GRAM

NEGATIVE
AEROBIC
COCCI
Neisseria and Moraxella
catarrhalis
Species to be considered

Pathogenic Nonpathogenic
◼ Neisseria gonorrhoeae ◼ Neisseria cinerea
◼ Neisseria meningitidis ◼ Neisseria flavescens
◼ Neisseria lactamica
◼ Neisseria mucosa
◼ Neisseria polysaccharea
◼ Neisseria sicca
◼ Neisseria subflava
◼ Neisseria elongata
◼ Neisseria weaveri

◼ Moraxella catarrhalis
 General Characteristics
◼ Family: Neissseriaceae

◼ Mx: Gram(-), diplococci resembling coffee beans or

kidney beans

◼ NF of the mucous membranes of the respiratory and

urogenital tracts EXCEPT N. gonorrhoeae and N.
meningitidis

Neisseria spp.
◼ Obligate aerobes but prefer increased CO2
Neisseria are typically diplococci
◼ Nonmotile and nonspore-forming EXCEPT the following w/c are
RODS:
◼ All are positive to cytochrome oxidase 1. N. elongata
2. N. weaveri
3. N. bacilliformis
◼ All are catalase positive EXCEPT Neisseria elongata

◼ Do not elongate when exposed to penicillin

 Pathogenic Neisseria
◼ VERY FASTIDIOUS!
▪ Neisseria gonorrhoeae requires chocolate agar and cysteine for growth
▪ Neisseria meningitidis and Moraxella catarrhalis require blood agar as the minimal
growth standard
▪ Both require iron for growth. They compete w/ their human hosts by binding
TRANSFERRIN to their specific receptors (reason why they are strict human
pathogens).

◼ VERY SENSITIVE to temperature changes and shd be protected from the

cold. Isolates shd be maintained at 37degC.

◼ Optimal growth is @ 35-37degC while commensals can grow on chocolate

agar @ RT and 35-37deg C on NA

◼ They establish disease through attachment to mucous membranes (pili).

 Epidemiology and Spectrum of Disease

◼ Normal inhabitants of the URT except for N. gonorrhoeae and Neisseria

meningitidis

◼ N. gonorrhoeae is sexually transmitted and causes gonorrhea (commonly

referred to as “clap”).

◼ N. meningitidis is spread by contaminated respiratory droplets and one the

leading causes of bacterial meningitis and meningococcemia.

◼ N. meningitidis can be a colonizer in the URT of carriers.

◼ Other Neisseria spp are not pathogenic, considered as saprophytic and

commensals but infections (if they occur) involve px’s endogenous strains.
ORGANISM HABITAT/RESERVOIR MODE OF
TRANSMISSION
Neisseria gonorrhoeae Not part of normal flora; only found Sexual (oral, anal)
(gonococcus) in mucous membranes during infection: Vertical transmission
•Genitalia
•Anorectal area
•Oropharynx
•Conjunctiva

Neisseria meningitidis Colonizes oro- and nasopharyngeal Contact with contaminated

(meningococcus) mucous membranes droplets

Other Neisseria spp Normal flora of URT Migration to sterile sites

Moraxella catarrhalis Normal flora URT, occasionally Migration to sterile sites

colonizes female GT
ORGANISM VIRULENCE FACTORS SPECTRUM OF DISEASE

Neisseria Receptors for human transferrin Gonorrhea

gonorrhoeae Capsule Opthalmia neonatorum
Pili
Pelvic inflammatory disease (PID)
Cell membrane proteins (Por, Opa, Rmp)
LOS (lipooligosaccharide)
IgA protease

Neisseria Pili Meningococcemia

meningitidis Capsule Waterhouse-Friderichsen Syndrome
Endotoxin

Other Neisseria Unknown Non – pathogenic, if implicated:

spp bacteremia, endocarditis, and
meningitis (very rare)

Moraxella Factors assoc w/ cell envelope w/c facilitate RT: otitis media, sinusitis, and pneumonia.
catarrhalis attachment to resp epi cells Bacteremia or meningitis (very rare)
Neisseria
gonorrhoeae
 Neisseria gonorrhoeae
◼ Agent of GONORRHEA – acute pyogenic infection mainly of the
mucous membranes of the endocervix (females) and urethra (males)

