Abstract
Zoono GermFree24 is a quaternary ammonium compound-based hand sanitiser. Its efficacy against planktonic bacteria is well established, but efficacy against biofilms has not been tested. We investigated the antibiofilm efficacy of Zoono GermFree24 hand sanitiser and a modified formulation against biofilms of Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 27853 grown invitro using the Centers for Disease Control (CDC) biofilm reactor and 96-pin lids. Biofilms were immersed in Zoono GermFree24 or Zoono B22-1402A (modified formulation) for 5 min, 1 h, 6 h or overnight (22 ± 2 h). The antiseptic was neutralised and the bacteria remaining after treatment were cultured and quantified. Zoono GermFree24 and Zoono B22-1402A caused time-dependent reductions in viable biofilms of both species with both methods of culture and testing, with more rapid biofilm eradication observed for Zoono B22-1402A. Biofilms grown on 96-pin lids were more quickly eradicated than those grown in the CDC biofilm reactor.
Keywords: Biofilm, Pseudomonas aeruginosa, quaternary ammonium compounds, Staphylococcus aureus
Introduction
Bacterial biofilms form in many wide-ranging settings. They may be found on biological surfaces in many chronic diseases, or on inanimate surfaces including in the natural environment and in settings of food preparation and healthcare (Vahdati etal. 2022). In healthcare, the presence of bacterial biofilms in patient surroundings may increase the risk of nosocomial infections (Fallon etal. 2022). In the food industry, they give rise to spoilage and issues of food safety while in other industrial settings, biofilm formation is associated with corrosion and degradation of equipment (Vahdati etal. 2022). The biofilm phenotype has been described as one of ‘tenacious survival’. This state provides protection against host immune defences and tolerance to antibiotics and biocides at concentrations orders of magnitude greater than the minimum inhibitory concentrations for planktonic cells of the same strain (Stewart and Costerton 2001; Fallon etal. 2022). Conventional antimicrobial approaches are therefore less effective against bacteria in biofilms than planktonic forms. There is a need for ongoing investigation of novel agents with antibiofilm action to better combat the problems caused by biofilm formation across a wide range of industries (Vahdati etal. 2022).
Zoono GermFree24 (Zoono Ltd., Auckland, New Zealand) is an aqueous non-alcoholic hand sanitiser in common use. Benzalkonium chloride, a quaternary ammonium compound, is the main active ingredient. Another proprietary ingredient, containing a silicon-based polymer, is surface-binding and acts in conjunction with benzalkonium chloride (Kim etal. 2020). These compounds are bactericidal and virucidal and act by membrane disruption (Gilbert and Moore 2005; Kim etal. 2020). In addition, the proprietary silicon-based polymer binds to surfaces and kills microbes with which it subsequently comes into contact (Kim etal. 2020). Zoono GermFree24’s efficacy against bacterial biofilms has not yet been investigated.
This study aimed to determine the efficacy of two formulations of this product against biofilms of S. aureus ATCC 6538 and P. aeruginosa ATCC 27853 grown invitro, using biofilms grown in the Centers for Disease Control Biofilm Reactor (BioSurface Technologies Corporation, Bozeman, MT, USA) and on 96-pin lids (analogous to the Calgary Biofilm Device). The first formulation being the original formulation, containing 0.13% benzalkonium chloride. The second being a modified formulation, Zoono B22-1402A, with the same concentration of benzalkonium chloride and an increased concentration of a quaternary ammonium silane (QAS) bonding agent, which is a component of the proprietary ingredient. If either formulation is effective in eradicating pre-formed biofilms, they may represent novel antibiofilm agents with broad potential applications.
