Neisseria

 Obligate anaerobes, non-motile and non-hemolytic.

 Fastidious and capnophilic and grows optimally at
moist temperature.
 Natural habitat: mucous membrane of the RT and
URT.
 Carbohydrate fermenters
 Grows best in media with blood and cholesterol.
Neisseria
 Non-pigmented colonies except N. flava, N.
flavescens and N. subflava.
 Sensitive to heat and drying (inoculation at the
bedside)
 Microscopy: Gram negative diplococci that are
coffee or kidney bean shaped except N.
bacilliformis, N. elongate and N. weaver (rod
shaped)
Neisseria
 Culture: Glistening colonies, small to large, grayish
white convex, some are mucoid with sticky to
granular appearance.
 Biochemical tests: (+) oxidase, (+) catalase except
N. elongate and N. bacilliformis
 Human pathogens: N. gonorrhoeae and N.
meningitidis
Neisseria gonorrhoeae
 Found on mucous membrane of URT, Anorectal area,
oropharynx and conjunctiva.
 Glucose fermenter
 Principal VF: Common pili
 Culture: CAP – small, shiny, gray to tan colored, translucent and
rasied.
 Colonies type – T1 and T2 are virulent with pili, T3 to T5 are
avirulent without pili.
 Other VF: Receptors for transferrin, capsule, IgA,
Lipooligosaccharides (LOS) endotoxin
Infections and Disease
 Gonorrhea – gonos “seed” ; rhoia “flux”, acute pyogenic
infection of non-ciliated, columnar, transitional epithelium.
Urethra, endocervix, conjunctiva, pharyngeal surface or
anorectal area.
Incubation period – 2 to 7 days
Symptoms: Purulent discharge, lower abdominal pain for men &
vaginal bleeding for women.
 Purulent urethritis and cervicitis
- untreated gonoccocal urethritis may cause perihepatitis
(Fitz-hugh-Curtis syndrome)
Infections and Disease
 Pharyngitis – chief complaint in symptomatic
oropharyngeal infection.
 Anorectal infections/Rectal gonorrhea – rectal pain and
bloody stool
 Opthalmia neonatorum – eye infection acquired by
newborns through infected birth canal.
 Purulent urethritis, epididymitis, and pelvic inflammatory
disease
General Guidelines on Spx colletion and
Handling
 Direct inoculation of samples onto plates is
acceptable
 If samples cannot be processed immediately, it
should be held immediately at RT and avoid
refrigeration since Neisseria species are sensitive to
cold temperature
 Swab should be placed in transport system (Amies
with charcoal if direct plating cannot be performed)
General Guidelines on Spx colletion and
Handling
 Temperature dependent – immediate incubation at 35C. (N.
gonorrhoeae and N. meningitides require prompt incubation in
increased CO2 after plating. Both requires iron as growth
enhancers.

