Neisseriaceae
The Gram-negative cocci of clinical
importance
Neisseriaceae
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Classification - Family Neisseriaceaeare all Gram negative aerobic rods and
cocci. Contains 4 genera:
Neisseria- cocci
 Moraxella, subgenus Branhamella- cocci;
other species within the genus are short
rods
 Acinetobacter- rods
 Kingella- rods
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Neisseriaceae
We will only be discussing Neisseria and
Branhamella which morphologically and
biochemically resembles Neisseria, but
shows DNA homology to Moraxella.
 Some are found as part of the normal flora
of the naso and oropharynx and are usually
considered to be non-pathogenic.
 Two species- N. meningitidis (MGC) and N.
gonorrhoeae (GC) are considered to be
pathogenic although N. meningitidis may
be found as part of the nasopharynx normal
flora in 10-15% of the population.
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Neisseria and Branhamella
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Morphology and General Characteristics
Gram negative diplococci that are coffeebean shaped with adjacent flattened
edges.
 In a direct smear GC may be found
intracellularly in PMNs or extracellularly.
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Neisseria species Gram stain
Intracellular GC
Morphology and General
Characteristics
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B. catarrhalis and Neisseria species other than
MGC and GC will grow on nutrient agar without
blood.
MGC and GC are more fastidious and require
serum or blood in addition to other growth factors.
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GC is more fastidious and requires chocolate agar,
whereas MGC may grow on CBA.
Colonies of both are small and translucent to
grayish-white.
Colonies of non-pathogenic species may
be dry and wrinkled, opaque or yellow, and they
tend to be self-adherent.
MGC colonies
N. sicca colonies
N. subflava colonies
Morphology and General
Characteristics
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A selective media is usually used to recover MGC and GC
when the specimen is likely to be contaminated with other
bacterial normal normal flora.
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Modified Thayer Martin (MTM) is the most common of these.
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It is chocolate agar containing Vancomycin (inhibits G +), Colistin
(inhibits G -), Nystatin (inhibits fungi), and Trimethoprim (inhibits
swarming Proteus).
Occasional strains of GC are sensitive to Vancomycin, therefore,
both chocolate agar and MTM should always be used.
The colonies may require 48-72 hours for growth.
Lewis Martin agar is similar to MTM, but it has anisomycin
substituted for nystatin for improved inhibition of Candida
albicans.
GC growth on MTM
Morphology and General
Characteristics
Members of the genus Neisseria and
B. catarrhalis are aerobic with little or no
growth under anaerobic conditions.
 MGC and GC require a CO2 concentration
of 3-10% for growth, particularly for primary
isolation.
 In the lab we will be growing the Neisseria
in candle jars.
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Growth in candle jars
Morphology and General
Characteristics
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GC and MGC are particularly
sensitive to temperature extremes and drying,
therefore successful cultivation depends upon
careful collection and careful processing of
specimens.
Ideally, specimens should be plated directly onto
culture media and incubated immediately.
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The reason for this is that MGC and GC may produce
autolytic enzymes at 250 C and an alkaline pH leading to
lysis of the cells.
Most MGC and GC produce capsules.
Neisseria and Branhamella
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Biochemical identification - need to
distinguish between B. catarrhalis and
both pathogenic and non-pathogenic
Neisseria.
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To distinguish from other organisms – use
colony morphology
 Gram stain (g-dc), and
 the fact that it is catalase and oxidase positive.
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Oxidase test on plate
Oxidase test on paper
Biochemical identification
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To differentiate between the different Neisseria
species and B. catarrhalis.
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Carbohydrate utilization tests - These are not
fermentation tests because the organisms use carbohy
drates oxidatively.
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Cystine-tryptic-agar (CTA) sugars are the reference tests,
but require a heavy inoculation, incubation in a non-CO2
incubator and observation for 24-72 hours.
Some fastidious GC won't grow or grow very poorly on CTA
and the small amount of acid produced is neutralized by
alkaline products formed from peptone utilization.
The sugars glucose, maltose, lactose, sucrose and
fructose are commonly used.
Pertinent results: GC + for glucose only; MGC + for
glucose and maltose; B. catarrhalis - for all.
CTA sugars
Biochemical identification
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We now have non growth dependent tests which
depend on preformed enzymes.
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Kits are now available that can identify the different
organisms by using enzyme profiles obtained using
chromogenic substrates.
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A heavy inoculation of organism is used with a small volume
of carbohydrate in a buffered salt solution.
Results are read in 4 hours.
