BACT211: CLINICAL BACTERIOLOGY
TOPIC: NEISSERIACEAE
2ND SEMESTER | S.Y 2023-2024
LECTURER: Ma’am Christy Gonzales
TOPIC
SUBTOPIC
SUB SUBTOPIC
• Gram + cocci: Staphylococcus spp., Micrococcus spp.
[Catalase Positive]; Streptococcus spp., Enterococcus spp.
[Catalase Negative]
- First Test: Catalase
• Gram + diplococci: Streptococcus pneumoniae
- No C carbohydrate; Merong C substance; autolytic
enzyme [like S. aureus]
• Gram – diplococci: Neisseria spp., Moraxella spp.
- First Test: Oxidase Test (+)
• Gram – bacilli: Vibrio spp., Aeromonas spp., Pleisiomonas
spp., Campylobacter spp., Helicobacter spp.
- First Test: Oxidase Test (+)
NEISSERIA
GENERAL CHARACTERISTICS
• Family Neisseriaceae
- Other members of this family are oxidase positive,
but catalase negative [like Edwarsiella spp.]
• Aerobic, nonmotile, non-spore-forming, gram-negative
diplococci except: Neisseria elongata, Neisseria weaeri,
and Neisseria bacciliformis
- These organisms are gram-negative bacilli
• Kidney-bean shaped
• All are cytochrome oxidase and catalase positive except
for N. elongata and N. bacilliformis [catalase negative]
• Capnophilic; can grow anaerobically if alternative
electron acceptors (e.g., nitrites) are available
• Normal inhabitants of the human respiratory tract
except Neisseria gonorrhoeae and Neisseria animaloris
[leading to infection; pathogenic]
• Pathogenic for humans and are found associated inside
polymorphonuclear cells
- PMNs: Neisseria gonorrhoeae and Neisseria
meningitidis [cause infection, but part of the
normal flora] = require iron for growth [fastidious]
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o Receptor for transferrin [transport protein
for iron]
o Compete with RBCs for the presence of iron
in our body
• Transmitted from person to person:
- N. gonorrhoeae: sexually transmitted
- N. meningitidis: respiratory droplets
VIRULENCE FACTORS
• Receptors for human transferrin
• Capsulated – N. meningitidis
• Pili (fimbriae)
• 5 Colonial Types:
- T1 and T2: virulent forms [have pili and fimbriae]
- T3 through T5: avirulent [no pili, no fimbriae]
• Cell membrane proteins:
- Protein 1 (Por): forms channels for nutrients to pass
into and waste products to exit the cell
o Por A [positive for N. meningitidis]
o Por B [positive for N. meningitidis, positive
for N. gonorrhoeae]
- Protein II (Opa): facilitate the adherence to
phagocytic and epithelial cells
- Protein III (reduction modified protein [Rmp]):
blocks the bactericidal effect of host IgG
• Lipooligosaccharide (LOS) or endotoxin
- The N. gonorrhoeae produces LOS that is similar to
human cell membrane glycosphingolipids
• Immunoglobulin A (IgA) protease that cleaves IgA on
mucosal surfaces [sinisira ‘yung antibodies natin]
- IgA [an antibody that can be seen in secretions]
- Have the same IgA as the Neisseria gonorrhoeae
o S. pneumoniae
o H. influenzae
• Other Proteins
- Lip (H8): a surface exposed protein that is heat
modifiable like Opa
- Fbp (ferric-binding protein): expressed when the
available iron supply is limited
Transcribed by: JAMARA
 NEISSERIA GONORRHOEAE
• Oxidize only glucose [in CHO utilization test]
• Require arginine, hypoxanthine, and uracil
• Anorectal and oropharyngeal infections
• Agent of the sexually transmitted disease gonorrhea
• Newborns: ophthalmia neonatorum – gonococcal eye
• Not part of normal microbiota
• Only found on mucous membranes of genitalia,
anorectal area, oropharynx, or conjunctiva at time of
infection
• In terms of carbohydrate utilization test, N.
gonorrhoeae can only oxidize glucose.
