Clostridium species - Microbiology

College Of Dentistry - Mosul University

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CLOSTRIDIUM SPECIES

Done By: Rami Bassam

3rd StageGroup – B-


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Clostridium

Gram-positive . obligate anaerobes capable of producing endospores which protect them in harmful environment . Individual cells are rod shaped.

The spores are usually wider than the rods, and are located terminally or sub terminally.

Most clostridia are motile by peritrichous flagella.while others have a capsule like

Clostridium.perfringens

Characteristics

Light Microscope Clostridium .tetani

http://en.wikipedia.org/wiki/Obligate_anaerobe


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Most Common Species

Clostridium consists of around 100 species that include common free-living bacteria as well as important pathogens There are five main species

responsible for disease in humans.C. perfringens: gas gangrene; food poisoningC. tetani: tetanusC. botulinum: botulismC. difficile: pseudomembranous colitisC.Sordellii : can cause a fatal infection in exceptionally rare cases after medical abortions


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SHAPES OF CLOSTIDIA The shape an position of spores varies in different species and is useful the identification of Clostridia *Central in Cl.bifermentans *Sub terminal in Cl.perfringens *Oval or terminal in Cl.tertium*Spherical and terminal giving drum stick appearance in Cl.tetani


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C. perfringens C. perfringens is a

relatively large Gram-positive short fat bacilli with blunt ends. It is capsulate and non-motile. Anaerobic. It grows quickly on laboratory media on blood agar ( B – Haemolytic )

Clostridium . PerfringensIn L.M

Clostridium . PerfringensIn E.M


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Media used for Cultivation

Liquid medium for cultivation cooked meat broth

Thiglyclolate broth

CMB contain unsaturated fatty acids which take up oxygen


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Clostridium. perfringens Culture & identification

1 =Control, 2 and 3= "Stormy clot fermentation“

Clostridium perfringens will produce acid and gases

B – HaemolysisOn blood agar media


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Litmus Milk TestIt distinguish between

different species of bacteria. The lactose (milk sugar), litmus (pH indicator), and casein(milk protein) contained within the medium can all be metabolized by different types of bacteria.

Milk is the first substrate used to maintain bacteria, this test allows for accurate depiction of bacterial types. The addition of litmus, other than explaining the pH type, acts as an oxidation-reduction indicator. The test itself tells whether the bacterium can ferment lactose, reduce litmus, form clots, form gas ,

Cl.perfringensStormy clot reaction


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Nagler’s Reaction(Lecithinase test )

This test is done to detect the lecithinase activity The M.O is streaked on the medium containing

egg yolk (contains lecithin) The plate is incubated anaerobically at 37 C

for 24 h Colonies of Cl. perfringens are surrounded by

zones of turbidity due to lecithinase activity and the effect is specifically inhibited if Cl. perfringens antiserum containing antitoxin is present on the medium

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C. perfringens Nagler Reaction

NOTE: Lecithinase (α-toxin; phospholipase) hydrolyzes phospholipids in egg-yolk agar around streak on right.

Antibody against α-toxin inhibits activity around left streak.

Nagler Reaction

College Of Dentistry - Mosul University 11

Positive Nagler ReactionProcedure of Nagler Reaction


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Specific Requirements for Anaerobic Cultivation

Anaerobic Jar


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Cl.perfringens Produce diseases

The organisms associated with gas gangrene attack soft tissues by producing toxins and aggressins, and some strains of the bacteria produce enterotoxins and cause food poisoning


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Collecting a Infected Tissues

If there are pieces of necrotic tissue in the wound, small pieces should be transferred aseptically into a sterile screw-capped bottle and examined immediately by microscopy and culture. Specimens of exudate should be taken from the deeper areas of the wound where the infection seems to be most pronounced.


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Virulence Factors Virulence factors

toxins – alpha toxin – causes RBC rupture, edema and tissue destruction

Enterotoxin collagenaseHyaluronidaseDNase


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Clostridium tetani Anaerobic bacteria of the genus species Clostridium it

is gram positive, slender bacillus and it has spherical terminal spores giving drum stick appearance

It is non capsulated & motile with peritrichus flagella

It produces a potent biological toxin, tetanospasmin, and is the causative agent of tetanus a disease

characterized by painful muscular spasms that can lead to respiratory failure and, in up to 40% of cases,

death.


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What is Tetanus? An infectious disease caused by

contamination of wounds from the bacteria Clostridium tetani, or the spores they produce that live in the soil, and animal feces

Infection follows when spores become activated and develop into gram-positive bacteria that multiply and produce a very powerful toxin

(tetanospasmin) that affects the muscles.


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Causes

Tetanus spores are found throughout the environment, usually in soil, dust, and animal waste.

Tetanus is acquired through contact with the environment; it is not transmitted from person to person.


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Causes The usual locations for the bacteria to enter the

body:

Puncture wounds (such as those caused by rusty nails, splinters, or insect bites.)

Burns, any break in the skin, and IV drug access sites are also potential entryways for the bacteria.


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Mode of action of tetanospasmin

1 .It inhibits the release of acetylcholine thus it interferes with neuromuscular transmission .

2 .Inhibition of postsynaptic spinal

neurons by blocking the release of an inhibiting mediator


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Laboratory Diagnosis:

Gram +ve stains grow on blood agar media aerobically

Inoculation of culture in to 2 mice one is protected with anti-toxin and the other is unprotected (dies with typical tetanic spasms )


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Clostridium difficile

Clostridium difficile " C. diff", is a

species of Gram-positive bacteria of the

genus Clostridium that causes

diarrhea and other intestinal disease when competing

bacteria are wiped out by antibiotics.

http://en.wikipedia.org/wiki/Gram-positive

http://en.wikipedia.org/wiki/Bacteria

http://en.wikipedia.org/wiki/Clostridium

Most common cause of nosocomial diarrhea.

