Evaluating the efficacy of disinfecting products against Bacillus subtilis spore using an optimized method

Abstract

Twenty disinfectant products were evaluated for sporicidal activity using an optimized method. Bacillus subtilis ATCC 6051 was sporulated in Difco sporulation medium for 3 days. The spores were purified using density centrifugation and heat treatment and then prepared for sporicidal efficacy assessment. The quality of the purified spores was verified using sodium hypochlorite solutions with free chlorine concentrations of 1,500 and 5,000 ppm. The active ingredients in disinfectant products included chlorine, hydrogen peroxide/peracetic acid, aldehydes, quaternary ammonium compounds, and phenols. Of the 20 products tested, 6 met the effectiveness criterion of greater than a 5 log₁₀ reduction after exposure of B. subtilis spores for the label-recommended contact time. Among the 11 products claiming sporicidal activity, 4 met the established criterion. Among the five categories of active ingredients evaluated, some chlorine-based and hydrogen peroxide/peracetic acid-based products demonstrated significantly higher sporicidal activity than products in the other three categories. Overall, products that did not meet the study criteria exhibited less than a 1 log₁₀ reduction. When these underperforming sporicidal products were tested with an extended contact time of 10 min, one additional product met the effectiveness criterion. Subsequent quality assessments using freshly prepared, laboratory-made products containing identical active ingredients demonstrated improved sporicidal efficacy compared with the original test items, indicating that defects or degradation of active ingredients may have occurred in some of the tested commercial disinfectant products.

One sentence summary Evaluation of 20 disinfectants against Bacillus subtilis spores using optimized methods.

Graphical Abstract

Graphical Abstract.

Introduction

Pharmacy compounding involves customizing medications for individual patients by blending or mixing various ingredients (FDA, 2017). Patients may choose compounded drugs for several reasons, such as being unable to take Food and Drug Administration (FDA)-approved medications due to allergies, certain dyes in the medication, or forms of medication (such as tablets or capsules) that are difficult for children and the elderly to consume. However, compounded drugs are not reviewed by FDA for safety, effectiveness, or quality before they are marketed. This creates risks for patients. For example, in 2001, the FDA sampled 29 compounded drugs and found that 34% (10 out of 29) did not meet the acceptance criteria. Of these 10 products, 9 contained 59%–89% less active ingredients than indicated on the product’s label (FDA, 2018). Moreover, the lack of sterility assurance in injectable compounded drugs, which are supposed to be sterile, poses significant threats to public health. There were 12 outbreaks related to contaminated drugs produced by compounding pharmacies from 2001 to 2012 (CDC, 2012; Staes et al., 2013). The causative agents of these contaminations included a broad range of fungi and bacteria. In 2012, an outbreak of fungal meningitis, attributed to Aspergillus spp. and Exserohilum spp., resulted in 751 confirmed cases and 64 fatalities (Outterson, 2013). Following this major fungal outbreak, the Drug Quality and Security Act (DQSA) was signed into law, amending the federal Food, Drug, and Cosmetic Act (FD&C Act) regarding human drug compounding. This update imposed restrictions on the compounding of drugs by pharmacies and physicians, intensifying oversight of such practices (Campbell et al., 2020). Effectively implementing sporicidal and other disinfection methods is essential for maintaining the necessary hygiene levels in aseptic pharmacy compounding facilities to ensure safety (Ben Belgacem et al., 2017).

Some bacteria, especially Gram-positive bacteria like Bacillus spp., can form endospores, which are dormant structures of bacteria (Zhu et al., 2022). Bacterial spores form in response to harsh environments, such as nutrient deficiency and extreme temperatures. These spores are more resistant to regular disinfectants or bactericidal products than vegetative bacterial cells due to their protective spore coats, which prevent disinfectants from penetrating and being effective. This resistance poses a considerable burden for public health (Russell et al., 1986). Additionally, these special structures allow bacterial spores to resist acid, heat, and radiation, making them more difficult to eliminate (Checinska et al., 2015). A previous study indicated that bacterial spores require the highest level of disinfectant, with their resistance to disinfection being significantly higher than that of other microorganisms, including non-spore-forming bacteria, viruses, and fungi (Russell, 1999). When spore-forming Bacillus spp. form biofilms, they can serve as persistent sources of contamination. Although spores deposited on stainless-steel surfaces may remain dormant, surviving spores can subsequently germinate when exposed to favorable environmental conditions and, depending on the context, may contribute to microbial contamination or infection (Faille et al., 2014).