Materials and methods
Bacterial strains
All testing was undertaken using S. aureus ATCC 6538 and P. aeruginosa ATCC 27853. Bacterial stocks were subcultured by streaking on tryptic soy agar (TSA) (BD Bacto Tryptic Soy Broth; BD Difco Agar, Bacteriological; Becton, Dickinson and Company, Franklin Lakes, NJ, USA), incubating at 37 °C overnight, then storing at 4 °C. Broth cultures were prepared by selecting single colonies from this plate to inoculate ∼10 mL tryptic soy broth (TSB) (BD Bacto Tryptic Soy Broth; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and incubating overnight at 37 °C, 200 revolutions per minute (RPM).
CDC biofilm reactor
Biofilm growth
Testing was undertaken according to modified versions of the US Environmental Protection Agency Office of Pesticide Programs Standard Operating Procedure EPA MLB SOP MB-19 (Growing a Biofilm using the CDC Biofilm Reactor) and EPA MLB SOP MB-20 (Single Tube Method for Determining the Efficacy of Disinfectants against Bacterial Biofilm) as previously reported by our group (Lux etal. 2022; Hale etal. 2023). In brief, a sterile reactor loaded with polycarbonate coupons and filled with ∼335 mL of TSB was incubated on a hot plate at 34 °C and stirred continuously at 120 rpm (HPS RT2 Advanced; Thermo Fisher Scientific, Waltham, Massachusetts, USA). This was inoculated with 1 mL of broth culture (∼109 colony forming units (CFU) mL−1). After 24 h, a continuous flow of dilute TSB (20% of usual concentration) through the reactor was commenced using a peristaltic pump at 11.1 mL min−1 (model 9000; New Era Pump Systems, Inc., Farmingdale, NY, USA) for a further 24 h. This pump rate allowed for replacement of the volume of TSB in the reactor every half hour. The medium was then changed to 10% TSB at the same rate for a final 24 h. Aliquots of sterile 20% and 10% TSB were spread-plated on TSA and incubated at 37 °C overnight to exclude contamination. After a total of 72 h of biofilm growth, 5 L of sterile 0.9% saline was flushed through the reactor to remove loosely attached or planktonic cells associated with the coupons prior to testing.
Antiseptic testing
Coupons were removed from the reactor aseptically and individually placed in 50 mL centrifuge tubes (Corning Inc., Tewksbury, MA, USA) containing 2 mL of sterile 0.9% saline (control) Zoono GermFree24, or Zoono B22-1402A, and incubated at room temperature for 5 min, 1 h, 6 h, or overnight (22 ± 2 h). Coupons were transferred to new centrifuge tubes containing Dey/Engley Neutralizing Broth (BD Difco Dey/Engley Neutralizing Broth; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and incubated for at least 1 min to neutralise the treatment solution. Coupons were then rinsed by dipping in 0.9% saline and transferred to new 30 mL centrifuge tubes (part number 19-6635, Omni International, Inc., Kennesaw, GA, USA), each containing 5 mL 0.9% saline, for recovery of surviving bacteria
Enumeration of surviving bacteria
Cells were released into suspension by shaking the centrifuge tubes containing the coupons in a bead ruptor using a 30 mL tube carriage (2.4 m s−1 for 30 s, twice with a 10 s intermission) (Omni Bead Ruptor 24, Omni International, Inc., Kennesaw, GA, USA). Coupons still in the centrifuge tubes were then sonicated in an ultrasonic bath for 10 min at 80 kHz (ELP030H; Elma Schmidbauer GmbH, Singen, Germany), and shaken again at the same settings in the bead ruptor.
A 10-fold dilution series was performed in triplicate on the resulting suspension and 10 µL aliquots were spot-plated on TSA and incubated overnight at 37 °C. Colonies were counted and the number of viable bacteria remaining on each coupon after treatment was calculated.
In total, 11 independent experiments were performed for S. aureus and 10 independent experiments for P. aeruginosa. Two coupons from each experiment underwent biofilm retrieval and bacterial enumeration immediately after being removed from the reactor to determine the number of bacteria in the biofilms at baseline. The remaining coupons were divided between treatments and saline control. Coupons for which colony counts could not be reliably determined were excluded.
