GRAM NEGATIVE COCCI

NEISSERIA
 Lecture objectives:
•Classification
•Morphology
•Neisseria gonorrhoeae
•Neisseria meningitidis
 Classification
 Family: Neisseriaceae
 5 Genus: Neisseria, Kingella, Simonsiella,
Alysiella, Eikenella
 Pathogens : Neisseria meningitidis,
Neisseria gonorrhoeae
• Commensals : N.lactamia, N.flavescens,
N.cattarhalis (Moraxella)
 Neisseria Associated Diseases

(ophthalmia neonatorum)
Manifestations of Neisseria Infections
Morphology:
 Gr negative diplococci, 0,6-0,8 µm in diameter
(coffee-beans or kidney-shaped in pairs)
 nonmotile, nonsporeforming, can have pills
 capsulated (N.menigitidis)
 non-capsulated (N.gonorrhea)
 require enriched media (chocolate agar)
 best grow at 5-7% CO2
 Oxidase positive
 t more than 50C and less than 22C kills bacteria,
sensitive to drying
Neisseria meningitidis
 Neisseria meningitidis
(Meningococcus )
• First isolated in 1887 by Weichselbaum

• Causes meningococcal meningitis,

septicemia

• Occurs sporadically or as epidemics

 Morphology
• Gram negative oval or spherical cocci arranged in
pairs with the adjacent sides flattened
• Non-motile, capsulated.
Neisseria meningitidis
in Cerebrospinal Fluid
 Culture
• Meningococci are fastidious organisms do not grow on
ordinary culture media.

• They are aerobic, no growth occurs anaerobically.

• The optimum temperature for growth is 35-36°C &

optimum pH is 7.4-7.6.

• It is essential to provide 5-10% CO2.

 Culture
a) Enriched media: Blood Agar
Chocolate agar,
Mueller-Hinton agar.

b) Selective media: Thayer Martin medium with

antibiotics (Vancomycin, Colistin & Nystatin.
 Colony Morphology

BloodAgar ChocolateAgar
 Biochemical Reactions
• Catalase positive

• Oxidase positive

• Glucose, maltose
acidified-peptone
serum agar slopes
 Pathogenecity

Sources of infection
• Asymptomatic nasopharyngeal carriers

Mode of transmission
• Airborne droplets, fomites
 Virulence factors

• Based on capsular polysaccharide antigens– 13

serogroups
• Group A, B, C are important
A – Epidemics
C – localised outbreaks
B – both epidemics and outbreaks

• Groups 29-E, W-135 and Y also cause meningitis

 Pathogenecity
Nasopharynx

Asymptomatic/local inflammation with

rhinitis,pharyngitis
Perineural sheath of
the olfactory
nerve/bloodstream

Subarachnoid space

Meningitis
 PATHOGENESIS
Mode of entry : Droplet infection.

a. Bacteraemic spread
(nasopharynx-meninges &subarachnoid space)
b. Direct spread:
Contiguous focus of infection (sinusitis, mastoiditis, skull, otitis media)

• Locally rhinitis and pharyngitis

• Dissemination In small proportion

• Organisms spread through perineural sheath of Olfactory nerve or
bloodstream or through conjunctiva
© 2007 Chettinad Hospital & Research
Institute
 Signs and symptoms

 INFANTS
onset: abrupt or insidious
rare signs of meningeal irritation
Irritability and refusal to take food
vomiting; dehydration
fever is typically absent in children less than
2 months old
hypothermia is more common
 Rash
 Petechiae
 purpura
 DAY 1 TO 3: 30 to 60% of patients with
meningococcal disease, with or without meningitis
 more prominent in areas of the skin subjected to
pressure
axillary folds
the belt line
back
 Rash

 Confluence of lesions results in

hemorrhagic patches, often with central
necrosis

 Impaired protein C anticoagulation

pathway: purpura fulminants
Rash of meningococcemia
Purpura
Purpura fulminans
Fulminant meningococcemia
 5 to 15% of patients
 high mortality rate
 ABRUPT ONSET
sudden high fever
chills
myalgias
weakness
nausea
vomiting
headache.
 Fulminant
meningococcemia
 Apprehension, restlessness, and delirium
occur within the next few hours
 Widespread purpuric and ecchymotic skin
lesions appear suddenly
 (+) or no signs of meningitis
Fulminant meningococcemia