Surface contamination with bacterial spores poses a significant risk in aseptic drug compounding facilities (Sandle, 2014). Currently, there are numerous commercial sporicidal products that claim sporicidal activity. The FDA generally relies on product labels when evaluating the disinfection efficacy in these facilities. However, the effectiveness of sporicidal products can be influenced by several factors, including the specific bacterial spore-forming strains, organic load, surface material being treated, and contact time. The quality of the spores also plays a crucial role in disinfection validation tests. Therefore, a systematic evaluation of various sporicidal products is necessary to understand their efficacy under different conditions.

Sporicidal disinfection is used to eliminate bacterial spores in pharmacy compounding facilities, thereby preventing contamination and reducing the risk of subsequent infections. Establishing a standardized method to evaluate the efficacy of commercially available sporicidal products is essential to ensure their effectiveness. However, several prerequisites must be addressed when developing a reliable sporicidal efficacy assessment. Previous studies have reported variations in sporicidal efficacy depending on the spore species and assay methodology used (Wesgate et al., 2016). A robust sporicidal efficacy assay begins with the preparation of high-quality spores for testing that are representative of other spore-forming bacteria. In addition, coupons that simulate surfaces commonly found in compounding facilities or other relevant application settings should be incorporated into the assay. To minimize false-positive results, the evaluation protocol must ensure that spores are exposed to the sporicidal product for the exact contact time specified on the product label. Furthermore, an effective neutralization procedure is required to terminate sporicidal activity immediately after the designated contact time. Based on our previous evaluation of seven Bacillus strains under standardized sporulation, purification, and sodium hypochlorite resistance testing conditions, Bacillus subtilis ATCC 6051 exhibited the greatest overall spore resistance and stability among the strains tested and was therefore selected for this study (Li et al., 2022). In this study, we evaluated the sporicidal efficacy of 20 commercially available disinfectants against B. subtilis ATCC 6051. The objective was to enhance aseptic control strategies and support public health efforts by ensuring effective disinfection practices in drug compounding.

Materials and methods

Bacterial strains and spore production

Bacillus subtilis ATCC 6051 and ATCC 19659 were obtained from the American Type Culture Collection (ATCC). The strains were streaked onto tryptic soy agar (TSA) plates for single-colony isolation and incubated at 30°C overnight. The following day, a single colony from a TSA plate was inoculated into a 50 ml tube containing 25 ml of tryptic soy broth (TSB). This 25 ml culture was then equally distributed into three 50 ml tubes and incubated at 30°C with shaking at 200 rpm overnight. The overnight cultures were transferred into 2 l flasks containing 250 ml of Difco sporulation medium (DSM). ATCC 6051 was incubated for 3 days and ATCC 19659 for 4 days, as previously reported (Li et al., 2022; Monteiro et al., 2014). After the respective incubation periods, 2 µl of culture was placed on a microscope slide to assess the sporulation rate by phase-contrast microscopy at 1,000 × magnification. A minimum of five fields per sample were examined to determine the sporulation rate.

Spore purification

Spores from both B. subtilis strains grown in Difco sporulation medium (DSM) were harvested and purified by centrifugation at 4,500 × g for 10 min (Tamir and Gilvarg, 1966). The spore pellets were washed with ice-cold sterile Milli-Q water three times, each followed by centrifugation at 4,500 × g for 10 min. To separate Bacillus endospores from cell debris, density centrifugation with a 50% w/v HistoDenz solution was performed at 4,500 × g for 10 min, as recommended by the U.S. Environmental Protection Agency (EPA) MLB SOP MB-28. After centrifugation, the upper three layers were carefully removed, and the pellets were resuspended in 1 ml of ice-cold PBST (0.1% v/v Tween-80). The suspension was centrifuged at 16,000 × g for 5 min, and the pellet was washed three times with 1 ml of ice-cold PBST, each wash followed by centrifugation at 16,000 × g for 2 min. The final spore pellets were resuspended in 1 ml of sterile Milli-Q water and stored at 4°C for maturation. Spore concentrations were determined by colony forming unit (CFU) enumeration on TSA plates following heat activation at 70°C for 20 min and serial dilution (15). Spore purity was assessed by comparing CFU counts before and after heat treatment at 70°C for 20 min to confirm the absence of vegetative cells.

