Chapter 16: Gram-Negative Cocci

Diseases

The genus Neisseria contains two important human pathogens: Neisseria meningitidis and Neisseria gonorrhoeae. Neisseria meningitidis mainly causes meningitis and meningococcemia (Figure 16–1). In the United States, it is the leading cause of death from infection in children. Neisseria gonorrhoeae causes gonorrhea (Figure 16–2), the second most common notifiable bacterial disease in the United States (Tables 16–1 and 16–2). It also causes neonatal conjunctivitis (ophthalmia neonatorum) (Figure 16–3) and pelvic inflammatory disease (PID). Note that N. meningitidis is also known as the meningococcus (plural, meningococci), and N. gonorrhoeae is also known as the gonococcus (plural, gonococci).

Additional information regarding the clinical aspects of infections caused by the organisms in this chapter is provided in Part IX entitled Infectious Diseases beginning on Osteomyelitis.

Important Properties

Neisseriae are gram-negative cocci that resemble paired kidney beans (Figure 16–4).

1. Neisseria meningitidis (meningococcus) has a prominent polysaccharide capsule that enhances virulence by its antiphagocytic action. The capsule also is the immunogen in the vaccine that induces protective antibodies (Table 16–3). Meningococci are divided into at least 13 serologic groups on the basis of the antigenicity of their capsular polysaccharides. Five serotypes cause most cases of meningitis and meningococcemia: A, B, C, Y, and W-135. Serotype A is the leading cause of epidemic meningitis worldwide. Serotype B accounts for most disease in the United States. This is because the group B polysaccharide is not immunogenic in humans and therefore is not part of the vaccines that contain the capsular polysaccharide of the other four groups. In 2014, a vaccine against group B meningococci containing factor H binding protein as the immunogen was approved.

2. Neisseria gonorrhoeae (gonococcus) has no polysaccharide capsule but has multiple serotypes based on the antigenicity of its pilus protein. There is marked antigenic variation in the gonococcal pili as a result of chromosomal rearrangement; more than 100 serotypes are known. Gonococci have three outer membrane proteins (proteins I, II, and III). Protein II plays a role in attachment of the organism to cells and varies antigenically as well.

Neisseriae are gram-negative bacteria and contain endotoxin in their outer membrane. Note that the endotoxin of neisseriae consist of lipooligo saccharide (LOS), in contrast to the lipopoly saccharide (LPS) found in enteric gram-negative rods. Both LPS and LOS contain lipid A, but the oligosaccharide part of LOS contains few sugars, whereas the polysaccharide part of LPS contains a long repeating sugar side chain.

The growth of both organisms is inhibited by toxic trace metals and fatty acids found in certain culture media (e.g., blood agar plates). They are therefore cultured on “chocolate” agar containing blood heated to 80°C, which inactivates the inhibitors. Neisseriae are oxidase-positive (Figure 16–5) (i.e., they possess the enzyme cytochrome c). This is an important laboratory diagnostic test in which colonies exposed to phenylenediamine turn purple or black as a result of oxidation of the reagent by the enzyme (see Figure 16–2).

The genus Neisseria is one of several in the family Neisseriaceae. A separate genus contains the organism Moraxella catarrhalis, which is part of the normal throat flora but can cause such respiratory tract infections as sinusitis, otitis media, bronchitis, and pneumonia. Moraxella catarrhalis and members of other genera, such as Branhamella, Kingella, and Acinetobacter, are described in Chapter 27. (Moraxella catarrhalis is the new name for Branhamella catarrhalis.)

1. Neisseria meningitidis

Pathogenesis & Epidemiology

Humans are the only natural hosts for meningococci. The organisms are transmitted by airborne droplets; they colonize the membranes of the nasopharynx and become part of the transient flora of the upper respiratory tract. Carriers are usually asymptomatic. From the nasopharynx, the organism can enter the bloodstream and spread to specific sites, such as the meninges or joints, or be disseminated throughout the body (meningococcemia).

About 5% of people become chronic carriers and serve as a source of infection for others. The carriage rate can be as high as 35% in people who live in close quarters (e.g., military recruits); this explains the high frequency of outbreaks of meningitis in the armed forces prior to the use of the vaccine. The carriage rate is also high in close (family) contacts of patients. Outbreaks of meningococcal disease also have occurred in college students living in dormitories.

