Title of Lecture: Gram +ve Bacilli: Corynebacteria. And Clostridia. Date of Lecture: November 16th. Sheet no: 15 Refer to slide no. : 7 (29-60)
8(1-15) Written by: Seema Salam Daradkeh.

Corynebacteria

Gram-positive

non motile

club shaped pleomorphic bacilli

They have a distinctive way of growth: when they grow , and divide, they snap, and appear in short chains like V or Y or π configurations or in clumps resembling Chinese letters. (charactaristis of the morphology of this bacteria)

Nonencapsulated, catalase positive, and oxidase negative

You can isolate Corynebacteria from the upper rispiratory tract which are nontoxic and sometimes known as Diphtheroids.

C. diphtheriae is fastidious while diphtheriods are not

When stained with special stain they show granules, known as metachromatic granules (polymetaphosphate) which stain bluish purple with methylene blue.

These granules sometimes give the impression of being spores, but they’re not spores, because spores do not stain(white).

There are both gram-positive and gram-negative species, although the majority of isolates are gram-positive.

Corynebacteria are aerobes or facultative anaerobes and generally grow slowly on enriched media.

Some of the morphologies of Corynebacteria: there are four distinct cultured types exist;

gravis

intermedius

mitis

belfanti.

These are not very important because supposedly they have something to do with the toxicity of the organism, but it is an old fashioned way of classification. Because when you grow them you get a fastidious shape, you can grow them on special medium known as Potassium tellurite. And you get these colonies which can be grey or black, etc.

These variants (biotypes) have been classified on the basis of growth characteristics, biochemical reactions and severity of disease.

They colonize: (found where):

skin

upper respiratory tract (most common)

Gastrointestinal tract and urogenital tract of humans.

The most important species is C. diphtheriae, the causative agent of diphtheria disease.

This causes disease by virtue of toxin production. This applies to the bacteria by a bacteriophage.

Bacteriophage can confirm onto the bacteria the ability to produce the toxin. so C. diphtheriae if it is infected by this lysogenic phage it acquires the activity or the ability to produce the diphtheria toxin

Diphtheria toxin is a very potent exotoxin that is lethal at doses as little as 100 to 150 ng /kg of body weight.

Nontoxigenic (avirulent) C. diphtheriae are morphologically indistinguishable from the virulent (toxigenic) strains

It is now recognized that nontoxigenic (avirulent-lack tox gene) strains may be converted to the toxigenic (virulent) phenotype following infection and lysogenization by one of a number of distinct corynebacteriophages that carry structural gene (Tox gene) for diphtheria toxin.

So the difference between the toxic and nontoxic is the Tox gene from the bacteriophage.

Lysogenic conversion may occur in situ as well as in vitro.

Pathogenesis

In 1883 Klebs demonstrated that C. diphtheriae is the agent of diphtheria.

The pathogenesis of diphtheria is based upon two primary determinants:

Colonization: The ability of a given strain to colonize in the nasopharyngeal cavity and/or on the skin (rarely).

Most common site of infection of diphtheria is the nasopharynx. it grows something like a pseudomembrane, whitish, covers the inside of the nasopharynx.and if you try to pull off the membrane it will bleed.

Its ability to produce the toxin.

Diphtheria Toxin

The regulation of diphtheria toxin gene expression is mediated by an iron-activated repressor which is encoded on the C. diphtheriae genome.

Production of the toxin is iron-dependant. If there is iron deficiency then the toxin will be produced.

The toxin itself is made of 2 units:

A: goes inside to produce the problem inside the cell

B: sticks to the membrane

The disease actually results from interference with the elongation of proteins and this produces unfunctional proteins and shorter proteins that are disrupt and you’ll get the disease.

Activation of the repressor gene derepresses the tox gene and diphtheria toxin is synthesized and secreted into the culture medium at maximal rates.

The disease mainly affects two variety of tissues:

The heart: it can produce myocarditis and this will lead to heart failure and death.