Spore quality assessment

The purified B. subtilis ATCC 6051 and ATCC 19659 spores were matured at 4°C between 7 and 21 days. The matured spores were heat-treated in an 80°C water bath for 20 min, followed by sonication at 37 kHz for 5 min. Coupons that simulate surfaces commonly found in compounding facilities or other relevant application settings should be incorporated into the assay. Spore suspensions were diluted in sterile Milli-Q water to ∼1.0 × 10⁹ spores/ml, and 10 µl of the diluted suspension was spotted onto sterile 25 × 25 × 1 mm glass and stainless-steel coupons. The inoculated coupons were dried in a biosafety cabinet for 1 h and then further dried overnight in a desiccator jar. After drying, each coupon was placed on top of an uncapped 50 ml conical tube. Subsequently, 50 µl of sterile Milli-Q water or sodium hypochlorite (NaOCl) solution containing 1,500 ppm or 5,000 ppm free chlorine—measured using a colorimeter (MD100: Chlorine/pH, Tintometer Inc.)—was applied to the spores in accordance with EPA MLB SOP-MB-31 (EPA, 2017a). Following a 10-min contact time, each coupon was transferred into a 50 ml conical tube containing 20 ml of PBS (pH 7.4)–based neutralization buffer (0.1% v/v Tween 80 and 0.6% w/v sodium thiosulfate) to neutralize NaOCl. The tubes were capped and incubated at 20°C with shaking at 230 rpm for 25 min. Neutralized samples were serially diluted in PBST and filtered through Microfil filtration funnels with a membrane filter system (100 ml capacity, 0.22 µm pore size, 47 mm white gridded membrane; Millipore-Sigma) according to the manufacturer’s instructions. After filtration of the diluted sample, 10 ml of PBS was added to the tube and poured through the filter twice to recover residual spores. The membrane filters were then placed onto TSA plates to allow germination and incubated at 37°C overnight. The following day, log₁₀ reduction was determined by colony counting

Sporicidal products efficacy assay

Based on spore quality assessment results using NaOCl, B. subtilis ATCC 6051 spores were selected for subsequent sporicidal product efficacy assays. A total of 20 commercially available sporicidal products or disinfectants were evaluated. After maturation at 4°C for 7–21 days, ATCC 6051 spores were heat-treated at 80°C for 20 min, followed by sonication at 37 kHz for 5 min. Following heat and sonication treatment, 10 µl of spores (≈ 1.0 × 10⁷ spores/coupon) were spotted on both glass and stainless-steel coupons and the same method described above for spore quality assessment was used. Spores on the coupons were exposed to each sporicidal product or disinfectant for the contact time specified on the product label. To determine the log₁₀ reduction for each product, an additional group of spore-inoculated coupons treated with water served as the control. After the manufacturer-recommended contact time, products were neutralized using a PBS-based universal neutralizing buffer (0.1% v/v Tween 80, 0.6% w/v sodium thiosulfate, 0.5% w/v sodium bisulfite, 0.1% w/v lecithin, and 100 µg/ml catalase). The neutralized samples were serially diluted in PBST and filtered, and the log₁₀ reduction was determined by colony counting.

Neutralization confirmation assay

A neutralization confirmation assay was conducted for each product used in this study to verify that the PBS-based universal neutralizing buffer effectively neutralized the products. Three groups of both glass and stainless-steel coupons were prepared as follows: a water control group, a product group, and a pre-neutralized product group. Spore inoculation on the coupons was performed in the same manner as described in the sporicidal efficacy assay. The pre-neutralized product was prepared by adding 50 µl of product to 20 ml of PBS-based universal neutralizing buffer, mimicking the neutralization procedure used in the efficacy assay for each sporicidal product. Subsequently, 50 µl of water, product, or pre-neutralized product was applied to the dried spores on each coupon and incubated for the manufacturer-recommended contact time. After incubation with shaking, samples were serially diluted and filtered. The log₁₀ reduction was determined based on colony counts.

