Anaerobes, Antibiotic Susceptibility

Question 1

Which Clostridium species have myonecrosis?

Answer 1

C. perfringens
C. septicum

Question 2

Concentration of gases for anaerobic conditions

Answer 2

90% nitrogen
5% CO2
5% hydrogen

Question 3

How are anaerobic conditions achieved?

Answer 3

Anaerobic boxes and jars to prevent O2 from getting in

Question 4

PRAS media

Answer 4

Pre Reduced Anaerobically Sterilized

Brucella, PEA, LKV

Question 5

Brucella media

Answer 5

• Nonselective, similar to SBA
• Prevotella (fluorescence)
• Hemolysis observation

Question 6

Phenyl Ethyl Agar (PEA)

Answer 6

• Selective for obligate anaerobes (both Gram pos and neg) and Gram (+) facultative anaerobes
• Inhibits G(-) rods - Enterobacteriaceae
• Inhibits swarming of Clostridia

Question 7

Laked Kanamycin Vanvomycin (LKV) agar

Answer 7

• Inhibits G(+) organisms and aerobic G(-) rods
• Inhibits Porphyromonas
• Isolates Bacteroides and Prevotella (black pigmentation)

Question 8

Bacteroides Bile Esculin (BBE) agar

Answer 8

• Bile resistance = growth on BBE agar
• Esculin hydrolysis = black pigmentation
• Bacteroides fragilis - growth and black
• Fusobacterium mortiferum - growth
• Bilophila wadsworthia - fish eye appearance
• Inhibits aerobic G(-) rods

Question 9

Egg Yolk Agar

Answer 9

• Detects presence of lecithinase and lipase
• Lecithinase: opaque precipitate around colonies (C. perfringence)
• Lipase: iridescent sheen on colony surface (F. nacrophorum)
• Proteolysis: clearing around colonies

Question 10

Chopped meat carbohydrate agar

Answer 10

Enrichment broth
- Detects organisms in low numbers (helps with recovery)

Question 11

Thioglycolate broth

Answer 11

Sodium thioglycollate and L-cysteine - reducing agents, helps save anaerobes from dying

• anaerobes grow at the bottom of the tube

Question 12

Acceptable specimens for anaerobes

Answer 12

NOT:
Sputum, feces, gastric contents, all swabs, urine

Question 13

Clostridium perfringens

Answer 13

• GI tract: food poisoning
• Non GI tract: myonecrosis (gas gangrene)
• Found in: soil/GI tract
• Gram stain: boxcar, G(+) straight rods
• Colony morphology: double zone beta hemolysis, lecithinase (+) opaque precipitate on Egg Yolk Agar
• Reverse CAMP with Group B Strep
• alpha and beta toxins

Question 14

Which organism has turbidity around colonies on Egg Yolk Agar?

Answer 14

Clostridium perfringens

Question 15

Alpha and beta toxins for C. perfringens

Answer 15

Type A/Alpha toxin:
- contaminated meat/gravies
- mild cramps, diarrhea, self limiting
- from spores

> alpha toxin for non GI tract/in muscle instead —> gas gangrene/myonecrosis

Type B/Beta toxin:
- life threatening, necrotizing enterocolitis (inflamed intestines)
- bloody diarrhea with vomiting

Question 16

Clostridium botulinum

Answer 16

• Botulism: food poisoning
• Ingestion of toxins in food, soil contaminated food or honey
• Virulence factor: neurotoxins, release of acetylcholine can cause paralysis
• “Tennis racket” gram stain
• Lipase (+) - iridescent on Egg Yolk Agar
• Adult botulism: toxin in food already
• Infant botulism: toxin released in GI tract (food has spores)

Question 17

Clostridioides difficile

Answer 17

• AAD (antibiotic associated diarrhea), colitis
• Virulence factors: A toxin (enterotoxin), B toxin (cytotoxin)
• watery diarrhea
• pseudomembranous colitis (PMC): yellow plaques through colonic mucosa

Media:
- CCFA
- Chartreuse fluorescence
- Barnyard odor

Question 18

Barnyard odor

Answer 18

Clostridioides difficile

Question 19

Clostridium septicum

Answer 19

• Myonecrosis
• Typhilitis (neutropenic colitis): sepsis in immunocompromised
• Uneven gram stain, Citron bodies
• Medusa Head on Brucella
• Beta hemolytic
• Swarming within 24 hours

