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Article

In Vitro Evaluation of the Effectiveness of a Commercial Enzymatic Product Against Pseudomonas aeruginosa Biofilms According to the Parameters of Use

by
Caroline Le Sénéchal
1,
Yoann Fautras
1,
Caroline Tokarski
1,2 and
Sébastien Vilain
1,*
1
CNRS, Bordeaux INP, CBMN, University Bordeaux, UMR 5248, F-33600 Pessac, France
2
Proteome Platform, Université de Bordeaux, F-33000 Bordeaux, France
*
Author to whom correspondence should be addressed.
Hygiene 2025, 5(3), 41; https://doi.org/10.3390/hygiene5030041
Submission received: 6 July 2025 / Revised: 16 August 2025 / Accepted: 29 August 2025 / Published: 4 September 2025
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Abstract

Biofilms are the source of numerous issues in the food, pharmaceutical, and production industries, making their control a major component of economic and public health. Among anti-biofilm strategies, enzyme-based products that target the biofilm matrix have proven effectiveness against multiple bacterial species. We tested the efficacy of a commercial product, Baso Bionil SL40® (SL40; Stockmeier France, Saint-Jacques de la lande, France), against biofilms of Pseudomonas aeruginosa under various conditions of temperature, concentration, pH, and incubation time. SL40 contains two enzymes: a subtilisin protease and an α-amylase glycosidase. Our results showed that SL40 removed up to 85% of the biofilm biomass compared to tris solutions. SL40’s efficacy was strongly influenced by the presence of the enzymes and both temperature and concentration. Enzymatic activity was maintained from 20 °C to 60 °C and at pH values ranging from 5 to 9, with effective concentrations corresponding to SL40 dilutions from 3/200 to 1/200 in 50 mM tris solutions. Additionally, we observed that the P. aeruginosa biofilm biomass after pH 9 tris solution treatment was twice compared to a H2O washing. Our results confirm the potential of enzymes against biofilms, highlight the need to define optimal application conditions, and support their integration into combined strategies for complete biofilm removal.

1. Introduction

Bacteria form multicellular structures named biofilms, defined as a set of microcolonies enclosed in a self-produced matrix that adhere to surfaces or develop at interfaces [1]. These structures provide resistance and resilience to the bacterial populations they contain. Indeed, compared to their planktonic counterparts, sessile bacterial cells are resistant/tolerant to numerous stresses, including antibiotic treatments [2] and, more broadly, to biocides or disinfectants [3,4]. In industrial facilities, biofilms are involved in biocorrosion, biofouling, and may contaminate agri-food products or pharmaceutical production lines [5]. The economic cost of biofilms is estimated at USD 5000 billion/year, primarily due to the corrosion [5,6], while food-borne pathogens alone are responsible for over one million deaths per year [7]. Controlling biofilms is therefore a key issue for industrials, and finding solutions to fight biofilms is both an economic and a public health issue. Strategies to combat biofilms aim to prevent bacterial attachment to surfaces, inactivate or eradicate sessile cells, and/or promote the dispersion of these cells or fragments of the biofilm [8]. Among the available approaches, enzyme-based products are used to disrupt and remove mature biofilms already established on surfaces [9,10]. These enzymes degrade key components of the extracellular matrix, such as polysaccharides, proteins, and eDNA [11]. Therefore, their effectiveness depends on the matrix composition, as well as on application parameters such as temperature, pH, enzyme concentration, application duration, and the presence of enzymatic co-factors [12,13]. In our study, we evaluated the effectiveness of the non-foaming di-enzymatic detergent Baso Bionil SL40® (Stockmeier France SAS, formerly Quaron SAS) against Pseudomonas aeruginosa biofilms under various application parameters. Baso Bionil SL40® (SL40), recommended by the manufacturer for cleaning and preventing biofilms in the food industry and authorized for use in Organic Farming (EC Regulation no. 834/2007), contains two enzymes: a protease (subtilisin) and a hydrolase (α-amylase). Based on the supplier’s recommendations, the activity of SL40 on 24-h-old P. aeruginosa PAO1 biofilms was tested at different concentrations (0.5%, 1.0%, and 1.5%); temperatures (10 °C, 20°C, 40 °C, and 60°C); and pH values (5, 7, and 9) while varying the application duration (5, 10, and 15 min). Pseudomonas aeruginosa, an opportunistic pathogen, was selected because it frequently contaminates industrial products and colonizes surfaces in healthcare facilities and because it is a reference organism for biofilm research [14,15]. Our results demonstrated that the SL40’s ability to remove P. aeruginosa biofilms was directly related to its enzymatic activity. SL40 effectiveness was strongly influenced by temperature and enzyme concentration and, in a temperature-dependent manner, was occasionally modulated by pH and application duration.

2. Materials and Methods

2.1. Bacterial Strain, Growth Medium, and Biofilms Preparation

Pseudomonas aeruginosa PAO1 strain (CIP 104116) was obtained from the Institut Pasteur (Paris, France). From a −80 °C glycerol stock, P. aeruginosa was steaked onto Lysogeny Broth agar plates (tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, and agar 15g/L) and incubated for 18 h at 37 °C. Colonies were harvested and suspended in sterile PBS (NaCl 8 g/L, KCl 0.2 g/L, Na2HPO4 2H2O 1.44 g/L, and KH2PO4 0.24 g/L) to obtain a bacterial suspension adjusted to OD600 ≈ 2.5 (i.e., 5.109 CFU/mL). Aliquots of 200 µL were dispensed into each well of untreated U-bottom 96-well microtiter plates (Caplugs Evergreen, Buffalo, NY, USA), and biofilms were obtained after 24 h at 37 °C. Before testing, plates containing 24-h-old biofilms were incubated 30 min at the target test temperature (10 °C, 20 °C, 40 °C, or 60 °C).

