Antimicrobial Effectiveness Testing (USFDA)

Antimicrobial Effectiveness testing is described in USP <51>.  Previously this chapter was known as “Preservative Effectiveness Testing”. Detailed procedure for the performance of the test can be found in USP <51>.

Antimicrobial Effectiveness Testing
  1. Media

For the cultivation of the test organisms, select agar medium that is favorable to the rigorous growth of the respective stock culture. The recommended media are Soybean Casein Digest Agar/Broth and Sabouraud’s Dextrose Agar/Broth.  Add a suitable inactivator (neutralizer) for the specific antimicrobial properties in the product to the broth and/or agar media used for the test procedure whenever needed.

  • Growth Promotion of the Media

Media used for testing needs to be tested for growth promotion by inoculating the medium with appropriate microorganisms. It is preferable that test microorganisms be chosen for growth promotion testing (Section D).

Solid media tested for growth promotion is to be set up using the method that will be used to analyze the product (pour plate or spread plate) to determine a microbial plate count (CFU) which must be ≥ 50% of the microorganism inoculum’s calculated value.

  1. Suitability of the Counting Method in the Presence of Product

For all product types, follow current USP methodology in chapter <51>, with the following additional instructions.

Prior to the Antimicrobial Effectiveness testing, determine if any antimicrobial properties exist by performing a Suitability testing utilizing microorganisms used for product testing (section D).  Should the Suitability Test fail the results of Suitability test are invalid and will need to be repeated with proper method modification to neutralize the inhibiting property.

If multiple samples of the same product from the same manufacturer (same amount and form) are collected, one sample may be used for method suitability for all the samples collected.

  • Test Organisms

All cultures must be no more than 5 passages removed from the original stock culture.

  1. Candida albicans (ATCC No. 10231)
  2. Aspergillus brasiliensis (ATCC No. 16404) 
  3. Escherichia coli (ATCC No. 8739)
  4. Pseudomonas aeruginosa (ATCC No. 9027)
  5. Staphylococcus aureus (ATCC No. 6538)

E. Preparation of Inoculum

Preparatory to the test, inoculate the surface of the appropriate agar medium from a recently grown stock culture of each of the above test microorganisms.

Use Soybean-Casein Digest agar for Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus ATCC 6538 and incubate at 32.5 ± 2.5° C for 3 – 5 days.  Use Sabouraud Dextrose agar for Candida albicans ATCC 10231 and Aspergillus brasiliensis ATCC 16404 and incubate at 22.5 ± 2.5° C for 3 – 5 days for Candida albicans and 3 – 7 days for Aspergillus brasiliensis.

Harvest the cultures by washing the growth with sterile saline to obtain a microbial count of about 1×108 CFU/mL (see Microbial Enumeration Tests <61> and Tests for Specified Microorganisms <62>). For the A. brasiliensis ATCC 16404 culture, use sterile saline containing 0.05% polysorbate 80.

Alternatively, cultures may be grown in a liquid medium, i.e. Soybean Casein Digest Broth or Sabouraud’s Dextrose Broth, (except for the A. brasiliensis ATCC 16404 culture) and harvested by centrifugation, washing and suspending in sterile saline to obtain a count of about 1 X 108   colony forming units (CFU) per mL.

The estimate of inoculum concentration may be obtained by turbidimetric procedures for the challenge microorganisms and later confirmed by plate count.

Refrigerate the suspension if not used within 2 hours at 2-8° C.

Determine the number of CFU/mL in each suspension using the appropriate media and recovery incubation times to confirm the CFU/mL estimate.

Use bacterial and yeast suspensions within 24 hr. of harvest. The mold preparation may be stored under refrigeration (2-8° C) for up to 7 days. Note:  Alternative commercially available standardized cultures may be used in lieu of in-house prepared cultures.

F. Procedure

The procedure requires that the test be conducted with a suitable volume of product.  It is advisable to begin with at least 20 mL of product.  Use the original product containers whenever possible or five sterile, capped bacteriological containers of suitable size into which a suitable volume of product has been transferred. If the diluted product exhibits antimicrobial properties, specific neutralizers may need to be incorporated into the diluents or the recovery media. For purposes of testing, products have been divided into four categories:

Category 1 – Injections, other parenteral including emulsions, otic products, sterile nasal products, and ophthalmic products made with aqueous bases or vehicles.

Category 2 – Topically used products made with aqueous bases or vehicles, non-sterile nasal products, and emulsions, including those applied to mucous membranes.

Category 3 – Oral products other than antacids, made with aqueous bases or vehicles.

Category 4 – Antacids made with aqueous bases or vehicles.

Inoculate each container with one of the prepared and standardized inoculums and mix.  The volume of the suspension inoculums used is 0.5% to 1.0% of the volume of the product. The concentration of the test organisms added to the product for Categories 1, 2 and 3 is such that concentration of the test preparation immediately after inoculation is between 1×105 and 1×106 colony forming organisms (CFU) per mL of product.  If no suitable neutralizing agent or method is found and method suitability requires significant dilution, a higher level of inoculum (e.g., 107-108) may be used so that a 3-log unit reduction can be measured.  For category 4 products (antacids) the final concentration of the test organisms is between 1×103 and 1×104 CFU/mL of product.  

Immediately determine the concentration of viable organisms in each inoculum suspension and calculate the initial concentration of CFU/mL by the plate count method (see Microbial Enumeration Tests <61>).

Incubate the inoculated containers between 22.5 ±2.5°C in a controlled environment (incubator) and sample the container at specified intervals.  The container sampling intervals include: Category 1 products are sampled at 7, 14, and 28 days and Category 2 – 4 products are sampled at 14 and 28 days.  Refer to table 3 within USP <51>. Record any changes in appearance of the product at these intervals.   Determine the number of viable microorganisms per mL present at each of these sampling intervals by the plate count method utilizing media with suitable inactivator (neutralizer).  Calculate the change in log10 values of the concentration per mL based on the calculated concentration in CFU/mL present at the start of the test for each microorganism at the applicable test intervals and express the changes in terms of log reductions.

NOTE: The USP does not require a specific volume of product to be added to each of the five sterile tubes.  It is recommended that 20 mL/tube be used to standardize testing for all ORS laboratories.

NOTE:  All plate counts should be performed in duplicate (2 plates per dilution), and in a dilution series to detect growth inhibited by the preservative system at the lower dilutions.  Carrying the test to the 10-3 dilution would be sufficient in most cases to overcome preservative inhibition. G. Interpretation

The criteria for microbial effectiveness are met if the specified criteria are met, see table below. No increase is defined as not more than 0.5 log10 unit higher than the previous value measured.

Antimicrobial Effectiveness testing Product Category:-

 Category 1 Products
Bacteria:Not less than 1.0 log reduction from the initial calculated count at 7 days, not less than 3.0 log reduction from the initial count at 14 days, and no increase from the 14-day count at 28 days.
Yeast and Molds:No increase from the initial calculated count at 7, 14, and 28 days.
 Category 2 Products
Bacteria:Not less than a 2.0 log reduction from the initial count at 14 days, and no increase from the 14-day count at 28 days.
Yeast and Molds:No increase from the initial calculated count at 14 and 28 days.
 Category 3 Products
Bacteria:Not less than a 1.0 log reduction from the initial count at 14 days, and no increase from the 14-day count at 28 days.
Yeast and Molds:No increase from the initial calculated count at 14 and 28 days.
 Category 4 Products
Bacteria, Yeast and Molds:No increase from the initial calculated count at 14 and 28 days.

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