Gonococci oxidize only glucose and differ antigenically from the other neisseriae. Gonococci usually produce smaller colonies than those of the other neisseriae. Gonococci that require arginine, hypoxanthine, and uracil (Arg−, Hyx−, and Ura− auxotype) tend to grow most slowly on primary culture. Gonococci isolated from clinical specimens or maintained by selective subculture have typical small colonies containing piliated bacteria. On nonselective subculture, larger colonies containing nonpiliated gonococci are also formed. Opaque and transparent variants of both the small and large colony types also occur; the opaque colonies are associated with the presence of a surface-exposed protein, Opa.
N gonorrhoeae is antigenically heterogeneous and capable of changing its surface structures in vitro—and presumably in vivo—to avoid host defenses. Surface structures include the following.
Pili are the hairlike appendages that extend up to several micrometers from the gonococcal surface. They enhance attachment to host cells and resistance to phagocytosis. They are made up of stacked pilin proteins (molecular weight [MW], 17–21 kDa). The amino terminal of the pilin molecule, which contains a high percentage of hydrophobic amino acids, is conserved. The amino acid sequence near the midportion of the molecule also is conserved; this portion of the molecule serves in attachment to host cells and is less prominent in the immune response. The amino acid sequence near the carboxyl terminal is highly variable; this portion of the molecule is most prominent in the immune response. The pilins of almost all strains of N gonorrhoeae are antigenically different, and a single strain can make many antigenically distinct forms of pilin.
Por protein extends through the gonococcal cell membrane. It forms pores in the surface through which some nutrients enter the cell. Por proteins may impact intracellular killing of gonococci within neutrophils by preventing phagosome–lysosome fusion. In addition, variable resistance of gonococci to killing by normal human serum depends on whether Por protein selectively binds to complement components C3b and C4b. The MW of Por varies from 32 to 36 kDa. Each strain of gonococcus expresses only one of two types of Por, but the Por of different strains is antigenically different. Serologic typing of Por by agglutination reactions with monoclonal antibodies was a useful method for studying the epidemiology of N gonorrhoeae. However, this method has been replaced by genotypic methods such as pulsed-field gel electrophoresis, Opa typing, and DNA sequencing.
These proteins function in adhesion of gonococci within colonies and in attachment of gonococci to host cell receptors such as heparin-related compounds and CD66 or carcinoembryonic antigen–related cell adhesion molecules. One portion of the Opa molecule is in the gonococcal outer membrane, and the rest is exposed on the surface. The MW of Opa ranges from 20 to 28 kDa. A strain of gonococcus can express no, one, two, or occasionally three types of Opa, but each strain has 11–12 genes for different Opas. PCR of the opa genes followed by restriction endonuclease digestion, and analysis of subsequent fragments by gel electrophoresis is a useful method of strain typing performed by reference laboratories.
This protein (MW, 30–31 kDa) is antigenically conserved in all gonococci. It is a reduction-modifiable protein (Rmp) and changes its apparent MW when in a reduced state. It associates with Por in the formation of pores in the cell surface.
In contrast to the enteric gram-negative rods (see Chapters 2 and 15), gonococcal lipopolysaccharide (LPS) does not have long O-antigen side chains and is called a lipooligosaccharide (LOS). Its MW is 3–7 kDa. Gonococci can express more than one antigenically different LOS chain simultaneously. Toxicity in gonococcal infections is largely attributable to the endotoxic effects of LOS. Specifically, in the fallopian tube explant model, LOS causes ciliary loss and mucosal cell death.
In a form of molecular mimicry, gonococci make LOS molecules that structurally resemble human cell membrane glycosphingolipids. A structure is depicted in Figure 20-3. The gonococcal LOS and the human glycosphingolipid of the same structural class react with the same monoclonal antibody, indicating the molecular mimicry. The presence on the gonococcal surface of the same surface structures as human cells helps gonococci evade immune recognition.
Structure of gonococcal lipooligosaccharide, which has lacto-N-neotetraose and a terminal galactosamine in a structure similar to the human ganglioside glycosphingolipid series. The basal oligosaccharide is in light red, and the lacto-N-neotetraose is in dark red. (Courtesy of JM Griffiss.)
