L19 - Medical Microbiology Laboratory (Neisseria spp.)

1. Medical Microbiology Laboratory
Gram Negative Cocci
(Neisseria spp.)
Hussein A. Abid
Medical Laboratory Scientist
Member at American Society of Microbiology
Chairman of Iraqi Medical Laboratory Association
Teacher at Middle Technical University

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OVERVIEW
 Neisseria is a large genus of bacteria that colonize the
mucosal surfaces of many animals.
 Of the 11 species that colonize humans, only two are
pathogens, N. meningitidis and N. gonorrhoeae.
 Most gonococcal infections are asymptomatic and self-
resolving, and epidemic strains of the meningococcus may be
carried in >95% of a population where systemic disease
occurs at <1% prevalence.
 Neisseria species are Gram-negative bacteria included
among the proteobacteria, a large group of Gram-negative
forms. Neisseria diplococci resemble coffee beans when
viewed microscopically.

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GENERAL CHARACTERISTIC
 Aerobic
 Gram-negative cocci often arranged in pairs,
diplococci, with adjacent sides flattened (like coffee
beans)
 Oxidase positive
 Most catalase positive
 Nonmotile
 Acid from oxidation of carbohydrates, not from
fermentation

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TAXONOMY
Scientific nameRank
• NeisseriaceaeFamily
• NeisseriaGenus
• N. meningitidis
• N. gonorrhoeae
Species
(medically important spp.)

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N. meningitidis
 Encapsulated small, gram-negative
diplococci
 Second most common cause (behind S.
pneumoniae) of community-acquired
meningitis in previously healthy adults;
swift progression from good health to life-
threatening disease
 Following dissemination of virulent organisms from the
nasopharynx (meningitis, Septicemia or meningococcemia)
with or without meningitis, meningoencephalitis, pneumonia,
arthritis, urethritis).

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N. meningitidis
 N. meningitidis is spread through
saliva and respiratory secretions
during coughing, sneezing,
kissing, chewing on toys and even
through sharing a source of fresh
water.
 It infects its host cells by sticking
to them with long thin extensions
called pili and the surface-
exposed proteins Opa and Opc
and has several virulence factors.
The picture shows the growth of
Neisseria meningitidis on NYC
media. It is used as a selective
media for gonococci.

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N. meningitidis
Clinical samples:
 Cerebrospinal fluid (CSF), Blood for culture, Swabs
from hemorrhagic skin lesions and nasopharyngeal
swab.
Blood for culture

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N. meningitidis (microscopy)
 Gram negative diplococci, resemble coffee beans.
Gram stain: in smears from clinical specimens, meningococci are found inside
pus cells (intracellular) as well as outside the pus cells (extracellular)

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N. meningitidis (culture)
 Grow on blood and chocolate agar, incubated at 37 ºC in
candle jar.
Transparent or grey, shiny, 1-2 mm colonies after incubation in 5-10% CO2

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N. meningitidis (culture)
Transparent or grey, shiny, 1-2 mm colonies after incubation in 5-10% CO2

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N. meningitidis (culture)
 Grow on blood and chocolate agar, incubated at 37 ºC in
candle jar.
Transparent or grey, shiny, 1-2 mm colonies after incubation in 5-10% CO2

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CLINICAL BIOCHEMISTRY OF CSF IN
GONOCOCCAL MENINGITIS
LEVELPARAMETER
IncreasedCSF pressure
DecreasedGlucose
IncreasedProtein
NormalChloride
Cloudy to purulentAppearance
> 200 × 106/ LW.B.C. count
Mostly neutrophilsType of W.B.Cs

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N. gonorrhoae
 Cause a sexually transmitted disease (gonorrhoea).
 In male appears as acute urethritis with purulent discharge.
 In female infect the endocervic resulting in vaginal discharge
and dysuria. 50% of females are a symptomatic carrier. Could
lead to pelvic inflammatory disease and sterility.
 Disseminated gonococcal infection appears as arthritis and
septicemia.
 gonococci occasionally isolated from mouth and anus from
homosexual and unusual sexual activities.
 Opthalmia neonatorum is an eye infection to a neonate during
delivery

