20 anaerobes
TRANSCRIPT
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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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CVM6202: Microbiology Rutherford
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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CVM6202: Microbiology Rutherford
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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CVM6202: Microbiology Rutherford
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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CVM6202: Microbiology Rutherford
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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CVM6202: Microbiology Rutherford
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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CVM6202: Microbiology Rutherford
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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CVM6202: Microbiology Rutherford
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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CVM6202: Microbiology Rutherford
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
Foul breathQuickTime and a
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QuickTime and a
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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