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Introduction  of O/129 Disk Susceptibility Testing

O/129 Disk Susceptibility Testing is useful for the identification of Vibrio and Aeromonas species.

Principle of O/129 Disk Susceptibility Testing

O/129 disk is the vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine phosphate. O/129 disks containing 10 and 150 µg are used in a method resembling disk susceptibility testing to differentiate Vibrio species from Aeromonas species. Aeromonas species are resistant, with no zone of inhibition at 24 hours, with both disks. Vibrio and Plesiomonas species will show susceptibility, with a distinct zone of inhibition with the 150 µg disk; results for the 10 µg disk will vary among the Vibrio and Plesiomonas species. Some Vibrio spp. require salt for growth. Therefore, the test is run in duplicate on Mueller-Hinton agar (MHA) with low salt (0.5%) and with added NaCl (4%) to ensure growth on at least one of the plates.

Requirements for O/129 Disk Susceptibility Testing

•  Test organisms may be of

1. Gram-negative rods which are oxidase-positive, indole positive, and non-pigmented and grow on MacConkey agar.
2. Microorganisms identified by kits as either Vibrio or Aeromonas species
3. Corynebacterium, to separate Corynebacterium amycolatum from Corynebacterium xerosis.
4. O/129 Disks for 10 µg and  O/129 for 150 µg

• MHA
•  MHA with 4% NaCl
• Swabs
• Broth for inoculum
• Control strains: For positive control (PC):Aeromonas hydrophila ATCC 7966

For Negative Control (NC): Vibrio fluvialis ATCC 33809

Procedure of O/129 Disk Susceptibility Testing

1. Remove O/129  disks from the freezer(-20°C) and equilibrate to room temperature.
2. Allow test MHA to equilibrate to room temperature. If the plates have excess moisture, dry them for 10 – 30 min at 35°C with lids slightly ajar.
3. Suspend an overnight growth of the organism and each of the controls in Tryptone soy broth (TSB) to turbidity of a no. 0.5 McFarland standard and divide each plate in half with a marker.
4. Using a swab dipped into the broth, inoculate the surface of one-half of each plate with organism suspension in three planes for antimicrobial susceptibility disk testing.
5. In the same manner, inoculate the other half of the MHA plate with 4% salt with the Vibrio control and the MHA plate without salt with the Aeromonas control.
6. Leave plates to dry for 3- 5 min but no longer than 15 minutes.
7. Put a 10 µg O/129 disk and a 150 µg O/129 disk on each half of the two plates in well-separated locations and press each disk gently onto the agar surface to ensure good contact Using sterile forceps.
8. Incubate MHA plates for 18 to 24 hours at 35°C in a non-CO2 incubator.
9. Finally, read the zone of inhibition on a plate with the best growth and most clear-cut zone.

Result and interpretation of O/129 Disk Susceptibility Testing

• The organism is O/129 resistant: no zone of inhibition around the respective disk
• The organism is O/129 susceptible: any zone around the respective disk
• Aeromonas hydrophila ATCC 7966: No zone of inhibition (10 and 150µg disks in 0.5% MHA) i.e. resistant
• Vibrio fluvialis ATCC 33809: Sensitive (Zone of inhibition for 10µg 7-15mm while 150µg  ≥20 mm in 4% MHA)
• The organism is generally salt requiring if it does not grow on Muller-Hinton agar.
• The organism is salt tolerant or enhanced if it grows on Muller-Hinton agar with 4% salt.
• The organism is inhibited by the salt if it does not grow on Muller-Hinton agar with 4% salt.

Keynotes of O/129 Disk Susceptibility Testing

1. Gram-negative bacteria that are oxidase-positive, grow on MacConkey agar, and ferment glucose (most are indole positive) are reported as follows.
2. Aeromonas, if resistant to 150 µg of O/129, grows without salt, and is arginine positive. Strains are also usually DNase positive. a. Aeromonas veronii biovar Veronii is arginine negative.
3. Vibrio cholerae is arginine negative, and some strains are resistant to O/ 129 and may not grow on media with 4% salt. Perform further testing on strains with these characteristics, or submit to a reference laboratory to separate V. cholerae from A. veronii bv. Veronii.
4. Vibrio fluvialis is arginine positive and may be resistant to 150 µg of O/ 129, but it usually grows only with 4% salt.
5. Plesiomonas shigelloides, if susceptible to 150 µg of O/129 and positive for indole, arginine dihydrolase, and ornithine and lysine decarboxylases. P. shigelloides is also inhibited on 4% salt agar and is DNase negative.
6. Vibrio spp., if susceptible to 150 µg of O/129 even if they do not grow on MHA with 4% salt but are either negative for arginine dihydrolase or negative for ornithine and lysine decarboxylases.
7. Some vibrios may be resistant to 150 µg of O/129, but they will be arginine negative.

Limitations of O/129 Disk Susceptibility Testing

1. Vibrio cholerae strains that are resistant to O/129 disk have been reported, which is probably related to antimicrobial therapy.
2. The 10 µg disk is not as important in the identification as the 150 µg disk and may be omitted.
3. Some vibrios do not grow on MHA with 4% salt, and some grow without added salt. All Aeromonas organisms grow without salt but may grow on a medium with salt.

Further Readings

1. https://himedialabs.com/td/dd047.pdf
2. https://www.scielo.br/j/bjid/a/GBq5hnfNgGNtXwXDMXjGpnL/?lang=en
3. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
4. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
5. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.