Mild Positive Pressure Improves the Efficacy of Benzalkonium Chloride against Staphylococcus aureus Biofilm

Abstract

Current protocols using liquid disinfectants to disinfect heat-sensitive hospital items frequently fail, as evidenced by the continued isolation of bacteria following decontamination. The contamination is, in part, due to biofilm formation. We hypothesize that mild positive pressure (PP) will disrupt this biofilm structure and improve liquid disinfectant/detergent penetration to biofilm bacteria for improved killing. Staphylococcus aureus biofilm, grown on polycarbonate coupons in the biofilm reactor under shear at 35 °C for 3 days, was treated for 10 min and 60 min with various dilutions of benzalkonium chloride without PP at 1 atmosphere (atm), and with PP at 3, 5, 7, and 10 atm. The effect on biofilm and residual bacterial viability was determined by standard plate counts, confocal laser scanning microscopy, and scanning electron microscopy. Combined use of benzalkonium chloride and PP up to 10 atm significantly increased biofilm killing up to 4.27 logs, as compared to the treatment using disinfectant alone. Microscopy results were consistent with the viability plate count results. PP improved disinfectant efficacy against bacterial biofilm. The use of mild PP is possible in many flow situations or if equipment/contaminated surfaces can be placed in a pressure chamber.

Full text

Citation: Tahir, S.; Emanuel, S.; Inglis,
D.W.; Vickery, K.; Deva, A.K.; Hu, H.
Mild Positive Pressure Improves the
Efficacy of Benzalkonium Chloride
against Staphylococcus aureus Biofilm.
Bioengineering 2022,9, 461.
https://doi.org/10.3390/
bioengineering9090461
Academic Editors: Fei Pan,
Yen-Hsun Su and Fangwei Guo
Received: 25 August 2022
Accepted: 5 September 2022
Published: 9 September 2022
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Copyright: © 2022 by the authors.
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4.0/).
bioengineering
Article
Mild Positive Pressure Improves the Efficacy of Benzalkonium
Chloride against Staphylococcus aureus Biofilm
Shamaila Tahir 1, Sarah Emanuel 1,2, David W. Inglis 2, Karen Vickery 1, Anand K. Deva 1
and Honghua Hu 1, *
1Surgical Infection Research Group, Faculty of Medicine, Health and Human Sciences, Macquarie University,
Sydney, NSW 2109, Australia
2School of Engineering, Macquarie University, Sydney, NSW 2109, Australia
*Correspondence: helen.hu@mq.edu.au
Abstract:
Current protocols using liquid disinfectants to disinfect heat-sensitive hospital items
frequently fail, as evidenced by the continued isolation of bacteria following decontamination. The
contamination is, in part, due to biofilm formation. We hypothesize that mild positive pressure (PP)
will disrupt this biofilm structure and improve liquid disinfectant/detergent penetration to biofilm
bacteria for improved killing. Staphylococcus aureus biofilm, grown on polycarbonate coupons in
the biofilm reactor under shear at 35
◦
C for 3 days, was treated for 10 min and 60 min with various
dilutions of benzalkonium chloride without PP at 1 atmosphere (atm), and with PP at 3, 5, 7, and
10 atm. The effect on biofilm and residual bacterial viability was determined by standard plate
counts, confocal laser scanning microscopy, and scanning electron microscopy. Combined use of
benzalkonium chloride and PP up to 10 atm significantly increased biofilm killing up to 4.27 logs,
as compared to the treatment using disinfectant alone. Microscopy results were consistent with the
viability plate count results. PP improved disinfectant efficacy against bacterial biofilm. The use of
mild PP is possible in many flow situations or if equipment/contaminated surfaces can be placed in
a pressure chamber.
Keywords:
disinfectant; positive pressure; benzalkonium chloride; synergy; Staphylococcus aureus; biofilm
1. Introduction
Pathogens that can form biofilms frequently cause healthcare-associated infections
(HAI) which impact patient morbidity and mortality. Transmission of pathogens and
development of HAIs is a complex interplay between healthcare workers, patients, envi-
ronmental contamination, medical devices/implants, antimicrobial regimens, and other
infection control measures. The hospital environment, equipment, and reusable medical
devices are known to be a source of HAI pathogens, including multidrug resistance or-
ganisms (MDROs) [
1
]. Adequate cleaning and disinfection are necessary to halt the cycle
of cross-contamination. Unfortunately, current cleaning and disinfection are frequently
suboptimal. Suboptimal decontamination leaves the organic matter in situ which often
leads to biofilm formation [2,3].
In one study, 9% of reusable tourniquets were found contaminated with Acinetobacter
baumannii and methicillin-resistant Staphylococcus aureus while another 32% of stethoscopes
were contaminated with MDRO. These items may serve as potential sources of pathogen
transmission [4,5].
