Lecture 9: Small Gram Negative Bacilli, Section C H.

aphrophilus - - -
ANTIGENIC STRUCTURE
I. HEMOPHILUS 1. Capsular polysaccharide – 6 antigenic types (a-f)
MORPHOLOGY 2. Lipopolysaccharide
 Pleomorhic, coccobacilli 3. Outer member protein
0.2 – 0.5 – 0.8 um MAJOR VIRULENCE FACTORS
 with faint refractile capsules 1. Polysaccharide capsule (PRP)
Demonstrable by quellung polyribitol phosphate – induce natural immunity after
 Gram negative but may appear gram infection or vaccination
variable 2. Lipopolysaccharide lipid A – induces meningeal irritation
 May exhibit bipolar staining 3. IgA, specific proteases – facilitate colonization of mucosal
surfaces
Haemophilus spp. 4. Outer-membrane proteins and pili – poorly defined roles

EPIDEMIOLOGY OF HAEMOPHILUS INFECTION

PHYSIOLOGY
 CULTURE MEDIA
1. Chocolate agar (heated blood) to 80°C.
Factor X and V released
2. Levinthal enriched agar - differentiating
- capsulated from non-capsulated
3. Fildes agar
4. Blood agar used if X-inoculated with
Staphylococcus
 Incubation at 10% CO2 recommended
 Growth apparent after 18-24 hours
- colonies 0.5 X 1.5 mm, encapsulated
varieties produce glistening, mucoid
colonies that are iridescent on
Levinthal agar
- convert spontaneously to rough colonies
- tendency to autolyze
HAEMOPHILUS INFLUENZA ON CHOCOLATE AGAR
HAEMOPHILUS INFLUENZA
ON CHOCOLATE AGAR
(close-up view)
• Disease/ Bacterial Factors
- H. influenzae type b causes meningitis,
epiglottitis, cellulites, arthritis, conjunctivitis
- H. influenzae type b is primarily a pediatric
pathogen (children younger than 5 y/o), although
the incidence is rapidly decreasing with
immunization
- H. ducreyi is an important cause of genital ulcer
- Nonencapsulated H. infuenzae type b causes
otitis media, sinusitis, bronchitis
- The type b polysaccharide capsule is
ANTIPHAGOCYTIC
- Increased exposure to nonencapsulated
Hemophilus strains is not associated with
increased infections because these organisms are
ubiquitous and have a low virulence
• Transmission
- Endogenous spread from upper respiratory tract
through the blood to meninges, epiglottis, skin,
joints; or direct extension to eye, inner ear,
sinuses, or lower respiratory tract
- H. ducreyi: sexually transmitted
• Who are at risk?
1. children < 5 years old without protective
antibodies against type-b capsular
polysaccharide
2. patients with depleted complement of after
splenectomy
3. elderly at greatest risk for pulmonary disease,
particularly those with obstructive pulmonary
disease or conditions predisposing to aspiration

