Do Plant Isolates Have a Role in Method Suitability and Growth Promotion Testing in the Microbiology Laboratory? Is It a Matter of Science versus Compliance?

Origin of the Use of Plant Isolates for Method Qualification and Growth Promotion Testing

What was the origin of the idea of using plant isolates for growth promotion testing in the pharmaceutical industry? The 1993 FDA Guide to Inspections of Microbiological Pharmaceutical Quality Control Laboratories (1) recommended the use of normal flora. The primary authors of this guide were the legendary FDA investigator Hank Avallone and, ironically, one of the authors, the young and now former FDA microbiologist Dennis Guilfoyle. The pertinent section of the Guide reads as follows: V. Methodology and Validation of Test Procedures You may find that dehydrated media are being used for the preparation of media. Good practice includes the periodic challenge of prepared media with low levels of organisms. This includes USP indicator organisms as well as normal flora.

This guide was written over 30 years ago and was very useful at the time but is now so dated that the FDA should either update or withdraw it. It is notable that the use of plant isolates for method suitability and growth promotion testing is not specifically recommended in the authoritative 2020 Second Edition of the FDA Pharmaceutical Microbiology Manual (2). Current opinion among FDA microbiological experts would appear not to support the use of plant isolates or to only use them when highly appropriate.

Support for the Use of Plant Isolates in Other Regulatory Documents

The authors searched other regulatory guidance documents that reference the use of plant isolates and found that it was not generally recommended.

The 2004 FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing—Current Good Manufacturing Practice (3) addresses growth promotion of media fill material as follows: A. Process Simulations 7. Media “In general, a microbiological growth medium, such as soybean casein digest medium, should be used. Use of anaerobic growth media (for example, fluid thioglycollate medium) should be considered in special circumstances. The media selected should be demonstrated to promote growth of gram- positive and gram-negative bacteria, and yeast and mold (for example, USP indicator organisms). The QC laboratory should determine if USP indicator organisms sufficiently represent production- related isolates. Environmental monitoring and sterility test isolates can be substituted (as appropriate) or added to the growth promotion challenge. Growth promotion units should be inoculated with a <100 CFU challenge. If the growth promotion testing fails, the origin of any contamination found during the simulation should nonetheless be investigated and the media fill promptly repeated.”

The authors believe that the use of standardized growth promotion organisms would always be the preferred approach to demonstrate satisfactory lot-to-lot performance with the attention on the media and not the inoculum preparation. Even USP <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests and USP <1111> Microbiological Examination of Nonsterile Products: Acceptance Criteria for Pharmaceutical Preparations and Substances for Pharmaceutical Use allow a twofold variance for the expected enumeration outcome, not uncommon for microbiological preparations. This would simplify the investigations by the media manufacturer and other industrial users in response to a reported failure. Because media fill simulation studies are not covered specifically by compendia requirements, the option for using plant isolates is not objectionable. However, the phenotypic stability of these noncharacterized in-house plant isolates may present a problem if they should change during laboratory storage. The use of the term for the compendial designated organisms, that is, specified microorganisms, as indicator organisms in the 2004 Guidance for Industry is confusing. These organisms are not identified as indicator organisms in the USP, which has unique meaning to food and water microbiology as indicative of recent fecal contamination.

What is the European thinking on this issue? The draft 2020 revision to the European Union Good Manufacturing Practices Annex 1 Manufacture of Sterile Medicinal Products (4) only recommends the use of local isolates for the growth promotion testing of the media fill material but not specifically in other circumstances. The pertinent section is as follows: 9 Viable and nonviable environmental & process monitoring. 9. 38 In developing the process simulation test plan, consideration should be given to the following: Vi. The selected nutrient media should be capable of growing a designated group of reference microorganisms as described by the relevant pharmacopeia and suitably representative local isolates and supporting recovery of low numbers of these microorganisms.

This appears to be a common recommendation from both United States (U.S.) and European Union (E.U.) regulators for media used for aseptic processing validation. Again, our position does not object to this growth promotion option, but is mindful that one may not obtain consistent growth results using plant isolates kept under poorly defined media and storage conditions. Most often, regulatory investigators see no growth for these plant isolates in industry laboratory records as an indicator of poor media growth promotion without the option that it could be an alternation of genotypic factors that may have reduced its growth promotion potential.