Two organisms cause more than 80% of cases of bacterial meningitis in infants older than 2 months of age: Streptococcus pneumoniae and N. meningitidis. Of these organisms, meningococci, especially those in group A, are most likely to cause epidemics of meningitis. Group B meningococci cause many cases of meningitis in developed countries because they are not present in the capsular polysaccharide vaccine (see “Prevention,” later). Overall, N. meningitidis ranks second to S. pneumoniae as a cause of meningitis but is the most common cause in persons between the ages of 2 and 18 years.

Meningococci have four important virulence factors:

1. A polysaccharide capsule that enables the organism to resist phagocytosis by polymorphonuclear leukocytes (PMNs). The capsule is the immunogen in several commonly used vaccines against meningococci.

2. Endotoxin, which causes fever, shock, and other pathophysiologic changes (in purified form, endotoxin can reproduce many of the clinical manifestations of meningococcemia).

3. An immunoglobulin A (IgA) protease that helps the bacteria attach to the membranes of the upper respiratory tract by cleaving secretory IgA.

4. Factor H binding protein (FHBP) on meningococci, which binds Factor H, an inhibitor of complement factor C3b. The presence of Factor H on the surface of meningococci reduces the opsonizing activity of C3b and reduces the amount of membrane attack complex produced (see complement action in Chapter 63). FHBP is the immunogen in the vaccine against group B meningococci.

Resistance to disease correlates with the presence of antibody to the capsular polysaccharide. Most carriers develop protective antibody titers within 2 weeks of colonization. Because immunity is group-specific, it is possible to have protective antibodies to one group of organisms yet be susceptible to infection by organisms of the other groups.

Complement is an important feature of the host defenses, because people with complement deficiencies, particularly in the late-acting complement components (C6–C9), have an increased incidence of meningococcal bacteremia. Patients receiving eculizumab, a terminal complement inhibitor used in the treatment of paroxysmal nocturnal hemoglobinuria, have a 1000-fold increase in meningococcal disease.

Clinical Findings

The two most important manifestations of disease are meningococcemia (see Figure 16–1) and meningitis. The most severe form of meningococcemia is the life-threatening Waterhouse–Friderichsen syndrome, which is characterized by high fever, shock, widespread purpura, disseminated intravascular coagulation, thrombocytopenia, and adrenal insufficiency. Bacteremia can result in the seeding of many organs, especially the meninges. The symptoms of meningococcal meningitis are those of typical bacterial meningitis, namely, fever, headache, stiff neck, and an increased level of PMNs in the spinal fluid.

Laboratory Diagnosis

The principal laboratory procedures are smear and culture of blood and spinal fluid samples. A presumptive diagnosis of meningococcal meningitis can be made if gram-negative cocci are seen in a smear of spinal fluid (see Figure 16–4). The organism grows best on chocolate agar incubated at 37°C in a 5% CO₂ atmosphere. A presumptive diagnosis of Neisseria can be made if oxidase-positive colonies of gram-negative diplococci are found (see Figure 16–5). The differentiation between N. meningitidis and N. gonorrhoeae is made on the basis of sugar fermentation: meningococci ferment maltose, whereas gonococci do not (both organisms ferment glucose). Immunofluorescence can also be used to identify these species. Tests for serum antibodies are not useful for clinical diagnosis. However, a procedure that can assist in the rapid diagnosis of meningococcal meningitis is the latex agglutination test, which detects capsular polysaccharide in the spinal fluid.

Treatment

Either ceftriaxone or penicillin G is the drug of choice for meningococcal infections. Strains resistant to penicillin have rarely emerged, but sulfonamide resistance is common.

Prevention

Chemoprophylaxis and immunization are both used to prevent meningococcal disease. Either rifampin or ciprofloxacin can be used for prophylaxis in people who have had close contact with the index case. These drugs are preferred because they are efficiently secreted into the saliva, in contrast to penicillin G.

The meningococcal vaccines are described in Table 16–4. The vaccines against groups A, C, Y, and W-135 meningococci contain the polysaccharide capsule as the immunogen. The vaccine against group B meningococci contains FHBP as the main immunogen.

In the United States, the vaccines against groups A, C, Y, and W-135 meningococci are conjugate vaccines, that is, the capsular polysaccharide is conjugated to a carrier protein.

There are three forms of the polysaccharide vaccine for use in the United States: (1) Menactra contains the four polysaccharides conjugated to diphtheria toxoid as the carrier protein; (2) Menveo contains the four polysaccharides conjugated to a nontoxic mutant of diphtheria toxin as the carrier protein; and (3) MenHibrix contains two polysaccharides (C and Y) plus the capsular polysaccharide of Haemophilus influenzae, all conjugated to tetanus toxoid.

