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Microbiology 2 > M10 Anaerobes > Flashcards

M10 Anaerobes Flashcards

1
Q

What type of site do we look for Anaerobes in?

A

Mostly sterile sites can have a possibility of anaerobes causing an infection.

Non-sterile sites are often not appropriate for anaerobe collection and there can be facultative anaerobes there as well as aerobes.

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2
Q

What is the one sterile site where anaerobes are not considered to be a possible cause of infection?

A

CSF

Anaerobes don’t tend to cross the blood/brain barrier.

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3
Q

How should a request for an anaerobe culture from a non-sterile site be treated by the lab?

A

For most non-sterile sites, ANO2 culture is
inappropriate and should be rejected

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4
Q

What type of infections do anaerobes most frequently cause?

A

Deep wound infections are frequently caused by
Anaerobes, in most
cases in combination with
other pathogens.
They involve the subcutaneous layers of skin.
Abscesses, necrotizing
fasciitis, gangrene, etc.
are some of the clinical
situations.

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5
Q

What area of the body are anaerobes in particular are the normal flora?

A

Most anaerobes are normal flora of our mucosas.

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6
Q

What anaearobe organisms are known to be soil and water inhabitants?

A

Pathogenic anaerobes are soil and water inhabitants.
1. Clostridium botulinum
2. Clostridium tetani

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7
Q

How do most anaerobes gain access to cause infections of sterile body sites?

A

Most anaerobes gain entry to sterile sites as a result of a
disruption of an anatomic barrier.

Note: Person-to-person nosocomial spread can occur.

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8
Q

What are some exogenous sources of infection by anaerobes?

A
  1. Soil is primary reservoir
    ie. C. botulinum, C. tetani
  2. Transmission from other patients - ie. Clostridioides difficile
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9
Q

What are some endogenous sources of infection by anaerobes?

A
  1. Primary source is normal flora of the gastrointestinal tract
    ie. C.difficile, C.perfringens
  2. Exist as normal flora in the oral cavity and gastrointestinal tract - i.e. Bacteroides fragilis
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10
Q

What anaerobe is the causative agent of infant botulism because of its production of neurotoxins?

A

C.botulinum

Produces a potent neurotoxin involved in food poisoning (preformed toxin is
ingested). In Infant botulism the organism produces the toxin after colonizing GI tract.

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11
Q

What anaerobe organisms produces neurotoxins that can result in lock-jaw?

A

C. tetani

Produces a potent neurotoxin that disrupts nervous impulses to muscles–> lockjaw to even respiratory failure.

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12
Q

What anaerobe organisms is known to make exotoxins that cause gangrene and food poisoning (endotoxin)?

A

C.perfringens

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13
Q

What can the anaerobe C. difficile produce and what problem does it cause?

A

Produces toxin A (endo) and B (cyto)= causes pseudomembranous colitis.
Not pathogenic if toxins are not present. Nosocomial and related to use of antimicrobials

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14
Q

What virulence factor does Bacteroides spp. have?

A

Produce capsules (inhibit phagocytosis), endotoxins and enzymes (damage tissue).

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15
Q

What g+ anaerobes are of concern in this module? Describe their gram stain morphology.

A

Clostridium spp.= Lge PB
Actinomyces = sm-med PB
Cutibacterium spp.= sm PB
Peptostreptococcus, Finegoldia & other many genera= PC

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16
Q

How does gangrene caused by Clostridium perfringens spread?

A

Gas gangrene: toxin-mediated breakdown of muscle + growth of the organism, life threatening. Also caused by other spp of Clostridium. It can evolve to sepsis & other infections.

Note: Clostridium perfringens
major cause of myonecrosis (gas gangrene) and other wounds.

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17
Q

What other conditions can Clostridium perfringens also cause?

A

Other infections/sicknesses include:
1. Pneumonia with empyema and septicemia.
2. Food poisoning

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18
Q

Where does Actinomyces cause infections in the body?

