micro sheet # ?(32/11/2010) - Mohammad Alshanteer

بسم الله الرحمن الرحيم

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Vibrionaceae (gram negative):

- Include one human pathogen : Cholerae
- Curve or straight bacilli that are facultative anaerobes, oxidase positive and non-spore- forming .
- Primarily found in water and are well known for their ability to produce gastrointestinal diseases .
- There are 3 genera that belong to this family :
 Vibrio (most important)
 Aeromonas
 Plesiomonas
Vibrio:
- Curved bacilli , motile due to presence of single polar flagellum.
- Present in surface water world wild , so they contaminate fresh water and water in lakes.
- Source of contamination is humans .
- They grow on alkaline media (pH 8.5 9.5).
- Alkaline water is required to enrich stool specimen .
- V.cholerae can be isolated on special media like thiosulphate
sucrose .
- There are different serogroups of V.cholerae:
 Serogroup O1 and O139 (O is somatic gene) : cause
classical cholera ……..epidemic
 Serogroup non O1 /non O139 : cause cholera-like disease…….
Sporadic
- Serotype available are Ogawa and Inaba
- Biotype are classic and ET tour (circulating).
- Infectious dose is very high (10 8 or more ) required to establish
infection because organisms don't survive gastric acidity.
- Patients with problems of acidity ; ahydrochlorial ( low secretion
of HCl ) are at high risk for developing cholera .
- Cholera as a disease is a result of toxins and the organism as such
doesn't cause invasion of intestinal tract.
- The organism colonizes the GIT and start producing heat labile toxins .
- The toxin has 2 subunits :


 B subunit : responsible for binding to the receptor on surface of brush border cells (epithelial cells in small intestine ).

 A subunit : responsible for activation of adenylate cyclase ,this leads to activation of cAMP ,resulting in prolonged hyper secretion of water and electrolytes .
- Diarrhea occurs with loss of 20-30 liters of fluid /day , leading to
dehydration , shock , acidosis and death .(if the water replaced>>no problem)
- 60-75% of infections are asymptomatic or subclinical.
Most infections are subclinical so what we see is the minority of
infection.
- Less then 35% of infections are symptomatic.
- The incubation period is 1-4 days , followed with sudden onset of
nausea ,vomiting and perfuse diarrhea with abdominal cramps.
- Stool resemble (rice water) , it contains mucus, epithelial cells and
large number of vibrio organisms.
- Dehydration with circulating collapse and anuria develop with
death in 25-50% of untreated cases.
- Diagnosis:
Made in the course of infection.
All cases with such diarrhea are considered as cholera cases.
Organisms can be isolated by use of alkaline peptone water as
selective media(TCBS) for culturing.
- Epidemiology:
Cholera is found in fresh water ponds and estuaries in Asia ,
Middle east ,Africa and along the coastal areas of south , central
and north America .
Major reservoir in infected humans (carriers).
There have been seven pandemic in last centuries
The last epidemic is still spreading in Africa and Latin America;
i.e. : cholera has eradicated in these countries in form of epidemic.
It is still spreading in many developing countries world wild .

- Vibrio parahemolyticus :
Causes acute gastroenteritis following ingestion of contaminated
sea food, it is an illness that is of short incubation and self limited.
It causes bloody diarrhea .


Eating raw oysters causes infection with Vibrio parahemolyticus .
- Vibrio vulnificus can cause :
4. Severe wound infections after exposure to contaminated
sea water .
5. Septicemia after consumption of raw oysters .
- Cholera is self limiting illness provided that water and electrolyte
are replaced
- No vaccination for cholera is available , because it is caused by
toxins , so vaccination will not provide immunity against cholera
Campylobacter:
- Curved , comma shaped , oxidase-positive , catalase - positive ,
microaerophilic gram negative bacilli that are motile by means of
a polar flagellum .
- They are commensals of cattle , sheep, dogs , cats , rodents and
fowl .
- Humans are infected due to consumption of contaminated food ,
milk or water .
- Contaminated poultry is responsible for > 50 % infections.
- Presents as acute enteritis with sudden onset of crampy abdominal
pain , profuse diarrhea that may be grossly bloody , headache
,malaise and fever .
- The illness is self limited within few days .
- Systemic disease may be caused by Campylobacter fetus in
immunocompromised individuals.