Biofilm is a complex multilayered community of micro-colonies where bacterial
phenotype adjusts to the limited availability of oxygen and nutrients [
6
]. Protected
within the coating of exopolymeric substances (EPS), biofilm bacteria stay safe against ad-
verse environmental conditions, displaying increased tolerance to desiccation, detergents,
and disinfectants [7].
Bioengineering 2022,9, 461. https://doi.org/10.3390/bioengineering9090461 https://www.mdpi.com/journal/bioengineering

Bioengineering 2022,9, 461 2 of 11
Removing and/or killing biofilm contaminating surfaces has been a significant area of
interest for researchers due to its challenges. While detergents effectively remove planktonic
bacteria, blood, and dirt from surfaces, they are relatively ineffective at removing biofilm
and, in some cases, have no efficacy at all [
8
,
9
]. Similarly, disinfectants are very good
at killing planktonic bacteria but have been shown to have decreased efficacy against
biofilms [
10
]. The effectiveness of disinfectants is diminished even more if the biofilm
is formed on channels within endoscopes that are subjected to multiple cycles of use
and decontamination [
11
,
12
]. Similarly, biofilms formed on dry surfaces, where they are
subjected to low water availability, are also exceedingly difficult to eradicate [
13
]. We
demonstrated that some bacteria within S. aureus dry surface biofilms survived a 10 min
treatment with sodium hypochlorite at concentrations of up to 20 times that used for
hospital disinfection [
14
]. We demonstrated that survived cells lacked genetic change
and so the dry surface biofilm tolerance to chlorine was due to the biofilm lifestyle [
14
],
especially the very thick EPS of the S. aureus dry surface biofilm [
15
], creating a diffusion
barrier around the bacterial cells. We suggested that physical wiping could improve
disinfection action by dispersing the biofilm, thus decreasing the diffusion distance and
improving the exposure of biofilm cells to the disinfectant.
The
in vitro
wound model combined with topical negative pressure therapy published
by our research group [
16
,
17
] showed that negative pressure therapy compressed traditional
Pseudomonas aeruginosa and S. aureus hydrated biofilm, reducing the biofilm thickness, thus
reducing the diffusion distance for antiseptics to penetrate the biofilm, and resulted in
improved bacterial cell killing in the biofilm. Ultrahigh pressures of 100 Mpa to 800 Mpa,
equivalent to 986.9
−
7895 atmospheres (atm), have also been shown to inactivate bacteria
when used to decontaminate food prepared for storage purposes [
18
]. In the current
paper, we hypothesize that the penetration of commonly used fluid disinfectants can be
improved with the synergistic use of mildly increased atmospheric pressures to disrupt
biofilm structure for the enhanced killing of biofilm cells. Here, we tested the synergistic
action of different mild positive pressure with various dilutions of a common disinfectant
benzalkonium chloride against a S. aureus biofilm.
2. Materials and Methods
2.1. Staphylococcus aureus Biofilm Production
AS. aureus biofilm was used as a model organism to test the efficacy of using a disinfec-
tant to kill biofilm cells in the presence or absence of PP. A CDC bioreactor (CBR, BioSurface
Technologies Corp, Bozeman, MT, USA) was used to grow the inter-experimental repro-
ducible S. aureus biofilm ATCC 25923 on 24 removable polycarbonate coupons.
The biofilm was grown over 3 days using batch phase (50% (15 g/L) Tryptone Soya
Broth TSB) for 24 h, then the growth media in CBR was replaced with fresh 20% (6 g/L) TSB,
followed by a flow-through phase (20% TSB introduced into the bioreactor at a flow rate of
80 mL/h) for 48 h under shear at 130 rpm at 35
◦
C. At the end of 3 days, the biofilm-covered
coupons were gently washed in phosphate-buffered saline (PBS) three times to remove
planktonic and loosely attached bacteria.
2.2. Strategy for Proof of Concept
The purpose of the study is to demonstrate the synergy effect of positive pressure and
the disinfectant as proof of concept.
To show the synergy between the disinfectant action and the application of positive
pressure, the tested disinfectant concentration had to be lower than that required to kill the
biofilm without the addition of pressure.
The test disinfectants were benzalkonium chloride 200 g/L [Bactex
®
Concentrate
quaternary ammonium compound (QAC), pH neutral hospital-grade disinfectant (Whiteley
Cooperation, North Sydney, NSW, Australia) – normal in-use concentration is 2 g/L or a
1/100 dilution of Bactex®concentrate].