96-Pin lid biofilm model
Biofilm growth
Biofilms were grown on 96-pin lids (Nunc Immuno TSP lids, part number 445497, Thermo Fisher Scientific, Waltham, MA, USA) using a previously described method with minor modifications (Harrison etal. 2010). Briefly, selected wells of a 96-well plate (Nunc Microwell, part number 167008, Thermo Fisher Scientific, Waltham, MA, USA) were inoculated with 150 µL of overnight culture diluted to ∼107 CFU mL−1 in TSB. Four wells with 150 µL of TSB were used as sterility controls. The 96-pin lid was placed on this plate (referred to as the ‘growth plate’), immersing the pins in the inoculum, and was incubated at 37 °C for 24 h, at 150 rpm for S. aureus and 125 rpm for P. aeruginosa.
Antiseptic testing
A new 96-well plate (referred to as the ‘treatment plate’) was prepared with 200 µL of Zoono GermFree24, Zoono B22-1402A, or 0.9% saline (for growth and sterility controls) in the appropriate wells. After 24 h of growth, the pin lid was transferred to this plate and incubated at room temperature in stationary conditions for the same treatment times as the CDC biofilm reactor experiments. The lid was then transferred to a 96-well plate containing 200 µL per well of either Dey/Engley Neutralizing Broth for antiseptic-treated pins, or 0.9% saline for controls, for at least 1 min. Pins were rinsed by transferring the lid to a 96-well plate containing 200 µL of 0.9% saline per well. Finally, the lid was transferred to a 96-well plate containing 200 µL of 0.9% saline per well for cell recovery (referred to as the ‘recovery plate’).
Enumeration of surviving bacteria
The pin lid and corresponding recovery plate were placed in a polypropylene tray and sonicated at 37 kHz for 30 min, in an ultrasonic bath containing ice to prevent excessive heating. Sonication was paused at 15 min to exchange the contents of the bath with fresh water and ice. This duration was used as preliminary experiments had demonstrated greater consistency in bacterial recovery at 30 min than at shorter durations using this equipment.
A 10-fold dilution series was performed on the resulting bacterial suspension, as well as the 0.9% saline from the treatment plate wells in which growth control pins were immersed. Aliquots of 10 µL of this were spot-plated on TSA and incubated overnight at 37 °C. The number of viable bacteria remaining on each pin after treatment, and the number of viable cells present in the saline from the treatment plates, were determined by colony counting. Aliquots were also spot-plated from all treatment plate wells containing Zoono GermFree24 and Zoono B22-1402A, and all sterility controls both from the wells of the growth plate and the recovery plate.
A total of 16 assays were performed to produce two biological replicates for each species at each time point (i.e. one assay per species per time point per biological replicate). Further pin lids were used to grow six additional biofilms per biological replicate. These were rinsed then recovered immediately to determine bacterial cell counts at baseline, representative of bacterial counts on pins prior to transfer to the treatment plate. Pins for which colony counts could not be reliably determined were excluded.
Scanning electron microscopy
Additional biofilms were grown in both the CDC biofilm reactor and on pin lids to confirm biofilm growth by scanning electron microscopy (SEM). These were fixed in 2.5% glutaraldehyde, rinsed in phosphate buffered saline, dehydrated by sequential immersion in ethanol then gold sputter coated (DSR1, Nanostructured Coatings Co., Tehran, Iran) prior to imaging (TM3030Plus, Hitachi Ltd., Tokyo, Japan).
Statistics
Colony counts were log10 transformed and for data from the CDC biofilm reactor, triplicates were averaged before statistical analysis. Data were summarised as mean ± standard deviation (SD). Samples from which no viable bacteria were recovered after treatment were recorded as being reduced to the limit of detection (LOD), i.e. the lowest number of viable cells detectable after treatment. The LOD was 500 (2.7 log10) viable cells per coupon following recovery in 5 mL 0.9% saline for biofilms grown in the CDC biofilm reactor, and 20 (1.3 log10) viable cells per pin following recovery in 200 µL 0.9% saline for biofilms grown on 96-pin lids.
