Agents of anaerobe Agents of anaerobe infectionsinfections

ClostridiaClostridia

General gropes of bacteria with General gropes of bacteria with respect to oxygen requirementsrespect to oxygen requirements

►Obligate (strict) aerobesObligate (strict) aerobes►Obligate (strict) anaerobesObligate (strict) anaerobes►Facultative anaerobesFacultative anaerobes►MicroaerophilesMicroaerophiles►Aerotolerant microorganisms Aerotolerant microorganisms ►Capnophiles Capnophiles

Obligate anaerobes Obligate anaerobes

There are microorganisms that cannot There are microorganisms that cannot multiply is any oxygen is present.multiply is any oxygen is present.

Some members are actually killed by traces of oxygen because they cannot modify the toxic forms of oxygen produced in metabolism.

Among the more important anaerobic pathogens are some species of Clostridium, Bacteroides

Enzyme content of bacteria with Enzyme content of bacteria with

different requirement for oxygendifferent requirement for oxygen

Name Name Enzyme content for OEnzyme content for O22 detoxification detoxification

Strict aerobeStrict aerobe Catalase – Catalase – HH22OO22 H H22O + OO + O22

Superoxide dismutaseSuperoxide dismutase

OO22- - +2H+ +2H+ OO22 + H + H22OO22 H H22O + OO + O22

Facultative Facultative anaerobe anaerobe

Catalase Catalase

Superoxide dismutaseSuperoxide dismutase

MicroaerophilMicroaerophilee

Small amount of catalase and superoxide Small amount of catalase and superoxide dismutasedismutase

AerotolerantAerotolerant Superoxide dismutaseSuperoxide dismutase

Strict Strict anaerobeanaerobe

Neither catalase nor superoxide Neither catalase nor superoxide dismutasedismutase

Cultivation of anaerobes Cultivation of anaerobes

Anaerobe microorganisms that are Anaerobe microorganisms that are medically important medically important

Spore-formingSpore-forming:: Gram-positive bacteriaGram-positive bacteria

ClostridiumClostridium

Nonspore-formingNonspore-forming::

Gram-positive bacteriaGram-positive bacteria :ActinomycesBifidobacteriumLactobacillusPropionobacterium

Gram-positive cocci:

PeptococcusPeptostreptococcus

Gram-negative bacteria and curved formsAnaerovibrioBacteroidesBacteroides (>40 species)FusobacteriumPrevatellaCampylobacterLeptotrichiaPorphyromonas TreponemaBorellia

Gram-negative cocci

Veilonella

General properties of ClostridiaGeneral properties of Clostridia

Gram-positive spore-forming rodsGram-positive spore-forming rods

Oval or spherical spores often have Oval or spherical spores often have diameter more than the vegetative cell diameter more than the vegetative cell (therefore is named Clostridium)(therefore is named Clostridium)

Anaerobes Anaerobes

Catalase-negativeCatalase-negative

Widely distributed in soil, vegetation, and Widely distributed in soil, vegetation, and commensals inhabit the bodies of humans commensals inhabit the bodies of humans and other animalsand other animals

There are over than 120 speciesThere are over than 120 species

General virulence factor of pathogenic General virulence factor of pathogenic clostridia is very powerful exotoxin clostridia is very powerful exotoxin

Clostridia that are implicated in Clostridia that are implicated in serious human diseaseserious human disease

Cl.tetaniCl.tetani causes tetanuscauses tetanus Cl.botulinumCl.botulinum causes botulismcauses botulism Cl.perfringensCl.perfringensCl.novyiCl.novyiCl.septicumCl.septicumCl.histolyticumCl.histolyticumCl.sporogenesCl.sporogenesCl.difficile cause Cl.difficile cause causes pseudomembranous causes pseudomembranous colitis (or antibiotic-associated colitis)colitis (or antibiotic-associated colitis)

cause gas gangrene

first threefirst three – in monoculture

last twolast two – only in association with one of first

Clostridium perfringensClostridium perfringens

The main causative agent of gas gangrene The main causative agent of gas gangrene (or anaerobe infection of wound).(or anaerobe infection of wound).Gram-positive rod with central or terminal Gram-positive rod with central or terminal endosporeendosporeEncapsulatedEncapsulatedNonmotile Nonmotile Anaerobes Anaerobes Commonly founded in human and animal intestines Commonly founded in human and animal intestines as well in soilas well in soilBesides gas gangrene causes Besides gas gangrene causes food poisoningfood poisoning with with diarrhea, abdominal cramps, nausea and vomiting diarrhea, abdominal cramps, nausea and vomiting ((symptoms are connected with spore germination in the symptoms are connected with spore germination in the intestine and enterotoxin production)intestine and enterotoxin production)