Helicobacter pylori :
- Spiral shaped , motile ( one polar flagellum ) gram negative rods
- Associated with antral gastritis , duodenal (peptic) ulcer disease ,
gastric ulcers and gastric carcinoma .
- Most important virulence factor is urease production ( protection
from gastric acids ), mucinase and adherence factors.
- It causes gastritis , peptic ulcers , and is associated with gastric
cancer .







Mycobacteria

Mycobacterium is one of the members of a big family called:
Mycobacteriaceae, this family contains two genera which are:
Mycobacterium and Nocardia.
Mycobacterium is characterized by:
-The presence of a long chain of fatty acids (60 carbons-90 carbons) called mycolic Acid that confirms the high resistance to the environmental conditions of the cell wall as we will see….
- Aerobic, slender (thin), curved rods that can be demonstrated by special stain (Carbol fuchsin, Rhodamine, Ziehl neelsen) in clinical specimens.
-The cell wall has mycolic acid, complex waxes and unique glycolipid. This unique composition of the cell wall provide a resistance to all environmental conditions (dryness, Heat, Chemicals, etc.) &many disinfection/sterilization procedures for a long time.

Cell wall components:
1) mycolic acid which contain a very long fattyacid chain
(60 C.- 90 C.) and these are join together by peptidoglycan and
arabinogalactan.
Although mycobacteria contains the peptidoglycan but they are
not similar to the gram positive or negative bacteria.
They don't take the gram stain so can't be classified as gram
positive or gram negative bacteria.
-The cell wall is acid (& Alcohol) - Fast and it's very difficult to stain without heating.
Acid-Fast stain procedure:
In this procedure, carbolfuchsin ( a bright red dye) is first driven into the bacterial cell using heat (usually by flooding the smear with carbolfuchsin and then holding a Bunsen burner Flame under the slide), the heat is necessary because the cell wall of mycobacteria contain waxes, which prevent the stain from penetrating the cells. The heat softens the waxes enabling the stain to penetrate. A decolorizing agent (a mixture of acid and alcohol) is then used in an attempt to remove the red color from the cells.

Because mycobacteria aren’t decolorized by acid-alcohol mixture, they are said to be acid fast. Finally by adding a counter stain like methylene blue red bacteria against blue stained background (organisms, cell.) will be formed.

The acid fast staining procedure are referred to as differential staining procedures because it's enable microbiologists to differentiate one group of bacteria from another, but not species and that’s why its called (Acid fast bacilli), After this procedure we apply other special biochemical test to determinate the different type of species whether it marinum, avium, TB,etc.).

2)Trehalose dimycolate: important component which is called cord factor because it believes to be responsible for serpentine cords arrangement of colonies on artificial media.
3)Mycobacterial sulfolipids which may play role in virulence.
4)Lipoarabinomannan (LAM): unusual hard complex structure of cell wall which endows mycobacteria with resistance to
dehydration, acid and alkalis, and this's important in the isolation of mycobacteria from contaminated clinical specimens such as sputum which contain other organisms that are capable to digest by acid (sensitive).
5) Adjuvant: are substances that increase the rate, level, and
duration of an immune response. For e.g. if we inject antigen in
someone the antibodies will be produced after 2 weeks & persist
for 2 weeks but injection of antigen with adjuvant will reduce the duration needed to produce antibody (for e.g. days) & elongation of antibody persistence (for 6 months) .. Also, adjuvant is important in vaccines. It was found that mycobactrial cell wall is an adjuvant alone or mixed with water (freund's incomplete adjuvant) or oil emulsion (Freund's complete adjuvant).

