Adv Exp Med Biol - Advances in Microbiology, Infectious Diseases and Public Health (2022) 16: 53–67

https://doi.org/10.1007/5584_2021_645
# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021
Published online: 26 June 2021

Antibiofilm Efficacy of Polihexanide,

Octenidine and Sodium Hypochlorite/
Hypochlorous Acid Based Wound
Irrigation Solutions against
Staphylococcus aureus, Pseudomonas
aeruginosa and a Multispecies Biofilm

Anne-Marie Salisbury, Marc Mullin, Rui Chen,

and Steven L. Percival

Abstract irrigation solution and the electrolysed water

Infection and the formation of biofilms have based wound care solution demonstrated
been shown to have a significant role in potent antibiofilm efficacy against S. aureus
increased inflammation and delayed wound and to a lesser extent P. aeruginosa. Overall,
healing. Wound irrigation solutions are used less efficacy was observed in the drip flow
to debride wounds, removing cell debris bioreactor model for all 3 test solutions,
and infecting microorganisms, therefore which may be attributed to the continuous
preventing infection. The aim of this study flow of nutrients during treatment, which
was to evaluate a Polihexanide (PHMB) may have diluted or washed away the solution.
based wound irrigation solution, Octenidine The data presented also highlights the impor-
HCl based wound irrigation solution and tance of testing antibiofilm activity in a range
electrolysed water based wound care solution of biofilm models and against different bacte-
for antibiofilm efficacy against Staphylococcus rial strains to get an overall representation of
aureus, Pseudomonas aeruginosa and a multi- efficacy.
species biofilm in several models to gain a
broad understanding of ability. The PHMB Keywords
based wound irrigation solution demonstrated Antimicrobial · Biofilms · Multispecies ·
broad range antibiofilm efficacy against Polihexanide · Wound irrigation
P. aeruginosa, S. aureus and the multispecies
biofilm. The Octenidine HCl based wound
1 Introduction

A.-M. Salisbury (*) · M. Mullin · R. Chen · S. L. Percival Multiple factors relating to a patient’s underlying
5D Health Protection Group Ltd, Centre of Excellence in physiology are thought to contribute to delayed
Biofilm Science (CEBS), Liverpool, UK wound healing, including age, sex hormones,
e-mail: [email protected];
stress, diabetes and nutrition (Guo and Dipietro
[email protected]; [email protected];
[email protected]

53
54 A.-M. Salisbury et al.