The terminal galactose of human glycosphingolipids is often conjugated with sialic acid. Sialic acid is a nine-carbon, 5-N-acetylated ketulosonic acid also called N-acetylneuraminic acid (NANA). Gonococci do not make sialic acid but do make a sialyltransferase that functions to take NANA from the human nucleotide sugar cytidine 5′-monophospho-N-acetylneuraminic acid (CMPNANA) and place the NANA on the terminal galactose of a gonococcal acceptor LOS. This sialylation affects the pathogenesis of gonococcal infection. It makes the gonococci resistant to killing by the human antibody–complement system and interferes with gonococcal binding to receptors on phagocytic cells.
N meningitidis and Haemophilus influenzae make many but not all of the same LOS structures as N gonorrhoeae. The biology of the LOS for the three species and for some of the nonpathogenic Neisseria species is similar. Four of the various serogroups of N meningitidis make different sialic acid capsules (see later discussion), indicating that they also have biosynthetic pathways different from those of gonococci. These four serogroups sialylate their LOS using sialic acid from their endogenous pools.
Several antigenically constant proteins of gonococci have poorly defined roles in pathogenesis. Lip (H8) is a surface-exposed protein that is heat modifiable like Opa. The Fbp (ferric-binding protein), similar in MW to Por, is expressed when the available iron supply is limited, such as in human infection. Gonococci elaborate an IgA1 protease that splits and inactivates IgA1, a major mucosal immunoglobulin of humans. Meningococci, H influenzae, and Streptococcus pneumoniae elaborate similar IgA1 proteases.
Genetics and Antigenic Heterogeneity
Gonococci have evolved mechanisms for frequently switching from one antigenic form (pilin, Opa, or LPS) to another antigenic form of the same molecule. This switching takes place in one in every 102.5–103 gonococci, an extremely rapid rate of change for bacteria. Because pilin, Opa, and LPS are surface-exposed antigens on gonococci, they are important in the immune response to infection. The molecules’ rapid switching from one antigenic form to another helps the gonococci elude the host immune system.
The switching mechanism for pilin, which has been the most thoroughly studied, is different from the mechanism for Opa.
Gonococci have multiple genes that code for pilin, but only one gene is inserted into the expression site. Gonococci can remove all or part of this pilin gene and replace it with all or part of another pilin gene. This mechanism allows gonococci to express many antigenically different pilin molecules over time.
The switching mechanism of Opa involves, at least in part, the addition or removal from the DNA of one or more of the pentameric coding repeats preceding the sequence that codes for the structural Opa gene. The switching mechanism of LPS is unknown.
Gonococci contain several plasmids; 95% of strains have a small, “cryptic” plasmid (MW, 2.6 mDa) of unknown function. Two other plasmids (MW, 3.4 and 4.7 mDa) contain genes that code for TEM-1 type (penicillinases) β-lactamases, which cause resistance to penicillin. These plasmids are transmissible by conjugation among gonococci; they are similar to a plasmid found in penicillinase-producing Haemophilus species and may have been acquired from Haemophilus or other gram-negative organisms. About 5–20% of gonococci contain a plasmid (MW, 24.5 × 106 kDa) with the genes that code for conjugation; the incidence is highest in geographic areas where penicillinase-producing gonococci are most common. High-level tetracycline resistance (minimum inhibitory concentrations [MICs] of ≥16 mg/L) has developed in gonococci by the insertion of a streptococcal gene tetM coding for tetracycline resistance into the conjugative plasmid.
Pathogenesis, Pathology, and Clinical Findings
Gonococci exhibit several morphologic types of colonies (see earlier discussion), but only piliated bacteria appear to be virulent. Opa protein expression varies depending on the type of infection. Gonococci that form opaque colonies are isolated from men with symptomatic urethritis and from uterine cervical cultures at midcycle. Gonococci that form transparent colonies are frequently isolated from men with asymptomatic urethral infection, from menstruating women, and from patients with invasive forms of gonorrhea, including salpingitis and disseminated infection. Antigenic variation of surface proteins during infection allows the organism to circumvent host immune response.
Gonococci attack mucous membranes of the genitourinary tract, eye, rectum, and throat, producing acute suppuration that may lead to tissue invasion; this is followed by chronic inflammation and fibrosis. Men usually have urethritis, with yellow, creamy pus and painful urination. The process may extend to the epididymis. As suppuration subsides in untreated infection, fibrosis occurs, sometimes leading to urethral strictures. Urethral infection in men can be asymptomatic. In women, the primary infection is in the endocervix and extends to the urethra and vagina, giving rise to mucopurulent discharge. It may then progress to the uterine tubes, causing salpingitis, fibrosis, and obliteration of the tubes. Infertility occurs in 20% of women with gonococcal salpingitis. Chronic gonococcal cervicitis and proctitis are often asymptomatic.