2.2. Preparation of Baso Bionil SL40® Solutions and Biofilm Treatment

Two stock solutions of Baso Bionil SL40® were provided by Stockmeier France SAS (Rennes, France): the commercial formulation containing enzymes (SL40) and an enzyme-free formulation (SL40 basis). These stock solutions were extemporaneously diluted to final concentrations of 0.5%, 1.0%, and 1.5% (v/v) in 50 mM tris solutions (trizma-HCl and/or trizma-base; Sigma, St. Louis, MO, USA) adjusted to pH 5, 7, or 9 (Figure 1). Before the addition of SL40 or SL40 basis, the tris solutions were incubated for 1 h at the target test temperature (10 °C, 20 °C, 40 °C, or 60 °C). Biofilm removal assays were performed by replacing the culture supernatant from each well of a 96-well microtiter plate with 250 µL of the test solution (SL40 or SL40 basis in tris buffer). After 5, 10, or 15 min of incubation at the test temperature, residual biofilms were quantified using the crystal violet (CV) staining method [16]. Briefly, the tested solution was removed, and each well was filled with 250 µL of 0.1% (w/v) CV (Sigma) in H2O. After 10 min at room temperature, the dye was discarded, and wells were washed 3 times with H2O. To solubilize bound CV, 250 µL of 33% (v/v) acetic acid (Fischer, Dover, DE, USA) was added to each well, and 50 µL were transferred to a flat bottom 96-well microtiter plate (Maxisorp, Thermo Scientific, Illkirch, France) already containing 150 µL per well of 33% (v/v) acetic acid (i.e., 1:4 dilution). Absorbance at 595 nm (OD595) was measured using a SPECTROstar nanoplate reader (BMG Labtech, Ortenberg, Germany). Two control conditions were included, the first with H2O, a treatment systematically applied on each 96-well plate for data normalization, and the second using the tris solutions without SL40/SL40 basis, to assess whether tris solution had the capacity per se to remove biofilms.

2.3. Data Analysis Methods

The effectiveness of the test solutions to remove biofilms was assayed as a function of 5 variables: presence of enzymes (yes/no); SL40 or SL40 basis concentration (0.5%, 1.0%, or 1.5%); pH (5, 7, or 9); temperature (10 °C, 20 °C, 40 °C, or 60 °C); and application duration (5, 10, or 15 min). For each condition, 3 biological replicates and 8 technical replicates per biological replicate were performed, yielding 24 experimental OD595 per condition (n = 24). Raw OD595 values were normalized (nOD595) by dividing an experimental OD595 by the mean OD595 of the 24 water controls from the same plate (Figure 1). This approach smoothed out differences of initial biofilm biomass between plates and enabled cross-plate comparison. The statistical tests were performed using Statgraphic Plus 5.1 software (StatPoint Technologies Inc., Warrenton, VA, USA). The non-parametric Mann–Whitney test was used to determine statistically significant differences (p-values < 0.05) between 2 sets of 24 data. This test was chosen because normality and homoscedasticity assumptions were not always met. Principal component analysis (PCA) was performed on centered-reduced values to identify correlations within the dataset. The PCA results were visualized as component plots.

3. Results

3.1. Description of the Dataset, Variability of the Results, and Criteria to Qualify SL40 Effectiveness Against Pseudomonas aeruginosa Biofilms

3.1.1. A Dataset of 8640 Values

The activity of SL40/SL40 basis against P. aeruginosa biofilms was evaluated by quantifying residual biofilms after treatment. Additionally, biofilms were treated with H2O, a reference applied on each 96-well plate to estimate the initial biofilm biomass and to normalize the OD595 values, and with the tris solutions, a reference condition for SL40/SL40 basis treatments, enabling the detection of potential pH effects (Figure 1). In total, 216 experimental conditions were tested with SL40 and SL40 basis, and 108 series of values were obtained with H2O and 36 with tris solutions. The merged dataset, combining all test and reference results, therefore contains 8640 data, corresponding to 360 series of 24 values each (Table S1).

3.1.2. Variability of the Data Obtained from Microtiter Plates

The variability of the dataset was assessed for the 360 series of 24 data by using the Relative Standard Deviation (RSD) and the ratio between the maximum and the minimum values (RMm). Both RSD and RMm included biological variability (e.g., variation in the initial biofilm biomass) and technical variations (e.g., differences in treatment effectiveness or CV staining).
Intra-plate variability: The average RSD was 8.7% and RMm was 1.4 (Table S2A,B), indicating a good repeatability of data obtained on the same plate. Among the 360 series, only 21 had an RSD ≥ 15.0% and 11 had an RMm ≥ 2. Principal component analysis (PCA) performed with RSD and RMm values revealed that the factors most strongly correlated with data repeatability were T °C and the SL40/SL40 basis concentration (Figure S1). The lowest RSD and RMm values were observed after biofilm treatment at 10 °C with solutions at concentrations below 1.5%, whereas the highest values were obtained for treatments at 20 °C with 0.5% solutions (Table S2A,B).
Inter-plates variability: The reproducibility between different plates was evaluated by analyzing the data obtained after H2O treatment (Table S3). RSD and RMm were calculated from the 216 OD595 values obtained on nine plates treated under the same conditions (temperature and incubation duration). The intra-plate variability in this subset was similar to that observed for the complete dataset, with an average RSD of 7.8% and an RMm of 1.4. However, the inter-plate variability was higher, with a mean RSD of 18.2% and an RMm of 2.6 (Table S3A). Temperature influenced this variability, with the highest RSD (32.1%) and RMm (4.4) observed at 60 °C (Table S3B). These results suggest some heterogeneity in the H2O treatment and/or in the initial biofilm biomass across plates.
Although this heterogeneity remained moderate, the OD595 values were normalized (nOD595) to enable comparisons across plates (detailed in Section 2.3) (Figure 1; Table S4). This normalization allowed straightforward visualization of treatment effects relative to H2O washing: an nOD595 of 1 indicated an effect identical to H2O, a value < 1 indicated greater biofilm removal than H2O, and a value > 1 indicated that H2O removed more biofilm than SL40/SL40 basis.