2010). Infection and the formation of biofilms in Centre for Disease Control (CDC) bioreactor
wounds has also been shown to have a significant model (ASTM E2871-19) allows growth of a
role in increased inflammation and delayed biofilm in a vessel under constantly stirred
wound healing (Banu et al. 2015; Percival 2017; conditions. The biofilm is grown under high
Malone et al. 2017). Wound biofilms are formed shear conditions, which provides a more chal-
when microbial cells adhere to a surface and each lenging biofilm to kill. The drip flow bioreactor
other and secrete extracellular polymeric model (ASTM E2647-20) allows growth of a
substances (EPS), encasing themselves in a biofilm close to the air/liquid interface under
matrix (Flemming 2016). Biofilms are difficult low shear conditions. Due to the continuous
to treat as once formed they are up to 1000x flow of proteinaceous media this model is more
more tolerant to antimicrobials than their plank- representative of an exuding wound environment
tonic counterparts (Fleming et al. 2017). and provides a further challenge to the efficacy of
Staphylococcus aureus and Pseudomonas wound care products. The multispecies biofilm
aeruginosa are the most common model involves growing the biofilm on filter
microorganisms isolated from chronic wounds discs using a hydrogel as a nutrient source to
(Serra et al. 2015). Additionally, S. aureus and provide a dry environment for biofilm growth.
P. aeruginosa are both on the list of ESKAPE Multispecies biofilms are often more challenging
pathogens, the most common multidrug resistant to eradicate, as different species in a biofilm often
(MDR) bacteria causing nosocomial infections work synergistically together and can also pro-
(Esposito and De Simone 2017; Santajit and vide an environment for genetic exchange of
Indrawattana 2016). The ability of S. aureus and antimicrobial resistant genes (O’Mar et al. 2017;
P. aeruginosa to form biofilms is also well Aguila-Arcos et al. 2017). The LabTek chamber
documented (De Oliveira et al. 2016; Billings slide model involves growing the biofilm under
et al. 2013; Stewart et al. 2015). Although batch conditions similar to the MBEC method;
biofilms can be single species, they are often however, allows for a qualitative evaluation
multispecies (O’Mar et al. 2017). Formation of rather than quantitative. Qualitative evaluation is
multispecies biofilms has the potential to create a useful as it provides information on extracellular
reservoir of antimicrobial resistance genes and an polymeric substance (EPS) breakdown/biofilm
environment for genetic exchange, potentially disruption and removal that may not be identified
contributing to MDR infections (Savage et al. from quantitative evaluation.
2013; Aguila-Arcos et al. 2017; Balcazar et al. Wound irrigation solutions are used to cleanse,
2015; Molin and Tolker-Nielsen 2003; Madsen rinse and moisturise wounds, effectively
et al. 2012; Stalder and Top 2016); therefore, debriding wounds of cell debris, slough, eschar
demonstration of antibiofilm efficacy of an anti- and microorganisms and therefore reducing
microbial against P. aeruginosa and S. aureus as wound healing time and preventing biofilm for-
well as multispecies biofilms is important for a mation and infection. Addition of antimicrobial
positive clinical outcome. agents and surfactants in wound irrigation
While there is currently no defined standard, solutions has been shown to enhance wound
representative of clinical treatment of a wound healing in comparison to normal saline solution
in vitro, there are standardised models that can (Percival et al. 2017; Bellingeri et al. 2016). The
be utilised for evaluating antibiofilm efficacy of aim of this study was to evaluate the antibiofilm
wound care products. The minimum bactericidal efficacy of a 0.1% Polihexanide (PHMB) based
eradication concentration (MBEC) model wound irrigation solution (Prontosan®, B Braun
(ASTM E2799-17) involves growing the biofilm Medical), Octenidine HCl based wound irrigation
on a peg lid, under batch conditions, in a 96 well solution (Octenilin®, Schülke & Mayr GmbH)
plate. The MBEC method is high throughput and and electrolysed water (sodium hypochlorite and
allows simultaneous testing of multiple hypochlorous acid) based wound care solution
concentrations of the same test solution. The (Microdacyn 60®, Sonoma™ Pharmaceuticals)
Antibiofilm Efficacy of Polihexanide, Octenidine and Sodium Hypochlorite. . . 55

in multiple in vitro models to gain a broad under- Following growth, the biofilm was washed
standing of their antibiofilm capability. twice with phosphate buffered saline (PBS) and
stained with the LIVE/DEAD™ BacLight™ kit
(ThermoFisher Scientific, UK) by preparing a
2 Materials and Methods 2 solution of SYTO 9 and propidium iodide
fluorescent stains and adding it to the LabTek
2.1 Test Articles chamber slides. The slides were incubated in the
dark for 15 min to allow staining of the cells,
A 0.1% Polihexanide (PHMB) based wound irri- before washing the biofilms twice with PBS and
gation solution containing 0.1% Betaine and adding each test solution at 100% concentration
purified water (Prontosan®, B Braun Medical), in triplicate. PBS only was added to the untreated
Octenidine HCl based wound irrigation solution control and the slides were incubated for 24 h.
containing aqua valde purificata, Glycerin and Following incubation, each well was
Ethylhexylglycerin (Octenilin®, Schülke & visualised using an LSM 780 imaging confocal
Mayr GmbH) and electrolysed water (sodium microscope and Zeiss software at an excitation/
hypochlorite and hypochlorous acid) based emission of 480/500 nm. For each chamber,
wound care solution containing super-oxidized 16 images were taken and collated into 1 image
water and 0.022% sodium chloride (Microdacyn to show the entire surface of each chamber.
60®, Sonoma™ Pharmaceuticals). Images were processed using Image J software
A broad-spectrum neutraliser consisting of (National Institutes of Health, USA).
30 g/L Tween 80, 3 g/L Lecithin, 1 g/L L-Histi-
dine, 2 g/L L-Cysteine and 15 g/L Saponin was
used to neutralise all of the wound irrigation 2.3 Minimum Biofilm Eradication
solutions throughout this study. All reagents Concentration (MBEC) Model
were purchased from Sigma-Aldrich, UK.
The antibiofilm efficacy of the 3 wound solutions
was evaluated against a 24 h biofilm of
2.2 LabTek Chamber Slide Biofilm P. aeruginosa ATCC 15442 and S. aureus
Model ATCC 29213 following an adapted version of
ASTM standard E2799-17 ‘Standard Test
The antibiofilm efficacy of the test solutions Method for Testing Disinfectant Efficacy against
against P. aeruginosa ATCC 15442 and Pseudomonas aeruginosa Biofilm using the
S. aureus ATCC 29213 was evaluated qualita- MBEC Assay.’
tively by growing the biofilms in LabTek cham- Briefly, overnight cultures of P. aeruginosa
ber slides and staining them with LIVE/DEAD™ ATCC 15442 and S. aureus ATCC 29213 were
BacLight™ bacterial viability kit (ThermoFisher set up by inoculating TSB with a single colony
Scientific, UK). The biofilms were then treated and incubating at 37
C and 125 rpm in an orbital
with each solution for 24 h before visualising shaking incubator. The following day the over-
them using confocal laser scanning microscopy night cultures were adjusted to 1  105 CFU/mL
(CLSM). in TSB and used to inoculate a 96 well plate. A
Overnight cultures of P. aeruginosa ATCC peg lid was placed onto the 96 well plate which
15442 and S. aureus ATCC 29213 were set up was incubated at 37
C and 110 rpm for 24 h.
by inoculating Tryptone Soya broth (TSB) with a The challenge plates for each test solution
single colony and incubating it overnight at 37
C were set up by adding the solution to a new
and 125 rpm. The following day, the overnight 96 well plate and serial diluting it 1:2 in PBS. A
cultures were adjusted to 1  106 CFU/mL and neutraliser effectiveness control was set up by
added to LabTek chamber slides. The slides were adding each solution to the neutraliser at a ratio
incubated at 37
C and 125 rpm for 24 h. of 1:1. A neutraliser toxicity control was set up by
56 A.-M. Salisbury et al.

