture.

Neisseria gonorrhoeae strain P9-2 was grown in iron-limited or replete continuous culture at a dilution rate of 0.05 h-1, in the presence and absence of Staphylococcus epidermidis. Gonococci maintained expression of pili (P+) and the transparent colony phenotype in pure culture during transitions of iron- and cystine-limited growth. They competed well with staphylococci during iron-limited co-culture and comprised greater than 95% of the population. Transition to cystine-limited growth allowed the staphylococcus to predominate but the gonococcus did not wash out. Furthermore, the gonococcal opaque colony phenotype (O+), indicating synthesis of outer membrane proteins II, was now expressed. Restoration of iron limitation returned the co-culture to its original composition but with P+O+ gonococci dominating. These results suggest that environments might exist in Man where gonococci can compete successfully with normal indigenous bacteria during infection.

Influence of iron-limited and replete continuous culture on the physiology and virulence of Neisseria gonorrhoeae.

Neisseria gonorrhoeae strains P9-2 (PenS) and KW2 (PenR) were grown in chemostats of nonferrous design at constant growth rate, pH and dissolved oxygen tension. Iron limitation (micromax 0.1 h-1) was imposed by omitting iron salts from the defined medium and titrating increasing concentrations of the non-metabolizable iron chelators ovotransferrin and Desferal, to progressively decrease the growth yield. Metabolic activity during iron limitation was very high, with a qGlc which was 2- or 11-fold greater than during cystine- or glucose-limited growth, respectively. More aspartate and isoleucine were metabolized during cystine-limited growth, while more glutamate, proline and serine were metabolized during glucose- or iron-limited growth. Significant concentrations of alanine or valine were excreted during cystine- or glucose-limited growth, respectively. Iron-limited growth of an initial inoculum of non-piliated, transparent colony-forming (P-O-) gonococci resulted in the selection of 100% piliated bacteria. Initial inocula of P+O- gonococci retained this phenotype for over 100 generations. Iron-limited gonococci were extremely virulent in the guinea-pig subcutaneous chamber model and inocula of even 12 bacteria grew rapidly and persisted. By contrast, cystine-limited (iron-replete) gonococci retained piliation but did not survive in the chambers. Transition from iron-limited to glucose-limited growth resulted in marked loss of piliation but the bacteria remained virulent. Loss of virulence did not correlate with susceptibility to killing by normal human serum, nor with changes in the content or composition of lipooligosaccharide, which contained 2.9, 3.7, 4.3 and 4.8 kDa moieties. Additional proteins were detectable in Sarkosyl-purified outer membranes of iron-limited gonococci but several proteins with molecular masses similar to those described in the literature for iron-restricted gonococci were detectable in cystine- or glucose-limited bacteria.

Plasmid stability and antibiotic resistance of Neisseria gonorrhoea during glucose-limited continuous culture.

Clinical isolates of Neisseria gonorrhoeae harbouring resistance (R) plasmids of mol. wts 4.4 x 10(6) (Asian) or 3.2 x 10(6) (African) were grown in prolonged glucose-limited continuous culture to determine the segregation efficiency of each type of plasmid and their expression of penicillinase activity in the absence of antibiotic selective pressure. One strain contained the African plasmid and cryptic and conjugative plasmids, which were all retained after 96 generations in the chemostat. By contrast, the other strain lost all plasmids after 100 generations. Both strains showed increased sensitivity to a range of antibiotics, particularly to the penicillins. Loss of penicillinase activity as minimal inhibitory concentration decreased was confirmed for both strains by assaying the enzyme spectrophotometrically. Activity decreased with the number of generations and none was detectable at the time of complete plasmid loss. This decrease was apparently due to individual bacteria ceasing to produce enzyme rather than a gradual decline in production by the whole population. The sensitivities to a broad range of antibiotics also generally increased during glucose-limited growth, but one strain became more resistant to clindamycin and the other to tetracycline.