20 Anaerobes

8/3/2019 20 Anaerobes

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CVM6202: Microbiology Rutherford

Anaerobic BacteriaAnaerobic Bacteria

Important Species

Gram-negative Gram-positive

Bacteroides Peptococcus

Fusobacterium Peptostreptococcus

Veillonella Eubacterium

Propionibacterium

Clostridium

Arachnia

Bifidobacterium

Many are commensals of the intestinal tract and are ubiquitous in nature Most are weak pathogens

Many genera produce toxins

Cause a wide range of diseases

Many infections are mixed

Two classes of anaerobes:

AerotolerantAerotolerant- resemble facultative organisms by growing with or without oxygen,

but their metabolism remains fermentative.

ObligateObligate- strict anaerobes that cannot tolerate O2, due to lack of superoxide

dismutase. Many lack catalase and peroxidases.

Obligate anaerobes are difficult to recover from samples:

poor transport technique

overgrowth by other organisms

collection of inappropriate samples

failure to consider anaerobes

2 O2- + 2H+ -----------------> H2O2 + O2Superoxide

dismutasecatalase

H2O + O2


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Suspect anaerobic involvement when:

tissue necrosis is observed

infection is near a mucous membrane

infection is secondary to a bite wound

the specimen has a foul odor

gas is present in tissue

blood is present in exudate

black discoloration to exudate (Bacteroides)

failure of all organisms observed in the Gram stain to grow in vitro

previous antibiotic therapy

Collection of samples

Critical for recovery and culture.

? Perform a direct smear for staining, especially if you are not culturing teh

sample immediately.

? Samples with normal flora are not suitable for culture.

? Aspirate material from lesion with a syringe, expel the air, then cap the

needle tightly.

? Swabs must be protected from drying by placing them in anaerobic broth.

? Tissue samples should be large or sealed in a bag under N2.

? Ship at 4 C.

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Culture of anaerobic organisms

If anaerobic involvement is suspected:

perform a direct exam first; this tells you the # and types of bacteria present.

inoculate both aerobic and anaerobic media.

use pre-reduced media. inoculate both solid and liquid media.

use platinum or pure nickel wire loops; nichrome is unacceptable.

place in anaerobic environment ASAP.

do not open to air for at least 2 days.

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Anaerobic Jar- a prepackaged kit contains a

catalyst that, when activated by water, will generate

hydrogen and carbon dioxide gases to exclude

atmospheric oxygen. The Gaspack system can

reduce the oxygen concentration to < 0.4% in 100

minutes.

naerobic Chamber- A non-mobile unit

at is self-enclosed. It is attached to a

trotgen gas source that will keep

mospheric oxygen out. Glove access

rmits a full working environment. A smallamber on the side can open to the outside

r sample entry. It is then flushed with

trogen prior to opening to the anaerobic

amber itself.


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Anaerobic Culture Media Anaerobic blood agar (ABA)

Non-selective

Blood agar plus:

yeast extract (amino acids, vitamins, minerals)

hemin- Fe+3 chelator

vitamin K1 L-cystine

cysteine-lowers redox potential

ABA with phenylethyl alcohol (APE or PEA)

Selective against Gram-negative facultative anaerobes

ABA plus:

phenylethyl alcohol (2.5 g/l) inhibits G-neg facultative aerobes

Kanamycin and vancomycin (KVA)

Selects for Gram-negative obligate anaerobes

ABA plus:

kanamycin (100 mg/l)

vancomycin (7.5 mg/l)

Bacteroides Bile Esculin (BBE)

Selects for pathogenic Bacteroides species

Trypticase agar plus:

gentamycin (100 g/ml)

20% bile- inhibits most non-pathogenicBacteroides spp.

0.1% esculin

Thioglycollate broth

Non-selective

TG broth plus:

hemin Vitamin K1 CaCO4 Indicator


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Genus: Clostridium

General characteristics of the genus

Large Gram-positive rods

Produce irregular, small colonies in 2-3 days Spore-forming

Easily decolorized

Catalase-negative

Motile

Ubiquitous distribution in soil

and associated with animals

At least 83 species recognized

Learning objectives

To learn the general characteristics of the genusTo know by which mechanism important species cause disease

Cause disease by two mechanisms:

I. Tissue invasion-produce weak toxins

Cl. chauvoei Cl. haemolyticum

Cl. perfringens Cl. difficile

Cl. novyi Cl. spiroforme

Cl. septicum Cl. colinum

II. Toxin forming, non-invasiveCl. tetani Cl. botulinum Cl. piliforme

To understand how toxins contribute to pathogenesis or clinical severity

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Clostridium spp. showing apparent Gram-negative rods due

over-decolorization.