- Aerobic and capnophilic
• Require Arginine, Hypoxanthine, and uracil [AHU strain:
N. gonorrhoeae in asymptomatic male; urethra] [not all
N. gonorrhoeae ay nagrrequire or merong AHU] [direct
contact]
GONORRHEA
• Acute pyogenic infection of non-ciliated columnar and
transitional epithelium
• Acquired through sexual contact
• Occur primarily in the urethra, endocervix, anal canal,
pharynx, and conjunctiva
• “flow of seed”
• “The clap”: French word clapoir meaning “brothel”
• Incubation Period: 2-7 days
CLINICAL MANIFESTATION
• Men: acute urethritis and dysuria. Asymptomatic is
uncommon (3-5% asymptomatic)
- Ascending infection – prostatitis (infection of
prostate glands) & epididymitis (infection of
epididymis – upper area of genital tract of men)
• Women: dysuria, cervical discharge, and lower
abdominal pain, 50%-Asymptomatic
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• Pelvic inflammatory disease – women
- Sterility, ectopic pregnancy, or perihepatitis (Fitz-
Hugh-Curtis syndrome)
infection
- When a mother is infected with N. gonorrhoeae, the
baby that will be birthed through the vaginal canal,
the baby could acquire Ophthalmia neonatorum
which is a gonococcal eye infection
LABORATORY DIAGNOSIS
SPECIMEN COLLECTION AND TRANSPORT
• Pus and secretions are taken from the urethra, cervix,
rectum, conjunctiva, throat, or synovial fluid for
culture and smear
• Specimen of Choice:
- Men: urethra
- Women: endocervix
• Swabs: Dacron or rayon swabs – preferred
- Cotton swabs are toxic for neisseria [contains
excessive fatty acids]
- Men urethra: 2 cm
- Women anal canal: 4-5 cm
- We do not use disinfectant to avoid non viability
• Calcium alginate and cotton swabs – inhibitory
• Direct Plating: James E. Martin Biological
Environmental Chamber (JEMBEC) plates, Gono-Pak
and Transgrow – BRAND NAMES
- Medium of Choice: Chocolate Agar Plate
- inoculate it with transport systems
• Amies Medium with charcoal [in case of unavailability
of specific transport systems]
• Within 6 hours, it should be plated already
DIRECT MICROSCOPIC EXAMINATION
• Urogenital specimens: gram-negative intracellular
diplococci
- Some of them are intracellular (nasa loob ng
cytoplasm ng cell)
• In pairs with adjacent sides flattened, kidney shaped
• Gram stain: not recommended for pharyngeal
specimens
- Spread the specimen
- We do not perform direct microscopic exam on
respiratory specimens (Due to many normal flora)
• More than five polymorphonuclear neutrophils per
field but no bacteria: nongonococcal urethritis with
other organisms, such as C. trachomatis or
Ureaplasma urealyticum
Transcribed by: JAMARA
 CULTURE
• Medium of Choice: CHOC Agar
• Trypticase soy agar with 5% sheep blood
• Not growing on sheep blood agar (SBA) = blood agar
plate (BAP)
INCUBATION
• 35°C in a 3% to 5% CO2 atmosphere
• Use of a CO2 generating pouch, or a candle extinction
jar (increase CO2) [Gaspak jar = ipapasok pa rin sa
loob ng incubator]
• Cultures are examined daily for growth and held for
72 hours
- Every 24 hours, you need to check it [daily]
- Slow growth
DEFINITIVE IDENTIFICATION
A. OXIDASE TEST
- Reagent: (1% dimethyl-pphenylenediamine
dihydrochloride or tetramethyl-p-
phenylenediamine dihydrochloride)
- Positive: Purple color within 10 seconds
• Modified oxidase: Micrococcus spp. (+) = blue color
• Acid: Yellow; No acid: Red
• Only glucose is positive
B. CARBOHYDRATE UTILIZATION
- Cystine trypticase agar (CTA)
- Glucose only (+)
- 1% individual carbohydrate
- pH indicator: Phenol red
- Result: Acid-Yellow color produced in 24- 72
hours

C. CHROMOGENIC SUBSTRATES
- Gonocheck II: detect enzymes that hydrolyze
colorless substates and produce colored end
products
- Only strains that are isolated on selective media
should be tested

D. MULTITEST METHODS
- Multitest conventional-chromogenic enzyme
methods
- Combine enzyme substrate tests with other
biochemical tests
- Allow for identification of strains isolated on
selective for nonselective media
- Immunologic assays

E. IMMUNOLOGIC ASSAYS
- Co-agglutination and fluorescent antibody testing
- Monoclonal antibodies against gonococcal
COLONIAL APPEARANCE protein I
- Do not require pure or viable organisms
• Small grayish white, convex, translucent, shiny
- N. gonorrhoeae attached to killed Staphylococcus
colonies with either smooth or irregular margins
aureus cells
• Staph – opaque
- No need for pure culture
• Strep – transparent
- Positive: Agglutination
• Neisseria – translucent
MICROSCOPIC MORPHOLOGY F. MATRIX-ASSIETED LASER
• Gram-negative diplococci - Desorption/Ionization-Time-of-flight Mass
- Wala nang ilalagay na arrangement Spectrometry (MALDI-TOF MS) [Most advance
equipment for the identification of bacteria]
- Identifies infectious pathogens by defining
unique protein signatures of the organism
- Popular
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 - Principle: The colony is ionized to vaporization of
proteins, the proteins separate based on size and
charge resulting to unique spectral signature and
compared to the identified pathogen.
