The antibiofilm effects of Byotrol™ G32

ORIGINAL ARTICLE

The antibiofilm effects of ByotrolTM G32N. Govindji1, P. Wills2, M. Upton1, N. Tirelli3, S. Yeates2 and M. Webb1

1 School of Medicine, University of Manchester, Manchester, UK

2 Organic Materials Innovation Centre, School of Chemistry University of Manchester, Manchester, UK

3 School of Materials and School of Medicine, University of Manchester, Manchester, UK

Keywords

antimicrobials, biofilms, infection.

Correspondence

Michelle Webb, School of Medicine,

University of Manchester, Oxford Road,

Manchester, M13 9PL, UK.

E-mail: [email protected]

2013/1896: received 19 October 2012,

revised 17 January 2013 and accepted 20

January 2013

doi:10.1111/jam.12162

Abstract

Aim: The purpose of this study was to evaluate a commercial antimicrobial

formulation, ByotrolTM G32, as a potential coating for impeding biofilm

formation on medical devices such as urinary catheters.

Methods and Results: The antimicrobial activity of ByotrolTM G32 and its

individual constituents has been tested on planktonic and biofilm cultures of

uropathogenic bacteria. The ByotrolTM G32 formulation was superior with

MICs ranging from 3 lg ml�1 to 15 lg ml�1 for planktonic cultures and 3–20lg ml�1 for biofilms. Furthermore, ByotrolTM G32 was able to remove

established biofilms and act as an antibiofilm surface coating.

Conclusions: ByotrolTM G32 displays impressive antimicrobial activity both in

suspension and as a coating. Pretreating medical devices with ByotrolTM G32

may significantly impede biofilm formation and prolong the lifetime of the

device.

Significance and Impact of the study: Medical devices are indispensable in

health care. They are, however, a predisposing factor in infection. This research

has demonstrated that ByotrolTM G32 reduces bacterial growth and subsequent

biofilm formation. Application of ByotrolTM G32 as a medical device coating

could have a significant impact on the costs associated with device replacement

and patient morbidity and mortality.

Introduction

It is estimated that over half of all nosocomial infections

are associated with medical devices, such as artificial

heart valves, prosthetic devices, surgically implanted

devices, contact lenses, wound drainage tubes, dressings,

intrauterine contraception devices, sutures, intravenous

catheters and urinary catheters (Smith et al. 1991; Coster-

ton et al. 1999; Richards et al. 1999). Systemic and

chronic infections are a result of biofilm formation on

the surface of medical devices (Costerton et al. 1999).

Biofilms are organized multicellular communities of

bacteria attached to a surface and embedded in a protec-

tive polymer matrix. The biofilm phenotype is a ubiqui-

tous characteristic of bacteria that constitutes a protected

growth mode to facilitate survival in hostile environments

(Costerton et al. 1987, 1995, 1999). It offers increased

resistance to host defences and antimicrobials, and

consequently, biofilms are notoriously difficult to treat

and commonly manifest as chronic or recurrent infec-

tions (Patel 2005; Vuong et al. 2005; Anderson and

O’Toole 2008). The most effective method to impede

biofilm development is to avoid or reduce the initial

adhesion of bacteria to the surface. The nonspecific

attachment of bacteria to any surface is a key determi-

nant in subsequent biofilm formation; therefore, many

approaches have been adopted to prevent bacterial

attachment to surfaces of medical devices (Banerjee et al.

2011; Knetsch and Koole 2011). Currently, the most

widely used antimicrobial biomaterials are those that have

silver-modified coatings (Boswald et al. 1999; Davenport

and Keeley 2005). These are now routinely used in

wound management (Davenport and Keeley 2005; Silver

et al. 2006; Atiyeh et al. 2007), and while they can reduce

the risk of infection, they introduce a host of problems.

For example, the uptake of silver ions by bacterial cells

Journal of Applied Microbiology 114, 1285--1293 © 2013 The Society for Applied Microbiology 1285

Journal of Applied Microbiology ISSN 1364-5072

has resulted in the emergence of silver-resistant strains

(Silver 2003; Percival et al. 2005; Silver et al. 2006),

indeed silver-resistant Pseudomonas aeruginosa has been

isolated from burn patients who have been treated with

silver-coated wound dressings (Modak and Fox 1981).

