Neisseria spp.

• Neisseriaceae


  Family of bacteria that includes Neisseria species

  
• Neisseria spp.


  • Aerobic, nonmotile (no flagella), non-spore-forming, gram (-) diplococci except: Neisseria elongata, Neisseria weaveri, and Neisseria bacilliformis
  • Optimum temperature 32-37°C
  • Majority are fastidious (require additional nutrients to grow)

  
• Genera under Neisseriaceae family

  • Neisseria
  • Kingella
  • Eikingella
  • Simonsiella

  
• Oxidase test


  First test performed for gram (-) organisms, presence of purple color indicates positive
• Modified oxidase test


  Performed for staphylococcus (micrococci), presence of blue color indicates positive
• Neisseria


  • Kidney-bean shaped
  • All are cytochrome oxidase and catalase (+) except for: N. elongata, and N. bacilliformis
  • Capnophilic (can grow anaerobically if alternative electron acceptors are available)
  • Normal inhabitants of human respiratory tract except: N. gonorrhoeae and N. animaloris
• Pathogenic Neisseria


  N. gonorrhoeae and N. meningitidis - require iron for growth, found associated inside polymorphonuclear cells (PMNs)
• Pathogenic Neisseria are transmitted from person to person
• N. gonorrhoeae transmission


  Sexually transmitted
• N. meningitidis transmission


  Respiratory droplets
• Pathogenic Neisseria compete with RBCs for iron and transferrin
• Virulence factors of Neisseria

  • Receptors for human transferrin
  • Capsule (N. meningitidis)
  • Pili (fimbriae)
  • Cell membrane proteins (Protein I, II, III)
  • Lipooligosaccharide (LOS) or endotoxin
  • Immunoglobulin A (IgA) protease
  • Other proteins (Lip, Fbp)
• Pili


  Determine virulent (T1, T2) or avirulent (T3-T5) forms of Neisseria
• Protein I (Por)


  Forms channels for nutrients and waste products in cell membrane
• Protein II (Opa)


  Facilitates adherence to phagocytic and epithelial cells
• Protein III (Rmp)


  Blocks the bactericidal effect of host IgG
• Lipooligosaccharide (LOS)


  Endotoxin in N. gonorrhoeae that resembles human cell membrane glycosphingolipid

  
• IgA protease


  Cleaves IgA on mucosal surfaces
• Fbp (ferric-binding protein)


  Expressed when iron supply is limited, allows Neisseria to compete for iron
• Neisseria gonorrhoeae


  • Slow growing
  • Oxidizes only glucose
  • Requires arginine, hypoxanthine, and uracil (AHU)
  • Agent of sexually transmitted disease gonorrhea
  • Not part of normal microbiota
  • Only found on mucous membranes at time of infection
• AHU strain of N. gonorrhoeae

  Usually seen in asymptomatic infections
• Gonorrhea


  • Acute pyogenic infection of nonciliated columnar and transitional epithelium
  • Acquired through sexual contact
  • Occurs primarily in urethra, endocervix, anal canal, pharynx, and conjunctiva
• Gonorrhea


  Incubation period: 2-7 days
• Clinical infections of gonorrhea

  • Men: acute urethritis and dysuria
  • Women: dysuria, cervical discharge, lower abdominal pain (50% asymptomatic)
  • Pelvic inflammatory disease in women
  • Anorectal and oropharyngeal infections
  • Newborns: ophthalmia neonatorum
• Specimen collection and transport for gonorrhea diagnosis

  1. Pus and secretions taken from urethra, cervix, rectum, conjunctiva, throat, or synovial fluid

  2. Swabs: dacron or rayon preferred, calcium alginate and cotton inhibitory

  3. Direct plating on selective media like JEMBEC, Gono-Pak, Transgrow

  4. Amies Medium with charcoal for transport
• Direct microscopic examination


  • Gram-negative intracellular diplococci in pairs with adjacent sides flattened, kidney shape
  • Not recommended for pharyngeal specimens due to normal flora
• Culture for gonorrhea diagnosis

  1. Medium of choice: CHOC agar, Trypticase soy agar with 5% sheep blood

  2. Incubation: 35°C, 3-5% CO2, 48-72 hours

  3. Colonial appearance: small, grayish white, convex, translucent, shiny colonies with smooth or irregular margins

  
• Definitive identification tests for gonorrhea

  1. Oxidase test: positive if purple color within 10 seconds

  2. Carbohydrate utilization: CTA agar with 1% carbohydrates, acid production indicated by yellow color

  3. Chromogenic substrates: Gonocheck II detects enzymes that produce colored end products

  4. Multitest conventional-chromogenic enzyme methods

  
• Immunologic assays like coagglutination and fluorescent antibody testing can identify N. gonorrhoeae without requiring pure viable organism
• Culture for Neisseria requires 72 hour incubation at 37°C with increased carbon dioxide
• Chromogen


  Determines the color
• Only strains that are isolated on selective media should be tested
• Multitest Methods


  • Multitest conventional-chromogenic enzyme methods
  • Combine enzyme substrate tests with other biochemical tests
  • Allow for identification of strains isolated on selective or nonselective media
  • Immunologic assays
• Immunologic Assays


  • Coagglutination and fluorescent antibody testing - serologic test
  • Monoclonal antibodies against gonococcal protein I right of gonorrhoeae - Por B
  • Do not require pure viable organism
  • N. gonorrhoeae attached to killed Staphylococcus aureus cells
  • Positive: agglutination
• Culture


  1. Incubate for 72 hours (Neisseria) at 37°C with increase carbon dioxide

  2. Amount of culture media depends on MT or lab protocol

  3. Quadrant streak to isolate pathogen from normal flora
• Characteristics for pathogen


  • Always present (continuously present in streak)
  • Line of streak (tube should be in streak, if not it's a contaminant)
  • Until Q3 or Q4, if not then contaminant or normal flora
• Mixed culture still needs to be purified by subculturing
• If pure culture, can proceed to bacterial ID
• Common names


  • N. gonorrhoeae (gonococcus)
  • N. meningitidis (meningococcus)
• MALDI-TOF MS


  • Identifies infectious pathogens by defining unique protein signatures of the organism
  • Does not require a long time
  • Principle: colony is ionized resulting in vaporization of the proteins, then proteins are separated based on size and charge resulting in unique spectral signature, which is compared to saved identity of pathogen