adding the neutraliser only and an untreated con- comparison growth control. The plates were
trol was set up by adding PBS only to the plate. then incubated for 24 h at room temperature.
The peg lid containing biofilm was transferred The following day, coupons were removed
to a rinse plate containing PBS before transferring from wells, added to neutraliser and sonicated
it to the challenge plate. The challenge plates on full power for 30 min. Samples were vortexed
containing P. aeruginosa were incubated at briefly, serial diluted 1:10 in PBS and plated onto
room temperature for 24 h. The challenge plates TSA. Plates were incubated at 37
C overnight
containing S. aureus were incubated at 37
C for and the following day colony counts were
24 h. enumerated.
Following 24 h treatment, the peg lids were
transferred to recovery plates containing
neutraliser and sonicated on full power (100 W)
in an Ultrawave water bath for 30 min. Samples 2.5 Drip Flow Bioreactor Model
were transferred to new 96 well plates and serial
diluted 1:10. Serial dilutions were spot plated The antibiofilm efficacy of the test solutions was
onto Tryptone Soya agar (TSA) and incubated evaluated in the drip flow bioreactor model fol-
overnight at 37
C. The following day counts lowing ASTM E2647-13 Quantification of Pseu-
were enumerated. domonas aeruginosa Biofilm Grown Using Drip
Flow Biofilm Reactor with Low Shear and
Continuous Flow.
The drip flow bioreactor was prepared by
2.4 Centers for Disease Control
adding a clean borosilicate microscope slide to
(CDC) Bioreactor Model
each channel and autoclaving at 121
C for
15 min. An overnight inoculum was set up by
The antibiofilm efficacy of the 3 wound solutions
inoculating TSB with a single colony of
was also evaluated in the CDC bioreactor model
P. aeruginosa ATCC 700888 and incubating at
against 24 h biofilms of P. aeruginosa ATCC
37
C and 125 rpm. The following day, the over-
15442 and S. aureus ATCC 29213.
night culture was adjusted to 1  108 CFU/mL
Overnight cultures of P. aeruginosa ATCC
and used to inoculate the drip flow bioreactor
15442 and S. aureus ATCC 29213 were set up
chambers. The drip flow bioreactor was incubated
by inoculating TSB with a single colony and
for 6 h in batch phase before connecting it to a
incubating at 37
C and 125 rpm. The overnight
nutrient carboy containing 270 mg/L TSB
culture of P. aeruginosa was adjusted to
operated continuously at a flow rate of 50 mL/h/
1  108 CFU/mL in TSB and used to inoculate
channel.
the CDC bioreactor. The CDC bioreactor was
After 24 h biofilm growth, sterile gauze was
then incubated at room temperature and 125 rpm
soaked in the test solutions for 30 min and added
for 24 h. The overnight culture of S. aureus was
to microscope slides in triplicate. The biofilms
pelleted by centrifugation, resuspended in TSB
were incubated in continuous phase for a further
and used to inoculate the CDC bioreactor. The
24 h. After the challenge period, each microscope
CDC bioreactor was incubated for 24 h on at
slide was scraped into 45 mL of neutraliser
37
C and 100 rpm.
washed with 5 mL neutraliser. Each sample was
Following 24 h incubation, coupons were
homogenised for 30 s before serial diluting it 1:10
washed twice in PBS and placed into 6 well
in PBS and plating it out in duplicate onto TSA.
plates. Each neat wound solution was added to
The plates were incubated overnight at 37
C and
wells in triplicate (4 mL/well). A set of untreated
the following day counts were enumerated.
coupons were also added to the wells for a
Antibiofilm Efficacy of Polihexanide, Octenidine and Sodium Hypochlorite. . . 57

