Neisseria and Moraxella

• Neisseriaspp.


  • Family: Neisseriaceae
  • Includes: Kingella, Eikenella, Simonsiella, Alysiella
  • Aerobic, nonmotile, non–spore-forming, capnophilic
  • Can grow anaerobically if nitrite is present (alternative electron acceptor)
  • Gram (-) diplococci (N. elongata, N. weaveri, N. bacilliformis)
  • Cytochrome oxidase (+) and catalase (+)
  • Normal flora: mucous membranes of the respiratory tract and urogenital tract

  
• Neisseria gonorrhoeae


  • Gonococci
  • Not part of normal flora
  • Always pathogenic
  • Fastidious organisms
  • Requires iron for growth
• Neisseria meningitidis


  • Meningococci
  • Commensal inhabitant of upper respiratory tract of carriers
  • Invasive pathogen
  • Fastidious organisms
  • Requires iron for growth
• Virulence factors of Neisseria

  • Receptors for human transferrin
  • Capsule (Nesseria meningitidis) (Polysaccharide capsule)
  • Pili (fimbriae)
  • Proteins in cell membrane (antigenic variation, major outer membrane porin protein, adherence to cells, blocks host serum bactericidal action)
  • Core lipooligosaccharide (LOS) or endotoxin
  • Lipid moiety
  • IgA protease
• Transferrin


  Blood-plasma glycoprotein, which plays a central role in iron metabolism and is responsible for ferric-ion delivery
• Neisseria gonorrhoeae


  • Only host: human
  • Gonorrhoea "flow of seed" "clap" meaning "brothel"
  • Causes acute pyogenic infection of nonciliated columnar and transitional epithelium (urethra, endocervix, anal canal, pharynx, and conjunctiva)
  • Sexually transmitted infection
  • Primary reservoir: asymptomatic carrier
  • Incubation period: 2-7 days

  
• Workflow: Neisseria gonorrhoeae


  1. Specimen collection (urethra for men, endocervix for women, avoid disinfectants, prefer Dacron or rayon swabs)

  2. Direct microscopic examination (Gram stain, Gram (-) intracellular diplococci in pairs with adjacent sides flattened)

  3. Culture (medium of choice: CAP, plated ASAP, susceptible to cold temperature, inhibitors: Vancomycin, lincomycin, Trimethoprim, Colistin, Nystatin, Anisomycin, Amphotericin B)

  4. Incubation (35°C, 3-5% CO2)

  5. Macroscopic examination (small, gray to tan, translucent, and raised colonies, autolytic enzyme)

  6. Microscopic examination (Gram (-) diplococci)

  7. Biochemical tests (Oxidase, Carbohydrate utilization)

  
• Neisseria meningitidis


  • Only found in human
  • Commensal & invasive pathogen
  • Incubation period: 1 to 10 days
  • Adhere to nasopharyngeal mucosa leading to colonization
  • Causes endemic and epidemic meningitis and meningococcemia
  • Complication: Waterhouse-Friderichsen syndrome, 12-48 hrs of onset: death, Meningococcal pneumonia: serogroup Y
• Workflow: Neisseria meningitidis


  1. Specimen collection (CSF, blood, nasopharyngeal swabs and aspirates, joint fluids, inhibited by SPS)

  2. Direct microscopic examination (Gram (-) intracellular and extracellular diplococci)

  3. Culture (BAP & CAP, 35°C, 3-5% CO2)

  4. Macroscopic examination (medium-sized, gray, and convex, mucoid if encapsulated)

  5. Microscopic examination (Gram (-) diplococci with adjacent sides flattened)

  6. Biochemical tests (Oxidase (+), Carbohydrate utilization: glucose and maltose, γ-glutamyl aminopeptidase)
• Moraxella catarrhalis


  • Family: Moraxellaceae
  • Genera: Moraxella, Acinetobacter, and Psychrobacter
  • Opportunistic pathogen, commensal of upper respiratory tract
  • Infections: upper respiratory tract infections, 3rd most common cause of acute otitis media and sinusitis in children
• Workflow: Moraxella catarrhalis


  1. Specimen collection (middle ear effusion, nasopharynx, sinus aspirates, sputum aspirates, or bronchial aspirates)

  2. Direct microscopic examination (intracellular gram (-) diplococci)

  3. Culture (BAP & CAP, grow well at 28°C, inhibited by colistin)

  4. Macroscopic examination (smooth, opaque, gray-to-white colonies)

  5. Microscopic examination (gram (-) diplococci)

  6. Biochemical tests (Oxidase & Catalase (+), asaccharolytic, Dnase & butyrate esterase (-))

  
• Commensal Neisseria species


  • N. polysaccharea
  • N. lactamica
  • N. cinerea
  • N. mucosa
  • N. sicca
  • N. subflava
  • N. flavescens
  • N. elongata
  • N. weaveri
  • N. bacilliformis
  • Kingella denitrificans

  
• Neisseria polysaccharea


  • Produces large amounts of extracellular polysaccharide when grown in media with 1% or 5% sucrose
  • To differentiate from N. gonorrhoreae: (+) growth at NA at 35° C, production of polysaccharide from 1% or 5% sucrose
• Neisseria mucosa


  • Large, mucoid colonies
  • Isolated in nasopharynx of children & young adults, and in airways of dolphins
  • Carbohydrate utilization: Glucose, maltose, sucrose, fructose
  • Reduction of nitrate & nitrite (+)
• Neisseria sicca