• Geography/ season
H. influenzae infections occur worldwide
H. ducreyi genital ulcers in Africa and Asia, less
IDENTIFICATION OF commonly in Europe and North America
HAEMOPHILUS INFLUENZA BY X No seasonal incidence
AND V STRIPS
LABORATORY DIAGNOSIS
1. Gram staining
2. Culture – blood culture should be performed on any
patient with meningitis or other invasive disease
3. Antigen detection
- CIE – counterimmunoelectrophoresis
- latex particle agglutination
- ELISA
 Recommended for patients with negative culture
Characteristics and growth requirements of some
Hemophilus species (X = heme; V = nicotinamide-adenine TREATMENT
dinucleotide) A. ANTIMICROBIALS
1. Meningitis – Chloramphenicol/+ Ampicillin
Requires
Species Hemolysis 2. Otitis media – Amoxycillin, TMP-SMX,
X V Erythromycin, Cefaclor
H. influenzae (H. aegyptius) + + - 3. Sinusitis – Ampicillin
H. parainfluenzae - + - B. PASSIVE IMMUNOTHERAPY
H. ducreyi + - -  adjunct to antibiotics – in treatment of severe
H. haemolyticus + + + infection
H. parahaemolyticus - + +
PREVENTION
1. Active immunization - + +
1985 – Purified capsular polysacc vaccine protective for 2 MacConkey
year old Agar
- + +
* Conjugated vaccines – with diphtheria and Neisseria
meningitides
2. Passive Immunization
Children with congenital or acquired deficiencies Virulence Factors Associated with Bordetella pertussis
3. Chemoprophylaxis – Rifampicin – Controversial Virulence Factors Biological Effects
ADP-ribosylation of
OTHER HEMOPHILUS SPECIES guanine nucleotide-
1. Haemophilus aegyptius (Koch-weeks) binding proteins,
- associated with purulent conjunctivities especially in Pertussis toxins lymphocytosis,
children hypoglycemia, mediates
2. H. para-influenza – part of the normal flora, causes of attachment of respiratory
opportunistic infections. Rare cause of meningitis epithelium
3. H. ducreyi – STD, worldwide in distribution, responsible Impairment of leukocyte
for 10% of venereal diseases in temperate countries, Adenylate cyclase toxin chemotaxis and killing,
more common among non-whites and in poor socio- local edema
economic and hygienic condition indurated, painful, Ciliastasis and then
single or multiple lesions, developing in the genitals and Tracheal cytotoxin extrusion of ciliated
perianal areas, accompanied by suppurative inguinal epithelial cells
buboes. Vascular smooth muscle
Dermonecrotic toxin contraction and ischemic
II. BORDETELLA necrosis
3 Species Disease Mediates attachment to
Filamentous hemaglutinin ciliated epithelial cells,
B. pertussis (man is only Pertussis or whooping agglutinates erythrocytes
natural host cough Lipopolysaccharide Exotoxin activity
B. parapertusis (man is Milder form of whooping
only natural host) cough CLINICAL PRESENTATION OF B. PERTUSSIS DISEASE
Bronchopulmonary Incubation Catarrhal Paroxysmal Convalescent
B. bronchiseptica disease in animals and 3-4 weeks (or
humans Duration 7-10 days 1-2 weeks 2-4 weeks
longer)
Diminished
PHYSIOLOGY AND STRUCTURE paroxysmal
Rhinorrhea, Repetitive cough,
 Size: 0.2 – 0.5 X um malaise, cough with development of
Strictly aerobic, gram negative coccobacilli non- Symptoms None fever, whoops, secondary
motile, highly communicable, multiply among cilia sneezing, vomiting, complications
of epithelial cells anorexia leokocytosis (pneumonia,
 Culture medium seizures,
encephalopathy)
 modified Bordet-Gengou medium (potato-
glycerol-blood agar)
 Regan- Lowe-medium-charcoal, oxoid medium Bacterial
with horse blood Culture
 Stainer – scholte agar – selective medium,
containing cyclodextrin and cephalexin
 Colonies are pinpoint in size, smooth convex, LABORATORY DIAGNOSIS
glistening, almost transparent and pearl like,  Specimen
produce zone of hemolysis that varies with cultural – optimum is nasopharyngeal aspirate (organism very
conditions. sensitive to drying, fatty acids on cotton swabs are
 Variation manifested with change from phase 1 – IV toxic)
variant - Direct inoculation on suitable medium
1. Serial passage on culture media recommended.
2. High level of magnesium in media
 MICROSCOPY
DFA – direct flourescent antibody
 CULTURE
(as long as 7 days) tiny colonies are observed only
after 3 days
B. bronchiseptica on
5% sheep blood agar III. Francisella tularensis
- causative agent of tularemia (glandular fever, rabbit fever,
tick fever, deerfly fever) both in rodents and humans

A. Physiology and Structure

- size: 0.2 x 0.2-0.7 um
Differential Characteristics of Bordetella Species - sometime bipolar on polychrome, faintly staining
Bordatella species - gram negative coccobacilli
B. B. B. - non-motile, non-piliated, with thin lipid capsule
Characteristics pertussis parapertussis bronchiseptica - pleomorphic obligate aerobe
Oxidase + - + - weakly catalase positive
Urease - + + - biochemical characterization of little value in
Motility - - + identification
Browning on: • Culture Characteristics
Mueller-Hinton - growth at 24°C- 39°C
Agar - + - - slow growing
Growth on: - requires cystine or cysteine
Sheep Agar - colonies noted after 2-4 days incubation on glucose-
 cysteine clood agar (CGB+) or peptone cysteine agar • Size: 0.5 x 0.6-1.5 um
(RCA) - non-motile, non-encapsulated
- greenish disc surrounds the colony but no true hemolysis - gram negative coccobacilli
occurs - grow slowly on culture
- aerobic
- some strains requiring CO2
- does not ferment lactose
B. Epidemiology of Tularemia
- catalase and oxidase (+)
• Disease/ Bacterial Factors - reduce nitrate
- Ulceroglandular, glandular, typhoidal, oculoglandular - with variable urease activity
and oropharyngeal tularemia Gram stain of Brucella bacill
- F. tularensis is a facultative intracellular pathogen and
can survive for prolonged periods in macrophages of Higher Magnification
the reticuloendothelial system
- Antiphagocytic capsule is present in pathogenic strains
- Most common reservoirs in the United States are
rabbits, ticks, and muskrats
• Transmission
- Bite from infected tick or contact with infected
animals
- Infection also acquired after ingestion of contaminated
meat or water, or inhalation of an infectious aerosol
• Who are at risk?
- hunters
- persons exposed to ticks
- laboratory personnel
• Geography/ Season Colonies of Brucella sp.
on 5% sheep blood agar
- Worldwide; in the United States most infections are in
Arkansas, Missouri, and Oklahoma
- Disease most frequent during summer and winter