A

Actinomyces: Oral infections, and pelvic & abdominal areas.

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19
Q

What organism typically causes acne? Is it part of skin normal flora? What other infections might it cause?

A

Cutibacterium spp: C. acnes causes Acne
Most of the time found as contaminant due to being NF of the skin, but it might cause infections of sterile sites: osteomielytis, etc.

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20
Q

This organism causes brain abscesses and other quite widespread infections because of where it is part of the normal flora. What is the organisms and where is it normal flora?

A

Peptostreptococcus: Brain abscesses, sinus infections, oral or pelvic infections, quite widespread as this organism is part of the NF of mucosas.

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21
Q

What type of organism is the most common cause of infections and where can it found to be in the normal flora?

A

GRAM NEGATIVES: most common cause of infections; NF of mouth, URT, GI and urogenital.

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22
Q

What bacteria organisms group is most resistant to many antimicrobials?

A

most common is Bacteroides fragilis Group
resistant to many antimicrobials

Infections below the diaphragm
Abscesses, decubitus ulcers,
septic arthritis, bacteremia

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23
Q

What is a gram negative cocci you are studying in this module on anaerobes?

A

Gram negative cocci:
Veillonella spp. (single cocci)

Note: Lab table says g-dc.

24
Q

What are the gram negative bacilli you are studying for this module on anaerobes?

A

Bacteroides fragilis group
Prevotella spp.
Porphyromonas spp.
Fusobacterium spp. (gram morph shows needle shape at end ‘fusiform’.)

25
Q

What are the two g- anaerobes that are pigmented on culture plates (although it takes time to show)?

A

Prevotella spp.
Porphyromonas spp.

26
Q

List three different anaerobe specimen collection systems?

A
  1. Rubber-stoppered collection vial for liquid specimens
  2. Oxygen-free collection tube for swab specimens (pre-reduced anaerobic transport-PRAS)
  3. Self-contained anaerobic bag for tissue specimens

In summary, any device that allows the specimen to not be in contact with O2

Normally Dr’s send the specimens to the lab ASAP (samples typically collected in hospital with lab).

27
Q

What type of specimens are appropriate for anaerobes?

A

In summary, any sterile site or enclosed area of the body would be appropriate, as long as the collection method is the proper one too (for example, bladder aspirated urine is ok, MSU is NOT).
- Sterile Tissues
- Aspirated Fluids
- Deep swabs

Look a list of appropriate sites in notes.

28
Q

What specimen location is always inappropriate for anaerobe culture?

A

Any specimens from mucosas are NOT appropriate

Look a list of inappropriate sites in notes.

29
Q

Describe four different types of anaerobes and the amount of O2 they can tolerate.

A

Obligate anaerobe (0% O2)
Aero-tolerant (5% O2)
Microaerophilic (2-10%)
Facultative anaerobes (capable of growing in O2 presence or not)

30
Q

What enzymes do strict anaerobes lack?

A

Strict ANO2’s lack superoxide dismutase & catalase= enzymes that breakdown reactive O2 which could damage them.

31
Q

List four different types of methods to create the anaerobic environment in the lab? Incubation temp?

A
  1. ANO2 Chambers
  2. Vacuum Pumps and gas
    (H2 CO2 N2 )
  3. Gas Packs (CO2, H2)
    - Catalyst 2H2 + 02 = 2H2 0
  4. Non-catalyst-requiring gas packs (most expensive)

Anaerobically – 33-35°C

32
Q

What’s important to check after setup before leaving a anaerobic jar to incubate for hours?

A

Very important to check that the jar looks humid (condensation) and warm after aprox. 10 minutes of set up: sign that things are working.

33
Q

What is important to have in anaerobic incubation environment for QC?

A

Indicators

Can be chemical (methylene blue or rezasurin=when white, Anaerobiosis has been reached) or biological= a very strict ANO2 on a plate

34
Q

What are the essential ingredients in BAK culture plates?