3.1.3. Three Criteria to Qualify the Effectiveness of SL40 Against P. aeruginosa Biofilms

Initially, two criteria were considered (Figure 1): (1) the statistical comparison of the “test condition” (SL40 or SL40 basis) vs. the “reference condition” (tris solution), with a significance threshold of p-value ≤ 0.05, and (2) the ratio between mean values (n O D ¯ 595) of the two conditions, referred to as the Effectiveness Index (EI). An EI ≤ 0.5 indicated that the test solution removed at least twice as much biofilm as its corresponding tris solution. However, the nOD595 values obtained with tris solutions led us to consider a third criterion. Indeed, application of pH 9 tris solution at 10 °C, 20 °C, and 40 °C resulted in nOD595 values ≈ 1.5–1.9 times higher than those obtained after H2O under the same conditions (Figure 2; Table 1).
This revealed a “protective” effect of pH 9 tris solution on P. aeruginosa biofilms, creating situations in which the p-value ≤ 0.05 and EI ≤ 0.5, yet n O D ¯ 595 ≥ 1. In such cases, SL40/SL40 basis removed some biofilm but was less effective than H2O. The third criterion was therefore defined as the n O D ¯ 595 value itself. A value ≤ 0.5 indicated that the residual biofilm quantity after treatment was at least two times lower than that after H2O treatment. A solution was considered effective in a given experimental condition only if all three criteria were met simultaneously: p-value ≤ 0.05; EI ≤ 0.5, and n O D ¯ 595 ≤ 0.5). Since the p-value was systematically ≤0.05 whenever the other two criteria were satisfied, it is not discussed further in the manuscript, although the values are reported in Table S5.

3.2. Effectiveness of the Di-Enzymatic Detergent SL40 to Remove P. aeruginosa Biofilms: Role of the Enzymes and Influence of the Temperature, Concentration, pH, and Incubation Time

3.2.1. Global Analysis of the Influence of the Tested Parameters on SL40 Activity

The SL40 ability to eliminate PAO1 biofilms was assessed using the significance criteria described above. All data used to calculate these criteria are presented in the Supplemental Data (Tables S1–S5), with the calculated values summarized in Table 1. The variation of these criteria across experimental conditions is shown in Figure 3, with high-resolution versions of each figure available in the Supplemental Excel File (Figures S2–S9).
A PCA was performed using p-values, EI, and n O D ¯ 595 values, along with the experimental conditions, to identify parameters potentially influencing the effectiveness of SL40/SL40 basis solutions against biofilms (Figure 4). The PCA identified two main components explaining > 83% of the data variability and clearly showed that the three criteria were strongly correlated with incubation temperature, SL40/SL40 basis concentration, and the presence of enzymes in the solutions. In contrast, pH and incubation time had only a limited impact on SL40/SL40 basis activity.
Based on these findings, the results presented below are presented by temperature, beginning with those obtained using SL40 basis (enzyme-free), followed by those obtained with SL40 (containing enzymes), with concentration effects considered in each case. The influence of pH and incubation time is addressed only when a notable effect was observed.

3.2.2. Activity of SL40/SL40 Basis on P. aeruginosa Biofilms at 10 °C

After treatment with SL40 basis, all EI and n O D ¯ 595 values exceeded 1, ranging from 1.05 to 1.32 and 1.18 to 2.21, respectively (Table 1), indicating no elimination of P. aeruginosa biofilms at 10 °C. Increasing the SL40 basis concentration from 0.5% to 1.0% produced a mean change of +2.5% in EI/n O D ¯ 595 values and a decrease of -1.6% of both criteria when increasing from 1.0% to 1.5%, showing a limited impact of concentration. Shifting the pH from 5 to 7 induced moderate decreases of EI (-9.2%) and n O D ¯ 595 (−4.2%), whereas moving from pH 7 to 9 caused a slight EI (+4.5%) increase but a marked rise in n O D ¯ 595 (+56.6%) (Table S5). This latter effect was reminiscent of the “biofilm preservative effect” observed with the tris solutions at pH 7 and 9 compared to a H2O treatment (Figure 3). At pH 9, the SL40 basis treatments yielded an average n O D ¯ 595 of 2.09, i.e., more than twice that of the H2O-treated biofilms.
With SL40, the EI/n O D ¯ 595 values ranged from 0.38 to 1.23 and 0.64 to 2.04, respectively (Table 1). Enzyme presence reduced both criteria in a concentration-dependent manner (−5.0%, −12.3%, and −31.7% for 0.5%, 1.0%, and 1.5%, respectively) and in a pH-dependent manner (−5.9%, −19.6%, and −23.4% for pH 5, 7, and 9, respectively). The lowest values were obtained with 1.5% SL40 at pH 7 or 9, with a maximal biofilm reduction (−64%) observed after 15 min with 1.5% SL40 at pH 9 compared to SL40 basis. In these conditions, EI and n O D ¯ 595 values decreased by ~50% between 5 and 15 min, illustrating a time-dependent effect. Despite these reductions, all but one value remained >0.5, indicating that SL40 did not achieve significant biofilm elimination at 10 °C.

