Peer Review Article | Open Access | Published 3rd July 2024
An Alternative Medium to Support Sterility Testing using the Growth Direct® Rapid Sterility System
DL Jones¹, K Volis ² 2 O Griffin ³, | EJPPS | 292 (2024) | https://doi.org/10.37521/29202 | Click to download pdf
Executive Summary
Recently, it has been shown that there are other media that perform equivalently or better than the compendial sterility test media, TSB and FTM1. The results presented here demonstrate that a new single medium, Rapid Sterility Medium (RSM), performs as well as or better for the growth promotion of test microorganisms compared to compendial sterility test media. In side-by-side tests of aerobic bacteria, yeast and molds and anaerobic organisms, RSM broth supported growth promotion comparable to TSB or FTM for a panel of microorganisms relevant to the pharmaceutical market. These results demonstrate that RSM broth can serve as a valid alternative for both TSB and FTM for sterility testing being performed by the Growth Direct® Rapid Sterility System.
Keywords: Sterility test, Alternative medium, Rapid Microbial Method, Growth Direct® System
1.Introduction
The compendial sterility test as described by the Pharmacopeia, USA (USP <71>²), European (EP 2.1.6³) and other guidance documents is performed to release sterile products. The test as carried out uses the liquid media trypticase soy broth (TSB), also known as soybean casein broth, and fluid thioglycollate medium (FTM) for supporting growth of microbial contaminants. FTM is designed to detect anaerobic and aerobic microorganisms, while TSB specifically targets aerobic microorganisms. Incubation is carried out at 20-25°C for TSB to support temperature sensitive species such as molds, and at 30-35°C for FTM for human derived microorganisms. Incubation is performed for 14 days having been extended from 7 days for products tested by membrane filtration in USP 24 (2000). The extended length of the incubation time was required to allow any stressed or injured microorganisms to recover and grow. Many papers have reported the effect of stress on extending lag time for organism recovery prior to moving to exponential growth⁴ ⁵ ⁶ ⁷ ⁸ . A positive result is determined by the development of visible turbidity in the test media due to the presence of microbial contaminants. Both TSB and FTM are clear and allow for growth to be easily observed as turbidity and is one of the main strengths of these media types in a visual based test. Some rapid micro methods (RMM) have limitations for the range of microorganisms detected. In a paper published in 2019, England et al⁹ working at National Institute for Health (NIH), reported an issue with Bac Tec and Bac T/Alert detecting mold growth which was clearly visible in the media. The test required the use of SDA based media to detect the growth by the system.
The Pharmacopeial harmonized guidance has two types of sterility methods, direct inoculation and membrane filtration. The direct inoculation test method is performed by directly inoculating the sample into each compendial medium. Alternatively, the membrane filtration method is performed by passing a sample through a 0.45µm membrane filter. After the sample is filtered the membrane is either transferred to each compendial medium or the medium is added to the filtration apparatus to immerse the membrane. For both compendial methods the test samples are visually monitored for turbidity (microbial growth) at regular intervals throughout the 14-day incubation period.
While both TSB and FTM have served well as the growth media for the sterility test, the possibility of using other media has been examined in the light of the development of new, rapid microbiological test methods (RMM). These alternative media have not been considered before due to the fact that the compendial test uses turbidity as the measure of growth and the alternative medium itself was turbid. The new RMM’s look for alternative indicators of growth e.g. colony formation directly on the surface of a membrane filter instead, so media clarity is not a requirement. As a result, discrete colonies may be detected either visually or by one of the RMMs. For example, Gray et al1 screened eight different nutrient media using both stressed and unstressed organisms comparing Schaedler blood agar, trypticase soy agar and broth, CDC anaerobic blood agar, and Difco Brewer anaerobic agar. Of these media, Schaedler blood agar exhibited either equal or better performance overall compared to the other media at both 20-25°C and 30-35°C, and under aerobic and anaerobic conditions. In contrast, TSB and FTM exhibited substandard performance. Thus, not only were possible replacement media identified for use in the sterility test application, but also, they appeared to exhibit better growth promotion properties than TSB and FTM. The Schaedler medium is now the basis of the Milliflex Rapid sterility test. The Biomerieux BacT/Alert¹⁰ also uses a modified media for its iFN plus and iFA plus sterility media options.
The use of an alternative media RSM was investigated for its ability to replace both FTM and TSB. RSM is a custom non-selective medium that supports growth of a broad range of aerobic and anaerobic microorganisms. The current paper demonstrates the growth promotion performance of the media with direct inoculation of microorganisms and compares the time taken to detect growth compared to the compendial media.
Testing Rationale
Microbial test samples used consisted of 33 microorganisms, including USP strains, from commercially obtained preparations and environmental isolates from cultures grown in-house (Table 1). The range of microorganisms were selected from previously published sterility test validations or papers describing problematic microorganisms e.g. molds. Microorganisms were evaluated as either “unstressed” or “stressed” prepared by heat treatment¹. The heat treatment duration varied by microorganism with the goal of at least 50% kill. The assumption being the remaining 50% were in some degree of stress. Stressed organisms were included due to the requirement for fast test turnaround time and stress is recognized as a significant cause of delayed microbial growth and detection. Growth promotion was performed by direct inoculation to the liquid broth and evaluation of turbidity to confirm growth.