◼ Gonorrhea is manifested:
▪ Males – acute urethritis w/ dysuria (painful urination) and urethral discharge
▪ Females – urethritis and cervicitis; long term effects may result to scarred
fallopian tubes, ectopic pregnancy and sterility
▪ Homosexuals and bisexuals – oropharyngeal and rectal infections

◼ Not all gonorrhea infections are symptomatic; it is then necessary to CULTURE

BOTH SYMPTOMATIC AND ASYMPTOMATIC SEXUAL CONTACTS!!

◼ It is highly recommended to screen individuals who are at high risk:

▪ Those w/ multiple sexual partners
▪ Those who are positive for other STIs
 Infectious process of gonorrhea
◼ Incubation period – 2 to 7 days
◼ The organism attaches by pili to cells of the mucous membranes
◼ Pili and capsule inhibit phagocytosis by interfering w/ the fxn of neutrophils

◼ Organism also has outer membrane CHONs that affect: Ab formation, leukocyte
response and cell-mediated immunity
▪ LPS produces endotoxic effects
▪ Por/Protein I – allows organism to insert into host cells
▪ Opa/Protein II – facilitates adherence to phagocytes and epi cells
▪ Rmp/Protein III- blocks IgG

▪ Por – porin protein

▪ Opa – opacity
▪ Rmp – reduction modified protein
Gonorrh0ea
Gonococcal disease in men

◼ Acute urethritis w/ dysuria (painful

urination), purulent urethral
discharge

◼ Can be asymptomatic

◼ Possible complications:
epididymitis, prostatitis,
urethral strictures

◼ Cured by a small dose of

CEFTRIAXONE
 Gonorrh0ea
Gonococcal disease in women
◼ Urethritis – painful & burning
urination w/ pus; more likely to be
asymptomatic w/ minimum purulent
discharge
◼ Cervicitis – cervix becomes
reddened, friable, w/ purulent
exudate
◼ Lower abdominal discomfort, pain
during sex (DYSPAREUNIA),
vaginal dicharge (pus)
◼ LARGE PERCENTAGE OF
WOMEN ARE
ASYMPTOMATIC!!!
 Complications of Gonorrhea in Female Pxs

◼ Untreated cervicitis leads to PID (pelvic inflammatory disease); involves:

▪ Endometritis – uteral infection
▪ Salpingitis – infection of the fallopian tubes
▪ Oophoritis – infection of the ovaries
▪ Perihepatitis (Fitz-Hugh-Curtis syndrome)

◼ Clinical manifestations of PID

▪ Fever, lower abdominal pain, abnormal menstrual bleeding
▪ Cervical motion tenderness (pain felt during IE)

◼ Menstruation spreads the bacteria fr cervix to UGT, explains why PID can occur one week
at the onset of menstruation

◼ Presence of IUD increases the risk of cervicitis!

 Ophthalmia neonatorum
◼ Gonocccal disease in infants

◼ Passed by infected mother to infant during

birth

◼ Conjunctivitis occurs on the 1st or 2nd day of

life; can damage cornea and may lead to
blindness

◼ Upon birth, infant is given w/ topical eye

drops containing Ag nitrate/erythromycin
eye drops
Auxotypes of Neisseria gonorrhoeae

◼ Atypical or nutritionally variant strains of Neisseria gonorrhoeae

◼ More than 30 auxotypes have been identified

▪ Example is AHU (arginine-hypoxanthine-uracil) strain – it
requires arginine,hypoxanthine and uracil
▪ AHU strain is penicillin-sensitive and is the more likely CA of
urethritis in males and asymptomatic gonorrhea and DGI
(disseminated gonococcal infection) in females
Neisseria
meningitidis
 Neisseria meningitidis
◼ Can cause bacterial meningitis and septicemia;
MENINGOCOCCEMIA

