Fe A. Bartolome, MD, DPASMAP
Department of Microbiology & Parasitology
Our Lady of Fatima University
HAEMOPHILUS
• Family Pasteurellaceae
• Small, gram negative, pleomorphic
• Require enriched media containing blood or its
derivatives
• Facultative anaerobes
• Obligate parasites
Haemophilus influenzae
• Found on mucus membrane of URT in humans
(noncapsular form)  encapsulated species uncommon
members of normal flora
• Short, coccoid bacilli in pairs or chains
Haemophilus influenzae
Classification:
1. Serotype – based on capsular antigen
2. Biotype – based on biochemical properties
a. indole production
b. urease activity
c. ornithine decarboxylase activity
3. Biogroup – useful for clinical purposes
Haemophilus influenzae
Culture:
• Chocolate agar – flat, grayish brown colonies after
24 hrs incubation
• Does not grow on sheep blood agar except around
colonies of Staphylococci  “satellite phenomenon”
Haemophilus influenzae
Growth Characteristics:
• Requires X factor (hemin) and V factor (NAD)
• Ferments carbohydrates poorly and irregularly
Haemophilus influenzae
Characteristics & Growth Requirements:
Species
X factor
V factor
Hemolysis
H. influenzae
H. parainfluenzae
H. ducreyi
H. haemolyticus
H. parahaemolyticus
H. aphrophilus
+
+
+
-
+
+
+
+
-
+
+
-
Haemophilus influenzae
Virulence Factors:
Capsule
• Antiphagocytic; impair ciliary function
• Main virulence factor
• With capsular polysaccharides (a to f)
 Type b – polyribose-ribitol phosphate (PRP)
Haemophilus influenzae
Virulence Factors:
Somatic antigen
• Outer membrane proteins  lipooligosaccharides (endotoxin)
IgA1 proteases
Haemophilus influenzae
Clinical Features:
H. influenzae type b
• Most common serotype causing systemic disease
1. Meningitis
2. Pneumonia & empyemia
3. Epiglottitis
4. Cellulitis
5. Septic arthritis
Haemophilus influenzae
Clinical Features:
Non-typeable (non-encapsulated) H. influenzae
• opportunistic
1. Chronic bronchitis
2. Otitis media
3. Sinusitis
4. Conjunctivitis
Haemophilus influenzae
Clinical Features:
Meningitis
• 20 to bacteremic spread from nasopharynx
• Peak incidence: 3 – 18 mos. Old
Epiglottitis
• Cellulitis & swelling of supraglottic tissues
• Pharyngitis, fever & dyspnea  complete airway
obstruction  death
Haemophilus influenzae
Clinical Features:
Cellulitis
• Reddish blue patches on cheeks or periorbital
areas
Haemophilus influenzae
Clinical Features:
Arthritis
• Infection of a single large joint
• Children < 2 y/o or immunocompromised patients
or those with previously damaged joints
Conjunctivitis
• Epidemic and endemic
• H. influenzae biogroup aegypticus
Haemophilus influenzae
Clinical Features: Sepsis with gangrene
Haemophilus influenzae
Prevention:
1. Chemoprophylaxis with Rifampin for non-immune
children < 4 y/o who are close contacts
2. Hib conjugate vaccine
• > 2 mos. Old  Hib conjugated with C.
