End-User or Third-Party Organism Identification and Purity

Introduction

Users of biological indicators (BIs) need to have an appropriate level of knowledge of the product and its intended use prior to purchase. Once achieved, users also need to have confidence in the quality of the product received prior to placing it into use. There are two general methods for gaining confidence in product quality, 1) performing a quality audit(s) of the manufacturer to verify compliance to relevant standards/regulations, and 2) performing quality testing on each incoming BI lot. There are several important BI qualities that can be evaluated by second/third parties including, BI spore count (population), BI resistance to the process (D-value and Survival/Kill), and identity/purity of the test organism.

This edition of Spore News will cover the various aspects of identity/purity activities, and the options for the end-user to consider. As detailed below, Mesa employs several methods to verify the identity/purity of the organism; the results of which can be verified during an audit. Once the manufacturer has received a successful audit, it is not recommended to perform second/third party verification.

Biological indicator manufacturers must be confident the organism being inoculated onto a BI carrier is the correct organism and that there are no interfering factors in order to ensure performance required by the end-user, consistent lot-to-lot performance, and BI stability throughout the shelf life. For this reason, Mesa uses numerous means to confirm the organism identity of source cultures and spore crops grown from the source cultures.

Additionally, Mesa verifies the purity of spore crops and spore suspensions used for carrier inoculation by inspection of the colonies on suitable media incubated at an appropriate temperature. End-users typically employ only a singular means of identification during either incoming acceptance or an investigation into unexpected results. The most common means of identification used by end-users is either a system that uses biochemical reactions or a system that uses the first 500 base pairs of the 16s rRNA sequence. Both are well-accepted methods but both can have limitations when performing identification of very closely related species within the same genus, such as Bacillus and Geobacillus. The result provided is simply the closest match in the system database and additional testing, or a polyphasic approach may be needed to resolve to the species level of these closely related organisms. If an unexpected identification result is obtained from one of the aforementioned methods, it is best to contact the third-party lab that performed the identification or the manufacturer of the identification system for further explanation about the result. Relevant standards and other resources are provided below.

Standards & References

ISO 11138-1:2017 Sterilization of health care products – Biological indicators – Part 1: General requirements

4.1 Manufacturing controls

4.1.1 Quality management systems A formal quality system (e.g., ISO 13485, BMPs or other national or regional requirements) to cover all operations required by the document shall be established, documented and maintained. In particular, precautions at all stages of production to minimize contamination that would adversely affect the performance of the biological indicator shall be taken.

4.2 Test organism

4.2.1.1 Test organisms shall be of a defined strain, available through a recognized culture collection, and shall be identified by appropriate test methods. . .

4.2.2 Originating inoculum for suspension

4.2.2.1 The initial inoculum for each batch of test organism suspension shall be

a) traceable to the reference culture and available through a recognized culture collection,
and
b) verified as to its identity and purity.

4.2.2.2 The methods used for maintaining test organism cultures shall be designed to protect them from contamination and to minimize any induced changes in the inherent properties of the test organisms.

4.2.2.3 Verification tests are specific for each strain of test organisms . . .

5.2.1 Carrier, primary and secondary packaging

5.2.1 The materials of the carrier and the primary and secondary packaging shall not contain any contamination (physical, chemical or microbial) that would adversely affect the performance of the biological indicator.

5.2.2 The carrier, the primary and secondary packaging, and the specified storage conditions shall be designed so that the performance characteristics of the biological indicator meet the requirements of this document throughout the stated shelf life of the product . . .

5.3 Inoculated carrier

5.3.2 Only one strain of test organism shall be used in a batch of inoculated carriers, unless the manufacturer has demonstrated that the use of multiple strains does not significantly affect test organism performance in the specified sterilization process.

 5.3.3 Prior to inoculation, the carrier shall be sterilized in accordance with ISO 17665-1, ISO 11135, and ISO 14937 or other relevant sterilization methods. If sterilization is not practicable, acceptable bioburden limits of the carrier prior to inoculation may be established in accordance with ISO 11737-1:2006, Annex B.

Mesa Labs’ Recommendations 

1. Perform a quality audit(s) of the manufacturer to verify compliance with relevant standards/regulations. 
2. Conduct second- or third-party identification verification studies only if a quality audit has not been conducted. 
3. Perform identification verification study according to the manufacturer’s recommendations. 
4. Utilize the Contract Studies Lab at Mesa Labs for all third-party testing.  Mesa Labs Contract Studies Lab provides a variety of testing services including biological indicator population, resistance (D-value, z-value, and Survival/Kill times), and organism identification verification.  Since many variables make it difficult to guarantee the ability to reproduce this testing, Mesa Labs, an experienced biological indicator manufacturer, offers these services to customers to support their compliance with standard requirements. The Contract Studies Lab is ISO 13485 certified for these services and functions as an independent lab because its reporting authority is separate from Mesa’s biological indicator manufacturing. 

Additional Resources

1. Spore News SN004 The Importance of 16S rRNA Bacterial Spore Identification
2. Spore News SN067 Sub-culturing a Positive Biological Indicator for Identification 
3. Position Statement -- TS-505 “Contamination” on Biological Indicators (BIs) with Non-liquid Carriers
4. ISO 11138-1:2017 Sterilization of health care products – Biological indicators – Part 1: General requirements
5. ISO 11138-7:2019 Sterilization of health care products Biological indicators – Part 7: Guidance for the selection, use and interpretation of results
6. United States Pharmacopeia, General Chapter <1113> MICROBIAL CHARACTERIZATION, IDENTIFICATION, AND STRAIN TYPING
7. United States Pharmacopeia 2022, General Chapter <1229.5> BIOLOGICAL INDICATORS FOR STERILIZATION
8. European Pharmacopoeia 10, 5.1.2 Biological indicators and related microbial preparations

SN077-V1