Gonococcal bacteremia leads to skin lesions (especially hemorrhagic papules and pustules) on the hands, forearms, feet, and legs and to tenosynovitis and suppurative arthritis, usually of the knees, ankles, and wrists. Gonococci can be cultured from blood or joint fluid of only 30% of patients with gonococcal arthritis. Gonococcal endocarditis is an uncommon but severe infection. Gonococci sometimes cause meningitis and eye infections in adults; these have manifestations similar to those caused by meningococci. Complement deficiency is frequently found in patients with gonococcal bacteremia. Patients with bacteremia, especially if recurrent, should be tested for total hemolytic complement activity.
Gonococcal ophthalmia neonatorum, an infection of the eye in newborns, is acquired during passage through an infected birth canal. The initial conjunctivitis rapidly progresses and, if untreated, results in blindness. To prevent gonococcal ophthalmia neonatorum, instillation of tetracycline, erythromycin, or silver nitrate into the conjunctival sac of newborns is compulsory in the United States.
Gonococci that produce localized infection are often serum sensitive (ie, killed by antibody and complement).
Diagnostic Laboratory Tests
Pus and secretions are taken from the urethra, cervix, rectum, conjunctiva, throat, or synovial fluid for culture and smear. Blood culture is necessary in systemic illness, but a special culture system is helpful because gonococci (and meningococci) may be susceptible to the polyanethol sulfonate present in standard blood culture media. Proprietary swabs may be required for diagnostic molecular assays. Clinicians should check with clinical laboratories regarding the appropriate collection devices for the assays used in a particular institution.
Gram-stained smears of urethral or endocervical exudates reveal many diplococci within pus cells. These give a presumptive diagnosis. Stained smears of the urethral exudate from men have a sensitivity of about 90% and a specificity of 99%. Stained smears of endocervical exudates have a sensitivity of about 50% and a specificity of about 95% when examined by an experienced microscopist. Additional diagnostic testing of urethral exudates from men is not necessary when the stain result is positive, but nucleic acid amplification tests (NAATs) or cultures should be done for women. Stained smears of conjunctival exudates can also be diagnostic, but those of specimens from the throat or rectum are generally not helpful.
Immediately after collection, pus or mucus is streaked on enriched selective medium (eg, modified Thayer-Martin medium [MTM]) and incubated in an atmosphere containing 5% CO2 (candle extinction jar) at 37°C. To avoid overgrowth by contaminants, the selective medium contains antimicrobial drugs (eg, vancomycin, 3 μg/mL; colistin, 7.5 μg/mL; amphotericin B, 1 μg/mL; and trimethoprim, 3 μg/mL). If immediate incubation is not possible, the specimen should be placed in a CO2-containing transport-culture system. Forty-eight hours after culture, the organisms can be quickly identified by their appearance on a Gram-stained smear; by oxidase positivity; and by coagglutination, immunofluorescence staining, or other laboratory tests. The species of subcultured bacteria may be determined by oxidation of specific carbohydrates (see Table 20-1). Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has potential to provide rapid (same-day) identification of cultured isolates. The gonococcal isolates from anatomic sites other than the genital tract or from children should be identified as to species using two different confirmatory tests because of the legal and social implications of a positive culture result. Most laboratories have abandoned culture in favor of NAATs. Because of this, it may be difficult to monitor for increasing multidrug resistance (see discussion below). Culture should be considered in a patient who appears to have failed standard treatment.
D. Nucleic Acid Amplification Tests
Several Food and Drug Administration–cleared nucleic acid amplification assays are available for direct detection of N gonorrhoeae in genitourinary specimens, and these are the preferred tests from these sources. In general, these assays have excellent sensitivity and specificity in symptomatic, high-prevalence populations. Advantages include better detection, more rapid results, and the ability to use urine as a specimen source. Disadvantages include poor specificity of some assays because of cross-reactivity with nongonococcal Neisseria species. Some of these assays are not approved for use in the diagnosis of extragenital gonococcal infections or for infection in children. NAATs are not recommended as tests of cure because nucleic acid may persist in patient specimens for up to 3 weeks after successful treatment. Patients who are believed to have failed treatment are best reevaluated using culture so that the organism can be tested for resistance (see discussion below under Treatment).