Distribution was assessed using normality plots and the Shapiro-Wilk test. Data were generally normally distributed in controls and samples treated with Zoono GermFree24 or Zoono B22-1402A for short durations, but became zero-weighted as viable cell counts following treatment approached the LOD. The Kruskal-Wallis test demonstrated significant differences between groups (p < 0.001), and the Pairwise-Wilcoxon test with Benjamini-Hochberg (BH) correction for multiple comparisons was performed to determine significant reductions in biofilm between control and treatment solutions. Two-way ANOVA with Tukey adjustment for pairwise comparisons was performed to assess biofilm dispersal from growth control pins into saline over time and by biological replicates during treatment for biofilms grown on 96-pin lids. Statistical analyses and data visualisation were conducted in the R software environment (v4.1.1) (R Core Team 2021) and GraphPad Prism (v9.0.2, GraphPad Software Inc., San Diego, CA, USA), respectively.
Results
Scanning electron microscopy
The presence of bacterial biofilm was confirmed by SEM for both species, in both models of biofilm growth (Figure 1).
Figure 1.
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CDC biofilm reactor
Time-dependent antibiofilm activity was observed with both Zoono GermFree24 and Zoono B22-1402A. Zoono B22-1402A yielded greater log10 reductions in viable cells than Zoono GermFree24 at each treatment duration, except 5 min against S. aureus. Both formulations had greater antibiofilm activity against biofilms of P. aeruginosa than against those of S. aureus.
Against S. aureus, Zoono GermFree24 gave only modest reductions in viable cells, with at most a 1.7 log10 reduction seen with overnight treatment (Table 1, Figure 2(A)). Against P. aeruginosa, a 1.2 log10 reduction was seen after 1 h and a 4.1 log10 reduction after 6 h. After overnight treatment, viable cells were reduced to near the LOD (Table 2, Figure 2(B)). Treatment with Zoono B22-1402A caused eradication to below the LOD by 6 h for both species.
Table 1.
Log10 colony forming units (CFUs) of Staphylococcus aureus ATCC 6538 biofilms grown in the CDC biofilm reactor recovered after treatment with Zoono GermFree24 and Zoono B22-1402A.
| Time | Treatment | Replicates | Log10 CFU recovered (mean ± SD) | Log10 CFU reduction compared to saline | p-Value (BH-corrected) |
|---|---|---|---|---|---|
| Baseline | – | 22 | 9.4 ± 0.4 | – | – |
| 5 Min | saline | 12 | 9.5 ± 0.4 | – | – |
| Zoono GermFree24 | 9 | 9.2 ± 0.2 | 0.3 | 0.3 | |
| Zoono B22-1402A | 14 | 9.2 ± 0.4 | 0.3 | 0.2 | |
| 1 Hour | saline | 12 | 9.3 ± 0.4 | – | – |
| Zoono GermFree24 | 9 | 9.0 ± 0.1 | 0.3 | 0.01 | |
| Zoono B22-1402A | 13 | 4.3 ± 1.4 | 5.0 | 0.0001 | |
| 6 Hours | saline | 12 | 9.7 ± 0.4 | – | – |
| Zoono GermFree24 | 9 | 9.1 ± 0.3 | 0.6 | 0.007 | |
| Zoono B22-1402A | 13 | 2.7 ± 0.0 | >7.0 | <0.0001 | |
| Overnight | saline | 12 | 9.6 ± 0.3 | – | – |
| Zoono GermFree24 | 9 | 7.9 ± 0.8 | 1.7 | 0.001 | |
| Zoono B22-1402A | 13 | 2.7 ± 0.0 | >6.9 | <0.0001 |
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Reductions in viable S. aureus compared to saline control are given. Samples from which no viable CFUs were recovered were recorded as being reduced to the limit of detection (2.7 log10). These results are summarised in Figure 2(A).