A

BAK (Brucella Anaerobic with Vitamin K)

Sheep’s blood, extra hemin, and Vit K are essential nutrients for certain obligate anaerobes.

35
Q

What kind of agar is BBE and what do some of the key ingredients do?

A

Selective & Differential

Bacteroides Bile Esculin (BBE) agar used for the presumptive
ID of B.fragilis group.

Ingredients include:
- gentamicin
- oxgall
- esculin

Gent and Oxgall inhibit facultative anaerobes and most gram neg anaerobes org

36
Q

What kind of agar is LKV and what do some of the key ingredients do?

A

Selective & Differential

LKV (BAK with supplements) agar
Laked blood
Kanamycin
Vancomycin)

Laked blood improves pigmentation; kanamycin inhibits aerobic gram – and some gram+; vancomycin inhibits G+

37
Q

What media (culture plate) inhibits aerobic and
facultative anaerobic GNB and lets Gram Positive cocci & bacilli grow? What does a tech need to be cautious about with this?

A

Phenylethyl Alcohol (PEA)
- Phenylethyl alcohol
- 5% sheep’s blood

Alcohol is bacteriostatic- it does not always kill the GNB, so techs need to be careful when subbing (they might still be there!)

38
Q

What agar can be used for the presumptive ID of Clostridium spp.?

A

Egg Yolk Agar (EYA) - differential
- egg yolk

It can be used aerobically too,
for Bacillus ID.

Note: Has yeast extract, aa digest, hemin for growth of anaerobes also.

39
Q

What enzymes do some bacteria have the produces a precipitate and iridescent sheen respectively on EYA? Length of time to see effects.

A

Lecithinase degrades lecithin in egg yolk producing a precipitate.

Lipase breaks down fat in egg yolk producing an “iridescent sheen”.

Lecithinase within 48 hrs, lipase up to 7 days; lipase hold plate at an angle.

40
Q

What does CCFA agar stand for and why is it not really used anymore in labs?

A

Cycloserine Cefoxitin Fructose Agar (CCFA)
- cycloserine
- cefoxitin
- fructose
- neutral red

For C.diff culture: not done in diagnostic anymore because of faster methods to detect toxin (which is the goal).

41
Q

What is a type of enrichment media that often used heart muscle?

A

Cooked Meat (CM)
muscle tissue granules

Note: Heart tissue is often used, source of aa (tissue provide sulfur groups; they act as reducing agents; media encourages sporulation by Clostridium, note proteolytic activity (eventually will look black showing protein breakdown).

42
Q

What are the requirements for incubating anaerobe culture plates?

A
  1. Plates are incubated under anaerobic conditions at 35º to 37ºC for 48 hours.
  2. Recommended not to open at 24 hrs.
  3. Plates showing no growth are incubated for 5 days.
  4. Always good to expose possible ANO2’s to air as little as possible. Plan time to work with them.

Note: Give them always 48 hours to grow. Some of them grow in 24 hours quite healthy.

43
Q

How is thioglycollate broth incubated?

A

Thioglycollate broth is aerobically incubated with the cap loose or anaerobically incubated with the cap tight (if used).

44
Q

What makes examining plates examined for anaerobes complicated?

A

Anaerobes are usually present in mixed culture.

45
Q

How do you work-up cultures for Anaerobes?

A
  1. Colonies should be characterized and subcultured to purity plates.
  2. Liquid broth should be Gram stained and subcultured if:
    Other cellular types are observed and no growth is observed on primary plates.
  3. First thing to do with any colony growing is the aerotolerance test: subbing to 2 BAK plates and incubate them in different atmospheres to compare, then Gram stain.
46
Q

For gram negative bacilli bacteria what are the things you look for on the culture plate and tests performed for presumptive ID?

