Transcript Fastidious Gram Negative Coccobacilli

‫د‪ .‬زينة مكي عبد الكريم اليوزبكي‬
‫‪Ms.C. in Microbiology‬‬
Fastidious Gram Negative Coccobacilli
Three genera are included in this group of bacteria.
They are the following:
1. Genus: Haemophilus
2. Genus: Bordetella
3. Genus: Brucella
Genus Haemophilus
 Haem = blood
 Philus = loving
These m.o are requiring blood for growth as enriched
media.
Classification:
There are 2 criteria for the classification of these m.o.:
1. Their ability to haemolyse blood.
2. Their requirments for X and/or V factors
Classification
Haemophilus influenzae (Pfeiffer’s
bacillus)
General characteristics:
 Gram negative coccobacilli.
 Facultativly anaerobic.
 Organism is sensitive to drying and extremes in
temperature
 Distinctive “mousy” or “bleach-like” odor
 Misnamed – originally thought to cause the “flu”
 Now know that flu is caused by viruses
 In some cases of flu, H. influenzae is secondary
bacterial infection
Haemophilus influenzae
 Have encapsulated and non encapsulated serotype.
 The encapsulated group are pathological types and the
non-encapsulated groups are normal flora of upper
respiratory tract .
 There are 6 serotypes of it from a-f the most important
one is type b.
Morphology:
 The typical m.o. are short bacilli (coccobacilli), but some times they are
long bacilli and even filamentous forms.
 This characteristic is called pleomorphism.
 From young cultures (6-8 hr.), the m.o. are coccobacilli and
capsulated, while from old cultures (18-24 hr), they are long bacilli with
filamentous forms and are non-capsultated.
 The capsule can be typed by anti-sera in a reaction called “Quellung
Reaction” which is “capsule swelling test”. The capsular antigen can be
identified by counter current electrophoresis (CCE) and
immunofluorescent (IF) test.
H. influenzae, in a Gram stain of a sputum sample,
appear as Gram-negative coccobacilli
H. influenzae, in a Gram stain from new culture
H. influenzae, in a Gram stain from new culture
H. influenzae, in a Gram stain from old cultures
gram negative long bacilli and even filamentous
form and non capsulated
Culture media:
 Have to contain blood from rabbits or horses and not
from human or sheep because it contains antiHaemophilus antibodies.
 The most important media used are:
A. Brain- heart infusion supplemented with blood.
B. Chocolate agar.
C. Levinthal’s agar which is nutrient agar supplemented
with blood
Cultural Requirements
1. Both X and V factors are required for growth of H.
influenzae.
X = Heam or heamatin factor
 Heat-stable substance
 Present in RBC and released with degradation of
hemoglobin
important for respiratory enzymes of the m.o. including
cytochrome oxidase, catalase.
V = (NAD: nicotinamide adenine dinucleotide)
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Heat- labile
Found in blood or secreted by certain organisms
2. Temperature: These m.o. tolerate temperatures of 25
- 40 C, but the optimum is 37 C.
3. CO2: These m.o. require 10% CO2 for their growth
which is provided by candle jar.
4. Isovitalex (1%): Added to the culture media can
enhance the growth of the m.o.
Colony Morphology
On choclate agar the m.o appear semi-opaque, graywhite, convex, mucoid
This organism would be identified as H. influenzae
because it is using both X and V factors.
This organism would be identified as H. parainfluenzae
because it is using V factor only.
Haemophilus influenzae b
identification:
 Gram stain
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Gram negative cocco-baccillus
Catalase +ve
Oxidase +ve
X and V factor strips or disks.
Facultatively anaerobic need 10% CO2 (candle jar)
Culture on eneriched media
Isolation of this bacteria:
Bacitracin: Can be added to the media and make
more selective .
