Immunoglobulin G antibodies directed against protein III block killing of serum-resistant Neisseria gonorrhoeae by immune serum

Neisseria gonorrhoeae that resist complement-dependent killing by normal human serum (NHS) are sometimes killed by immune convalescent serum from patients recovering from disseminated gonococcal infection (DGI). In these studies, killing by immune serum was prevented or blocked by IgG isolated from NHS. Purified human IgG antibodies directed against gonococcal protein III, an antigenically conserved outer membrane protein, contained most of the blocking activity in IgG. Antibodies specific for gonococcal porin (protein I), the major outer membrane protein, displayed no blocking function. In separate experiments, immune convalescent DGI serum which did not exhibit bactericidal activity was restored to killing by selective depletion of protein III antibodies by immunoabsorption. These studies indicate that protein III antibodies in normal and immune human serum play a role in serum resistance of N. gonorrhoeae.     

Diagnosis and treatment of Neisseria gonorrhoeae infections

The most common site of Neisseria gonorrhoeae infection is the urogenital tract. Men with this infection may experience dysuria with penile discharge, and women may have mild vaginal mucopurulent discharge, severe pelvic pain, or no symptoms. Other N. gonorrhoeae infections include anorectal, conjunctival, pharyngeal, and ovarian/uterine. Infections that occur in the neonatal period may cause ophthalmia neonatorum. If left untreated, N. gonorrhoeae infections can disseminate to other areas of the body, which commonly causes synovium and skin infections. Disseminated gonococcal infection presents as a few skin lesions that are limited to the extremities. These legions start as papules and progress into bullae, petechiae, and necrotic lesions. The most commonly infected joints include wrists, ankles, and the joints of the hands and feet. Urogenital N. gonorrhoeae infections can be diagnosed using culture or nonculture (e.g., the nucleic acid amplification test) techniques. When multiple sites are potentially infected, culture is the only approved diagnostic test. Treatments for uncomplicated urogenital, anorectal, or pharyngeal gonococcal infections include cephalosporins and fluoroquinolones. Fluoroquinolones should not be used in patients who live in or may have contracted gonorrhea in Asia, the Pacific islands, or California, or in men who have sex with men. Gonorrhea infection should prompt physicians to test for other sexually transmitted diseases, including human immunodeficiency virus.     

Gonococci causing disseminated gonococcal infection are resistant to the bactericidal action of normal human sera.

The susceptibility of strains of Neisseria gonorrhoeae to the bactericidal action of normal human sera was determined for isolates from patients with disseminated gonococcal infection and uncomplicated gonorrhea. Serum susceptibility was correlated with penicillin susceptibility and auxotype. 38 of 39 strains (97%) of N. gonorrhoeae from Seattle patients with disseminated gonococcal infection were resistant to the complement-dependent bactericidal action of normal human sera. 36 of these were inhibited by less than or equal to mug/ml of penicillin G and required arginine, hypoxanthine, and uracil for growth on chemically defined medium (Arg-Hyx-Ura- auxotype). 12 of 43 isolates from patients with uncomplicated gonorrhea were also of the Arg-Hyx-Ura-auxotype, inhibited by less than or equal to 0.030 mug/ml of penicillin G, and serum resistant. Of the 31 remaining strains of other auxotypes isolated from patients with uncomplicated gonorrhea, 18 (58.1%) were sensitive to normal human sera in titers ranging from 2 to 2,048. The bactericidal action of normal human sera may prevent the dissemination of serum-sensitive gonococci. However, since only a small proportion of individuals infected by serum-resistant strains develop disseminated gonococcal infection, serum resistance appears to be a necessary but not a sufficient virulence factor for dissemination. Host factors such as menstruation and pharyngeal gonococcal infection may favor the dissemination of serum-resistant strains. Since serum-resistant Arg-Hyx-Ura strains are far more frequently isolated from patients with disseminated gonococcal infection than serum-resistant strains of other auxotypes, Arg-Hyx-Ura-strains may possess other virulence factors in addition to serum resistance.     

Antibodies to opacity proteins (Opa) correlate with a reduced risk of gonococcal salpingitis.

