Chapter 18

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• Chapter 18
• The Gram-Negative Cocci

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Chapter 18- Neisseria Species and Moraxella
catarrhalis

• General characteristics
• Aerobic, gram-negative diplococci
• Nonmotile, capnophilic
• Oxidase positive (one of the most important
  features)
• Catalase positive
• Primary pathogens
• N. gonorrhoeae (ALWAYS pathogenic)
• N. meningitidis (May be carried as commensal in
  URT)

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Chapter 18- Neisseria Species and Moraxella
catarrhalis

• Habitat
• Upper respiratory tract
• Genitourinary tract
• Alimentary(Digestive) tract

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Chapter 18- Neisseria gonorrhoeae

• Virulence factors
• Fimbrae (common pili)- enhance the ability of
  bacterial cells to adhere to host cells and to
  each other
• Lipopolysaccharide resembles that of other
  gram-negative organisms prevents phagocytosis
• Capsule
• Cell membrane proteins
• IgA protease- cleaves IgA on mucosal surfaces

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Chapter 18- Neisseria gonorrhoeae Clinical
Infections

• Pyogenic (pus-producing) infection of columnar
  and transitional epithelial cells anywhere they
  are found (urethral, endocervix, anal canal,
  pharynx, and conjunctiva)
• Disease in the male
• Incubation period 2 to 7 days
• Transmitted only by intimate sexual contact
• 95 show symptoms of acute infection
• Symptoms include dysuria, urethral discharge
• Complications include epididymitis and urethral
  stricture, and prostatitis
• Specimen is purulent discharge after urinating

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Chapter 18- Neisseria gonorrhoeae Clinical
Infections

• Disease in the female
• 20 to 80 are asymptomatic
• Symptoms (if symptomatic) include burning or
  frequency of urination, vaginal discharge
• Fever and abdominal pain
• Complications include pelvic inflammatory disease
  (PID), sterility and ectopic pregnancy

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Chapter 18- Neisseria gonorrhoeae Infections in
Other Sites

• Disseminated gonococcal disease ( 1 of
  infections)
• Acute form has the following symptoms fever,
  chills, malaise, intermittent bacteremia, and
  skin lesions
• If untreated will progress to septic joint form
  of the disease (inflamed joints, swollen, hot,
  full of pus and fluid)
• Gonococcal arthritis occurs as a result of
  disseminated gonococcal bacteremia

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Chapter 18- Neisseria gonorrhoeaeInfections in
Other Sites

• Disease in children
• In infancy, an eye infection (ophthalmia
  neonatorum) may occur during vaginal delivery
• May cause blindness if not treated
• Infection is preventable with the application of
  antibiotic eye drops at birth
• Extragenital infections
• Pharyngitis
• Anorectal infections

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Chapter 18- Laboratory DiagnosisNeisseria
gonorrhoeae

• Clinical specimens
• Genital sites
• Female endocervix
• Male urethra
• Anal
• Oral/pharyngeal
• Eye
• Blood/joint fluids
• If specimen is from site other than genital, lab
  must be notified, as N. gonorrhoeae is not
  ordinarily looked for as a pathogen in other body
  sites (i.e. throat)

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Chapter 18

• Specimen collection
• Dacron/ Rayon swabs preferred
• Swabs transported in Amies medium with charcoal
• Inoculate media within 6 hours of collection,
  avoid drying

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Chapter 18- Laboratory DiagnosisNeisseria
gonorrhoeae

• Transport media
• Transgrow or JEMBEC
• JEMBEC James E Martin Biological Environmental
  Chamber

JEMBEC
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Chapter 18- Laboratory Diagnosis Neisseria
gonorrhoeae

• Identification
• Morphology
• Gram-negative, kidney-beanshaped diplococci

A direct gram-stained smear of male urethral
discharge showing intracellular gram-negative
diplococci
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Chapter 18- Laboratory Diagnosis Neisseria
gonorrhoeae

• Intracellular g dc in symptomatic males
  correlates 95 with positive culture for N.
  gonorrhoeae
• Intracellular g- dc in females results in
  positive N. gonorrhoeae culture only 50-70, due
  to other g- dc vaginal flora
• Urethral g.s. with gt 5 PMN/oif but NO bacteria
  may indicate other genital infection, such as
  Chlamydia or Ureaplasma

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Chapter 18- Laboratory Diagnosis Neisseria
gonorrhoeae

• Identification
• Inoculated culture media must be incubated at 350
  C in 3 to 5 CO2 or candle jar
• Colony morphology on modified Thayer-Martin (MTM)
  agar
• Small, gray
• Translucent, raised
• Fresh growth must be used for testing, because N.
  gonorrhoeae produces autolytic enzymes

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Chapter 18- Laboratory Diagnosis Neisseria
gonorrhoeae

• Thayer-Martin agar is chocolate agar with
  vancomycin, colistin, and nystatin
• MTM contains the above trimethoprin
• These antibiotics will inhibit the growth of MOST
  bacteria, but N. gonorrhoeae is catalase and
  oxidase
• Specimen MUST be plated on warmed media ASAP
  (need for transport media if not close to a lab)

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Chapter 18- Laboratory Diagnosis Neisseria
gonorrhoeae
Candle extinction jar with inoculated MTM agar
plates Only white candles must be used colored
ones may inhibit growth
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Chapter 18- Laboratory Diagnosis Neisseria
gonorrhoeae