Results are in 4 hours and are non growth dependent.
This is what we will do in the lab.
DNAse test - B. catarrhalis is + for DNAse , all Neisseria
are -.
Nitrate reduction - GC and MGC are - for nitrate
reduction, B. catarrhalis is +.
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Some nonpathogenic Neisseria species are + and others are
-.
Neisseria and Branhamella
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Serologic and Other Identification methods –
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Many kits are available for serologic identification of
MGC and GC
Auxotyping distinguishes GC on the basis
of nutritional requirements.
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Gonococcal strains that are associated with disseminated
disease have a multiple requirement for arginine,
hypoxanthine and uracil.
Nucleic acid based tests – both probe based and
PCR based tests have been developed for the
identification of GC directly from clinical samples.
Neisseria and Branhamella
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Mechanisms of Pathogenicity –
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Neisseria gonorrhoeae –
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Pili- important in attachment to human columnar epithelial cellsmay inhibit phagocytosis.
Protein I (Por) - the principal protein found on the outer
membrane- functions as a porin to allow small molecules to
diffuse thru the membrane and is important for intracellular
survival.
 Is protective against host’s inflammatory response and
serum complement mediated killing
Protein II (Opa)- a minor protein found on the outer membrane.
 It is involved in attachment to PMNs and other leukocytes
where it induces the endocytic uptake of GC
 After endocytosis, GC is transported in vesicles to the base
of the cells and released into subepithelial tissue.
 Both pili and protein II are subject to antigenic
variation;even if antibodies are formed against them
they are not effective.
Induced uptake
Neisseria and Branhamella
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Protein III (rmp) – an outer membrane protein that may
associate with Protein I to form pores.
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Blocks host serum bacteriocidal (IgG) action against the
organism by eliciting formation of blocking antibodies
Lipopooligosaccharide- (LOS) - comparable to LPS of
Gram negative bacilli (endotoxin). May undergo antigenic
variation and causes mucous membrane damage, fever
and toxicity. Specific activities include:
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Adherence and invasion
Molecular mimicry of host structures
Release in membrane blebs
Suppression of leukotreine B4 synthesis→ inhibits PMN
activation
Activates alternative complement pathway → inflammation
Activates TNF → inflammation
LOS and Membrane blebs
Neisseria and Branhamella
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Capsule – helps prevent phagocytosis and protects
organism from antimicrobial peptides
IgA1 protease- cleaves IgA to allow mucosal attachment
of GC.
Ability to acquire iron from the host by producing outer
membrane proteins that capture it under conditions of iron
limitation.
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Enhanced capacity occurs in those that cause disseminated
gonococcal infection (DGI).
Iron acquisition
GC cell wall structure
Neisseria and Branhamella
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Strains of GC that cause DGI possess certain types of
Protein I, are resistant to phagocytosis and serum
bactericidal activity, and are able to multiply unchecked in
the presence of PMNs.
Neisseria meningitidis –
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Pili- allow binding to specific nasopharyngeal receptors.
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Type IV pili are essential for the interaction of MGC with
brain endothelial cells
Neisseria and Branhamella
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Capsule (polysaccharide) – antibodies, if made, are
protective.
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Only certain serotype antigens are found during epidemics.
Ability to acquire iron from human transferrin
IgA1 Protease
Class 1,2, and 3 outer membrane proteins ( Por) that
function as porins and are important for intracellular
survival
Class 5 outer membrane protein (Opa) involved in
attachment, induced uptake (invasion).
Class 4 outer membrane protein (rmp) important in
blocking host serum bacteriocidal (IgG) action against the
organism by eliciting formation of blocking antibodies
Opc protein – important for attachment and invasion
LOS with same functions as GC LOS
Pili, Opa, LOS, and to a lesser extent, Opc, undergo
antigenic variation
Neisseria and Branhamella
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Clinical Significance –
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Neisseria gonorrhoeae - usually transmitted by
direct sexual contact.
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Used to be the most common reportable disease in
the U.S.
Those infected commonly have a concomitant Chlamydia
trachomatis infection (most common reportable disease in
the U.S. today).
Asymptomatic infections- Males= 10%, Females= 20-75%.
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The problem with asymptomatic individuals is that they are
carriers and can transmit the disease to others.
Clinical significance of GC
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Uncomplicated infections - in males incubation= 1-4 days
followed by acute urethritis with profuse purulent
discharge filled with GC.
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If untreated may spread to cause prostitis and epididymitis
- with proper antibiotic treatment this is rare.
