Using stressed microorganisms as part of alternative microbial test method validation is expected of pharma manufacturers by the United States Pharmacopeia (USP) and U.S. Food and Drug Administration (FDA).
As pressures to accelerate time-to-result (TTR) in QC Micro labs intensify, researchers at Rapid Micro Biosystems have turned their attention to the incubation duration required for standard environmental isolates. “All microorganisms are going to be stressed when you recover them from the manufacturing environment,” notes Owen Griffin, Director of Microbiology at RMB. Selecting the right incubation duration is critical to recovery of microorganisms from the manufacturing environment. “Therefore, we decided to evaluate various microorganisms under stress conditions and unstressed conditions to understand the impact to TTR and provide recommended incubation durations.”
Now available in webinar or poster form, the new research data offers valuable support for users of the Growth Direct® System as they establish environmental monitoring (EM) programs. Here are some key takeaways.
TAKEAWAY #1: Stress conditions negatively impact Time-to-Result (TTR). Environments such as cleanrooms are hostile to microorganisms by design, denying them nutrients and water while subjecting them to disinfectants. Such stresses typically discourage growth of bacteria, mold, or yeast, so it becomes more difficult to recover them on high-nutrient media. This isn’t a problem with traditional methods of microbial quality control (MQC), because a 5- to 7-day incubation period allows even stressed organisms enough time to grow and be detected. But when you’re reducing that time by 50% or more with the Growth Direct® System, there’s less margin for error.
By studying 39 commonly recovered microorganisms under stressed and unstressed conditions, our researchers found that a typical TTR for detecting at least 85% of colony forming units was approximately 116 hours compared to 72 hours for unstressed organisms. While response of individual organisms to stress conditions varied, Griffin says that “Overall, there’s a delta of roughly a day when you stress an organism.”
TAKEAWAY #2: Including stressed microorganisms in EM makes sense if you use rapid methods. Given the importance of effective cleaning and sanitation practices in controlled pharma environments, the risk of under-recovery can severely compromise regulatory compliance and accreditation.
“If you’re looking at rapid methods, you need to set up your environmental monitoring program and incubation scheme for worst-case scenarios,” Griffin points out. That means establishing parameters in which an unstressed cell determines the minimum incubation time you need, while the stressed cell defines the maximum time. But in all cases, the TTR of stressed microorganisms should still be shorter than the standard USP incubation durations.
TAKEAWAY #3: Incubation temperature affects TTR for stressed populations. Most stressed organisms exhibited a decreased time-to-result as temperature increased between 27.5°C and 32.5°C, with the impact becoming less pronounced as incubation times lengthened. This inverse relationship means that recovery of stressed cells can be improved by adjusting the incubation temperature; Growth Direct® System users, for example, have the potential to optimize TTR by selecting and validating an optimal temperature for the microorganisms found in their cleanroom environments.
TAKEAWAY #4: Selection of growth media has a lesser effect on TTR. The use of Trypticase Soy Agar with Lecithin and Polysorbate 80 and/or Trypticase Soy Agar with Lecithin, Polysorbate 80, Histidine, and Sodium Thiosulfate are common general-purpose growth media in standard environmental monitoring programs. While not nearly as impactful as temperature, the difference in these two media types can have some effect on TTR. “In most cases, Trypticase Soy Agar with Lecithin and Polysorbate 80 (TSALP80) tends to be the better media for each temperature tested in our study,” says Griffin. “Neutralizers or additives can sometimes have a negative effect on organism growth or TTR, so you should consciously tailor the selection to disinfectants used in your cleanroom environment for optimal performance.”
A few moments is all it takes to access our free on-demand webinar with Owen Griffin. Or you can download RMB’s technical poster on the same topic. In a little more than 20 minutes, you’ll gain insights on:
It’s that simple. If you have further questions, contact Rapid Micro Biosystems for expert assistance with your environmental monitoring needs.