diphtheriae toxin protein or N. meningitidis outer
membrane complex
• > 15 mos. Old  Hib conjugated with diphtheria
toxoid
Haemophilus aegypticus
• H. influenzae biotype III
• Koch-Weeks bacillus
• Resembles H. influenzae closely
• Diseases:
1. Conjunctivitis – highly communicable
2. Brazilian purpuric fever – fever, purpura, shock
and death
Haemophilus ducreyi
• Causes chancroid (soft chancre)
• Ragged ulcer on genitalia with marked swelling
and tenderness
• Lymph nodes enlarged and painful
• Organism grows best on chocolate agar incubated
in 10% CO2
• No permanent immunity
Haemophilus ducreyi
Bordetella pertussis
• Small, coccobacillary, encapsulated, gram (-)
• With bipolar metachromatic granules (toluidine blue
stain)
• Non-motile; strict aerobe
• Forms acid from glucose and lactose
• Requires enriched media
 Bordet-Gengou medium (potato-blood-glycerol agar)
 Contains Pen G 0.5 ug/mL
• Virulence genes – bvgA and bvgS
Bordetella pertussis
Gram stain
Culture on chocolate agar
Bordetella pertussis
Virulence Factors:
1. Filamentous hemagglutinin
• Protein on pili; adhesion to ciliated epithelial cells
2. Pertussis toxin
a. promote lymphocytosis via inhibition of signal
transduction by chemokine receptors 
lymphocytes do not enter lymphoid tissues
b. promote sensitization to histamine
c. enhance insulin secretion
d. stimulate adenylate cyclase via ADP-ribosylation
Bordetella pertussis
Virulence Factors:
3. Adenylyl cyclase toxin – inhibit phagocytosis
4. Tracheal cytotoxin
• Fragment of bacterial peptidoglycan
• Induce nitric oxide  destroy ciliated epithelium
5. Dermonecrotic toxin
6. Hemolysin
Bordetella pertussis
Pathogenesis:
• Adheres to and multiplies rapidly on epithelial surface
of trachea and bronchi  interfere with ciliary action
• No invasion of blood
Bordetella pertussis
Clinical:
• MOT: airborne droplets
• Source of infection: patients in early catarrhal
stage
• Disease: Pertussis or Whooping Cough  acute
tracheobronchitis
• Incubation period: approx. 2 weeks
Bordetella pertussis
Clinical: Stages of Disease
1. Catarrhal
• Mild coughing and sneezing
• Highly infectious but not very ill
2. Paroxysmal (1-4 weeks)
• Series of hacking coughs, accompanied by copious
amts. of mucus, ending with inspiratory “whoop” 
exhaustion, vomiting, cyanosis and convulsions
• High wbc count (16,000-30,000/uL) with absolute
lymphocytosis
3. Convalescence - slow
Bordetella pertussis
Laboratory Diagnosis:
Specimen: saline nasal wash (preferred) or nasopharyngeal
swab
1. Direct fluorescence antibody test – 50% sensitivity
2. Culture of saline nasal wash fluid
3. PCR – most sensitive
4. Serology – (+) only on third week of illness  of little
diagnostic value
Bordetella pertussis
• First defense is antibody that prevents attachment
• Recovery from disease or immunization is followed
by immunity
• Second infection may occur but is mild
• Re-infection occurring years later in adults may be
severe
• Vaccine-induced immunity not completely protective
Bordetella pertussis
Prevention:
1. Chemoprophylaxis – Erythromycin  for exposed,
unimmunized individuals OR exposed, immunized
children < 4 years old
2. Vaccine – two vaccines available:
a. acellular vaccine – contains 5 purified antigens 
main immunogen is inactivated pertussis toxin;
first vaccine to contain a genetically inactivated
toxoid  ADP-ribosylating activity removed
b. DPT x 3 doses
BRUCELLA
• Zoonotic  obligate parasite of animals & humans
• Intracellular organism
• Gram negative coccobacilli
• Aerobic; non-motile; nonspore-forming
• Catalase (+); oxidase (+)
• Produces H2S
• Culture: trypticase soy agar OR blood culture media;
B. abortus requires 5-10% CO2 for growth
BRUCELLA
• Route of infection in humans:
1. Intestinal tract – ingestion of infected milk &
contaminated dairy products (cheese from
unpasteurized goat’s milk)
2. Mucous membranes – droplets
3. Skin – contact with infected tissues of animals
• Pathogenesis: endotoxin – O antigen polysaccharide
BRUCELLA
Species
Animal
Pathology
B. melitensis
Goats
Acute & severe infection
B. suis
Swine
