Chapter 50
Gram-Negative Cocci in Ocular Disease
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Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria catarrhalis are the gram-negative cocci of interest to the ophthalmologist.The appearance of these species on Gram stain and in culture can be indistinguishable. These organisms often occur in pairs or in short chains, and their adjacent sides may be flattened, giving them a coffee-bean shape. They are nonmotile, do not form spores, inhabit the mucous membrane surfaces of warm-blooded hosts, and are oxidase-positive and catalase-positive.1 The specific species can be determined on the basis of biochemical or serologic differences. The infections caused by these pathogenic organisms are uncommon, but they can rapidly progress and can cause significant ocular morbidity.
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N. gonorrhoeae is the agent responsible for gonorrhea and is one of the most commonly reported infectious diseases. Although the incidence steadily increased between 1960 and 1975, the last 10 to 15 years have shown a steady decline in the incidence of this disease.2 Symptoms occur 1 to 10 days (average 3 to 5 days) after acquiring the infection. In men, the organism causes an acute urethritis with dysuria. A thick, creamy, purulent discharge is present in more than 95% of males and usually abates within 6 months. If untreated, complications such as prostatitis, seminal vesiculitis, infections of the vas deferens, epididymitis, periurethral abscess, and urethral stricture can occur. In females, the primary infection occurs in the endocervix, with associated urethral infection occurring in more than 80% of patients. A yellow vaginal discharge is often present, with associated dysuria, labial tenderness, and dyspareunia. Retrograde spread can result in endometritis and pelvic inflammatory disease. In both males and females, the infection can include other sites, such as the anorectal area, pharynx, skin, joints, synovium, endocardium, myocardium, pericardium, liver, and eye.3


Gonorrheal ophthalmia neonatorum usually presents as a unilateral conjunctivitis with an incubation period of a few hours to 2 to 3 days. The disease is characterized by lid edema, severe bulbar conjunctival injection, and chemosis, with a watery or serosanguineous discharge. After 4 or 5 days, the disease enters the purulent stage, with increasing, copious, purulent discharge and formation of a pseudomembrane on the tarsal conjunctiva. Untreated, the conjunctival inflammation will slowly decrease, but conjunctival scarring may occur. The cornea can be involved, displaying peripheral corneal infiltrates that can ulcerate.

Adults usually present with an acute conjunctivitis characterized by a purulent discharge and associated lid edema. Corneal involvement is more common in adults than in neonates or prepubertal children. Complications involving the uveal tract and retina, as well as a dacryoadenitis, can occur.

The diagnosis should be suspected on the basis of the clinical presentation and is confirmed by finding gram-negative cocci in the urethral discharge, cervical smears, or conjunctival discharge.


Naturally occurring gonococcal infections are limited to humans; rarely does N. gonorrhoeae affect the mucosal tissue of other species.4 The organisms attach to the mucosal cells by their pili; this appears to occur rapidly and involves a specific receptor on human cells as well as nonpili surface antigens.5,6 The receptor for gonococcal pili is the B1-3-N-acetylgalactosamine-4-galactose portion of a ganglioside. The attachment is facilitated at 37°C under acidic conditions. Thus, the number of pili on the organism, the density of the receptors, and the local conditions appear to play a role in the adherence of the organism to a particular site.

Pili are hairlike protein polymers projecting from the surface of the cell wall. They are composed of repeated peptide subunits, which have an approximate molecular weight of 20,000 daltons7; each subunit contains 165 amino acids.8 The amino-terminal end of the pili protein has a constant amino acid sequence, whereas the middle region and carboxy-terminal end have variable amino acid sequences. The antigenic heterogenicity of the pili isolated from different strains of N. gonorrhoeae appears to be caused by the middle and carboxy-terminal end of the pili protein.7 In addition, the pili may function to overcome electrostatic forces that occur between negatively charged mucosal surfaces, as well as similarly charged bacterial cells.9 They may also be involved in phagocytosis by neutrophils and in the exchange of genetic material. Antibodies directed at the pili can inhibit adherence and infection.10

The surface of the outer membrane of the Neisseria organisms also contains proteins that can affect antibody formation, cell-mediated immune response, and penetration of human cells.11,12 Protein I (32,000 to 36,000 daltons) is the most abundant outer membrane protein. This protein covers the width of the outer membrane and functions in the entrance and exit of small molecules to and from the periplastic space.11 Protein I is also termed a porin. These proteins allow the passage of hydrophilic molecules through the hydrophobic outer membrane. Protein I molecules move rapidly from the outer membrane of N. gonorrhoeae to the cytoplasmic membrane of human cells during endocytosis.12 Antibodies directed against this protein are used for various serologic typing tests for gonococci.13