2. Satellite phenomenon (Satellitism):
around the growth of Staphylococcus aureus (or
Strep. pneumoniae or Neisseria gonorrhoeae) the
colonies of H. influenzae will be larger. This is
because the former growth provides an extra amount
of V factor which enhances the size of the colonies of
the latter m.o.
1.
Satellitisim phenomena of H. influanzae around and
between the large, white, hemolytic staphylococci
Satellitisim phenomena of H. influanzae around
and between the large, white, hemolytic
staphylococci
Antigenic structures (virulent
factors)
 Capsule
Which is of polysaccharides in nature. It is composed of
polyribose ribitol phosphate (PRP). According to the
serological types of PRP , the m.o. can be typed into 6
serotypes (a – f). It has Antiphagocytic
 IgA Protease
Cleaves IgA on mucosal surfaces
 Lipid A
Effects ciliated respiratory epithelium
 Pili
Attachment
Clinical conditions caused by
H.infuanzae
1.
Acute epiglottitis or laryngotracheal infection in small
children
Can cause airway obstruction needing immediate tracheostomy
2.
Cellulitis/arthritis
cheek and upper extremities
3.
Meningitis
Children under 6 years
Contagious, vaccine has decreased incidence
4. Pneumonia/septicemia
In children
5.
Conjunctivitis “pink eye”
very contagious
Diagnostic laboratory tests
1. Specimens: Nasopharyngeal swabs, pus, blood,
and spinal fluid.
2. Direct identification; by immunofluorescent (FA),
Quellung reaction for capsule swelling test, or
commercial kits for identification of H. influenzae
antigens in the CSF.
3. Culture
Treatment
Mortality rate from H. influezae meningitis may reach up to
90%. Many strains of this m.o. are sensitive to:
1. ampicillin and 25% of them are beta lactamases producer.
2. Most strains are susceptible to chloramphenicol. So,
combined use of ampicillin and chloramphenicol in the
treatment of meningitis is important to prevent
neurological complications and intellectual impairment.
3. Moreover, cefotaxime may also give excellent results .
Subdural fluid accumulation after meningitis requires
surgical drainage
Genus: Bordetella
There are 3 important species of Bordetella:
1. B. pertussis.
2. B. Parapertussis.
3. B. Bronchiseptica.
B. pertussis is the causative agent of a highly infectious
disease which is called pertussis (whooping cough).
B. parapertussis can cause a disease similar to whooping
cough, but ofter subclinical of mild.
B. bronchisepica is important in canines, produces smaller m.o.,
and infrequently is responsible for chronuic RTI in humans.
General characteristics
 Very small gram negative cocco –bacilli with toludine blue
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appear as bi chromatic granules .
Strictly aerobic.
Strictly human pathogen.
Transmission by aerosolized droplets
Heamolytic m.o.
Catalase and oxidase positive
Ferment glucose with acid and no gas production.
Do not require X and V factor for growth.
Require a special media for growth with prolonged
incubation.
Culture media :
1.
2.
3.
4.
5.
For primary isolation; an enrichment media is used. This
medium is called Bordet-Gengou (potato-blood-glycerol agar).
The medium contains 20-30 % sheep blood cells and penicillin
-G or methicillin to make more selective.
Buffered charcoal yeast extract agar (BCYE): this medium can
also be used for the isolation of Legionella pneumophila .
Blood Charcoal Agar (BCA) to which an antibiotic may be
added as cephalexin to make more selective ( inhibits
respiratory flora).
Lowe-Regan agar which is similar to BCA, but contains a
half strength of charcoal.
Charcoal and cephalexin
All media should be kept in moist environment (sealed
plastic bag).
Cultural characteristics
The colonies appear as rounded, convex,
1-2 mm in diameter, and look like mercury drops or
pearl-appearance. The colonies have iridescence and
surrounded by narrow zones of haemolysis
B. pertussis
Small, transparent hemolytic colonies on
Bordet-Gengou medium
Gram stain of B. pertussis
Antigenic structures:
1.
2.
Capsule.