◼ Virulence factors are similar to gonococci

◼ Asymptomatic carriers are the reservoir of the

infection; they spread the infection through their
infected aerosols, close contact in overcrowded and
unsanitary environments

◼ It is encapsulated; w/ 9 serogroups: A, B, C, D, X,
Y, Z, 29E, W135 MENINGOCOCCEMIA
Group A – most commonly seen as the CA in poor countries
Group B – most commonly seen as the CA in developed countries
 Meningococcal Disease
Meningococcemia
◼ Fatal meningitis
◼ Abrupt onset of spiking fever, chills, joint pain,
muscle pain
◼ PETECHIAL RASH due to the release of
endotoxin causing:
▪ vascular necrosis,
▪ inflammatory rxn, and
▪ hemorrhage to surrounding skin

Waterhouse Friderichsen Syndrome

◼ Hemorrhage into the adrenal glands
◼ Fulminant meningococcemia
◼ Septic shock
◼ Enlarging petechiae
◼ Disseminated intravascular coagulation (DIC)
◼ Coma
◼ Death may occur in 12 to 48 hrs from onset
Waterhouse
Friderichsen Syndrome
(WFS)
• Fulminant
meningococcemia
• Hemorrhaging in the
adrenal glands
caused by N.
meningitidis
 ◼ Chronic meningococcemia
▪ px is mild to moderately ill
▪ Recurrent episodes of
meningococcemia, petechiae
▪ There is eventual development of
arthritis

◼ Has been involved in

epidemics!

Disseminated intravascular
coagulation (DIC)
Treatment and Prevention

◼ Meningococcal disease remains susceptible to

PENICILLIN; chloramphenicol is used in those
who are penicillin sensitive

◼ Carriers and close contacts of infected individuals

can be treated w/ oral rifampin

◼ Vaccination:
▪ Univalent – for Group A and Group C
▪ Quadrivalent – for Groups A, C, Y and W135
▪ Poor response in children less than 2y/o
Moraxella
catarrhalis
 Moraxella catarrhalis
◼ Similar to genus Moraxella in DNA composition but it
morphologically and biochemically resembles Neisseria

◼ NF of the URT but can cause:

▪ Otitis media, sinusitis and pneumonia
▪ Endocarditis, conjunctivitis, meningitis
▪ Bacteremia, wound infections

◼ Infections are usually seen in immunosupressed Moraxella catarrhalis (gram stain)

pxs (esp w/ underlying condition of diabetes
mellitus or pulmonary problems)

Important Features:
◼ Grows on NA, BAP and Thayer Martin
◼ Colonies: nonpigmented, smooth, “hockey puck”
◼ Oxidase and catalase positive
◼ Dnase positive
◼ Nonsaccharolytic
◼ Positive butyrate esterase (tributyrin as substrate)
◼ Often produces beta-lactamase Moraxella catarrhalis (“hockey puck”)
 Test Reaction
Gram stain Gram negative
diplococci

BAP/CHOC agar “Hockey puck”

colonies

“wagon-wheel “
appearance

Catalase Positive

Oxidase Positive

BUTYRATE ESTERASE TEST: rapid test to DNase Positive

identify and differentiate Moraxella from Neisseria.
Positive result is formation of blue color w/in 5 mins. Beta-lactamase Positive (often)
Positive: Moraxella
Negative: Neisseria CHO utilization Nonsaccharolytic
(Cystine Trypticase Soy
Agar)

Butyrate esterase Positive

NONPATHOGENI
C NEISSERIA
Differentiation of Nonpathogenic Neisseria
SPECIES KEY CHARACTERISTICS GROWTH ON CHO
SELECTIVE UTILIZATION
MEDIA
N. lactamica The only lactose positive species in + G,M,L
Neisseria

N. polysaccharea Hyperproducer of extracellular + G, M

polysaccharide

N. cinerea Resembles T3 colonies of gonococcus; + Negative

sensitive to colistin

N. mucosa Produces very mucoid and large - G,M,S,F

colonies; isolated from dolphins; can
cause pneumonia; can reduce NO3 to
NO2
N. sicca Produces dry, breadcrumb like colonies - G,M,S,F