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Clostridium chauvoei

Associated with Blackleg in ruminants

Infection and pathogenesis

Exposure is via ingestion of spores or through puncture wounds or duringteething.

Spores germinate and the organism replicates.

The organism moves through the lymph to the blood.

Organism localizes in muscle and liver.

Cl. chauvoei remains dormant in tissue until trauma occurs.

Replication and production of toxins.

Sudden onset of fever and tissue necrosis with swelling and gas.

? Death can occur in 1-3 days due to toxemia. The infection can self-cure, withthe tissue damage repairing itself.

Toxins

alpha toxin: lethal and necrotizing

beta toxin: DNase activity

gamma toxin: hyaluronidase delta toxin: hemolytic

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Tissue samples from cattle suffering from Blackleg.

Vaccination Vaccines against either the whole bacterium or the alpha toxin are available,

but are recommended to be used yearly.


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Clostridium septicum

Associated with malignant edema in sheep, horses and cattle and (rarely)with gastroenteritis in carnivores. Recently associated with necroticdermatitis in poultry.

Infection and PathogenesisMalignant edema

Intestinal commensal and therefore a common soil contaminant.

Enters via cuts or wounds.

Induces a rapidly spreading watery swelling; tissue appears gelatinous due to

connective tissue degradation.

Little or no gas is produced.

High fever induction with appearance of intoxication. Toxemia induces a rapid death in 12-48 hours.

Toxins

alpha: necrotizing and lethal

beta: DNase

gamma: hyaluronidase

delta: hemolytic

Necrotic dermatitis

Intestinal commensal and therefore a common contaminant of litter.

Enters via cuts or wounds, but occurance increases with stress/crowding.

Disease is sporadic.

Observe lameness, red/black patches on skin.

Prostration. Rapid death in 12-24 hours.

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Tissues from animals suffering

from Cl. septicum


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Clostridium novyi

Associated with gas gangrene and infectious necrotic hepatitis (Black disease) incattle, sheep and humans, andbig head in rams.

Infection and Pathogenesis

Two types of disease:

Type A- Gas gangrene and Big Head

Invasion of spores or bacterium via wounds, particularly in yearling rams as aresult of fighting

Replication and toxin production

Type A produces alpha, gamma, and epsilon toxins

Capillary damage exacerbates swelling

Type B- Black Disease

The organism grows in liver infarcts caused by liver damage (flukes?).

Local liver damage widens

Toxins are produced and get into bloodstream

Type B produces alpha, beta and zeta toxins

Death is due to alpha toxin which is a phospholipase C (lecithinase). The toxindestroys host cell membranes, especially on RBCs, causing hematuria. Death is

due to anoxia resulting from a loss of RBCs.

NOTE- this disease is very similar in appearance, mechanism of pathogenesis, and etiology

as Red water disease in cattle caused by Cl. haemolytica.

There is a Type C Cl. novyi that does not produce toxins and is not pathogenic (?)


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Clostridium perfringens

Associated with gas gangrene and fatal enterotoxemia in sheep, pigs, horses, dogs,and humans.

Commensal of the large intestine and skin and is found in the soil.

Exposure is due to ingestion of or exposure to fecal material.

Sanitation is most important for control.

Infection and pathogenesis The organism usually exists in the intestinal tract. Sporulation can occur

spontaneously, in alkaline conditions, following antimicrobial therapy,

immunosuppression, or dietary alterations.

Toxins are released during sporulation and enter bloodstream.

The hallmark ofCl. perfringens infection is diarrhea that lasts a few days.

There are 5 types ofCl. perfringens that produce up to 15 different exotoxins:

Type A:produces a lethal alpha toxin that kills muscle cells and immune cells:

associated with gas gangrene and avian necrotic enteritis (Yellow lamb disease).

This is probably the most commonly encountered type. Infection can be fatal

within 6-12 hours.

Type A is also a common cause of necrotizing myositis in horses at wounds orinjection sites.

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Cl. perfringens on BA plate showing

complete hymolysis.

Gangrenous tissue caused by Cl. perfringen

Tissue smear from Cl. Perfringens-

infected tissue.


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Type B: produces alpha, beta, and epsilon toxins; causative agent of lamb dysentery

but is not found in the US.

Type C: produces alpha and beta toxins; hemorrhagic enterotoxemia (bloody scours)

in piglets, calves, and lambs that ranges from subclinical to fatal. Babies dont

nurse, and there is increased incidence of piglet crushing.

Vaccinating sows can reduce incidence.