3.2.3. Activity of SL40/SL40 Basis on P. aeruginosa Biofilms at 20 °C

With SL40 basis, EI/n O D ¯ 595 values decreased compared to 10°C, ranging from 0.70 to 1.26 and 0.80 to 1.93, respectively (Table 1 and Table S5). Temperature effects varied with pH and concentration of the solution. At pH 9, the EI/n O D ¯ 595 values for the 1.0% and 1.5% solutions decreased by −24.0% and −34.9%, respectively, whereas the 0.5% solutions produced a +6.4% increase in EI and a −9.7% decrease in n O D ¯ 595. Increasing the concentration from 0.5% to 1.0% reduced both criteria by −20.1%, while the shift from 1.0% to 1.5% had a minimal effect (−0.6%). All EI and n O D ¯ 595 values remained >0.5, with pH 9 values exceeding 1, confirming a “preservative” effect similar to that seen at 10 °C.
With SL40, EI values ranged from 0.22 and 1.07 and n O D ¯ 595 from 0.28 and 0.99. With 1.0% and 1.5% solutions, most EI and n O D ¯ 595 values were <0.5, indicating significant biofilm elimination, except for 1.0% at pH 5 after 5 min (EI = 0.51). At 1.5%, SL40 reduced biofilm biomass by an average of 67% compared to tris or H2O controls. Similar reductions were observed with 1.0% SL40 at pH 7 and 9. Time dependence was evident, e.g., with 0.5% SL40 at pH 9, where EI decreased from 0.51 to 0.27 and n O D ¯ 595 from 0.85 to 0.40 between 5 and 15 min, whereas, at pH 5 or 7, all values remained >0.5 (Table 1 and Table S5).
Overall, SL40 at 1.0% or 1.5% significantly eliminated biofilms at 20 °C, with similar activity for 0.5% at pH 9 only. In all cases, this activity was linked to the presence of the enzymes. Enzymatic activity was temperature-dependent, as raising the temperature from 10 °C to 20 °C reduced the EI and n O D ¯ 595 by −64.1% and −69.0% for 1.0% and 1.5% SL40, respectively (Figure 3).

3.2.4. Activity of SL40/SL40 Basis on P. aeruginosa Biofilms at 40 °C

After SL40 basis treatment, the EI and n O D ¯ 595 values ranged from 0.53 to 1.11 and 0.50 to 1.73, respectively (Table 1 and Table S5), with no significant biofilm elimination in any condition (Figure 3). However, compared to 20 °C, both indices decreased again in a pH-dependent manner for n O D ¯ 595 (mean values: 0.61, 0.86, and 1.26 at pH 5, 7, and 9, respectively). At pH 9, all values exceeded 1, while, at pH 5 and incubation over 10 min, the values approached but did not cross the 0.5 threshold (Table 1).
With SL40, the EI values ranged from 0.17 and 0.40 and n O D ¯ 595 from 0.24 and 0.41, indicating significant elimination of biofilms under all conditions. The effect was linked to enzyme presence, with mean reductions of −50.0%, −67.0%, and −73.2% for pH 5, 7, and 9, respectively (Figure 3; Table 1). Concentration effects were also clear, with the lowest EI values at 1.0% and 1.5%, especially at pH 9 (mean EI = 0.17, i.e., −83% vs. tris pH 9). Temperature impact varied with concentration: from 20 °C to 40 °C, EI and n O D ¯ 595 decreased by −57.9%/−55.9% for 0.5%, −19.0%/−18.5% for 1.0%, and −12.9%/−13.5% for 1.5% solutions.

3.2.5. Activity of SL40/SL40 Basis on P. aeruginosa Biofilms at 60 °C

For SL40 basis, EI/n O D ¯ 595 values ranged from 0.22 to 1.00 and 0.27 to 1.11, respectively (Table 1 and Table S5). The impact of temperature from 40 °C to 60 °C depended on pH and concentration of the solutions. The largest decreases were observed with 1.0% and 1.5% solutions at pH 9 (EI/n O D ¯ 595 reduction of −62.4% and −72.0%, respectively), whereas 0.5% solution at pH 5 increased the EI and n O D ¯ 595 by +40.6% and +38.4%, respectively. Notably, SL40 basis significantly eliminated biofilms for the first time but only at 1.0% and 1.5% pH 9, with ~70% biomass reduction compared to a simple H2O treatment.
With SL40, all EI and n O D ¯ 595 values were <0.5 (ranges: 0.13–0.48 and 0.19–0.47, respectively), confirming significant elimination under all conditions (Table 1). Enzyme contribution remained dominant at pH 5 and 7 but was only partial at pH 9 due to the strong activity of SL40 basis at this pH. Shifting pH from 5 to 7 reduced EI/n O D ¯ 595 by −52.3%/−34.8%, while, from pH 7 to 9, the reduction was minimal (−2.7%/−2.3%). Temperature effects varied: at pH 7 and 9, most 60°C values were lower than those at 40 °C, whereas, at pH 5, values increased by +35.1%, +29.0%, and +17.7% for 0.5%, 1.0%, and 1.5%, respectively. This increase was modest up to 10 min but increased sharply after 15 min (+79.8%, +93.9%, and +61.7%, respectively), though still below 0.5.
In summary, SL40 significantly removed biofilms at 60 °C in all conditions, with the enzyme presence being the main driver at pH 5 and 7 and a partial driver at pH 9, where SL40 basis also displayed strong activity.

