Bacterial Spore Formers in Disinfectant Efficacy Testing

Disinfectant efficacy testing is performed to qualify a wet contact time for disinfectants for use within classified areas of aseptic manufacturing facilities (e.g., Biopharma, Pharma, Medical Device, etc.). Disinfectant efficacy testing is a regulatory expectation and designing a disinfectant efficacy study can be complex, with many different potential study variables to consider. Periodically, the question is raised when designing a disinfectant efficacy study: Should bacterial spore formers be tested against disinfectants that are not considered sporicidal agents? Considering this possibility, we will evaluate the question and provide a reasoned approach. Industry regulators are also interested in bacterial spore testing as noted in a recent FDA 483, “There was no analysis to determine if the microbial population used consisted of spores and/or dividing vegetative microorganisms.”¹

From a disinfectant efficacy testing regulatory guidance perspective, USP 43-NF38 <1072> Disinfectants and Antiseptics defines a chemical disinfectant as, “A chemical agent used on inanimate surfaces and objects to destroy infectious fungi, viruses, and bacteria, but not necessarily their spores…” USP 43-NF38 <1072> Disinfectants and Antiseptics, goes on to define a Sporicidal Agent as, “An agent that destroys bacterial and fungal spores when used in sufficient concentration for a specified contact time. It is expected to kill all vegetative microorganisms.” This demonstrates that a disinfectant that is not considered a sporicidal agent is not expected to exhibit efficacy against bacterial spores, which are one of the most challenging forms of microorganisms to chemically inactivate. Table 1 demonstrates the challenge presented by bacterial spores.

Formulated chemical disinfectants are registered for marketing in a geographical area with the applicable regulatory authority and should have label claims to support the expected range of microbicidal efficacy (e.g., bactericidal, fungicidal, sporicidal, etc.). Label claims for a chemical disinfectant should be reviewed and evaluated to help guide an end-user to test against an appropriate range of microorganisms in a disinfectant efficacy study as well as how to incorporate it into the facility’s contamination control program. For example, a use dilution of a phenolic or quaternary ammonium compound is not expected to exhibit efficacy against bacterial spores. Negative log10 reductions can even be observed for non-sporicidal chemical disinfectants against bacterial spores in a disinfectant efficacy coupon study if the test coupons recover at a slightly higher value than the positive control coupons, due to standard error associated with cultural microbial recovery.

Attempting to test against a vegetative form of bacterial spore former is also not recommended for a litany of reasons. A bacterial spore presents a significantly greater challenge to disinfection than a bacterial spore-former vegetative cell. If efficacy data is generated against a suspension with a significant portion comprised of vegetative cells, the sporicidal efficacy of a product could be overestimated. Additionally, the spore form of a bacterial spore former is the most likely form to be present in a classified area since it may be present and can survive indefinitely on a variety of surfaces that may be introduced into a classified area. Vegetative cells of bacterial spore formers are 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 unpredictable nature of sporulation, an aliquot of a bacterial spore-former 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. GMP Trends, September 15, 2021.
2. Errington, J. (2003) Regulation of endospore formation in Bacillus subtilis. Nature Reviews Microbiology 1, 117–126.
3. 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. United States Pharmacopoeia USP 43 (2022). General Information Chapter <1072>Disinfectants and Antiseptics. United States Pharmacopeial Convention/National Formulary, Rockville, MD.
5. McDonnell, G. and Russell A.D. (1999) Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev 12 (1): 147-179.

Author Details 

David Shields, Senior Technical Service Manager - STERIS Corporation; Jim Polarine, Jr., MA., Senior Technical Service Manager - STERIS Corporation

Publication Details 

Subscribe to our e-Newsletters
Stay up to date with the latest news, articles, and events. Plus, get special
offers from American Pharmaceutical Review delivered to your inbox!
Sign up now!