2.6 Multispecies Biofilm Model untreated control and the treated biofilms
one-way ANOVA using Dunnett’s multiple com-
The antibiofilm efficacy of the 3 wound solutions parison test was carried out using Prism
was evaluated against a 24 h multispecies biofilm 7 software.
of P. aeruginosa ATCC 15442, S. aureus ATCC
29213 and E. faecalis ATCC 29212. The biofilm
was grown on filter discs using a hydrogel as a 3 Results
nutrient source.
The hydrogel was prepared by dissolving 3.1 Antibiofilm Efficacy
3-sulfopropyl acrylate potassium salt (polymer) in the LabTek Chamber Slide
in PBS and then adding PEG dissolved in PBS, Model
foetal bovine serum (FBS) and 1% 1-hydroxy
cyclohexyl phenol ketone prepared in 70% etha- The antibiofilm efficacy of the test solutions was
nol (photo-initiator) to it. The mixture was added evaluated qualitatively against 24 h biofilms of
to a 12 well plate (2 mL/well) and set by exposing S. aureus ATCC 29213 and P. aeruginosa ATCC
the hydrogel to 366 nm UV light. 15442.
Overnight cultures of P. aeruginosa ATCC All 3 test solutions demonstrated efficacy
15442, S. aureus ATCC 29213 and E. faecalis against P. aeruginosa (Fig. 1) and S. aureus
ATCC 29212 were set up by inoculating TSB (Fig. 2) biofilms, with evidence of biofilm disrup-
with a single colony and incubating at 37
C tion and removal of the biofilms being observed
and 125 rpm. Overnight cultures were adjusted in all the treated wells in comparison to the
to 1  108 CFU/mL before adding all 3 strains untreated control.
together in TSB at a final concentration of
1  106 CFU/mL. Durapore 13 mm (1 μM) mem-
brane filter discs (Merck, UK) were incubated 3.2 Antibiofilm Efficacy in the MBEC
with the culture for 2 h at 37
C and 125 rpm. Model
Following this, the filters were transferred to a
12 well plate containing the hydrogel and Following treatment of a 24 h P. aeruginosa bio-
incubated at 37
C for 24 h. film and S. aureus biofilm with the PHMB based
Following 24 h biofilm growth, the filter discs wound irrigation solution at 100% concentration,
were transferred to fresh 12 well plates and complete eradication of the biofilms were found
treated with each solution in triplicate. PBS was (Fig. 3). In comparison the untreated growth
added to the untreated control in triplicate. controls had a bacterial cell density of
Biofilms were treated for 24 h at 37
C. Following 2.84  104 CFU/mm2 ( p < 0.0001) and
24 h treatment, filter discs were transferred to 7.29  104 CFU/mm2 ( p 0.0003), respectively
neutraliser and sonicated on full power for showing a 4 log reduction in both biofilms. The
30 min. Samples were vortexed briefly, serial PHMB based wound irrigation solution also
diluted 1:10 in PBS and plated onto TSA. The demonstrated efficacy against the P. aeruginosa
plates were incubated overnight at 37
C and the biofilm at 50%, 25% and 12.5% concentration,
following day counts were enumerated. showing a 2–4 log reduction in bacterial cell
density ( p  0.0308) and at 50%, 25%, 12.5%
and 6.25% concentration against the S. aureus
biofilm, showing a 1.5–3 log reduction in bacte-
2.7 Statistical Analysis rial cell density ( p  0.0321).
Following treatment with the Octenidine HCl
Raw data was entered into Microsoft Excel and based wound irrigation solution at 100%, 50%,
average CFU/mL was calculated. To determine if 25%, 12.5%, 6.25% and 3.13% antibiofilm effi-
there was a statistical difference between the cacy was observed against a 24 h biofilm of
58 A.-M. Salisbury et al.

