anaerobes - from notes

facultative anaerobes

grows in either presence or absence of oxygen

aerotolerant anaerobes

might see small growth in aerobic environment (oxygen) but enhanced growth in anaerobic environment (no oxygen)

obligate anaerobes

do not use oxygen and are inhibited by oxygen presence

there are varying degree of oxygen tolerance

what are the two subtypes of olbigate anaerobe

moderately and strict

moderately obligate anaerobe

generally produce enzymes that help break down the oxygen and protects them

growth in 2-8% oxygen

strict obligate anaerobes

cannot grow in oxygen

little as 0.5% oxygen will inhibit growth

what are preferred specimen for anaerobes

needle aspiration of fluid or tissue biopsy

why are swabs not preferred for collection of anaerobes

swabs have normal flora from surrounding sites and limit amount of specimen picked up

exposes the specimen to oxygen

preferable specimens

abscesses of liver, lung, brain and abdomen

necrotic tissues

wound infections proximal to mucous membranes

serous fluids

cerebrospinal fluid

infected human bites

food ssupected of botulism

stool or feces

avoid or reject specimen

nasal, pharyngeal, baginal, cervical, rectal swabs

voided or cathereized urine

surface skin

sputum

feces

what are spores helpful to ID which species

Clostridium

terminal spores

end of organism

central spores

middle of organism

subterminal spores

between middle and end

what fixation is used to enhance gram stain appearance

methanol

what supplements do anaerobic media contain

hemin, sodium bicarbonate, blood and vitamin k

what helps absorb oxygen in anaerobe media

reducing agents

what broth can grow anaerobes

thioglycollate

where should anaerobic media be stored

in oxygen filled environment but ideally before inoculation should be moved to aerobic condition and held in this “reduced state”

CDC anerobic blood agar (ABAP) - key ingredients

supplemented sheep blood agar

CDC anerobic blood agar (ABAP) - purpose

non-selective for isolation of all anaerobes including both obligate and facultative

anaerobic phenylethyl alcohol blood agar (CDC-PEA) - key ingredients

ABAP with phenylethyl alcohol added

anaerobic phenylethyl alcohol blood agar (CDC-PEA) - purpose

isolation of all obligate anaerobes (GP and GN)

GP facultative anaerobes

inhibits Proteus swarming and most Enterobacteriaceae

Anaerobic kanamycin-vancomycin blood agar (KV) - key ingredents

ABAP plus antibiotics - kanamycin and vancomycin

Anaerobic kanamycin-vancomycin blood agar (KV) - purpose

antibiotic inhibits growth:

vancomycin - facultative and obligate anaerobic GP organisms

kanamycin - facultative GNB

anaerobic paromycin-vancomycin laked blood agar - key ingredients

similar to KV but blood is “laked” meaning frozen and then thawed

anaerobic paromycin-vancomycin laked blood agar - purpose

isolation of GN obligate anaerobes, pigment production by Prevotella is detected sooner bc of laked blood

enriched thioglycollate broth - key ingredients

broth with hemin and vitamin K

enriched thioglycollate broth - purpose

isolate more fastidious anaerobes and also actinomyces; used in conjunction with plated media

bacteroides bile-esculin (BBE) agar - key ingredients

bile, esculin, supplements, agar base, gentamicin

bacteroides bile-esculin (BBE) agar - purpose

genetamicin inhibits most facultative anaerobes

bile inhibits anaerobic GNB except for bile resistant including B. fragilis group

black/brown pigment = B. fragilis

McClung toabe / egg yolk agar -- key ingredients

egg yolk, gelatin peptone with agar base

McClung toabe / egg yolk agar -- purpose

egg yolk allows detection for lecithinase and lipase activity

lecithinase = opaque precipitate

lipase = “oil in water”

what is an anaerobic jar

plates are placed into a jar,

hydrogen gas formed that reacts with oxygen water is from as th oxygen is removed “condensation”

needs palladium catayst in lid for reaction

what is an anaerobic chamber

external gas thank are hooked up to chambers

the chambers are filled with an anerobic environment (85,10,5)

what is anaerobic bags

disposable, and portable

for low volume or transporting of anaerobic plates

indicator used

are cultures for anaerobes fast or slow growing

slow growing, incubate at 35-37C for atleast 48hrs before 1st examination

how long should cultures be incubated for before reporting as negative

7 days

why is AnaeroGRO plates helpful for preliminary growth

are pre-reduced and packaged in oxygen free pouch

quickly inoculated then returned to pouch

then incubated in anaerobic chamber

examined pouch as to no introduce oxygen

what are oxyplates

uses standard incubator

media creates the anaerobic environment

what is aerotolerance test

tawking an anaerobically growing organism and plating it to an aerobic plate/environment