N. subflava Colonies are less yellow than N. - G,M

flavescens; has 3 biovars

N. flavescens Yellow colonies - Negative

N. elongata Rod, catalase negative; has 3 - Negative

subspecies
N. weaveri Rod; normal oral microbiota of dogs - Negative
LAB DIAGNOSIS
FOR
MENINGOCOCCI
Specimen Collection, Processing and Identification

◼ Specimen – depends on the stage or type of ◼ CSF

meningococcal infection ▪ NOT REFRIGERATED until
1. Nasopharyngeal swab – for identification is complete since
suspected carriers
organism is VERY SENSITIVE TO
TEMPERATURE CHANGES
2. Blood - for meningococcemia
3. CSF – for meningitis ▪ Clear CSF that is less than 1mL shd
4. Skin lesions – if petechial rash is be centrifuged to concentrate the
present sample; sediment is vortexed; plated
on chocolate agar; placed in FT and
5. Synovial fluid – for suspected septic Gram stained
arthritis
▪ Turbid CSF – centrifugation is not
necessary; follow procedure for clear
CSF

▪ REPORT also the presence and

number of PMNs and if
organism is found inside or
outside the PMNs
◼ Use Modified Thayer-Martin or Martin
Lewis agar when culturing contaminated
specimens or specimens w/ normal flora
(nasopharyngeal aspirate)
◼ Incubate @ 35-37deg C w/ 5-10% CO2
◼ Unlike N. gonorrhoeae, N. meningitidis can
grow on BAP
◼ Colonies: round, smooth, glistening and
gray in chocolate agar; encapsulated
strains are mucoid
◼ Perform oxidase test on any growth from
culture media (shd be oxidase positive)
◼ CONFIRMATORY TEST: organism
will utilize GLUCOSE AND MALTOSE
in CHO utilization test using Cystine
Trypticase Soy agar (CTA)
RAPID TESTS - antigen can be
detected even in the absence of viable
organism
•Latex agglutination - Capsular
polysaccharide can be directly detected in
CSF, serum and urine
•Coagglutination – latex beads or S.
aureus colonies are coated w/ the
organism’s Abs; binding occurs in the
presence of the organism as manifested
by agglutination
•Counterimmunoelectrophoresis –
migration of Ag and Ab in an electrical
field; specific serotypes can be detected
w/ this test
Key Tests for Neisseria meningitidis
Test Reaction
Gram stain Gram negative diplococci (coffee/kidney bean
shaped)
Nutrient agar Negative growth

BAP/CHOC agar Mucoid (encapsulated strains)

TM, MTM, ML Maybe greenish under colonies

Catalase (3% H2O2) Positive

Superoxol (30% H2O2) Negative

Oxidase Positive

CHO utilization Glucose – positive

(Cystine Trypticase Soy Agar) Maltose – positive
Lactose – negative

γ-glutamyl aminopeptidase Positive (yellow)

ONPG Negative
N. lactamica can also grow on selective Positive: N. lactamica
media

Production of polysaccharide in 1-5% Negative

sucrose Positive: N. polysaccharea
N. polysaccharea is a hyperproducer of
polysaccharide
ONPG TEST γ-GLUTAMYL AMINOPEPTIDASE TEST
Ortho-nitrophenyl-β-d-galactopyranoside
Tablet contains 3 substrates:
Rapid test to differentiate meningococcus from Neisseria 1. gamma-glutamyl nitroanalide (yellow)
lactamica, since N. lactamica mimics meningococcus and 2. bromo-chloro-indol-beta-D galactopyranoside (blue)
can also grow on selective media. 3. proline-naphthylamide (red)

Beta-galactosidase hydrolyzes ONPG (colorless) into Test to differentiate:

galactose and o-nitrophenol (yellow). 1. Neisseria meningitidis - yellow (γ-glutamyl aminopeptidase)
2. Neisseria lactamica – blue (B-d-galactosidase)
ONPG------------------------- galactose + o-nitrophenol 3. Neisseria gonorrhoeae – red (hydroxyprolylaminopeptidase)
β-galactosidase