The outer membrane also contains opa proteins (opacity proteins), formerly called protein II. These proteins have a molecular weight of 20,000 to 27,000 daltons. Each organism has 10 to 12 complete opa genes in the chromosome. This protein plays a role in the attachment of the gonococcal outer membrane to epithelial cells and polymorphonuclear leukocytes, in the adhesion of gonococci with each other, and in the susceptibility to killing by normal serum.14 Thus, whereas the pili are responsible for the attachment of the bacteria to the epithelial cells, components of the outer membrane are involved in penetration into the cells.

A third type of protein, protein III or Rmp (reduction modifiable protein), has a molecular weight of 30,000 to 31,000 daltons. It is closely associated with the lipo-oligosaccharide and porin proteins of the outer membrane. This protein shows little intra- or interstrain variation, but it does share a similarity with Escherichia coli's outer membrane proteins, called OmpA (outer membrane protein A).15 In addition, several other outer membrane proteins have been described. These additional proteins may function as receptors for human transferrin and lactoferrin, which are important in acquiring the necessary iron for bacterial metabolism.16

Other substances are also important in the virulence of N. gonorrhoeae. Gonococcal lipopolysaccharide (LPS) is composed of a lipid A portion (a component of the outer membrane) and a polysaccharide portion that protrudes from this membrane and has endotoxin activity, which is probably responsible for local killing of the cells and the initiation of inflammation.17

Four hydrolases have been identified in suspensions of N. gonorrhoeae cultures.18 Gonocosin, an endopeptidase, is an alkaline proteinase that cleaves elastin. This capability could aid in the invasiveness of gonococci in ophthalmia neonatorum and in the tenosynovitis of disseminated gonococcal infections. Gonococcal aminopeptidase-P, an exopeptidase, cleaves the peptide bond between the NH2-terminus and a following proline residue. This enzyme may help in the hydrolysis of internal proline-containing peptides. Gonococcal proline aminopeptidase hydrolyzes the tripeptide Pro-Gly-Gly. These two enzymes may be important in helping gonococci to use proline, and only secondarily may cause tissue damage. Gonococcal asparaginase inhibits protein synthesis and decreases immune responses.18 These functions may aid in cellular invasion by gonococci.

N. gonorrhoeae, in addition to N. meningitidis, produces a proteolytic enzyme, IgA 1 protease, that is capable of selectively cleaving human serum and secretory IgA 1 into both the Fab and the Fc fragment, thus neutralizing the effect of secretory IgA.18,19 This protease is immunogenic, and antibodies appear both during infection and in asymptomatic carriers. Antibody activity is lost after cleavage.20

After gaining access to the bloodstream, the organisms develop thicker capsules. The capsules themselves do not appear to play a role in the adherence of the organism to mucosal epithelial cells; however, they may help in diminishing phagocytosis by both macrophages and polymorphonucleocytes. The organisms tend to attack and penetrate stratified squamous epithelial cells.6 The organism penetrates the cells by endocytosis after extension of cytoplasmic processes around the bacteria. The organism may multiply within the cell, or multiple bacteria may be engulfed by the same cell. Infection can then spread along the mucous membranes by proliferation in the mucosal film, which also allows retrograde spread. Gonococcal LPS aids in the penetration of the organism into epithelial cells after adherence by the pili and transmembrane channel formation by protein I. The various hydrolases that cleave peptides aid in tissue penetration and blunt the immune response. The presence of large numbers of polymorphonuclear leukocytes, as well as activation of complement, may be a result of the production of gonococcal LPS.6 This substance may also be responsible for destroying cells that the organism fails to penetrate.

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N. meningitidis can cause a spectrum of infection ranging from mild disease with rapid recovery to fulminating disease causing death. Systemic infections are characterized by a petechial rash, signs of meningitis, shock, and disseminated intravascular coagulation.21 Humans are the only hosts. Although the disease can affect any age group, most infections occur in children younger than 5 years, probably initiated by way of the respiratory tract. The ocular manifestation depends on the severity of the disease. Lid edema is common, with petechial hemorrhages developing on the skin. An acute purulent conjunctivitis with papillary hypertrophy and subconjunctival hemorrhages can occur. Corneal involvement is less common than with gonorrheal infections. Peripheral grayish corneal infiltrates, which can ulcerate and vascularize, may occur. In addition, orbital abscesses, iridocyclitis, endophthalmitis, and panophthalmitis have been described.