Menomune, the unconjugated vaccine, contains only the four polysaccharides (not conjugated to a carrier protein). It is not available in the United States but is used in other countries. Another vaccine created for use in the meningitis belt of Africa, called MenAfriVac, is a conjugate vaccine that contains only the group A polysaccharide.

The conjugate vaccines induce higher titers of antibodies in children than does the unconjugated vaccine. The vaccines induce similar antibody titers in adults. Note that none of these vaccines contain the group B polysaccharide because it is not immunogenic in humans. The conjugate vaccine is recommended for children at the age of 11 to 12 years, which will reduce the incidence of meningococcal disease in teenagers and young adults. The conjugate vaccine is also recommended for children younger than 11 years with high-risk conditions, such as asplenia and HIV infection. Travelers to areas where an epidemic of meningococcal disease is occurring should receive the conjugate vaccine. College students living in dormitories are encouraged to receive the conjugate vaccine.

The vaccine against group B meningococci contains FHBP as the immunogen. It induces antibody against the binding protein, thereby inhibiting the ability of the bacteria to bind Factor H on its surface. This enhances the action of complement, an important host defense, because Factor H blocks complement component C3b from binding to the bacterial surface. Stated another way, if Factor H cannot bind to the surface of the bacteria, that allows C3b, an important opsonizer, to bind.

The FHBP used in the vaccine is made by recombinant DNA techniques in Escherichia coli. The vaccine is approved for use in people age 10 to 25 years. In 2015, a second vaccine against group B meningococci containing four surface proteins (fHbp, NadA [neisserial adhesin A], NHBA [neisserial heparin binding antigen], and OMV [outer membrane vesicle; PorA]) was approved.

2. Neisseria gonorrhoeae

Pathogenesis & Epidemiology

Gonococci, like meningococci, cause disease only in humans. The organism is usually transmitted sexually; newborns can be infected during birth. Because gonococcus is quite sensitive to dehydration and cool conditions, sexual transmission favors its survival. Gonorrhea is usually symptomatic in men but often asymptomatic in women. Genital tract infections are the most common source of the organism, but anorectal and pharyngeal infections are important sources as well.

Pili constitute one of the most important virulence factors, because they mediate attachment to mucosal cell surfaces and are antiphagocytic. Piliated gonococci are usually virulent, whereas nonpiliated strains are avirulent. Two virulence factors in the cell wall are endotoxin (lipooligosaccharide, LOS) and the outer membrane proteins. The organism’s IgA protease can hydrolyze secretory IgA, which could otherwise block attachment to the mucosa. Gonococci have no capsules.

The main host defenses against gonococci are antibodies (IgA and IgG), complement, and neutrophils. Antibody-mediated opsonization and killing within phagocytes occur, but repeated gonococcal infections are common, primarily as a result of antigenic changes of pili and the outer membrane proteins.

Gonococci infect primarily the mucosal surfaces (e.g., the urethra and vagina), but dissemination occurs. Certain strains of gonococci cause disseminated infections more frequently than others. The most important feature of these strains is their resistance to being killed by antibodies and complement. The mechanism of this “serum resistance” is uncertain, but the presence of a porin protein (porin A) in the cell wall, which inactivates the C3b component of complement, appears to play an important role.

The occurrence of a disseminated infection is a function not only of the strain of gonococcus but also of the effectiveness of the host defenses. Persons with a deficiency of the late-acting complement components (C6–C9) are at risk for disseminated infections, as are women during menses and pregnancy. Disseminated infections usually arise from asymptomatic infections, indicating that local inflammation may deter dissemination.

Clinical Findings

Gonococci cause both localized infections, usually in the genital tract, and disseminated infections with seeding of various organs. Gonococci reach these organs via the bloodstream (gonococcal bacteremia).

Gonorrhea in men is characterized primarily by urethritis accompanied by dysuria and a purulent discharge (see Figure 16–2). Epididymitis can occur.

In women, infection is located primarily in the endocervix, causing a purulent vaginal discharge and intermenstrual bleeding (cervicitis). The most frequent complication in women is an ascending infection of the uterine tubes (salpingitis, PID), which can result in sterility or ectopic pregnancy as a result of scarring of the tubes.

Disseminated gonococcal infections (DGIs) commonly manifest as arthritis, tenosynovitis, or pustules in the skin. Disseminated infection is the most common cause of septic arthritis in sexually active adults. The clinical diagnosis of DGI is often difficult to confirm using laboratory tests because the organism is not cultured in more than 50% of cases.