Figure 2.
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Table 2.
Log10 colony forming units (CFUs) of Pseudomonas aeruginosa ATCC 27853 biofilms grown in the CDC biofilm reactor recovered after treatment with Zoono GermFree24 and Zoono B22-1402A.
| Time | Treatment | Replicates | Log10 CFU recovered (mean ± SD) | Log10 CFU reduction compared to saline | p-value (BH-corrected) |
|---|---|---|---|---|---|
| Baseline | – | 20 | 9.0 ± 0.2 | – | – |
| 5 Min | saline | 11 | 9.0 ± 0.2 | – | – |
| Zoono GermFree24 | 9 | 8.8 ± 0.3 | 0.2 | 0.1 | |
| Zoono B22-1402A | 9 | 4.8 ± 1.3 | 4.2 | <0.0001 | |
| 1 Hour | saline | 11 | 8.9 ± 0.2 | – | – |
| Zoono GermFree24 | 9 | 7.7 ± 0.4 | 1.2 | <0.0001 | |
| Zoono B22-1402A | 10 | 2.8 ± 0.2 | 6.1 | 0.0002 | |
| 6 Hours | Saline | 12 | 8.9 ± 0.4 | – | – |
| Zoono GermFree24 | 9 | 4.8 ± 0.7 | 4.1 | <0.0001 | |
| Zoono B22-1402A | 13 | 2.7 ± 0.03 | 6.2 | <0.0001 | |
| Overnight | saline | 12 | 9.0 ± 0.8 | – | – |
| Zoono GermFree24 | 9 | 3.2 ± 0.7 | 5.8 | 0.0003 | |
| Zoono B22-1402A | 13 | 2.7 ± 0.0 | >6.3 | <0.0001 |
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Reductions in viable P. aeruginosa compared to saline control are given. Samples from which no viable CFUs were recovered were recorded as being reduced to the limit of detection (2.7 log10). These results are summarised in Figure 2(B).
96-Pin lid biofilm model
Effective antibiofilm activity was observed when Zoono GermFree24 and Zoono B22-1402A were tested against biofilms grown on 96-pin lids. Treatment of biofilms of S. aureus for 5 min yielded reductions of 3 log10 by Zoono GermFree24, and 3.2 log10 by Zoono B22-1402A. Both formulations eradicated S. aureus biofilms to below the LOD by 1 h (Table 3, Figure 2(C)). Biofilms of P. aeruginosa were reduced by 1 log10 by Zoono GermFree24 at 5 min and 1.6 log10 at 1 h, with near eradication at 6 h; Zoono B22-1402A gave a 2.7 log10 reduction at 5 min and eradication to below the LOD by 1 h (Table 4, Figure 2(D).
Table 3.
Log10 colony forming units (CFUs) of Staphylococcus aureus ATCC 6538 biofilms grown on 96-pin lids recovered after treatment with Zoono GermFree24 and Zoono B22-1402A.
| Time | Treatment | Replicates | Log10 CFU recovered (mean ± SD) | Log10 CFU reduction compared to saline | p-Value (BH-corrected) |
|---|---|---|---|---|---|
| Baseline | – | 12 | 5.6 ± 0.4 | – | – |
| 5 Min | saline | 8 | 4.5 ± 0.4 | – | – |
| Zoono GermFree24 | 30 | 1.5 ± 0.4 | 3.0 | <0.0001 | |
| Zoono B22-1402A | 30 | 1.3 ± 0.2 | 3.2 | <0.0001 | |
| 1 Hour | Saline | 8 | 4.3 ± 0.4 | – | – |
| Zoono GermFree24 | 32 | 1.3 ± 0.0 | >3.0 | <0.0001 | |
| Zoono B22-1402A | 32 | 1.3 ± 0.0 | >3.0 | <0.0001 | |
| 6 Hours | Saline | 8 | 3.9 ± 0.4 | – | – |
| Zoono GermFree24 | 32 | 1.3 ± 0.0 | >2.6 | <0.0001 | |
| Zoono B22-1402A | 32 | 1.3 ± 0.0 | >2.6 | <0.0001 | |
| Overnight | Saline | 8 | 2.7 ± 0.5 | – | – |
| Zoono GermFree24 | 32 | 1.3 ± 0.0 | >1.4 | <0.0001 | |
| Zoono B22-1402A | 32 | 1.3 ± 0.0 | >1.4 | <0.0001 |
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Reductions in viable S. aureus compared to saline control are given. Samples from which no viable CFUs were recovered were recorded as being reduced to the limit of detection (1.3 log10). These results are summarised in Figure 2(C).
