LAB - Anaerobic Bacteriology Flashcards
aerotolerant anaerobes
can proliferate in the presence of trace oxygens
factors contributing to O2 sensitivity may include:
- direct toxic effect of molecular oxygen
- toxic products of oxygen metabolism
- oxidation-reduction potential (Eh in millivolts)
- oxidized organic constituents of culture media
toxic products of O2 metabolism
- superoxide anion (O2-): accumulates when bacteria don’t have superoxide dismutase (SOD) to convert superoxide to hydrogen peroxide; anaerobic bacteria do not have this
- formation and accumulation of hydrogen peroxide: due to lack of catalase
> formerly believed that H2O2 toxicity was an important factor in oxygen intolerance but some anaerobes do possess catalase activity and some facultative organisms capable of growth in air are devoid of catalase activity
media used for the growth of anaerobes must have a low __ which is usually achieved by the addition of ______ ____. the presence of molecular oxygen hinders the achievement of a low __
Eh
reducing agents
Eh
these form in media stored in air and may be inhibitory to certain anaerobes
oxidized organic constituents of culture media
- ex: C. haemolyticum will not grow on blood agar which is exposed to air for 1-2 hrs but C. perfringens will still grow on blood agar which has been stored in presence of air for several weeks
- optimal recovery = fresh media or media that have been stored anaerobically are required
major genera of anaerobes in clinical infection are …
Actinomyces, Bacteroides, Clostridium, Fusobacterium, and Peptostreptococcus (exception = Peptococcus niger)
Certain hints that a given specimen is likely to contain anaerobic bacteria:
- foul odor
- location of infection = mucosal surface
- infections secondary to human or animal bite
- gas
- previous therapy with aminoglycoside antibiotics (gentamicin, tobramycin, amikacin)
- black discoloration of blood-containing exudates (may fluoresce red)
- sulfur granules in discharges (Actinomyces)
- unique gram stain morph
- failure to grow, aerobically, organisms seen on Gram stain of original exudate
- growth anaerobically
- characteristic colonies
good specimen in anaerobic bacteriology
- aspirated pus
- tissue
- body fluids
- transtracheal aspirates
- direct lung aspirates
- ‘sulfur granules’ from patients with suspected actinomycosis
these specimens are NOT cultured for anaerobic bacteria
specimens contaminated with normal flora
use of swab is often not suitable for these organisms
anaerobes
transport systems for anaerobes
designed to avoid exposure of specimen to O2
- tubes containing oxygen-free gas
- swabs sterilized under O2 free CO2 in tubes containing a non-nutritive transport medium, for example, Cary-Blair transport
media for anaerobes
- must be freshly prepared OR pre-reduced supplemented media (fastidious anaerobes with low redox potential)
supplemented media
media for cultivation of anaerobes
- 5% sheep blood yeast extract, hemin and menadione (vit. K) added to a rich nutrient agar bse such as brain-heart infusion agar
- reducing agents
these are added to media to provide a low redox potential for the growth of anaerobes
reducing agents
- beef heart tissue in chopped meat medium
- glucose
- ascorbic acid
- sodium thioglycolate
- cysteine hydrochloride
most commonly used reducing agent in anaerobic media
cysteine
These are used in fluid media for the cultivation of anaerobes
Eh indicators such as resazurin
media for primary isolation of anaerobes
- liquid = small #s in OG; backup cultures
- plated agar media
> non-selective enriched = hemin, vit. K1, yeast extract, reducing agent (cysteine)
> selective media = to prevent overgrowth of facultative bacteria
menadione
vitamin K
examples of non-selective enriched media for anaerobes
Columbia agar
Brucella agar
BHI bases
examples of selective media for anaerobes
Phenyl ethyl alcohol blood agar (PEA)
kanamycin-vancomycin-laked blood agar (KVLB)
indicators of anaerobiosis
- methylene = blue; colourless when reduced; oxidized when coloured
- resazurin = colourles when reduced and pink when oxidized at Eh - 40 mv
**inclusion of strict and fastidious anaerobe is alternative **
evacuation of air vs gas generation
- evacuation of air = replaced w gas mixture containing H2; O2 reacts with H2 = water; Pallidum as catalyst
- gas gen = GasPak use disposable envelopes wth H2 and CO2 generators; no evac of air; H2 and CO2 generated when H2O added to chemicals in GasPak envelope
** add indicators **
prolonged incubation for anaerobes is essential!!!
at least 48 hrs without opening anaerobic jar
- most sensitive to O2 during log phase
- 72 hrs optimal
test to confirm suspect isolate is indeed an anaerobe
aerotolerance test
- tests whether it is obligate or facultative anaerobe
- from primary plate to two fresh BAPs; one will go to CO2, one will go to anaerobic jar PLUS a gram stain to ensure that the colony you picked was pure
simplest method for measuring the end products of glucose metabolism
GLC
- non-sporulating anaerobes
- patterns of short-chain fatty acid end products of glucose and AA metabolism
- “fingerprinting”
GLC
- gas-liquid chromatography
- elution chromatography which employs an inert carrier gas (mobile phase)
- carries heat vaporized solute into contact with liquid stationary phase
- differences in molecular weights, bp, affinities of different substances for stationary phase = retention time (time required to pass through column)
what do GLC instruments consist of?