Bioengineering 2022,9, 461 3 of 11
Preliminary Titration of Disinfectants
Biofilm-coated coupons were treated with 2 mL of the prepared chemical dilutions
(n = 6/dilution) for 10 min or 60 min of contact time. On completion of the test duration,
coupons were washed three times with 3% bovine serum in PBS to remove and inactivate
residual disinfectant. Coupons were placed in 4 mL of PBS and subjected to sonication at
43 kHz for 5 min, followed by 2 min of vigorous shaking to disrupt and release bacteria
from the biofilm, followed by serial 10-fold dilutions and standard plate culture for colony
forming units (CFU) determination.
Preliminary experiments with the recommended in-use concentration of benzalko-
nium chloride QAC (2 g/L) killed all the bacterial cells within the S. aureus biofilm. At
this concentration, we were unable to demonstrate synergy between antiseptics and PP,
therefore, we needed to test a concentration of antiseptic that failed to kill all biofilm cells.
The disinfectants were serially diluted in sterile water and the concentrations of benza-
lkonium chloride tested were 4 g/L, 2 g/L, 200 mg/L, 100 mg/L, 40 mg/L,
20 mg/L
, and
10 mg/L, which were 200%, 100%, 10%, 5%, 2%, 1%, and 0.5% of the in-use concentration
(IUC), respectively.
Untreated positive control coupons (n = 24 in total) had an average of Log
10 7.16 ±0.39
CFU/coupon. Coupons treated with 200 mg/L (10% of the in-use concentration) resulted in a
4Log
10
reduction in titer, but growth was easily detected (data not shown). Therefore, to test for
synergism between disinfectants and PP, 200 mg/L was the maximum concentration tested.
2.3. Combined Positive Pressure and Disinfectant Efficacy Testing
Six biofilm-coated coupons were incubated in water and served as a positive control.
To test for synergism between benzalkonium chloride QAC and positive pressure (PP)
treatments, disinfectant dilutions just lower than the one showing the complete killing of
biofilm bacteria (sub-lethal doses) were selected. Coupons were placed in 2 mL of diluted
disinfectant or water (positive control coupons, n = 6; positive pressure coupons) for 10- or
60-min treatments, as described below.
Based on the results obtained in the preliminary titration, the benzalkonium chloride
QAC dilutions tested were 200 mg/L, 100 mg/L, 40 mg/L, and 20 mg/L, which were 10%,
5%, 2%, and 1% IUC, respectively.
(1)
Diluted test disinfectant only without PP (1 atm).
(2)
PP only, at 3, 5, 7, and 10 atm in a positive pressure chamber designed and custom
made by Dr. David Inglis (Figure 1).
(3)
Combined disinfectant and PP at 3, 5, 7, and 10 atm.
Bioengineering 2022, 9, x FOR PEER REVIEW 4 of 11
(a) (b)
Figure 1. Positive pressure (PP) chamber. (a) From outside and (b) from inside with 5 mL sterile
serum tubes with loose lids containing biofilm coated coupons for various treatments.
The positive control coupons, PP, and/or benzalkonium chloride QAC treated cou-
pons were individually placed in 2 mL of PBS and sonicated in an ultrasonic bath (Soni-
clean; Dudley Park, SA, Australia) for 10 min at 42–47 kHz, followed by a 2 min vortex.
The viability of bacterial cells was counted by standard plate culture and colony forming
units (CFU).
CFU log reduction was calculated as the CFU Log10 value of positive control minus
the CFU Log10 value in each treatment condition. Each treatment condition was tested in
triplicates in two independent experiments. The standard deviation was calculated from
the CFU Log10 value of the six replicates in each treatment condition.
2.4. Confocal Laser Scanning Microscopy
Additional polycarbonate coupons were used for confocal laser scanning microscopy
(CLSM) and scanning electron microscopy (SEM) for the positive control and each treat-
ment condition, i.e., disinfectant only, PP only, and combined disinfectant and PP at 10
atm for 10 min. Following each treatment, the coupon was washed as above with PBS and
then stained with a LIVE/DEAD BacLight bacterial viability kit (Molecular Probes, Invi-
trogen, Carlsbad, CA, USA), as per the manufacturer’s instructions. Live bacteria were
stained green and dead bacteria were stained red. Biofilm grown on the coupon was then
fixed for one hour with 4% paraformaldehyde and washed three times with PBS for 10
min each. Images were scanned for 2D and 3D imaging on a Fluoview FV1000 CLSM
(Olympus Cooperation, Shinjuku, Japan) within 24 to 48 h of staining.
The 3D Images were built with 0.2 µm optical sections and analyzed for average
thickness, biofilm mass, and percentage of viable cells using the IMARIS 7.7.2 software
(Bitplane, Zurich, Switzerland) and the ImageJ program (Java application for scientific
image processing, https://imagej.nih.gov/ij/ (accessed on 25 August 2022), U. S. National
Institutes of Health, Bethesda, Maryland, USA), as described in our previous publication
[17].