Cationic compounds represent a suitable alternative as

they define a structurally diverse class of antimicrobials

(Banerjee et al. 2011). Such chemical diversity leads to a

broad spectrum of activity and different modes of action.

Furthermore, despite over a century of use, only trace

levels of cross-resistance have been observed in the clini-

cal environment (Gilbert and Moore 2005; Jaglic and

Cervinkova 2012). Antibiofilm coatings containing novel

cationic compounds could, therefore, have a significant

impact on the clinical setting.

ByotrolTM G32 is a successful commercial antimicrobial

hygiene product that is based on a novel proprietary mix-

ture of poly(hexamethylene biguanide) chloride (PHMB);

didecyldimethylammonium chloride (DDQ) and alkyl

(C12, 70%; C14, 30%) dimethyl benzyl ammonium chlo-

ride (BAC). In this study, we evaluated the in vitro

efficacy of ByotrolTM G32 for impeding biofilm formation.

Methods

Bacterial strains, media and growth conditions

The well-characterized laboratory strain Escherichia coli

K12 (XL1 blue phage) and clinical isolates of E. coli,

Klebsiella pneumoniae and Ps. aeruginosa obtained from

urinary tract infections and characterized by Vitek� 2

(BioM�erieux, Inc., Basingstoke, UK) were used in this

study. The staff at the Central Manchester Foundation

Trust, Clinical Sciences Building 2, Manchester, UK,

kindly provided the clinical isolates used in this study.

Stocks were stored at �80°C in 80% glycerol. Stock bac-

teria were cultured for 18 h on Luria–Bertani (LB) agar

plates every two weeks. Plates were stored at 4°C. Over-night cultures were prepared by inoculating LB broth

with several colonies from the working culture plates and

incubating for 18 h with shaking at 200 rev min�1. The

inoculum was standardized to 1 9 108 CFU ml�1 using

the Miles and Misra method for the determination of via-

ble cell counts. All cultures were incubated in an aerobic

atmosphere at 37°C.

ByotrolTM G32

The composition of ByotrolTM G32 (ByotrolTM Technology

Ltd., Daresbury, UK) is PHMB (poly(hexamethylene

biguanide) chloride); DDQ (didecyldimethylammonium

chloride) and BAC (alkyl (C12, 70%; C14, 30%) dimethyl

benzyl ammonium chloride).

Poly(hexamethylene biguanide) was obtained from

Arch Chemicals (Tradename-Vantocil TG) as a 20 wt%

solution in water. DDQ was obtained from Lonza

(Tradename - Lonza Bardac 2240) and received as a

40 wt% solution in water. BAC was obtained from Thor

(Tradename – Acticide BAC 50 mol l�1) and received as

a 50 wt% solution in water.

Determination of the minimum inhibitory concentration

The minimum inhibitory concentration (MIC) of

ByotrolTM G32 and its constituents was determined by

incubating increasing concentrations of each with 100 llof a 1 : 50 dilution of overnight cultures prepared in LB

in 96-well flat-bottomed nontissue culture–treated poly-

styrene microtitre plates (Greiner Bio-one Ltd., Glouces-

tershire, UK). Eight technical replicates were prepared on

each plate for each concentration of antimicrobial tested.

The microtitre plates were incubated under aerobic, static

conditions for 18 h at 37°C.Positive growth controls were prepared by inoculating

eight wells with 100 ll of 1 : 50 dilutions of bacteria and

100 ll sterile distilled water in which the antimicrobial

was prepared. Negative controls were prepared by dis-

pensing 100 ll LB broth and 100 ll sterile distilled water

into a further 8 wells. The microtitre plates were incu-

bated under aerobic, static conditions for 18 h at 37°C.After the incubation period, the optical density at

595 nm (OD595) of planktonic growth was measured to

quantify the MIC using a spectrophotometer (BMG Lab-

tech FLUOstar OPTIMA). The average optical density

(OD) from the eight negative control wells was sub-

tracted from the average OD from the eight technical

replicates of each concentration in the test wells. The

MIC is determined as the lowest concentration of antimi-

crobial that completely inhibits visual bacterial growth, or

an OD595 < 0�05.