In addition, the Pharmaceutical Inspection Co-operation Scheme (PIC/S) Recommendation for Sterility Tests (5) makes the following recommendation: 11.6.1 Growth promotion test Challenge organism strains that are used to verify the fertility of each batch of standard test media should be selected from those reference strains specified by the pharmacopoeial method. Environmental or fastidious organisms may be used, if alternative nonselective enrichment media have been selected for the sterility test.

The authors believe that it would be highly unlikely that plant isolates, most frequently isolated using Trypticase Soy Agar (TSA), would not grow in Trypticase Soy Broth (TSB). Microorganisms of the same genera and species, which are compendial quality control (QC) organisms, grow in TSA and TSB without issues. Plant isolates, by the time they are isolated, subcultured, stored frozen in a culture library, reisolated, and cultured to create standardized inocula for growth promotion and method suitability testing, are no longer stressed microorganisms (6). It should be noted that this latter PIC/S recommendation to use plant isolates is directed toward alternative method validation not routine USP <71> sterility testing. This may make sense in the case of validation of an alternative method to replace the conventional USP <71> Sterility Tests.

The Instability of Plant Isolates in QC Laboratories

The growth and maintenance of USP QC challenge microorganisms are evaluated and maintained from various well-established repository institutions that function as major national culture collections or commercial companies supplying QC challenge organisms derived from these collections. These referenced organisms cited by the Compendia are distributed with a certificate of analysis as to their authenticity and identification. The Compendia describe the manner of culturing for these organisms and mandates the number of allowable subcultures before the isolate potentially may lose its phenotypic/genotypic characteristics. These compendial reference cultures are critical to the establishment of media growth promotion and method suitability testing. However, this fundamental systematic approach for culture control is not described in the USP or any regulatory guidance. There are no instructions on how to culture, maintain, and characterize these collected plant isolates, which may be later used as additional challenge microorganisms for microbiological method/procedure validation and/or suitability testing.

There is a risk from using these plant isolates. There are published reports that the stability of recovered environmental isolates will change over the course of laboratory storage. Studies indicate that to maintain natural isolates in a stable state the transition of strains to the laboratory should minimize culture cycles and extended stationary phase (7). A second report (8) revealed that during laboratory storage, the studied strains of Lactococcus lactis exhibited 6, 7, and 28 mutations in the three strains, including point mutations in loci related to amino acid biosynthesis and transport; two strains lost a conjugative transposon containing genes important in the plant microorganisms natural niche. Lastly, a plasmid carrying an extracellular protease was introduced by transformation. These evolving traits in domesticated microbial metabolisms do not lend well to plant isolates usefulness as a stable reference microorganism for test method response and consistency. It was the opinion of Liu (7) that “the transfer of a plant bacterium to laboratory medium and standard growth environments provides a selection condition for mutations and changes in properties”. Again, not a suitable condition for plant isolates used in validation and growth promotion studies for compendia methods.

The Authority Given to the USP as a U.S. Standard-Setting Organization in the U.S. Federal Food, Drug, and Cosmetics Act

Does the confusion around the use of plant isolates arise from a failure to recognize that the USP and FDA have different roles with the pharmaceutical industry? Simply put, the USP is the standard-setting organization directed to providing product monographs and the corresponding test methods to meet the monograph specifications, whereas the FDA approves drug product submission from pharmaceutical manufacturers and enforces compliance to good manufacturing practices (GMP). The USP General Notices fully describe the legal authority of the compendia. The USP General Notices states: “In the United States under the Federal Food, Drug, and Cosmetic Act (FDCA), both USP and NF are recognized as official compendia.”

In addition, USP General Notices 8.90 Microbial Strain states: “A microbial strain cited and identified by its American Type Culture Collection (ATCC) catalog number shall be used directly or, if subcultured, shall be used not more than five passages removed from the original strain.”

