MEDT 14 | CLIN.

BACTERIOLOGY (LEC)
Jonnel P. Andaya || 3rd Year LESSON 10. FAMILY
Transcribed by: Ybanez, Trisha Mae C. | De Los Reyes,
John Dominic M.
NEISSERIACEAE
 Capnophilic bacteria: Neisseria spp., Moraxella spp.
OUTLINE:
I. Introduction NEISSERIA SPP.
II. Neisseria spp. GENERAL CHARACTERISTIC
A. General Characteristics  Often arranged in pairs with flattened adjacent surface
giving the appearance of kidney bean shape.
B. Clinical Manifestations and Pathogenesis
 Gram-negative diplococci that do not elongate when
C. Laboratory Diagnosis exposed to sub-inhibitor concentrations of penicillin.
a. Specimen Collection and Transport  Catalase positive (+) EXCEPT: N. elongata
b. Culture Medium and Incubation  Oxidase positive (+)
c. Characteristics on Chocolate Agar  Capnophilic (grows best in high conc. of carbon dioxide
d. Direct Detection Method – about 5%-10%).
D. Identification Approach  Through the use of a candle jar, the CO2 concentration is
E. Antimicrobial Therapy and Prevention increased. The candle is used to consume oxygen and
III. Moraxella spp. generate carbon dioxide. CO2 enhances the growth of
capnophilic bacteria.
A. Species
 Plates are placed inside the candle jar where Neisseria
B. Direct Detection Method spp. and Moraxella spp. are isolated.
C. Moraxella Cultivation  There are only selected species of Neisseria app. That
D. Identification Approach can pose as a threat to humankind, but not general.
E. Prevention and Control There are Neisseria app. Colonizers in our body that
IV. Other members of Family Neisseriaceae does not cause infection except to those
immunocompromised individuals (except N. gonorrhoeae
and N. meningitidis). They are opportunistic
pathogens.
INTRODUCTION  N. gonorrhoeae – causes gonorrhea
 The Genus Neisseria contains the two gram-negative  N. meningitidis – causes meningococcemia
cocci (diplococci) which are established human  They are referred to as saprophytic Neisseria spp.
pathogens. The genus also contains many commensal  Saprophytic Neisseria spp. are low irulent species and do
species, most of which are harmless inhabitants of the not cause infection to healthy hosts. For example: N.
upper respiratory and alimentary tracts. The pathogenic sicca, N. mucosa, N. subflava
species are  Saprophytic bacteria are decaying agents.
a. N. meningitidis (meningococcus) – main cause of
meningitis and bacteremia CLINICAL MANIFESTATION AND PATHOGENESIS
b. N. gonorrhoeae (gonococcus) – leading cause of
 Inhabitants of upper respiratory tract (normal
gonorrhea
flora/colonizers) because it is where we exhale carbon
 They typically appear in pairs (diplococcic) with the
dioxide, except for N. gonorrhoeae.
opposing sides flattened, imparting a “kidney bean”
appearance. They are non-motile, non-spore forming, o N. gonorrhoeae always causes infection to
and non-fastidious. humans and is not a colonizer/normal flora.
 Their cell walls are typical of gram-negative bacteria, with  It is the primary pathogen of urogenital tract,
a peptidoglycan layer and an outer membrane containing and is always clinically significant.
polysaccharides complexed with lipid and protein. The  N. gonorrhoeae is a gram-positive
structural elements of N. meningitidis and N. diplococci and is causing gonorrhea.
gonorrhoeae are the same, except that the  It is sexually transmitted disease. Also if
meningococcus has a polysaccharide capsule external to unsafe oral sex is done with the infected
the cell wall. individual, N. gonorrhoeae can cause infection
to the pharynx, called pharyngitis.
Family Neisseriaceae
1. Neisseria spp.
2. Branhamella/Moraxella spp.
3. Acinetobacter spp.
4. Kingella spp.

