APR March 2024 - 43

« MICROBIOLOGY
typically associated with soil (dirt) or materials of organic origin that
are not often a part of manufacturing processes. Vegetative cells do
not survive indefinitely. Vegetative cells become non-viable outside
of the required environmental conditions, and even when under ideal
conditions auto-lyse and release a spore, upon cell maturity.
A lack of sporulation cannot be accurately predicted within a culture.
The mechanism for sporulation is highly complex with many genes and
transcription factors being involved in the induction of sporulation to
the degree that there are cell-specific transcription factors.2
Sporulation
typically will begin within an incubation period that is required
to grow up a vegetative culture (18-24 hours) in a laboratory. The
initiation of sporulation is known to occur at variable times amongst
different species. For instance, Bacillus cohnii has been demonstrated
to sporulate within three hours and achieve 98% sporulation by six
hours.3
Figures 1-2 exhibit varying degrees of spore formation and
release in 18-hour cultures of Bacillus thuringiensis and Bacillus subtilis,
with arrows pointing to some examples of spores. Subpopulations
within a culture sporulate at different times rather than sporulating
in a synchronized manner. Sporulating quickly in a subpopulation of
a culture is a sound survival strategy that allows the microorganism to
survive. This strategy is important if extreme environmental conditions
occur that will result in the inactivation of the vegetative cells. This all
demonstrates that creating a suspension comprised of only vegetative
cells for a spore-forming bacteria is highly improbable.
When considering the viability of testing a vegetative bacterial
spore-former culture in a disinfectant
efficacy study, given the
Figure 1. Bacillus thuringiensis 18-hour Culture Malachite
Green Spore Stain
unpredictable nature of sporulation, any aliquot of a bacterial sporeformer
suspension may or may not contain spores. The absence or
presence of spores cannot be verified through established laboratory
techniques without compromising the aliquot of inoculum, rendering
it unusable for the actual disinfectant test (e.g., spore staining).
Therefore, any results generated with a suspension containing some
unknown number of spores would not lead to results that are of value,
as it would not be known if the results were based upon the presence
or absence of spores for each individual test and positive control
(water control) coupon. Having an inconsistent ratio of vegetative cells
to spores on individual test and positive control coupons could also
lead to differential levels of survival of the inoculum drying process on
individual coupons within a single test parameter as spores are able
to survive complete desiccation better than some vegetative cells.
This would lead to variability in results attributable to a variable that is
outside the scope of the study's evaluation.
Demonstrating an ability to effectively inactivate bacterial spores
is a critical component of a disinfectant efficacy study and an
organization's contamination control strategy, verifying that the
contamination control program is able to maintain microbial control
within the classified areas. However, if the study is not based upon
a scientifically sound approach, specious conclusions can be made
about the disinfectants in the contamination control program, which
could ultimately lead to an increased contamination risk for the
product. As demonstrated here, bacterial spore formers should only
be tested in spore form, against sporicidal agents, when performing
a disinfectant efficacy study. Testing bacterial spore formers against
disinfectants that are not sporicidal and attempting to test bacterial
spore formers in vegetative form does not lead to generating data that
adds value to a disinfectant efficacy study but does take up valuable
resources and time.
References
1.
2.
3.
GMP Trends, September 15, 2021.
Errington, J. (2003) Regulation of endospore formation in Bacillus subtilis. Nature Reviews
Microbiology 1, 117-126.
Sharma, T.K., Alazhari, M., Heath, A., Pine, K., and Cooper, R.M., (2017) Alkaliphilic
Bacillus species show potential application in concrete crack repair by virtue of rapid
spore production and germination then extracellular calcite formation. Journal of Applied
Microbiology, 122: 1233-1244.
4.
Figure 2. Bacillus subtilis 18-hour Culture Malachite
Green Spore Stain
5.
United States Pharmacopoeia USP 43 (2022). General Information Chapter <1072>
Disinfectants and Antiseptics. United States Pharmacopeial Convention/National
Formulary, Rockville, MD.
McDonnell, G. and Russell A.D. (1999) Antiseptics and disinfectants: activity, action, and
resistance. Clin Microbiol Rev 12 (1): 147-179.
www.americanpharmaceuticalreview.com |
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APR March 2024