Fig. 1 Representative images of P. aeruginosa biofilm on treatment with the PHMB wound irrigation solution (b),
the surface of the Lab Tek chamber slide. Biofilms were the Octenidine HCl based wound irrigation solution (c)
stained with LIVE/DEAD BacLight stain. The images and the electrolysed water based wound care solution (d)
show the untreated biofilm (a) and biofilm following

P. aeruginosa, with a bacterial cell density rang- log reduction in bacterial cell density
ing from 5.83 x 103 to 1.36  104 CFU/mm2. In ( p  0.0423).
comparison the untreated growth control had a Following treatment of a 24 h P. aeruginosa
bacterial cell density of 1.66  105 CFU/mm2 biofilm with the electrolysed water based wound
showing a 1–1.5 log reduction in biofilm care solution at 100% and 50% concentration,
( p  0.0376). Following treatment with the complete eradication of the biofilm was found.
Octenidine HCl based wound irrigation solution In comparison the untreated biofilm growth
at 100% and 50% complete eradication of a control had a bacterial cell density of
24 h S. aureus biofilm was found. In comparison 4.02  104 CFU/mm2 showing a 4 log reduction
the untreated biofilm growth control had a bacte- following treatment ( p 0.0005). The electrolysed
rial cell density of 2.90  105 CFU/mm2 showing water based wound care solution at 25% and
a 5 log reduction ( p < 0.0001). At 25%, 12.5% 12.5% concentration also showed antibiofilm effi-
and 6.25% concentration, the Octenidine HCl cacy with a 3–4 log reduction ( p  0.0221). Fol-
based wound irrigation solution also lowing treatment of a 24 h S. aureus biofilm with
demonstrated antibiofilm efficacy showing a 2–5 the electrolysed water based wound care solution
Antibiofilm Efficacy of Polihexanide, Octenidine and Sodium Hypochlorite. . . 59

Fig. 2 Representative images of S. aureus biofilm on the treatment with (b), the Octenidine HCl based wound irri-
surface of the Lab Tek chamber slide. Biofilms were gation solution (c) and the electrolysed water based wound
stained with LIVE/DEAD BacLight stain. The images care solution (d)
show the untreated biofilm (a) and biofilm following

at 100% concentration, complete eradication of 3.3 Antibiofilm Efficacy in the CDC

the biofilm was found. In comparison the Bioreactor Model
untreated biofilm growth control had a bacterial
cell density of 1.98  105 CFU/mm2 showing a The antibiofilm efficacy of the 3 test solutions
5 log reduction ( p < 0.0001). The electrolysed was also evaluated in the CDC bioreactor model
water based wound care solution at 50% and 25% and all showed antibiofilm efficacy against 24 h
concentration also showed antibiofilm efficacy biofilms of P. aeruginosa ATCC 15442 (Fig. 4)
against S. aureus, with a 1 log reduction being and S. aureus ATCC 29213 (Fig. 5) to different
found following treatment ( p  0.0058). extents.
The neutraliser effectiveness and neutraliser Following 24 h growth of P. aeruginosa
toxicity controls in this model demonstrated that ATCC 15442 biofilm in the CDC bioreactor
it neutralised all 3 of the wound irrigation model, the untreated growth control had a bacte-
solutions and that it was also non-toxic to the rial cell density of 4.53  107 CFU/mL. The
bacteria. biofilm treated with the PHMB based solution,
60 A.-M. Salisbury et al.