Note: Acinetobacter spp. and Kingella spp. are

coccobacilli but resembles a diplococci.
 The clinical manifestation of this pathogen can
 Catalase positive (+): Neisseria spp., Moraxella spp., be acute with a pronounced purulent
Acinetobacter spp. ; Catalase negative (-): Kingella spp. response, or they may be asymptomatic.
 Oxidase positive (+): Neiserria spp., Moraxella spp.,  If this pathogen causes infection to the eyes, it
Kingella spp. ; Oxidase negative (-): Acinetobacter spp. will cause conjunctivitis.
 If there is bacterial growth in Mac Conkey
Agar, always conduct an oxidase test to
determine if the bacteria is a member of
Enterobacteriaceae because those non-
fermentative like Acinetobacter spp. are
oxidase (-).
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 Usually, female and male genitals have
discharges, the reason why in Tagalog, it is
termed as “tulo”. Literal na tumutulo yung
nana.
 It can be transmitted through person to person
spread by sexual contact, including rectal
intercourse and urogenital sex. It can also be
spread vertically from infected mother to
newborn during birth. If the baby passes
through the infected female’s genitals, the
baby may acquire the infection. The reason
why after birth, the newborns are given eye
drops within 4 hours (silver nitrate, 1%
tetracycline, erythromycin eye drop).
o N. meningitidis is the leading cause of fatal
bacterial meningitis and meningococcemia (Water
house- Friedrichsen Syndrome)
 Can be transmitted through aerosol,
contaminated respiratory droplets. It is
very contagious.
 Its virulence factors responsible for the
spread of this organisms from patient
upper respiratory to the blood-stream and
meninges to cause-life threatening
infections.
 It causes disseminated petechiae in the
body.
 Petechiae – purpuric rash. A
maculopapular blanching rash.
These are pinpoint round spots
appearing on the skin as a result
of bleeding. Also may cause
kidney bleeding and meningitis.
It is very contagious.
 It can be transmitted through human
carriage. There are people with very high
bactericidal antibodies in their body the
reason why even if they acquire the
bacteria, they will not exhibit symptoms or
infections. They are asymptomatic but
they still harbor the pathogen and can
transmit it to other people.
 In other organisms, before meningitis,
bacteremia comes first and the infected
blood will circulate to the body back to the
heart and then causes endocarditis, then
travels to the brain and causes
meningitis. (E.coli, S. pneumoniae)
 In N. meningitidis, without the presence of
bacteremia, it can still cause meningitis
because it was found in the upper
respiratory tract. When you inhale from
the nose, the bacteria can be transported
to the brain.
 It can be transmitted through direct
contact (due to inhalation of
contaminated respiratory droplets),
sharing of utensils and water bottles,
kissing, coughing, sneezing.
 Other Neisseria spp. are not considered pathogens and
are often referred to as saprophytic Neisseria. Although
they are most commonly encountered as contaminants in
clinical specimens, they can occasionally be involved in
bacteremia and endocarditis. These species are not
common cause of human infections and low virulence but
may rarely involve in bacteremia, endocarditis and
meningitis.
Organism Virulent Factors
N. gonorrhoeae  Pili
 Invasion of host cells
 Survival in inflammatory
response
N. meningitidis  Pili
 Lipopolysaccharides –
causes aggregation of
platelets ; anti-
phagocytic
 Moraxella catarrhalis is from genus Moraxella. It is a
normal flora of the upper respiratory tract. It only causes
infection to immunocompromised individuals.
 Rarely isolated
 Virulence factor is uncertain
 Infections: otitis media, sinusitis, pneumonia, and rare
causes of bacteremia, endocarditis and meningitis.
LABORATORY DIAGNOSIS
SPECIMEN COLLECTION AND TRANSPORT
 Have to carefully follow the protocol as this requires
special considerations.
 JEMBEC transport system
o Used for N. gonorrhoeae
o Other Neisseria spp. can grow on Thayer Martin
Agar (TMA).
o It can be bedside collection
o A rectangle with chocolate-colored agar inside
(TMA), plus, a cotton swab (Calcium alginate and
rayon fiber).
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o N. gonorrhoeae will die in a usual cotton swab
during collection because it is sensitive in drying of
cotton swab and temperature.
o After collection, it is needed to be subjected to the
JEMBEC transport system and put inside the
ziplock bag with CO2 generating tablet. Then it will
be transported to the lab and upon receipt, it will be
placed to the candle jar.