Table of Contents for the Digital Edition of APR March 2024

Message from the Editor
Editorial Advisory Board
BIOPHARMACEUTICALS - Getting to GMP-Quality Biotherapeutics From Today’s Bench-Scale Continuous Manufacturing Systems: A Gap Analysis
MICROBIOLOGY - Critical Behavioral Attributes and the Application of COM-B Framework in Aseptic Processing
FORMULATION AND DEVELOPMENT - Use of AUC in AAV Analysis in a GMP Setting
FORMULATION AND DEVELOPMENT - Precision Medicine in Clinical Trials: A Statistical Perspective
An Interview with Dan Smithey, PhD President & CEO, Serán
FORMULATION AND DEVELOPMENT - Still Early Days for AI in Drug Discovery...Says Who?
QC Corner - Enhancing Material and Equipment Availability in Production Isolators
BIOPHARMACEUTICALS - Technologies for Aseptic Filling: The Choice is Clear
Vendor Viewpoint - Data Integrity and Rapid Micro Methods: Transforming to a Modern Microbiology Lab
MICROBIOLOGY - Bacterial Spore Formers in Disinfectant Efficacy Testing
Partner Perspective - Nanoparticle Technologies: Enablers for Ocular Drug Delivery
DRUG DELIVERY - Your Nails and You
DRUG DEVELOPMENT - Battling Exorbitance: High Costs in Sickle Cell Gene Therapies and the Imperative of Global Patient Registries for Equity
FORMULATION AND DEVELOPMENT - How Pharma Companies Are Solving Regulatory Challenges with AI-based Technology
Event Preview - CPHI North America
Event Preview - Excipient World 2024
P.I.N. Points
Advertiser's Index
APR March 2024 - CoverTip01
APR March 2024 - CoverTip02
APR March 2024 - Cover1
APR March 2024 - Cover2
APR March 2024 - 1
APR March 2024 - 2
APR March 2024 - 3
APR March 2024 - 4
APR March 2024 - 5
APR March 2024 - Message from the Editor
APR March 2024 - Editorial Advisory Board
APR March 2024 - BIOPHARMACEUTICALS - Getting to GMP-Quality Biotherapeutics From Today’s Bench-Scale Continuous Manufacturing Systems: A Gap Analysis
APR March 2024 - 9
APR March 2024 - 10
APR March 2024 - 11
APR March 2024 - 12
APR March 2024 - 13
APR March 2024 - MICROBIOLOGY - Critical Behavioral Attributes and the Application of COM-B Framework in Aseptic Processing
APR March 2024 - 15
APR March 2024 - 16
APR March 2024 - 17
APR March 2024 - 18
APR March 2024 - FORMULATION AND DEVELOPMENT - Use of AUC in AAV Analysis in a GMP Setting
APR March 2024 - 20
APR March 2024 - 21
APR March 2024 - 22
APR March 2024 - 23
APR March 2024 - FORMULATION AND DEVELOPMENT - Precision Medicine in Clinical Trials: A Statistical Perspective
APR March 2024 - 25
APR March 2024 - 26
APR March 2024 - 27
APR March 2024 - An Interview with Dan Smithey, PhD President & CEO, Serán
APR March 2024 - 29
APR March 2024 - 30
APR March 2024 - FORMULATION AND DEVELOPMENT - Still Early Days for AI in Drug Discovery...Says Who?
APR March 2024 - 32
APR March 2024 - 33
APR March 2024 - QC Corner - Enhancing Material and Equipment Availability in Production Isolators
APR March 2024 - 35
APR March 2024 - BIOPHARMACEUTICALS - Technologies for Aseptic Filling: The Choice is Clear
APR March 2024 - 37
APR March 2024 - 38
APR March 2024 - 39
APR March 2024 - Vendor Viewpoint - Data Integrity and Rapid Micro Methods: Transforming to a Modern Microbiology Lab
APR March 2024 - 41
APR March 2024 - MICROBIOLOGY - Bacterial Spore Formers in Disinfectant Efficacy Testing
APR March 2024 - 43
APR March 2024 - Partner Perspective - Nanoparticle Technologies: Enablers for Ocular Drug Delivery
APR March 2024 - 45
APR March 2024 - 46
APR March 2024 - 47
APR March 2024 - DRUG DELIVERY - Your Nails and You
APR March 2024 - 49
APR March 2024 - 50
APR March 2024 - 51
APR March 2024 - DRUG DEVELOPMENT - Battling Exorbitance: High Costs in Sickle Cell Gene Therapies and the Imperative of Global Patient Registries for Equity
APR March 2024 - 53
APR March 2024 - 54
APR March 2024 - 55
APR March 2024 - FORMULATION AND DEVELOPMENT - How Pharma Companies Are Solving Regulatory Challenges with AI-based Technology
APR March 2024 - 57
APR March 2024 - Event Preview - CPHI North America
APR March 2024 - 59
APR March 2024 - Event Preview - Excipient World 2024
APR March 2024 - 61
APR March 2024 - P.I.N. Points
APR March 2024 - 63
APR March 2024 - Advertiser's Index
APR March 2024 - Cover3
APR March 2024 - Cover4
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