 Pulmonary insufficiency
 death within 24 hours of being hospitalized
despite appropriate antibiotic therapy and
intensive care.
 Clinical features
Children:(<18yrs)
• Temperature,
• instability, irritability,
• poor feeding, vomiting

Adults:
• Fever, headache, Increased intracranial pressure –
papilledema
• Nuchal rigidity - Neck stiffness
• Kernig’s sign positive
• Nuchal rigidity - is the inability to flex the neck forward due
to rigidity of the neck muscles.(If flexion of the neck is painful but
full range of motion is present, nuchal rigidity is absent)

• Kernig sign : indicating is one of the physically demonstrable

symptoms of meningitis.

• The test for Kernig sign is done by having the person lie flat on
the back, flex the thigh so that it is at a right angle to the trunk, and
completely extend the leg at the knee joint.

• Kernig's sign positive - Severe stiffness of the hamstrings causes

an inability to straighten the leg

when the hip is flexed to 90 degrees.

MENINGOCOCCAL
MENINGITIS
CEREBROSPINAL
MENINGITIS
 Clinical Features
Meningitis-Suppurative lesion of meninges
• Involve spinal cord as well as the base and cortex of the
brain

• Cocci found in CSF outside and within leucocytes

• Case fatality if untreated – 80%

• Sequelae – Blindness and deafness

• Chronic or recurrent meningites

 Clinical Features
Meningococal septicemia
• Acute fever with chill, malaise and prostration

• Petechial rash [meningococci isolated in rash]

• 10% develop pneumonia

• Fulminant meningococcemia – Waterhouse Friderichsen
syndrome- Shock, DIC

• LPS released during autolysis sensitises vascular epithelium

• C5-C9 deficiency favour infection

Clinical Features
 Meningitis

Meningitis
Inflammation of the meninges (membranes
surrounding the brain and the spinal cord) is called
as Meningitis .

Encephalitis:
Inflammation of the parenchyma of brain

Meningoencephalitis:
Inflammation of brain & meninges
 Types of meningitis
• Pyogenic meningitis:

Evidence of Pathogenic bacteria in CSF.

(Streptococcus pneumoniae, Neisseria meningitidis, Hemophilus
influenzae)

• Aseptic meningitis:
Without the usual evidence of
pathogenic bacteria in the CSF.

(viral, tuberculosis, leptospiral, fungal/protozoal)

 Laboratory diagnosis

• Primary agents causing purulent bacterial meningitis :

Meningococci
Pneumococci
Haemophilus influenzae type b

• Others:
Group B streptococcus
Staphylococci
E. coli
Listeria monocytogenes
 Laboratory diagnosis

• Specimen collected-CSF, blood culture, nasopharyngeal

swab (carriers), petechial lesions

• CSF- increased pressure, turbid

Centrifuged deposit Culture on BA/CA Direct incubation/

Gram Stain Glucose broth
Supernatant-Ag detection
Lumbar
punture
Gram Stain
 Laboratory diagnosis
CSF- lumbar punture
Collection- sterile container,
Incubator with in 1 hour

3 separate sterile vials

1 - cell count
2 - chemical analysis
3 - culture

a. Grams staining
b. Culture
c. refregirate
Parameter Normal CSF Pyogenic Tuberculous Viral
Meningitis Meningitis Meningitis
Chemical

Total Protein 30-45mg% Highly Slightly increased Slightly

increased (100- (80-120mg %) increased(60-
600 mg %) 80 mg %)
Sugar 40-80 mg% Diminished (10- Diminished Normal
20 mg %)

Cell Count

Total Count 1-3/cumm 500- 50-500/cumm 4-10/cumm

10,000/cumm
Type of Cell Lymphocytes Neutrophils (95 Lymphocytes Lymphocytes
%) (90%)
Bacteriological
analysis
Gram Stain Nil GNC/GPC/GNB Nil Nil