Type D: produces alpha and epsilon toxins; associated with overeating disease

(pulpy kidney disease) in sheep, an enterotoxemia The epsilon toxin is

converted by gastric juices, gets into the blood, and damages kidneys. The

brain can also be affected. Diagnose by neural signs, no fever, no vomiting,

and history. Post-mortum, kidneys are swollen and soft.

Vaccinate against epsilon toxin, but treatment is difficult once neural signs are

noted.

Type E:produces alpha and iota toxins; a very uncommon cause of an

enterotoxemia in calves and lambs.

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Sheep showing leaning and head-thrust behavior associated with Cl. perfringens toxemia.

Toxins-

alpha toxin is lethal and is produced by all types ofCl. perfringens

beta toxin is produced by types B, C, and E and is responsible for intestinal mucosa

loss leading to diarrhea

epsilon toxin produced by types B and D, it is converted to a lethal toxin in the gut

kappa and lambda toxins break down collagenase


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Clostridium botulinum

Causative agent of food poisoning in a wide variety of hosts.

Member of toxin forming, non-invasive group of Clostridia.

Forms highly resistant spores that are released during vegetative growth.

There are at least 7 types ofC. botulinum.

Humans, horses, and cattle are particularly susceptible. Cats have no reported natur

cases of disease.

Infection and Pathogenesis

C. botulinum makes at least 7 neurotoxins (A-G) which are used to type strains. Most

animal infections are caused by types C and D.

The toxins can be detected in feces, blood, and food source. Expsoure is usually viaingestion of preformed toxins, but vegetative growth of bacteria in gut can occur.

Ingestion of toxins

Toxins move to blood

Invasion of peripheral nerves (hours to days)

Blocks release ofacetylcholine resulting in flaccid paralysis, usually of ocular

and respiratory muscles first

Animals remain aware and can feel pain Limberneckin birds

quadraplegia in mammals

blurred vision

swallowing difficulty

mortality depends on toxin dose; recovery length depends on dose but ca

be as long as 1 month. Recovery is usually complete.

Diagnosis/Vaccination/TreatmentDiagnosis is based on finding the toxin in foodsource, urine, feces, or vomitus. An

electromyography can show abnormal nerve transduction.

Toxoid vaccines are of questionable efficacy, but administration of neutralizing

antibodies can help clear unbound toxins.

Supportive care involves feeding, preventing secondary infections, and expression of

body wastes.

Penicillin is effective for killing Cl. botulinum.


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Clostridium tetani

ausative agent oflockjaw in mammals, especially horses and humans.

Member of the toxin-forming, non-invasive group.

Ubiquitous distribution of spores in soil and feces.

Ten types based on flagella, but the toxin is the same in all types.

Toxins

tetanospasmin- a neurotoxin that causes clinical disease; highly lethal

hemolysin- tissue necrosis

nonspasmogenic toxin- function?

nfection and Pathogenesis Spores enter deep tissues with low oxygen tension.

Spores germinate and toxins are produced.

In 3-21 days, tetanospasmin toxin blocks neurotransmitter (glycine and gamma-

aminobutyric acid) release.

Toxins spread along:

* Peripheral nerves: ascending tetanus (from wound to trunk)* Hematogenous through lymph: descending tetanus (lockjaw)

Vascillating spasms occur; respiratory distress; victim remains conscious.

? Respiratory arrest, and death occur if toxin dose is high enough.

Diagnosis/Vaccination/Treatment Diagnosis based on clinical signs and organism in smears from lesion.

Antitoxins can neutralize unbound toxins, but bound toxins must be cleared naturall

Supportive care includes removal of stimulation, giving food and fluids, irrigating s

of infection, and antibiotics to kill vegetative organisms. Muscle relaxants can help

minimize discomfort. Can also vaccinate with toxoid (?). But recovery can take lot

of time and $.

Yearly vaccination is recommended for horses.

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Cattle showing clinical signs

of tetanus. Note the

sawhorse stance and rigid

tail (left) and the facial

convulsions with protrusion

of third eyelid (right).

Cl. tetani showing spores

one end of cell.


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Clostridium piliforme

Causative agent ofTyzzers Disease in mammals, especially laboratory rodents.

Gram-negative; used to be in the genusBacillus.

Obligate intracellular parasite.

Commensal of the intestinal tract of rodents.

Dogs and cats susceptible, but most cases are associated with rodent contact.

Infection and Pathogenesis Infection is linked to immunosuppression.

Bacteria or spores are ingested.

Bacteria grow in intestine until stress or some event permit entry into liver.

Clinical appearance is characterized by: Rodents- diarrhea, necrotic intestinal epithelium, and infiltration of

inflammatory cells.