2.5. Scanning Electron Microscopy
Following CLSM imaging, the coupons were dehydrated through serial dilutions of
ethanol and hexamethyldisilazane (HMDS, Sigma, St. Louis, MO, USA) for 10 min each,
aspirated dry, and air-dried for more than 48 h. The coupons with a dehydrated biofilm
were then mounted on specimen stubs, gold coated, and examined at low and high mag-
nification using JOEL 6480LA scanning electron microscopy (JOEL, Tokyo, Japan).
Figure 1.
Positive pressure (PP) chamber. (
a
) From outside and (
b
) from inside with 5 mL sterile
serum tubes with loose lids containing biofilm coated coupons for various treatments.

Bioengineering 2022,9, 461 4 of 11
The positive control coupons, PP, and/or benzalkonium chloride QAC treated coupons
were individually placed in 2 mL of PBS and sonicated in an ultrasonic bath (Soniclean;
Dudley Park, SA, Australia) for 10 min at 42–47 kHz, followed by a 2 min vortex. The
viability of bacterial cells was counted by standard plate culture and colony forming
units (CFU).
CFU log reduction was calculated as the CFU Log
10
value of positive control minus
the CFU Log
10
value in each treatment condition. Each treatment condition was tested in
triplicates in two independent experiments. The standard deviation was calculated from
the CFU Log10 value of the six replicates in each treatment condition.
2.4. Confocal Laser Scanning Microscopy
Additional polycarbonate coupons were used for confocal laser scanning microscopy
(CLSM) and scanning electron microscopy (SEM) for the positive control and each treatment
condition, i.e., disinfectant only, PP only, and combined disinfectant and PP at 10 atm for
10 min. Following each treatment, the coupon was washed as above with PBS and then
stained with a LIVE/DEAD BacLight bacterial viability kit (Molecular Probes, Invitrogen,
Carlsbad, CA, USA), as per the manufacturer’s instructions. Live bacteria were stained
green and dead bacteria were stained red. Biofilm grown on the coupon was then fixed
for one hour with 4% paraformaldehyde and washed three times with PBS for 10 min
each. Images were scanned for 2D and 3D imaging on a Fluoview FV1000 CLSM (Olympus
Cooperation, Shinjuku, Japan) within 24 to 48 h of staining.
The 3D Images were built with 0.2
µ
m optical sections and analyzed for average thick-
ness, biofilm mass, and percentage of viable cells using the IMARIS 7.7.2 software (Bitplane,
Zurich, Switzerland) and the ImageJ program (Java application for scientific image process-
ing, https://imagej.nih.gov/ij/ (accessed on 25 August 2022), U. S. National Institutes of
Health, Bethesda, Maryland, USA), as described in our previous publication [17].
2.5. Scanning Electron Microscopy
Following CLSM imaging, the coupons were dehydrated through serial dilutions
of ethanol and hexamethyldisilazane (HMDS, Sigma, St. Louis, MO, USA) for 10 min
each, aspirated dry, and air-dried for more than 48 h. The coupons with a dehydrated
biofilm were then mounted on specimen stubs, gold coated, and examined at low and high
magnification using JOEL 6480LA scanning electron microscopy (JOEL, Tokyo, Japan).
2.6. Statistical Analysis
ANOVA (univariate analysis of variance), followed by Dunnett’s multiple compar-
isons test, was performed to check significant differences in the biofilm bacteria CFU log
reduction, biofilm thickness, and biomass of various treatment groups in comparison to the
control group using GraphPad Prism version 9.3.1 for Windows (GraphPad Software, San
Diego, CA, USA, www.graphpad.com, accessed on 25 August 2022).
3. Results
Enhanced killing of bacterial cells occurred when in-vitro grown biofilm was exposed
to PP along with chemical treatment, as compared to the treatment with test disinfectant or
PP alone.
3.1. Effect of Positive Pressure on Benzalkonium Chloride Treatment against
Staphylococcus aureus Biofilm
Treatment with 10% IUC (200 mg/L) or 5% IUC (100 mg/L) of benzalkonium chloride
only without PP (1 atm) for 10 min resulted in a 0.86
±
0.07 or 0.67
±
0.08 log
10
reduction
in biofilm CFU, respectively. Subjecting biofilm to 3, 5, 7, and 10 atm of PP for 10 min
during the 200 mg/L (10% IUC) or 100 mg/L (5% IUC) benzalkonium chloride treatment
resulted in a significant increase in the killing of the S. aureus biofilm (p< 0.001) (Figure 2a).
Applying 10 atm of PP for a 10 min treatment of 10% IUC (200 mg/L) or 5% IUC (100 mg/L)

Bioengineering 2022,9, 461 5 of 11
benzalkonium chloride resulted in a 5.13
±
0.39 or 3.69
±
0.51 log
10
reduction, respectively
(p< 0.001) (Figure 2a).
