Colstridium

Anaerobes of clinical Importance

Oxygen

ClassDefinitionExamples

Obligate aerobe

Grow only in the presence of O2

Mycobacterium tuberculosis

Obligate anaerobe

Can not grow in the presence of O2

Clostridia

Facultative anaerobe

Can grow in the presence or absence of O2

Most bacteria of medical importance

Microaerophilic bacteria

Require low O2 tension

Campylobacter

22222 OO2H2OO2H

In the presence of oxygen, two toxic substances to the bacteria are produced which are hydrogen peroxide and superoxide anion.

In obligate aerobes and facultative anaerobes:

Catalase and peroxidase enzymes degrade hydrogen peroxide.

Superoxide dismutase enzyme degrades superoxide anion.

BUT

In obligate anaerobes: These enzymes are not present. So, the presence of oxygen is toxic to them.

CLASSIFICATION

Anaerobic spore forming bacilli (Clostridia)Gram negative bacilli non-sporing forming

(Bacteroides)Anaerobic streptococci (Peptostreptococcus)Anaerobic staphylococcus (Peptococcus)Gram negative diplococci (Veillonella)Gram positive bacilli (Actinomyces)

HABITAT I:

These organism are normal flora in:• A. Oropharynx eg. . Fusobacteria and

Veillonella • B. Gastrointestinal tract

– Found mainly in the large colon in large numbers– Total number of anaerobes = 10 11

– While all aerobes (including E. coli) = 10 4

– examples are (1) B acteroides fragilis (2) Bifidobacterium species

• C. Female genital tract (mainly in the vagina)

Features of anaerobic infections• Infections are always near to the site of

the body which are habitat.1.Infection from animal bites.2.Deep abscesses3.The infections are also polymicrobial 4.Gas formation, foul smell5.Detection of "Sulphur granules"' due to

actinomycosis 6.Failure to grow organism from pus if not

culture anaerobically.7.Failure to respond to usual antibiotics.

Infections begin :• Disruption of barriers

–Trauma–Operations–Cancerous invasion of tissues

• Disruption of blood supply–Drops oxygen content of tissue–Tissue necrosis

what are the infection caused by these anaerobic organisms

Post operative wound infectionBrain, dental, lung abscessIntra abdominal abscess, appendicitis,

diverculitis Infection of the female genital tract: Septic

abortion, endometritis , , puerperal infection and endometritis , pelvic abscess or breast abscess

Diabetic foot infections

Laboratory diagnosis:

• When anaerobic infection is suspected;a) Specimens have to be collected from the site

containing necrotic tissue.b) Pus is better than swabs.c) Specimens has to be send to the laboratory

within 1/2 hour d) Fluid media like cooked meat broth are the

best culture media.e) Specimens have to incubated anaerobically

for 48 hours.

Organism groupsGram negative rods

BacteroidesPrevotellaPorphyromonasFusobacterium ButyrivibrioSuccinomonas

Bacteroides

Strict anaerobePleomorphicGram negative bacilli (cocco bacilli)Normal flora in

Oropharynx Gastrointestinal tractVagina

Bacteroides

• B. Fragilis, B. Vulgaris, B. Uniformis–Account for 1/3 of all isolates–Resistant to 20% bile–Resistant to many antibiotics

• Penicillin, kanamycin, vancomycin, colistin – and many more

–No pigmentation of colonies or fluorescence

•  

Bacteroides other species

Bacteroides species other than b. Fragilis groupBile sensitiveResistant to kanamycin onlySome pigmented

Other gram negative rods• Fusobacterium necrophorum• Gram negative bacilli• Peritonisillar intrnal jugular vein

thrombosisemboli to the lung• Peptococcus • Gram positive cocci in clusters• Peptostreptococcus• Gram positive cocci in chains• Brain abscess• Veillonella parvula• Gram negative cocci

Clostridium SPP

Clostridium spp. Anaerobic Gram-Positive Spore-Forming Bacilli Four broad types of pathogenesis:

1. Histotoxic group — tissue infections (C. perfringens type A, exogenously acquired more commonly than endogenously) (C. septicum; endogenously-acquired)

a. cellulilitis b. myonecrosis c. gas gangrene d. fasciitis

2. Enterotoxigenic group — gastrointestinal disease a. clostridial foodbome disease (8-24h after ingestion of large numbers of organisms on con-taminated

meat products, spores germinate, enterotoxin produced (C. perfringens type A) b. necrotizing enteritis (beta toxin-producing C.perfringens type C)