 Cotton swabs should not be used since it may

inhibit the growth.
Laboratory Diagnosis
 Microscopy – presence of intracellular gram negative
diploccoci, coffee bean shaped or kidney bean shaped.
Laboratory Diagnosis
 Culture (For Confirmation)
- CAP, TMA, MTM, ML, NYC and GC-ELECT
- Direct inoculation at the bedside is optimal.
- Prompt incubation of plates at 35C to 37C for 72 hours in
increased CO2 atmosphere (Candle jar and JEMBEC
system) with citric acid bicarbonate tablet is required.
- TYPES: T1 – T5
SPECIMEN TRANSPORT
 Transport media: Cary Blair and Amies with charcoal
 Transport system: Transgrow, JEMBEC, Gono Pak
SELECTIVE CULTURE MEDIA
 THAYER MARTIN AGAR (TMA)
- Inhibitors: Vancomycin, colistin and nystatin
- N. meningitides, N. lactamica, N. flavescens
 MODIFIED THAYER MARTIN AGAR (MTM) agar
- has all TMA components + Trimethoprim lactate
 MARTIN LEWIS (ML) medium
- contains all ingredients of MTM agar except Nystatin.
 NEW YORK CITY (NYC) AGAR
- Vancomycin, colistin, trimethoprim and amphotericin B
- Adv: Growth of Mycoplasma and Ureaplasma
 GC – LECT MEDIUM
- Contains the same NYC antibiotic and lincomycin
BIOCHEMICAL TESTS
A. Carbohydrate Utilization test (Cystine Trypticase Agar/CTA test)
- detects acid production from glucose, maltose, fructose, lactose
and sucrose)
- Medium: CTA with 1% carbohydrate
- ph indicator: Phenol red
- Control: Carbohydrate free medium
- (+) result: yellow color within 24 to 72 hours of incubation at
35C
- N. gonorrhoeae and N. meningitidis
BIOCHEMICAL TESTS
B. QuadFerm +
- Components: Glucose, maltose, lactose and sucrose
- Additional tests: DNAse and beta-lactamase
- Substrate: 4 sugars, DNA and penicillin
- ph indicator: Phenol red
- (+) result: Red to yellow
QC:
(+) for Glucose; (-) DNAse: N. gonorrhoeae ATCC 31426
(+) Glucose, Maltose, Lactose: N. lactamica ATCC 23971
(+) Glucose, Maltose, Sucrose: N. mucosa ATCC 19695
(-) Acid Production & (+) DNAse: M. catarrhalis ATCC 25240
BIOCHEMICAL TESTS
C. Oxidase Test
- detects the presence of cytochrome oxidase.
- Reagent: 1% tetramethyl-p-phenylenediamine dihydrochloride
- (+) resut: Dark purple color within 10 seconds
QC:
(+) control: Pseudomonas aeruginosa ATCC 27853
(-) control: Escherichia coli ATCC 25922
BIOCHEMICAL TESTS
D. Superoxol Test
- Rgt: 30% H2O2
- (+) vigorous bubbling (N. gonorrhoeae)
BIOCHEMICAL TESTS
E. DNAse Test
- culture medium: DNAse agar with
methyl green
- (+) result: clear halo around the colonies
after 18 hrs to 24 hrs
of incubation
- (+) M. catarrhalis, (-) N. gonorrhoeae
QC:
(+) SAU ATCC 25923
(-) E.coli ATCC 25922
BIOCHEMICAL TESTS
F. B-lactamase test (Cephalosporin/Nitrocefin test)
- (+) result: Positive deep pink or red within 10 minutes.
QC:
(+) Haemophilus influenza ATCC 33533
Staphyloccus aureus ATCC 43300
(-) Branhamella catarrhalis ATCC 25240
BIOCHEMICAL TESTS
G. Chromogenic substrate
- detects atypical strains of Neisseria with differentiated results
in the carbohydrate utilization test.
- Identifies bacterial enzymes that hydrolyze the colorless
substrate to colored end products.
MULTITEST
- Identifies colonies from both selective and nonselective media.
- Detects M. catarrhalis and Haemophilus
Ex: API NH, Microscan HNID Panel, Vitek and BBL crystal
ANTIMICROBIAL SUSCEPTIBILITY
TESTING
Antibiotics such as
Extended spectrum
cephalosporins and
quinolones are utilized.
E-test may be used as
alternative
Medium: GC agar
CLSI recommendation:
Disk diffusion or Agar
dilution
IMMUNODIAGNOSIS
A. FLUORESCENT ANTIBODY TEST (FAT)
- uses monoclonal antibodies that recognized epitopes
on the principal outer membrane protein (Por) of N.
gonorrhoeae.

