The genetics of low-level resistance to penicillin and other antibiotics in a clinical isolate and a multistep laboratory mutant of Neisseria gonorrhoeae was studied by transformation. Mutations at three loci affected sensitivity to penicillin. Mutation atpenA resulted in an eightfold increase in resistance to penicillin without affecting response to other antimicrobial agents. Mutation at ery resulted in a twoto fourfold increase in resistance to penicillin and similar increases in resistance to many other antibiotics, dyes, and detergents. Mutation at penB resulted in a fourfold increase in resistance to penicillin and tetracycline, the phenotypic expression of which was dependent on the presence of mutation at ery. The cumulative effect of mutations at penA, ery, and penB was an approximate 128-fold increase in penicillin resistance, to a minimum inhibitory concentration of 1.0 ,ug/ml. Low-level resistance to tetracycline or chloramphenicol was due to similar additive effects between mutations at the nonspecific ery and penB loci and a locus specific for resistance to each drug (tet and chl, respectively). No evidence was found for penicillinases or other druginactivating enzymes.We previously presented evidence that lowlevel resistance ofNeisseria gonorrhoeae to penicillin (Pen), tetracycline (Tet), chloramphenicol (Chl), and erythromycin (Ery) was determined by separate loci, designated penA, tet, chl, and ery, respectively (29). Introduction of these loci by transforming deoxyribonucleic acid (DNA) individually into a wild-type (sensitive) recipient resulted in phenotypic levels of resistance considerably less than exhibited by the multiply antibiotic-resistant donor strains, however (29). In contrast, introduction of loci for high-level resistance to rifampin (Rif), streptomycin (Str), or spectinomycin resulted in phenotypic resistance in the transformants equal to the donor's (29). These observations suggested that the genetic control of low-level resistance to Pen and other drugs was complex, either due to additive effects between the known loci or existence of other undescribed loci.In this communication, we have utilized genetic transformation to show that low-level resistance to each of Pen, Tet, and Chl is acquired in small increments as the result of additive effects between mutations at several previously described loci and a new locus, designated penB. Both ery and penB are shown to result in low-level cross-resistance between unrelated drugs. Evidence is also presented that pheno-74C typic expression of mutation at penB depends on the presence of mutation at ery.MATERIALS AND METHODS Bacterial strains. Bacterial strains are listed in Table 1. Several have been described previously (16,17,29). Strain FA19 is strain Ceylon 3 from the collection of A. Reyn. Strain FA48 is a two-step mutant from FA19, initially selected for high-level Str resistance, and subsequently for Pen resistance, as described (17). Strain FA5 was a clinical isolate from a patient in Los Angeles, obtained as strain P955 from th...

Loci for resistance to several antibiotics in laboratory-derived strains of Neisseria gonorrhoeae were mapped by genetic transformation. Genes for high-level resistance to streptomycin (str) and spectinomycin (spc) and for low-level resistance to tetracycline (tet) and chloramphenicol (chl) were linked. Also, a locus for high-level resistance to rifampin (rinf was linked to str and tet. The apparent order was rif . .. str . . tet . . . chi. . . spc. Loci for resistance to other antibiotics (penicillin, erythromycin) were transferred independently of each other and were not linked to the cluster around str. Similar linkage relationships were found with str, tet, chl, and spc loci obtained from naturally occurring (clinical) isolates of N. gonorrhoeae.suspended to a density of approximately 5 x 107 colony-forming units per ml (after vigorous agitation to disrupt most clumps) in 0.90 ml of GCBB containing 0.002 M CaCl,. No attempt was made to selec-1284 on July 31, 2020 by guest

The ability of 107 Neisseria isolates to compete for iron with human lactoferrin (LF) was examined. Each of 15 meningococci, 53% of 59 selected gonococci, and 24% of 33 commensal Neisseria could use LF-bound iron for growth. Isolates which could not obtain iron from LF were growth inhibited when sufficient LF was added to defined agar medium to bind available free iron. No difference was observed in the ability of colony type 1 and colony type 4 gonococci of the same strain to compete with LF for iron. LF was growth inhibitory for 50%o of 22 disseminated disease isolates (DGI strains) and 51% of 35 local urogenital disease isolates (UGI strains). Only 14% of gonococcal isolates requiring arginine, hypoxanthine, and uracil for growth were able to compete with LF for iron, whereas 87% of all other gonococcal isolates could do so (P < 0.005). Ability to obtain iron from LF does not appear to be required for survival of Neisseria on mucosal surfaces, nor essential for invasion of the bloodstream by gonococci. However, ability to utilize LF as a source of iron may contribute to differences in pathogenicity among certain gonococcal isolates.

Certain gonococci, which heretofore have lacked a conjugal mating system, can sexually transfer a small plasmid (4.5 x 10)6) daltons) which carries the gene for beta-lactamase production. Frequencies of conjugal transfer were similar into diverse recipients (other gonococci, Neisseria flava, and Escherichia coli), which suggests that gonococci may transfer the plasmid promiscuously in nature.

Mutations (env) which resulted in increased sensitivity of gonococci to diverse compounds were studied by transformation. Strains carrying an env mutation were more sensitive than wild-type strains to several antibiotics, dyes, and detergents. The env mutations resulted in complete phenotypic suppression of low-level resistance to these same drugs determined by mutation at ery. Recombination was observed in transformation crosses between various env mutants. The env locus was not linked to the cluster of antibiotic resistance genes near str and spc.