Neisseria: Morphology, Identification, and Culture

• Neisseria are Gram-negative cocci that occur in pairs (diplococci).
• Neisseria gonorrhoeae (gonococci) and Neisseria meningitidis (meningococci) are exclusively pathogenic for humans.
• They are typically found associated with or inside polymorphonuclear cells (PMNs).

Morphology and Identification

• Neisseria are aerobic, Gram-negative, nonmotile diplococci, approximately 0.8 µm in diameter.
• Individual cocci are kidney bean shaped, with flat or concave sides adjacent when in pairs.

Culture

• They grow on sheep blood agar, chocolate agar, and selective agar media e.g., modified Thayer-Martin agar, Martin-Lewis agar and New York City medium.
• N. meningitidis grows on sheep blood agar as well as selective media.
• N. gonorrhoeae requires enriched chocolate agar and/or selective media for optimal growth.
• Selective media contain vancomycin (suppression of Gram-positive bacteria) and colistin (suppression of Gram-negative bacteria).
• It also has inhibitory substances to suppress the growth of many commensal microorganisms from clinical sites.
• N. gonorrhoeae, N. meningitides, and N. lactamica are colistin-resistant and can grow on these selective media.

Growth Characteristics

• Neisseriae grow best under aerobic conditions, but some, like N. gonorrhoeae, can grow anaerobically.
• They produce acid but not gas by oxidation of various carbohydrates, and the oxidase test is key for identification.
• All Neisseria species, except N. elongata, are catalase-positive.
• Species grow best on media with complex organic substances like heated blood, hemin, and animal proteins, in a 5% CO2 atmosphere.
• They are killed rapidly by drying, sunlight, moist heat, and disinfectants.
• They produce autolytic enzymes, leading to rapid swelling and lysis in vitro at 25°C and alkaline pH.

Neisseria Gonorrhoeae

• Gonococci oxidize only glucose and differ antigenically from other neisseriae.
• N. gonorrhoeae is antigenically heterogeneous and can change its surface structures in vitro and in vivo to avoid host defenses.

Antigenic Structure

• Surface structures include pili, Por, Opa proteins, Rmp, and lipooligosaccharide (LOS).
• Pili (fimbriae) are hairlike appendages that enhance attachment to host cells and resistance to phagocytosis.
• They are made of stacked pilin proteins.
• Por protein extends through the gonococcal cell membrane, forming pores for nutrient entry.
• They impact intracellular killing of gonococci within neutrophils by preventing phagosome-lysosome fusion.
• Opa proteins mediate adhesion of gonococci within colonies and attachment to host cell receptors.
• Rmp (Protein III) is a reduction-modifiable protein; it associates with Por in pore formation.
• Lipooligosaccharide (LOS) lacks long O-antigen side chains, unlike enteric Gram-negative rods, and LOS toxicity is significant in gonococcal infections.
• Lip (H8) is a surface-exposed protein, heat-modifiable like Opa, and Fbp is a ferric-binding protein.

Pathogenesis, Pathology, and Clinical Findings

• Opaque colonies are isolated from men with symptomatic urethritis and from uterine cervical cultures at midcycle.
• Transparent colonies are frequently isolated from men with asymptomatic urethral infection, menstruating women, and patients with invasive gonorrhea.
• It includes salpingitis and disseminated infection.
• Gonococci attack mucous membranes of the genitourinary tract, eye, rectum, and throat.
• It results in acute suppuration that may lead to tissue invasion, followed by chronic inflammation and fibrosis.
• Men usually have urethritis, with yellow, creamy pus and painful urination.
• Gonococcal bacteremia leads to skin lesions and arthritis.
• Gonococcal endocarditis is uncommon but severe.
• Gonococcal ophthalmia neonatorum is an eye infection in newborns acquired during birth.