The basic structure of N. meningitidis is similar to that of N. gonorrhoeae. This organism also contains pili, which help it adhere to certain epithelial cells, especially those of the posterior pharynx.21 In addition, meningococcal LPS appears to be similar to gonococcal LPS in its function. Hydrolases are also produced by N. meningitidis, and they appear to function in a similar fashion. However, meningococci produce more gonocosin (78 times) and asparaginase (37 times) than do gonococci. N. meningitidis also produces IgA1 protease that cleaves serum and secretory IgA1.

In contrast to N. gonorrhoeae, N. meningitidis spreads hematogenously, resulting in either meningitis or vascular collapse. The differences in the clinical pathogenesis of these two organisms are probably caused by the unique features of the capsule of the meningococcus, the differing production of gonocosin and asparaginase, and the action of meningococcal LPS. The polysaccharide capsule on the surface of N. meningitidis helps the organism to resist phagocytosis. In addition, the capsular material of N. meningitidis (serogroup B) is indistinguishable from the neuraminic acid found in the human central nervous system. Thus, the organism's capsule is not recognized as foreign by the human immune system and is completely nonimmunogenic.22 The major antigens of N. meningitidis are associated with the group's specific capsular polysaccharide, so it has been possible to develop vaccines to protect against meningococcal disease. However, after immunization, the antibody titer falls rapidly over time.23,24 In the United States, the vaccine is not recommended for general use, but only for persons at high risk. The vaccines are poorly immunogenic in children younger than 2 years.

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Immune responses have been studied in sera and cells from the systemic circulation and local secretions. Most observations suggest a key role for antibodies and a small role, if any, for cell-mediated immune responses. Gonococci capable of affecting local mucous membranes, but not causing disseminated infections, are readily killed by antibody and complement in normal human sera.25 In contrast, gonococci that cause disseminated infections are resistant to such killing. This peculiar resistance correlates with certain protein I antigens.26

Most persons with mucous membrane infections produce antibodies to protein I and protein II, as well as to the pili.27 Both IgG and secretory IgA antibodies have been recovered from the genital tracts of patients with gonococcal infection. These antibodies block the attachment of pili to human epithelial cells.28–30 Gonococci release the IgA1 protease, which inactivates the antibodies to secretory IgA1 class by cleaving them at the hinge region.31,32 This blocks attachment of the organism to epithelial cells; inactivation of this immune response may be a key factor in the pathogenesis of acute gonorrhea. The immune response to LPS is typically bactericidal.33

The immune response to N. meningitidis infections is similar to those initiated by gonococcal infections. Asymptomatic nasopharyngeal meningococcal infections result in the production of bactericidal antibodies against most pathogenic strains of N. meningitidis.34 This probably accounts for the presence of these antibodies in infants up to age 6 months (secondary to maternal antibodies) and in persons age 10 years and older. The lack of bactericidal antibodies in children is probably the most important risk factor in their susceptibility to meningococcal infection. The immune response to both the outer membrane proteins and LPS, as well as to capsular polysaccharides, indicates that human immune responses are most important; however, cell-mediated immune responses can be demonstrated as well.35

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This organism, a gram-negative diplococcus, is also referred to as Neisseria catarrhalis or Branhamella catarrhalis. Despite its morphologic similarities to N. gonorrhoeae, most microbiologists argue that the name of this organism should be Moraxella catarrhalis.1 Although considered a normal constituent of the conjunctival flora, it has been reported to cause extensive dermatitis of the face and eyelids, purulent conjunctivitis in children and adults with pseudomembrane formation, and acute or chronic catarrhal conjunctivitis. Central or paracentral corneal involvement consisting of an epithelial defect with underlying stromal infiltrate has been described.1 In addition, lacrimal involvement and endophthalmitis have been reported.36,37

M. catarrhalis is responsible for infections of the respiratory tract and is a common cause of otitis media and sinusitis.38,39 In adults, it may cause acute exacerbations of bronchitis and septicemia in immunocompromised patients.40,41