4. Discussion

Biofilms developing in industrial installations can cause long-term problems for colonized equipment, notably through biocorrosion [17]. They can also compromise the quality of finished products or, in rare but significant cases, affect the health safety of products sold by food or health manufacturers [18]. It is estimated that 40–60% of foodborne outbreaks are associated with biofilms [19,20]. Pseudomonas cepacia has even been detected in a batch of disinfectant intended for clinical use [21]. Biofilms therefore represent a major concern for manufacturers, who seek effective methods, under any condition, to prevent their formation or eliminate them, ideally without dismantling their installations. This demand has driven the development of numerous anti-biofilm products in recent decades [5], supporting a global market expected to reach USD 3.3 billion by 2030 [22].
The effectiveness of Baso Bionil SL40® (SL40) against Pseudomonas aeruginosa biofilms was evaluated under various conditions of temperature, pH, concentration, and application time. Such analyses require a repeatable initial immobilized biomass to treat. This problem of repeatability and reproducibility is well known in the biofilm field, as many parameters influence the quality and quantity of biofilms formed by a given bacterial species [23,24,25]. We retrospectively assessed variability in our multi-month dataset, both within a single 96-well plate (intra-plate variability) and between different 96-well plates (inter-plate variability). Overall, the intra-plate variability yielded a mean RSD of 8.7% and RMm of 1.4. The inter-plate variability of the initial biofilm biomasses was approximately twice as high (RSD = 18.2% and RMm = 2.6). For comparison, a study analyzing 960 data from six laboratories over 2 days published a few year ago reported a mean RSD of 20.2% and an average RMm of 2.3 [24]. An RSD threshold of 15% is commonly cited as indicative of good reproducibility, notably by the International Council for Harmonization (ICH), which defines this value as a standard for ensuring precision and reproducibility in analytical methods [26].
Anti-biofilm strategies target various stages of the biofilm life cycle [27,28,29]. These approaches can be preventive, avoiding the formation of new biofilms, or curative, destabilizing and removing established biofilms. Preventive methods include chemically modified surfaces to avoid/limit/delay the colonization [30] and anti-quorum sensing molecules perturbing the biofilms maturation [31]. Preventive methods include chemically modified surfaces to limit colonization [30] and anti-quorum sensing molecules to interfere with maturation [31]. Curative approaches often focus on disrupting the extracellular polymeric substances (EPS) that form the biofilm matrix, which mainly consist of polysaccharides, proteins, and eDNA [32]. Matrix destabilization facilitates mechanical removal or enhances the activity of antimicrobial agents such as antibiotics [33]. Enzymatic treatments are a common curative strategy, with carbohydrases, proteases, and DNases being the most frequently used.
SL40 contains two hydrolases: a protease, the subtilisin, and a glycosidase, the α-amylase. No information on the exact origin of these enzymes is available in the product documentation. Since their discovery in 1954, subtilisins have been used in wound care and dental hygiene [34,35], as well as in industrial applications in detergents and degreasers, especially for “cleaning-in-place” processes [36]. Subtilisins (EC 3.4.21.62) are serine protease active at 50–60 °C and pH of 8–10, under moderate ionic strength and, for some, in the presence of calcium [37,38]. They are naturally produced by Bacillus sp., and some are recognized as GRAS by the FDA and can remain active under extreme temperatures and pH conditions. Subtilisin degrades both the proteins of the biofilm matrix but also those of the membrane of sessile bacteria, weakening their physical integrity and making them as vulnerable to additional treatments [39]. Amylase, the first enzyme discovered in 1833 [40], is a metalloenzyme requiring metals (e.g., Ca2+) for molecular stability. Amylases are glycoside hydrolases and act on α-1,4-glycosidic bonds [41] and are classified as α-, β-, and γ-amylases. The two last classes led to the release of maltose and glucose, respectively, whereas α-amylases (EC 3.2.1.1) converts polysaccharides, mainly starch and glycogen, into D-glucose oligosaccharides of various lengths, including dextrins [33]. Amylases are present in human saliva and the pancreas, as well as in plants and numerous bacteria [42,43]. Important sources of α-amylases are Bacillus sp. and Aspergillus oiyzae [44]. Optimal activity ranges from 37 °C for human amylases to 50–70 °C for bacterial enzymes, with pH optima between 5 and 8 [45]. The detergent industries need alkali-stable, surfactant-stable, thermostable, and Ca2+-independent amylases [46] for applications in industries (textiles, agri-food products productions, etc.) and clinical, medicinal, and analytical chemistry [42].
Based on our significance criteria (Figure 1), the ability of SL40 and its enzymes-free basis (SL40 basis) to eliminate 24-h-old P. aeruginosa biofilms was evaluated. A principal component analysis (PCA) revealed that temperature, solution concentration, and enzyme presence were the main factors influencing efficacy. With SL40 basis, we observed a progressive decrease in residual biofilms as the temperature increased from 10 °C to 60 °C. Nevertheless, biofilm elimination was significant only at 60 °C for pH 9 solutions at 1.0% and 1.5%. Our study showed that SL40 basis had a limited intrinsic capacity to remove PAO1 biofilms. SL40 basis contains sodium ethylhexyl sulfate, a surfactant commonly used against biofilms, as well as methylchloroisothiazolinone and methylisothiazolinone, biocides active against P. aeruginosa and widely used in many cosmetics and household and disinfection products [47]. Therefore, the significant biofilm reduction observed at 60 °C with pH 9 SL40 basis solutions may be linked to the activity of these compounds and/or their degradation products, as these molecules are known to be degraded in basic environments. While pH 9 SL40 basis solutions used at 60 °C reduced biofilm biomass by −70%, the opposite effect was observed at 10 °C. Indeed, the same SL40 basis solutions “preserved” the biofilms compared to water washing, resulting in more than twice the biofilm biomass. This was also observed at 20 °C and 40 °C, notably for 0.5% solutions. This was consistent with the results obtained with tris solutions. At 10 °C, 20 °C, and 40 °C and, to a lesser extent, at 60 °C, we observed that residual biofilms after treatment with a pH 9 tris solution were 1.5–1.9 times greater than after treatments with pH 5 or pH 7 tris solutions. While the promoting effect of a basic pH on P. aeruginosa biofilm formation has been reported in the literature [48], this is, to our knowledge, the first report of a “preservative” effect of a basic solution on PAO1 biofilms. The results obtained with the tris and SL40 basis solutions therefore indicate that the initial quantity of biofilms to be treated is variable and depends on both pH and temperature. This highlights the importance of systematically standardizing data when comparing a large number of conditions.
Compared to SL40 basis, we demonstrated that SL40’s ability to reduce PAO1 biofilm biomass was due to the enzymes it contains. Previous in vitro studies have described the ability of amylases and subtilisins to prevent biofilm formation or disrupt established biofilms in bacterial species, including P. aeruginosa [33,39,41]. The PCA showed that the effectiveness of SL40 was temperature- and concentration-dependent. At 60 °C and 40 °C, the presence of enzymes led to a decrease of -57.7% in EI/n O D ¯ 595 values, with a maximum reduction of 82.4%. Our results indicated that the enzymes contained in SL40 were active from 20 °C to 60 °C, enabling a significant biofilm reduction. While non-significant at 10 °C, some enzyme activity appeared to be present in 1.5% solutions at pH 9 as the n O D ¯ 595 and EI values decreased after 5 to 15 min of incubation. It is conceivable that longer treatment durations could achieve EI/n O D ¯ 595 < 0.5, thereby meeting our significance criteria. A “concentration effect” that doubled the SL40’s effectiveness was observed only at 20 °C for pH 5 and 7 solutions when the concentration increased from 0.5% to 1.0%, with a reduction of 62.2% in EI/n O D ¯ 595. This effect was noticeable after 5 min of incubation, suggesting that the enzyme/substrate ratio was optimal at 1.0%, as the substrate quantity was relatively constant according to our data variability analysis (RSD < 15%). Our results indicated that the maximum enzymatic activity was reached at 60 °C as the lowest EI/n O D ¯ 595 values were observed, with 1.0% and 1.5% pH 9 solution corresponding, respectively, to a biofilm biomass reduction of 85% and 80% compared to tris or H2O. Although very significant, the reduction in biofilm biomass did not reach 100%, indicating that part of the initial biofilm remained. The lowest mean experimental OD595 obtained at 60 °C was 0.072, almost double the background OD595 (≈0.040).
Thus, despite important progress in biofilm control, the complete elimination of biofilms from surfaces remains a field of investigation in biofilm research, as well as the development of in situ studies of the effectiveness of anti-biofilm products.