Acute salpingitis complicating cervical gonococcal infection is a significant cause of infertility. Relatively little data are available concerning the pathophysiologic mechanisms of this disease. A cohort of 243 prostitutes residing in Nairobi were followed between March 1985 and April 1988. Gonococcal cultures were performed at each visit, and acute salpingitis was diagnosed clinically. Serum at enrollment was tested by immunoblot for antibody to gonococcal outer membrane proteins. 8.6% (146/1689) of gonococcal infections were complicated by salpingitis. Increased risk of salpingitis was associated with younger age, shorter duration of prostitution, HIV infection, number of gonococcal infections, and episodes of nongonococcal salpingitis. Rmp antibody increased the risk of salpingitis. Antibody to Opa decreased the risk of salpingitis. By logistic regression analysis, antibody to Opa was independently associated with decreased risk of gonococcal salpingitis (adjusted odds ratio [OR], 0.35; 95% confidence interval [95%CI], 0.17-0.76); HIV infection (adjusted OR, 3.5; 95% CI, 0.96-12.8) and episodes of nongonococcal salpingitis (adjusted OR, 3.4; 95% CI, 1.8-6.4) were independently associated with an increased risk of salpingitis. Antibody to Opa appears to protect against ascending gonococcal infection, perhaps by interfering with Opa mediated adherence and endocytosis. The demonstration of natural immunity that protects against upper genital tract infection in women suggests that a vaccine to prevent gonococcal salpingitis is possible.     

Disseminated gonococcal infection.

The most frequent systemic complication of acute, untreated gonorrhea is disseminated infection, which develops in 0.5 to 3 percent of the more than 700,000 Americans infected with Neisseria gonorrhoeae each year. The classic triad of features consists of dermatitis, tenosynovitis and migratory polyarthritis. Disseminated gonococcal infection is most common in young women but may develop in sexually active persons of any age. The diagnosis often is not suspected because the initial mucosal infection is frequently asymptomatic, providing no clue to an infectious etiology. Prompt identification and treatment are essential to prevent complications such as endocarditis, meningitis, perihepatitis and permanent joint damage.     

Regulation of catalase in Neisseria gonorrhoeae. Effects of oxidant stress and exposure to human neutrophils.

We studied the effects of oxidant stress on the catalase activity and hydrogen peroxide sensitivity of Neisseria gonorrhoeae. N. gonorrhoeae is an obligate pathogen of man that evokes a remarkable but ineffective neutrophil response. Gonococci make no superoxide dismutase but express high catalase activity. Gonococcal catalase activity increased threefold when organisms were subjected to 1.0 mM hydrogen peroxide. This increase in catalase activity was marked by a parallel increase in protein concentration recognized by a rabbit polyclonal antibody raised against the purified gonococcal enzyme. Catalase was primarily localized to the gonococcal cytoplasm in the presence or absence of stress; only a single isoenzyme of catalase could be identified. Exposure of gonococci to neutrophil-derived oxidants was accomplished by stimulating neutrophils with phorbol myristate acetate or by using gonococcal Opa variants that interacted with neutrophils with different degrees of efficiency. Gonococci exposed to neutrophils demonstrated a twofold increase in catalase activity in spite of some reduction in viability. Exposure of gonococci to 1.0 mM hydrogen peroxide made the organisms significantly more resistant to higher concentrations of hydrogen peroxide and to neutrophils than control organisms. These results suggest that catalase is an important defense for N. gonorrhoeae during attack by human neutrophils. The rapid response of this enzyme to hydrogen peroxide should be taken into consideration in studies designed to evaluate the interaction between neutrophils and gonococci.     

Acquired and natural immunity to gonococcal infection in chimpanzees.

Despite the fact that gonorrhea is our most common reportable infectious disease, little is known about natural and acquired resistance to Neisseria gonorrhoeae. With the chimpanzee model, which mimics human gonococcal infection in signs, symptoms, and host response, a natural resistance to gonococcal challenge was found. One aspect of this natural resistance became evident when the cervix and oral pharynx resisted more gonococci than the urethra. Natural resistance was also shown when environmental factors were found to influence resistance to gonococcal pharyngitis. In addition to natural resistance a postinfection-acquired immunity to the gonococcus was demonstrated. Following gonococcal pharyngitis, this anatomical location successfully resisted more gonococci than were initially resisted. Similarly, more gonococci were successfully resisted in rechallenging the urethra. These findings are related to the clinic situation and suggest possible new approaches to gonorrhea control.     