 N. gonorrhoeae is more fastidious and requires an

enriched chocolate agar for growth on primary agars.
o Supplementary of isovelatex or horse blood
 Because Gonococci, and sometimes meningococci
must be isolated from sites that contain large number
of normal flora (e.g. genital, upper respiratory tracts),
 If the specimen received is swab, in the collection, the selective media been developed to facilitate their
specimen shall immediately be placed on the transport recovery:
medium (e.g. Stuart and Cary Blair) but make sure to 1. Thayer Martin Medium Chocolate agar with:
put charcoal. Agar  Isovelatex -
o The transport medium should contain charcoal supplement
to inhibit toxic fatty acid present in the cotton  Colistin- Inhibits
fibers. gram negative
o These fatty acids are toxic to N. gonorrhoeae.  Nystin - Inhibits
Upon receipt in the lab, it shall be plated fungi
immediately within 6 hours.  Vancomycin-
 The recovery of N. gonorrhoeae or N. meningitidis from Inhibits gram
normally sterile body fluids requires no special positive
methods. Blood culture being a notable exception. Both 2. Modified Thayer Chocolate agar with:
organisms are sensitive to Sodium Polyanethol Martin Medium Agar  Isovelatex -
supplement
Sulfonate (SPS). So that content of SPS in blood
 Colistin- Inhibits
culture broth should not exceed 0.025% and must
gram negative
contain some gelatin.
 Nystin - Inhibits
o SPS can be bought commercially in different
fungi
concentrations: 0.025% to 0.05%.  Vancomycin-
o Toxic concentrations to Neisseria spp.: Inhibits gram
0.03% to 0.05% positive
o We shall use the lowest concentration for N.  Trimethoprim
gonorrhoeae. lactate- to inhibit
o The vacuum tube to be used for the culture proteus
shall be given a drop of 1% gelatin (also 3. Martin– Lewis Agar  Isovelatex-
prepared in the lab, and is commercially Supplement
prepared for bacterial culture).  Colistin- Inhibits
 Any volume of clear fluid greater than 1 mL suspected gram negative
of containing either of N. gonorrhoeae or N. meningitidis  Anisomycin -
pathogens should be centrifuged at room temp for 15 Inhibits fungi
mins.  Vancomycin
 - Any specimen of culture which N. meningitidis is a (Increased
Concentration) -
consideration should be handled in biological safety
Inhibits gram
cabinet to avoid laboratory-acquired infections. -
positive
Pathogenic Neisseria spp. suspected direct inoculum of  Trimethoprim
specimen at the bedside followed by prompt incubation lactate- to inhibit
at 35 degrees Celsius in CO2 is optimal. Proteus spp.
 This can be done by several ways: (According to book 4. New York City Agar  Yeats dialysate
of Henry’s)  Horse Blood and
1. Placing medium into a candle jar. Plasma
2. Placing the medium in a sealed bag with citric acid  Vancomycin
bicarbonate  Colistin
3. Using a medium contained within a bottle having a  Trimethoprim
CO2 atmosphere. lactate
 Amphotericin B (
CULTURE MEDIUM AND INCUBATION Inhibits fungi )
 N. meningitidis, M. catarrhalis and saprophytic
Neisseria spp. grows well on 5% sheep blood agar and ***M. hominis and U.
chocolate agar. urealyticum may grow also
o All Neisseria spp. grows in CAP except for N. at NYCA***
gonorrhoeae 5. GC-LECT Same as NYC agar added
with Lincomycin.
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Lincomycin – inhibits gram

+/- bacteria, protein
synthesis. It is a bactericidal
antibiotic.

