Gram negative cocci

Neisseria species

aerobic gram negative diplococci

nonmotile

oxidase positive

catalase positive

fastidious, capnophilic

families

 Neisseriaceae
◦ Neisseria
◦ Kingella
◦ Eikenella
◦ Simonsiella
 Moraxellaceae
◦ Moraxella
◦ Acinetobacter

Neiseeria species and morazella catarrhalis

Habitat:

• upper respiratory tract

• genitourinary tract

• alimentary (digestive) tract

primary pathogens:

• N. gonorrhoease

• N. meninigitidis

virulence factors

 Fimbrae (common pili)-
◦ enhance the ability of
bacterial cells to adhere to
host cells and to each other
 Lipooligosaccharide:
◦ endotoxin involved in
damage to host tissue
 Capsule
 Cell membrane proteins
 IgA protease-
◦ cleaves IgA on mucosal
surfaces

clinical conditions Neisseria gonorrhoeae

 Pyogenic (pus-producing) infection of
columnar and transitional epithelial
cells
◦ urethral, endocervix, anal canal,
pharynx, and conjunctiva
 Incubation period: 2 to 7 days
 NOT Normal Flora
 Transmitted by sexual contact

Clinical infection in female and male Neisseria gonorrhoeae

 Disease in the male
◦ 95% show symptoms of
acute infection
◦ Symptoms include
dysuria, urethral
discharge
◦ Complications include
epididymitis and
urethral stricture, and
prostatitis
 Disease in the female
◦ 20% to 80% are
asymptomatic
◦ Symptoms include:
 Burning or frequency of
urination, vaginal
discharge, fever and
abdominal pain

disseminated gonococcal disease - N. gonorrhoeae

◦ Acute form has the following symptoms: fever, chills,
malaise, intermittent bacteremia, and skin lesions
◦ If untreated will progress to septic joint form of the
disease (inflamed joints, swollen, hot, full of pus and
fluid)
◦ Gonococcal arthritis occurs as a result of disseminated
gonococcal bacteremia

N. gonorrhoeae in children

 Disease in children
◦ In infancy, an eye infection (ophthalmia
neonatorum) may occur during vaginal delivery
◦ May cause blindness if not treated
◦ Infection is preventable with the application of
antibiotic eye drops at birth
 Extragenital infections
◦ Pharyngitis
◦ Anorectal infections

clinical lab diagnosis of Neisseria gonorrhoeae

 Clinical specimens
◦ Genital sites
 Female: endocervix
 Male: urethra
◦ Anal
◦ Oral/pharyngeal
◦ Eye
◦ Blood/joint fluids

specimen collection

◦ Dacron/ Rayon swabs preferred
◦ Swabs transported in Amies medium with
charcoal
◦ Inoculate media within 6 hours of
collection, avoid drying

transport media for Neisseria gonorrhoeae

Transgrow or JEMBEC
JEMBEC= James E Martin Biological
Environmental Chamber

patient side inoculation of selective media

 Optimal recovery of gonococci obtained when
species inoculated onto growth medium
immediately after collection
 JEMBEC plate: Inoculation of selective agar
(GC-Lect agar), placement of a CO2-generating
citric acid and sodium bicarbonate tablet in the
plate, and plate covered for transport to
laboratory
 Moisture in the covered JEMBEC plate
sufficient to generate CO2 from the citric
acid/sodium bicarbonate tablet
 Candle jar: Agar plates placed in a jar, a candle
lit, and jar sealed; candle extinguished at 3%
CO2 atmosphere

lab diagnosis of Neisseria gonorrhoeae morphology

◦ Gram-negative,
kidney-bean–shaped
diplococci

media selection

 Chocolate agar
◦ Enriched, non selective- Haemophilus & Neisseria
GC.
◦ Subject to overgrowth of normal flora
 Thayer-Martin agar is chocolate agar with
vancomycin, colistin, and nystatin
 Modified TM contains the above plus
trimethoprin
 Martin-Lewis medium
 New York City (NYC) medium
 GC agar, GC lect
 Specimen MUST be plated on warmed media
ASAP

