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Anaerobic Bacteriology


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What TYPE of anaerobes are most anaerobic bacteria?
Aerotolerant. not obligate anaerobes; O2 is toxic to them, but they can tolerate it while we work on them.
Clinical symptoms unique to anaerobes:
Foul odor
Gas in tissues/discharge
Necrotic tissue
Black discolor
Sulfur granules (not really, but YELLOW)
Infection proximal to mucosal surface
Infection follows:
1. abdom. wound/surgery 2. human bite, 3. aspirationfrom oral to lungs.
Are anaerobes normal or pathogenic in humans?
mostly NORMAL - everywhere and anywhere in the body.

can be pathogenic - often in mixed infections, because aerobic pathogens create a microaerophilic environment suitable.
why do anaerobes produce a foul odor?
because in anaerobic metabolism volatile acids are produced.
what type of anaerobic infection do sulfur granules appear in?
only one - actinomycosis; these arent really sulfur, but YELLOW granules
6 types of specimens that should be cultured anaerobically:
body fluids
bladder urines (suprapubic aspirations)
Which Specimens should you NEVER culture for an anaerobe?
Eye, ear, nose, throat
Sputum unless transtracheal

Vaginal, cervical, urethral, stool, urine.
How are specimens for anaerobic bacterial cultures collected?

(3 methods)
Preferred: Biopsy, or Aspiration

Less Preferred: Swabs - they tend to collect surface fluids, which will not contain anaerobic bugs that prefer deep, anaerobic environments.
In what type of tube are anaerobic specimens transported?

What is in the media
in Transport tubes, which have no O2.

In case O2 does get in, an indicator is in the semisolid media in the tubes.
3 special methods to transport anaerobic specimens:
-Transport tube
-Use syringe, aspirate air out. (outdated)
-Use Swab transport biobag with tablet to react with air and remove it.
What is Rezazarin?
pink indicator of O2 found in swab transport systems
4 types of plate media used to grow/identify obligate anaerobic bacteria:
1. Anaerobic BA + Vit K1 & Hemin
2. KV agar w/ laked blood
3. PEA agar
4. aerobic BA
Describe anaerobic BA ingredients, what grows on it:
Blood Agar
Vitamin K1

All anaerobes grow - facultative and obligate. From a stool, that's bad.
Describe KV agar ingredients, what grows on it.
Kanamycin - aerobic gnb inhibitor
Vancomycin - gpos inhibitor
Laked blood - shows black pigment of some anaerobic bugs.

Only Anaerobic gnb will grow
Describe PEA agar ingredients, what grows on it.

what does PEA stand for?
Phenylethyl alcohol agar

-Inhibits facultative anaerobes
-only obligate anaerobic bugs will grow from an enteric specimen.

Alternate to CNA; selective for gPOS bugs
Why is an aerobic plate useful in identifying/isolating obligate anaerobic bacteria?
Because if it grows on the aerobic, you know it's not the kind of anaerobe WE'RE lookin fowr.
When would a bug grow on both aerobic and anaerobic blood agar?
If it is facultatively anaerobic, often the case in enteric specimens.
What are 2 Types of anaerobic broth media:
1. Sodium Thioglycollate w/ Vit K1 & hemin

2. Cooked meat broth - a protein source
2 Things that must be done to prepare media for anaerobic cultures:
Plates: reduce for 24 hours to take out any oxygen.

Broths: Boil for 10 min to remove any O2 that was absorbed.
What is Sodium Thioglycollate?
a REDUCER; keeps the oxidation/reduction potential low in anaerobic broths
What's required of LOOPS for anaerobe culture setups?
Type of Loop
-If metal, only Tungsten or Platinum
-Plastic loops best

Inoculate quickly to prevent O2 absorption
List four different systems for incubating obligate anaerobes:
1. GasPak jar
2. Glovebox
3. Biobag
4. Oxyrase system
what reaction takes place in a Gaspak jar?

What is the visible result?

Components of the jar:
2H2 + 02 -> 2H2O

Condensation forms, CO2 also released.

H2 generator
Palladium pellet catalyst
gasket, screw clamp, indicator ensures it is an anaerobic environment
With a gaspak jar, what should you wait to see, and how soon?
Condensation formation within 2 hours.

The indicator will not probably ensure an anaerobic environment that soon.
What's a Glove box?
like a fume hood, with a sliding door to keep anaerobic bugs in anaerobic cond.
What are the incubation conditions for anaerobic bugs in Broths vs. Plates?
Broths - can be incubated as usual, with caps screwed tight.

Plates - must be in anaerobic environment, for at least 48 hours.
If you only test for anaerobic bugs now and then, what type of incubation system should you use?

If often?
Biobag - cheaper, easier, can see through plastic. What we use.

GasPak jar.
What is the Oxyrase system?
Plates that can be incubated for anaerobic bugs, but not in any special bag or jar.

