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BIOBALL® Burkholderia cepacia Complex Strains Performance Testing for USP <60> Growth Promotion Requirements

Introduction

Over the years, presence of objectionable microorganisms has been identified as the leading cause of non-sterile drug product recalls in the U.S. Burkholderia cepacia is one of the most prominent objectionable microorganism associated with 102 non-sterile drug recalls from 2012 to 2019(1,2). It is an opportunistic pathogen and has a distinctive ability to overcome antimicrobial preservative systems. Moreover, Burkholderia cepacia complex (Bcc) comprises of about 24 closely related species which are widely found in the environment and have the ability to form biofilms in pharmaceutical water systems(3). More specifically, 2 members of the Bcc group, Burkholderia cenocepacia and Burkholderia multivorans account for >85% of the cystitis fibrosis infections(1,4).

USP <60 > Microbiological Examination of Non-sterile Products – Tests for Burkholderia cepacia complex was officialised in December 2019 in the second supplement to USP 42/NF 37(5). It illustrates mandatory testing of aqueous based non-sterile products for the absence of Bcc test strains, i.e. B. cepaciaB. cenocepacia and B. multivorans. All 3 test strains are required for growth-promoting and indicative properties testing of Burkholderia cepacia selective agar (BCSA) and method suitability testing, as described in the chapter. Growth Promotion Testing acceptance criteria for USPis based on the growth of microorganisms on new batch of BCSA comparable to the previously tested and approved batch of same culture media(5). This suggests compendial test requirements for selective media are not necessarily quantitatively evaluated. Bcc test strains are available in BIOBALL® MultiShot 550 format for USP <60 > testing needs.

 

Purpose

Evaluate BIOBALL® MultiShot 550 Bcc range recovery levels on bioMérieux BCSA (Ref 33631) ensuring they are acceptable as per growth promotion and indicative properties testing requirements of USP <60 > chapter.

 

Materials

BIOBALL® MultiShot 550 Bcc Range- It consists B. cepacia NCTC 10743 (Ref 416589), B.cenocepacia NCTC 13227 (Ref 423580) and B. multivorans NCTC 13007 (Ref 423581). 3 BIOBALL® batches of each of these Bcc strains were tested in this study.

Alternative Re-Hydration Fluid (Ref 417843) – It is used to enhance BIOBALL® recovery on selective culture media. BIOBALL® MultiShot 550 is dispensed and rehydrated in the tube containing 1.1 mL of fluid, which yields 10x100 µL aliquots. BIOBALL® rehydrated in alternative re-hydration fluid can be used for upto 8 hours, if stored in a refrigerator at 2°C to 8°C(6).

 

Method

Total 9 batches of BIOBALL® MultiShot 550 Bcc range (3 each of B.cepaciaB.cenocepacia and B.multivorans) were tested on 3 different BCSA batches. To prepare aliquots, BIOBALL® was dispensed into a 1.1 mL tube of Alternative Re-Hydration Fluid to make 10 doses of 100 µL each. Thereafter, wait for 30 seconds, followed by vortexing for 5 seconds. The tube was then incubated at 32±2°C for atleast 30 minutes. 100 µL aliquots were drawn and plated on the center of BCSA plates. Testing involved 25 replicates per batch, i.e., 5 replicates from 5 balls. Plates were incubated at 30-35°C for 48 to 72 hours and colonies were manually counted by a single operator.

The results from all tested BIOBALL® batches were summarized and a mean CFU count per BIOBALL® and per 100 µL aliquot was determined. The mean CFU for each batch from the study was compared with the mean CFU stated on the corresponding batch “Certificate of Analysis” to determine recovery rates in %. The quantification data reported on Certificate of Analysis for BIOBALL® Bcc range is based on rehydration using standard BIOBALL® Re-Hydration Fluid (Ref 56021) and QC testing on Nutrient agar plates.

