Review of Medical Microbiology and Immunology, 13th Edition

16. Gram-Negative Cocci

CHAPTER CONTENTS

NEISSERIA

1. Neisseria meningitidis

2. Neisseria gonorrhoeae

Self-Assessment Questions

Summaries of Organisms

Practice Questions: USMLE & Course Examinations

NEISSERIA

Diseases

The genus Neisseria contains two important human pathogens: Neisseria meningitidis and Neisseria gonorrhoeae. N. meningitidis mainly causes meningitis and meningococcemia (Figure 16–1). In the United States, it is the leading cause of death from infection in children. N. 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 Neisseria meningitidis is also known as the meningococcus (plural, meningococci), and Neisseria gonorrhoeae is also known as the gonococcus (plural, gonococci).

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FIGURE 16–1 Meningococcemia. Note purpuric lesions on leg caused by endotoxin-mediated disseminated intravascular coagulation (DIC). (Reproduced with permission from Wolff K, Johnson R (eds): Fitzpatrick’s Color Atlas & Synopsis of Clinical Dermatology. 6th ed. New York: McGraw-Hill, 2009. Copyright © 2009 by The McGraw-Hill Companies, Inc.)

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FIGURE 16–2 Gonorrhea. Note purulent urethral discharge caused by Neisseria gonorrhoeae. (Reproduced with permission from Wolff K, Johnson R (eds): Fitzpatrick’s Color Atlas & Synopsis of Clinical Dermatology. 6th ed. New York: McGraw-Hill, 2009. Copyright © 2009 by The McGraw-Hill Companies, Inc.)

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FIGURE 16–3 Neonatal conjunctivitis (ophthalmia neonatorum) caused by Neisseria gonorrhoeae. Note purulent exudate, especially on lower right eyelid. The other common cause of neonatal conjunctivitis is Chlamydia trachomatis.

TABLE 16–1 Neisseriae of Medical Importance1

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TABLE 16–2 Important Clinical Features of Neisseriae

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Important Properties

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

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FIGURE 16–4 Neisseria gonorrhoeae—Gram stain. Arrow points to typical “kidney bean”–shaped gram-negative diplococci within a neutrophil. (Used with permission from Professor Shirley Lowe, University of California, San Francisco School of Medicine.)

(1) N. meningitidis (meningococcus) has a prominent polysaccharide capsule that enhances virulence by its antiphagocytic action and 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.

TABLE 16–3 Properties of the Polysaccharide Capsule of the Meningococcus1

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(2) N. 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 lipooligosaccharide (LOS), in contrast to the lipopolysaccharide (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 (Figure 16–2).

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FIGURE 16–5 Oxidase test—A drop of the oxidase reagent was placed on the left and right side of the filter paper. Bacteria from a colony of Neisseria gonorrhoeae were rubbed on the drop on the left, and the purple color indicates a positive test (i.e., the organism is oxidase-positive). Bacteria from a colony of Escherichia coli were rubbed on the drop on the right, and the absence of a purple color indicates a negative test (i.e., the organism is oxidase-negative). (Used with permission from Professor Shirley Lowe, University of California, San Francisco School of Medicine.)

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. M. catarrhalis and members of other genera, such as Branhamella, Kingella, and Acinetobacter, are described in Chapter 27. (M. 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 it is not present in the 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 three important virulence factors:

(1) A polysaccharide capsule that enables the organism to resist phagocytosis by polymorphonuclear leukocytes (PMNs).

(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.

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.

Clinical Findings

The two most important manifestations of disease are meningococcemia (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 a typical bacterial meningitis, namely, fever, headache, stiff neck, and an increased level of PMNs in 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 (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 (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

Penicillin G is the treatment of choice for meningococcal infections. A third-generation cephalosporin such as ceftriaxone can also be used. Strains resistant to penicillin have rarely emerged, but sulfonamide resistance is common. In 2007–2008, strains of N. meningitidis resistant to ciprofloxacin emerged.

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.

There are three forms of the meningococcal vaccine for use in the United States, all of which contain the capsular polysaccharide of groups A, C, Y, and W-135 as the immunogen. There are two forms of the conjugate vaccine: Menactra contains the four polysaccharides conjugated to diphtheria toxoid as the carrier protein, whereas Menveo contains the four polysaccharides conjugated to a nontoxic mutant of diphtheria toxin as the carrier protein. Menomune, the unconjugated vaccine, contains only the four polysaccharides (not conjugated to a carrier protein). 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 the vaccines contain the group B polysaccharide because it is not immunogenic in humans. A fourth vaccine created for use in the meningitis belt of Africa called MenAfrivac is a conjugate vaccine that contains only the group A polysaccharide.