C. Manifestations of Tularemia
Infections Colonies of Brucella
Type of disease Characteristics
(%) spp. on chocolate agar.
Ulcers at the site Growth of Brucella spp.
of exposure and on chocolate agar after 4
Ulceroglandular 75-85 adenopathy of the days of incubation.
draining lymph Colonies require
nodes prolonged incubation to
Adenopathy but no attain sufficient growth for
Glandular 5-10 further biochemical
ulcers
testing.
Systemic signs but
Typhoidal 5-15 no adenopathy or
Epidemiology of Brucellosis
ulcers
• Disease/ Bacterial factors
Oculoglandular 1-2 Eye involvement
- Brucella is a facultative intracellular pathogen and can
Orapharyngeal
Oropharyngeal <1 survive for prolonged periods in macrophages of
involvement
reticuloendothelial system
D. Laboratory Diagnosis
- Reservoirs are animals: cattle, goats, sheep, swine, dogs,
• Specimen collection – hazardous for both physician and foxes, and coyotes
laboratory worker
- small organism can penetrate thru the skin and mucous • Transmission
membrane
- ingestion of contaminated milk or cheese
• Microscopy – direct staining of clinical specimen with - contact with infected animals
flourescin labeled antibodies
• Cultures • Who are at risk?
- prolonged incubation specimen – sputum and aspirates - persons in direct contact with infected animals:
from lymph nodes or draining sinuses veterinarians, meat handlers, farmers
- blood culture are usually negative - persons eating unpasteurized milk or cheese
E. Modes of Control - laboratory personnel
1) Streptomycin (drug of choice), gentamicin
2) Avoid reservoirs and vectors infection(e.g., rabbits, ticks) • Geography/ Season
3) Live, attenuated vaccines reduce severity of disease
- worldwide; in the United States, most common in
4) Lab personnel: use of gloves and biohazard hood when
California and Texas; less disease in the United States
handling specimens
attributable to control of disease in animals
- no seasonal incidence
IV. Brucella
- Bang’s disease
- Malta fever Characteristic of Human Brucellosis
- Undulant fever Growth on dyes
- Mediterranean remittent fever Animal CO2 Basic
- Rock fever of Gibraltar Thioni Clinical
Species Reservoi require fuchsi
n disease
- Fever of Crete r d n
- Country fever of Constantinople B.
Severe, acute
- 6 species known, 4 are associated with human disease – Goats, disease with
melitensi – + +
abortus, melitensis, suis, and canis sheep complications
s
common
B. Cattle + + – Mild disease
A. Physiology and Structure
abortus with
 suppurative - Ferment glycerol – (orientalis, medievalis and antigua)
complications having differences in geographic distribution
uncommon
Growth on dyes B. Epidemiology of Yersinia infection
Animal CO2 Basic
Thioni Clinical • Disease/ Bacterial factors
Species Reservoi require fuchsi
n disease Y. pestis – plague
r d n
Suppurative, Y. enterocolitica – enterocolitis, transfusion-related
destructive septicemia
B. suis Swine – + + disease with Y. psuedotuberculosis – enterocolitis
chronic Y. pestis – present in animal reservoir, fleas
manifestations Other Yesinia – present in domestic animals (GI tract) and
Mild disease contaminated food products
with
Numerous virulence factors
B. conis Dogs – – + suppurative
complications
uncommon • Transmission
- Y. pestis – spread from mammalian reservoir (rats,
Antibody Responses in untreated Brucellosis prairie dogs, dogs, mice, rabbits) via fleas or contact
with contaminated animal tissues
- Other Yersinia: ingestion of contaminated food
products, infusion of contaminated blood products