USP Requirements for Method Suitability and Growth Promotion Testing in USP <60>, <61>, <62>, and <71>

The compendial QC strains including their designation when obtained from other major national culture collections are found in the official test chapters USP <60> Microbiological Examination of Nonsterile Products: Tests for Burkholderia cepacia Complex, USP <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests, USP <62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms, and USP <71> Sterility Tests. Table I summarizes these organisms as to the general description, species, and individual test chapter requirements. As one can see in the table, the microorganisms chosen describe a very broad range of cell morphology classifications; this was intentional and rational.

Experience in Clinical Microbiology Related to Growth Promotion Testing

What is the common practice concerning growth promotion testing among our clinical microbiology counterparts? According to Sutton (9), U.S. clinical microbiology laboratories are not required to test the most common microbiological media under National Committee for Clinical Laboratory Standards (NCCLS) Standard M22-2 Quality Assurance for Commercially Prepared Microbiological Culture Media. The understanding was based on a survey performed in the early 1980s of 1164 clinical laboratories that media could be safety accepted based on the manufacturer's certificates of analysis (10). These results confirmed an earlier study (11) that questioned the growth-promotion testing of commercially prepared media that examined 900 lots of 46 different media representing 350,000 plates or tubes of purchased culture media and found that only 17 lots of specialized media containing labile chemical components were found to be unsatisfactory. This collective experience strongly suggests that routine growth promotion testing of general microbiological growth media is largely unnecessary.

History of Citation of the Absence of the Use of Plant Isolates during Regulatory Inspections

Unfortunately, it is difficult to search the FDA 483 observations and warning letters to determine the frequency of observations that indicate the failure to use plant isolates as a deficiency. As summarized by Westney (12), the FDA has cited pharmaceutical manufacturers in Warning Letters for their lack of use of in-house isolates in microbiological culture media growth promotion testing. They include the following:

CP Pharmaceutical, Ltd. October 2010 Warning Letter

“–your firm does not perform challenge testing to sterility media with environmental isolates from the environmental monitoring program –”

Centocor, Inc. July 1998 Warning Letter

“Growth promotion qualification of the media used for environmental monitoring does not include a challenge with mold isolates.”

American Pharmaceutical Partners, Inc. January 2001 Warning Letter

“Growth promotion testing performed on media fill vials does not include evidence the media is capable of detecting environmental isolates found in class 100 filling areas. For example, mold organisms are not used to challenge media, even though mold isolates have been identified in filling room 1.”

Why Are These Citations Misguided?

The readers are reminded that FDA 483 observations, which are sometimes elevated to Warning Letters, are the opinion of individual FDA investigators, who may not be microbiologists. In addition, the observations are subject to review by subject matter experts and regulatory specialists at the FDA Headquarters. The carryover of minor 483 citations into warning letters may be viewed as compliance piling-on and should be discouraged. As experienced auditors, the authors believe that the failure of the FDA to classify citations as critical, major, and minor, which is supported by PIC/S, is a shortcoming, as not all GMP deviations have the same potential impact on patient safety. However, the authors would expect media fill material to be growth-promotion tested with at least one mold, representing a potentially slow-growing microorganism.

Microbial Species Most Frequently Isolated during Environmental and Water Monitoring and Product Release Testing

The readers of this article cannot readily determine the plant isolates that are most frequently isolated in our industry. The authors found that the microbial species most frequently isolated may be obtained from the published literature, plant isolates that are commercially prepared as standardized inoculums for customers, or the frequency of microbial identification by major contact testing laboratories (Table II). When comparing the compendial designated QC organisms with these categories, it is striking how representative the former QC organisms are of these plant isolates. It becomes more obvious when one makes the comparison between these data sets that there is little or no benefit from adding plant isolates to the list of growth-promotion organisms, especially in the compendia chapters.

The relevance of the emerging understanding of the human microbiome to pharmaceutical microbiology is outside the scope of this technology review. The reader is referred to recent discussions of this topic (13, 14).

What Are the Phenotypic and Genotypic Differences between Compendial Strains and Plant Isolates?