Table 4.
Log10 colony forming units (CFUs) of Pseudomonas aeruginosa ATCC 27853 biofilms grown on 96-pin lids recovered after treatment with Zoono GermFree24 and Zoono B22-1402A.
| Time | Treatment | Replicates | Log10 CFU recovered (mean ± SD) | Log10 CFU reduction compared to saline | p-Value (BH-corrected) |
|---|---|---|---|---|---|
| Baseline | – | 12 | 7.0 ± 0.2 | – | – |
| 5 Min | Saline | 8 | 6.6 ± 0.3 | – | – |
| Zoono GermFree24 | 32 | 5.6 ± 0.5 | 1.0 | <0.0001 | |
| Zoono B22-1402A | 32 | 3.9 ± 1.0 | 2.7 | <0.0001 | |
| 1 Hour | saline | 8 | 5.9 ± 0.3 | – | – |
| Zoono GermFree24 | 32 | 4.2 ± 0.6 | 1.7 | <0.0001 | |
| Zoono B22-1402A | 32 | 1.3 ± 0.0 | 4.6 | <0.0001 | |
| 6 Hours | saline | 8 | 5.7 ± 0.3 | – | – |
| Zoono GermFree24 | 32 | 1.3 ± 0.2 | 4.4 | <0.0001 | |
| Zoono B22-1402A | 32 | 1.3 ± 0.0 | >4.4 | <0.0001 | |
| Overnight | saline | 8 | 6.1 ± 0.5 | – | – |
| Zoono GermFree24 | 32 | 1.3 ± 0.0 | >4.8 | <0.0001 | |
| Zoono B22-1402A | 32 | 1.3 ± 0.0 | >4.8 | <0.0001 |
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Reductions in viable P. aeruginosa compared to saline control are given. Samples from which no viable CFUs were recovered were recorded as being reduced to the limit of detection (1.3 log10). These results are summarised in Figure 2(D).
A reduction in viable biofilm cells on control pins with increasing durations of treatment was observed compared to baseline, most notably with S. aureus (F = 60.5 for S. aureus, F = 29.9 for P. aeruginosa [p < 0.0001], indicating a statistically significant effect of treatment time on the number of cells in biofilms on control pins for each species). Total cell counts between pins and wells remained relatively stable, suggesting that this observation may be due to dispersal of biofilm cells from control pins into saline during treatment (Figures S1 and S2). No significant difference between biological replicates was identified with either species.
No growth was observed in either Zoono GermFree24 or Zoono B22-1402A cultured on TSA after treatment of S. aureus biofilms. Growth was seen in Zoono GermFree24 used to treat P. aeruginosa for 5 min from 7 of 32 wells, with scant growth in 1 of 32 wells used to treat P. aeruginosa for 1 h. No other growth was observed. This demonstrates that reductions in viable cell counts following treatment with either formulation in this model are due to true biofilm eradication and not simply biofilm dispersal into solution.