G. NUCLEIC ACID AMPLIFICATION TESTS (NAAT)
- Amplify a specific nucleic acid sequence before
detecting the target sequence with a probe
- Increased sensitivity
- Specificity
- Ability to test with a noninvasive urine specimen
ANTIMICROBIAL RESISTANCE
• Susceptible to penicillin
• Plasmid-mediated penicillin resistance: penicillinase-
producing Neisseria gonorrhoeae (PPNG)
• Chromosome-mediated penicillin resistance (PenR) –
Beta – lactamase negative
TREATMENT
• Ceftriaxone (Cephalosporin)
• Ceftriaxone plus azithromycin – co-infection with C.
trachomatis
NEISSERIA MENINGITIDIS
• Commensal as well as an invasive pathogen
• Important etiologic agent of endemic and epidemic
meningitis and meningococcemia and rarely
pneumonia, purulent arthritis, or endophthalmitis
• MOT: close contact with respiratory droplet secretions
• Serogroups A,B,C,Y and W-135: account for most cases
of disease in the world
• Capsule: sialic acid moieties
- S. pyogenes: hyaluronic acid
- S. agalactiae: sialic acid
- S. pneumoniae: polysaccharide
• Incubation period: 1-10 days
- After incubation, there are none or mild
manifestation
- But in some cases, there could be manifestation of
meningitis or sepsis
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CLINICAL MANIFESTATION
FULMINANT MENINGOCOCCEMIA
• Purpura with petechial skin
• Tachycardia
• Hypotension
• Thrombosis (concurrent blood clots)
• Intravascular coagulation
• Septic shock
• Hemorrhage in the adrenal glands (Waterhouse-
Friedrichsen syndrome)
MENINGITIS
• Characterized by an abrupt onset of front headache,
stiff neck (nuchal rigidity), confusion, and
photophobia
LABORATORY DIAGNOSIS
SPECIMEN COLLECTION AND TRANSPORT
• Cerebrospinal fluid (CSF), blood, nasopharyngeal
swabs and aspirates, joint fluids, and, less commonly,
sputum and material from urogenital sites
• Inhibited by SPS [anti-coagulant; 0.035% sodium
polyanethole sulfonate], when commercial blood
culture systems are used, you need to add gelatin to
neutralize the effect
DIRECT MICROSCOPIC EXAMINATION
• Intracellular and extracellular gram-negative
diplococci
CULTURE AND INCUBATION
• SBA and CHOC agar
• Same atmospheric conditions described for N.