Immune responses to Helicobacter pylori colonization: mechanisms and clinical outcomes

Helicobacter pylori colonizes the stomachs of half of the world's population and usually persists in the gastric mucosa of human hosts for decades or life. Although most H. pylori-positive people are asymptomatic, the presence of H. pylori is associated with increased risk for the development of peptic ulcer disease, gastric adenocarcinoma and gastric lymphoma. The development of a sustained gastric inflammatory and immune response to infection appears to be pivotal for the development of disease. During its long co-existence with humans, H. pylori has evolved complex strategies to maintain a mild inflammation of the gastric epithelium while limiting the extent of immune effector activity. In this review, the nature of the host immune response to H. pylori infection and the mechanism employed by the bacterium to evade them is considered. Understanding the mechanisms of colonization, persistence and virulence factors of the bacterium as well as the innate and adaptive immune responses of the host are critically important for the development of new strategies to prevent the development of H. pylori-induced gastroduodenal disease.     

Comparison of the penicillin-binding proteins of different strains of Neisseria gonorrhoeae.

This study was undertaken to determine if the categorization of Neisseria gonorrhoeae strains into disseminated gonococcal infection (DGI) and non-DGI types was also paralleled by some common characteristic in their penicillin-binding proteins (PBPs). Fluorography of sodium dodecyl sulfate-containing polyacrylamide gels, on which the [14C]penicillin-labeled PBPs had been separated, was used to visualize the PBPs. No common characteristic PBP for genital or DGI strains was observed. Apart from PBPs 1 and 4, which were observed in all cases, there was an apparently random distribution of other PBPs in both types of N. gonorrhoeae. It was concluded from these data that the penicillin susceptibility associated with most DGI strains of N. gonorrhoeae cannot be correlated with any specific changes in their PBP patterns.     

Antimicrobial interference with bacterial mechanisms of pathogenicity: effect of sub-MIC azithromycin on gonococcal piliation and attachment to human epithelial cells.

The effects of subinhibitory concentrations of azithromycin (CP-62,993) on the piliation and attachment properties of Neisseria gonorrhoeae were examined. Subinhibitory concentrations of azithromycin significantly reduced the percentage of gonococci that expressed assembled pili on their surfaces by decreasing pilin subunit synthesis and substantially decreased gonococcal adherence to human mucosal cells.     

The bitter taste of infection

The human innate immune response to pathogens is complex, and it has been difficult to establish the contribution of epithelial signaling in the prevention of upper respiratory tract infection. The prevalence of chronic sinusitis in the absence of systemic immune defects indicates that there may be local defects in innate immunity associated with such mucosal infections. In this issue of the JCI, Cohen and colleagues investigate the role of the bitter taste receptors in airway epithelial cells, and find that these are critical to sensing the presence of invading pathogens. The participation of respiratory mucosal epithelial cells in innate immune defense has been increasingly appreciated. Not only do airway cells express the full complement of pattern recognition receptors and corresponding adaptor proteins to signal the recruitment of professional immune cells in response to perceived infection, they also participate directly in pathogen eradication. Mucociliary clearance is activated in response to bacterial components, and bacterial killing is mediated through epithelial production of NO and antimicrobial peptides. Although major defects in ciliary function (e.g., Kartagener syndrome) are clearly associated with increased respiratory infection rates, more subtle epithelial abnormalities that might be important in susceptibility to common conditions such as chronic sinus infection have not been fully characterized. Mutations in cystic fibrosis transmembrane conductance regulator (CFTR) that do not cause cystic fibrosis have been associated with chronic rhinosinusitis, although the specific pathogenetic mechanisms involved have not been determined (1). Given the complexity of the human innate immune response to pathogens, it has been difficult to establish the contribution of epithelial signaling in the prevention of upper respiratory tract infection. Nonetheless, given the large number of patients with chronic sinusitis, in the absence of any clinically apparent systemic immune defect, it seems likely that there must be local defects in innate immunity associated with such mucosal infections. In this issue of the JCI, Cohen and coworkers explore unexpected players in innate immune defense: the bitter taste receptors (2).     

Oral ciprofloxacin versus ceftriaxone for the treatment of urethritis from resistant Neisseria gonorrhoeae in Zambia.