With oropharyngeal infection mild pharyngitis.
With rectal infection - proctitis;
Uncomplicated infections - in females following the
incubation may have increased vaginal discharge, burning
or frequency of urination and menstrual abnormalities.
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Infected mothers can transmit GC to their babies at
birth causing gonococcal ophthalmia neonatorum.
This can lead to blindness, therefore all babies are
given erythromycin eye drops at birth to prevent this.
Uncomplicated GC infection
Pus containing discharge
Neonatal ophthalmia neonatorum
Clinical significance of GC
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Complicated infections
Occur more frequently in women - spread from the
cervix into the fallopian tubes resulting in
endometritis, salpingitis, and peritonitis= pelvic
inflammatory disease (PID).
 Occurs in 10-20% of infected women.
 Symptoms include lower abdominal pain,
abnormal vaginal and cervical discharge,
and uterine tenderness.
 This can result in ectopic pregnancy and infertility.
Salpingitis
Complicated infections of GC
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Spread to the bloodstream may result in DGI in both
men and women (1%).
 Can result in endocarditis, arthritis, maculopapular
rash, and meningitis.
Clinical significance
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Neisseria meningitis- causes endemic and epidemic epidemic
cerebral meningitis.
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Most common in children under 1 year, adolescents, and young
adults.
Can be part of the normal flora of the nasopharynx and cause
disease when there is a failure in host resistance or when a
particularly virulent strain colonizes the mucous membranes.
Asymptomatic carriers are the primary source of spread thru
aerosols.
Colonization of the nasopharynx usually results in a subclinical
infection or a mild upper respiratory tract infection like the
common cold.
Invasion of the bloodstream occurs only in individuals lacking
bactericidal antibodies or deficient in certain complement
components (C5-C8).
Chronic irritation or damage to the respiratory mucosa may be
predisposing factors.
Invasion of the bloodstream may result in septicemia
(meningococcemia) or meningitis.
MGC infections
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Meningococcemia- symptoms include high fever, shaking,
chills, muscle pain, and petechial rash (hallmark of MGC
infections).
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Disease may be chronic, moderate or fulminant.
The fulminant type is called the Waterhouse-Friderichsen
syndrome and it is characterized by disseminated
intravascular coagulation (DIC), with hemorrhaging into the
skin, adrenal glands and other internal organs.
This results in rapid death due to acute generalized
toxemia and shock.
Most of these symptoms are due to the activity of the
endotoxin (LOS).
Patients that survive may lose their limbs from tissue
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necrosis (gangrene of the skin and soft tissues)
Petechial rash
Waterhouse-Friderichsen
syndrome
Waterhouse-Friderichsen syndrome
MGC infections
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Meningitis- symptoms include fever, headache,
stiffness of back and neck, and petechial rash.
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In severe cases there is severe cerebral hyperemia
(accumulation of blood) and tissue swelling.
May progress to convulsions and coma
This is invariably fatal without rigorous antimicrobial
therapy.
10%-14% of cases are fatal even with treatment
For patients who recover 11%-19% have permanent
hearing loss or mental retardation
Meningitis
Clinical significance
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Other Neisseria species - rarely associated with
endocarditis, septicemia, and meningitis.
B. catarrhalis - may be a significant cause of otitis
media and maxillary sinusitis in children
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May cause pneumonia and bronchitis in
immunocompromised individuals.
May also occasionally cause endocarditis, meningitis and
septicemia.
Neisseriaceae
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Treatment and antimicrobial susceptibility –
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GC - used to use only penicillin.
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Resistant strains are emerging so -lactamase testing
should be done.
For penicillin resistant strains or penicillin sensitive
individuals substitute ceftriaxone or spectinomycin for
penicillin (1 dose to treat GC) plus 7 days of doxycycline to
treat possible coexisting C. trachomatis infection.
Amoxicillin or ampicillin or Penicillin G plus probenecid
can be used for uncomplicated cases
MGC- I.V. penicillin or chloramphenicol and
erythromycin or ceftriaxone.
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Prophylaxis with rifampin, ciprofloxacin, or ceftriaxone for
household and other close contacts
Treatment
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Vaccines (tetravalent to polysaccharide
capsular antigens A, C, Y, W135) are
available for the virulent types of MGC.
Most human infections are due to types A, B, C,
Y, and W135.
 They are given to those at risk, including college
students living in dorms.
 A group B vaccine consisting of OM antigens
has been developed, but is not available in the
United States.
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