Agar contains fragments of bacterial cell membranes that convert oxygen to waer.
What are the 2 Anaerobic GPC?
Which anaerobic GPC is more important?
What does it resemble?
Resembles streptococcus - especially viridans because can cause SBE.
Where are these peptos mostly found as normal flora?
mucous membranes
where is peptostreptococcus pathogenic?
almost anywhere in the body
if you see GPC on a gram stain, what do you know?
they could be either aerobic streptococus or anaerobic peptostreptococcus.. never should assume you know
What are the 3 Anaerobic GNC genera?
1. Veillonella
2. Acidaminococcus
3. Megasphaera
where are veillonella, acidaminococcus, and megasphaera located in the body?
mucous membranes and intestine, but rare.
How can you differentiate anaerobic GNC from aerobic?
They are much smaller and tend to be in clusters.

Neisserias and M. catarrhalis tend to be diplococci that are big and round.
What are the three genera of anaerobic gnb?
Prevotella and Porphyromonas
Bacteroides is broken down into what two groups?
B. fragilis group
Bacteroides sp. - not B. fragilis
What 2 specimens of B. fragilis group do you need to know?
Bacteroides fragilis
Bacteroides thetaiotamicron
What species of Fusobacterium do you know?
Fusobacterium nucleatum
What is the unique characteristic of Prevotella and Porphyromonas
they are black pigmented
Which anaerobic bacteria are normal flora in the mouth?
Fusobacterium, Prevotella, and Porphyromonas
Which anaerobic bacteria are normal flora in the intestine?
B. fragilis - it is the PREDOMINANT group in the intestine - more than 1000x greater than even E. coli!
Which anaerobic bacteria are normal flora in the vagina?
Fusobacterium and Bacteroides species
where is bacteroides potentially pathogenic in the body?

B. fragilis group?
almost anywhere.

B. fragilis species can cause peritonitis after abdominal wounds and surgery
Where do Fusobacterium, Prevotella, and Porphyromonas typically cause infection?

What five types of infections?
In the oral cavity.

head and neck
human bite wound
aspiration pneumonia
Fusospirachaetal disease - Vincent's Angina with Borellia species
What are the two symptoms of Vincent's angina? caused by what two bugs?

often this is confused with:
Pharyngeal membrane and gingivitis

Borrelia species and Fusobacterium sp.

Confused with Diphtheria
What are two ways to identify anaerobic gnb on a gram stain?
Bacteroides, Prevotella/Porphyrom. may balloon out and look weird.

Fusobacterium fuses in long chains and they're very thin, long, tapered at ends.
what is the characteristic colonial morphology of Bacteroides?
very similar to enterococcus, difficult to differentiate on a plate - off white, milky, etc.
what is the colonial morphology that distinguishes PREVOTELLA AND PORPHYROMONAS?

how can you best see them?
Black-pigmented colonies.

best seen on KV agar with laked blood, after shining with a flourescent light (this speeds pigmentation to 48 hrs instead of 5-7 days.
what is the characteristic colonial morphology of fusobacterium?
ground-glass appearance, breadcrumb-like.
What are three Antibiotics that the anaerobic GNB are susceptible to,

At what concentrations?
Kanamycin - 1mg
Vancomycin - 5 microg
Colistin - 10 microg
what does the 20% bile sensitivity show, how?
B. fragilis - it is resistant to it.
what is the "anaerobic gram variable bacilli", really?
really, it's an aerobic gram positive bacilli. it's just that the gram stain is variable due to a thinner than normal peptidoglycan layer, and it grows better on anaerobic agar.
what is the name of this gram variable species?
Gardnerella vaginalis
What is the characteristic gram stain of a gardnerella vaginal swab?
-decreased lactobacillus
-clue cells
-gram variable bugs with GULL-WING shape
-various bacteria adhere to the cells
What is bacterial vaginosis?
an annoying infection with burning discharge but no pain really.
what are the 3 complications of bacterial vaginosis?
-premature birth
-Septicemic mothers or babies
what is the role of another organism in bacterial vaginosis, what is its name?
mobiluncus is another anaerobic bug that is associated with the disease - it's just found there.
what are the 3 main methods of diagnosing bacterial vaginosis?
-gram stain shows clue cells

-alkaline vaginal discharge = cloudy, runny.
-Discharge has fishy odor when mixed with 10% KOH.
what is used to treat bacterial vaginosis?
what are the 7 non-sporeforming anaerobic gram positive bacilli?
Propionibacterium acnes
which non-spore anaerobic GPB is found on skin normally?
propionibacterium acnes
which anaerobic gpb (nonspore) is actually a very aerotolerant anaerobe so that is classified as aerobic?
which anaerobic gpb nonspore forms sulfur granules?
what are its other characteristics?
Results in actinomycosis, with symptoms of Lumpy jaw - swelling under the jawline.
It's actually a fungi.
what anaerobic gpb nonspore is a commmon cause of gingival plaque?
Where is Propionibacterium acnes normally flora?
on the skin
Where is Bifidobacterium normal flora?
in the intestine.

ironic, B goes with B - bifidobacterium and bacteroides
What four gpb anaerobic non-spore formers are normal flora in the mouth?
Lactobacillus, Eubacterium, Actinomyces, and Rothia.