Discussion
This is the first study to investigate the effect of Zoono GermFree24 on bacterial biofilms. Zoono GermFree24 and Zoono B22-1402A caused time-dependent reductions in viable S. aureus and P. aeruginosa biofilms grown in both the CDC biofilm reactor and on 96-pin lids. However, the effect of Zoono GermFree24 on S. aureus biofilms grown in the CDC biofilm reactor was limited. Zoono B22-1402A acted more rapidly than Zoono GermFree24 against biofilms of both species; this observation was consistent across both methods of biofilm culture, with eradication to the limit of detection within 6 h in all experiments.
Biofilms grown on 96-pin lids showed more rapid reductions in viable cell counts than those grown in the CDC biofilm reactor, in keeping with previous studies comparing the CDC biofilm reactor with other models (Buckingham-Meyer etal. 2007; Percival and Salisbury 2018). The CDC biofilm reactor generates greater shear force across the surfaces on which biofilms grow compared to other models, leading to a more densely cellular biofilm and stronger adherence to the underlying substrate (Figure 1) (Buckingham-Meyer etal. 2007). Further, cell counts were much higher in biofilms grown in the CDC biofilm reactor. This may be due to differences in the greater surface area of coupons (127 mm2 each side) compared with pins (95 mm2) upon which biofilm can form, as well as differences in growth times and conditions: biofilms are grown for 72 h with decreasing nutrient availability in the CDC biofilm reactor but only 24 h on 96-pin lids.
In biofilms grown in the CDC biofilm reactor, both formulations demonstrated more rapid reductions in viable P. aeruginosa biofilm than in S. aureus. In biofilms grown on 96-pin lids, viable S. aureus biofilm cells were reduced to below the limit of detection more rapidly than P. aeruginosa. However, S. aureus biofilms in this model contained fewer cells at baseline than biofilms of P. aeruginosa, consistent with previous data from other authors (Harrison etal. 2010, Supplementary Material) (Harrison etal. 2010). The biofilms grown in this model are therefore not equivalent between species and these results should be interpreted in the context of the results produced using the CDC biofilm reactor.
In the 96-pin lid model, biofilms of both species exhibited some degree of dispersal from the growth control pins into the saline in the wells of the treatment plates (Figures S1 and S2). S. aureus biofilms dispersed to a greater degree over time. The lower baseline cell counts observed with this species and its rate of dispersal into solution over time meant there were fewer cells remaining in the biofilm on the pins after overnight incubation. Previous protocols have recommended breaking pins off after biofilm growth and before placing the pin lid in treatment solution, equivalent to the ‘baseline’ pins in the present study (Harrison etal. 2010). This feature of S. aureus biofilms grown and treated on 96-pin lids may therefore have been previously underappreciated.
There was an apparent reduction in bacterial counts in saline controls compared to baseline even after 5 min treatment. Baseline pins were rinsed once prior to recovery, whereas control pins had been placed in three different wells of fresh saline in their course from the growth plate through treatment, neutralisation and rinse plates and onto the recovery plate. It has been previously shown that pins rinsed multiple times in this model yield lower bacterial counts than pins rinsed only once (Ali etal. 2006). In the present study, we note that total cell counts from pins and incubation solution (saline) for controls were closer to counts on the baseline pins. This apparent reduction in counts may therefore be due to a combination of the differences in rinses between controls and baseline pins, and dispersal into solution over time.
Previous studies have investigated the antibiofilm efficacy of benzalkonium chloride and organosilanes, such as QASs independently. To our knowledge, the antibiofilm efficacy of benzalkonium chloride and organosilanes in combination has not yet been studied.
Studies investigating the antibiofilm effect of benzalkonium chloride have demonstrated varying degrees of activity when this has been applied alone. One study observed 4.6 and 6 log10 reductions in S. aureus Collection de l’Institut Pasteur (CIP) 53154 and P. aeruginosa CIP A 22 respectively after 5 min exposure to 5 mmol L−1 (∼0.2%) benzalkonium chloride, using biofilms grown in Tygon tubing (Campanac etal. 2002). Another study observed eradication of P. aeruginosa ATCC 15442 biofilms grown on 96-pin lids with 5% benzalkonium chloride at 5 min, and near eradication of biofilms grown in 96-well plates with 0.5% benzalkonium chloride at 25 min (Bridier etal. 2011). A third study demonstrated eradication of P. aeruginosa ATCC 15442 biofilms grown on 96-pin lids with 0.02% benzalkonium chloride at 24 h (Harrison etal. 2008).