Sporicidal quality check

Six sporicidal products—four chlorine-based and two hydrogen peroxide/peracetic acid–based—that did not achieve greater than a 5 log₁₀ reduction were further evaluated to verify product quality (Fraise et al., 2015; Li et al., 2022). These products were retested using an extended contact time of 10 min. In addition, the pH and free chlorine levels of three NaOCl-based products were measured using a pH meter and a colorimeter (MD100: Chlorine/pH, Tintometer Inc.), respectively. The NaOCl-based products were replicated using NaOCl solution (Millipore-Sigma) at the concentrations stated on the product labels, with the pH adjusted to match that of the original formulations. Similarly, two hydrogen peroxide/peracetic acid–based products were replicated using hydrogen peroxide and peracetic acid solutions (Millipore-Sigma). These replicated sporicidal formulations were tested under the same conditions as the original products, using the manufacturer-recommended contact times.

All experiments were performed in triplicate and repeated three times.

Results

To minimize false positive results in the sporicidal products efficacy assay, a representative Bacillus strain (ATCC 6051) was chosen for the assays based on a previous study from our group and the initial results from this study (Li et al., 2022). The acceptable spore quality criteria followed the EPA MLB SOP MB-28 guidelines, which include three requirements: i) spore titer >5.0 × 10⁸ viable spores/ml, ii) spore purity >95%, and iii) spore showing <3 log₁₀ reduction with 1,500 ppm NaOCl solution and >5 log₁₀ reduction with 5,000 ppm NaOCl solution (EPA, 2017b). Bacillus subtilis ATCC 6051 exhibited ∼1 log₁₀ reduction against 1,500 ppm NaOCl solution even after spores were heated at 80°C for 20 min. In contrast, B. subtilis ATCC 19659, the reference strain for the EPA sporicidal efficacy assay for Bacillus spores, showed >3 log₁₀ reduction under the same conditions. Therefore, the B. subtilis ATCC 6051 strain spore was used as the reference strain in this study.

Sporicidal products efficacy assay

In this study, a total of 20 products with five different categories of main active ingredients were tested: chlorine-based (n = 8), hydrogen peroxide/peracetic acid-based (n = 6), aldehyde-based (n = 1), quaternary ammonium-based (n = 3), and phenol-based (n = 2) (Table 1, 2, 3). The effective sporicidal activity was determined if the products could meet the criteria of exceeding a log₁₀ reduction of 5, based on EPA SOP MB-21–02. Of the 20 products, 11 claimed to be sporicidal, 6 claimed to be broad disinfectants, and 3 claimed to be hospital-grade disinfectants.

Table 1.

Category I products label information.

Products			Labeling	Dilution	Recommended contact time (minutes)	Passed criteria (≥5 log10)
Category	No.	Ingredients
Ⅰa	A	NaOCl (52,500 ppm)	Broad disinfectant	Ready to use	5	Yes
	B	NaOCl (13,120 ppm)	Sporicidal agent	Ready to use	4	Yes
	C	NaOCl (9,400 ppm)	Sporicidal agent	Ready to use	4	Nod
	Db	NaOCl (3,900 ppm)	Sporicidal agent	Ready to use	2	No
	E	NaOCl (2,500 ppm)	Sporicidal agent	Ready to use	5	No
	F	SDSTc (chlorine: 4,306 ppm)	Sporicidal agent	1 tablet/946 ml ddH2O	4	No
	G	SDST (chlorine: 1,988 ppm)	Broad disinfectant	1 tablet/757 ml ddH2O	10	No
	H	Hypochlorous acid (170 ppm)	Hospital grade disinfectant	Ready to use	10	Yes

^aChlorine-based disinfectants.

^bAs a result of colorimeter measurement, product that didn’t contain as much free chlorine as stated on the label.

^cSDST: Sodium dichloro-S-triazinetrione.

^dThis product could pass the criteria when contact time was 10 min.

Table 3.

Category III, IV, V products label information.