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N. gonorrhoae
 Readily transmitted by sexual contact
 Gram-negative diplococci flattened along the adjoining side
 Fastidious, capnophilic and susceptible to cool
temperatures, drying and fatty acids
 Requires complex media pre-warmed to 35-37 ºC
 Soluble starch added to neutralize fatty acid toxicity
 Grow best in moist atmosphere supplemented with CO2
 Produce acid from glucose, but not from other sugars

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N. gonorrhoae
Clinical samples:
 Urethral swab, Endocervical swab (and exudate), Eye
swab, Throat swab, Rectal swab and urine
 Avoid using cotton or calcium alignment swab use
Rayon or Dacron swab

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N. gonorrhoae (microscopy)
 Microscopy of a direct smear of the discharge stained
with Gram stain reveals Gram-negative diplococci within
polymorphonuclear leucocytes.
 The sensitivity of the microscopy is highest in urethral
samples in men, reaching 90–95%, whereas for
endocervical smears sensitivity drops to 30–50%. In
asymptomatic patients sensitivity is extremely low (20%).

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N. gonorrhoae (microscopy)
Neisseria gonorrhoeae in Urethral Exudates

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N. gonorrhoae (culture)
 Chocolate agar with a 5-10% CO2.
 Selective media for gonococci:
1- Thayer-Martin media chocolate agar contain:
 Vancomycin to inhibit gram +ve
 Colistin to inhibit gram –ve
 Nystatin for fungi and Yeast
2- Modified Thayer-Martin media contain:
 Trimethoprim which kill swarming Proteus spp.

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N. gonorrhoae (culture)
3- Martin-Lewis
 Contain Anisomycin instead of Nystatin
4- Modified New York City media contain:
 Vancomycin
 Colistin
 Amphotericin B
 Trimethoprim

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N. gonorrhoae (culture)
Growth on chocolate agar: colorless, raised, 1-2 mm in diameter colonies

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N. gonorrhoae (culture)
Growth on modified New York City (MYNC) agar: Small, raised, translucent
colonies

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N. gonorrhoae (culture)
Growth on Thayer-Martin agar: Small, raised, grey colonies

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N. gonorrhoae (culture)

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BIOCHEMICAL TESTS
1. Catalase: positive (+ve)
2. Oxidase: positive (+ve)
3. Carbohydrate utilization:
4. Api for Neisseria and Haemophilus (NH):
MaltoseGlucoseSpecie
(– ve)Acid (+ve)N. gonorrhoeae
Acid (+ve)Acid (+ve)N. meningitidis

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BIOCHEMICAL TESTS
Carbohydrate utilization
For N. gonorrhoeae

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SEROLOGICAL TESTS
1. Latex slide agglutination test: directly detects antigens
to Neisseria meningitidis serogroups A, C, Y, W135 present
in human cerebrospinal fluid (CSF), serum, urine or blood
cultures.

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SEROLOGICAL TESTS
2. Slide agglutination test: for N. meningitidis serogroups

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SEROLOGICAL TESTS
3. Latex slide co-agglutination test: slide co-agglutination
test for confirmative identification of Neisseria gonorrhoea
and distinguishes between its 3 serogroups WI ,WII and
WIII.

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OTHER METHODS
1. Enzyme immunoassays (EIAs) can be used for quick
identification of gonococcal urethritis with good sensitivity
and specificity. These tests lack sensitivity and specificity
when used for cervical, pharyngeal and rectal samples.
2. Nucleic acid detection methods enable the detection of
N. gonorrhoeae from clinical samples without the
requirement of bacterial viability. These are the nucleic acid
amplification tests (NAATs) that are used in pharyngeal and
rectal samples, with greater sensitivity than cultures.