Neisseria gonorrhoeae strains resistant to treatment with penicillin, tetracycline, and/or spectinomycin are increasing in prevalence in many parts of the world. In Zambia, 52% of N. gonorrhoeae isolates produced beta-lactamase in 1986. Few oral regimens have proven effective for treatment of resistant N. gonorrhoeae. We conducted a prospective, double-blind, randomized clinical trial of 250 mg of ciprofloxacin given orally versus 250 mg of ceftriaxone given intramuscularly for treatment of uncomplicated gonococcal urethritis in adult males. Two hundred men were enrolled and treated. The two groups were comparable in age (27.5 years), prevalence of latent syphilis (14 and 10%), and human immunodeficiency virus infection (32 and 38%). Of 165 patients with cultures positive for N. gonorrhoeae who returned for follow-up, ciprofloxacin cured 83 of 83 (100%), including 26 with penicillinase-producing N. gonorrhoeae (PPNG) and 21 with N. gonorrhoeae with chromosomally mediated resistance to multiple antibiotics (CMRNG), and ceftriaxone cured 81 of 82 (98.7%), including 30 with PPNG and 19 with CMRNG. Both treatment regimens were well tolerated. Chlamydia trachomatis in urethral exudate was found by direct fluorescent-antibody microscopic examination or by culture in 10 (5%) participants. All N. gonorrhoeae isolates were inhibited by ceftriaxone at 0.06 micrograms/ml, except one which was inhibited at 0.125 micrograms/ml, while ciprofloxacin inhibited all isolates at 0.03 micrograms/ml. Ciprofloxacin is a safe and effective therapy for uncomplicated gonococcal urethritis, including that caused by PPNG and CMRNG in human immunodeficiency virus-infected men.     

Nucleic acid amplification testing for Neisseria gonorrhoeae: an ongoing challenge

Nucleic acid amplification tests (NAATs) for the detection of Neisseria gonorrhoeae became available in the early 1990s. Although offering several advantages over traditional detection methods, N. gonorrhoeae NAATs do have some limitations. These include cost, risk of carryover contamination, inhibition, and inability to provide antibiotic resistance data. In addition, there are sequence-related limitations that are unique to N. gonorrhoeae NAATs. In particular, false-positive results are a major consideration. These primarily stem from the frequent horizontal genetic exchange occurring within the Neisseria genus, leading to commensal Neisseria species acquiring N. gonorrhoeae genes. Furthermore, some N. gonorrhoeae subtypes may lack specific sequences targeted by a particular NAAT. Therefore, NAAT false-negative results because of sequence variation may occur in some gonococcal populations. Overall, the N. gonorrhoeae species continues to present a considerable challenge for molecular diagnostics. The need to evaluate N. gonorrhoeae NAATs before their use in any new patient population and to educate physicians on the limitations of these tests is emphasized in this review.     

Epidemiologic evidence for the development of serovar-specific immunity after gonococcal infection

We tested the hypothesis that strain-specific immunity occurs after gonococcal infection in a longitudinal study of 227 prostitutes resident in one small community who experienced frequent gonococcal infections. Women were examined and cultured for Neisseria gonorrhoeae at 2-wk intervals. Gonococcal isolates were typed according to protein 1 serovar, auxotype, and beta-lactamase plasmid type, and classified as to serovar and strain. The hypothesis was tested by comparing the predictions of the hypothesis with the observations of the study. Over the 14-mo period of the study, major changes in the prevalence of specific serovars were observed in the gonococcal population infecting these women. Women with HIV infection experienced a higher rate of gonococcal infection (0.56 +/- 0.03 vs. 0.46 +/- 0.04, P less than 0.05, t test) compared with HIV-negative women and were more likely to experience multiple infections with the same strain. The duration of prostitution was inversely related to the frequency of gonococcal infection. Women experiencing an infection with a specific gonococcal serovar were at a 2- to 10-fold reduced risk of reinfection with the same serovar, except for the 1B-1 serovar. The results of the study were consistent with all four predictions of the hypothesis. Infection with a specific gonococcal serovar results in specific but incomplete protection against subsequent infection with the homologous serovar. The mechanism of this protection remains to be determined.     