Bioengineering 2022, 9, x FOR PEER REVIEW 6 of 11
200mg/L
100 mg/L
40 mg/L
20 mg/L
0 mg/L
0
2
4
6
Benzalkonium chloride concentration
1 atm
3 atm
5 atm
7 atm
10 atm
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱
✱✱
✱✱✱
✱✱✱✱
(a)
200mg/L
100 mg/L
40 mg/L
20 mg/L
0 mg/L
0
2
4
6
8
Benzalkonium chloride concentration
1 atm
3 atm
5 atm
7 atm
10 atm
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱
✱✱
✱✱✱✱
(b)
Figure 2.
Biofilm CFU Log
10
reduction after treatment with benzalkonium chloride in various
concentrations without PP (at 1 atm) and with PP (at 3, 5, 7, and 10 atm) for 10 min (
a
) and 60 min (
b
).
*p< 0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001.

Bioengineering 2022,9, 461 6 of 11
Treatment with 10% IUC (200 mg/L) or 5% IUC (100 mg/L) of benzalkonium chloride
only without PP for 60 min resulted in a 4.69
±
0.12 or 3.88
±
0.14 log
10
reduction in
biofilm CFU, respectively. Subjecting the biofilm to 3, 5, 7, and 10 atm of PP during the
200 mg/L (10% IUC) or 100 mg/L (5% IUC) benzalkonium chloride 60 min treatment
resulted in a significant increase in the killing of the S. aureus biofilm (p< 0.001) (Figure 2b).
Applying 10 atm of PP during the 60 min treatment of 10% IUC (200 mg/L) and 5%
IUC (
100 mg/L
) benzalkonium chloride resulted in the complete killing of biofilm cells
(p< 0.001) (Figure 2b).
Even only 1% IUC (20 mg/L) and 2% IUC (40 mg/L) of benzalkonium chloride
resulted in significantly greater log reductions when combined with PP of 10 atm. However,
increasing the contact time from 10 min to 60 min did not show increased biofilm killing
(Figure 2a,b).
There was no significant reduction in CFU when S. aureus biofilm coated coupons were
treated only with PP of 3, 5, 7, and 10 atm for 10 or 60 min (p> 0.05) without antiseptics or
disinfectants (Figure 2a,b). This shows that mild PP itself does not kill the biofilm bacterial
cells, but it facilitates the process of benzalkonium chloride killing the bacterial cells inside
the biofilms.
3.2. Confocal Laser Scanning Microscopy and Scanning Electron Microscopy
Both biofilm thickness and biomass were reduced after being treated with 5% IUC
(100 mg/L) or 10% IUC (200 mg/L) benzalkonium chloride for 10 min. Applying 10 atm
of PP during the 10 min benzalkonium chloride treatment further reduced the biofilm
thickness and biomass. The addition of 10 atm of PP also enhanced the killing of bacteria
cells (Figures 3–5).
Bioengineering 2022, 9, x FOR PEER REVIEW 7 of 11
Figure 2. Biofilm CFU Log
10
reduction after treatment with benzalkonium chloride in various con-
centrations without PP (at 1 atm) and with PP (at 3, 5, 7, and 10 atm) for 10 min (a) and 60 min (b).
* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
3.2. Confocal Laser Scanning Microscopy and Scanning Electron Microscopy
Both biofilm thickness and biomass were reduced after being treated with 5% IUC
(100 mg/L) or 10% IUC (200 mg/L) benzalkonium chloride for 10 min. Applying 10 atm of
PP during the 10 min benzalkonium chloride treatment further reduced the biofilm thick-
ness and biomass. The addition of 10 atm of PP also enhanced the killing of bacteria cells
(Figures 3−5).
(a)
(b)
Figure 3. The effect of 10 min benzalkonium chloride (BC) treatment without or with 10 atm positive
pressure on S. aureus biofilm thickness (a) and biomass (b). ** p < 0.01, **** p < 0.0001.
Figure 3. Cont.

Bioengineering 2022,9, 461 7 of 11
Bioengineering 2022, 9, x FOR PEER REVIEW 7 of 11
Figure 2. Biofilm CFU Log
10
reduction after treatment with benzalkonium chloride in various con-
centrations without PP (at 1 atm) and with PP (at 3, 5, 7, and 10 atm) for 10 min (a) and 60 min (b).
* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
3.2. Confocal Laser Scanning Microscopy and Scanning Electron Microscopy
Both biofilm thickness and biomass were reduced after being treated with 5% IUC
(100 mg/L) or 10% IUC (200 mg/L) benzalkonium chloride for 10 min. Applying 10 atm of
PP during the 10 min benzalkonium chloride treatment further reduced the biofilm thick-
ness and biomass. The addition of 10 atm of PP also enhanced the killing of bacteria cells
(Figures 3−5).