• some strains of gc are susceptible to some of

incubation

 Inoculated culture media
must be incubated at 350 C
in 3% to 5% CO2 or candle
jar
 Candle jar must use white
wax candles

modified thayer-martin agar

 Modified Thayer-Martin (MTM) agar an
enriched and selective medium for
pathogenic N. gonorrhoeae and N.
meningitidis
 Nutritive base of MTM is chocolate agar
◦ Colistin: Inhibits gram-negative flora (N.
gonorrhoeae and N. meningitidis resistant to
colistin, most saprophyic species of
Neisseria susceptible)
◦ Vancomycin: Inhibits gram-positive flora
◦ Nystatin: Inhibits yeast flora
◦ Trimethoprim: Inhibits swarming Proteus

Neisseria gonorrhoeae colony morphology on MTM agar

 Colony morphology on
modified Thayer-Martin
(MTM) agar
◦ Small, beige- gray
◦ Translucent, smooth
 Fresh growth must be
used for testing,
because N.
gonorrhoeae produces
autolytic enzymes

oxidase test for Neisseria gonorrhoeae

 Test on filter paper or directly on plate
 Oxidase reagent =Dimethyl or tetramethyl oxidase
reagent
 Violet-purple color indicates a positive result

carbohydrate utilization

Cystine trypticase agar
(CTA)
◦ Contain 1% of a single
carbohydrate
 Glucose, maltose,
lactose, sucrose
◦ Phenol red is pH indicator
 Read in 24-72 hours

immunologic methods, non-culture methods, PCR/Nucleic acid probe testing

 Immunologic methods
◦ Use colonies from primary plate
◦ Organisms do not need to be viable
 Fluorescent antibody technique
 Coagglutination
 Non-culture methods
◦ Use direct patient specimen
◦ ELISA
 PCR/Nucleic acid probe testing
◦ Multiplex testing with Chlamydia trachomatis
◦ Denaturation, Annealing & Primer Extension
◦ Thermal cycler
◦ Primers, nucleotides, buffers, probes
◦ Good for large volume of testing
◦ Unable to perform on all sources

treatment and antimicrobial resistance: Neisseria gonorrhoeae

 PPNG = Penicillinase Producing Neisseria gonorrhoeae
◦ First seen in 1976
◦ Plasmid-mediated
◦ Beta-lactamase testing should always be done on N.
gonorrheoae
 Treatment: CDC recommends dual therapy
◦ two antimicrobials with different mechanisms of action
 Penicillin
 Recommended Regimen
◦ Cephalosporins and Flouroquinolones
◦ Ceftriaxone 250 mg IM in a single dose
◦ Azithromycin 1g orally in a single dose
◦ Cefixime 400 mg
 Tetracycline if beta-lactamase positive strain

Neisseria meningitidis

 Commensal of carriers in the nasopharynx
 Cross the epithelium and enter the circulatory
system
◦ Primarily affects the immunocompromised, young
children, trauma victims
 Leads to septicemia and localization to the
meninges causing inflammation of the brain
 Meningitis
 Highly fatal (25% even if treated)
◦ Encapsulated strains A, B, C, Y, W-135

Virulence factors of Neisseria meningitidis

 Pili
 Polysaccharide capsule
 Cellular membrane proteins
 Lipooligosaccharide/endotoxin

bacterial meningitis from Neisseria meningitis

◦ Transmission is by respiratory droplets and requires
both close contact (ex: dormitories, military barracks,
in institutions) and lack of specific antibody
(susceptibility)
◦ Symptoms include fever, headache, stiff neck, nausea,
vomiting, and purulent meningitis with increased
WBCs
◦ Serotypes B and C most common in US
 Other infections include meningococcemia, pneumonia,
purulent arthritis, & endophthalmitis
 May be seen in genital tract with oral-genital contact

types of infectious disease caused by Neisseria

 N. meningitidis causes a rapidly
progressive meningitis in school-aged
children, adolescents, and young adults
with a mortality of 7-13%
 N. meningitidis bacteremia (mortality of
19-70%) a cause of Waterhouse-
Friderichsen syndrome with petechiae,
purpura, adrenal hemorrhage, dissemin-
ated intravascular coagulation, and
shock

causes: hemorrhage in the adrenal glands in Waterhouse-Fredericksen syndrome

identification of Neisseria meningitidis

◦ Examine direct smear
from CSF for intra & extra
cellular gram negative
diplococci
◦ Examine smear for halo
◦ Other body sites include
nasopharyngeal swabs,
sputum, and urogenital
specimens