Smear of Cl.perfringensSmear of Cl.perfringens

Cl.perfringens in the smear Cl.perfringens in the smear from wound exudatefrom wound exudate

Cl.perfringens in the smear Cl.perfringens in the smear from wound exudatefrom wound exudate

Sporulation ofSporulation of Cl. perfringensCl. perfringens

Five different types of Cl.perfringens are Five different types of Cl.perfringens are recognized, depend on which toxins they recognized, depend on which toxins they produce – A, B, C, D, Eproduce – A, B, C, D, E

Cl.perfringens virulence factorsCl.perfringens virulence factors

exotoxinexotoxin - lecithinase (the most potent one) kills - lecithinase (the most potent one) kills leukocytes, causes red blood cell rupture, edema leukocytes, causes red blood cell rupture, edema and tissue destruction by degrading the lecithin and tissue destruction by degrading the lecithin component of their membranes. On blood agar component of their membranes. On blood agar causes causes -hemolysis-hemolysis

exotoxinexotoxin causes causes -hemolysis on the blood agar-hemolysis on the blood agar

Enzymes: Enzymes: CollagenaseCollagenase Hyaluronidase Hyaluronidase DNaseDNase

Lecithinase and hemolytic activities Lecithinase and hemolytic activities ofofCl. perfringensCl. perfringens

Lecithinase and hemolytic activities Lecithinase and hemolytic activities ofofCl. perfringensCl. perfringens

Absence of lecithinase effect when antitoxin is used

Absence of lecithinase effect when antitoxin is used

Hemolysis Hemolysis Lecithinase effect Lecithinase effect

Absence of hemolysis when antitoxin is used

Absence of hemolysis when antitoxin is used

Pathogenesis of gas gangrene Pathogenesis of gas gangrene

Agent Agent – Cl.perfringens: other Clostridia less – Cl.perfringens: other Clostridia less frequently frequently

Sours of infection.Sours of infection. The natural habitat of The natural habitat of C.perfringens included both the soil and the human C.perfringens included both the soil and the human intestine.intestine.

Transmission.Transmission. Ednospores commonly contaminate Ednospores commonly contaminate wounds (especially puncture and gunshot), crushing wounds (especially puncture and gunshot), crushing injuries (if anaerobic condition)injuries (if anaerobic condition)

Incubation periodIncubation period – 1 to 5 days – 1 to 5 days

Spores germinate, vegetative bacilli multiply and Spores germinate, vegetative bacilli multiply and release enzymes for invasion and release enzymes for invasion and exotoxinexotoxin

Gas gangrene therefore the gas forms in tissues due to fermentation of muscle carbohydrates, amino acids and glycogen.

Changes in the infected Changes in the infected tissue tissue

The gas formed in the tissue can destroy muscle structure

Pathogenesis of gas gangrene Pathogenesis of gas gangrene

Symptoms: pain, edema, and a bloody exudate in the lesion, fever, tachycardia, and blackened necrotic tissue filled with bubbles of gas.

Treatment Treatment

Surgical removalSurgical removal of all dead and infected tissues of all dead and infected tissues

Hyperbaric oxygenHyperbaric oxygen treatment (it inhibits growth of treatment (it inhibits growth of the clostridia, thereby stopping release of toxin, the clostridia, thereby stopping release of toxin, and it also improves oxygenation of injured tissue)and it also improves oxygenation of injured tissue)

Antibiotics Antibiotics administration (penicillin)administration (penicillin)

Laboratory diagnosisLaboratory diagnosis

Bacterioscopy.Bacterioscopy. Digested muscle, Gram- Digested muscle, Gram-positive rods, and few of no leukocytes positive rods, and few of no leukocytes

Bacteriological methodBacteriological method. Cultivation bacilli in . Cultivation bacilli in anaerobic condition. Identification according anaerobic condition. Identification according morphology, biochemical properties; toxin morphology, biochemical properties; toxin production. production.

Botulism is the most feared type of food Botulism is the most feared type of food poisoning because it can result in poisoning because it can result in paralysis and deathparalysis and death

Gram-positive anaerobe rod with Gram-positive anaerobe rod with subterminal sporesubterminal spore

Widely distributed in soils around the Widely distributed in soils around the worldworld

Has very powerful exotoxin Has very powerful exotoxin

Cl. botulinum morphologyCl. botulinum morphology

Cl.botulinum sporulation Cl.botulinum sporulation processprocess

Virulence factorVirulence factor

Exotoxin.Exotoxin. It is neurotoxin, meaning that it acts It is neurotoxin, meaning that it acts against the nervous system, and is one of the most against the nervous system, and is one of the most powerful poisons known. powerful poisons known.