Dogs/cats- show rapid onset of lethargy anorexia, and abdominal pain.

Hepatomegaly and focal lesions on liver are apparent at necropsy. Liver

enzymes (AAT, SDH) are often elevated.

Death can occur within 24 hours of clinical signs.

Diagnosis/Vaccination/Treatment Diagnosis is usually based on necropsy results. Culture of the organism is

difficult and time-consuming.

Treatment is as yet not successful, in part due to rapid progression of the disease.

Vaccination is not practiced, although formalin-inactivated vaccines are successful

in mice.


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Genus: Bacteroides

General characteristics of the genus Numerous species exist, but few are pathogenic

Gram-positive rods

Non-spore forming.

Many species produce black pigment on KVA plates.

Many fluoresce under long-wave UV.

Commensals of the GI tract.

Require complex media (vitamin K1, hemin) for growth.

Extremely intolerant of O2.

Can be speciated by gas chromatography by determining the fatty acids formed

when grown on glucose or peptone. Usually, indirect immunofluorescence assays

are used.

Associated with necrotic infections or abscesses.

Learning objectives

To learn the general characteristics of the genus.

To understand the mechanism of pathogenesis for the important species.Important species Other species

B. nodosus B. melaninogenicus

B. fragilis B. pyogenes

B. suis

To understand the synergism with Fusobacterium.

Bacteroides fragilis

The most commonly isolated anaerobe from human infections. Found in >80% of

human peritonitis, and is very often found in soft tissue abscesses.

Reported neonatal infections in sheep.

Constitutes 1-2% of normal flora in humans, and is an intestinal commensal in

many mammals.

Secretes an enterotoxin that has metalloprotease activity that damages tight

junctions, leading to diarrhea or blood leakage.


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Bacteroides or Dichelobacter nodosus

Causative agent ofvirulant foot rotin ruminants.

Pili are required for virulence.

There are at least 14 serotypes.

Data suggests a susceptibility link to the MHC.Infection and Pathogenesis

Enters wound in hoof from soil.

Is aided by coinfection with Fusobacterium necrophorum and/orActinomycepyogenes, wet weather, or rocky terrain.

Produces a protease which dissolves the keratin of the hoof.

Also produces a factor which enhances growth ofF. necrophorum.

The horn will rapidly separate from the foot; very painful.

A foul smell is produced.

Many animals recover spontaneously.

Diagnosis/Vaccination/Treatment Diagnosis is usually based on clinical signs, culture of the organism, or PCR.

Treatment involves clearing dead tissue, footbaths, and killing the organism.

Vaccines are available for many serotypes. Administer prior to pasture.

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Normal (left) sheep hooves. The middle shows a hoof during virulent foot rot infection. Note the hornseparation and matted tissue and and soil. On right is the same hoof after cleaning to raise the oxygen

tension in the lesion.

Sheep with virulent foot rot demonstrating

knee-walking due to painful hooves.


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Genus: Fusobacterium

General characteristics of the genus

F. necrophorum Gram-negative long rods

Commensal of the alimentary canal and therefore shed in feces.

Requires complex media for growth.

Colonies are fuzzy and show complete hemolysis.

Speciated by gas chromatography of fermentation products or

IFA assay.

Causative agent of necrotic infections in mammals and occaisionally poulty.

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Infection and PathogenesisCauses a number of necrotic lesions:

Interdigital necrobacillosis in sheep and cattle.

Non-invasive; requires sut or lesion to intiate infection.

Mixed infection withA. pyogenes, B. nodosus, or B. melaninogenicus.

Swelling between digits, resulting in lameness, but disease is not as

severe asB. nodosus.

Produces a foul smell.

F. necrophorum on blood agar.

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Footrot due to Fusobacterium in a

sheep (left) and cow (arrow, right).

Note the absence of purulence and

keratin damage as withB. nodosus.


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Oral necrobacillosis in pigs and cattle.

Abscess forms in mouth or on tongue, often following penetration of a

foreign object

Animals lose appetite

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re needed to use this pictureFocal liver necrosis due to Fusobacterium.

Oral necrobacillosis on tongue due to Fusobacterium.

Bullnose in pigs

Necrotic rhinitis initiates from ringing or clipping needle teeth

Strong cellular response results in severe facial swelling

Foul-smelling nasal discharge

Liver necrobacillosis in cattle, usually neonates

Organism invades at navel

Septicemia for 5-7 days Localizes in liver and spleen causing focal necrosis

Anorexia and fever observed.

Calf diphtheria

Initiated as oral necrobacillosis Material is aspirated into lungs

Fatal supperative pneumonia

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