Gram Negative UPH

7/27/2019 Gram Negative UPH

1/41

1

HAEMOPHILLUS


2/41

2

Haemophilus Influenzae:

Clinical Infections

Infections caused by typable (encapsulated) strains

Acute epiglottis or laryngotracheal infection in small

children

Cellulitis/arthritisMeningitis

Pneumonia/septicemia (in children)

Conjunctivitis


3/41

3

Haemophilus Influenzae:

Clinical Infections

Infections caused by Nontypable strains

Otitis media

Sinusitis

Pneumonia, bronchitis (in adults)


4/41

4

Haemophilus Species

Haemophilus species require hemoglobin for growth:

X-factor ( hemin): Heat-stable substanceV-factor (NAD): Heat- labile, coenzyme I, nicotinamide

adenine dinucleotide, found in blood or secreted by

certain organisms

H. influenzae

satellitism around

and between the

large, white,hemolytic

staphylococci


5/41

5

Haemophilus Species

Direct smear of H. influenzaein CSF in acase of meningitis. Note the intracellular

and extracellular pleomorphic gram-

negative bacilli.


6/41

6

Haemophilus species

H.influenzaegrowing on

chocolate agar. Notice the

semi-opaque, gray-white,

mucoid colonies characteristic

of encapsulated strains.

Gram stain of H. influenzae

from colony


7/41

7

Haemophilus Species:

Identification

This organism would be identified as H. influenzae

because it is using both X and V factors.


8/41

8


Identification

This organism would be identified as H.parainfluenzaebecause it is using V factor only.


9/41

9


Identification

This organism would be identified as H. aphrophilus because it is

using X factor only.


10/41

10


Identification

Under ultraviolet light, the organism on the bottomis showing a positive porphyrin reaction, whereas

the organism on the top is demonstrating a

negative porphyrin reaction.


11/41

11

BORDETELLA


12/41

12

Bordetella

Classificationthe genus contains three medially

important species

B. pertussisB. parapertussis

B. bronchoseptica

Morphology and cultural characteristics

Small g-cb

B. parapertussisandB. bronchosepticaboth grow on

sheep BA (SBA) in 1-2 days


13/41

13

BordetellaB. pertussisfor initial isolation (The best clinical

specimen is a nasopharyngeal swab.) theorganism requires special media with additional

nutrients for growth and absorbents to removetoxic substancesfound in complex media such as

fatty acids and sulfides.Borget-Gengou mediacontains glycerol, potato

infusion, albumin (binds fatty acids), and up to 50%defibrinated SRBCs

Charcoal agarsupplemented with 10% horse bloodwith or without cephalexin.

May take 3-7 days for growth and colonies aresmooth, raised, and glistening (phase 1 colonies).

They are also hemolytic and produce toxin.


14/41

14

Charcoal-horse blood agar


15/41

15

Bordetella

Virulence factors (B. pertussis)

Pili for attachmentPertactin, an outer membrane protein also acts

as an adhesion

Filamentous hemagglutininis found on the

cell surface of and is also secreted.It attaches to cilia by binding to exposed lactose

receptors.


16/41

16

Bordetella


17/41

17

BordetellaPertussis toxin

Secreted by type IV secretion system

Has one A subunit (toxic part), plus four different kinds of B

subunits (involved in binding).


18/41

18

Bordetella

Clinical significanceB. pertussiscauses whooping cough

Acquired by inhalation of droplets containing theorganism

The organism attaches to the ciliated cells of therespiratory tract.

During an incubation period of 1-2 weeks, the organismmultiplies and starts to liberate its toxins.

Next the catarrhal stage occurs - the patient has a

mild cough and sneezing whereby large numbers oforganisms are spread through the respiratory

secretions.

This last ~ 2 weeks.


19/41

19


20/41

20

Bordetella

Next is the paroxysmal stage that lasts 4-6 weeks.The patient has rapid, consecutive coughs with a rapid intake

of air between the coughs (has a whooping sound).

The ciliary action of the respiratory tract has beencompromised, mucous has accumulated, and the patient is

trying to cough up the mucous accumulations.

The coughs are strong enough to break ribs!

Other symptoms due to the activity of the released toxinsinclude:

Increased peripheral lymphocytes due to a blocking ofhoming of lymphocytes to the spleen and lymph nodes.

Metabolic alteration such as increased insulin release andthe resulting hypoglycemia

Increased capillary permeability and increasedsusceptibility to histamine, serotonin, and endotoxin

shock


21/41

21

Bordetella

Finally there is a convalescent stage during which

symptoms gradually subside.

This can last for months.

B. pertussis rarely spreads to other sites, but a lot of

damage may occur, such as CNS dysfunction which

occurs in ~10 % of the cases and is due to an

unknown cause.

Secondary infections such as pneumonia and otitismedia are common.


22/41

22

Bordetella

B. parapertussiscauses a mild form ofwhooping cough

B. bronchosepticaWidespread in animals where it causes

kennel cough.

Occasionally causes respiratory or wound

infections in humans.