Fig. 3 MBEC of the PHMB based wound irrigation solu- ATCC 15442 (left) and S. aureus ATCC 29213 (right).
tion (a), the Octenidine HCl based wound irrigation solu- Error bars represent the standard error of the mean. * ¼ a
tion (b) and the electrolysed water based wound care statistically significant reduction in biofilm in comparison
solution (c) against a 24 h biofilm of P. aeruginosa to the untreated control ( p  0.0423)

Octenillin solution and electrolysed water solu- 3.4 Antibiofilm Efficacy in the Drip
tion had a bacteria cell density of 3.00 x 102 CFU/ Flow Bioreactor Model
mL, 2.80  104 CFU/mL and 3.33  106 CFU/
mL, showing a 5 ( p 0.0005), 3 ( p 0.0005) and The antibiofilm efficacy of the test solutions was
1 ( p 0.0009) log reduction in biofilm compared to evaluated against a 24 h biofilm of P. aeruginosa
the untreated control, respectively. ATCC 700888 in the drip flow bioreactor model,
Following 24 h treatment of the S. aureus by growing the biofilm and applying the treat-
ATCC 29213 biofilm, the growth control had a ment with a continuous supply of nutrients
bacterial cell density of 4.93  106 CFU/mL. (Fig. 6).
Treatment with each solution resulted in complete Following 24 h treatment of the P. aeruginosa
eradication of the biofilm, showing a 6 log reduc- biofilm with the PHMB based wound irrigation
tion in bacterial cell density in comparison to the solution, a bacterial cell density of
untreated control ( p 0.0001). 2.54  106 CFU/mL was found. In comparison
Antibiofilm Efficacy of Polihexanide, Octenidine and Sodium Hypochlorite. . . 61

Fig. 4 Log10 bacterial cell density of P. aeruginosa water based wound care solution. Error bars represent the
ATCC 15442 24 h biofilm following treatment with the standard error of the mean. * ¼ a statistically significant
PHMB based wound irrigation solution, the Octenidine reduction in biofilm in comparison to the untreated control
HCl based wound irrigation solution and the electrolysed ( p  0.0009)

Fig. 5 Log10 bacterial cell density of S. aureus ATCC wound care solution. Error bars represent the standard
29213 24 h biofilm following treatment with the PHMB error of the mean. * ¼ a statistically significant reduction
based wound irrigation solution, the Octenidine HCl based in biofilm in comparison to the untreated control
wound irrigation solution and the electrolysed water based ( p 0.0001)

the untreated biofilm had a bacterial cell density wound irrigation solution and the electrolysed
of 2.15  109 CFU/mL, showing a 3 log reduc- water based wound care solution resulted in a
tion. Treatment with the Octenidine HCl based bacterial cell density of 5.40  105 CFU/mL
62 A.-M. Salisbury et al.

Fig. 6 Log10 bacterial cell density of P. aeruginosa HCl based wound irrigation solution and the electrolysed
ATCC 700888 24 h biofilm following treatment with the water based wound care solution. Error bars represent the
PHMB based wound irrigation solution, the Octenidine standard error of the mean

and 9.93  107 CFU/mL, showing a 3.5 log and 3 log reduction in biofilm in comparison to the
1.5 log reduction in comparison to the untreated untreated control ( p 0.0016).
control, respectively.

4 Discussion
3.5 Antibiofilm Efficacy
in the Multispecies Biofilm Model In this study, the antibiofilm efficacy of a PHMB
based wound irrigation solution in comparison to
The antibiofilm efficacy of the test solutions was an Octenidine HCl based wound irrigation solu-
evaluated against a 24 h multispecies biofilm of tion and the electrolysed water based wound care
P. aeruginosa, S. aureus and E. faecalis by grow- solution was evaluated in several different models
ing the biofilm on filter discs with a hydrogel as a against P. aeruginosa, S. aureus and a multispe-
nutrient supply. cies biofilm of P. aeruginosa, S. aureus and
Following 24 h treatment with the PHMB E. faecalis. The solutions were applied at 100%
based wound irrigation solution and the concentration and at diluted concentrations for a
electrolysed water based wound care solution no contact time of 24 h to represent clinical applica-
colonies were observed, showing complete eradi- tion of wound irrigation solutions when they are
cation of the biofilm ( p 0.0016; Fig. 7). In com- used longer term in comparison to short contact
parison the untreated biofilm had a bacterial cell times such at 15 min.
density of 1.07  106 CFU/mL showing a 6 log The PHMB based wound irrigation solution
reduction with the treatments. Following treat- contains the PHMB as an antimicrobial preserva-
ment with the Octenidine HCl based wound irri- tive and the surfactant Betaine. PHMB has been
gation solution a bacterial cell density of shown to have a broad spectrum of activity
3.10  103 CFU/mL was found, showing a against bacteria, yeast and fungi through cell
Antibiofilm Efficacy of Polihexanide, Octenidine and Sodium Hypochlorite. . . 63

