MLAB 2434:
Microbiology
Keri Brophy-Martinez
The Gram-Negative Cocci
Families

Neisseriaceae
◦
◦
◦
◦
◦
Neisseria
Kingella
Eikenella
Simonsiella
Alysiella

Moraxellaceae
◦ Moraxella
◦ Acinetobacter
General Characteristics:
Neisseria Species
Aerobic, gram-negative diplococci
 Nonmotile
 Oxidase positive
 Catalase positive
 Fastidious, capnophilic

Neisseria Species and Moraxella
catarrhalis

Habitat
◦ Upper respiratory tract
◦ Genitourinary tract
◦ Alimentary(Digestive) tract

Primary pathogens:
◦ N. gonorrhoeae
◦ N. meningitidis
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

Transmitted by sexual contact
Clinical Infections:
Neisseria gonorrhoeae

Disease in the male

Disease in the female
◦ 95% show symptoms of
acute infection
◦ 20% to 80% are
asymptomatic
◦ Symptoms include dysuria,
urethral discharge
◦ Symptoms include:
 Burning or frequency of
urination, vaginal discharge,
fever and abdominal pain
◦ Complications include
epididymitis and urethral
stricture, and prostatitis
◦ Complications include pelvic
inflammatory disease (PID),
sterility and ectopic
pregnancy
Clinical Conditions:
Neisseria gonorrhoeae:

Disseminated gonococcal disease
◦ 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
Clinical Conditions:
Neisseria gonorrhoeae:

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
Laboratory Diagnosis:
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
Transgrow or JEMBEC
JEMBEC= James E Martin Biological Environmental
Chamber
JEMBEC
Laboratory Diagnosis:
Neisseria gonorrhoeae

Morphology
◦ Gram-negative, kidneybean–shaped
diplococci
Laboratory Diagnosis:
Neisseria gonorrhoeae
Media Selection

Chocolate agar
◦ Subject to overgrowth of normal flora
Thayer-Martin agar is chocolate agar with
vancomycin, colistin, and nystatin
 MTM contains the above plus trimethoprin


Specimen MUST be plated on warmed media ASAP
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
Laboratory Diagnosis:
Neisseria gonorrhoeae

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
Laboratory Diagnosis:
Neisseria gonorrhoeae
◦ Oxidase Test
 Test on filter paper or directly on plate
 Oxidase reagent =Dimethyl or tetramethyl oxidase reagent
 Violet-purple color indicates a positive result
Laboratory Diagnosis:
Neisseria gonorrhoeae


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
Laboratory Diagnosis:
Neisseria gonorrhoeae

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, nucleic acid probe, and PCR testing
◦ Expensive; usually used in high-risk populations with large
volume of testing
◦ Unable to perform on all sources
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 = Penicillin
 Tetracycline if beta-lactamase positive strain. Can also
use cephalosporins and flouroquinolones

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:
Neisseria meningitidis
Pili
 Polysaccharide capsule
 Cellular membrane proteins
 Lipooligosaccharide/endotoxin

Clinical Infections:
Neisseria meningitidis:

Bacterial 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

Clinical Infections:
Neisseria meningitidis
Hemorrhage in the adrenal
glands in WaterhouseFridericksen syndrome
Laboratory Diagnosis:
Neisseria meningitidis

Identification
◦ Examine direct smear from
CSF for intra & extra
cellular g- dc
◦ 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
Laboratory Diagnosis:
Neisseria meningitidis

Examine cultures on
blood agar & chocolate
agar after incubation in
increased CO2
 Colony Morphology
◦ Small
◦ Tan-grey color
◦ Smooth
Neisseria meningitidis growing
on sheep blood agar (right) and
chocolate agar (left)
Laboratory Diagnosis:
Neisseria meningitidis
◦ Oxidase-test positive
◦ Conventional CTA
carbohydrates for biochemical
identification (glucose+ and
maltose+)
◦ Immunologic methods
Antibiotic Therapy:
Neisseria meningitidis
Penicillin
 Other options: rifampin or sulfonamide


Vaccine
◦ For use with people aged 11-55
◦ Does not protect against all serotypes
Nonpathogenic Neisseria species

Normal flora of upper respiratory tract

Some members
◦
◦
◦
◦
◦
Neisseria cinera
Neisseria lactamica
Neisseria mucosa
Neisseria sicca
Neisseria subflava
Moraxella catarrhalis

Previously known as Branhamella 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 factors:
Moraxella catarrhalis
Endotoxin
 Pili
 Beta-lactamase

Laboratory Diagnosis:
Moraxella catarrhalis
Direct smear from an otitis
media sample showing
intracellular gram-negative
diplococci
Laboratory Diagnosis:
Moraxella catarrhalis
Colonies appear smooth with a grayishwhite color
 When colonies pushed with loop, they
“scoot” across media

Moraxella catarrhalis
growing on chocolate agar
after 24 hours of incubation
Laboratory Diagnosis :
Moraxella catarrhalis
Oxidase positive
 Catarrhalis Disc

◦ Positive= blue-gren

All CTA sugars negative

Produce beta- lactamase
Identification of
Selected Neisseria Species & Moraxella
Species
BAP
Growth
R.T
T/M
Acid production
Gluc Mal Lac Suc
N. gonorrhoeae
=/+
=
+
+
=
=
=
N. meningitidis
+
=
+
+
+
=
=
N. lactamica
+
v
+
+
+
+
=
N. sicca
+
+
=
+
+
=
+
M. catarrhalis
+
+
=
=
=
=
=
References
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Engelkirk, P., & Duben-Engelkirk, J. (2008). Laboratory
Diagnosis of Infectious Diseases: Essentials of Diagnostic
Microbiology . Baltimore, MD: Lippincott Williams and Wilkins.
http://www.awinhospitalproducts.com/product/35collection-swab-amies-medium-w-charcoal-wwcsam-3450
https://new.fishersci.com/ecomm/servlet/fsproductdetail_10
652_606366_29104_-1_0
https://picasaweb.google.com/pia8628/0411microlab#559610
4725803822690/
Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook
of Diagnostic Microbiology (4th ed.). Maryland Heights, MO:
Saunders.