The peripheral nerves: it will lead to peripheral neuritis.

So the main symptoms that will appear are due to the effect on the heart and the peripheral nerves.

You may have fever and sore throat from the presence of the bacteria in the body.

It is composed of a single polypeptide chain of 535 amino acids which consists of 3 structural functional domains:

An N-terminal ADP- ribosyl transferase (catalytic domain)

A region which facilitates the delivery of the catalytic domain across the cell membrane (transmembrane domain).

The eukaryotic cell receptor binding domain.

Following mild digestion with trypsin and reduction under denaturing conditions, the toxin is cleaved into two polypeptide fragments (A and B).

Fragment A is the N-terminal containing the catalytic center for the ADP- ribosylation of elongation factor 2 (EF-2).

Fragment B (the C terminal) carries the transmembrane and receptor binding domain of the toxin.

One molecule can inactivate the whole factor.

The intoxication of eukaryotic cells by diphtheria toxin involves at least four distinct steps:

Binding of toxin to its cell surface receptor.

Clustering of charged receptors into coated pits and internalization of toxin by receptors mediated endocytosis.

Insertion of transmembrane domain into the membrane and delivery of the catalytic domain into the cytosol.

ADP- ribosylation of EF-2 irreversibly inhibiting protein synthesis.

Clinical Manifestations

Incubation of 2-6 days.

There are two types of clinical diphtheria; nasopharyngeal and cutaneous.

Diphtheria is most commonly an infection of the upper respiratory tract and causes fever, sore throat and malaise.

A thick, gray-green fibrin membrane, the pseudomembrane, often forms over the site(s) of infection as a result of the combined effects of bacterial growth, toxin production, necrosis of underlying tissue and the host immune response.

This membrane can sometimes grow a lot and extend down to larynx causing blockage to airway passages causing death, or asphyxiation because of the blockage of airway passages.

Symptoms of pharyngeal diphtheria vary from mild pharyngitis to hypoxia due to airway obstruction by the pseudomembrane.

The involvement of cervical lymph nodes may cause profound swelling of the neck (bull neck diphtheria).

The skin lesions in cutaneous diphtheria are usually covered by a gray-brown pseudomembrane.

Life-threatening systemic complications, principally loss of motor function (as difficulty in swallowing) and congestive heart failure may develop as a result of the action of diphtheria toxin on peripheral motor neurons and the myocardium.

Fully immune individual have asymptomatic colonization and partially immune persons develop a mild respiratory illness.

Disease is followed by long lasting immunity which is also achieved by immunization using diphtheria toxoid (protective level is 0.01 1U/ml)/

The bacteria is sensitive to penicillin. So giving it will help with curing diphtheria.

The problem is with the toxin, if a patient has the toxin then the treatment is with an antiserum/antitoxin.

The antiserum can be from animal origin or human origin.

An antiserum is a serum that contains antibodies against the toxin, which will neutralize the toxin (IgG).

To make an antiserum: the toxin is injected into a horse in small doses and increase the doses and then the horse will produce antibodies against the toxin, the serum is then taken from the horse and it is known as horse serum. And now it can be given to people who are infected with the diphtheria or tetanus and the patient will survive.

In other cases you can use human serum that contains immunoglobulins against the toxin. This human serum is taken from people who are vaccinated against the disease.

You use a pool serum to make sure the antitoxin is present 100% .

Advantages and disadvantages of pool serum and animal serum:
Advantages:

Human serum can be used in tetanus, hepatitis B or some viral diseases. It doesn’t produce any allergic reactions. It lasts longer because it’s a similar protein to humans’ and it can be a better protection.

Animal serum: it protects from animal bites (snakes, scorpions, etc.)

Disadvantages:

It is difficult to use human serum in snake bites or scorpion bite, because it is hard to find other humans with antitoxins for it. In this case animal serum must be used. It can be infected especially if you have pool serum for many people it increase the risk of having Hepatitis B or HIV.