Fig. 7 Log10 bacterial cell density of a 24 h multispecies care solution. Error bars represent the standard error of the
biofilm following treatment with the PHMB based wound mean. * ¼ a statistically significant reduction in biofilm in
irrigation solution, the Octenidine HCl based wound irri- comparison to the untreated control ( p 0.0016)
gation solution and the electrolysed water based wound

membrane disruption; although the ability to ulcers, with the PHMB based wound irrigation
selectively enter bacterial cells and bind solution has been associated with an improved
chromosomes has also been shown (Ali and clinical outcome, with wounds healing faster
Wilson 2017; Chindera et al. 2016; and more wounds completely healing
Kamaruzzaman et al. 2016; Rembe et al. 2016). (Andriessen and Eberlein 2008).
Treatment of wounds with PHMB has been Octenidine has demonstrated broad spectrum
associated with a positive clinical outcome antimicrobial activity in vitro (Alvarez-Marin
(Webster et al. 2017; To et al. 2016; Villela- et al. 2017; Assadian 2016). Additionally, follow-
Castro et al. 2018). Surfactants, such as Betaine, ing central venous catheter insertion in a double-
are widely used in wound care to aid cleaning and blind randomized controlled trial, the Octenidine
debridement of wounds (Percival et al. 2017; HCl based wound irrigation solution significantly
Bellingeri et al. 2016). The PHMB based wound reduced insertion site skin colonisation and
irrigation solution has shown the ability to break colonisation of the catheter tip (Dettenkofer
down dried human plasma, representative of et al. 2010). The Octenidine HCl based wound
wound coatings in vitro and also clean, moisten irrigation solution has also been shown to effec-
and decontaminate encrusted chronic wounds in a tively reduce MRSA colonisation in observa-
small clinical study of 10 patients (Horrocks tional studies (Krishna and Gibb 2010). The
2006; Kaehn and Eberlein 2009). In a previous electrolysed water based wound care solution
study, the PHMB based wound irrigation solution contains 0.004% sodium hypochlorite and
demonstrated antimicrobial activity against 0.004% hypochlorous acid as preservative agents.
S. aureus (Hirsch et al. 2011). Additionally, treat- The electrolysed water based wound care solution
ment of chronic wounds, such as venous leg primarily debrides wounds, decreasing infection
64 A.-M. Salisbury et al.