(a)
(b)
Figure 3. The effect of 10 min benzalkonium chloride (BC) treatment without or with 10 atm positive
pressure on S. aureus biofilm thickness (a) and biomass (b). ** p < 0.01, **** p < 0.0001.
Figure 3.
The effect of 10 min benzalkonium chloride (BC) treatment without or with 10 atm positive
pressure on S. aureus biofilm thickness (a) and biomass (b). ** p< 0.01, **** p< 0.0001.
Bioengineering 2022, 9, x FOR PEER REVIEW 8 of 11
(a) (b) (c)
Figure 4. CLSM images of stained S. aureus biofilm with LIVE/DEAD
®
BacLight™ Bacterial Viability
Kit. Live bacteria are stained green and dead bacteria are stained red. (a) Control without any treat-
ment; (b) treated with 10% IUC (200 mg/L) of benzalkonium chloride only for 10 min; (c) treated
with 10% IUC (200 mg/L) of benzalkonium chloride + 10 atm of PP for 10 min.
(a)
(b)
(c)
Figure 5. 3D CLSM images of stained S. aureus biofilm with LIVE/DEAD
®
BacLight™ Bacterial
Viability Kit. Live bacteria are stained green and dead bacteria are stained red. (a) Control without
any treatment; (b) treated with 10% IUC (200 mg/L) of benzalkonium chloride only for 10 min; (c)
treated with 10% IUC (200 mg/L) of benzalkonium chloride + 10 atm of PP for 10 min.
SEM visually confirmed the biofilm viability results obtained by CFU and CLSM.
Fewer cocci in the biofilm can be seen after the 10 min treatment with 10% IUC (200 mg/L)
of benzalkonium chloride and 10 atm of PP (Figure 6c) when compared to 10% IUC (200
mg/L) of benzalkonium chloride only ((Figure 6b) or untreated S. aureus biofilm (Figure
6a).
Figure 4.
CLSM images of stained S. aureus biofilm with LIVE/DEAD
®
BacLight
™
Bacterial Viability
Kit. Live bacteria are stained green and dead bacteria are stained red. (
a
) Control without any
treatment; (
b
) treated with 10% IUC (200 mg/L) of benzalkonium chloride only for 10 min; (
c
) treated
with 10% IUC (200 mg/L) of benzalkonium chloride + 10 atm of PP for 10 min.
SEM visually confirmed the biofilm viability results obtained by CFU and CLSM.
Fewer cocci in the biofilm can be seen after the 10 min treatment with 10% IUC (
200 mg/L
)
of benzalkonium chloride and 10 atm of PP (Figure 6c) when compared to 10% IUC
(
200 mg/L
) of benzalkonium chloride only ((Figure 6b) or untreated S. aureus biofilm
(Figure 6a).

Bioengineering 2022,9, 461 8 of 11
Bioengineering 2022, 9, x FOR PEER REVIEW 8 of 11
(a) (b) (c)
Figure 4. CLSM images of stai ned S. aureus biofilm with LIVE/DEAD
®
BacLight™ Bacterial Viability
Kit. Live bacteria are stained green and dead bacteria are stained red. (a) Control without any treat-
ment; (b) treated with 10% IUC (200 mg/L) of benzalkonium chloride only for 10 min; (c) treated
with 10% IUC (200 mg/L) of benzalkonium chloride + 10 atm of PP for 10 min.
(a)
(b)
(c)
Figure 5. 3D CLSM images of stained S. aureus biofilm with LIVE/DEAD
®
BacLight™ Bacterial
Viability Kit. Live bacteria are stained green and dead bacteria are stained red. (a) Control without
any treatment; (b) treated with 10% IUC (200 mg/L) of benzalkonium chloride only for 10 min; (c)
treated with 10% IUC (200 mg/L) of benzalkonium chloride + 10 atm of PP for 10 min.
SEM visually confirmed the biofilm viability results obtained by CFU and CLSM.
Fewer cocci in the biofilm can be seen after the 10 min treatment with 10% IUC (200 mg/L)
of benzalkonium chloride and 10 atm of PP (Figure 6c) when compared to 10% IUC (200
mg/L) of benzalkonium chloride only ((Figure 6b) or untreated S. aureus biofilm (Figure
6a).
Figure 5.
3D CLSM images of stained S. aureus biofilm with LIVE/DEAD
®
BacLight
™
Bacterial
Viability Kit. Live bacteria are stained green and dead bacteria are stained red. (
a
) Control without
any treatment; (
b
) treated with 10% IUC (200 mg/L) of benzalkonium chloride only for 10 min;
(c) treated with 10% IUC (200 mg/L) of benzalkonium chloride + 10 atm of PP for 10 min.