Animal serum: it can produce hypersensitivity reactions (allergy). Also it doesn’t last long, the antibodies are catabolized quickly in the human body.

Diagnosis: Requires demonstration of toxigenic C. Diphtheria in lesions (throat, nasopharynx, nose) by Elek test.

Laboratory diagnosis

Specimen: A throat swab

Culture: The swab is inoculated on Loeffler's serum medium and/or on blood potassium tellurite agar aerobically at 37C for 24.

On Loeffler's serum medium:

Corynebacteria grow much more readily than other respiratory pathogens

Used to enhance the characteristic microscopical appearance of corynebacteria

The colonies of C. diphtheriae are small, granular, grey, smooth, and creamy with irregular edges

Cultural characteristics

On blood tellurite agar (McLeod's blood agar)

It is a selective medium for the isolation of C. diphtheriae (Potassium tellurite)

3 biotypes of C. diphtheriae are characterized on BTA

i.e. Gravis, mitis and intermedius biotypes

The most severe disease is associated with the gravis biotype

Colony of gravis biotype is large, non-hemolytic & grey.

Colonies of mitis biotype are small, hemolytic and black, the least dangerous.

Colonies of intemedius biotype are intermediate in size, non-hemolytic with black center & grey margin.

In Vitro: Elek’s Test

When you isolate the Corynebacteria some areas are nontoxigenic, you’ll get a bacteria but it is unable to produce a toxin. But also there’s toxigenic areas where the toxin is produced and it is important when you isolate it to see wither its toxigenic or not and the Elek test will determine that.

Principle:

It is a toxin/antitoxin reaction

Toxin production by C.diphtheriae can be demonstrated by a precipitation between exotoxin and diphtheria antitoxin

Procedure:

Here we have inoculated perpendicularly the corynebacteria diphtheriae.

A strip of filter paper impregnated with diphtheria antitoxin is placed on the surface of serum agar at a 90 degree with the bacteria (like a cross)

Incubate the plate at 37C for 48 hrs

Epidemiology

Worldwide distribution.

Incidence has dropped dramatically since the introduction of the vaccine. The vaccines are made of toxoid: toxin treated with formaline. Toxoids lose their toxicity but attain their antigenicity, so it can be injected into people.

This vaccine is part of the MMR vaccination. It is attenuated. Given to babies to start producing the antibodies and protecting them.

Crowding and low immunity promote spread and it is maintained in the population by asymptomatic carriage in the oropharynx or on the skin of immune individuals.

Infections is acquired by direct (person-to-person) spread via respiratory droplets or skin contact.

30-60% of adults have antitoxin antibodies below the protective level and are at risk.

Epidemics in Russia; since 1994, 80.000 cases and 2000 deaths.

Immunity can be assessed by serology or the Shick test.

Control is based on immunization (adults should be reimmunized every 10 years)

Treatment

Antitoxin (20.000-100.000 units)

Antibiotics (Erythromycin and penicillin)

Related Organisms:

Listeria monocytogenes

Listeria: Because Lister discovered it.

Monocytogenes: it affects monocytes.

Gram positive rods, but may be coccoid in direct smears, sometimes arranged in short chains, motile at room temperature, and beta hemolytic.

It is an intracellular organism.

Widely distributed in nature.

They can multiply at low temperature and may accumulate in refrigerated food. (Meat, refrigerated cheese and dairies) and these are the most common sources of infection.

L. monocytogenes is ingested with raw contaminated food.

Listeria monocytogenes

No capsules

Resistant to cold, heat, salt, pH extremes and bile

Virulence attributed to ability to replicate in the cytoplasm of cells after inducing phagocytosis; avoids humoral immune system

If someone is infected with it, it will be taken up by the monocytes and macrophages inside, and it produce a chemical substance known as listeriolysin this will dissolve the phagolysosome and the bacteria escapes into the cytoplasm and still live inside the cytoplasm without being killed by the phagolysosomes.