- an inlet port and vaporization chamber
- a glass or stainless column packed with the stationary phase = usually high molecular weight polymer coated onto a support phase of inert particulate matter
- detector/recorder at elution end of column
anaerobic gram negative bacilli with peritrichous flagella or non-motile
- Fusobacterium = butyric acid
- Bacteroides = produce only lactic acid
- Prevotella
anaerobic gram negative bacilli with polar flagella
FERMENTATIVE
- Butyrivibrio = butyric acid
- Succinivibrio = succinic acid (Spiral)
- Succinimonas = succinic acid (Ovoid)
NON-FERMENTATIVE
- Vibrio succinogenes = succinic acid
- Selenomonas = tufts of flagella
- Trepnoema, Borrelia = spiral-shaped cells
anaerobic gram positive cocci
- pairs & chains = fermentable carb as main energy source
- Coprooccus = butryic acid
- Ruminococcus = NO butyric acid - do not require a fermentation carb
- Streptococcus = lactic acid sole major product
- Peptostreptococcus, Peptococcus
anaerobic gram negative cocci
- Veillonella = produce propprionic + acetic acids
2. Acidaminacoccus - butyric and acetic acids
T or F. Most clostdial infections are of endogenous origin
T, but exogenous sources may be involved as well such as botulism or tetanus or C. perfringens food poisoning and sometimes gas gangrene
absence of visible spores on large bacilli with blunt ends (boxcars)
C. perfringens
EYA
- egg yolk agar
- for detection of lecithinase + lipase
- Clostridia sp. can produce one, both, or neither
lipase rxn on EYA
- organisms break down neutral lipids to fatty acids
- shallow precipitate
- pearly iridescence
- C. sporogenes and botulinum
lecithinase rxn on EYA
- splits lecithin on egg yolk = insoluble diglycerides = wide zone of opacity
- C.perfringins = lecithinase AKA alpha toxin
Nagler inhibition test
- antitoxin neutralizes lecithinase or alpha toxin
- one half of EYA plate gets C. perfringens alpha-ANTItoxin; dry
- inoculate plate with C. perfringens
- lecithin will precipitate only on the half without antitoxin
Reverse CAMP test
- C. perfringens perpendicular to GBS; act synergistically w CAMP factor of GBS = enhanced hemolyis
- incubated anaerobically on a BAP
- will enhance hemolysis with a characteristic arrowhead pattern
Clostridia sp. biochemical rxns
- initial separation base on proteolytic activity (gelatin0 and
- saccharolytic activity (glucose)
major case of antibiotic-associated diarrhea and pseudomembranous colitis
C. difficile
C. difficile toxins
- most but not all strains = two toxins in vitro
- toxin A = enterotoxin
- toxin B = cytotoxin
CYCLOSERINE-CEFOXITIN-EGG YOLK-FRUCTOSE AGAR (CCFA)
isolation of C. diff
- yellow w regular light
- yellow-green fluorescence with UV light
C. diff in anaerobic BAP
non-hemolytic colonies, grey-translucent with a rhizoid edge; ground-glass
most frequent isolate in human anaerobic infections
Bacteroides (43%)
- normal flora of mouth, intestinal tract, urogenital tract
- more often found in mixed infection
- acquired endogenously; contiguous spread
- end products of glucose fermentation is mixed
- speciation achieved by GC and biochemical tests
“B. fragilis group”
- abdominal cavity
- cell-bound B lactamase = resistant to penicillin
- other species but all are pleomorphic gram neg bacilli faintly stained with safranin
- gry and shiny
- encapsulated = abscess formation
- rapid growing
- relatively aerotolerant
- non-pigmented
- saccharolytic
- resistant to kanamycin, Vancomycin, Colistin
- BBEA growth (presumptive ID)
Prevotella
- pigmented and non-pigmented colonies
- ALL bile sensitive
- oral cavity, URT, colon
- dental, sinus infections, pulmonary, abscesses, abdominal + pelvic lesions
pigmented Prevotela sp.
- saccharolytic
- black pigmented = protohemin; laked blood media incubated for more than 7 days
- younger colonies = red fluorescence under long UV
- resistant to kanamycin and vancomycin
non-pigmented Prevotella
may fluoresce pink, orange, or chartreuse colour
Fusobacterium
- inhabit oral cavity, colon, sometimes female genital tract
- long, slender rods with tapered ends (may not be present)
> F. mortiferum = pleomorphic with spheroid swellings… - 5% of clinical specimens
- butyric acid major end product of Fuso but not Bacteroides
antibiotic susceptibility of Fusobacterium that can be used to differentiate from Bacteroides
- susceptible to kanamycin
- susceptible colistin (but also resistant to vancomycin)
- definitive = GLC for end products of glucose metabolism
Although several genera of gram-neg cocci are found in indigenous microflora, only these are implicated as pathogens
Veillonella