Bioengineering 2022, 9, x FOR PEER REVIEW 9 of 11
(a) (b) (c)
Figure 6. Scanning electron microscopy images (magnification 1000×) showing relative biofilm on
(a) untreated control; (b) 10 min treatment with 10% IUC (200 mg/L) of benzalkonium chloride only;
(c) 10 min treatment with 10% IUC (200 mg/L) of benzalkonium chloride + 10 atm of PP.
4. Discussion
4.1. Key Findings of the Study
The current study demonstrates that synergism between benzalkonium chloride and
the application of a mild increase in pressure is possible. Benzalkonium chloride used at
10% IUC (200 mg/L) (sublethal dose) killed 4.27 logs more bacteria in the S. aureus biofilm
when 10 atm of PP was applied over 10 min. The enhanced killing of biofilm cells was
significantly seen with all treatments of 5% and 10% IUC by using 3 to 10 atm of PP for
only 10 min. Further diluting benzalkonium chloride QAC (2% and 1% IUC) showed less
of an impact of PP on bacterial killing, even upon extending the PP treatment time to 60
min. No significant clinically relevant effect on biofilm eradication was seen at lower con-
centrations of disinfectants.
4.2. How Does the Positive Pressure Improve the Disinfectant Killing of Biofilm Cells?
Bacteria are known to be some of the most resilient primitive living organisms on
earth. Inside biofilms, they are protected by thick exopolysaccharides (EPS) and can be up
to 1500 times more tolerant/resistant to antibiotics [19]. Biofilms also show increased tol-
erance to many disinfectants [10,20,21]. Previous scientific studies found that a 5-log re-
duction in CFU resulted when planktonic S. aureus was treated with 10−20 mg/L of ben-
zalkonium chloride for 5 min while a much higher dose of 2000 mg/L of benzalkonium
chloride produced a similar reduction when treating biofilm S. aureus cells. In the current
study, biofilm cell killing was enhanced by 5 logs using a sub-lethal dose (200 mg/L) of
benzalkonium chloride for 10 min when 10 atm of PP was added.
Previously, benzalkonium chloride efflux-resistant pumps have been identified in
some S. aureus strains encoded by two gene family sets. The qacA and qacB genes set
encodes for high resistance while another gene set with qacC and qacD genes induces
low-level resistance against benzalkonium chloride and ethidium bromide [22]. A whole-
genome analysis of the S. aureus strain ATCC 25923 used in this study demonstrated the
absence of these qac resistance genes [23], hence intrinsic genetic resistance to ben-
zalkonium chloride in S. aureus was ruled out. Therefore, the increased tolerance of bio-
film cells is thought to be due to the biofilm lifestyle of which the thick biofilm EPS is a
major component. EPS forms a barrier, thus hindering disinfectant diffusion into the bio-
film. Benzalkonium chloride causes cell lysis by the physical disruption and solubilization
of bacterial cell membranes and cell wall structures. However, to do this, benzalkonium
chloride and other disinfectants need to contact the bacterial cell to kill it, so if disinfect-
ants are unable to diffuse deep enough into the biofilm, cells deep in the biofilm would
remain alive.
Previously, Ngo et al. demonstrated the physical disruption of an in-vitro grown P.
aeruginosa biofilm due to compression using negative pressure wound therapy [16]. Con-
sequently, the biofilm thickness was reduced, resulting in decreased diffusion distance
Figure 6.
Scanning electron microscopy images (magnification 1000
×
) showing relative biofilm on
(
a
) untreated control; (
b
) 10 min treatment with 10% IUC (200 mg/L) of benzalkonium chloride only;
(c) 10 min treatment with 10% IUC (200 mg/L) of benzalkonium chloride + 10 atm of PP.
4. Discussion
4.1. Key Findings of the Study
The current study demonstrates that synergism between benzalkonium chloride and
the application of a mild increase in pressure is possible. Benzalkonium chloride used at
10% IUC (200 mg/L) (sublethal dose) killed 4.27 logs more bacteria in the S. aureus biofilm
when 10 atm of PP was applied over 10 min. The enhanced killing of biofilm cells was
significantly seen with all treatments of 5% and 10% IUC by using 3 to 10 atm of PP for only
10 min. Further diluting benzalkonium chloride QAC (2% and 1% IUC) showed less of an
impact of PP on bacterial killing, even upon extending the PP treatment time to 60 min. No
significant clinically relevant effect on biofilm eradication was seen at lower concentrations
of disinfectants.