(C. difficile endogenously-acquired or exogenously-acquired person-to-person in hospital) c. antibiotic-associated diarrhea d. antibiotic-associated pseudomembrane colitis

3. Tetanus (exogenously acquired) — C. tetani neurotoxin

a. generalized (most common) b. cephalic(primary infection in head, comnnonly ear) c. localized e. neonatal (contaminated umbilical stump)

4. Botulism (exogenously acquired) — C. botulinum neurotoxin

a. foodborne (intoxication,1-2days incubation period) b. infant (ingestion of spores in honey) c. wound (symptoms similar to foodborne, but 4 or more days incubation)

Spores Clostridium form endospores under

unfavourable environmental conditions

Spores are a survival mechanism

Spores are characterized on the basis of position, size and shape

Most Clostridium spp., including C. perfringens and C. botulinum, have ovoid subterminal near an end) ( (OST) spores

C. tetani have round terminal (RT) spores

Clostridium Associated Human Disease

Clostridium perfringens

Clostridium perfringens — histotoxic or enterotoxigenic infections

Morphology and Physiology • large, rectangular bacilli (rod) staining gram-positive • spores rarely seen in vitro or in clinical specimens (ovoid, subterminal) • non-motile, but rapid spreading growth on blood agar mimics growth of motile organisms • aerotolerant, especially on media supplemented with blood • grow at temperature of 20-50°C (optimum 45°C) and pH of 5.5-8.0

Pathogenicity Determinants (note that toxins include both cytolytic enzymes and bipartite exotoxins) • four major lethal toxins (alpha (), beta (), epsilon (), and iota () toxins) and an enterotoxin • six minor toxins (delta(), theta(), kappa(), lambda(), mu(), nu()toxins) & neuraminadase • C. perfringens subdivided into five types (A-E) on basis of production of major lethal toxins • C. perfringens Type A (only major lethal toxin is alpha toxin) responsible for histotoxic and

enterotoxigenic infections in humans; Type C causes necrotizing enteritis (not in U.S.)

Lab Identification • direct smear and Gram stain, capsules upon direct examination of wound smears • culture takes advantage of rapid growth in chopped meat media at 45° C to enrich and then

isolate onto blood agar streak plate after four to six hours • gas from glucose fermentation • in vivo toxicity testing and identification of the specific toxin types involved • double zone of hemolysis on blood agar (p-hemolytic theta(e) toxin, a-hemolytic alpha(oc) toxin) • Nagler rxn; precipitation in serum or egg yolk media; oc -toxin (phospholipase C) is a lecithinase • "stormy" fermentation (coagulaltion) of milk due to large amounts of acid and gas from lactose

Diagnosis/Treatment of systemic infection — Early diagnosis and aggressive treatment essential • removal of necrotic tissue (surgical debridement) • Penicillin G in high doses if more serious infection

Of poorly defined clinical value are: • administration of antitoxin • hyperbaric oxygen (dive chamber) adjunct therapy (??inhibit growth of anaerobe??)

Summary of C. perfringens

Infections

Micro & Macroscopic C. perfringens

NOTE: Large rectangular gram-positive bacilli

Inner beta-hemolysis = θ toxin Outer alpha-hemolysis = α

toxin

NOTE: Double zone of hemolysis

Clostridium perfringens • Pathogenesis: Traumatic open wounds or

compound fractures lead to muscle damages and contamination with dirt etc,

• Mainly in war wounds, old age, low blood supply and amputation of thigh (required prophylaxis with penicillin

• Prevention and Treatment• Remove dead tissue , debris and foreign

bodies .Penicillin and hyperbaric oxygen in some cases

Clostridium perfringens • Can leads to the following diseases • 1) Wound Contamination• 2) Wound infection• 3) Gas Gangrene - most important disease• 4) Gas Gangrene of the uterus in criminal

abortion• 5) Food Poisoning : Spores are swallowed

Germinate in gut after 18 hours(Toxin production) abdominal pain and diarrhoea

Clostridial Cellulitis

C. perfringens Virulence FactorsM

ajor

Min

or

Exotoxins Associated with C. perfringens Types A-E

Major

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.