Remember Rothia causes gingival plaque, and just goes with three others in the mouth
What is normal flora in the vagina?
What is the anaerobic GPB spore-former?
how many species of clostridium are there?

which is the biggest?

C. perfringens

it infringens on the others
What are the characteristics of Clostridium?
(gram stain, natural habitat, virulence factors)
GS: Spore-former GPB
Nat'l Habitat: soil/intestine
Virulence: Exotoxin
where in nature is clostridium normally found?
in the soil
what are the characteristic virulence factors of clostrdiium?
what are the 3 most commonly isolated species of clostridium?
c. difficile
c. perfringens
c. ramosum --> like abby ranum only mos instead of n
what is the number one isolated clostridium species? what diseases does it cause?
clostridium perfringens

causes gas gangrene and foodborne illness
what is clostridium dificile associated with?
antibiotic associated colitis
what are the characteristics of c. perfringens?
(Gstain, colonies, emissions, special agar)
-large boxy gpc w/ spores
-large gray colonies with double zone of beta hemolysis.
-produces considerable gas
-stormy fermentation in milk media
-Lec+ Lip- on egg yolk agar
what is the course of gas gangrene? what causes it?
caused by c. perfringens
bug in soil, enters wound, proliferates, produces exotoxin, necrosis/blebs start, enters bloodstream and because systemic.
how is gas gangrene treated?
with penicillin and wound debredement
What clostridium species cause
-Foodborne illness
-Food poisoning
Foodbn illness = perfringens; bug proliferates after eating.
Food poisoning = botulinum; toxin ingested causes sx.
what is the course of foodborne illness caused by c. perfringens?

how to test for it?
the cafeteria germ is ingested with SPORE-containing meat, after incubating 6-8 hrs it causes diarrhea.

it's self-limiting.
to test for it, must culture food, not stool.
what does Clostridium tetani cause?
what type of toxin is produced by c. tetani?
a neurotoxin
what is the course of tetanus as a disease?
bug in soil enters wounds. it germinates and produces the neurotoxin, which then acts on the CNS and causes muscle spasms - first local then systemic.
how is tetanus treated?
with antitoxin and debridement
how can tetanus be prevented?
with a tetanus shot - toxoid - once every 10 years
List 3 diseases caused by C. botulinum:
which is most common?
-food poisoning
-wound botulism
-infant botulism - most common
what is the course of food poisoning by botulinum?
the bug in soil gets in fruits/veggies, then gets canned. forms gas - blows lids off like in holiday inn. passes to intestine, then CNS and causes paralysis.
what is special about the botulism toxin?
-most toxic toxin known

-inhibits acetylcholine

-no real good specimen to culture, so test serum.

-can be used to treat facial ticks/crossed eyes
what is the course of disease of wound botulism?
from the soil, the bug enters a wound. then it produces the toxin, it enters the CNS and causes paralysis.
what is the course of infant botulism?
through honey, the bacteria colonizes the small intestine and produces toxin, which enters the CNS and causes paralysis.
what age of infants develop botulism?
under 6 mo
For which Clostridium species are antitoxins available?
which infections are caused by the bug entering a puncture wound?
clostridium tetani
clostridium perfringens
clostridium botulinum - wound botulism
other clostridia
what disease is caused by ingestion of preformed toxin?
Food poisoning caused by C. botulinum.
what diseases are caused by colonization of the intestine by the bug?
infant botulism
C. perfringens food poisoning
though it's a spore-former, why is clostridium perfringens difficult to ID?
only produces spores in direct smear - after culturing, not often seen on gram stain.
what causes antibiotic associated colitis?
c. difficile
what is the pathogenic mechanism of antibiot assoc. colitis?
use antibiots, reduce normal intestinal flora, c. difficile proliferates/produces toxins a/b, colon lesions develop and diarrhea results
what are the symptoms of aac?
-diarrhea, bloody w/ WBCs
-abdominal pain and fever
-plaquey lesions and pseudomemb. on intestinal mucosa.
how is AAC diagnosed?
-toxin detection from tissue culture and immunologic tests.
what antibiotics are used for anaerobic infections?
combos of beta-lactam drugs with beta-lactam inhibitors
how are anaerobes identified?
(4 ways)
1. traditional biochem testing
2. GLC for volatile acids
3. high-resol GLC - fatty acids
4. disc differentiation - kanamycin/vancomycin/colistin

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