5. Conclusions

Our in vitro study demonstrated SL40 effectiveness against 24-h-old P. aeruginosa biofilms and showed that its efficacy is linked to the presence of subtilisin and α-amylase enzymes contained in SL40. We showed that SL40’s activity depends on temperature and concentration. While enzymatic activity was detectable at 10 °C, significant biofilm biomass reduction occurred at 20 °C using 1.0% and 1.5% solutions and at 40 °C and 60 °C under any experimental condition. At 60 °C, a significant biofilm reduction was observed with SL40 basis at higher concentrations and/or longer incubation times. The enzymes cocktail was functional from pH 5 to pH 9 and from 20 °C to 60 °C. However, at 20 °C, SL40’s effectiveness depended on the concentration and/or the incubation time, and at 60 °C, the maximal biofilm biomass reduction was 85%. These findings highlight the importance of tailoring application conditions to maximize the anti-biofilm efficacy of a product and to provide optimal guidance for industrial hygiene practices.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/hygiene5030041/s1, Figure S1: Correlation between experimental conditions and the variability of the data; Figures S2–S9 (in the Excel File): Evolution of the effectiveness of SL40 basis and SL40 against Pseudomonas aeruginosa PAO1 24-h-old biofilms (zoomed images corresponding to Figure 3); Tables S1–S5 (in the Excel File)—Table S1: Raw OD595 data; Table S2: Intra-plate variability data; Table S3: Inter-plate variability data; Table S4: Normalized OD595; Table S5: Analysis of the results and guide for interpretation.

Author Contributions

Conceptualization, S.V.; methodology, S.V. and C.T.; validation, S.V. and C.T.; formal analysis, S.V.; investigation, C.L.S. and Y.F.; resources, S.V.; data curation, S.V.; writing—original draft preparation, S.V.; writing—review and editing, C.L.S., Y.F. and C.T.; visualization, S.V.; supervision, S.V. and C.T.; project administration, S.V.; funding acquisition, S.V. and C.T. All authors have read and agreed to the published version of the manuscript.

Funding

The Stockmeier France SAS (formerly Quaron SAS) Company funded the consumables and provided Baso Bionil SL40® stock solutions (with and without enzymes). All other expenses were financed from the laboratory’s own funds.

Data Availability Statement

All data used in this article are presented in the Supplementary Data Excel File.

Acknowledgments

We thank Marie-Pierre Laurent and Marc Lefeuvre (Stockmeier France SAS) for responding favorably to our request for study. We also thank the director of ENSTBB, Pr Bakalara, for giving us access to certain materials and the availability of Laurent Bonneau for setting them up.

Conflicts of Interest

This study was initiated at the request of Pr. Vilain. The Stockmeier France SAS Company funded the consumables but did not participate in the strategic or methodological choices of the experimental work, nor in the interpretation or presentation of the results. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be interpreted as a potential conflict of interests.

Abbreviations

The following abbreviations are used in this manuscript:
CVCristal Violet
EIEffectiveness Index
PCAPrincipal Component Analysis
RSDRelative Standard Deviation
SL40Baso Bionil SL40® with enzymes
SL40 basisBaso Bionil SL40® without enzymes