The microorganism panel followed the growth promotion requirements specified in USP Ch <71>:
“Using a separate portion of medium for each of the following species of microorganism: Clostridium sporogenes, Pseudomonas aeruginosa, and Staphylococcus aureus. Inoculate portions of thioglycollate medium with a small number (not more than 100 cfu) of Clostridium sporogenes. Inoculate portions of Soybean–Casein Digest Medium with a small number (not more than 100 cfu) of the following microorganisms, using a separate portion of medium for each of the following species of microorganism: Aspergillus brasiliensis, Bacillus subtilis, and Candida albicans.”
The testing also included the alternative pharmacopeial microorganisms Micrococcus luteus ATCC 9341 and Bacteroides vulgatus ATCC 8482. The other microorganisms were selected as defined by published data on rapid sterility test validations and key organisms in relevant market segments e.g. Cutibacterium acnes for the biologics industry. Special focus was also given to the detection of molds following the paper by England et al⁹ highlighting the difficulty detecting some of the species with certain detection technology.
Methods
Direct Inoculation method
Replicate 10mL aliquots of media were dispensed into 15mL conical tubes. Inoculum was added to each tube to achieve ≤100 CFU. A positive control on relevant media, TSA, SDA, BAP or R2A, was prepared for each microorganism inoculated to verify dose level met specification. Negative controls were prepared for each incubation and media type. Test samples were incubated for 14 days at 20-25°C or 30-35°C, depending on organism, with reads once a day every day to determine when each sample became positive.
Table 1
Table 1. Microorganisms tested for media Growth Promotion, incubation conditions and growth detection
Results and Discussion
Table 1 shows the results for the microorganism growth in the compendial media compared to the new Rapid Sterility Test formulation. All microorganisms, both as stressed and unstressed form, grew to create a turbid medium.
Overall the RSM medium used in the direct inoculation mode is equivalent to the compendial media in its ability to grow all the test microorganisms used in this study in <14 days. The results show growth with 90% of the microorganisms detected in <5 days for both the single RSM and the compendial TSB/FTM media and 100% of the microorganisms were detected within 10 days. This will be evaluated further in a following paper on defining Time To Result (TTR) using the advanced optics and microcolony detection facilitated by the Growth Direct® technology.
Conflict of interest
The authors all work for Rapid Micro Biosystems the manufacturer of the Growth Direct® Rapid Sterility system
References
1. Gray, J. C.; Staerk, A.; Berchtold, M.; Hecker, W.; Neuhaus, G.; and Wirth, A; Growth-promoting properties of different solid nutrient media evaluated with stressed and unstressed micro-organism: pre-study for validation of a rapid sterility test. PDA J. Pharm. Sci. & Technol. 2010, 64 (3) 249-263
2. Chapter <71> Sterility Tests, USP 37--NF32, pg. 71-77; U.S. Pharmacopoeial Convention: Rockville, MD, 2014
3. European Pharmacopoeia Working Group. EP 2.6.1. Sterility. London, UK: EMA;.
4. Sage, A; Use of stressed populations for application validation to better represent real world testing. American Pharmaceutical Review. Aug 2013. 15-21.
5. Dupont, C.; Augustin, Influence of stress on single-cell lag time and growth probability for Listeria monocytogenes in half fraser broth. App. Environ Microbiol, 2009; 75; 3069-3076
6. Stephens, P. J.; Joynson, J. A.; Davies, K. W.; Holbrook, R.; Lappin-Scott, H. M.; Humphrey, T. J. The use of an automated growth analyser to measure recovery times of single heat-injured Salmonella cells, J. App. Microbiol. 1997; 83; 445-455
7. Wesche, A.M.; Gurtler, J. B.; Marks, B. P.; Ryser, E.T. (2009); Stress, Sublethal injury, resuscitation, and virulence of bacterial foodborne pathogens 2009 J. Food Prot. 72, 5 121-138
8. Cundell. T.; Tidswell.E.C.; Massaro. C.; Live, Stressed, and Dead Microorganisms – Their Role in Microbial Test Method Validation. American Pharmaceutical Review. Apr 2021. 18-24
9. Matthew R.; England, A; Frida Stock, A.; James E. T. Gebo,A.; Frank. K.A; and Lau. A F; J Clin Microbiol. 2019 Feb; 57(2): e01548-18. Comprehensive Evaluation of Compendial USP<71>, BacT/Alert Dual- T, and Bactec FX for Detection of Product Sterility Testing Contaminants
Authors
DL Jones¹, K Volis ², O Griffin³
Rapid Micro Biosystems®, Lowell, Boston, Massachusetts. USA.
* Corresponding author:
Owen Griffin
E-mail:ogriffin@rapidmicrobio.com
Tel: +1 339 223 2174
Address: Rapid Micro Biosystems
1001 Pawtuckett Av West
Lowell
MA 01854
USA
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