Rate and severity of C. difficile-associated diarrhea (CDAD) increasing.

Clostridium difficile is a bacterium that can cause symptoms ranging from diarrhea to life-threatening inflammation of the colon.

Illness from C. difficile most commonly affects older adults in hospitals or in long

term care facilities and typically occurs after use of antibiotic medication

characteristic features:

Clostridia are anaerobic, spore-

forming rods (bacilli) .

Clostridia

habitantC. difficile bacteria can be found

throughout the environment — in soil, air, water, and human and animal feces. A

small number of healthy people naturally carry the bacteria in their large intestine.

But C. difficile is most common in hospitals and other health care facilities,

where a much higher percentage of people carry the bacteria.

Reservoir

Infectious AgentC.difficile

Means of Transmission

Portal of entry

Susceptible Host

Chain of infection Bowel and

Contaminated environment

Contact transmission from contaminated

hands,equipment or the

environmentFaecal/Oral

>65 years History of antibiotic use

Recent received healthcare

Underlying conditions Abdominal surgery

Weakened immunity

PathogenesisDisruption of

normal colonic flora

Colonisation with C. difficile

Production of toxin A +/- B

Mucosal injury and inflammation

Toxin production is cause of Pathogenesis

Toxigenic strains produce 2 major

toxins:toxin A

(enterotoxin)toxin B

(cytotoxin)Neutralised by C.

sordellii antitoxin

Signs and symptomsWatery diarrhea three or more times a day for two

or more daysMild abdominal cramping and tendernessWatery diarrhea 10 to 15 times a dayAbdominal cramping and pain, which may be

severeFeverBlood or pus in the stoolNauseaDehydrationLoss of appetiteWeight loss

Complications of CDADPseudomembraneous colitis

Perforation of the colon

Sepsis

Death

The SpecimenFresh is best (test within 2 hours)Liquid or loose, not solidIf unable to test within 2 hours, refrigerate at 4°C for up to 3 daysFreeze at -70°C (not -20°C) if testing will be delayedSpecimen quality will influence test results

Diagnosis of CDADEndoscopy

(pseudomembranous colitis)

CultureCell culture

cytotoxin testEIA toxin testPCR toxin gene

detection

ENDOSCOPY PICTURE

preventation

Hand washing

Contact precautions

Thorough cleaning

Perform Hand Hygiene after removing gloves.

Note : alcohol does not kill C. difficile spores, use of soap and water is more

efficacious

Clostridium Botulinum

Characteristic

Gram positiveObligate anaerobic bacillusSpores

Resistant to heat, light, drying and radiationSpecific conditions for germination

Anaerobic conditionsWarmth (10-50oC)Mild alkalinity

Transmission

IngestionOrganismSporesNeurotoxin

Wound contaminationInhalationPerson-to-person not documented

HUMAN DISEASEThree formsFoodborneWoundInfant

All forms fatal and a medical emergencyIncubation period: 12-36 hours

Diagnosis

The symptoms of botulism are similar to those of Guillain-Barré syndrome,

stroke, and myasthenia gravis.As a result, botulism is probably

substantially under-diagnosed.Serum electrolytes, renal and liver

function tests, complete blood tests, urinalysis, and electrocardiograms will

all be normal unless secondary complications occur.

The incubation period varies according to the mode of transmission, rate of

absorption of the toxin, and the total amount and type of toxin.

Foodborne botulism usually takes 24-36 hours to manifest itself.

Wound botulism often takes 3 or more days to appear.

Inhalation botulism has occurred very rarely, but incubation times may range

from several hours to perhaps days, again depending upon the type and

amount of toxin inhaled.

All four types of botulism result in symmetric descending flaccid paralysis of motor and autonomic nerves always

beginning with the cranial nerves. These symptoms are preceded by

constipation in cases of infant botulism.

Symptoms include:Double or blurred visionDrooping eyelidsDry mouthDifficulty SwallowingMuscle weakness

If left untreated symptoms may expand to include paralysis of

respiratory muscles as well as the arms and legs.

Asphyxiation due to respiratory paralysis is the most common cause of

death in botulism cases.

Prevention

Proper food preparation is one of the most effective ways to limit the risk of

exposure to botulism toxin.Boiling food or water for ten minutes can

eliminate some strains of Clostridium botulinum as well as neutralize the toxin

as well. However, this will not assure 100% elimination.

Limiting growth of Clostridium botulinum and the production of botulism toxin is an

alternative to their outright destruction.

Medical uses of BoNT

Now manufactured under the name “Botox”

Experimentally used for treating migraine headaches, chronic low back

pain, stroke, cerebral palsy, and dystonias (neurologic diseases

involving abnormal muscle posture and tension)

Frequent injections allows an individual to develop antibodies

Studies carried out to determine feasibility of other strains of BoNT

BoNT B manufactured for treatment of cervical dystonia in 2000 as “Myobloc”

BoNT A (Botox)

Botox injection patient 13 weeks after injection

Sadick, N. and A.R. Herman (2003). “Comparison of Botulinum Toxins A and B in the Aesthetic Treatment of Facial Rhytides.” Dermatologic Surgery 29:340-347.

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