Gram-Negative Cocci in Ocular Disease
Peter C. Donshik
William Ehlers
Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria catarrhalis are the gram-negative cocci of interest to the ophthalmologist. 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 appearance of these species on Gram stain and in culture is so similar as to be indistinguishable, but the specific species can be determined on the basis of biochemical and serologic differences. The infections caused by these pathogenic organisms are uncommon, but they can rapidly progress causing significant ocular morbidity.
Neisseria Gonorrhoeae
N. gonorrhoeae (GC) is the agent responsible for gonorrhea and is the second most often reported sexually transmitted disease (STD) in the United States. Each year, approximately 600,000 individuals are affected in the United States, but worldwide more than 60 million new cases occur yearly.2,3 Although the incidence of gonococcal infection steadily increased between 1960 and 1975, the rate declined by approximately 74% between 1975 and 1997, followed by a small increase in 1998. Since then, the rate of gonococcal infection has steadily declined.3,4
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, but this usually abates within 6 months even if left untreated. If untreated, complications such as prostatitis, seminal vesiculitis, epididymitis, periurethral abscess, urethral stricture and infections of the vas deferens 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 involve other sites, such as the anorectal area, pharynx, skin, joints, synovium, endocardium, myocardium, pericardium, liver, and eye.5
Ocular Manifestations
Gonorrheal ophthalmia neonatorum, the most common ophthalmic manifestation, occurs in children born to mothers with GC infection of the genital tract.2 It usually presents as a unilateral conjunctivitis with an incubation period that may be as short as a few hours or as long as 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 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 with single or multiple peripheral corneal infiltrates that may ulcerate and usually occur adjacent to areas of severe conjunctival inflammation. They can progress circumferentially and appear as a ring ulcer. Central corneal ulceration rarely occurs.
Adults usually present with an acute conjunctivitis characterized by a purulent discharge and associated lid edema. The inflammation of the conjunctiva can be so severe that it restricts ocular motility, mimicking orbital cellulitis.6 Corneal involvement is more common in adults than in neonates or prepubertal children, and it can be severe, leading to ulceration, scarring, perforation, and loss of vision. Many other complications can occur, including involvement of the uveal tract and retina, endophthalmitis, dacryoadenitis, and rarely preseptal or orbital cellulitis.7,8,9 Ocular infection usually results from autoinoculation of infected secretions from the genital tract of either the patient or their partner. However, infection has also been reported from direct ejaculation into the conjunctiva.10 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.
Pathogenesis
Naturally occurring gonococcal infections are limited to humans; but rarely GC may affect the mucosal tissue of other species.11 The organisms attach to the mucosal cells by their pili, and this appears to occur rapidly and involves a specific receptor on human cells, as well as nonpili surface antigens.12,13 The receptor for gonococcal pili is the B1-3-N-acetylgalactosamine-4-galactose portion of a membrane ganglioside. The attachment is facilitated at 37C 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 hair like protein polymers projecting from the surface of the cell wall. They are composed of repeating peptide subunits, which have an approximate molecular weight of 20,000 daltons;14 and each subunit contains 165 amino acids.15 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 GC appears to be caused by the middle and carboxy-terminal end of the pili protein.14 In addition, the pili may function to overcome electrostatic forces that occur between negatively charged mucosal surfaces, as well as similarly charged bacterial cells.16 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.17 Certain pili can influence CD4+T-cell activation. In addition, the adhesion mediated by pilus components can aid in the regulation of the T-cell response to GC.18
The outer membrane surface of the Neisseria organisms also contains proteins that can affect antibody formation, cell-mediated immune response, and penetration into human cells.19,20 Protein I (32,000 to 36,000 daltons) is the most abundant outer membrane protein and is also termed a porin. This protein covers the outer membrane and facilitates in the entrance and exit of small molecules to and from the periplastic space.19 They also allow the passage of hydrophilic molecules through the hydrophobic outer cell membrane. Protein I molecules move rapidly from the outer membrane of GC to the cytoplasmic membrane of human cells during endocytosis.20 Antibodies directed against this protein are used for various serologic typing tests for gonococci.21 The outer membrane porin molecules can bind to complement protein C4b-binding protein, affecting the complement systems ability to kill the organism.22
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 its 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 organisms susceptibility to killing by normal serum.23
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).24 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.25
Other membrane components are also important in the virulence of GC. Gonococcal lipopolysaccharide (LPS) is composed of a lipid portion (a component of the outer membrane) and a polysaccharide portion that protrudes from this membrane and has endotoxin activity. This endotoxin activity is probably responsible for local killing of host cells and the initiation of inflammation.26
Four hydrolases have been identified in suspensions of GC cultures.27 Gonococcin, an endopeptidase, is an alkaline proteinase that cleaves elastin. This capability contributes to 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 play a part in the hydrolysis of internal proline-containing peptides. Gonococcal proline aminopeptidase hydrolyzes the tripeptide Pro-Gly-Gly. Together, these two enzymes may be important in helping gonococci to utilize proline and may only secondarily cause tissue damage. Gonococcal asparaginase inhibits protein synthesis and decreases the hosts immune response.27 These functions may aid in cellular invasion by gonococci.
GC, 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 Fab and the Fc fragment, thus neutralizing the effect of secretory IgA.27,28 This protease is immunogenic, and antibodies appear during infection and in asymptomatic carriers. Antibody activity is lost after cleavage.29
After gaining access to the bloodstream, gonococcal organisms develop thicker capsules. Although the capsules themselves do not appear to play a role in the adherence of the organism to mucosal epithelial cells, they may help diminish phagocytosis by both macrophages and polymorphonucleocytes. The organisms tend to attack and penetrate stratified squamous epithelial cells.13 They penetrate cells by endocytosis after the epithelial cells extend cytoplasmic processes around the bacteria. The induction of specific genes by the organism appears necessary for invasion into the cell during the initial stages of infection.30 The organism may multiply within the cell or the same cell may engulf multiple bacteria. Infection can then spread along the mucous membranes by proliferation in the mucosal film, and this allows retrograde spread. The organisms adhere to epithelial cells by their pili. After adherence occurs, protein I aids in forming transmembrane channels and gonococcal LPS aids in the penetration of the organism into epithelial cells. 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 activity of gonococcal LPS.13 This substance may also be responsible for destroying cells that the organism fails to penetrate. Thus, although the pili are responsible for the attachment of the bacteria to the epithelial cells, other components of the outer membrane are involved in penetration into host cells and the propagation of infection.
These factors may play a part in other pathologic conditions because GC appears to have the capacity to enhance HIV replication in dendritic cells during coinfection by secreting a peptidoglycan that activates a bacterial lipoprotein, stimulating a strong enhancement of HIV infection in human dendritic cells.31
Neisseria Cinerea
Neisseria cinerea was first identified in 1906 but was initially misclassified as a subtype of Branhamella catarrhalis. N. cinerea is a gram-negative diplococcus similar to GC with regard to its cultural and biochemical characteristics.32 However, it can be differentiated from GC based on specific biochemical characteristics, culture characteristics, such as failure to grow subcultures on gonococcal selective medium, and a negative GC fluorescent monoclonal antibody test.33 This organism usually colonizes the upper respiratory tract of humans, rarely colonizes the vagina, and was first recognized as a cause of ophthalmia neonatorum in 1990.
Neisseria cinerea has now been recovered from the eyes of young children, especially during the neonatal period.33 This organism can cause a variety of ocular infections, such as conjunctivitis and orbital cellulitis. It has also been reported to be responsible for meningitis and septicemia.34 It is important for social and possibly legal reasons to differentiate this species from GC.35
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