Pertussis toxin which have the following affects:
A. Prolonged immunity because it is an immunogenic
factor.
B. Anaphylactic –like reaction due to histamine
sensitization.
C. Paroxysmal cough that is followed by deep
inspiration .
D. enhances insulin secretion
E. It reduces the migratory and phagocytic activity of
macrophages and neutrophils.
F. Absolute lymphocytosis may reach up to 30000
/cmm.
3. Filamentous haemagglutinin that mediates
adhesion to ciliated epithelial cells.
4. Leucocytes promoting factor that promote
lymphocytosis.
5. Lethal toxin that causes local necrosis.
6. Tracheal cytotoxin which affects the activity of
epith. and cilia of respiratory tract.
7. Adenylate cyclase complex that can impair
phagocytosis.
Pathogenesis
Clinical manifestation of pertussis
disease:
 Spread by aerosol/direct contact with infected and carrier persons
 Early symptoms - nonspecific
- seldom diagnosed until paroxysmal stage
- most contagious early
Stages
- Incubation period: 7-10 days
 Catarrhal stage:
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Symptoms like common cold, lasts 1-2 weeks
 Paroxysmal stage:
 dry nonproductive cough, paroxysmal
 excess mucus production, vomiting, convulsions, cyanosis, paroxysms
separated by inspiratory whoop
 Lasts 4-6 weeks
Children who are too young
to be fully vaccinated and
those who have not
completed the primary
vaccination series are at
highest risk for severe illness
Diagnostic tests
1. Specimens: A saline nasal wash is the preferred
specimen. Nasopharyngeal swabs or cough droplets
expelled onto a “ cough plate” held in front of the
patient’s mouth during a paroxysm are some times
used.
2. Direct fluorescent antibody (FA) test to examine
nasopharyngeal swab specimens with sensitivity of
50%. Also, it is most useful in identifying B. pertussis
after culture on solid media
3. Cultures: Mentioned previously.
4. Polymerase chain reaction (PCR): It is the most
sensitive method for the diagnosis of pertussis.
5. Serology: Serological tests are of limited value because
specific antibodies ( agglutinating or precipitating)
rise does not occur until the third week of illness
Treatment
 B. pertussis is sensitive to many drugs in vitro. During
the catarrhal stage erythromycin promotes the
elemination of the m.o. and may have prophylactic
value. Treatment during the paroxysmal stage rarely
alters the clinical picture.
Prevention:
1. Vaccination (part of DTP): 3 doses in the first year
of life, followed by booster of a total of 5
injections.
2. New acellular vaccine of 5 antigens is also
available.
3. Prophylactic erythromycin for 5 days is given for
unimmunized infants or heavily exposed adults
Genus: Brucella
 Gram-negative coccobacillus
 Facultative intracellular parasites
 Six species
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B. abortus
B. suis
B. melitensis
B. canis
B. ovis
B. neotomae
- cattle
- pigs
- goats
- dogs
- sheep
- desert wood rats
 Cause zoonoses worldwide
 Disease in human: Brucellosis, Malta fever, Undulant Fever
 Mediterranean Fever, Rock Fever of Gibraltar, Gastric Fever
Sir David Bruce (1855-1931) isolated
the bacterium of Malta fever in 1887
Morphology :
The typical m.o. are predominantly gram negative
coccobacilli, and from young cultures they vary from
cocci to short bacilli and are capsulted , and stain
irregularly. They are aerobic and nonmotile m.o.
Culture media
1. Trypticase soy agar.
2. Castaneda biphasic medium (broth & agar). This medium
is prepared in bottles and has two portions; solid one
(bottom), and liquid (top). The m.o. can grow on the
surface of the solid portion and in the liquid rendering it
turbid.
3. Blood agar or blood culture media.
4. MacConkey’s agar supplemented with 5% of heated horse
or rabbit serum.