In general, the conjugate vaccines are preferred over the unconjugated version. The unconjugated vaccine is effective in preventing epidemics of meningitis and in reducing the carrier rate, especially in military personnel. Travelers to areas where epidemics are occurring should receive the vaccine. College students living in dormitories are encouraged to receive the vaccine. No booster dose is recommended for either form of the vaccine if given after the age of 16 years. 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. A booster dose is recommended for those who received the conjugate vaccine prior to the age of 16. Vaccine Adverse Events Reports describe several cases of Guillain-Barré syndrome following immunization with Menactra. A causal relationship between the immunization and Guillain-Barré syndrome has not been established.

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 (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 (DGI) 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) (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 (Figure 16–1). However, nucleic acid amplification tests are widely used as screening tests (see below).

In men, the finding of gram-negative diplococci within PMNs in a urethral discharge specimen is sufficient for diagnosis (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 (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.

Two rapid tests that detect the presence of gonococcal nucleic acids in patient specimens are widely used as a screening test. These tests are highly sensitive and specific. In one type of test, the gonococcal nucleic acids are amplified (amplification tests), and in the other type, they are not amplified. The amplification tests, often abbreviated NAAT, 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. Azithromycin or ciprofloxacin should be used if the patient is allergic to penicillins or cephalosporins. Because mixed infections with C. trachomatis are common, azithromycin or doxycycline should be prescribed also. 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.

Prevention

The prevention of gonorrhea involves the use of condoms and the prompt treatment of symptomatic patients and their contacts. Cases of gonorrhea must be reported to the public health department to ensure proper follow-up. 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 less frequently. No vaccine is available.

SELF-ASSESSMENT QUESTIONS

1. Regarding the differences between Neisseria meningitidis (meningococci) and Neisseria gonorrhoeae (gonococci), which one of the following is the most accurate statement?

(A) Meningococci are oxidase-positive, whereas gonococci are not.

(B) Meningococci have a thick polysaccharide capsule, whereas gonococci do not.

(C) Meningococci have lipid A, whereas gonococci do not.

(D) Meningococci produce penicillinase, whereas gonococci do not.

(E) Meningococci synthesize IgA protease, whereas gonococci do not.

2. Your patient is a 14-year-old girl who was sent home from school because she had a fever of 102°C, a severe headache, and was falling asleep in class. When her fever rose to 104°C, her mother took her to the emergency room, where a blood pressure of 60/20 and several petechial hemorrhages were found. Gram-negative diplococci were seen in a Gram stain of the spinal fluid. Which one of the following is most likely to cause the fever, hypotension, and petechial hemorrhages?

(A) Endotoxin

(B) IgA protease

(C) Oxidase

(D) Pilus protein

(E) Superantigen

3. Regarding the patient in Question 2, which one of the following is the best antibiotic to treat the infection?

(A) Azithromycin

(B) Doxycycline

(C) Penicillin G

(D) Rifampin

(E) Trimethoprim-sulfamethoxazole

4. Regarding the differences between Neisseria meningitidis (meningococci) and Neisseria gonorrhoeae (gonococci), which one of the following is the most accurate statement?

(A) Humans are the reservoir for both organisms.

(B) Many clinical isolates of meningococci produce β-lactamase, but clinical isolates of gonococci do not.

(C) Meningococci have multiple antigenic types, but gonococci have only one antigenic type.

(D) The conjugate vaccine against gonorrhea contains seven types of the pilus protein as the immunogen.

(E) The main mode of transmission for both organisms is respiratory droplets.

5. Your patient is a 20-year-old man with a urethral exudate. You do a Gram stain of the pus and see gram-negative diplococci with neutrophils. Which one of the following is the best antibiotic to treat the infection?

(A) Ceftriaxone

(B) Gentamicin

(C) Penicillin G

(D) Trimethoprim-sulfamethoxazole

(E) Vancomycin

ANSWERS

1. (B)

2. (A)

3. (C)

4. (A)

5. (A)

SUMMARIES OF ORGANISMS

Brief summaries of the organisms described in this chapter begin on page 663. Please consult these summaries for a rapid review of the essential material.

PRACTICE QUESTIONS: USMLE & COURSE EXAMINATIONS

Questions on the topics discussed in this chapter can be found in the Clinical Bacteriology section of PART XIII: USMLE (National Board) Practice Questions starting on page 693. Also see PART XIV: USMLE (National Board) Practice Examination starting on page 731.