aerobic choco agar is inoculated and incubated aerobically at the same time anaerobic blood agar is inoculated and incubated anaerobically

what % does anaerobe organism grow

20%

what is lipase

if produces will degrade fat from fatty acids and glycerol

what does a positive reaction of lipase look like

“oil in water”

what is lecithinase

if organism produces the enzyme to degrade the lecitin, water insolube diglycerides are formed

where is lecithin present in

egg yolk

what is a positive reaction of lecithinase look like

opaque white halos around growing colony

what does the clostridium species form

spores

how to spores help clostrium survive

they help in unfavorable environemtns and then germinate to produce active organisms again

where are clostridium species found in

environment - soil and water

clostrium species are normal flora to where

GI tract

what do clostridum species produce

exotoxin that are very potent

most clostridum species are catalase what

negative

differentiate from bacillus species

what agar is helpful to differentate clostridum

egg yolk agar

spores of clostridium tetani

round and terminal

where is clostridium tetani found in

environment

what is clostridium tetani commly associated with

rusty old nails

what does clostridium tetani produce

potent neurotoxin

gelatinase and indole

clostridium tetani: lecithinase and lipase

negative

how is tetanus contracted

spores enter through skin puncture wound or other open wound

what acts as reservoir for germination of spores: tetanus

host necrotic cells (no longer getting oxygen from the body)

what is tetanospasmin neurotoxin

travels along nerves from the wound area to the spinal cord and causes convulsions

what does tetanus affect the first

the neck and jaw region “lockjaw”

what does tetanus also cause

backward arching of the back muscles fro mthe toxin and eventually respiratory paralysis

what is the rate of fatality if tetanus is untreated

>50%

what is the treatment of tetanus

antitoxin antibody as wells as antibiotics

what toxin does clostridium botulinium produce

botulism toxin

where is clostridium botulinium found in

environment soil/water

what are the spores look like in clostridium botulinium

oval and subterminal

clostridium botulinium - lipase

positive

clostridium botulinium - lecithinase and indole

negative

what is clostridium botulinium isolation

must be reported to state health department

grows on routine anaerobic media

colony morphology of clostridium botulinium

white beta hemolytic colony that can have smooth or rough edges

what does clostridium botulinium cause

botulism

what is botulism toxin

powerful neurotoxin several different forms of toxin (A-G)

what is the most common botulism toxin

A,B & E

what does botulism toxin bind to

synapse of cholinergic nerve fibers

what does botulism toxin cause to the body

acute and flaccid paralysis to muscle of the face, head, and throat, and eventually thorax diaphram and limbs

what is the treatment of botulism toxin

anti-toxin

what is food borne botulism

ingestion of performed toxin-home canned products are common source

in food borne botulism where does spores germinate

in the oxygen free stored products leading to toxin producing organism

what are the symptoms of food borne botulism

12-72 post consumption as nausa constipation and eventurally neurological symptoms

5-10% mortality rate

what is wound botulism

very rare: spores get into wound and then germinate

what is infant botulism

most common <1yo

what causes infant botulism

spores ingested and germinated in the infant GI tract which has no normal flora to compete with the organism

symptoms of infant botulism

contipation followed by trouble swallowing and eventually muscle weakness, loss of head control and respiratory failure

what is infant botulism linked with

honey

what is the most common clinical clostrium spp.

clostridium perfringens

clostridium perfringens - gram stain

large GPB, line up “box car”

clostridium perfringens - spores

do not usually sporulate in culture, rarely seen in gram stain

toxin production in clostridium perfringens

alpha-toxin, cardiotoxin, enterotoxin

what is the virulence mechanism of clostridium perfringens

lecithinase, hemolysins, fibrionlysin, DNase, RNase, other proteolytic enzyme

clostridium perfringens - normal flora

vaginal and GI tract

found in environment too

what media does clostridium perfringens grow in

routine anaerobic media

clostridium perfringens - hemolysis

double zone of hemolysis

innerzone - complete hemolyzed

outerzone - incomplete hemolyzed

clostridium perfringens - lecithinase

positive, opaque yellow halo on egg yolk agar

clostridium perfringens - lipase

negative

clostridium perfringens - reverse CAMP test

positive

streak the clostrium down the blood agar, then streak Group B Strep at a right angle

arrow head of hemolysis will form

what is myonecrosis aka “gas gangrene”'

sereve muscle/tissue damage and gas pocket formation in tissue

leads to an aerobic environment where pathogen can flourish

myonecrosis is most common in

people with decrease ciruclation diabetes

uterine gangrene induced abortions