Positive: Neisseria lactamica

Negative: Neisseria meningitidis
LAB DIAGNOSIS
FOR
GONOCOCCI
 Specimen Collection and Transport
Specimens: Dacron swab
▪ Female – endocervical
▪ Male – urethral

Other Specimens:
▪ w/ suspected DGI – blood or synovial fluid
▪ Rectal fluid
▪ Pharynx
▪ Joint fluid

Collection
▪ Dacron or rayon fibers (preferred swabs) Rayon swabs
▪ Calcium alginate and cotton swabs are
inhibitory to gonococci
▪ Cotton swabs are acceptable ONLY if plated
w/in 6hrs and shd contain charcoal to inhibit
toxic effect of fatty acids in cotton fibers
 Specimen Collection and Transport

◼ TRANSPORT -BEST METHOD:

plate specimen immediately after JEMBEC SYSTEM
collection and place medium in increased
CO2 for transport (JEMBEC plates);
upon receipt @ the lab, incubate @ 35deg
C in 3-5% CO2

◼ Blood culture broths: sodium

polyanethol sulfonate (SPS) content
shd not exceed 0.025% conc;
Neisseria are sensitive to SPS

JEMBEC (John E. Martin Biological

Environmental Chamber): Plate contains Thayer
Martin. The CO2 generating tablet is composed of
Na bicarbonate and citric acid.
Inoculation
▪ Inoculate specimen @ RT immediately since
organism is susceptible to drying
▪ Plates are streaked in a “Z” motion by
rolling the swab over the agar surface, then
streaked with a loop for isolation.

◼ Common isolating media are:

▪ Chocolate agar plate
▪ Modified Thayer-Martin agar
▪ Martin – Lewis agar
▪ New York City agar (NYC)
▪ GC-LECT
“Z” motion

Specimen Processing
◼ Clear fluid specimens greater than 1ml shd be
centrifuged, sediment is vortexed and inoculated
to TM, MTM, ML, NYC agar
Incubation conditions: plates are
placed @ 35-37deg C for 72hrs in a
CO2 enriched, humid atmosphere
(candle jar)
Gram Stain
Gram (-) diplococci inside PMNs recovered
fr male’s urethral discharge - DIAGNOSTIC
FOR GONORRHEA (MALES)
 Cultivation for Pathogenic Neisseria
Selective Medium Inhibitory Agents Suppressed Remarks
Organisms
Thayer-Martin Vancomycin Gram positive CHOC-base with
VCN Colistin Gram negative IsoVitaleX
Nystatin Yeast

Modified Thayer-Martin Vancomycin Gram positive CHOC-base with

VCNT Colistin Gram negative IsoVitaleX
Nystatin Yeast
Trimethoprim Swarming of Proteus

Martin-Lewis Vancomycin Gram positive Concentration of

VaCAnT Colistin Gram negative Vancomycin is increased
Anisomycin Yeast
Trimethoprim Swarming of Proteus

New York City Vancomycin Gram positive Can also support the
VaCAmT Colistin Gram negative growth of:
Amphotericin B Yeast Mycoplasma hominis
Trimethoprim Swarming of Proteus Ureaplasma urealyticum

GC-LECT Vancomycin Gram positive

VLCAT Lincomycin Gram positive
Colistin Gram negative
Amphotericin B Yeast
Trimethoprim Swarming of Proteus
Capnocytophaga spp
Key Tests for Neisseria
gonorrhoeae
Test Reaction
Gram stain Gram negative diplococci (coffee/kidney bean shaped)
Diagnostic for male: urethral discharge

Nutrient agar Negative growth

BAP

CHOC agar Grayish – white, raised

TM, MTM, ML

Catalase (3% H2O2) Positive

Superoxol (30% H2O2) Positive

Oxidase Positive

CHO utilization Glucose – positive; Maltose – negative; Lactose –

(Cystine Trypticase Soy Agar) negative

Hydroxyprolylaminopeptidase Positive (red)

