Neisseria and Moraxella

    Cards (56)

    • 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
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