Acid fast Stain Nil Nil AFB may be seen Nil

 Treatment

• Intravenous Penicillin G

• Chloramphenicol
• Ceftriaxone, ceftazidime (cephalosporins) before etiology
is known

• Eradicative therapy with Rifampicin to remove

pathogen from nasopharynx

• Vaccines available
 Epidemiology

• Reservoir – human nasopharynx

• Asymtomatic nasopharyngeal carriers

• Common in children between 3 months and 5 years

• Epidemic occurs in crowded conditions like jails,
ships and army camps

• “Meningitis belt of Africa”– Ethiopia to Senegal

Neisseria gonorrhoeae
 Neisseria gonorrhoeae (Gonococcus)

• N. gonorrhoeae causes the sexually transmitted

disease Gonorrhoea.

• The gonococcus was first described by Neisser in

1879 in gonorrheal pus.

• Gonococci resemble meningococci very closely in

many properties.
 MORPHOLOGY
• Gram negative diplococci with adjacent sides

concave, being typically kidney shaped.

• They are usually found within the polymorphs.

• They possess pili on their surface.

Gram Staining  The demonstration
of intracellular
Gram negative
diplococci in
stained smears
provides a
presumptive
evidence of
gonorrhea in men.
 Specimens are
inoculated onto
culture plates
freshly prepared
 CULTURE & CULTURAL CHARACTERISTICS

• Gonococci are fastidious organisms do not grow on

ordinary culture media.

• They are aerobic but may grow anaerobically also.

• The optimum temperature for growth is 35-36°C &

optimum pH is 7.2-7.6.

• It is essential to provide 5-10% CO2.

36
 Media used

a) Non selective media: Chocolate agar,

Mueller-Hinton agar.

b) Selective media: Thayer Martin medium with

antibiotics (Vancomycin, Colistin & Nystatin.
Colony morphology: Colonies are small, round,
translucent, convex or slightly umbonate with finely
granular surface & lobate margins.
 Biochemical reactions

1) Oxidase test: Positive

2) Acidifies only
glucose
but not maltose.
Antigenic structure & virulence factors
1. Pili: They help in adherence of bacteria to host
epithelial cells & they are antiphagocytic.

2. Lipo oligosaccharide: Endotoxic.

3. Outer membrane proteins: 3 types
a) Protein I (por)- it is a porin & helps in adherence.
b) Protein II (opa)- helps in adherence.
c) Protein III (rmp)- it is associated with protein I.
4. IgA1 protease: Splits & inactivates IgA.
 PATHOGENICITY

Source of infection:
1. Asymptomatic carriers
2. Patients

Mode of infection:
1. Venereal infection (sexual contact)
2. Non venereal infection
 PATHOGENICITY
• Mode of infection – Venereal
• Incubation period 2-8 days
• Source of infection is human patient
or carrier

• Primary Site of infection – urethra

• Female - Endocervix
• Acute urethritis – purulent urethral
discharge

© 2007 Chettinad Hospital & Research

Institute
 Mechanism of pathogenesis

Gonococci adhere to epithelial cells of urethra or

other mucosal surface through pili

Cocci penetrate through the intercellular space

They reach the sub epithelial connective tissue &

causes inflammation

Leads to clinical manifestations

Incubation period: 2-8 days.

43
 Disease
A) In men:
The disease starts as an acute urethritis with a
mucopurulent discharge

The infection extends to the prostate, seminal

vesicles & epididymis

In some it may become chronic urethritis leading

to stricture formation

The infection may spread to the periurethral tissues,

causing abscesses & multiple discharging sinuses
(Watercan perineum)
B) In women:
The initial infection is urethritis & cervicitis but vaginitis
does not occur in adult female (vulvovaginitis can occur in
prepubertal girls)

The infection may extend to Bartholin’s glands, endometrium &

fallopian tubes causing Pelvic Inflammatory Disease (PID)

Rarely peritonitis may develop with perihepatic inflammation

(Fitz-Hugh-Curtis syndrome)
Pelvic Inflammatory Disease Fitz-Hugh-Curtis syndrome
 CLINICAL FEATURES