The toxin acts by blocking the transmission of nerve The toxin acts by blocking the transmission of nerve signals to the muscles, producing paralysis.signals to the muscles, producing paralysis.

100 milligrams of the toxin would be sufficient to kill all 100 milligrams of the toxin would be sufficient to kill all population of the Earth. population of the Earth.

1 gram of purified crystallized botulinum toxin contains 1 gram of purified crystallized botulinum toxin contains 10101212 fatal doses for a human fatal doses for a human

There are clinical forms of botulism: There are clinical forms of botulism:

Botulinum food poisoningBotulinum food poisoning is intoxication associated is intoxication associated with eating poorly preserved food, contaminated by with eating poorly preserved food, contaminated by Cl.botulinum spores. These spores later germinate (in Cl.botulinum spores. These spores later germinate (in anaerobic condition) and growth of the bacteria results in anaerobic condition) and growth of the bacteria results in the release of exotoxin into the food. the release of exotoxin into the food. The neurotoxin survives stomach acid and pepsin, is The neurotoxin survives stomach acid and pepsin, is absorbed into the blood stream, and is carried to nerves. absorbed into the blood stream, and is carried to nerves. Incubation period 12-36 hours.Incubation period 12-36 hours.

Wound botulism.Wound botulism. The spores enter a wound or The spores enter a wound or puncture, germinate, and produce toxinpuncture, germinate, and produce toxin

Infant botulism,Infant botulism, in which the bacilli grow in the gut and in which the bacilli grow in the gut and produce toxin (in contrast the infant, the adult intestinal produce toxin (in contrast the infant, the adult intestinal tract normally inhibits this sort of infection)tract normally inhibits this sort of infection)

Wound and infant botulism are generally milder than first form

SymptomsSymptoms

Diplopia, dysphagia, weakness, nausea, vomiting Diplopia, dysphagia, weakness, nausea, vomiting and diarrhea.and diarrhea.

Nerve involvement leads to generalized paralysis Nerve involvement leads to generalized paralysis and respiratory insufficiencyand respiratory insufficiency

Respiratory paralysis is the most common cause of Respiratory paralysis is the most common cause of death. death.

Despite treatment, about one fourth of the Despite treatment, about one fourth of the victims of botulism die.victims of botulism die.

Serotypes ofSerotypes of Cl.botulinum and its Cl.botulinum and its connection with toxigenicityconnection with toxigenicity

SerotypeSerotype Human Human sensitivity sensitivity Location of tox-genLocation of tox-gen

АА ++ Bacterial chromosome Bacterial chromosome

ВВ ++ Bacterial chromosomeBacterial chromosome

С1С1 -- Temperate bacteriophage Temperate bacteriophage

С2С2 -- Temperate bacteriophageTemperate bacteriophage

DD -- Temperate bacteriophageTemperate bacteriophage

EE ++ Temperate bacteriophageTemperate bacteriophage

FF ++ Bacterial chromosomeBacterial chromosome

GG -- Bacterial chromosomeBacterial chromosome

Treatment and preventionTreatment and prevention

Botulism is treated by administering the Botulism is treated by administering the antitoxin specific for the causative serotype antitoxin specific for the causative serotype of Cl.botulinum.of Cl.botulinum.

Infectious botulism is treated with antibiotics Infectious botulism is treated with antibiotics – penicillin (addition to antitoxin)– penicillin (addition to antitoxin)

Immunization with toxoid is not generally Immunization with toxoid is not generally availableavailable

Laboratory diagnosisLaboratory diagnosis

Biological method.Biological method. Botulinum toxin is demonstrable Botulinum toxin is demonstrable in uneaten food and the patient’s serum by in uneaten food and the patient’s serum by neutralization test in mouse (protection test) – mice neutralization test in mouse (protection test) – mice are inoculated with a sample of the clinical are inoculated with a sample of the clinical specimen and will die unless protected by antitoxin.specimen and will die unless protected by antitoxin.

Susceptibility of animals to Susceptibility of animals to botulinum toxinbotulinum toxin

► SusceptibleSusceptible:: Mouse Mouse Guinea pigGuinea pig Cat Cat Hors Hors DuckDuck

Unsusceptible:RatDog Chicken

Guinea pig, effected by Guinea pig, effected by botulinum toxin botulinum toxin

Clostridium tetaniClostridium tetaniCause tetanus or lockjawCause tetanus or lockjaw

Gram-positive motile anaerobic rod with spherical Gram-positive motile anaerobic rod with spherical terminal sporeterminal spore

Does not ferment carbohydrates but ferment Does not ferment carbohydrates but ferment proteins proteins