Microtitre plate biofilm formation assay

After determining the MIC, the microtitre plate biofilm

formation assay was used according to the method by

Christensen et al. (1985). Briefly, excess media and any

planktonic cells were decanted from the microtitre plate,

and each well was washed with 200 ll sterile phosphate-

buffered saline (PBS) (Sigma-Aldrich Company Ltd.). The

plate was left in an inverted position to air dry overnight at

room temperature. Each well was stained with 150 ll 0�4%(w/v) crystal violet (Sigma-Aldrich Company Ltd.) at room

temperature for 10 min and washed with running tap

water until the excess stain was removed and the running

water appeared colourless. The plate was inverted and left

to dry overnight at room temperature. The biofilm density

1286 Journal of Applied Microbiology 114, 1285--1293 © 2013 The Society for Applied Microbiology

Preventing biofilm-related infections N. Govindji et al.

was quantified by solubilizing the ammonium crystal violet

stain with 200 ll 99�5% ethanol (Fisher Scientific UK Ltd.,

Loughborough, UK) and measuring the OD595 of solubi-

lized crystal violet in each well using a spectrophotometer

plate reader (BMG Labtech FLUOstar OPTIMA). The aver-

age OD from the eight negative control wells was sub-


replicates of each concentration in the test wells.

Bright field microscopy

Eight-well glass chamber slides (Lab-TekTM Chamber

slideTM; Nunc, Fisher Scientific, Loughborough, UK) were

used to analyse biofilms by bright field microscopy. Each

slide had six increasing concentrations of antimicrobial

agent, one in each well. The concentration of antimicrobial

agent in each well was prepared by dispensing 150 ll of theantimicrobial agent and 150 ll of a 1 : 50 dilution of inoc-

ulum prepared from an overnight culture as described pre-

viously. The remaining two wells contained a positive

growth control prepared with 150 ll inoculum and 150 llwater, and a negative control with 150 ll media and 150 llwater. Slides were prepared in triplicate. After incubation,

the slides were washed with 1 ml PBS and immediately

stained with 0�4% crystal violet for 10 min. Excess stain

was washed with running water and left to dry in an

inverted position overnight. The chambers were removed

and microscopic images were collected on an Olympus

BX51 upright microscope using a 1009/1�30 UPlanFLN

objective. Images were captured using a CoolSnap HQ

camera (Photometrics) through MetaVue Software

(Molecular Devices). Images were then processed and anal-

ysed using ImageJ (http://rsb.info.nih.gov/ij).

Minimum Biofilm Eradication Concentration assay

This assay determines the effect of ByotrolTM G32 on

biofilm cells. 200 ll of a 1 : 100 dilution of an overnight

culture was dispensed into wells of a microtitre plate.

The Minimum Biofilm Eradication Concentration

(MBEC) assay is a modification of the method described

by Ceri et al. (1999). A lid with protruding pegs (trans-

ferable solid-phase screening system, Nunc) was placed

into the inoculated wells and incubated under static con-

ditions for 18 h. The lid carrying pegs were transferred to

a microtitre plate containing 200 ll sterile PBS, shaken

to remove any nonadhered bacterial cells and then placed

in a microtitre plate containing a concentration range of

each antimicrobial, which was prepared in the same man-

ner as described for the MIC assay and microtitre plate

biofilm production assay. The plates were incubated

under static conditions for 18 h. The pegs were then

placed in 200 ll PBS, shaken briefly and immediately

placed in 200 ll fresh sterile media and incubated for a

further 18 h to allow for regrowth of viable bacterial cells

on the pegs. After incubation, the OD595 of the plates

containing any planktonic growth was read. The pegs

were washed in 200 ll PBS and allowed to dry overnight

at room temperature before placing the pegs in 200 ll0�4% crystal violet for 15 min. The pegs were washed in

running water and left to air dry overnight at room tem-

perature. The biofilm density was quantified by solubiliz-

ing the ammonium crystal violet stain with 200 ll 99�5%ethanol and measuring the OD595 of solubilized crystal

violet using a spectrophotometer plate reader. The aver-

age OD from the eight negative control wells was sub-


replicates of each concentration in the test wells.

Effect of pretreatment of glass chamber slides on biofilm

formation

The wells of a glass chamber slide were pretreated by

incubating overnight at 37°C with a 5 lg ml�1 and

1 mg ml�1 solution of ByotrolTM G32. After incubation,

excess ByotrolTM G32 was decanted and slides were

re-incubated for a further 18 h at 37°C to allow for evap-

oration of water. A 1 : 100 dilution of overnight cultures

of bacteria was prepared in LB broth, 100 ll of which

was added to pretreated slides and incubated for 8 h at

37°C. A minimum of four biological replicates were pre-

pared for each concentration. After the incubation per-

iod, the slides were washed with 1 ml PBS, stained with

250 ll crystal violet and solubilized with 300 ll ethanol.The OD595 measurements were determined as for the

microtitre plate biofilm formation assay.