B. Coagglutination
- confirmatory test
- employs Monoclonal antibodies to identify the gonococci.
- (+) agglutination
MOLECULAR DIAGNOSIS
A. NUCLEIC ACID AMPLIFICATION (NAAT)
- detects gonococcal antigen or nucleic acid directly in
specimens.
- Spx: endocervical or urethral swabs and urine
- Methods: Abbott Real time CT/NG, BD Probetec ET,
Cobas CT/NG, and Xpert CT/NG
- Adv: High sensitivity and able to detect C. trachomatis
- DA: Lower detection ate in extragenital infections
MOLECULAR DIAGNOSIS
B. CHEMILUMINISCENT NUCLEIC ACID PROBE
- detects gonococcal rRNA in genital and
conjunctival spx.
- Spx: endocervical and urethral swabs
- DA: Not recommended for pharyngeal and rectal
spx.
Neisseria meningitides (Meningococci)
 Agent of meningococcemia or spotted fever.
 Leading cause of fatal bacterial meningitis
 Isolated from nasopharynx and oropharynx
 Commensal and a true pathogen of the URT.
 MOT: Respiratory droplets
 Glucose & maltose fermenter, beta lactamase
producer, requires iron
 VF: Lipooligosaccharides (LOS) endotoxin complex
Neisseria meningitides (Meningococci)
 Culture:
BAP – Colonies appear large, smooth, bluish gray,
and convex with greenish dots under colonies
CAP – Small gray to tan colored, convex and mucoid
TMA – Smooth, convex, colorless to gray
 Serogroups: A,B,C,Y and W-135
 Other VF: Pili, Polysaccharide cpsule, IgA1, Cellular
membrane proteins (Por A and Por B)
Infection and Diseases
 Pleuritis, Pericarditis, Arthritis
 Waterhouse-Friderichsen syndrome
 Meningitis
 Meningococcemia
- blood infection can be acute or chronic.
- Oral secretions or Respiratory droplet
- Incubation: 10 days
- S/S: Frontal headache, stiff neck and fever,
petechial skin lesions
LABORATORY DIAGNOSIS
 Microscopy – gram negative intracellular and
extracellular diplocci.
LABORATORY DIAGNOSIS
 Culture – BAP, CAP, TMA
- NPS should be plated immediately in the JEMBEC
system
- Culture plates should be incubated in environment with
increased CO2 at 35C for 24 hrs to 72 hrs
- N. meningitides is sensitive to SPS.
- TMA allows growth of N. gonorrhoeae and N.
meningitides.
- Both will not grow on BAP or CAP incubated at RT
LABORATORY DIAGNOSIS
 Oxidase Test
- (+) result: Purple color
QC:
(+) Pseudomonas aeruginosa ATCC 27853
(-) E.coli ATCC 25922
LABORATORY DIAGNOSIS
 Antimicrobial Susceptibility Testing
- Broth microdilution or an agar dilution MIC test
with cation-supplemental MH broth and laked horse
blood or MHA with 5% sheep blood.
- Third generation cephalosporins
- Penicillin G for meningococcal meningitis
LABORATORY DIAGNOSIS
 IMMUNODIAGNOSIS

A. Latex agglutination – direct ID of antigens in CSF,

serum, urine.
B. Counterimmunoelectrophoresis – accelerates
antibody and antigen reaction through a buffered
diffusion medium.
LABORATORY DIAGNOSIS
 Gamma-glutamyl aminopeptidase (GCAP) test)
- reagent: Gamma-glutamyl-p-nitroanilide (GPNA)
- (+) result: yellow color (p-nitroaniline product)

QC:
(+): Neisseria meningitides ATCC 13077
(-): Moraxella catarrhalis ATCC 25240
Moraxella catarrhalis
 Part of the indigenous microbiota of the oropharynx
 Opportunistic pathogen
 Non-motile, fastidious, B-lactamase producer,
encapsulated with common pili
 Cause of upper respiratory tract infection in healthy
and elderly.
 Third most common cause of otitis media and
sinusitis in children
Moraxella catarrhalis
 Microscopy –
Small, gram
negative cocci that
tend to grow in
pairs from end to
end with their
adjacent sides
flattened
Moraxella catarrhalis
 Culture
BAP – Smooth,
opaque, grayish white
with hockey puck
appearance, old colonies
with “wagon wheel”
appearance
Moraxella catarrhalis
 Biochemical test – oxidase and catalase (+)
LABORATORY DIAGNOSIS
 Microscopy – gram negative intracellular diploccocci
 Culture – BAP & CAP
- Growth at 25C and NA at 35C
- CAP: Pink to brown colonies
- BAP: Hockey puck consistency
- inhibited by gonoccocal media by colistin
Moraxella catarrhalis
 Carbohydrate Utilization Test – does not utilize
 DNAse/DNA hydrolysis test
- definitive tests
- (+) clear or colorless halo around the colonies
- QUADFERM+, exhibits yellow color
QC:
(+) SAU ATCC 25923; M.catarrhalis ATCC 25240
(-) E.coli ATCC 25922; N. gonorrhoeae ATCC 31426
Moraxella catarrhalis
 Butyrate Esterase/Tributyrin Test
- Rgt substrate: Bromo-chlor-indolyl butyrate or
tributyrate
- (+) blue or indigo
- (-) No color change
QC:
(+) M. catarrhalis ATCC 25240
(-) N. gonorrhoeae ATCC 43069
Moraxella catarrhalis
 Antimicrobial Susceptibility Testing
- sensitive to cephalosporins and trimethoprim-
sulfomethoxazole
- susceptible to amoxicillin/clavulanic acid
combination in the presence of beta-lactamase
production.
Moraxella catarrhalis
 Molecular Diagnosis
- PCR reveals production of beta lactamases BRO-1
and BRO-2 (penicillin resistance)