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Introduction of Anaerobes Presumptive Identification using Antibiotic disks

Anaerobes Presumptive Identification using Antibiotic disks is useful to determine an anaerobe’s inhibition that can be used for presumptive identification based on its characteristic susceptibility pattern to colistin (10 µg), vancomycin (5 µg), and kanamycin (1 000 µg ).

Principle of  Anaerobes Presumptive Identification using Antibiotic disks

Normally, gram-positive anaerobes are susceptible to vancomycin while resistant to colistin. Gram-negative anaerobes are resistant to vancomycin. These disks are principally useful in differentiating between Bacteroides and Fusobacterium species. With a few exceptions, Bacteroides spp. is resistant to kanamycin and vancomycin and show variable susceptibility to colistin, while Fusobacterium spp. are generally resistant to vancomycin and susceptible to kanamycin and colistin.

Test Requirements for anaerobes Presumptive Identification using Antibiotic disks

• Test organisms
• McFarland Densitometer
•  Test tubes
• Blood agar/Mueller Hinton Agar supplemented with 5%
  Sheep blood (preferred)
• Antimicrobial disks, colistin (10 µg), vancomycin (5µg), and kanamycin (1 000µg )
• Swab stick
• Bunsen burner
• Inoculating wire
• Incubator
• Disk applicator/ forceps
• Control strains-Bacteroides fragilis ATCC 25285, Fusobacterium nucleatum
  ATCC 25586

Test Procedure for anaerobes Presumptive Identification using Antibiotic disks

1. Let the antibiotics disks adjust to room temperature prior to use.
2. Blood agar/Mueller Hinton Agar supplemented with 5%
   Sheep blood is used in these antibiotic susceptibility tests.
3. Emulsify heavy colonies in sterile saline matching the turbidity that standard (0.5 McFarland).
4. Place a sterile cotton swab in the bacterial suspension and remove the excess fluid by pressing and rotating the cotton against the inside of the tube above the fluid level.
5. The swab is streaked in three directions over the surface of the antimicrobial sensitivity testing agar (Blood agar/MHAsupplemented with 5%
   Sheep blood to obtain uniform growth.
6. Using sterile forceps or a suitable disk dispenser, place paper disks impregnated with a fixed concentration of an antibiotic, on the surface agar plates having distance disc to disc 25 mm while plate border to disk 15 mm.
7. Incubate the plates at 35°C for 48 hours.
8. Following incubation, measure the diameter of the zone of inhibition in millimeters (mm) around each disk.

Result Interpretation of anaerobes Presumptive Identification using Antibiotic disks

Antibiotic Disks for the Presumptive Identification of Anaerobes

Susceptible (S) – zone of inhibition is equal and greater than (≥) 10 mm

Resistant(R) – zone of inhibition less than (<) 10 mm

Control strains-Bacteroides fragilis ATCC 25285 is resistant to all three disks.  Fusobacterium nucleatum ATCC 25586 is sensitive to colistin and kanamycin but resistant to vancomycin.

Type of organism -Vancomycin-Kanamycin-Colistin

B. fragilis group- R -R- R
B. ureolyticus group- R-S- S
Fusobacterium spp. R- S- S
Porphyromonas spp. S- R- S
Veillonella spp. R- S- S

Keynotes

1. Results of these antimicrobial disks susceptibility testing cannot be used to accurately determine in vivo antibiotic treatment since it’s just for diagnostic purposes.
2. Antibiotic disks should be stored at -20°C.

Bibliography 

1. Vargo V, Korzeniowski M, Spaulding EH. Tryptic soy bile-kanamycin test for the identification of Bacteroides fragilis. Appl Microbiol 1974; 27:480-3.
2. Sutter VL, Citron DM, Edelstein MAC et al.Wadsworth anaerobic manual. 4th ed. Belmont, CA: Star Publishing Company, 1985.
3. Balows A, Hausler WJ, Hermann KL et al. Manual of clinical microbiology. 5th ed.Washington, DC: ASM, 1991.
4. Isenberg HD, Ed. Clinical microbiology procedures handbook, Vol 1. Washington, DC: ASM, 1992.
5. MacFaddin JF. Biochemical tests for identification of medical bacteria. 3rd ed. Philadelphia, PA: Lippincott Williams & Wilkins, 2000.