Gram-stained smear of CSF
showing the extra cellular and
intracellular gram-negative
diplococci

examination of agars for Neisseria meningitidis

Neisseria meningitidis growing
on sheep blood agar (right) and
chocolate agar (left)

 Examine cultures on
blood agar & chocolate
agar after incubation in
increased CO2
 Colony Morphology
◦ Small
◦ Tan-grey color
◦ Smooth

oxidase test, CTA, and immunologic methods Neisseria meningitidis

Oxidase-test positive
◦ Conventional CTA
carbohydrates for biochemical
identification (glucose+ and
maltose+)
◦ Immunologic methods

antibiotic therapy Neisseria meningitidis

 Treatment
 Penicillin
◦ Penicillin G (Benzylpenicillin)
 Chloramphenicol – for Penicillin
allergy
 Cephalosporins
◦ Cefotaxime or Ceftriaxone
 Other options: rifampin or sulfonamide
 Vaccine
◦ For use with people aged 11-55

vaccines

 Two vaccines are currently commercially available
◦ Conjugate & recombinant protein
 Conjugated protein-polysaccharide vaccines
 Quadrivalent (covers 4 serogroups)
 for serogroups A, C, W and Y
◦ Conjugated meningococcal vaccines offer a number of important
advantages over purified polysaccharide vaccines.
◦ Most importantly they stimulate memory T lymphocytes (meaning that
they offer long-term protection and that the immune response can be
boosted by repeat exposure)
 Recombinant Protein Vaccine
◦ Monovalent (only 1 serogroup)
◦ Booster required to retain immunity

safe laboratory handling of Neisseria meningitidis

 Use of biosafety cabinet for mechanical manipulation of
samples with risk of aerosolization or droplet formation
(grinding, centrifuging, blending)
◦ Use of a biosafety cabinet for manipulation of sterile site isolates of
N. meningitidis
 Informed decision by microbiology workers of
vaccination by quadrivalent meningococcal
polysaccharide vaccine (A, C, Y, W-135)
 Antimicrobial prophylaxis (rifampin, ciprofloxacin, or
ceftriaxone) for potential inhalational exposure
(aerosolization or droplet formation), percutaneous
exposure, or mucosal exposure to invasive N.
meningitidis isolate

nonpathogenic Neisseria species

 Normal flora of upper respiratory tract
 Some members
◦ Neisseria cinera
◦ Neisseria lactamica
◦ Neisseria mucosa
◦ Neisseria sicca
◦ Neisseria subflava

Moraxella catarrhalis

 Normal commensal of the respiratory tract
 Has become an important opportunistic pathogen
◦ Predisposing factors
 Advanced age, Immunodeficiency,
Neutropenia, Other debilitating diseases
 Clinical infections
◦ Pneumonia
◦ Sinusitis
◦ Otitis media (3rd most common cause)

Virulence Moraxella catarrhalis

• Endotoxin

• Pili

• Beta-lactamase

Moraxella catarrhalis observation of plate and smear

Direct smear from an otitis
media sample showing
intracellular gram-negative
diplococci

 Colonies appear smooth with a
grayish- white color with characteristic
wagon-wheel appearance
 When colonies pushed with loop, they
“scoot” across media

lab results from Moraxella catarrhalis

 Oxidase positive
 Catarrhalis Disc
◦ Butyrate Esterase Positive= blue-green
 All CTA sugars negative
 Nitrate reduction +
◦ (all Neisseria spp = 0)
 DNAse +
 Produce beta- lactamase +
 Butyrate Esterase (Tributyrin) +

identification of selected Neisseria species and Moraxella

selected biochemical reaction for identification of Neisseria and Moraxella catarrhalis