The genetic basis of multiple drug resistance of Neisseria gonorrhoeae was investigated by the technique of transformation. Six different genetic loci were characterized by the type and amount of antibiotic resistance they controlled, and also by the degree of linkage to other resistance markers. A streptomycin resistance locus is linked to separate loci determining resistance to tetracycline, chloramphenicol, and erythromycin. A multiple resistance locus was identified. This genetic locus determines resistance to a variety of antibacterial agents. Lastly, a locus determining resistance to the penicillins was found which is unlinked to any other resistance locus.The emergence of gonococci with increased drug resistance has rendered many antibiotics ineffective or barely effective in the treatment of gonorrheae (17,20). With growing frequency, multiply drug-resistant strains are encountered which exhibit increased resistance to penicillin, tetracycline, and streptomycin, as well as to other antimicrobial agents (10,11,15).We have undertaken studies to investigate the genetic basis of multiple drug resistance in naturally occurring strains of Neisseria gonorrhoeae. It was possible to analyze antibiotic resistance using transformation, since the gonococcus has been shown previously to be a competent species (16 Media and cultivation of gonococci. GC agar consisting of GC medium base (Difco), and 1.0% chemically defined supplements 1 and 2 (17) were used for routine culturing of gonococci and as the base medium when antibiotics were incorporated. The GC broth used in these experiments was prepared following the formula of GC medium base with the omission of starch and agar. Gonococci were incubated at 36 C under 5% CO2 in a CO2 incubator.MIC technique. The minimal inhibitory concentration (MIC) of a gonococcal isolate was determined by an agar dilution technique. Gonococci to be tested were streaked twice for isolation on GC agar. Colonies (18 to 24 h) were suspended in GC broth, vortexed vigorously, and diluted to contain approximately 5 x 107 colony-forming units (CFU) per ml, as judged against a borium sulfate turbidity standard. The desired concentrations of antibiotic were incorporated into 20 ml of GC agar in a 100-mm Petri dish.

The sensitivities of gonococcal isolates to six antibiotics were determined for gonococci isolated in Philadelphia in 1972. The degree of association between susceptibilities to any two antibiotics was determined (coefficient of correlation). The correlation between penicillin and tetracycline (r = 0.75) was almost as good as that between two penicillins, penicillin G and ampicillin (r = 0.85), but the difference was statistically significant. The lowest correlation found was between erythromycin and chloramphenicol (r = 0.62), two antibiotics seldom used in gonorrhea therapy. In addition, gonococci most resistant to one antibiotic were the most likely to be multiply resistant. This was found with respect to penicillin, tetracycline, erythromycin, and chloramphenicol. Approximately 40% of gonococci classified as "most resistant" (exceeding the resistance of 75% of all isolates) to one antibiotic were also "most resistant" to three others. Finally, multiply resistant mutants were isolated by selection for resistance to either penicillin or tetracycline. These results provide evidence for the existence of a common mechanism for multiple antibiotic resistance in the gonococcus.

Studies on the stability of the normal human fecal flora. J. Bacteriol. 83:968-974. 1962.-The results of two series of stool cultures show that members of the genus Bacteroides constitute the most numerous group of bacteria in the normal human adult fecal flora. Together with the enterococci, coliform bacilli, diphtheroids, and lactobacilli, these major components account for more than 99%7o of the total counts. Wide fluctuations in the number and types of minor organisms observed suggest the probability that they are held in check by these major components which may also possess mechanisms for preventing

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