rates and improving wound healing. A PHMB based wound irrigation solution showed
randomised single-blind clinical control study of complete eradication of the P. aeruginosa and
patients with diabetic foot ulcers demonstrated S. aureus biofilms at 100% concentration. Com-
the electrolysed water based wound care solution plete eradication of the P. aeruginosa biofilm was
to be more efficacious in infection control, odour also found with the electrolysed water based
reduction and erythema reduction than conven- wound care solution at 50% and complete eradica-
tional disinfectants (Martinez-De Jesus et al. tion of the S. aureus biofilm was found with the
2007). However, some studies have demonstrated Octenidine HCl based wound irrigation solution at
that sodium hypochlorite/hypochlorous acid 50% and the electrolysed water based wound care
wound care solutions with low total chlorine solution at 100%. The Octenidine HCl based
have low antimicrobial and antibiofilm efficacy wound irrigation solution was less efficacious in
(Severing et al. 2019; Krasowski et al. 2021). this model against S. aureus but showed some
In this study, the antibiofilm efficacy of all activity with a 1.5 log reduction in biofilm in
3 solutions was evaluated in multiple models comparison to the untreated control. The MBEC
against S. aureus and P. aeruginosa, which are of the wound irrigation solutions has been
commonly associated with wound infections evaluated elsewhere recently using a 96-well
(Serra et al. 2015). The LabTek chamber slide plate method and 1% tetrazolium chloride staining
model involves growing the biofilm in batch method (Krasowski et al. 2021). Krasowski et al.
phase and staining the biofilm with SYTO 9 and showed the PHMB based wound irrigation solu-
propidium iodide fluorescent stains, to allow tion eradicated the S. aureus and P. aeruginosa
visualisation of the biofilm and qualitative analy- biofilms at approx. 20% and 35% concentration,
sis following treatment with test solutions. Using respectively, whilst the Octenidine solution
this method, disruption and removal of both eradicated them at approx. 10% and 40%, respec-
P. aeruginosa and S. aureus biofilms could be tively. The electrolysed water based wound care
observed following treatment with all 3 test solution did not eradicate the biofilm at up to 50%
solutions. It has been shown previously that in concentration. The differences in concentration
models such as the LabTek chamber slide model, required to fully eradicate the biofilms between
pipette-based wash steps can cause random holes the 2 studies may be as a result of differences in
and alterations within the biofilms as it is quite an the methodology, such as the sensitivity of bacte-
aggressive method (Tasse et al. 2018). To account rial detection methods and that different bacterial
for possible biofilm removal through use of the strains used.
pipette washing technique, an untreated biofilm The CDC bioreactor model involves growing a
control that was subject to the same washing and biofilm under high shear conditions and adding
staining steps was used as a comparison when the test samples in a dry environment to the bio-
visually analysing the effects of each treatment. film containing coupons. In this model, the
Additionally, each group was tested in triplicate PHMB based wound irrigation solution showed
to allow consistent results to be drawn from each the greatest antibiofilm efficacy against a
treatment group compared to the untreated group. P. aeruginosa biofilm, reducing it by 5 log in
Although these measures were taken to account comparison to the untreated control. The
for any biofilm disruption caused by the method- Octenidine HCl based wound irrigation solution
ology rather than the treatment, the limitations of and the electrolysed water based wound care
this model should be taken into consideration solution showed some antibiofilm activity with a
when reviewing the data. 3 log and 1 log reduction, respectively. All 3 test
The MBEC model involves growing the bio- solutions demonstrated greater efficacy against
film under batch conditions, so there is no flow of S. aureus in this model, with complete eradication
nutrients and allows high throughput testing so of the biofilm being found following treatment.
that multiple concentrations of test solution can The drip flow bioreactor model involves grow-
be evaluated simultaneously. In this model, the ing a P. aeruginosa biofilm close to the air/liquid
Antibiofilm Efficacy of Polihexanide, Octenidine and Sodium Hypochlorite. . . 65

interface in an environment with continuous biofilm, showing potent efficacy against these
nutrient flow under low shear conditions. The biofilms; however, less efficacy was observed
nutrient flow is continued during treatment appli- against P. aeruginosa in the CDC bioreactor
cation and is designed to represent a highly exu- model and the drip flow bioreactor model. Over-
dative wound environment. In this model, the all, less efficacy was observed in the drip flow
PHMB based wound irrigation solution and the bioreactor model for all 3 test solutions, which
Octenidine HCl based wound irrigation solution may be attributed to the continuous flow of pro-
both showed antibiofilm efficacy, reducing the teinaceous media during treatment, which may
bacterial cell density by 3 log and 3.5 log, respec- have diluted or washed away the solution.
tively. The electrolysed water based wound care The data presented in this study shows the
solution was less efficacious, showing a 1 log PHMB based wound irrigation solution to have
reduction in the P. aeruginosa biofilm. the greatest broad range antibiofilm activity
The test solutions were also evaluated against against both P. aeruginosa, S. aureus and a mul-
a multispecies biofilm model of P. aeruginosa, tispecies biofilm in comparison to the other
S. aureus and E. faecalis, as clinical studies have solutions tested. The Octenidine HCl based
shown that biofilms are often multispecies rather wound irrigation solution demonstrated potent
than single species (Alexiou et al. 2017). Addi- antibiofilm activity against S. aureus, but to a
tionally, all 3 strains are commonly associated lesser extent against P. aeruginosa and the multi-
with nosocomial infections and wound biofilms species biofilm and the electrolysed water based
(Krishna and Gibb 2010; Banu et al. 2015; Serra wound care solution demonstrated potent
et al. 2015; Obermeier et al. 2018; Faron et al. antibiofilm activity against S. aureus and the
2016). Therefore, the biofilm in this model may multispecies biofilm, but to a lesser extent against
be more representative of a wound environment. P. aeruginosa. The data presented also highlights
In this model, the PHMB based wound irrigation the importance of testing antibiofilm activity in a
solution and the electrolysed water based wound range of biofilm models and against different
care solution eradicated the multispecies biofilm bacterial strains to get an overall representation
showing potent antibiofilm efficacy. The of efficacy.
Octenidine HCl based wound irrigation solution
also demonstrated antibiofilm efficacy, with treat-
ment resulting in a 3 log reduction of the biofilm.
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