CHARACTERISTICS ON CAP
 Moraxella catarrhalis: Large non-pigmental of gray,
opaque smooth friable “Hockey Puck” (moving over)
consistency, colony may be moved intact over surface
of agar.
2. Nucleic Acid Amplified Assay
 For urine, Urethral swan, and cervical swab
and more sensitive than non-amplified assay.
 Examples are Roche Diagnostic System and
Belton Dickson and Company. They are just
like PCR where amplification of the specific
DNA of bacteria is done in order to identify it.

 N. meningitidis: Medium smooth found moist, gray to

white encapsulated strains are mucoid; may be
greenish cast agar underneath colonies

 It is more sensitive than biochemical tests in

 N. lactamica: Small non-pigmented or yellowish, lab.
smooth transparent  Disadvantage: You can still be tested
ppositive even after three days of recovery
from the bacterial infection because the DNA
of the lysed bacteria still exists in your system.
It is not recommended to monitor treatment.
3. Non-amplified DNA probe assay (from Bailey’s and
Scott Book 1)
 Direct detection of gonococcal ribosomal
RNA in genital and conjunctival specimens.
 N. gonorrhoeae: Small, grayish white convex,  We do not amplify genes of bacteria for
translucent shiny colonies with either smooth or detection
irregular margins may be up to five different colony  RNA gene of bacteria is detected through
types on primary plates. complementary DNA with label. This DNA will
attach to the bacteria with label such as
chemiluminescent, fluorescent dye, and when
it attaches, it will send a signal that RNA is
detected.

Neisseria spp. cannot grow in Mac Conkey Agar except from

some saphrophytic Neisseria spp. those who grow poorly: N.
flavacens, N. mucosa, N. sicca, N. subflava.
The reason why oxidase test is important because not all
bacterial growth in Mac Conkey is enteric bacilli. It is used for
confirmation.