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Figure 1. Summary of the preparation steps of the solutions tested on Pseudomonas aeruginosa biofilms under various experimental conditions, of the exploitation of the results, and of the significance criteria used.
Figure 1. Summary of the preparation steps of the solutions tested on Pseudomonas aeruginosa biofilms under various experimental conditions, of the exploitation of the results, and of the significance criteria used.
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Figure 2. Effect of tris solutions (without SL40 or SL40 basis) on 24-h-old Pseudomonas aeruginosa biofilms. First, 50 mM tris solutions at pH 5, 7, or 9 were placed in contact with P. aeruginosa biofilms for 5, 10, or 15 min (5, 10, and 15 on the x-axis) at 10 °C (blue), 20 °C (orange), 40 °C (brown), or 60 °C (purple). The effect of H2O treatment is indicated by a red dashed line (n O D ¯ 595 = 1). Means of OD595 (n O D ¯ 595) were calculated from 24 experimental data (n = 24; bar = SD).
Figure 2. Effect of tris solutions (without SL40 or SL40 basis) on 24-h-old Pseudomonas aeruginosa biofilms. First, 50 mM tris solutions at pH 5, 7, or 9 were placed in contact with P. aeruginosa biofilms for 5, 10, or 15 min (5, 10, and 15 on the x-axis) at 10 °C (blue), 20 °C (orange), 40 °C (brown), or 60 °C (purple). The effect of H2O treatment is indicated by a red dashed line (n O D ¯ 595 = 1). Means of OD595 (n O D ¯ 595) were calculated from 24 experimental data (n = 24; bar = SD).
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Figure 3. Evolution of the effectiveness of SL40 basis (without enzymes, left panels) and SL40 (with enzymes, right panels) against Pseudomonas aeruginosa PAO1 24-h-old biofilms. Effectiveness is shown by incubation temperature (10 °C, 20 °C, 40 °C, or 60 °C) as a function of concentration (0.5%, triangle; 1.0%, circle; or 1.5%, square) and pH (5, black; 7, blue; or 9, yellow) of the SL40/SL40 basis solutions. The x-axes represent the Effectiveness Index (EI; ratio n O D ¯ 595 test/n O D ¯ 595 tris). An EI ≤ 0.5 (vertical red dotted line) indicates that the tested solution reduced biofilm biomass at least twofold compared to the tris reference (i.e., confirming biofilm removal activity). An EI > 1 (vertical blue dotted line) indicates that the biofilm biomass after treatment was greater than that observed with tris control (i.e., indicating a “biofilm-preserving” effect). The y-axes represent the n O D ¯ 595, the mean of 24 OD595 values obtained after SL40/SL40 basis treatment, normalized by the mean of 24 OD595 values obtained after H2O treatment under the same experimental condition. A value ≤ 0.5 (horizontal red dotted line) indicates a ≥two-fold biofilm biomass reduction compared to H2O (i.e., confirming biofilm removal activity), whereas a value > 1 (horizontal blue dotted line) indicates that the tested solution was less effective than a simple H2O rinse. For each condition, the triplet of points (same form/color) corresponds to incubation times of 5, 10, and 15 min, generally ordered from right to left, respectively. Significant reductions in biofilm biomass by SL40 are characterized by an EI ≤ 0.5 and an n O D ¯ 595 ≤ 0.5 (position illustrated on upper left panel, green square). All figures are available in large format in the Supplemental Data (Figures S2–S9).
Figure 3. Evolution of the effectiveness of SL40 basis (without enzymes, left panels) and SL40 (with enzymes, right panels) against Pseudomonas aeruginosa PAO1 24-h-old biofilms. Effectiveness is shown by incubation temperature (10 °C, 20 °C, 40 °C, or 60 °C) as a function of concentration (0.5%, triangle; 1.0%, circle; or 1.5%, square) and pH (5, black; 7, blue; or 9, yellow) of the SL40/SL40 basis solutions. The x-axes represent the Effectiveness Index (EI; ratio n O D ¯ 595 test/n O D ¯ 595 tris). An EI ≤ 0.5 (vertical red dotted line) indicates that the tested solution reduced biofilm biomass at least twofold compared to the tris reference (i.e., confirming biofilm removal activity). An EI > 1 (vertical blue dotted line) indicates that the biofilm biomass after treatment was greater than that observed with tris control (i.e., indicating a “biofilm-preserving” effect). The y-axes represent the n O D ¯ 595, the mean of 24 OD595 values obtained after SL40/SL40 basis treatment, normalized by the mean of 24 OD595 values obtained after H2O treatment under the same experimental condition. A value ≤ 0.5 (horizontal red dotted line) indicates a ≥two-fold biofilm biomass reduction compared to H2O (i.e., confirming biofilm removal activity), whereas a value > 1 (horizontal blue dotted line) indicates that the tested solution was less effective than a simple H2O rinse. For each condition, the triplet of points (same form/color) corresponds to incubation times of 5, 10, and 15 min, generally ordered from right to left, respectively. Significant reductions in biofilm biomass by SL40 are characterized by an EI ≤ 0.5 and an n O D ¯ 595 ≤ 0.5 (position illustrated on upper left panel, green square). All figures are available in large format in the Supplemental Data (Figures S2–S9).
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Figure 4. Principal component analysis (PCA) performed on SL40 application conditions and the significance criteria parameters. The PCA indicated that the three criteria of significance, i.e., the p-value, the Effectiveness Index (EI), and the n O D ¯ 595, were mainly correlated to the temperature (T°C), the SL40 concentration ([C]), and to the absence/presence of the enzymes. To a more moderate extent, the criteria were also correlated with the pH and, to a lesser extent, with the application duration of the SL40 solutions.
Figure 4. Principal component analysis (PCA) performed on SL40 application conditions and the significance criteria parameters. The PCA indicated that the three criteria of significance, i.e., the p-value, the Effectiveness Index (EI), and the n O D ¯ 595, were mainly correlated to the temperature (T°C), the SL40 concentration ([C]), and to the absence/presence of the enzymes. To a more moderate extent, the criteria were also correlated with the pH and, to a lesser extent, with the application duration of the SL40 solutions.
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Table 1. Effectiveness of SL40 basis (without enzymes) and SL40 (with enzymes) against Pseudomonas aeruginosa PAO1 24-h-old biofilms. The SL40/SL40 basis solutions were considered as having eliminated significant P. aeruginosa biofilms when the following three significance criteria were respected: a p-value ≤ 0.05 (green background; see Table S5 for values), an Effectiveness Index (EI) ≤ 0.5 (bold), and a normalized OD595 (n O D ¯ 595) ≤ 0.5 (bold). An EI value ≈ 1 indicated an effect similar to tris treatment, and an n O D ¯ 595 value ≈ 1 indicated an effect similar to H2O treatment. All details regarding these criteria are presented in the Section 2 (and summarized in Figure 1) and in Tables S4 and S5.
Table 1. Effectiveness of SL40 basis (without enzymes) and SL40 (with enzymes) against Pseudomonas aeruginosa PAO1 24-h-old biofilms. The SL40/SL40 basis solutions were considered as having eliminated significant P. aeruginosa biofilms when the following three significance criteria were respected: a p-value ≤ 0.05 (green background; see Table S5 for values), an Effectiveness Index (EI) ≤ 0.5 (bold), and a normalized OD595 (n O D ¯ 595) ≤ 0.5 (bold). An EI value ≈ 1 indicated an effect similar to tris treatment, and an n O D ¯ 595 value ≈ 1 indicated an effect similar to H2O treatment. All details regarding these criteria are presented in the Section 2 (and summarized in Figure 1) and in Tables S4 and S5.
Experimental Conditions EI / n O D ¯ 595 Values Obtained with
SL40 Basis (Without Enzymes)		 EI / n O D ¯ 595 Values Obtained with
SL40 (with Enzymes)
TemperaturepHTime (min)0.5%1.0%1.5%0.5%1.0%1.5%
10 °C551.21/1.321.32/1.451.32/1.441.19/1.301.23/1.341.23/1.34
101.16/1.341.24/1.431.32/1.511.13/1.301.18/1.351.10/1.27
151.07/1.291.18/1.421.20/1.451.08/1.301.10/1.331.11/1.34
751.24/1.391.08/1.201.06/1.181.10/1.230.96/1.070.86/0.96
101.14/1.461.08/1.381.07/1.381.03/1.320.92/1.180.50/0.64
151.06/1.311.13/1.401.11/1.381.03/1.281.01/1.260.60/0.75
951.25/2.091.27/2.131.18/1.971.15/1.911.07/1.780.81/1.36
101.17/2.171.19/2.211.10/2.051.10/2.040.97/1.800.65/1.21
151.05/2.021.11/2.131.07/2.051.02/1.970.87/1.670.38/0.73
20 °C551.13/1.050.97/0.901.04/0.961.07/0.990.51/0.470.41/0.38
100.98/0.990.80/0.800.89/0.890.94/0.940.34/0.340.33/0.33
151.04/1.040.86/0.860.89/0.890.88/0.870.28/0.280.29/0.29
751.07/1.220.89/1.020.86/0.980.82/0.930.31/0.350.26/0.30
101.23/1.181.05/1.010.94/0.900.96/0.920.34/0.330.36/0.34
150.89/1.110.70/0.870.73/0.920.75/0.940.28/0.350.27/0.34
951.16/1.930.91/1.530.91/1.530.51/0.850.22/0.370.24/0.40
101.26/1.891.00/1.490.86/1.300.39/0.580.25/0.380.29/0.44
151.25/1.850.81/1.190.77/1.130.27/0.400.23/0.340.27/0.39
40 °C550.78/0.730.72/0.680.77/0.720.40/0.380.33/0.310.32/0.30
100.70/0.650.58/0.530.54/0.500.31/0.290.35/0.320.36/0.33
150.59/0.590.53/0.530.55/0.550.24/0.240.24/0.250.29/0.30
751.11/0.990.89/0.800.89/0.800.37/0.330.28/0.250.30/0.27
100.91/0.930.79/0.810.80/0.820.26/0.270.28/0.290.31/0.31
150.84/0.950.71/0.810.73/0.820.22/0.250.24/0.270.26/0.29
951.01/1.730.77/1.320.63/1.090.22/0.370.17/0.290.18/0.31
100.81/1.500.62/1.140.56/1.040.22/0.410.17/0.320.18/0.33
150.75/1.340.62/1.100.62/1.110.21/0.370.17/0.300.17/0.31
60 °C551.00/0.930.78/0.730.59/0.550.42/0.400.32/0.300.32/0.30
100.95/0.850.59/0.520.51/0.460.38/0.340.34/0.300.33/0.29
150.93/0.920.64/0.630.51/0.500.43/0.430.48/0.470.48/0.47
750.92/1.110.73/0.870.73/0.870.29/0.350.19/0.230.18/0.22
100.82/1.060.73/0.950.64/0.830.19/0.250.16/0.210.16/0.20
150.77/1.060.66/0.900.48/0.660.14/0.190.14/0.200.17/0.23
950.58/0.780.27/0.360.23/0.310.18/0.240.14/0.190.15/0.20
100.46/0.670.25/0.360.23/0.330.13/0.190.16/0.230.16/0.24
150.30/0.360.22/0.270.22/0.270.17/0.210.19/0.230.21/0.25
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MDPI and ACS Style