Products			Labeling	Dilution	Recommended contact time (minutes)	Passed criteria (≥5 log10)
Category	No.	Ingredients
Ⅲa	O	OPAd (6,000 ppm)	Sporicidal agent	Ready to use	12	No
Ⅳb	P	DDACe (105,000 ppm)	Broad disinfectant	1:64 ddH2O	10	No
	Q	ODDACf (65,100 ppm), DIDACg (26,040 ppm), DDAC (39,060 ppm), ADBACh (86,800 ppm)	Hospital grade disinfectant	1:64 ddH2O	10	No
	R	ODDAC (260 ppm), DIDAC (130 ppm), DDAC (130 ppm), ADBAC (340 ppm)	Broad disinfectant	Ready to use	10	No
Vc	S	O-phenylphenol (60,000 ppm), O-benzyl-p-chlorophenol (127,500 ppm)	Broad disinfectant	1:128 ddH2O	10	No
	T	O-phenylphenol (60,000 ppm), O-benzyl-p-chlorophenol (130,000 ppm)	Broad disinfectant	1:64 ddH2O	10	No

^aAldehyde-based disinfectants.

^bQuaternary ammonium-based disinfectants.

^cPhenol-based disinfectants.

^dOPA: Ortho-Pathalaldehyde.

^eDDAC: Didecyl dimethyl ammonium chloride.

^fODDAC: Octyl Decyl Dimethyl Ammonium Chloride.

^gDIDAC: Dioctyl Dimethyl Ammonium Chloride.

^hADBAC: Alkyl (C₁₄ 50%, C₁₂ 40%, C₁₆ 10%) dimethyl benzyl ammonium chloride.

Table 2.

Category II products label information.

Products			Labeling	Dilution	Recommended contact time (minutes)	Passed criteria (≥5 log10)
Category	No.	Ingredients
Ⅱa	I	H2O2 (73,500 ppm), Peracetic acid (2 300 ppm)	Sporicidal agent	Ready to use	15	Yes
	J	H2O2 (44,000 ppm), Peracetic acid (2,300 ppm)	Sporicidal agent	Ready to use	3	Yes
	K	H2O2 (10,000 ppm), Peracetic acid (800 ppm)	Sporicidal agent	Ready to use	30	Yes
	L	H2O2 (275,000 ppm), Peracetic acid (58,000 ppm)	Sporicidal agent	2.3% v/v ddH2O	3	No
	M	H2O2 (53,400 ppm), Peracetic acid (13,600 ppm)	Hospital grade disinfectant	2.3% v/v ddH2O	10	No
	N	H2O2 (78,000 ppm)	Sporicidal agent	Ready to use	7	No

^aHydrogen peroxide & peracetic acid-based disinfectants.

Of the eight chlorine-based products, three (37.5%) met the effectiveness criteria, achieving around a 7 log₁₀ reduction, while the other five products failed to meet the criteria, with four displaying less than a 1 log₁₀ reduction (Figure 1). Similarly, of the six hydrogen peroxide/peracetic acid-based products, three (50.0%) met the criteria with around a 7 log₁₀ reduction, while the other three products did not meet the criteria, showing less than a 1 log₁₀ reduction (Figure 2).

Figure 1.

Log₁₀ reduction of B. subtilis ATCC 6051 spores by category I products. Experiments were performed in triplicate and repeated three times. Error bars represent the standard deviation of the mean.

Figure 2.

Log₁₀ reduction of B. subtilis ATCC 6051 spores by category II products. Experiments were performed in triplicate and repeated three times. Error bars represent the standard deviation of the mean.

Among the other three categories of disinfectants, the single aldehyde-based sporicidal product tested, which was composed of ortho-phthalaldehyde, achieved less than a 1 log₁₀ reduction (Figure 3). The three quaternary ammonium-based products did not meet the criteria, and the two phenol-based products also showed less than a 1 log₁₀ reduction (Figure 3).

Figure 3.

Log₁₀ reduction of B. subtilis ATCC 6051 spores by category III, IV, V products. Experiments were performed in triplicate and repeated three times. Error bars represent the standard deviation of the mean.

Overall, six products (6/20; 30%)—three each from the chlorine-based and hydrogen peroxide/peracetic acid-based categories—passed the criteria. Four of these products were among the 11 (36.4%) that claimed to be sporicidal, achieving greater than a 5 log₁₀ reduction. The other two products that satisfied the criteria were listed as a broad disinfectant and a hospital-grade disinfectant, respectively. No significant difference was detected (p ≥ 0.05) between glass and stainless-steel coupons.