Phagolysosomes usually produces hydrolytic enzymes that will kill the bacteria. The listeria monocytogenes escapes from the phagolysosome into the cytoplasm and grows there intracellular without being killed.

The macrophages must be activated in order to produce free oxygen radicals and this will eventually kill the bacteria.

They produce an invasion factor that enables them to penetrate host cells of the epithelial lining.

They are facultative intracellular parasites.

Normally, the immune system eliminates the infection before it is spread.

L. monocytogenes infection is acquired from food (vegetable or animal products).

Most cases occur in immunocompromised hosts; newborns, elderly, cancer patients and transplant recipients. Pregnant women as well.

It may be transmitted congenitally (mild flue like in mother) and the fetus develops a serious illness.

Depending on the stage of gestation, the fetus is either stillborn or born with signs of congenital manifestations, a condition known as granulomatosis infantiseptica.

Listeriosis is a serious disease of humans with a mortality of more than 25%.

There are two main clinical manifestations, sepsis and meningitis(mainly in newborns).

Meningitis is often complicated by encephalitis which is exceptional among bacterial infections.

Occasionally, pyogenic infections of various organs have been found.

Relapses may occur after apparent recovery.

Diagnosis is made by isolation of the organism.

Control is achieved by hygienic food processing and storage. For example: pasteurization.

Treatment: most antibiotics except cephalosporins can be given (e.g.. ampicillin and aminoglycosides)

Epidemiology and Pathology

Primary reservoir is soil and water; animal intestines

Can contaminate foods and grow during refrigeration

Listeriosis - most cases associated with dairy products, poultry, and meat

Often mild or subclinical in normal adults

Immunocompromised patients, fetuses and neonates; affects brain and meninges

20% death rate

Diagnosis and Control

Culture requires lengthy cold enrichment process.

Rapid diagnostic tests using ELISA available

Ampicillin and trimethoprimsulfamethoxazole (Cotrimoxazole)

Prevention – pasteurization and cooking

(Slides from 61-66 are not included)

CLOSTRIDIA

Introduction

All medically important anaerobic gram-positive, spore- forming bacilli are classified in the genus clostridium.

The genus consists of more than 100 species that have diverse biochemical properties and limited genetic relatedness.

Although most members of the genus are strict anaerobes, some are aerotolerant.

The organisms are ubiquitous (widely distributed in nature); present in soil, water, sewage, and as part of the normal microbial flora in the gastrointestinal tract of animals and humans.

Most clostridia are harmless saprophytes, although some are well-recognized human pathogens with a clearly documented history of causing disease such as:

Tetanus (clostridium tetani)

Botulism (Clostridium botulinum)

Gas gangrene (clostridium perfringens)

(Clostridium difficile) which is the most commonly encountered of all these clostridium.

These diseases became rare and clostridia are more commonly associated with skin and soft tissue infections, food poisoning, and antibiotic associated with diarrhea and colitis.

They are known for producing numerous histolytic toxins, enterotoxins, and neurotoxins. And according to the species that produce them you’ll get different diseases.

It destroys tissues and produces gangrene, and because in the process of destruction of all these enzymes there’s fermentation, which will lead to release gases, so there will be necrosis of tissues and at the same time there will be gas in the tissues. That’s why it’s called gas gangrene.

Five types A-E

It is the Clostridial species most frequently isolated in clinical specimens.

It either can be associated with simple colonization or can cause severe life-threatening disease.

It produces four major lethal toxins, seven minor toxins, and an enterotoxin that alters membrane permeability (cytotoxic and enterotoxic).

In order for this bacteria to grow, there must be the right conditions, normally there is an injury, surgical procedure, wound cut, and the tissues is low on oxygen: the bacteria gets in and finds the right conditions to grow and produce toxins.

Toxins will kill more tissue and produce more necrosis, and more anaerobic conditions for the organism to grow.

Major Toxins:

Alpha (phospholipase C): the most traumatic/potent, it destroys RBCs, WBCs and so on. It increases vascular permeability and it is hemolytic.