The clinically significant isolates of M. catarrhalis produce beta-lactamase enzymes, which render the organism resistant to penicillin, ampicillin, and amoxicillin.40 M. catarrhalis strains are piliated, which helps their adherence to pharyngeal epithelial cells.42 The gene code responsible for the pili is similar to that of Moraxella bovis. Thus, although the organisms are similar to Neisseria, there is a genetic relation to the other Moraxella species.43 This organism also contains membrane proteins. UspA has a molecular weight of 300 to 400 daltons and is closely associated with the outer membrane lipo-oligosaccharide. It has been found to be antigenic, and antibodies directed against this material can be recovered.44 A surface protein designated CopB, with a molecular weight of 81 daltons, has also been described in about 70% of the strains.45 This has also been found to be antigenic, and antibodies to this protein appear to enhance pulmonary clearance of the organism in a mouse model. M. catarrhalis also has membrane-associated proteins that can bind lactoferrin and transferrin, enabling the organism to acquire iron for growth.46

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M. catarrhalis grows on most media and can be differentiated from gonococci and meningococci by fermentation reactions. M. catarrhalis does not ferment glucose or maltose. It is oxidase- and catalase-positive and hydrolyzes tributyrin. M. meningitidis grows on both chocolate and blood agar, as well as Thayer-Martin media incubated in 5% to 10% CO2. The organism is oxidase- and catalase-positive and ferments both glucose and maltose. It does not hydrolyze tributyrin. Gonococci or presumed gonococcal infectious materials should be cultured on modified Thayer-Martin media containing vancomycin, colistin, and nystatin or New York City media, which contains vancomycin, colistin, amphotericin B, and trimethoprim. These media inhibit other bacteria, including other Neisseria species, except N. meningitidis. Specimens should be plated on chocolate or blood agar with the culture media in a CO2 incubator at 35°C, with 70%or more humidity. The plates should be examined periodically over the next 3 days. If growth occurs, they should be examined by Gram stain, which would show uniform, characteristic gram-negative diplococci. The organisms should also be tested to determine if they are oxidase-positive.

There are numerous tests available to differentiate N. gonorrhoeae, N. meningitidis, and M. catarrhalis:

  • Carbohydrate utilization tests, which use rapid fermentation methods
  • Chromogenic enzyme substrate tests
  • Immunologic tests using fluorescent monoclonal antibody methods that recognize the specific Por outer membrane protein
  • Coagglutination tests.1
  • Further, nucleic acid probes are being evaluated for their ability to confirm and differentiate Neisseria species.47
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19. Mulks MK, Plaut AG: IgA protease production as a characteristic distinguishing pathogenic from harmless Neisseriaceae. N Engl J Med 299:973, 1978

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27. Zak K, Diaz JL, Jackson D et al: Antigen variations during infection with Neisseria gonorrhoeae: Detection of an antibody to surface proteins in sera of patients with gonorrhea. J Infect Dis 149:166, 1984

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32. Koomey JM, Gill RE, Falkow LS: Genetic and biochemical analysis of gonococcal IgA1 protease: Cloning in Escherichia coli and construction of mutants of gonococci that fail to produce the activity. Proc Natl Acad Sci USA 79:7881, 1982

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39. Marchant CD: Spectrum of disease due to Branhamella catarrhalis in children with particular reference to acute otitis media. Am J Med 80 (Suppl 5A):155, 1990

40. Catlin BW: Branhamella catarrhalis: An organism gaining respect as a pathogen. Clin Microbiol Rev 3:293, 1990

41. Saito H, Annaissie EJ, Khardori N et al: Branhamella catarrhalis septicemia in patients with leukemia. Cancer 61:3215, 1988

42. Ahmad K: Fimbriae of Branhamella catarrhalis as possible mediators of adherence to pharyngeal epithelial cells. APMIS 100:1066, 1992

43. Marrs CF: Pili (fimbriae) of Branhamella species. Am J Med 88(suppl 5A):36S, 1990

44. Helminen ME, Maciver I, Latimer JL et al: A large antigenically conserved protein on the surface of Moraxella catarrhalis as a target for protective antibodies. J Infect Dis 170:867, 1994

45. Helminen ME, Maciver I, Latimer JL et al: A major outer membrane protein Moraxella catarrhalis as a target for antibodies and enhanced pulmonary clearance of the pathogen in an animal model. Infect Immun 61:2003, 1993

46. Murphy TF: Studies on the outer membrane proteins of Branhamella catarrhalis. Am J Med 88(suppl 5A):41S, 1990

47. Lewis JS, Fakile O, Foss E et al, Direct DNA probe assay for N. gonorrhoeae pharyngeal and rectal specimens. J Clin Microbiol 31:2783, 1993

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