Neutralization confirmation

All 20 products were tested for successful neutralization. When the products were pre-neutralized with a PBS-based neutralizing buffer, all products lost their sporicidal activity, showing less than a 0.2 log₁₀ reduction after neutralization (Figure 4). This indicates that the six products that had achieved greater than a 5 log₁₀ reduction before could be neutralized quickly and effectively. The universal neutralizing buffer used in this study had neither a positive nor negative effect on the spores when the spores were incubated with only the universal neutralizing buffer. Additionally, no significant difference was detected between glass and stainless-steel coupons in the neutralization confirmation assay.

Figure 4.

Log₁₀ reduction of B. subtilis ATCC 6051 spores by pre-neutralized category I-V products on glass coupons (A) and stainless-steel coupons (B). Experiments were performed in triplicate and repeated three times. Error bars represent the standard deviation of the mean.

Sporicidal quality check

Sporicidal products that did not meet the criteria following their label-recommended contact time were tested for a longer contact time of 10 min. One product from the chlorine-based category subsequently met the criteria, while the other five products still could not reach a 5 log₁₀ reduction (Figures 5 and 6). As a quality check, NaOCl-based products and hydrogen peroxide-based products were replicated by their active ingredients. Among five freshly prepared laboratory-made products containing identical active ingredients, three exhibited a slightly or significantly better sporicidal effect compared to their corresponding commercial counterparts (Figures 5 and 6). One of the NaOCl-based products was found to not contain free chlorine, contrary to what was stated on the label, based on free chlorine measurement (data not shown).

Figure 5.

Log₁₀ reduction of B. subtilis ATCC 6051 spores when some category I sporicidal products that did not meet the criteria were replicated as household products and tested with longer contact time of 10 min (A, B, C, and D). Experiments were performed in triplicate and repeated three times. Error bars represent the standard deviation of the mean.

Figure 6.

Log₁₀ reduction of B. subtilis ATCC 6051 spores when some category II sporicidal products that did not meet the criteria were replicated as household products and tested with longer contact time of 10 min (A and B). Experiments were performed in triplicate and repeated three times. Error bars represent the standard deviation of the mean.

Discussion

Bacillus species are spore-forming bacteria commonly found in soil, air, and some animal intestines. In their dormant form, these spores can survive much longer than vegetative cells (Markland et al., 2013). One traditional method to eliminate spores involves inducing all spores to germinate, making the germinated cells more susceptible to inactivation methods (Ghosh and Setlow, 2010). However, this method has some limitations since not all spores germinate simultaneously, and in some cases, toxins can be produced during germination (De Vries et al., 2004). Furthermore, some species form superdormant spores, which are characterized by slow germination with nutrients and a better survival rate than other spores (Rodriguez-Palacios and LeJeune, 2011). Bacterial spores, formed by infectious agents associated with medical facility-related infections, often require effective, high-level disinfection for removal. Aseptic drug manufacturing facilities need to control bacterial spore contamination, as drug compounding is typically done for patients. Therefore, sporicidal products must ensure they can effectively kill bacterial spores. The sporicidal efficacy assessment assay used in this study was modified from EPA SOP MB-21–02 (EPA, 2014). This method tests the efficacy of sporicidal products against Bacillus spores; however, the hard, non-porous coupons recommended in their SOP, which are 5 × 5 × 1 mm, are too small in size and there is a risk of contamination between each coupon or tubes containing neutralizing buffer when the coupons are transferred. Therefore, larger coupons (25 × 25 × 1 mm) were used in this study, and the coupons were placed on top of 50 ml; tubes to minimize any additional transfer time, which could inadvertently increase contact time with spores and disinfectant. The neutralization method is a key factor for a reliable sporicidal assay. If the neutralizing buffer either boosts the number of spores or negatively impacts spores, it can lead to false results. Both Letheen medium and D/E neutralizing medium are common neutralizing buffers for organism recovery after disinfectant treatment, as they can neutralize a broad range of disinfectants, including quaternary ammonium compounds, phenols, iodine and chlorine compounds, mercurial, formaldehyde, and glutaraldehyde (Dey and Engley, 1995). However, these media contain nutrients, such as amino acids and sugars, which can help spores germinate and thus affect overall sporicidal results (Delbrück et al., 2021). To minimize any additional factors that could promote spore germination, a universal PBS-based neutralizing buffer was used, which does not contain any nutrients but can neutralize each of the active ingredients tested in this study. A larger volume of neutralizing buffer was used to ensure effective neutralization and dilution of disinfectants. The results of this study indicated that the neutralization method used was effective and did not affect spore germination.