A

Gram negative bacilli
Presumptive Identification

  1. Growth on media - pigmentation, fluorescence on BAK/LKV or hydrolysis on BBE
  2. Cellular Morphology: regular bacilli, fusiform bacilli
  3. Biochemical Testing
    high potency antimicrobic discs (Vancomycin, Kanamycin, Colistin)
47
Q

What are the purposes of presumptive ID for gram neg or pos anaerobes?

A

Presumptive ID: good enough for non-critical specimens and as preliminary for critical ones. Most times good for Genus.

48
Q

What can be used for definitive ID of gram negative or positive bacteria?

A
  1. Gas Liquid Chromatography (not used- not practical for clinical labs) organism are identified based upon end-products of fermentation
  2. Biochemical systems: Vitek has an ANO2’s card, API, RapidAna, etc.
  3. Maldi-tof: works great + the organism could die and will still be ID’ed.
49
Q

When is a definitive ID pursued for gram neg or pos anaerobes?

A

Definitive ID used for sterile specimens, otherwise sometimes not worth the time/effort/resources.

50
Q

What characteristics presumptively ID the Bacteroides fragilis group?

A
  1. Growth anaerobically after 48 hours
  2. Sm-med gy on BAK
  3. Presumptively identified on BBE: grows on bile and hydrolyses Esculin: black colonies
  4. Typically resistant to 3 high potency discs:
    Kanamycin (K),
    Vancomycin (Va),
    Colistin (C)

Note: BBE can be used as a primary plate if highly suspicious, or as a way of identification after confirming it is an anaerobic GNB.

51
Q

What characteristics indicate presumptively Porphyromonas spp.?

A

Porphyromonas spp.
1. Black pigment strains (after a few days)
2. Fluorescence under UV light (brick red)
3. Kanamycin “R”, Vancomycin “S”, Colistin “R”
4. No growth on BBE and LKV (Van S)

52
Q

What characteristics indicate presumptively Prevotella spp.?

A

Prevotella spp.
1. No growth on BBE
2. Black pigmented strains (after a few days)
3. Fluorescence under UV light
4. Kanamycin “R”, Vancomycin “R”, Colistin “V” (can grow on LKV)

53
Q

What characteristics indicate presumptively Fusobacterium spp.?

A

Fusobacterium spp.
1. Gram negative bacilli with tapered ends (not all species)
2. Kanamycin “S”, Vancomycin “R”, Colistin “R”
3. Growth on BBE (very ppt) but no hydrolysis
4. No growth on LKV

54
Q

What characteristics indicate presumptively Veillonella spp.?

A

Veillonella spp. :
1. Gram negative cocci
2. Kanamycin “S”, Vancomycin “R”, Colistin “S”
3. No growth on BBE
4. No growth on LKV

55
Q

What are the general characteristics of Clostridium spp.?

A

Clostridium spp.
1. Spore producers (in ANO2), can be central, subterminal, terminal
2. Sometimes aerotolerant (some species) but grow better
in ANO2 conditions
3. Gram positive but over-decolorize easily
4. Almost always cat neg
(Bacillus spp usually pos)
5. Large bacilli

56
Q

What characteristics (8) indicate presumptively C. perfringens?

A

C. perfringens

  1. Subterminal spores
  2. Moderate anaerobe
  3. usually catalase +
    (15% H2O2)
  4. Lge GPB, blunt ends
    (boxcar)
  5. Double zone of hemolysis (DZH), produced by the presence of two toxins:
    a) Hemolysin (outer zone)
    b) Beta-toxin (inner zone)
  6. Lecithinase positive
    –> toxin (phospholipase C) hydrolyzes lecithin in egg yolk agar= white precipitate
  7. Lipase negative (no zone of
    Iridescence)
  8. Reverse Camp - Pos.

Note: May see Pinpt. growth on BA Co2 for C. perfringens;

57
Q

What is Propionibacterium is now called?

A

Cutibacterium

Microbiology 2 (22 decks)

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