Clostridium tetani

Clostridium tetani — agent of tetanus

Morphology and Physiology- • long thin gram-positive organism that stains gram negative in old cultures • round terminal spore gives drumstick appearance • motile by peritrichous flagella • grow on blood agar or cooked meat medium with swarming • beta-hemolysis exhibited by isolated colonies • spores resist boiling for 20 minutes

Antigenic Structure- flagella (H), somatic (0), and spore antigens. Single antigenic toxin characterizes all strains.

Pathogenicity Determinants" • play a role in local infection only in conjunction with other bacteria that create suitable

environment for their invasion • systemic-acting, plasmid-mediated A-B neurotoxin (tetanospasmin) produced intracellularly

Mode of Action — one of most poisonous substances • binds gangliosides in synaptic membranes (synapses of neuronal cells) and blocks

release of inhibitory neurotransmitters; continuous stimulation by excitatory transmitters • muscle spasms (spastic paralysis) (trismus (lockjaw), risus sardonicus, opisthotonos),

cardiac arrhythmias, fluctuations in blood pressure

Lab Identification" • use characteristics of resistance to heat, motility, and toxin production to help identify

Diagnosis/Treatment/Prevention • empirical diagnosis on basis of clinical manifestations • treat to prevent elaboration and absorption of toxin

clean wound (debridement), control spasms metronidazole administered to eliminate vegetative bacteria that produce neurotoxin passive immunity (human tetanus immunoglobulin); vaccination (active) as preventative antitoxin administered to bind free tetanospasmin

C. tetani Infections

Summary of Clostridium tetani Infections (cont.)

Clostridium tetani Gram Stain

NOTE: Round terminal spores give cells a “drumstick” or “tennis racket”

appearance.

Clinical Forms of Tetanus

Opisthotonos in Tetanus Patient

Risus Sardonicus in Tetanus Patient

Clostridium botulinum

CLOSTRIDIUM BOTULINUIM

• Found in soil ponds and lakes• Toxin is exotoxin (protein) heat labile at 100

OC and resist gastrointestinal enzymes• It is the most powerful toxin known Lethal

dose 1 µg human and 3 kg kill all population of the world .It dictated for by lysogenic phage

• Botulism• From canned food., sea food e_g. salmon

when it is not well cooked (Spores resist heat at 100 oC ) then multiply and produce toxin

CLOSTRIDIUM BOTULINUIM

• Symptoms• Abnormal eye movement as if cranial nerve

affected when bulbar area of the brain affected. Finally the patient might develop respiratory and circulatory collapse

• Enfantile Botulism• Ingestion of Spores germination in the

gutBotulism .• Botulism Patogenesis• Attacks neuromuscular junctions and prevents

release of acetylcholine that can leads to paralysis

CLOSTRIDIUM BOTULINUIM

• Laboratory diagnosis• Suspected food and the patient faeces culture

or serum toxin detection by mice inoculation after weeks paralysis and death

• Treatment • Maily supportive and horse antitoxin in sever

cases• Prevention• Adequate pressure cooking autoclaving and

heating of food for 10 minutes at 100 OC

Clostridium dificile

Clostridium Difficile

• Normal flora in gastroentestinal tract after exposure to antibiotics and killing of other normal flora, this organism will multiply witch then produce toxin that has two components– A–Subunit enterotoxin (cause diarrhea) – B-Subunit Cytotoxic ( kill the cells ie necrosis)– Hyaluronidase and spore forming so bacteria can

survive for a month in hospital environment • PSEUDOMEMBRANE COLITIS is the clinical

manifestation of this disease which composed of bacteria , fibrin , WBCs and dead tissue cells

• Sever dehydration , intestinal obstruction and perforation are some of complication of this syndrome

Clostridium Difficile

Laboratory diagnosis: this organism hard to grow in the laboratory required special media and growth of the organism in solid media required cell line culture to demonstrate cytotoxicity of the organism.

The simplest method for diagnosis by detection of the toxin in the stool by immunological testing (ELISA)

Clostridium Difficile

• Treatment : Metronidazole or and oral vancomycin in sever cases

• Prevention: This organism form spores and hard to control in the hospital because they are resistant to alcohol decontamination ( use Na hypochloride instead).

• Patient need to be isolated and contact need to be screened to find out if they carrying the toxic strain of the bacteria.