23/41

23

Bordetella

Treatment

Erythromyinonly effective in early stages of

the disease before the toxin(s) have beenreleased

Vaccination P part of DPT (killed, encapsulatedorganism); a subunit vaccine has also been

developed (purified pertussis toxin).


24/41

24

BRUCELLA


25/41

25


26/41

26

1. Brucellosis is considered as the most wide spreadzoonosis in the world and it is considered as True

zoonosis ( That mean it is Basically transmitted from

animal to human).

2. The importance of this contagious disease is the economic

impacton livestock industry.

3. Causes sever hazard to human health, through eitherdirect contact with infected animals or the consumption of

contaminated milk and dairy products.


27/41

27

Causative bacteria of the disease

Brucellosisis named after Sir David Bruce, who is in 1886

isolated the causative agent from a soldier inMalta.

Brucella species are recognized based on the natural

animal hostto the following species as shown in table ( 1).


28/41

28

Human

disease

Other animal

species affected

Natural

hostBiovarSpeciesNO

Less sever

Wild animals,

water buffalo,

camels

Cattle1-9Brucel la abortus1.

SeverWild ruminant

cattle, camels

Sheep

and Goat

1-3Brucel la meli tensis2.

NoneNoneSheep

(Ram)1Brucel la ovis3.

Sever

( except

biovar 2)

Various wild

speciesSwine1-5Brucel la suis4.

BenignNoneDog1Brucel la canis5.

NoneNoneWood rat1Brucel la neotomae6.

Table (1) show Brucella species :


29/41

29

1. The bacteria are strictly parasitic and prefer theintracellular habit.

2. The species of the genus Brucella are small non

motile, non spore forming, Gram negative rodsand they do not produce true capsules.

3. They aresomewhat resistant to decolonization by

weak acids and thus stain red by the modified

Ziehl - Neelsen method.

Morphology and staining


30/41

30

Gram stain of Brucella

(Gram ve ) Coccobacilli


31/41

31

Antigenic Structure


32/41

32

The designation of the antigens in cultures composed of smooth

and rough coloniesare shown in the table 2.

NO. Species Type of colony

Type of

surface antigen

1. Brucella abortus Smooth A m ---

2. Brucella melitensis Smooth a M ---

3. Brucella suis Smooth A m ---

4. Brucella ovis Rough --- --- R

5. Brucella canis Rough --- --- R

The Production of monospecific antiserato A and M antigen

can be used in the identification of the Brucella species.

Br.canis and Br.ovis grow as rough colonies that do not

possess either of the surface antigens Aand M, but instead of

that they have Rantigen.


33/41

33

1.Culture media

There are two major types of media for cultivation of Brucella.A. Basal Medium

Direct isolation and culture of Brucella are usually performed on solidmedia.

This enables the developing colonies to be isolated and limit thedevelopment of contaminants.

There is many Kinds of commercial media ,e.g. Brucella medium base,Trypticase soy agar, Columbia agar, Serum- dextrose agar or Glycerol-dextrose agar.

The addition of 2-5 % Bovine or Equine serumis necessary for the growthof strains such as B. abortus biovar 2.

B. Selective Media

Appropriate antibiotics are added in order to suppress the growth oforganisms other than Brucella. The most widely used medium is Farrellsmedium, which is prepared by the addition of six antibiotics :

Polymyxin B sulphate,Bacitracin, Cycloheximide, Nalidixic acid, Nystatin,

Vancomycin

Cultural characteristics


34/41

34

2. Colony Morphology

Brucella colonies are visible after 3-5 daysincubation period at 37 C on suitable solidmedia, and they are aerobic or microaerophilic.

Cultures should not be discarded as negativeuntil 8-10 days have elapsed.

Brucella colonies are 1-2 mm in diameter,round, entire, smooth, glistening, translucent,and a pale honey color when plates are viewedin the daylight through a transparent medium.


35/41

35

Brucella colonies on blood agar


36/41

36

Brucella colonies on blood agar


37/41

37

Epidemiology of the disease

1.Transmission of the disease

Animal to animal Transmission

The oral route, Contamination of the udder during milking

and contact with aborted fetusesand infected newborn lambs

are considered to be common methods of spread, also thevenereal transmissionof the disease is occur due to infectedmale or contaminated semen.

Animal to human Transmission

Infected tissues, and contaminated materials must behandled under (biosafty 3)conditions. Transmission could beeither by contaminated food, invasion by intact skin,inhalation of aerosols containing the bacteria and aerosol

contamination of the conjunctiva.


38/41

38

One of the most important route of animal to

human transmission of Brucella


39/41

39

Brucella melitensis( biovar 1, 2 or 3) is the main

causative agent of caprine and ovine brucellosis.

Sporadiccases caused by B.abortus have been

observed. The infection is widespread world-wide.

Brucella abortus is usually causes bovine

brucellosis, less frequentlyby brucella melitensis.


40/41

40

Brucellosis: Edema and swelling of scrotum


41/41

41

Documents

Gram Negative UPH