Is a common resident of cultivated soil and the Is a common resident of cultivated soil and the gastrointestinal tract of animalsgastrointestinal tract of animals

Has potent exotoxinHas potent exotoxin

Ziehl-Neelsen staining

Red acid-fast spore at the end of bacillus

Cl.tetani morphologyCl.tetani morphology

Spherical terminal spores

Cultural properties of Cl.tetani

Swarming growth on the solid media

Cl.tetani sporulation processCl.tetani sporulation process

Virulence factorVirulence factor Extremely potent exotoxinExtremely potent exotoxin with neurotoxic with neurotoxic

activity activity It consist of 2 components: tetanospasmin and It consist of 2 components: tetanospasmin and

tetanolysin (causes lysis of erythrocytes)tetanolysin (causes lysis of erythrocytes)

TetanospasminTetanospasmin attaches to motor nerves where they join attaches to motor nerves where they join the muscles. The toxin is taken into the nerve by the muscles. The toxin is taken into the nerve by endocytosis and is carried intra-axonally to the central endocytosis and is carried intra-axonally to the central nervous system, where it binds to ganglioside receptors nervous system, where it binds to ganglioside receptors and blocks release of inhibitory madiators at spinal and blocks release of inhibitory madiators at spinal synapses. synapses.

So tetanospasmin alters the usual regulation mechanisms So tetanospasmin alters the usual regulation mechanisms for muscle contraction. As result, the muscle are released for muscle contraction. As result, the muscle are released from normal inhibition and begin to contract from normal inhibition and begin to contract uncontrollably.uncontrollably.

Clinical symptomsClinical symptoms

The first symptoms are clenching of the jaw, followed in succession by extreme arching of the back, flexion of the arms, and extension of the legs.

Death is most often due to paralysis of the respiratory muscles and respiratory collapse. The fatality rate ranging from 10% to 70%.

PathogenesisPathogenesis Spores usually enter the body through accidental Spores usually enter the body through accidental puncture wounds, burns, frostbite, and crushed puncture wounds, burns, frostbite, and crushed body parts. Spores germinate and release exotoxin.body parts. Spores germinate and release exotoxin.

Neonatal tetanus occurs when spores enter the Neonatal tetanus occurs when spores enter the newborn through umbilical wound after delivery. newborn through umbilical wound after delivery.

Baby with neonatal tetanus

Laboratory diagnosisLaboratory diagnosis has has limited valuelimited value

Bacteriological method.Bacteriological method.

Biological method. Infecting of mouse causes Biological method. Infecting of mouse causes tetanus and death. Neutralization test in mice can tetanus and death. Neutralization test in mice can be used – mice protected by antitoxin (antibodies to be used – mice protected by antitoxin (antibodies to botulinum exotoxin) remain live. botulinum exotoxin) remain live.

Tetanus in animalsTetanus in animals

TreatmentTreatment and prevention and prevention

Tetanus is treated by administering tetanus Tetanus is treated by administering tetanus antitoxin to neutralize any toxin not yet attached to antitoxin to neutralize any toxin not yet attached to motor nerve cellsmotor nerve cells

An antibiotics such as penicillinAn antibiotics such as penicillin

For prevention use toxoidFor prevention use toxoid (formaldehyde-treated exotoxin)

CClostridiumlostridium difficile difficile

Cl.defficile causes pseudomembranous or antibiotic-associated colitis.

Antibiotics suppress drug-sensitive normal flora, allowing Cl.defficile to multiply and produce exotoxins A and B:

toxin Atoxin A is an enterotoxin that causes watery diarrhea toxin Btoxin B is a cytotoxin that causes damage to the colonic

mucosa, leading to pseudomembrane formation

Clindamycin and ampicillin are 2 of many antibiotics that cause this colitis

Action of B cytotoxin to colonic Action of B cytotoxin to colonic mucosamucosa

Yellow-white plaques formation on the

colonic mucosa.

Diagnosis, treatment, Diagnosis, treatment, preventionprevention

Diagnosis.Diagnosis. Exotoxin B is detected in filtrates of stool samples by Exotoxin B is detected in filtrates of stool samples by its cytotoxin effect on cultured cells. It is identified by its cytotoxin effect on cultured cells. It is identified by inhibition of cytotoxicity by specific antibody.inhibition of cytotoxicity by specific antibody.An ELISA (immuno-enzyme test) is available for An ELISA (immuno-enzyme test) is available for detection both exotoxins A and B.detection both exotoxins A and B.

Treatment.Treatment. The causative antibiotics should be The causative antibiotics should be withdrawn. Oral metronidazole should be given and withdrawn. Oral metronidazole should be given and fluids replaced. fluids replaced.

Prevention.Prevention. There are no vaccines There are no vaccines