Gonozyme Positive

Colistin Resistant
(differentiates gonococcus from N. cinerea Susceptible: N. cinerea
since both show similar morpho on CHOC)
Colonial types of N. gonorrhoeae
Colonies Morphology Indication
Type 1 colonies Small, raised and moist; bright and w/ pili; virulent
reflective indicating fresh colonies

Type 2 colonies Small, raised and dry; bright and w/ pili; virulent
reflective indicating fresh colonies

Type 3 colonies Large, flat and nonreflective Lack pili; avirulent

Type 4 colonies Large, flat and nonreflective Lack pili; avirulent

Type 5 colonies Large, flat and nonreflective Lack pili; avirulent

AHU strains are more fastidious and are smaller in size

Colonial appearance on Chocolate agar
ORGANISM COLONIAL APPEARANCE

Moraxella catarrhalis Large, non-pigmented or gray, opaque,

smooth, “hockey puck” appearance

Neisseria gonorrhoeae Small, grayish-white, shiny, translucent w/

either smooth or irregular margins

Neisseria meningitidis Medium, smooth, round, moist, gray-white,

mucoid (encapsulated), may be greenish
under colonies
Moraxella catarrhalis Neisseria gonorrhoeae Neisseria meningitidis

On Chocolate Agar

Neisseria (+) on OXIDASE

TEST
Reactions on Cystine Trypticase Soy
Agar
ORGANISM Glucose Maltose Lactose

Moraxella catarrhalis - - -

Neisseria gonorrhoeae + - -

Neisseria meningitidis + + -

Neisseria lactamica + + +
 Other Tests:
1. Rapid Fermentation Test (RFT) – system can be read in a shorter incubation
time (2-4hrs) for results
▪ Kellog and Turner (1st to devise this system)
▪ Minitek - uses CHO impregnated disks
▪ RapID NH – uses biochemical and substrate tests
▪ RIM-N Neisseria - employs microtubes w/ CHOs
▪ QuadFERM+ - determines CHO utilization by beta-lactamase and deoxyribonuclease

2. Direct Identification of N. gonorrhoeae

▪ ELISA (urethral swab for males and endocervical swabs for females)
▪ Gonozyme – direct detection of gonococcal Ag; color produced is proportional to the
amount of Ags present

3. Nucleic acid probe – sample is collected and lysed, and a gene probe is
added
4. Serological Methods – coagglutination method; Phadebact Monoclonal
GC OMNI test
5. Fluorescent Ab Technique
 Treatment and Prevention
◼ Moraxella catarrhalis – beta-lactams: cephalosporins, macrolides, quinolones
◼ Neisseria gonorrhoeae – ceftriaxone or quinolones
◼ Neisseria meningitidis – penicillin, ceftriaxone, chloramphenicol

◼ PREVENTION
▪ Single-dose vaccine to the polysaccharide capsular Ags of N. meningitidis
▪ Chemoprophylaxis – rifampin/ciprofloxacin/ceftriaxone; given to those close
in contact w/ meningococcal pxs

▪ For opthalmia neonatorum:

▪ 1% silver nitrate
▪ 0.5% erythromycin ointment
▪ 1% tetracycline
▪ 2.5% povidone-iodine soln
 Penicillin Resistance
◼ All N.gonorrhoeae isolates shd be tested for the productionn of
BETA-LACTAMASE since penicillin-resistant strains have been found (penicillin
has been used for 50 years in gonorrhea treatment)

◼ In cases of penicillin resistance, alternative treatments include:

▪ Spectinomycin or ceftriaxone plus tetracycline, doxycycline
▪ Erythromycin for penicilinase-producing N. gonorrhoeae (PPNG)

◼ For uncomplicated cases:

▪ Amoxicillin w/ probenecid
▪ Ampicillin w/ probenecid
▪ Ceftriaxone
Assigned Readings:

◼ Cystine Trypticase Soy Agar

◼ Cytochrome Oxidase Test
◼ Other Neisseria species

◼ Bailey and Scott

◼ Delost, Maria (Introduction to Diagnostic Microbiology)