Beta: It is necrotizing and it induces hypertension by release of catecholamines.

Epsilon: It increases permeability of the gastrointestinal wall.

Iota: It is necrotizing and it increases vascular permeability.

The most important is alpha toxin which lyses erythrocytes, platelets, leukocytes, and endothelial cells.

Alpha toxin is associated with increased vascular permeability, massive hemolysis, bleeding, tissue destruction, hepatic toxicity, and myocardial dysfunction (bradycardia and hypotension).

Beta toxin is responsible for the necrotic lesions in necrotizing enterocolitis(enteritis necroticans, pig- bel).

Minor toxins (read only)

Delta (hemolytic)

Theta (hemolytic, cytolysin)

Kappa (collagenase, gelatinase)

Lambda (protease)

Mu (hyaluronidase)

Nu (DNAse)

Neuraminidase.

Clinical Syndromes:

Myonecrosis (Gas gangrene):

Gangrene is the death of tissues; the most commonly encountered gangrene in clinical practice is due to diabetes. (diabetic foot) due to compromised blood supply.

It involves the skin and subcutaneous tissue and muscles,

Initial trauma to host tissue damages muscle and impairs blood supply.

myonecrosis in skin, subcutaneous tissue and muscles Lack of oxygenation causes the oxidation-reduction potential to decrease and allows the growth of anaerobic clostridia. To produce toxins which will destroy tissues and fermentation will cause gas bubbles.

Infected muscle becomes discolored (purple mottling) and edematous and produces a foul-smelling exudate.

Gas bubbles are generated as a result of anaerobic fermentation.

As capillary permeability increases, the accumulation of fluid increases and venous return eventually is curtailed.

Septicemia although rare, may occur in the late stages of the disease.

Septic shock may happen due to the large number of cytokines not due to the presence of bacteria.

Severe shock with massive hemolysis and renal failure is usually the ultimate cause of death.

It is primarily caused by C. perfringens but C. septicum and C. novyii may cause it.

It illustrates the full virulence potential of histotoxic clostridia.

Onset is characterized by intense pain, generally 1 week after introduction by trauma or surgery.

The progression from the time of onset through extensive muscle necrosis, shock, renal failure, and death is rapid, frequently occurring in less than 2 days.

Macroscopic examination of muscle reveals devitalized necrotic tissue with the presence of gas.

Extensive bleeding and hemolysis are characteristic.

Most common with gun shots wounds and accident wounds.

The Perferenges mostly cause gas gangrene these other cases are less common

Soft tissue Infections (Cellulitis, fasciitis)

Differs from myonecrosis by the lack of muscle involvement but it shares a dismal outcome. Because muscles have good blood (oxygen) supply.

Surgical intervention is generally unsuccessful because of the rapidity with which the organisms spread.

In cellulitis, the infecting organisms invade only tissue that is already dead, the infection does not spread to healthy undamaged tissue.

No systemic toxemia (usually) but, it could lead to tissue destruction, and septicemia and septic shock, might lead to death.

Food poisoning

Abdominal cramps and watery diarrhea in 5-24 hours but no fever, nausea or vomiting.

Disease is due to ingestion of meat products contaminated with enterotoxin producing type A

C. perfrengens. (self limited in 24 hours).

Enteritis Necroticans

Acute necrotizing process in the small intestine that is characterized by abdominal pain, bloody diarrhea, shock, and peritonitis. (the intestines will rot because of the beta toxin)

Beta toxin is responsible for it.

Bacteremia

More than half of cases represent transient bacteremia, or more likely contamination and therefore are insignificant.

Treatment

Prevention must be taken in consideration, by cleaning wounds and getting rid of dead tissue completely.

Aggressive surgical debridement of all dead tissue and high dose penicillin therapy, and imipenem will kill the bacteria.

Hyperbaric treatment: putting the gangerenic part of the body under high pressure of oxygen, and it should kill the bacteria

Mortality is 40-100%

Good luck.

Seema Daradkeh.