 

Results and discussion

The batches of BIOBALL® MultiShot 550 Bcc range tested with 3 different batches of BCSA showed recovery rates between 50 to 200% of the mean CFU counts obtained on the reference non-selective growth media (Nutrient agar) as reported on batch-specific BIOBALL® Certificates of Analysis (Table 1). BCSA showed a mean recovery of 46.8±6.5 CFU per 100 µL aliquot for B. cepacia batches, whereas for B. cenocepacia and B. multivorans batches, mean recovery per 100 µL aliquot was found to be 36.7±7.4 CFU and 44.4±5.3 CFU, respectively. All 3 BIOBALL® batches of each Burkholderia strain tested consistently grew well on BCSA as indicated by the high mean CFU recovery rate of >50%, demonstrating reliability, reproducibility and robustness of these batches.

 

CFU Counts

Table 1: Summary table of mean CFU counts obtained (per BIOBALL® and per 100 µL aliquot) for every Bcc range BIOBALL® batch tested. # Nutrient agar, non-selective reference growth media, reporting batch-specific CFU recovery on Certificate of Analysis ; BCSA, selective growth media, used in this study.

In addition to recovery rates, colony appearance of the BIOBALL® Bcc range on BCSA was also analyzed. Following incubation at 30 to 35°C for 48 to 72 hours; B. cepacia appeared as greenish-brown colonies with yellow halos, whereas, B. cenocepacia and B. multivorans colonies were purple in color surrounded pink-red zone (Figure 1). Acid production from the oxidation of carbohydrates creates a yellow zone in the area surrounding the growth, whereas peptone utilization results in a pink zone. Absorption of the crystal violet dye will cause colonies to appear purple to purple-gray.

Furthermore, after 48 hours of incubation colony size of B. cepacia on BCSA was comparable to that on a non-selective growth media, whereas, colony sizes of B. cenocepacia and B. multivorans were found to be relatively smaller on BCSA compared to their corresponding colony sizes on a non-selective growth media upon 72 hours of incubation.

 

Figure 1: BIOBALL Bcc range colony appearance on BCSA. (a) B.cepacia (b) B.cenocepacia and (c) B.multivorans

 

Results and discussion

All BIOBALL® Bcc range batches tested on BCSA demonstrated recovery rates within 50 to 200%. The range was defined using mean CFU counts reported on batch-specific BIOBALL® Certificates of Analysis where Bcc BIOBALL® batches were tested on a non-selective media, Nutrient agar. The study demonstrates batch-to-batch consistency, strong reproducibility and robustness of Bcc BIOBALL® regardless of the type of culture media used for testing. Selective culture medium, due to its composition and purpose, is sometimes known to cause recovery difficulties. However, the results of this study indicate that BIOBALL®, being a quantitative Certified Reference Material, ensures consistent recovery batch-after-batch even on culture media that may be highly selective in nature. This helps in significantly minimizing risk of QC failure, ensuring timely release of culture media and overall boosting user’s confidence in their culture media QC methods.

 

References

  1. Cundell, T. 2019 Excluding Burkholderia cepacia complex from Aqueous, Non-Sterile Drug Products. Posted February 12, 2019 Amer. Pharm. Rev.
  2. L. Jimenez, 2019 Analysis of FDA Enforcement Reports (2012-2019) to Determine the Microbial Diversity in Contaminated Non-Sterile and Sterile Drugs Posted October 24 2019 Amer. Pharm. Rev.
  3. Akers J.E., Agalloco J.P. and Madsen R.E. B. Cepacia: A Case Study For Determining Objectionableness In Drug Manufacturing Posted on August 9, 2021 Outsourced Pharma.
  4. Reik R, T. Spilker, and J.J. LiPuma 2005 Distribution of Burkholderia cepacia complex species among isolates recovered from persons with or without cystic fibrosis. J Clin Microbiol 43:2926–2928
  5. USP Microbiological examination of non-sterile products – Tests for Burkholderia cepacia complex.
  6. BIOBALL® MultiShot 550 Instructions for Use.