Serum and genital fluid contain IgG and IgA antibodies against gonococcal pili, outer membrane proteins, and LPS. Some IgM of human sera is bactericidal for gonococci in vitro.
In infected individuals, antibodies to gonococcal pili and outer membrane proteins can be detected by immunoblotting, radioimmunoassay, and enzyme-linked immunosorbent assay (ELISA) tests. However, these tests are not useful as diagnostic aids for several reasons, including gonococcal antigenic heterogeneity, the delay in development of antibodies in acute infection, and a high background level of antibodies in the sexually active population.
Repeated gonococcal infections are common. Protective immunity to reinfection does not appear to develop as part of the disease process, because of the antigenic variety of gonococci. Although antibodies can be demonstrated, including the IgA and IgG on mucosal surfaces, they either are highly strain specific or have little protective ability.
Since the development and widespread use of penicillin, gonococcal resistance to penicillin has gradually risen, owing to the selection of chromosomal mutants and to increased prevalence of penicillinase-producing N gonorrhoeae (PPNG) (see earlier discussion). Chromosomally mediated resistance to tetracycline (MIC ≥2 μg/mL) is common. High-level resistance to tetracycline (MIC ≥ 32 μg/mL) also occurs. Spectinomycin resistance as well as resistance to fluoroquinolones has been noted. Single-dose fluoroquinolone treatment was recommended for treatment of gonococcal infections from 1993 until 2006. Since 2006, rates of quinolone resistance among gonococcal isolates have exceeded 5% in men who have sex with men and in heterosexual men. Because of the problems with antimicrobial resistance in N gonorrhoeae, the Centers for Disease Control and Prevention (CDC) recommended that patients with uncomplicated genital or rectal infections be treated with ceftriaxone (250 mg) given intramuscularly as a single dose or 400 mg of oral cefixime as a single dose. Additional therapy with 1 g of azithromycin orally in a single dose or with 100 mg of doxycycline orally twice a day for 7 days is recommended for possible concomitant chlamydial infections. Unfortunately, new data from CDC’s Gonococcal Isolate Surveillance Project (GISP) have noted an increase in the percentage of isolates exhibiting elevated MICs to both oral cefixime and ceftriaxone. This observation, combined with reports of cefixime treatment failures in other countries, has resulted in revised treatment guidelines. Since ceftriaxone is more potent than cefixime, the CDC no longer recommends cefixime as an effective treatment. Injectable ceftriaxone 250 mg IM once plus either azithromycin or doxycycline as written above is recommended for treatment of uncomplicated urethritis, cervicitis, and proctitis. Azithromycin has been found to be safe and effective in pregnant women, but doxycycline is contraindicated. Modifications of these therapies are recommended for other types of N gonorrhoeae infection. See the CDC’s website for the update to the 2010 treatment guidelines (http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6131a3.htm?s_cid=mm6131a3_w).
Because other sexually transmitted diseases may have been acquired at the same time as gonorrhea, steps must also be taken to diagnose and treat these diseases (see discussions of chlamydiae, syphilis, and so on).
Epidemiology, Prevention, and Control
Gonorrhea is worldwide in distribution. In the United States, its incidence rose steadily from 1955 until the late 1970s, when the incidence was between 400 and 500 cases per 100,000 population. Between 1975 and 1997, there was a 74% decline in the rate of reported gonococcal infections. Thereafter, the rates plateaued for 10 years and decreased from 2006 to 2009, but since 2009 rates have once again increased slightly each year. Gonorrhea is exclusively transmitted by sexual contact, often by women and men with asymptomatic infections. The infectivity of the organism is such that the chance of acquiring infection from a single exposure to an infected sexual partner is 20–30% for men and even greater for women. The infection rate can be reduced by avoiding multiple sexual partners, rapidly eradicating gonococci from infected individuals by means of early diagnosis and treatment, and finding cases and contacts through education and screening of populations at high risk. Mechanical prophylaxis (condoms) provides partial protection. Chemoprophylaxis is of limited value because of the rise in antibiotic resistance of the gonococcus.
Gonococcal ophthalmia neonatorum is prevented by local application of 0.5% erythromycin ophthalmic ointment or 1% tetracycline ointment to the conjunctiva of newborns. Although instillation of silver nitrate solution is also effective and is the classic method for preventing ophthalmia neonatorum, silver nitrate is difficult to store and causes conjunctival irritation; its use has largely been replaced by use of erythromycin or tetracycline ointment.