Are there significant differences between plant isolates and compendial strains? The data appears to demonstrate that there is no difference. For example, a standard strain employed in USP <61>, <62>, and <71> is the Gram-positive, spore-forming, rod-shaped bacterium Bacillus subtilis. The genus Bacillus contains the order of 160 species with only a few species being of clinical significance. Clinical microbiologists differentiate among the B. subtilis (B. subtilis, Bacillus pumilus, Bacillus atrophaeus, Bacillus licheniformis, and Bacillus amyloliquefaciens), B. cereus (B. anthracis, B. cereus, B. thuringiensis, B. weihenstephanensis, B. pseudomycoides, and B. mycoides), and B. circulans groups. Among these three groups, the small phenotypic and genotypic differences (15) between individual species make their identification challenging and suggest that the use of plant isolates to supplement the compendial QC organisms B. subtilis is not necessary to establish method suitability or growth promotion capability (Tables III and IV).

The Role of Plant Isolates in Formulation Development—Especially Preservative Effectiveness Testing

Does it make sense to use plant isolates for preservative effectiveness testing during product development? Definitely yes. USP <51> Antimicrobial Effectiveness Testing states: Challenge organisms are generally based on likely contaminants to a drug product while considering its physical attributes, formulation, and intended use. The standard battery of challenge organisms described in this test need not prevent the inclusion of other species of microorganisms if deemed useful to measure the biological activity of the preservative system for a specific product. These supplemental challenge organisms are not within the scope of this chapter, but may be added in addition to the described test organisms. Use cultures of the following microorganisms: Candida albicans (ATCC No. 10231), Aspergillus brasiliensis (ATCC No. 16404), Escherichia coli (ATCC No. 8739), Pseudomonas aeruginosa (ATCC No. 9027), and Staphylococcus aureus (ATCC No. 6538). The viable microorganisms used in the procedure should be part of a freshly growing culture (e.g., in logarithmic growth phase) with the exception of A. brasiliensis spores.

What would be considered a supplemental challenge organism? The microorganisms prominent in product recalls for microbial contamination would be obvious choices. An example of such challenge organisms for a multiple-use, aqueous nonsterile drug product especially targeted to immune-compromised infant and aged populations would be the Gram-negative, oxidase-positive, waterborne bacterium Burkholderia cepacia, a species responsible for multiple product recalls (16, 17).

To demonstrate that the compendial QC strains are highly representative of the most frequently isolated bacteria from pharmaceutical and consumer health product manufacturing facilities, the authors have in Table II compared the compendial organisms with the frequency of the commercial preparation of standard inoculum of plant isolates from major suppliers and the frequency of identification of the top 12 submitted cultures for identification by the customers of a major contract testing laboratory. What becomes apparent is that the compendial QC organisms are represented by genus and species in all three lists.

Relationship between Compendial Designated Strains and Common Plant Isolates

What do these data in Table II tell us? The top ten plant isolates prepared as standard inoculums for their customers as reported by the company Microbiologics website are familiar environmental and water source monitoring isolates. There are two Gram-positive cocci and a Corynebacterium associated with human skin (M. luteus, S. epidermidis, and C. tuberculostearicum), three Gram-positive, spore-forming rods (B. cereus, B. licheniformis, and B. thuringiensis), two environmental fungi (P. rubens and P. chrysogenum), and two waterborne Gram-negative, nonfermentative rods (Ralstonia pickettii and Burkholderia multivorans). The most frequently identified organisms identified by the contract testing laboratory Accugenics did not change greatly from 2010 to 2020-2021 (Christine Farrance, personal communication, 2021). With a significant overall increase in the number of submitted isolates and some minor taxonomic revisions, the number of species identified to encompass 50% of the identifications grew from 15 to 30 species, which is largely a function of the increased number and diversity of the submitted isolates for identification.

The critical question is whether there is any value in adding plant isolates to the growth-promotion and method suitability organisms specified in the official compendial tests? Obviously, adding these organisms is not a compendial requirement. With USP Chapters <61> and <71> that employ general microbiological growth media like soybean casein digest broth and agar, Sabouraud dextrose agar, and fluid thioglycolate broth, the addition of very similar phenotypic and genotypic species would have little or no value in assuring a broader range of recovery and detection capability. For example, the 16S rRNA base sequencing similarity for common members of the genus Bacillus may be >99% and minor phenotypic differences that would not affect growth in the compendial media. A similar argument can be made for members of the genus Staphylococcus.