The study evaluated a diverse range of disinfectant products categorized by their main active ingredients. Notably, a significant proportion of chlorine-based and hydrogen peroxide/peracetic acid-based products met the effectiveness criteria, achieving >5 log₁₀ reduction. However, a substantial number of products in these categories failed to meet the criteria, with some displaying minimal sporicidal activity. In contrast, the aldehyde-based, quaternary ammonium-based, and phenol-based products generally exhibited <1 log₁₀ reduction, indicating limited sporicidal efficacy. The findings are consistent with previous studies showing that chlorine-releasing compounds, hydrogen peroxide, and peracetic acid exhibit strong sporicidal activity when used under appropriate conditions. In contrast, quaternary ammonium and phenolic disinfectants are generally ineffective against dormant spores. The substantial variability observed among products with similar active ingredients highlights the influence of formulation, concentration, and exposure conditions, emphasizing the need to verify individual product performance rather than relying solely on disinfectant classification (McDonnell and Russell, 1999; Rutala and Weber, 2021).

The quality check experiment from this study indicated that, overall, sporicidal products stated inappropriate contact times, which were too short to kill Bacillus spores. From the lab-produced products, it was apparent that some of the active ingredients in various products were either degraded or malfunctioning. Therefore, product labels need to provide more accurate information. Some products claimed to be a sterilant, which requires a few hours of contact time; this duration works only for small medical devices that can be submerged in disinfectant. Realistically, it is difficult to keep the surface area wet for several hours, especially in drug compounding areas that require hygiene to maintain clean surfaces in the workplace.

The considerable variability among products containing similar active ingredients underscores the need for standardized evaluation of individual compounds and their combinations. While some chlorine- and hydrogen peroxide/peracetic acid-based products achieved >5 log₁₀ spore reduction, others showed minimal activity despite belonging to the same chemical class. These differences may reflect variations in ingredient concentration, formulation, stability, and ingredient interactions. Future studies should identify key factors influencing sporicidal efficacy to support more accurate product performance claims. Additionally, biofilms formed by single or multiple microbial species can provide a protective matrix that enhances resistance to disinfectants and may alter the susceptibility of embedded spores, warranting further investigation. Furthermore, studying the stability of active chemical ingredients, and determining a more accurate expiration date and shelf life for the products will be essential to ensure the generation of dependable data regarding the effectiveness of sporicidal products. Moreover, disinfectant efficacy should be assessed under soiled conditions and in conjunction with standardized wiping procedures to better simulate real-world applications in aseptic drug manufacturing facilities. These efforts are crucial for enhancing the safety evaluation of drug compounding.

In conclusions, a total of 20 sporicidal or disinfecting products, containing five categories of active ingredients, were tested. Of these, six achieved more than a 5 log₁₀ reduction, while the others could only kill about half of the tested B. subtilis spores, resulting in less than a 1 log₁₀ reduction. Chlorine- and hydrogen peroxide and peracetic acid-based ingredients are common in sporicidal products, and our data indicated that products not based on chlorine or hydrogen peroxide and peracetic acid were ineffective in spore killing. This study revealed that certain commercial disinfectant products claiming sporicidal activity do not meet the effectiveness criteria when tested against Bacillus spores. Since Bacillus species are commonly found in aseptic compounding facilities, it is critical to assess the efficacy of sporicidal products available in the market against Bacillus spores.

Data availability

The authors declare that all the data supporting the findings of the presnet study are available within the manuscript.

Funding

This work was supported by the U.S. Food and Drug Administration (E0771701) and in part by appointments (YH) to the Postgraduate Research Fellowship Program by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration.

Conflicts of interest

The authors declare no conflicts of interest.