Atomic force microscopy

Wells of a glass chamber slide were precoated with 300 llof a 1 mg ml�1 solution of ByotrolTM G32 overnight at

37°C. After incubation, excess ByotrolTM was decanted

and the slides were re-incubated for a further 18 h at

37°C to allow for evaporation of water.

Topographies were recorded using atomic force

microscopy (AFM) (PSIA Inc, XE100, Surrey, UK) in

noncontact mode. A commercial silicon cantilever (PSIA

Inc, 910M-NSC15) with a nominal spring constant of

about 40 N/m was used.

The glass slides with precoated ByotrolTM G32 were

secured to a metal disc using double sided tape and

installed on the AFM scanner. An area of 20 lm2 was

scanned. A scratch was made across the bottom of the

glass chamber using a 0�8 9 40 mm needle. The resulting

topography image of this scratch gives an indication of

the film thickness on the precoated glass slides.


N. Govindji et al. Preventing biofilm-related infections

Results

ByotrolTM G32 as an antimicrobial

The antimicrobial activity of the ByotrolTM G32 formula-

tion and its individual constituents was tested on a

planktonic laboratory E. coli K12 strain and clinical

strains of E. coli, Kl. pneumoniae and Ps. aeruginosa, all

isolated from patients with urinary tract infections. For

the ByotrolTM G32 formulation, antimicrobial activity was

observed against all isolates (Fig. 1) with MICs ranging

from 3 lg ml�1 for the two E. coli isolates to 15 lg ml�1

for Kl. pneumoniae and Ps. aeruginosa. A comparison of

the MICs for the individual constituents of ByotrolTM

G32, displayed in Table 1, reveals that the G32 formula-

tion outperforms the individual constituents.

Next, the amount of biofilm produced by the remain-

ing viable planktonic cells after 18 h was determined by

crystal violet staining of the adhered bacterial cells.

Figure 2 illustrates that low concentrations of ByotrolTM

G32 (3–20 lg ml�1) reduce the biofilm development of

all isolates. To confirm these results, biofilms stained with

crystal violet were visualized by bright field microscopy

(Fig. 3). In this instance, bright field microscopy of

Table 1 The minimum inhibitory concentrations of ByotrolTM G32

and its individual components; poly(hexamethylene biguanide)

chloride (PHMB), didecyldimethylammonium chloride (DDQ) and

benzyl ammonium chloride (BAC) against Escherichia coli K12 and

clinical isolates of E. coli, Klebsiella pneumoniae and Pseudomonas

aeruginosa grown for 18 h

Minimum inhibitory concentration

(lg ml�1)

ByotrolTM G32 PHMB DDQ BAC

E. coli K12 3 15 20 60

E. coli clinical isolate 3 60 30 100

Kl. pneumoniae clinical isolate 15 100 100 200

Ps. aeruginosa clinical isolate 15 200 100 400

All data represent the mean of over 24 replicates, involving three

biological replicates for each strain.

1·00·80·6

0·20·0

0·4

1·5

1·0

0·5

0·0

1·00·80·6

0·20·0

0·4

0·50·4

0·10·0

0·20·3

Control 0·3 0·6 3 5 13 20 30

Control 0·3 0·6 3 5 13 20 30

Control 0·3 0·6 3 5 13 20 30

Control 0·3 0·6 3 5 13 20 30

ByotrolTM concentration (µg ml–1)

OD

595

nm

E. coli K12

E. coli

K. pneumoniae

P. aeruginosa

Figure 1 Minimum inhibitory concentrations determining the activity

of increasing concentrations of ByotrolTM G32 on planktonic bacterial

cells grown under static conditions for 18 h. All data represent the

mean of over 24 replicates, involving three biological replicates for

each strain. Error bars indicate the standard error of the mean.