The recently introduced USP <60> Microbiological Examination of Nonsterile Products—Test for Burkholderia cepacia Complex designed as a test for the 22 members of B. cepacia complex specifies three species, that is, B. cepacia, B. multivorans, and B. cenocepacia, that are the most clinically significant members of the complex. Additional experience with the test in industrial settings may justify to the USP Microbiology Expert Committee the reduction of the challenge organisms from three to one, not the addition of plant isolates. The performance of the selective enrichment and diagnostic media specified in USP <62> are backed by the long history of use of these media in water, food. and clinical microbiology, supported by publications in the peer-reviewed literature, so adding plant isolates as challenge organisms would not be necessary. Historic review of decades of data from published research and collective experience, by the authors, eliminates the need to repeatedly revalidate already proven compendial methods. The cGMPs in Section 21 CFR 211.194 recognized the USP and AOAC International official methods as valid and ready-to-use, and industry is just required to make “reference will suffice”.

Common Plant Isolates from Pharmaceutical Environments

It is well established that the most common microorganisms isolated from cleanrooms are Gram-positive bacteria with human-derived members of the genera Staphylococcus and Micrococcus predominating (18). For ease of review, the species are listed in Tables V and VI in their descending order of frequency of isolation.

The environmental microorganisms most frequently isolated from 2006 to 2017 in the U.S. Federal National Institutes of Health (NIH) sterile compounding facility are useful to our discussion (19). The data collected during the study period were unremarkable and the authors, based on our experience, agree. They reported that on average, positive environmental cultures were identified in 1%, 10%, and 40% of samples sent from ISO 5, 7, and 8 zones, respectively. These frequencies of recovery were higher than the recommended recoveries of less than 1%, 5%, and 10% for the three ISO zones in USP <1116> Microbiological Control and Monitoring of Aseptic Processing Environments. Between 56% and 69% of these isolates were categorized as skin or oral flora, 13% to 23% were environmental organisms, and 8% to 29% were reported as objectionable (defined as yeasts, mold, Gram-negative rods, and coagulase-positive Staphylococcus). The authors question that characterization of isolates as objectionable, as any viable microorganism found in a compounded sterile preparation should be viewed as objectionable. Among the bacteria (N = 469), Micrococcus luteus was most commonly identified (19%), followed by Kocuria spp. (10%), Staphylococcus hominis (7%), Staphylococcus epidermidis (5%), and Streptococcus mitis (4%). Common fungi (N = 114) included Aspergillus sydowii (20%), Aspergillus fumigatus (13%), Cladosporium spp. (12%), Penicillium spp. (10%), and Aspergillus niger (9%).

In a single case, the Environmental Monitoring (EM) isolate identified as M. luteus had also been cultured from a manufactured product at around the same time as the environmental isolation. This product was not infused for reasons unrelated to sterility testing results. Again, the isolated microorganisms reported (19) include those cited as most common in Table II and would be well represented by the compendial designed QC organisms. An exception is the Gram-positive coccus Streptococcus mitis, which is prevalent in the human oral cavity, suggesting facemasks were not being consistently worn in the NIH facility. The authors of this review understand that environmental controls have been significantly improved in the facility since 2017, and that the frequency of fungal isolation has decreased.

How prevalent is microbial contamination of cell therapy products? A recent review article emphasized that data differs by cell types, national origin, and time period reported, so it is difficult to establish the prevalence of microbial contamination (20). The most common contaminants are mycoplasma, bacteria, fungi, and viruses. Prominent bacteria found in contaminated cell cultures are Gram-positive spore-forming rods like Bacillus cereus, B. coagulans, and B. brevis, Gram-positive cocci like Enterococcus malodoratus, E. casseliflavus, and S. epidermidis and Gram-negative bacteria such as E. coli.

What Are the Growth Promotion Tests Used by Media Manufacturers?

This section contains a summary of the growth promotion testing of media commonly used in the pharmaceutical industry. Information found in the authoritative Difco/BBL Manual and other sources may not always meet the USP requirements in terms of organism selection, inoculum size, incubation conditions, and acceptance criteria. This occurs because media manufacturers have a range of customers outside the pharmaceutical industry with different requirements. Examples of the growth promotion testing requirements used by media manufacturers are found in Tables VII⇓⇓⇓⇓–XIII and include:

Conflict of Interest Declaration

The authors declare no personal conflict of interest.