Bioengineering 2022,9, 461 9 of 11
4.2. How Does the Positive Pressure Improve the Disinfectant Killing of Biofilm Cells?
Bacteria are known to be some of the most resilient primitive living organisms on
earth. Inside biofilms, they are protected by thick exopolysaccharides (EPS) and can be
up to 1500 times more tolerant/resistant to antibiotics [
19
]. Biofilms also show increased
tolerance to many disinfectants [
10
,
20
,
21
]. Previous scientific studies found that a 5-log
reduction in CFU resulted when planktonic S. aureus was treated with 10
−
20 mg/L of
benzalkonium chloride for 5 min while a much higher dose of 2000 mg/L of benzalkonium
chloride produced a similar reduction when treating biofilm S. aureus cells. In the current
study, biofilm cell killing was enhanced by 5 logs using a sub-lethal dose (200 mg/L) of
benzalkonium chloride for 10 min when 10 atm of PP was added.
Previously, benzalkonium chloride efflux-resistant pumps have been identified in some
S. aureus strains encoded by two gene family sets. The qacA and qacB genes set encodes
for high resistance while another gene set with qacC and qacD genes induces low-level
resistance against benzalkonium chloride and ethidium bromide [
22
]. A whole-genome
analysis of the S. aureus strain ATCC 25923 used in this study demonstrated the absence of
these qac resistance genes [
23
], hence intrinsic genetic resistance to benzalkonium chloride
in S. aureus was ruled out. Therefore, the increased tolerance of biofilm cells is thought
to be due to the biofilm lifestyle of which the thick biofilm EPS is a major component.
EPS forms a barrier, thus hindering disinfectant diffusion into the biofilm. Benzalkonium
chloride causes cell lysis by the physical disruption and solubilization of bacterial cell
membranes and cell wall structures. However, to do this, benzalkonium chloride and other
disinfectants need to contact the bacterial cell to kill it, so if disinfectants are unable to
diffuse deep enough into the biofilm, cells deep in the biofilm would remain alive.
Previously, Ngo et al. demonstrated the physical disruption of an in-vitro grown
P. aeruginosa biofilm due to compression using negative pressure wound therapy [
16
].
Consequently, the biofilm thickness was reduced, resulting in decreased diffusion dis-
tance and improved killing of P. aeruginosa biofilm cells with silver ions [
16
]. Synergist
inactivation of methicillin-resistant S. aureus,S. epidermidis, and P. aeruginosa biofilm cells
was also demonstrated between compression pressure and silver ions [
24
]. A similar phe-
nomenon is observed in our study after applying 10 atm of PP where more than a 55%
reduction was seen in live S. aureus biofilm thickness, while the percentage of dead cells
increased from 34% to 72% when 10 atm of PP was added to the 10 min treatment with
10% IUC of benzalkonium chloride. We strongly propose that the PP forces benzalkonium
chloride through the physically disrupted biofilm and thus contacts more bacterial cells
for the improved kill, which is demonstrated by a two-fold higher dead cell mass in the
S. aureus biofilm after combined treatment. With the further dilution of the benzalkonium
chloride disinfectant, biofilm killing was reduced, which can be explained by the reduced
availability of chemicals for disinfection. Therefore, a plateau effect was seen with 10 min
of PP treatments after diluting benzalkonium chloride (2% and 1%) owing to decreased
availability of the chemical and its sluggish diffusion.
4.3. Practical Implications and Future Directions
High positive pressure alone is currently used to decontaminate food while disin-
fectants are used to sterilize contaminated surfaces and equipment [
18
]. Some medical
equipment and certainly human tissue could not withstand the higher pressure. This
in vitro
study was looking at positive pressure as proof of concept that slightly increased
positive pressure in conjunction with antiseptics or disinfectants increased perfusion into
the biofilm and thus increased killing. Our study establishes synergism between mild
PP and disinfectants to microbial decontamination focusing on biofilm eradication. To
combat biofilm tolerance, heat-sensitive equipment, such as endoscopes, tourniquets, and
other medical equipment with hard-to-reach surfaces, can be decontaminated in a pressure
chamber using the combined mild PP and disinfectants at in-use concentrations.
Due to funding restrictions, this study has only tested benzalkonium chloride against
S. aureus biofilms. Future studies investigating the effect of mild positive pressure with

Bioengineering 2022,9, 461 10 of 11
other anti-biofilm agents against both hydrated biofilms and dry surface biofilms, and
biofilms of other bacterial species, are warranted.
Author Contributions:
Conceptualization, K.V., D.W.I., A.K.D. and H.H.; investigation, S.T. and S.E.;
data curation, S.T. and S.E.; methodology, S.T., K.V., D.W.I. and H.H.; formal analysis, S.T., K.V. and
H.H.; supervision, K.V., A.K.D. and H.H.; writing—original draft preparation, S.T., K.V. and H.H.;
writing—review and editing, all authors. All authors have read and agreed to the published version
of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Data Availability Statement: Data available on request.
Acknowledgments: The authors thank Anna Guller for her help and support in this study.
Conflicts of Interest: The authors declare no conflict of interest.
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