• Urethritis - acute and chronic

• Watercan perineum in chronic

urethritis
• Proctitis

• Males – Prostate, testes, Seminal

vesicles epididymis

• Females – Cervicitis, Salpingitis,

Endometritis, Bartholin’s gland
infection,

Research Institute
© 2007 Chettinad Hospital & Research Institute
C) In both the sexes:
Proctitis, pharyngitis, conjunctivitis,
bacteremia which may lead to metastatic infection
such as arthritis, endocarditis, meningitis, pyemia &
skin rashes.
D) In neonates:
Opthalmia neonatorum (a non venereal
gonococcal conjunctivitis in the newborn) results
from direct infection during passage through birth
canal.
Gonococcal bacteremia
 Skin lesions on hands, forearms, feet
 Tenosynovitis
 Suppurativ arthritis
 Gonococcal endocarditis
 Gonococcal meningitis
 Gonococcal eye infections
Gonorrhea can manifest as Oral Infection

Changing sexual
practices and
oral sex
predisposes the
sex partners with
involvement of
oropharyngeal
regions
GONORRHEAL CONJUNCTIVITIS
• Non venereal disease - Ophthalmia neonatorum

• Non venereal gonococcal conjunctivitis in newborn

through infected birth canal
Gonococcal Opthalmia
 Babies born to infected
women suffer, can lead to
a serious condition of
Ophthalmia neonatorum
 Manifest with sever
purulent discharge with
periorbital oedema within
a few days of birth
 Topical application of 1%
silver nitrate has
drastically reduced the
incidence.
 In present contest Topical
erythromycin is used.
 LABORATORY DIAGNOSIS
Specimens collected:

A) In men:
a) Acute infection- Urethral discharge
b) Chronic infection-
i) Morning drop
ii) Discharge collected after prostatic massage
iii) Centrifuged deposit of urine

B) In women:
i) Urethral discharge
ii) Cervical swabs
C) When necessary: Blood, CSF, synovial fluid, throat
swab, rectal swab & material from skin rashes.
Transport: If there is delay in processing than the
specimens should be sent in “ Stuart’s medium”.
 Diagnosis
 Microscopy: Gr- diplococci intracellular
 Blood agar + CO2+ ristomycin (Thyer-Marthin
agar- vancomycin, colistin, amphotheracin B,
trimethoprim - N. gon)
 Biochemical testing: N. gonorrheae fermented
only glucose, N. meningitidis fermented glucose
and maltose
 AB resistance testing
 All neisseria catalase positive except N. elongata
 serology: agglutination test, IFA, ELISA etc
 LAB DIAGNOSIS

Gram’s staining

• Intra cellular Gram negative cocci

• Non motile
• Capsulated diplococci with
concave adjacent sides

© 2007 Chettinad Hospital & Research Institute

Microscopy

A) Direct microscopy:

Gram staining:
2. Immunofluorescence:
 B) Culture:

Media used:
Acute: Chocolate agar, Mueller-HintonAgar
Chronic: Thayer-Martin medium
C) Serology:

• Complement fixation test,

• Precipitation,

• Passive agglutination,

• Immunofluorescence,

• Radioimmunoassay.
 TREATMENT

• Previously Penicillin was drug of choice but resistance

developed rapidly.

• Penicillin resistance is due to production of penicillinase

enzyme & the strains are called as penicillinase producing
Neisseria gonorrhoeae (PPNG).

• Now Ceftriaxone or Ciprofloxacin plus Doxycycline or

Erythromycin is useful.
Controlling Gonorrhea

 The key control measures in gonorrhea are

1. Rapid diagnosis
2. Use of effective antibiotics
3. Tracing, examination and treatment of
contacts.
4. Inappropriate self medication has
contributed to widespread antimicrobial
resistance.
 EPIDEMIOLOGY

• Gonorrhoea is an exclusively human disease.

• The only source of infection is a human carrier or less

often a patient.

• Asymptomatic carriage in women makes them a reservoir

to spread infection among their male contact.

• Gonorrhoea is an venereal disease (STD).

 PROPHYLAXIS

• Early detection of cases,

• Tracing of contacts,

• Health education,

• General measures,

• Vaccination has no role in prophylaxis.

THANK YOU