• Identification
• Oxidase test ()

Carbohydrate utilization test acid produced
only in the glucose tube indicates that the
isolate is N. gonorrheoae
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Chapter 18-Laboratory Diagnosis Neisseria
gonorrhoeae

• Immunologic methods
• Use colonies from primary plate
• Organisms do not need to be viable
• Fluorescent antibody technique
• Coagglutination
• Non-culture methods
• Use direct patient specimen
• ELISA, nucleic acid probe, and PCR testing
• Expensive usually used in high-risk populations
  with large volume of testing
• Unable to perform on all sources

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Chapter 18- Laboratory Diagnosis Neisseria
gonorrhoeae

• If N. gonorrheoae is cultured from children or in
  suspected sexual abuse cases, at least two
  different forms of organism identification should
  be performed to withstand legal scrutiny
• PPNG Penicillinase Producing Neisseria
  gonorrhoeae
• First seen in 1976
• Plasmid-mediated
• Beta-lactamase testing should always be done on
  N. gonorrheoae
• Treatment Penicillin
• Tetracycline if beta-lactamase positive strain.
  Can also use cephalosporins and flouroquinolones

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Chapter 18- Neisseria meningitidis

• General characteristics
• Exclusively human parasite
• Exist as harmless member of normal upper
  respiratory flora
• Similar to Neisseria gonorrhoeae
• Antigenic structures
• Capsular polysaccharide nine serotypes
  A, B, C, D, X, Y, Z, W135, 29E.
• Contribute to invasive properties by inhibiting
  phagocytosis
• Same virulence factors as N. gonorrhoeae
• Vaccine available, but poorly antigenic in
  children lt 2 years old

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Chapter 18- Neisseria meningitidisClinical
Infections

• Bacterial meningitis
• Transmission is by respiratory droplets and
  requires both close contact (ex dormitories,
  military barracks, in institutions) and lack of
  specific antibody (susceptibility)
• Symptoms include fever, headache, stiff neck,
  nausea, vomiting, and purulent meningitis with
  increased WBCs
• Serotypes B and C most common in US
• Other infections include meningococcemia,
  pneumonia, purulent arthritis, endophthalmitis
• May be seen in genital tract with oral-genital
  contact

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Chapter 18- Neisseria meningitidisClinical
Infections

• Bacteremia
• (meningococcemia)
• Appearance of skin petechiae due to hemorrhage

Hemorrhage in the adrenal glands in
Waterhouse-Fridericksen syndrome
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Chapter 18- Laboratory DiagnosisNeisseria
meningitidis

• Identification
• Examine direct smear from CSF for intra extra
  cellular g- dc
• Examine smear for halo
• Other body sites include nasopharyngeal swabs,
  sputum, and urogenital specimens

Gram-stained smear of CSF showing the extra
cellular and intracellular gram-negative
diplococci
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Chapter 18- Laboratory Diagnosis Neisseria
meningitidis

• Identification
• Examine cultures on blood agar chocolate agar
  in CO2
• Oxidase-test positive
• Conventional CTA carbohydrates for biochemical
  identification (glucose and maltose) or
  immunologic methods are available for serogrouping

Neisseria meningitidis growing on sheep blood
agar (right) and chocolate agar (left)
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Chapter 18- Laboratory Diagnosis Neisseria
meningitidis

• Other tests
• DNase negative
• Nitrate reduction negative
• Latex agglutination from CSF
• Counterimmunologic electrophoresis
• Other molecular diagnostics

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Chapter 18- Neisseria meningitidisTreatment

• Penicillin
• Other options rifampin or sulfonamide

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Chapter 18- Neisseria lactamicaHabitat

• Normal flora of nasopharynx

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Chapter 18- Neisseria lactamicaLaboratory
Diagnosis

• Identification
• Colony morphology on chocolate is grayish white
• Carbohydrate utilization
• Dextrose, maltose, lactose positive
• Sucrose negative
• DNase negative

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Chapter 18- Moraxella catarrhalis (Branhamella)

• Normal commensal of the respiratory tract (humans
  only)
• Has become an important opportunistic pathogen

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Chapter 18- Moraxella catarrhalis
(Branhamella)Clinical Infections

• Clinical infections
• Pneumonia
• Sinusitis
• Otitis media (3rd most common cause)

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Chapter 18- Moraxella catarrhalis (Branhamella)

• Predisposing factors
• Advanced age
• Immunodeficiency
• Neutropenia
• Other debilitating diseases

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Chapter 18- Moraxella catarrhalis

• Colonies appear smooth with a grayish- white
  color
• When colonies pushed with loop, they scoot
  across media

Moraxella catarrhalis growing on chocolate agar
after 48 hours of incubation
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Chapter 18- Moraxella catarrhalis
Direct smear from an otitis media sample showing
intracellular gram-negative diplococci M.
catarrhalis was identified from cultures. M.
catarrhalis may resist decolorization.
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Chapter 18- Moraxella catarrhalis
(Branhamella)Laboratory Diagnosis

• Oxidase positive
• Catalase positive
• Catarrhalis Disc positive
• All CTA sugars negative
• Produce beta- lactamase
• DNase positive

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Chapter 18- Moraxella catarrhalisTreatment

• penicillin

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Chapter 18- Identification of Selected
Neisseria Species Moraxella