Question 20

Clostridium tetani

Answer 20

• Wound/puncture contaminated in soil
• Tetanus: trismus (lockjaw) and Rudy’s sardonicus (distorted grin)
• G(+) but turns G(-) after 24 hours
• Oval spores, drumstick appearance
• Narrow zone of beta hemolysis

Question 21

Cutibacterium (Propionibacterium) acnes

Answer 21

• Joint infection
• “Palm of Hands” in bunches on gram stain
• Catalase and Indole (+)
• Gram (+) non-spore forming rods

Question 22

Bacteroides fragilis

Answer 22

• Mucosal surfaces of oral cavity and GI tract
• Abscesses
• Foul odor
• Bacteremia, septic arthritis, aspiration pneumonia, chronic sinusitis, decubitus ulcers
• Black colonies on BBE agar (bile esculin resistant and hydrolyzes esculin

Question 23

Prevotella

Answer 23

• Normal flora of oral cavity
• Black colonies on agar
• Brick-red fluorescence
• Colistin S (KV - R)
• Lipase (+)

Question 24

Porphyromonas

Answer 24

• UV fluorescence (pink/orange/red)
• Some species pigmented/black and mask UV pigment
• Asaccharolytic
• Vancomycin S, KC - R
• Does not grow on BBE/LKV

Question 25

Fusobacterium nucleatum

Answer 25

• Isolated from oral cavity (biofilms)
• KC (S), V (R)
• Beta hemolytic
• Chartreuse fluorescence
• Ground glass/bread crumb colonies
• Indole (+)
• No growth on PEA/LKV/BBE except F. mortiferum (also indole (-))
• Long, tapered thin rods

Question 26

What are the Gram (+) anaerobic cocci?

Answer 26

Peptostreptococci (brain abscess)
Finegoldia
Parvimonas
Peptoniphilus
Murdochiella
Staphylococcus
Anaerococcus

Question 27

What are the Gram (-) anaerobic cocci?

Answer 27

Veillonella
Megasphaera
Anaeroglobus
Negativicoccus
Acidaminococcus

Question 28

How MIC is performed

Answer 28

MIC - minimum inhibitory concentration, the first tube without organisms growing

Broth dilution
Same inoculum, different concentrations of antibiotics.

Question 29

Bactericidal

Answer 29

Kills bacteria

Question 30

Bacteriostatic

Answer 30

Inhibits bacteria, reversible

Question 31

Difference between Prevotella and Porphyromonas?

Answer 31

Prevotella: vancomycin resistant, brick red fluorescence

Porphyromonas: vancomycin susceptible, pink red orange fluorescence

Question 32

Beta lactam resistance

Answer 32

Beta lactams bind enzymes (PBPs - penicillin binding proteins) which inhibit cell wall synthesis
- weaken cell wall and cause cell death

Question 33

ESBLs (Extended Spectrum Beta- lactamases)

Answer 33

Organisms can hydrolyze beta lactam rings

Resistance to penicillin, cephalosporin and aztreonam

Question 34

CRE - carbapenem resistant enterobacteriales

Answer 34

WITH carbapenemase enzyme: Inactivate carbapenems and other beta lactam antibiotics

WITHOUT carbapenemase: mutations in porin gene prevent carbapenems/antibiotic from entering bacterial cell wall

Question 35

Protein synthesis inhibitors

Answer 35

Aminoglycosides: gentamycin, kanamycin, tobramycin, streptomycin, amikacin

Macrolides: erythromycin, clindamycin

Streptogramins: dalfopristin, quinopristin

Oxazolidinones: linezoid

Chloramphenicol: chloramphenicol

Tetracyclines: doxycycline, tigecycline, tetracycline

Glycylglycines: improved tetracyclines

Question 36

What do beta lactams inhibit?

Answer 36

Cell wall

Question 37

Examples of beta lactams

Answer 37

Penicillin, cephalosporin, carbapenems, monobactams

Question 38

Cell wall inhibitors

Answer 38

Beta lactams

Glycopepetides

Question 39

Cell membrane inhibitor

Answer 39

Lipopeptides

Question 40

Inhibitors of DNA/RNA synthesis

Answer 40

Disrupt DNA -
- Fluoroquinolones: ciprofloxacin, levofloxacin
- Metronidazole

Inhibits RNA/protein synthesis -
- Rifamycin

Question 41

Metabolic inhibitors

Answer 41

Folic acid inhibitors -
- Sulfonamide
- Trimethoprim

• Nitrofurantoin