Studies investigating the antibiofilm activity of organosilanes including QASs are limited. One study has demonstrated inhibition of the formation of multi-species bacterial and fungal biofilms (including S. aureus) on silicone tracheo-oesophageal shunt prostheses invitro, when prostheses are coated with a QAS prior to microbial inoculation (Oosterhof etal. 2006). The majority of research on QAS antibiofilm activity has focused on the use of this antimicrobial on dental materials. One study tested compounds derived from a QAS by reaction with tetraethoxysilane, using biofilms of Streptococcus mutans ATCC 35668 and Lactobacillus acidophilus ATCC 4356 grown on dentine ex vivo. Marked reductions in biofilmwere observed in this model, though the chemical modification of the QAS makes these results difficult to compare with our study (Daood etal. 2020).
The current study has some significant limitations. Both formulations were tested against single-species biofilms of laboratory reference strains invitro. Pathogenic and environmental biofilms may contain multiple species, all with characteristics that vary from these reference strains. Future studies may overcome this limitation by testing against multi-species biofilms and testing against biofilms of clinical or environmental isolates. Further, tolerance to quaternary ammonium compounds has been described, often in association with antibiotic resistance, and future studies could include testing against such organisms. It is recognised that the presence of organic material reduces the antimicrobial efficacy of Zoono used as a surface coating on dry fomites (Aranega-Bou etal. 2023). Future antibiofilm testing may therefore also include investigation of the effect of organic material in the liquid phase. Testing of benzalkonium chloride and the QAS bonding agent separately may clarify the relative contributions of each to the antibiofilm effect observed in this study. In addition, further investigation using other methodologies such as live-dead staining with confocal laser scanning microscopy, assessment of biomass using crystal violet assays, and investigation of antibacterial mechanisms and potential post-antibiotic effects of the QAS may be helpful to further characterise the antibiofilm activity of these formulations. SEM was used to confirm the growth of biofilms in these invitro models for each species. Future studies may also use SEM to qualitatively assess changes in biofilmmorphology following treatment. This study is strengthened by the use of two distinct, well-establishedmodels of invitro antibiofilm testing. Theresults from each model may be compared andcontrasted with each other, providing a greater richness of information with which to consider their potential real-world significance.
Conclusion
We have demonstrated time-dependent reductions in viable biofilm of both S. aureus and P. aeruginosa with two different Zoono formulations. Zoono B22-1402A in particular has a rapid and potent biofilm eradicating effect. P. aeruginosa is more susceptible to these antiseptics than S. aureus. Biofilms grown on 96-pin lids are more easily eradicated than those grown in the CDC biofilm reactor.
Supplementary Material
Supplementary material.docx
GBIF_A_2467078_SM8891.docx (121.8KB, docx)
Acknowledgements
The authors are grateful to Satya Amirapu and Yohanes Nursalim (Biomedical Imaging Research Unit, Faculty of Medical and Health Sciences, University of Auckland) for assistance with scanning electron microscopy, and to Jessica McLay (Statistical Consultant, Statistical Consulting Centre, Department of Statistics, University of Auckland) for help with data analysis. We are also grateful to the Garnett Passe and Rodney Williams Memorial Foundation and the Auckland Medical Research Foundation for their generous financial support.
Funding Statement
This work was supported by The Garnett Passe and Rodney Williams Memorial Foundation under [grant numbers 2021_ASSRS_HALE, 2020_ASSF_KIM, and 2022_MCF_BISWAS]; the Auckland Medical Research Foundation under [grant number 1120008]; and Zoono Ltd.
Disclosure statement
This study was performed under contract with Zoono Ltd.
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