  • Dry, wrinkled, adherent, and breadcrumb-like colonies
• Neisseria lactamica


  • Nasopharynx of infants and children
  • similar to N. polysaccharea (encountered in meningococcal carrier surveys)
  • Similar colonial morphology to N. meningitidis
  • Carbohydrate utilization: Glucose, maltose, lactose
  • ONPG (+)
• Neisseria subflava

  • “less yellow”

  • part of the upper respiratory microbiota
• Core lipooligosaccharide (LOS) or endotoxin 


  • Major in vivo virulence factor

  • mediates damage to body tissues and elicits an inflammatory response

  • “blebs” : outer membrane fragments released during rapid growth containing the LOS
• Lipid moiety 


  • differentiation from the lipopolysaccharide found in most gram (-) bacilli
• IgA protease 


  • cleaves IgA on mucosal surfaces
• Pili (fimbriae) • N. gonorrhoeae

  Colony types: Type 1-5

  Basis: Presence or absence of pili

  Virulent forms: Type 1 & 2
• Pili (fimbriae)


  initial attachment of the organism to host tissues

  resist phagocytosis of leukocytes
• Proteins in CM 


  • Antigenic variation
• Protein I: major outer membrane porin protein (Por) 


  porA & porB: N. meningitidis

  porB: N. gonorrhoeae
• Protein II (Opa)


  adherence to phagocytic and epithelial cells
• Protein III (reduction modified protein [Rmp]) 


  • blocks host serum bactericidal (IgG) action
• N. Gonorrhoeae


  • Men (Urethra): 3-5% asymptomatic AHU strain (Arginine, hypoxanthine, uracil)
  • Women (Endocervix): 50% asymptomatic, complications: sterility, ectopic pregnancy, or perihepatitis (Fitz-hugh-Curtis syndrome)
  • Inhibited by SPS (Sodium polyanethole sulfonate), not recovered in blood
  • Newborns: ophthalmia neonatorum, gonococcal eye infection, Connatal infection, result in blindness
• Workflow: Neisseria gonorrhoeae (Specimen Collection)

  Specimen of choice:

  (Men: urethra)

  (Women: endocervix)

  • Avoid use of disinfectants

  • Calcium alginate and cotton swabs: inhibitory

  (Dacron or rayon swabs are preferred)

  • extremely susceptible to drying and temperature changes

  (direct plating)
• Workflow: Neisseria gonorrhoeae (Direct Microscopic Examination)

  Urogenital specimen: Gram stain

  • Pharyngeal specimens: Gram stain NOT recommended (Commensal Neisseria spp.)

  • Gram (-) intracellular diplococci in pairs with adjacent sides flattened “kidney bean shape”

  • Gram stain with > 5 PMN/ field but no bacteria:

  (nongonococcal urethritis, C. trachomatis or Ureaplasma urealyticum)
• Workflow: Neisseria gonorrhoeae (Culture)

  Medium of choice: CAP

  o no growth in BAP

  Plated ASAP

  • Susceptible to cold temperature (Medium should be warmed at RT before inoculation)

  • Acinetobacter spp., Capnocytophaga spp., & Kingella denitrificans

  (+) growth on gonococcal media

  o oxidase and catalase tests

  Inhibitors:

  o Vancomycin, lincomycin: Gram (+)

  o Trimethoprim: Swarming Proteus spp.

  o Colistin: Gram (-)

  o Nystatin, Anisomycin, Amphotericin B: yeast
• Workflow: Neisseria gonorrhoeae (Culture)

Thayer-Martin


  Vancomycin, colistin, nystatin
• Workflow: Neisseria gonorrhoeae (Culture)


  Modified Thayer-Martin



  Vancomycin, colistin, nystatin, trimethoprim
• Workflow: Neisseria gonorrhoeae (Culture)


  Martin-Lewis


  Vancomycin, colistin, anisomycin, trimethoprim
• Workflow: Neisseria gonorrhoeae (Culture)


  New York City

  Vancomycin, colistin, amphotericin B, trimethoprim
• Workflow: Neisseria gonorrhoeae (Culture)


  GC-LECT


  Vancomycin, lincomycin, colistin, amphotericin B, trimethoprim
• Workflow: Neisseria gonorrhoeae (Culture)


  Incubation

  35°C in a 3% to 5% CO2 atmosphere

  CO2 incubator

  CO2 generating pouch

  Candle jar
• The lysis of RBC during the heating process releases intracellular coenzyme nicotinamide adenine dinucleotide (NAD or V Factor) into the agar for utilization by fastidious bacteria (the heating process also inactivates growth inhibitors).

  Hemin (factor X) is available from non-hemolyzed as well as hemolyzed blood cells
• Workflow: Neisseria gonorrhoeae (Macroscopic Examination)

  • Small, gray to tan, translucent, and raised

  T1 & T2: Bright, smaller and raised

  T3 & T5: larger, flatter colonies

  • autolytic enzyme
  • make the isolate nonviable on prolonged incubation
• Workflow: Neisseria gonorrhoeae (Biochemical Test)Oxidase test


  • done on all suspected isolates of N. gonorrhoeae

  • Reagent: 1% dimethyl-p-phenylenediamine dihydrochloride or tetramethyl-pphenylenediamine dihydrochloride

  • Result: (+): purple to black color after 10 mins (-): no change in color
• Workflow: Neisseria gonorrhoeae (Biochemical Test) Carbohydrate Utilization

  • Cystine Trypticase Agar (CTA)

  • 1% of the individual carbohydrate o phenol red (indicator)

  • Result: (+) yellow: acid production (-): no change: no acid produced

  • N. gonorrhoeae: glucose (Dnase & Butyrate esterase (-))

  • N. meningitidis: glucose & maltose (Dnase & Butyrate esterase (-))

  • M. catarrhalis: asaccharolytic