Diagnostic Laboratory Tests

• Specimens are pus and secretions from the urethra, cervix, rectum, conjunctiva, throat, or synovial fluid; blood culture is needed in systemic illness.
• Gram-stained smears of urethral or endocervical exudates reveal diplococci within PMNs, providing a presumptive diagnosis.
• After collection, pus or mucus is streaked on enriched selective medium like modified Thayer-Martin medium (MTM) and incubated in 5% CO2 at 37°C.
• Selective media contains antimicrobial drugs like vancomycin, colistin, nystatin, and trimethoprim to avoid contaminant overgrowth.
• If immediate incubation is not possible, place the specimen in a CO2-containing transport-culture system.
• Presumptive identification is achieved after 48 hours by appearance on a Gram-stained smear and a positive oxidase test.
• The ability to produce acid from carbohydrates by oxidation is used to determine the definitive species level and N. gonorrhoeae uses only glucose.

Neisseria Meningitidis

Antigenic Structure

• Capsular polysaccharides include A, B, C, X, Y, and W-135, the six most important serogroups associated with disease worldwide.
• Human sialic acid derivatives like NANA incorporated into meningococcal capsules allow the organism to be overlooked by the host immune system "molecular mimicry".
• The outer membrane consists of proteins and LPS that play major roles in virulence; porin proteins (Por A and Por B) interact with host cells.
• The opacity proteins (Opa) are comparable to Opa of the gonococci and play a role in attachment.
• Meningococci are piliated, initiating binding to the nasopharyngeal cells and other cells.
• The Lipid A disaccharide of meningococcal LPS causes toxic effects found in disease; it has the highest levels of endotoxin.

Pathogenesis, Pathology, and Clinical Findings:

• The nasopharynx portal of entry where organisms attach to epithelial cells using pili, and may form part of the transient microbiota without producing symptoms or disease.
• Invasive meningococcal disease (IMD) occurs in a small number of individuals and transient carriers; infants and adolescents have the highest incidence of IMD in developed countries.
• From the nasopharynx, organisms reach the bloodstream, causing meningococcal bacteremia; initial symptoms may be similar to a "flu-like" infection.
• Meningitis, sepsis (meningococcemia), or a combination of both is how IMD typically presents.
• Meningitis is the most common complication of bacterial infections.
• Begins suddenly with an intense headache, vomiting, photophobia, confusion, and stiff neck; and it may progress to coma within a few hours.
• Fulminant meningococcemia presents with high fever and hemorrhagic rash, possible disseminated intravascular coagulation, and circulatory collapse with bilateral hemorrhagic necrosis of the adrenal glands with (Waterhouse-Friderichsen syndrome).
• In meningitis, the meninges are acutely inflamed with thrombosis of blood vessels and purulent exudate covers the surface of the brain.
• The exact mechanisms are not well understood but they transform an asymptomatic colonization of the nasopharynx into meningococcal bacteremia, subsequently leading to meningococcemia and meningitis.

Diagnostic Laboratory Tests:

• Specimens for isolation of N. meningitidis include blood for culture and cerebrospinal fluid (CSF) for smear and culture.
• Puncture material or biopsies from petechiae may be taken for smear and culture and nasopharyngeal swab cultures.
• Gram-stained smears of the sediment of centrifuged spinal fluid or of petechial aspirate will often show typical Neisseriae within polymorphonuclear leukocytes or extracellularly.
• Although neisseriae are inhibited by toxic factors and polyanethole sulfonate in commercial blood culture broths, there seems to be a lesser problem to recover N. meningitis from blood cultures.
• CSF specimens are plated on sheep blood agar and chocolate agar, then incubated at 37°C in 5% CO2.
• MTM agar favors the growth of Neisseriae, inhibiting many other bacteria, and is used for nasopharyngeal cultures.
• Colonies of N. meningitidis are gray, convex, and glistening, with entire edges; a positive oxidase test and Gram-stain-showing Gram-negative diplococci provides presumptive organism identification found in spinal fluid.
• Spinal fluid and blood generally typically yield pure cultures that can be identified by carbohydrate oxidative reactions and agglutination with type-specific or polyvalent serum.
• Antibodies to meningococcal polysaccharides can be measured by latex agglutination or hemagglutination tests or by their bactericidal activity.