gonorrhoeae
• Examined daily for 72 hours
COLONIAL APPEARANCE
• Medium, smooth, round, moist, gray to white;
encapsulated [mucoid colony] strains are mucoid;
may be greenish cast in agar underneath colonies
IDENTIFICATION
• Oxidase Positive (+) & Coagulase (+)
• Carbohydrate methods – uses glucose and maltose
• Molecular techniques
• MALDI-TOF
TREATMENT
• DOC: Penicillin
• Do not develop resistance with penicillin
• For Meningococcemia: third generation
cephalosporins
• Chemoprophylaxis: rifampin or ciprofloxacin
Transcribed by: JAMARA
 • For ciprofloxacin resistance: azithromycin
COMMENSAL NEISSERIA SPECIES
Neisseria cinerea
• Glucose negative in CTA sugars
• Grows on SBA
• Positive hydroxyprolyl aminopeptidase reaction
• Reduction of nitrate and negayive DNase reaction – to
differentiate from M. catarrhalis
• Lack of yellow pigment production – to differentiate
from Neisseria flavescens (meaning yellow)
Neisseria lactamica
• Found in the nasopharynx of infants and children
• Only Neisseria species that uses lactose (glucose &
maltose)
• Can exhibit delayed lactose utilization and be confused
with N. meningitidis
• Lactose utilization or positive ONPG
- The only Neisseria that has the capable in using
lactose = ONPG [ortho nitrophenyl-B-D-
galactopyranoside
Neisseria mucosa
• Large, often adherent to the agar, and very mucoid
• Documented to cause pneumonia in children
• Same carbohydrate patter as N. sicca and N. subflava
biovar perflava
• Ability to reduce nitrite to nitrogen gas
• Lack of pigment production
Neisseria subflava
• Less yellow lesser than N. flavescens
• Part of the upper respiratory microbiota
• Cause serious infectious, such as a bacteremia,
meningitis, and septicemia
• Resemble N. meningitidis infection, including septic
shock, petechial hemorrhage and purpura
• Reduced sensitivity to penicillin, cefixime, and
ciprofloxacin.
Neisseria elongata
• Gram-negative bacilli
• Contains three subspecies – elongata, glycolytica, and
nitroreducens
• Commensals in the upper respiratory tract
• Opportunistic pathogens
• Neisseria elongata subsp. elongata
Neisseria weaveri
• Bacilli
• Normal oral microbiota in dogs
• Found in humans in infections following dog bites
• Catalase positive
• Does not produce acid from any of the carbohydrates
• Does not reduce nitrate but does reduce nitrite to gas
• Weakly phenylalanine deaminase positive
• Rare case of septicemia in immunocompromised
patients have been reported
• Sensitive to penicillin

Neisseria sicca
• Dry, wrinkled, adherent, and breadcrumb-like colony
• Sicca means “dry”
• N. sicca and N. subflava biovar perglava
- Two most common Neisseria spp. found in the
respiratory tract of adults.
• Cause endocarditis
• A variant of N. mucosa

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 MORAXELLA CATARRHALIS
• Family Moraxellaceae
• Three genera – Moraxella, Acinetobacter, and
Psychrobacter
• Moraxella catarhhalis
- Isolated only from humans
- A commensal of the upper respiratory tract (similar
with Neisseria)
- Third most common cause of acute otitis media
and sinusitis in children
- Oxidase and catalase positive (similar with
Neisseria)
- Asaccharolytic [no carbohydrate is utilized]
o Differentiate neisseria from Moraxella:
DNase Test or Butyrate esterase reaction
- Positive DNase and butyrate esterase reaction (to
differentiate Moraxella & Neisseria) – Tributyrin is
used as the substrate to detect butyrate esterase
activity [negative moraxella]
- Antimicrobial agents: amoxicillin-clavulanic acid,
extended-spectrum cephalosporins, azithromycin,
quinolones, and trimethoprim-sulfamethoxazole • Additional Test:
- Gamma-glutamyl-aminopeptidase test = + N.
LABORATORY DIAGNOSIS meningitidis
SPECIMEN COLLECTION AND TRANSPORT
• Middle ear effusion, nasopharynx, sinus aspirates,
sputum aspirates, or bronchial aspirates
CULTURE AND INCUBATION
• SBA and CHOC agar
• Can tolerate lower temperatures and grow well at 28
degrees Celsius
COLONIAL APPEARANCE
• Smooth, opaque, gray-to-white colonies
• “hockey puck” – remains intact when pushed across
the play with a loop
- When the colony is moved, it will not be
destroyed, glide across the plate
• Older colonies: “wagon-wheel” appearance

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