Other infected sites include the anorectal area, throat, and eyes. Anorectal infections occur chiefly in women and homosexual men. They are frequently asymptomatic, but a bloody or purulent discharge (proctitis) can occur. In the throat, pharyngitis occurs, but many patients are asymptomatic. In newborn infants, purulent conjunctivitis (ophthalmia neonatorum) (see Figure 16–3) is the result of gonococcal infection acquired from the mother during passage through the birth canal. The incidence of gonococcal ophthalmia has declined greatly in recent years because of the widespread use of prophylactic erythromycin eye ointment (or silver nitrate) applied shortly after birth. Gonococcal conjunctivitis also occurs in adults as a result of the transfer of organisms from the genitals to the eye.

Other sexually transmitted infections (e.g., syphilis and nongonococcal urethritis caused by Chlamydia trachomatis) can coexist with gonorrhea; therefore, appropriate diagnostic and therapeutic measures must be taken.

Laboratory Diagnosis

The diagnosis of urogenital infections depends on Gram staining and culture of the discharge (see Figure 16–4). However, nucleic acid amplification tests are widely used as screening tests (see later).

In men, the finding of gram-negative diplococci within PMNs in a urethral discharge specimen is sufficient for diagnosis (see Figure 16–4). In women, the use of the Gram stain alone can be difficult to interpret; therefore, cultures should be done. Gram stains on cervical specimens can be falsely positive because of the presence of gram-negative diplococci in the normal flora and can be falsely negative because of the inability to see small numbers of gonococci when using the oil immersion lens. Cultures must also be used in diagnosing suspected pharyngitis or anorectal infections.

Specimens from mucosal sites, such as the urethra and cervix, are cultured on Thayer-Martin medium, which is a chocolate agar containing antibiotics (vancomycin, colistin, trimethoprim, and nystatin) to suppress the normal flora. The finding of an oxidase-positive colony (see Figure 16–5) composed of gram-negative diplococci is sufficient to identify the isolate as a member of the genus Neisseria. Specific identification of the gonococcus can be made either by its fermentation of glucose (but not maltose) or by fluorescent antibody staining. Note that specimens from sterile sites, such as blood or joint fluid, can be cultured on chocolate agar without antibiotics because there is no competing normal flora.

Nucleic acid amplification tests, often abbreviated NAAT, detect the presence of gonococcal nucleic acids in patient specimens. These tests are widely used for screening purposes, produce results rapidly, and are highly sensitive and specific. They can be used on urine samples, obviating the need for more invasive collection techniques. Note that serologic tests to determine the presence of antibody to gonococci in the patient’s serum are not useful for diagnosis.

Treatment

Ceftriaxone is the treatment of choice in uncomplicated gonococcal infections. If the patient is allergic to penicillins or cephalosporins, a regimen such as gentamicin plus azithromycin can be used.

Because mixed infections with C. trachomatis are common, azithromycin or doxycycline should be prescribed in addition to ceftriaxone. A follow-up culture should be performed 1 week after completion of treatment to determine whether gonococci are still present. Treatment of complicated gonococcal infections, such as PID, typically requires hospitalization. Treatment regimens are complex and beyond the scope of this book.

Prior to the mid-1950s, all gonococci were highly sensitive to penicillin. Subsequently, isolates emerged with low-level resistance to penicillin and to other antibiotics such as tetracycline and chloramphenicol. This type of resistance is encoded by the bacterial chromosome and is due to reduced uptake of the drug or to altered binding sites rather than to enzymatic degradation of the drug.

Then, in 1976, penicillinase-producing (PPNG) strains that exhibited high-level resistance were isolated from patients. Penicillinase is plasmid-encoded. PPNG strains are now common in many areas of the world, including several urban areas in the United States, where approximately 10% of isolates are resistant. Isolates resistant to fluoroquinolones, such as ciprofloxacin, have become a significant problem, and fluoroquinolones are not recommended as treatment. Isolates resistant to sulfonamides and tetracyclines have also been recovered. In 2017, the World Health Organization (WHO) reported that several strains of gonococci resistant to all known antibiotics have been isolated.

Prevention

The prevention of gonorrhea involves the use of condoms and the prompt treatment of symptomatic patients and their sex partners. Cases of gonorrhea must be reported to the public health department to ensure proper follow-up and contact tracing. A major problem is the detection of asymptomatic carriers. Gonococcal conjunctivitis in newborns is prevented most often by the use of erythromycin ointment. Silver nitrate drops are used in some countries. No vaccine is available.