DIRECT DETECTION METHOD

1. Gram-staining
 Member genus Neisseria and Moraxella
cattarhalis appear as Gram negative
diplococci with adjacent sides flattened. The
direct gram stain of body fluids for best
accomplished using cytocentrifuge.
 Presumptive test for N. gonorrhoeae just like
oxidase test.
o Commercial Molecular Assay (Henry’s Book)
 Chemiluminescent nucleic acid probe:
For detection of N. gonorrhoeae can be
used for culture or direct detection of the
organisms in endocervical or urethral swab
specimen.
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 Nucleic Acid amplification assay: For use
on endocervical and urethral swab
specimen with nucleic acid probe and
culture techniques, largely because the
problem with organism viability is not an
issue.
 Latex agglutination assay: Available for
direct detection of N. meningitidis antigens
is CSF, serum and urine. The sensitivity of
this method has been found to less than or
equal to that gram stain; therefore many
laboratories have stopped offering this test.
4. Rapid Enzymatic Test
 They are valuable for differentiating maltose
negative strains of N. meningitidis.
 N. meningitidis are glucose (+) and maltose
(+) in CO2 utilization test but there are strains
that are maltose (-).
IDENTIFICATION APPROACH
1. PRIMARY ISOLATION PLATE
2. OBSERVE THE GROWTH IN PLATE
3. GRAM STAIN
4. OXIDASE TEST AND CATALASE TEST
o Oxidase test is used to separate Acinetobacter
spp. because it resembles as diplococci
o Catalase test is used to separate Kingella spp.
Because it also appears like diplococci
5. TRIBUTYRIN TEST
CARBOHYDRATES UTILIZATION TEST
NUTRIENT BROTH GROWTH
NITRATE REDUCTION TEST
DNASE TEST
o Test used to separate Moraxella from
Nesseria spp. since both are oxidase and
catalase positive.
When the result of Tributyrin test is positive, it is presumptive
Moraxella catarrhalis and supplemented with the following test
results below:
 Non fermenter of carbohydrates
 With growth in nutrient broth
 Nitrate reduction test positive
 Dnase test positive
 Beta galactoside test negative
When the result of Tributyrin test is negative, it is Nesseria spp.
and supplemented with the following test results below:
 Carbohydrates Utilization Test (Definitive test only)
o Principle: The medium has pH indicator
called “phenol red”. Once the bacteria utilized
carbohydrates, it will produce acid. The
changes in pH level allows the medium to
change its color from red to yellow.
(POSITIVE COLOR: YELLOW)
(NEGATIVE COLOR: RED)
o Medium: Cystein Tryptic Soy Agar (CTA)
TYPES OF CTA
1. CTA with glucose
2. CTA with maltose
3. CTA with lactose
4. CTA with sucrose
5. CTA with fructose
 Nutrient Broth Incubated for room temperature
o Nutrient broth – became turbid, means there
is growth of N. mucosa, N. sicca, and N.
subflava
 Nitrate reduction test
o N. mucosa is the only positive in this test
Note: To separate N. sicca to N. subflava, use the
carbohydrates utilization test.
Result:
N. sicca – glucose, maltose, sucrose, and fructose positive
N. subflava – glucose and maltose positive
When the result are Tributyrin test negative and non-
carbohydrates fermenter, subculture the growth in selective
medium such as Thayer Martin Medium:
Non-carbohydrates fermenters: N. cinerea and N. flavescens
To distinguish these two, get the colonies and subculture
using Thayer Martin medium.
N. cinerea - do not grow in Thayer Martin Agar
N. flavescens – grows in Thayer Martin Agar
When the result is only glucose weak positive, bacteria
subculture in Thayer Martin
Weak positive in glucose: N. cinerea and N. gonorrheae
To distinguish these two:
N. cinerea – do not grow in Thayer Martin Agar
N. gonorrhea – grows in Thayer Martin Agar
Glucose and Maltose positive: N. meningitidis
Note: Observe also the nutrient broth because N. meningitidis
cannot grow in nutrient broth. If there is growth it can be N.
subflava
Glucose, Maltose, Lactose positive: N. lactamica
Note: Supplementary test is beta-galactosidase test positive
ANTIMICROBIAL THERAPY AND PREVENTION
ANTIMICROBIAL THERAPY
 Moraxella catarrhalis – beta lactams
 Penicillin resistant Neisseria gonorrheae –
ceftriaxone and quinolones
 Neisseria gonorrheae AUH strain – vancomycin
PREVENTION
 N. meningitidis A, B, C, Y, W135 – single dose
vaccine
 Chemoprophylaxis for close contact to
meningoceccemia – rifampin, ciprofloxacin, and
ceftriaxone
● Vertical transmission prevention – 2.5% povidone
iodine, 1% tetracycline eye ointment, 0.5% erythromycin
eye ointment, 1% silver nitite eye drop (within 1 hour of
delivery)
MORAXELLA SPP.
 Non fermenter of carbohydrates
 Gram negative diplococci / can be gram-negative rod
or bacilli
 DNase positive
 Aerobic
 Catalase positive
 Oxidase positive
 Non-motile
 M. catarrhalis may be carried in the oropharynx of
healthy children and adults
Additional Information
 General Characteristics
o Coccobacilli or short to medium-sized, gram-
negative rods.
o Moraxella catarrhalis: is the species most
commonly associated with human infections,
primarily of the respiratory tract. However,
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because the cellular morphology of this species is 3. UREASE TEST AND OBSERVE PITTING IN THE
more similar to that other Moraxella spp. details of MEDIUM
this organisms are discussed in Neisseriaceae. o To separate M. atlantae to M. osloensis
o M. osloensis is rare to pit therefore, M. osloensis
LABORATORY DIAGNOSIS: No special considerations for is urease test negative while M. atlantae is
collection and transport of the organisms urease test positive since it has capability of
pitting.
SPECIES
1. Moraxella nonliquifaciens, Moraxella lacunata, M.
osloensis, M. lincolnii. M. canis and M. atlantae -Normal 1. WITHOUT GROWTH IN MAC CONKEY BUT WITH
flora that inhabits mucous membranes covering the nose, throat GROWTH IN BAP/CAP
other parts of the upper respiratory tract, conjunctiva (M. o Gram stain (if gram-negative diplococci and
osloensis, may colonize urogenital tract and skin) oxidase negative) proceed to
 Infections are to those organisms are rare, when they o Tributyrin test (to confirm if it is Moraxella)
are probably caused by the patient’s endogenous o Oxidase test and Catalase test (to confirm that the
strains. spp. is not acinetobacter and kingella)
 Virulence factor of those organisms: Unknown o If the result is gram-negative diplococci, oxidase
 M. lacunata has been associated to eye infection and catalase test positive, and tributyrin positive,
this is MORAXELLA.
2. M. elongata – rare infection of endogenous strain.
 They are low virulence and virulence factor is still 2. NITRATE TEST AND GROWTH IN DIGEST
unknown LOEFFFLERS SERUM SLANT