5. Brucella selective medium (good medium but expensive).
Cultural characteristics
Colonies are small, convex, smooth, translucent,
nonhaemolytic, slightly yellowish in young
cultures but brownish in old ones, and appear in 25 days (may be delayed up to 3 weeks).
Clinical picture and pathogenesis of
Brucellosis
 Method of transmission:
1. Ingestion of raw or un boilled milk or milk products in
particular white cheese.
2. Mucous membrane (droplets).
3. Skin (contact with infected animals or tissues as meat
for workers in slaughter houses, butchers
,veterinarians, or house wives).
4. Conjunctiva rarely.
5. Breast milk lactating woman
the organisms are phagocytosed by macrophages and
monocytes phagocytosed bacteria are carried to the spleen,
liver, bone marrow, lymph nodes. the bacteria secrete
proteins that induce granuloma formation
 Incubation period 1-6 weeks
 Insidious onset start as flue like symptoms.The fever usually
rises in the afternoon; its fall during the night is accompanied by
drenching sweat. There may be GI and nervous symptoms. L.N.
enlarge, and the spleen palpable
 The manifestations may subside in weeks or months , although
localized lesions and symptoms may continue.
 chronic stage may develop; characterized by weakness, aches and
pains, low grade fever, nervousness or depression.
Brucellae usually can not be isolated from such patients except
in only 2% of them. The diagnosis of “chronic brucellosis” is
difficult to established.
Diagnostic tests:
A. Specimens: Blood and biopsy material
B. Culture: Mentioned above. Subcultures are made from at
intervals from blood culture or broths.
C. Serology:
IgM antibody levels rise during the FIRST week of acute
illness, peak at 3 months, and may persist during chronic
disease.
IgG antibody levels rise 3 weeks after onset of acute disease,
peak at 6- 8 weeks and remain high during chronic disease.
IgA levels parallel the IgG levels; could be blocking antibody
(causes prozone pheno.).
Serological tests:
1. Agglutination test (tube or slide); using:
a. Heat-killed phenolized smooth
standerized brucella antigens.
b. Rose bengal antigens (stained red).
The significant titre = 1/160 or higher.
Cross reaction may occur with cholera vaccine, S.typhi
/paratyphi , or Yersinia.
2. 2-Mercaptoethanol test; 2-ME can destroy IgM and leaves
IgG. This may be used to differentiate between current
/recent brucellosis from previous /old brucellosis. Also, is
useful in chronic active brucellosis
3.Blocking antibodies: These are IgA antibodies that interfere
with agglutination by IgG & IgM and cause false negative
result (prozone phenomena). These antibodies appear
during the subacute stage of infection tend to persist for
many years, and are detected by Coomb’s antiglobulin
method. Also, high dilution agglutination test can
overcome the problem of prozone phenomena.
4. Skin test (Brucellergen or Brucellin): it is DHS reaction to
I.D. Injection of a protein brucella extract. Erythema,
oedema, and induration develop within 24 hours. It is
unreliable test & is rarely used.
5. ELISA , RIA or Immunofluorescent ; for Ab detection.
Treatment:
Combined anti-microbial drugs are used, taking in
consideration that the m.o. is an intracellular one:
1. Ampicillin or tetracycline with Streptomycin give good
result. Cotrimoxazole may be added.
2. Refampcin (900 mg once daily) with doxycycline (200
mg once daily) gives 97% cure rate. Also, an
aminoglycoside drug could be added.
Treatment should be continued for at least 6 weeks and
for bone or joint infections the treatment continues for
at least 8 weeks.
For neuro-Brucellosis chloramphenicol or ampicillin
could be used with other drugs
Francisella tularensis
Fastidious gram negative bacilli
Intracellular pathogen
Strictly aerobic
Transmitted from animals to human
Cause ulceroglandular disease
Grow on media contain cystein-supplemented as sensitive
and specific for their growth with prolonged incubation
Dx: By culture and serological methods.
Rx: Streptomycein
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