Vancomycin hypersusceptibility in Neisseria gonorrhoeae isolated from patients involves diverse mutations

We investigated the genetic determinants of hypersusceptibility to vancomycin and erythromycin found in Neisseria gonorrhoeae strains isolated from patients. In terms of resistance (highest concentration of antibiotic permitting growth), the levels of vancomycin resistance of six strains ranged from 0.2 to 1.0 microgram/ml, and the level of erythromycin resistance of these strains was 0.02 or 0.05 micrograms/ml. DNA from these strains was used to introduce their hypersusceptibility determinants into partially isogenic derivatives of N. gonorrhoeae 89 which initially had wild-type levels of resistance to vancomycin (greater than or equal to 3.0 micrograms/ml) and erythromycin (greater than or equal to 0.1 microgram/ml). The recombination frequencies found in reciprocal transformation tests of six isogenic strains indicated that the mutations responsible for vancomycin hypersusceptibility were located at different sites. The transformants selected for increased resistance to vancomycin were also resistant to erythromycin. This evidence, together with DNA concentration-response curves, indicated that the mutations affected either one gene locus or closely linked loci. The recombination indices obtained in crosses between our hypersusceptible strains and DNAs from reference strains carrying the envelope mutations env-1, env-2, env-3, and env-10 showed that the mutation (designated env-12) responsible for erythromycin hypersusceptibility in one strain (89-954) was located in close proximity to env-2. The determinant of vancomycin hypersusceptibility in strain 89-954 was distinct from env-12, but the two were linked. In the other five isogenic strains, the hypersusceptibilities to both vancomycin and erythromycin could be annulled by spontaneous mutations in a locus provisionally designated vel because of its likely effects on the envelope. Vel+ mutants obtained by selection with either vancomycin alone or erythromycin alone gained increased resistance to both antibiotics.