Chronic w/ suppurative lesions;
caseating granulomas
B. abortus
Cattle
Mild disease w/o suppuration;
non- caseating granulomas of
the RES (LN, liver, spleen, BM)
B. canis
Dogs
Mild disease
BRUCELLA
Clinical: Brucellosis (Undulant or Malta Fever)
1. Acute
• Malaise, fever, weakness, aches & sweats
• Fever rises in the afternoon  fall during the
night with drenching sweats
• (+) lymphadenopathy w/ palpable spleen; +
hepatitis with jaundice
2. Chronic
• With psychoneurotic symptoms
• Weakness, aches & pains, low grade fever
BRUCELLA
Diagnosis:
1. Culture
• BM & blood – commonly used specimen
• Brucella agar, trypticase soy medium, brain
heart infusion medium, chocolate agar
2. Serology – inc. IgM during 1st week of illness;
peak at 3 months
Francisella tularensis
• Small, gram (-) pleomorphic rod
• Widely found in animal reservoirs (rabbits, deer,
rodents)
• Humans are accidental “dead-end” hosts
• Two biotypes:
1. Jellison type A – more virulent ; US
2. Jellison type B – less virulent ; Europe
• Culture: glucose cysteine blood agar OR glucose
blood agar
Francisella tularensis
Gram stain
F. tularensis
colonies on agar
plate
Francisella tularensis
Mode of transmission:
1. Contact with animal tissue
2. Bite of vector (Dermacentor tick)
3. Ingestion of infected meat
4. Inhalation
Symptoms caused by endotoxin
Francisella tularensis
Clinical:
1. Ulceroglandular – 75%; ulceration at site of
entry with swollen & painful LN
2. Glandular
3. Oculoglandular
4. Typhoidal
5. GI & pulmonary
Disease confers lifelong immunity
Francisella tularensis
Cutaneous tularemia
Francisella tularensis
Diagnosis: culture not done due to high risk to lab
workers
1. Agglutination test – most frequently used
2. Fluorescent antibody staining of infected tissue
Treatment: Streptomycin (DOC)
Prevention: live, attenuated vaccine – partial immunity;
not available commercially
YERSINIA
• Short, pleiomorphic gram (-) rods with bipolar
staining
• Catalase and oxidase (+)
• Microaerophilic or facultative anaerobe
• All with LPS that have endotoxic activity
Yersinia pestis
• Non-motile, facultative anaerobe
• Growth more rapid in media containing blood or
tissue fluids at 300C  gray and viscous colonies
Yersinia pestis
Virulence Factors:
1. LPS – endotoxin
2. Envelope – with protein (fraction I) 
antiphagocytic
3. Coagulase
4. V-W antigens (virulent, wild type) – essential for
virulence
5. Pesticin - bacteriocin
Yersinia pestis
Pathogenesis:
1. Bite of vector (Xenopsylla cheopis)  organism
phagocytosed by PMNs & monocytes  multiply in
monocytes  lymphatics  (+) intense
hemorrhagic inflammation in enlarged LN 
bloodstream  hemorrhagic & necrotic lesions in
all organs
2. Inhalation of infective droplets from coughing
patients  primary pneumonic plague with
hemorrhagic consolidation, sepsis and death
Yersinia pestis
Clinical: Plague
• I.P.: 2 – 7 days
• High fever & painful lymphadenopathy (buboes)
• Vomiting & diarrhea may develop with early sepsis
• Later  DIC  hypotension, altered mental status,
renal and cardiac failure
• Terminal: signs of pneumonia & meningitis
Yersinia pestis
Bubo on neck
Septicemic plague
Yersinia pestis
Diagnosis:
1. Smear – Giemsa stain or Wayson’s stain (+ bipolar
appearance)
2. Culture – blood agar or MacConkey’s agar plates;
infusion broth; all cultures highly infectious
3. Serology - examination of acute and convalescent
sera for antibody levels
Treatment: Streptomycin (DOC)
Pasteurella multocida
• Primarily animal pathogens
• Non-motile gram (-) coccobacilli with bipolar
appearance on stained smears
• Occurs worldwide in respiratory tract and GIT of
many domestic and wild animals
• Most common organism in human wounds inflicted
by bites from cats and dogs
• Virulence factors include capsule and endotoxin
Pasteurella multocida
Clinical:
• Rapidly spreading cellulitis at site of animal bite
• Incubation period < 24 hours
• May present as bacteremia or chronic
respiratory infection
• Complication: osteomyelitis (cat bites)
Treatment: Penicillin G (DOC)
Pasteurella multocida
Culture
P. multocida infection
Bacteremic P. multocida cellulitis