Le Sénéchal, C.; Fautras, Y.; Tokarski, C.; Vilain, S. In Vitro Evaluation of the Effectiveness of a Commercial Enzymatic Product Against Pseudomonas aeruginosa Biofilms According to the Parameters of Use. Hygiene 2025, 5, 41. https://doi.org/10.3390/hygiene5030041

AMA Style

Le Sénéchal C, Fautras Y, Tokarski C, Vilain S. In Vitro Evaluation of the Effectiveness of a Commercial Enzymatic Product Against Pseudomonas aeruginosa Biofilms According to the Parameters of Use. Hygiene. 2025; 5(3):41. https://doi.org/10.3390/hygiene5030041

Chicago/Turabian Style

Le Sénéchal, Caroline, Yoann Fautras, Caroline Tokarski, and Sébastien Vilain. 2025. "In Vitro Evaluation of the Effectiveness of a Commercial Enzymatic Product Against Pseudomonas aeruginosa Biofilms According to the Parameters of Use" Hygiene 5, no. 3: 41. https://doi.org/10.3390/hygiene5030041

APA Style

Le Sénéchal, C., Fautras, Y., Tokarski, C., & Vilain, S. (2025). In Vitro Evaluation of the Effectiveness of a Commercial Enzymatic Product Against Pseudomonas aeruginosa Biofilms According to the Parameters of Use. Hygiene, 5(3), 41. https://doi.org/10.3390/hygiene5030041

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