• Who is at risk?
- Y. pestis: communities with endemic plague and
exposure to infected animals
- Y. enterocolitica: individuals eating contaminated
food, recipients of contaminated blood products
C. Laboratory Diagnosis
• Microscopy – direct fluorescent antibody techniques • Geography/ Season
• Culture – slow growing, fastidious MacConkey agar - Y. pestis : primarily in Asia and Africa
- 3 or more days identification by biochemical reactions - Y. pestis: disease is cyclical, as reservoir population
and growth in the presence of basic dyes, fuchsin and increases/ decreases
thionin
• Serology – fourfold increase in antibody titer, IgM
- Other Yersinia: infections worldwide but primarily in
cold climates
followed by IgG and IgA
C. Determinants of Pathigenicity
D. Modes of Control 1. Ca2+ dependency
1) Tetracycline combined with streptomycin or with 2. V and W antigen
gentamicin 3. outer membrane proteins
2) Long-term therapy with high doses of 4. envelope antigen
trimethoprimsulfamethoxazole 5. pesticin, coagulase and plasminogen activation
3) Control of disease in animal reservoir 6. pigment binding and iron regulated surface proteins
4) Avoidance of unpasteurized dairy products
5) Protective clothing when working with animals or D. Clinical Syndromes
specimens 1. Bubonic Plague
- 7 days incubation, after bite from infected flea
V. Yersinia - high fever
- consists of 7 species, 3 are best known human pathogens, - painful bubo (inflammatory swelling of lymph node) groin
the rest can occasionally cause opportunistic infections or axilla
- Y. pestis - 75% mortality in absence of treatment
- Y. pseudotuberculosis 2. Pneumonic plague
- Y. enterocolita - 2-3 days incubation period
- fever, malaise, pulmonary signs within one day
A. Morphology and Physiology - fatality rate – 90% in untreated cases
- gram negative, non-motile, coccobacillus
- marked bipolar staining on tissue impression safety pin E. Treatment
appearance
- faculatative anaerobes, does not ferment • Chemotherapy
- lactase oxidase (-) – streptomycin, alternative drugs as tetracyclines and
- catalase (+) chloramphenicol
- can grow in ordinary media, small mucoid colonies (on D2 of - Kanamycin appears as effective as streptomycin
incubation)
- no hemolysis on blood agar F. Prevention and Control
1. Vaccination
Yersinia pestis in blood (Wright-Giemsa stain) – 3 doses, in series recommended to concerned personnel
- laboratory and fields with enzootic plague efficacy of
High Magnification vaccine is also uncertain hence prophylactic antibiotics is
recommended for people with definite exposure
2. Quarantine of persons or ships and aircraft carrying
persons
3. Flea control before rat control
4. Proper garbage disposal and good personal hygiene

• Strain identification
- thru ability to reduce
nitrates
VI. Yersinia enterolica and Y. pseudotuberculosis VII. Pastuerella multocida
- primarily parasite of domestic animals and birds – but
A. Morphology capable of a variety of human diseases
- motile, with paripolar and peritrichous- produced at
22°C and not at 37°C A. Morphology and Physiology
- usual media for enteric bacteria • Size: 0.3-1 um x 1-2 um
- identification based on biochemical differences – the - gram negative
results are markedly affected by temperature - show bipolar staining
- common cause of enterocolitis in cold countries – - pleomorphic in primary cultures
Scandinavia, Europe, and North America - produce capsule
- outbreaks associated with contaminated meat or milk • Facultatively anaerobic
- catalase and oxidase positive
B. Pathogenic determinants - metabolism is fermentative
1. Inv (invasion) gene • Cultural Characteristics
2. Ail (attachment-invasion locus) - non-hemolytic on blood agar
3. V and W Ag - does not grow on bile containing media
4. Gene for Ca2+ concentration and temperature - show round grayish colonies 1-3 minutes within 24
5. Enterotoxin hours
– produced by Y. enterocolitica • Antigenic Structure
- role still uncertain, but physico-chemical and antigenic
- 4 different serotypes (A, B, D, E)
properties is similar to that of heat stable of E. coli
- A and D are most associated with human infections
C. Clinical Syndrome
B. Epidemiology
1. Diarrhea, fever and abdominal pain lasting for as long as
- Present as normal flora in domestic animals (dogs and cats)
1-2 weeks
- Transmitted by direct contact, usually animal bite
2. Chronic form can develop and persist for months to one
- Those causing respiratory tract infections in man are caused
year
by Type A
3. Involves the ileum, with enlargement of mesenteric
lymph nodes. Can mimic acute appendicitis
C. Clinical Manifestations
4. Other manifestations may include septicemia, arthritis,
1. Infection via bites or scratches
intraabdominal abscess, hepatitis and osteomyelitis
2. Superinfection of chronically diseased lung
5. 1087 – first reported cause of BT and related Bacteremia
3. Other foci of infection secondary to septicemia
and endotoxic shock
D. Laboratory Diagnosis
D. Diagnosis
1. Culture
1. Culture of organisms from uncontaminated samples
2. Widal type agglutination test
E. Treatment and Prevention
1. Antibacterial - Penicillin and Tetracyclines
E. Treatment
2. Cleaning of wound, drainage of abscess
1. Aminoglycosides
3. Limiting contact to wild and domestic animals
2. Trimetoprim and Sulfamethoxazole
3. Supportive – fluid and electrolytes

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