Mycoloacteria are fastidious in their growth (very difficult to grow) they require certain factors (e.g. glycerol, mineral, vitamins) to be added to a certain media and when they grow, the doubling (generation) time is very long (18-24 hours), in contrast to other organism that have doubling time (18-24 min) that why mycobacteria should be incubated for a very long period of time up to 6 weeks to produce visible growth.





This extremely slow growth has two consequences of clinical
significance:
1-The infection or disease caused by mycobacteria is slow chronic process, which may take several weeks or months to become clinically patent.
2-On solid media inoculated with clinical material, identifiable
myobacterial colonies may not appear befor 4-6 weeks.
With the exception of M.liprae which is the causative agent of leprosy (Hansen's disease (HD) الجذام ), the mycobacteria are classified into two broad categories:

1)Typical Mycobacteria or M.tuberculosis complex which include :
* M. tuberculosis
* M. bovis (tuberculosis in cattle)
* M. microti
* M. africanum
2)A typical or Nontuberculous myobacteria : are described based on their growth rate (growth occur in days (Fast growers) & pigment production that can take place in the:
*light (photochromogen)
*Dark (scotochromogen)
*non chromogen (not produce pigment).
Avium intracellulare complex is the most species that belongs to atypical mycobacteria as a human pathogen.

Pathogensis
The infection is airborne meaning that small nuclei (aerosol
<5microns) transmit the infection by deposition deep in the
lung(it's not direct like respiratory droplet in influenza).
The bacilli are deposited in the alveolar spaces of the lungs where they are engulfed by alveolar macrophages. A portion of the infectious inoculum resists intracellular destruction and persists, eventually multiplying and killing the macrophage.
Most of the tissue destruction associated with tuberculosis results from delayed type hypersensitivity mediated by type one T helper cells which secrete cytokines (gamma-interferon) which activates macrophages and other mononuclear cells. The whole mark of infection is the formation of granuloma.





So granuloma formation is characteristic for many organisms,
parasitic infection and some viral infection this is important
because it differs from humeral immunity where antibody areimportant but in our case the granulomatous lesion characterized by mononuclear cell infiltrate surrounding a core of degenerating epitheloid and multinucleated giant (Langerhans cells).
The immune response which causes the pathogenicity not the organism itself.
-This lesion which is called the tubercle (primany lesion in the
lung) may become enveloped by fibroblasts and its center often
progresses to caseous necrosis which then calcified without
causing any problem.
-Liquefaction of the caseous material and erosion of the tubercle
into an adjacent airway may result in cavitations and the release of massive numbers of bacilli into the sputum.
-All of the manifestation above we called it the primary TB, you all have been exposed to this .exposure to M.tuberculosis lead to
formation of primary lesion (granuloma) and there are two paths for it either:
1)Calcified in immune competent host and this is marked by a
change in the status of individual (became sensitive to organism)
and can be discovered by skin testing, doesn’t cause any harm.
2)Releases of the content out side by open air ways like suputem, the patient remain healthy for decades without any harmful event.
-Early in infection, myobacteria may spread distally, either directly or indirectly; indirectly through the lymphatics to the Hilar or mediastinal lymph nodes and then via thoracic duct into the circulation by erosion of the developing tubercle into a pulmonary vessel.
-Extrapulmonary hematogenous dissemination results in the
seeding of other organs (spleen, liner, kidneys) and eventually reinoculation of the lungs.
-Primary lesion in (95% or more) are followed by nothing it just
give immunity to TB, (5% or less) of patient may disseminated or may develop pulmonary disease.
-Almost always, patients with active (TB) have had an infection in the past its not new infection. It is the infection during childhood that remain dormant in calcified legion for long time and for a reason or the other the patients immunity declined as a result secondary tuberculosis will be formed.