Susceptibilities of Neisseria gonorrhoeae and Ureaplasma urealyticum isolates from male patients with urethritis to several antibiotics including telithromycin

BACKGROUND: The minimal inhibitory concentrations (MICs) of erythromycin, azithromycin, clarithromycin, telithromycin, tetracycline, doxycycline, ciprofloxacin, ofloxacin, norfloxacin, levofloxacin, gemifloxacin and moxifloxacin against 78 Neisseria gonorrhoeae and 31 Ureaplasma urealyticum strains, which were isolated from patients with urethritis in Istanbul, were determined and compared. Additionally, the activities of penicillin and ceftriaxone against N. gonorrhoeae strains were explored. METHODS: MICs were determined by agar and broth dilution methods for N. gonorrhoeae and U. urealyticum, respectively. RESULTS: The susceptibility rates for penicillin and tetracycline in N. gonorrhoeae strains were 35.9 and 24.3%, respectively. All gonococcal strains were susceptible to ceftriaxone, with very low MICs (MIC90 0.008 microg/ml). Telithromycin was highly active against N. gonorrhoeae and U. urealyticum strains (MIC90 0.25 microg/ml for both). Ciprofloxacin was the most active quinolone against N. gonorrhoeae (MIC90 0.008 microg/ml) while quinolone resistance was detected in a single strain (1.3%). CONCLUSIONS: Tetracycline and penicillin should not be the option in empirical treatment of N. gonorrhoeae infections due to the very low susceptibility rates. Ceftriaxone continues to be the first choice antibiotic in the treatment of gonococcal urethritis.     

Evaluation of difloxacin in the treatment of uncomplicated urethral gonorrhea in men.

Difloxacin is a new quinolone antimicrobial agent with in vitro activity against both Neisseria gonorrhoeae and Chlamydia trachomatis and a long (26-h) half-life. A single oral dose of 200 mg of difloxacin was used to treat 30 men with uncomplicated urethral gonorrhea in an open trial. Of the isolates of N. gonorrhoeae, three produced penicillinase and two were resistant to tetracycline. N. gonorrhoeae was eradicated from all 29 evaluable patients. The geometric mean MIC of difloxacin for 30 pretreatment N. gonorrhoeae isolates was 0.014 (range, less than or equal to 0.0039 to 0.03) microgram/ml. Four (13.3%) of the 30 subjects with gonococcal urethritis also had C. trachomatis recovered from their pretreatment cultures. Treatment with difloxacin was associated with the eradication of C. trachomatis from all four men. In addition, C. trachomatis was isolated from the posttreatment culture of only one man who had a negative culture before treatment. Nineteen patients (65.5%) reported adverse experiences, and 17 of them (58.6%) developed symptoms suggestive of central nervous system dysfunction. An oral dose of 200 mg of difloxacin is effective treatment for uncomplicated urethral gonorrhea and may also eliminate a coexisting infection with C. trachomatis. Side effects may limit the utility of this agent.     

Attachment role of gonococcal pili. Optimum conditions and quantitation of adherence of isolated pili to human cells in vitro.

Gonoccocal pili facilitate attachment of virulent Neisseria gonorrhoeae to human cells. To characterize this attachment function, purified gonococcal pili isolated from four strains possessing antigenically distinct pili were radiolabeled with 125I and used to measure the attachment of pili to various human cells in vitro. Human buccal and cervical-vaginal mucosal epithealial cells, fallopian tube mucosa, and sperm bound pili in greater numbers per micrometer2 of surface area (1--10) than fetal tonsil fibroblasts, HeLa M cells, erythrocytes, or polymorphonuclear leukocytes. This cell specificity of attachment suggests a greater density of membrane pili binding sites on cells similar or identical to cells from natural sites of infection. The pili binding sites were quantitated as 1 X 10(4) per cervical-vaginal squamous cell. Pili of all antigenic types attached equally to a given cell type, implying that the attachment moiety of each pilus was similar. Attachement of gonoccocal pili to human cells occurred quickly with saturation of presumed receptor sites within 20--60 min. Attachment was temperature dependent (37 degrees greater than 20 degrees greater than 4 degrees C), and pH dependent (3.5 less than 4.5 less than 5.5 less than 7.5). Attachment was inhibited by antibody to pili (homologous pili Ab greater than heterologous Ab). The extent of possible protection against gonococcal infection due to inhibition of pili-mediated attachment might prove limited as a result of the considerable antigenic heterogeneity among pili and the observation that blockage of pili attachment is maximal only with antibody to pili of the infecting strain.     