3. M. weaverii – infection associated with dog bite. Moraxella spp. Nitrate Digest
 Virulence factor is still unknown Reduction Loefflers
Tests Slant
DIRECT DETECTION METHOD M. lacunata (+) (+)
GRAM STAINING M. lincolnii (-) (-)
M. atlantae, M. Coccobacilli or short rods M. nonliquifaciens (+) (-)
nonliquifaciens, M. that can be gram variable
osloensis CULTIVATION
M. canis Short chains or in pairs 1. 5% sheep blood and chocolate agar should be incubated
M. lacunata Coccobacilli or medium- at 35 degrees Celsius in carbon dioxide or ambient air
sized rods for a minimum of 48 hrs.
M. lincolnii Coccobacilli in chains
Note: Moraxella has better growth inside the candle jar
MORAXELLA CULTIVATION
 Moraxella spp. grow well on 5% Sheep Blood agar and 2. For those species that may grow on Mac Conkey agar,
Chocolate Agar the medium should be incubated at 35 degrees Celsius
 Most strains are slow grower in Mac Conkey that in ambient air.
resembles slow grower non-lactose enteric bacilli
PREVENTION AND CONTROL
MORAXELLA SPP. THAT GROWS IN MAC CONKEY BAP  Because these organisms do not generally do a threat
AND CAP to human health, there are no recommended
1. M. atlantae – pitting on blood agar vaccination or prophylaxis
2. M. osloensis – smooth translucent opaque in blood
agar OTHER MEMBERS OF FAMILY NEISSERIACEAE
3. M. canis – resembles as Enterobacteriaceae
Acinetobacter spp.
MORAXELLA SPP. THAT DOES NOT GROW IN MAC  Gram negative short bacilli
CONKEY BUT GROW IN BAP  Oxidase negative
1. M. lacunata – small colonies that pit the agar  Catalase positive
2. M. lincolnii – smooth translucent opaque  Non- motile
3. M. nonliquefaciens – smooth translucent opaque in  Growth on Mac Conkey agar
BAP
Kingella spp.
IDENTIFICATION APPROACH  Gram negative bacilli
 Oxidase (+)
o Gram stain (if gram-negative diplococci and  Capnophilic
oxidase negative) proceed to
 Catalase (-)
o Tributyrin test (to confirm if it is Moraxella)
o Oxidase test and Catalase test (to confirm that the
spp. is not acinetobacter and kingella)
o If the result is gram-negative diplococci, oxidase
and catalase test positive, and tributyrin positive,
this is MORAXELLA.

2. DNASE TEST
o Dnase positive – M. canis
o Dnase negative – either M. atlantae and M.
osloensis