The resulting secondary lung lesions may serve as the origin of
reactivation of clinical disease years or decades later owing to the persistence of viable bacilli.
Primary disease is usually characterized by a single lesion in the
middle or lower right lobe with enlargement of draining lymph
nodes.
Endogenous reactivation is often accompanied by a single
(cavitary) lesion in the apical region, with unremarkable lymph
nodes and multiple secondary tubercles.
Direct infection of the GIT can take place if the individual
consumes unpasteurized milk infected cows (M.bovis), or it may
swallow sputum heavily infected with mycobacteria that resist the stomach acidity so establish the infection in GIT.
In either case, the principal site of involvement is the mesenteric
lymph node with subsequent dissemination. Innate susceptibility to pulmonary infection with M. Tuberculosis is clearly influenced by genetic and ethnic (most common in black and malnourished individual).
Acquired immunity following mycobacterial infection usually develops with in 4 to 6 weeks (4 to 6 weeks from the primary infection) the individual become positive or sensitive to the organism, and the individual immunity to mycobacteria can be
detected by skin test (tuberculin test).

Tuberculin test or PPD test (purified protein derivative): A
standard dose of tuberculin is injected intradermally and read 48 to 72 hours later. A person who has been exposed to the bacteria is expected to mount an immune response (erythema + induration) in the skin containing the bacterial proteins.

The reaction is read by measuring the diameter of indurations
(thickness) across the forearm. If there is no indurations, the result should be recorded as "0mm" . Erythema (Redness) should not be measured. The result of this test considered to be +ve if the thickness become 5mm or more. Successful acquired resistance is mediated by T lymphocyte ,Antimycobacterial antibodies although present in many patients, do not play a protective role. Since the vast majority (90-95%) of other wise healthy individuals who are infected with M. tuberculosis never develop clinically apparent disease, acquired resistance must be quite effective so they don't affected by M. TB unless the immune responses become weak.



However, the immune response to mycobacterium is double – edged sward meaning that the immunity protect the individual against TB but at the same time the intense cell mediated hypersensitivity is responsible for much of the pathology associated with clinical tuberculosis. Reactivation is usually associated with deterioration of the cell mediated immune response due to going or to some associated clinical condition (AIDS, cancer).

Clinical manifestation
The first sign of a new infection is often conversion PPD ,if an individual start to express very strong PPD it doesn’t indicate immunity its indicate the progression of disease& then Antimycobacterial agents are recommended.
So, strong PPD marks the onset of illness and a lesion can be detected by X-Ray. Clinical signs and symptoms develop in only small proportion (5- 10%) of infected healthy people.
These patients usually present with pulmonary disease, prominent symptoms are chronic, productive cough, low-grade fever, night sweats, easy fatigability and weight less.
TB may present with extrapulmonary manifestations including lymphadenitis, kidney, bone or joint involvement, CNS (meningitis) or disseminated (military) disease.
Lymphadenitis and meningitis are more common among childhood, and all extrapulmonary manifestation are increased in frequency among immunocompromised individuals like HIV patients.

Epidemiology
TB increases in the Era of HIV and other immunosuppressive
modalities. Its common in poor, malnourished individuals
Diagnosis

1)PPD test : can't distinguish between past and recent infection, however the increase in postivity indicate the progression of diseases.
2)Smears: Prepared from clinical specimens can demonstrate the presence of organism by flourecent stain (Fluorochorme, Ziehl Neelsen).
3)Culture which should be examined for 8 weeks .




Treatment and control
1) Intensive case finding is important by PPD test.
2) Aggressive prophylactic chemotherapy of converters (isoniazid is recommended)
3) Vaccination (BCG) has a variable efficacy; there are studies
which reports it as good protection others don't .
-It decreases the severity of infection , doesn't prevent it.
4)Antimycobacterial treatment
-Combination therapy (Multi drug resistance)

Done By:
Mohammad Al-Shantir
23/11/2010