Conjugal transfer of penicillin resistance plasmids to proline-citrulline-uracil dependent strains of Neisseria gonorrhoeae

Proline-, citrulline-, uracil (PCU)-dependent Neisseria gonorrhoeae represented only 2% of all Neisseria gonorrhoeae isolated in Greece during 1984-1987. We investigated whether these strains could be recipients of gonococcal plasmids. We carried out bacterial conjugation experiments and obtained the transfer of the African (3.2 Mdal) and of the Asian (4.5 Mdal) penicillin resistance plasmid from clinical penicillinase producing N. gonorrhoeae (PPNG) isolates to PCU gonococcal strains. The transfer frequency was very low, which suggests an explanation of the fact that the wild type PCU strains do not harbour either of these two plasmids. The conjugative plasmid of 24.5 Mdal was not transferred to any of the transconjugants carrying either the African or the Asian type plasmids.     

Gonococcal Invasion of Epithelial Cells Driven by P.IA,a Bacterial Ion Channel with GTP Binding Properties

The neisserial porin P.I is a GTP binding protein that forms a voltage-gated channel that translocates into mammalian cell membranes and modulates host cell signaling events. Here, we report that P.I confers invasion of the bacterial pathogen Neisseria gonorrhoeae into Chang epithelial cells and that this event is controlled by GTP, as well as other phosphorus-containing compounds. Bacterial invasion was observed only for strains carrying the P.IA subtype of porin, which is typically associated with the development of disseminated neisserial disease, and did not require opacity outer membrane proteins, previously recognized as gonococcal invasins. Allelic replacement studies showed that bacterial invasiveness cotransferred with the P.IA (por1A) gene. Mutation of the P.I-associated protein Rmp did not alter the invasive properties. Cross-linking of labeled GTP to the porin revealed more efficient GTP binding to the P.IA than P.IB porin subtype. GTP binding was inhibited by an excess of unlabeled GTP, ATP, and GDP, as well as inorganic phosphate, but not by UTP or beta-glycerophosphate, fully in line with the respective invasion-inhibitory activities observed for these compounds. The P.IA-mediated cellular invasion may explain the more invasive behavior of P.IA strains in the natural infection and may broaden the basis for the development of a P.I-based gonococcal vaccine.     

Dihydrofolate Reductase from Neisseria sp

Members of the genus Neisseria are relatively nonsusceptible to trimethoprim, an inhibitor of dihydrofolate reductase. For example, the minimal inhibitory concentration (MIC) of trimethoprim for N. gonorrhoeae ranges from 2 to 70 mug/ml, whereas the MIC for Escherichia coli is 0.2 mug/ml or less. In an effort to understand this difference, dihydrofolate reductase was partially purified from five Neisseria species and compared with the enzyme from E. coli. N. gonorrhoeae dihydrofolate reductase was similar to that from E. coli in molecular weight (18,000) and affinity for the substrates reduced nicotinamide adenine dinucleotide phosphate and dihydrofolate (K(m) = 13 and 8 muM, respectively). However, the gonococcal enzyme had a decreased affinity for trimethoprim, with an apparent K(i) of 45 x 10(-9) M, some 30-fold greater than the E. coli value of 1.2 x 10(-9) M. These enzymes also differed in their isoelectric points and pH activity profiles. Within the genus Neisseria, the dihydrofolate reductase isolated from N. meningitidis and N. lactamica resembled the N. gonorrhoeae enzyme, and only small differences were detected for the N. flavescens and Branhamella catarrhalis dihydrofolate reductases. These data indicate that the relatively poor affinity of trimethoprim for the dihydrofolate reductase from these organisms may be largely responsible for the relative nonsusceptibility of Neisseria sp. to trimethoprim. The contribution of other resistance mechanisms to the overall nonsusceptibility was assessed. Strains of N. gonorrhoeae with altered cell envelope permeability had MIC values less than twofold different from those of isogenic wild-type strains. Also, a direct relationship was observed between the affinity of trimethoprim analogs for gonococcal dihydrofolate reductase and the MIC of these compounds for the gonococcus. These observations suggest that the cell envelope of N. gonorrhoeae is not impermeable to trimethoprim. Changes in the amount of dihydrofolate reductase activity could cause alterations in the susceptibility of the gonococcus to trimethoprim, as demonstrated with N. gonorrhoeae strains selected for trimethoprim resistance after chemical mutagenesis. However, the level of dihydrofolate reductase activity in wild-type N. gonorrhoeae was similar to that of E. coli, indicating that the difference in the susceptibility of these organisms is not due to greater amounts of enzyme in N. gonorrhoeae.