0·6

0·4

0·2

0·0

2·0

1·5

1·0

0·5

0·0

0·3

0·1

0·0

0·2

0·4

0·3

0·1

0·0

0·2

Control 0·3 0·6 3 5 15 20 30

Control 0·3 0·6 3 5 15 20 30

Control 0·3 0·6 3 5 15 20 30


Control 0·3 0·6 3 5 15 20 30

OD

595

nm

E. coli K12

E. coli

K. pneumoniae

P. aeruginosa

Figure 2 Effect of ByotrolTM G32 on biofilm development under static

conditions. Increasing concentrations of ByotrolTM G32 were incubated

with each bacterial strain at 37°C for 18 h, and the extent of biofilm

formation was determined by crystal violet staining. All data represent

the mean of over 24 replicates, involving three biological replicates

for each strain. Error bars indicate the standard error of the mean.



bacterial cells stained with crystal violet was preferable to

fluorescence microscopy, as it avoided nonspecific bind-

ing of dyes to ByotrolTM G32, which we observed for the

standard fluorescent biofilm labelling dye FilmTracerTM

SYPRO� Ruby biofilm matrix stain (InvitrogenTM, Life

Technologies Ltd., Paisley, UK), and for the Live/Dead�

stain (InvitrogenTM). Figure 3 demonstrates that the num-

ber of adherent bacteria is substantially reduced in rela-

tion to the growth control, at concentrations of ByotrolTM

G32 as low as 3 lg ml�1, confirming the ability of Byo-

trolTM G32 to impede biofilm development. Consistent

with previous results, reducing Ps. aeruginosa biofilm

development required higher concentrations of ByotrolTM

G32. Furthermore, comparison to the minimum biofilm

inhibitory concentrations for the individual constituents

displayed in Table 2 reveals that again the G32 formula-

tion outperforms any of the individual constituents in its

ability to reduce the degree of bacterial growth.

The impact that ByotrolTM G32 had on established bio-

films was also determined by performing the MBEC

assay. The results, which are presented in Fig. 4, show

that ByotrolTM G32 was effective against biofilms of E. coli

and Kl. pneumoniae; >50% of the biofilm was eradicated

at concentrations of 60 lg ml�1. Ps. aeruginosa was more

difficult to eradicate requiring 400 lg ml�1 to reduce the

biofilm mass by 50%.

ByotrolTM G32 as a biofilm reducing device coating

ByotrolTM G32 was used to coat glass surfaces by incubat-

ing a solution at 37°C and then drying. Atomic force

microscopy was used to verify the extent of coating and

access surface morphology. Figure 5(a) shows the atomic

force micrographs of a glass surface coated with 5 lg ml�1

and 1 mg ml�1 ByotrolTM G32. A scratch drawn along the

surface allowed the depth of the coatings to be measured

(Fig. 5b). At a concentration of 5 lg ml�1, the coating

depth was measured as 20 nm. At a concentration of

1 mg ml�1, the surface is evenly coated and the depth of

the coating was measured as 60 nm (Fig. 5b). Biofilm

development on the coated surfaces was determined by

crystal violet staining of a biofilm grown for 8 h. On the

surface coated with 1 mg ml�1 ByotrolTM G32, no biofilm

development was observed for the two E. coli strains and

Control

E. c

oli

E. c

oli

K. p

neum

onia

eP.

aer

ugin

osa

0·3 µg ml–1 0·6 µg ml–1 13 µg ml–13 µg ml–1 20 µg ml–16 µg ml–1


Figure 3 Bright field microscopy showing the effects of increasing concentrations of ByotrolTM G32 on biofilm formation. Bright field images

(1009 magnification) of Escherichia coli K12 and the clinical isolates of E. coli, Klebsiella pneumoniae and Pseudomonas aeruginosa grown for

18 h in the presence of increasing concentrations of ByotrolTM G32 and stained with crystal violet.

Table 2 The minimum biofilm inhibitory concentrations of ByotrolTM

G32 and its individual components; poly(hexamethylene biguanide)

chloride (PHMB), didecyldimethylammonium chloride (DDQ) and

benzyl ammonium chloride (BAC) against Escherichia coli K12 and

clinical isolates of E. coli, Klebsiella pneumoniae and Pseudomonas

aeruginosa grown for 18 h

Minimum biofilm inhibitory

concentration (lg ml�1)

ByotrolTM G32 PHMB DDQ BAC

E. coli K12 3 20 20 60

E. coli clinical isolate 5 60 30 100

Kl. pneumoniae clinical isolate 15 100 30 60

Ps. aeruginosa clinical isolate 20 400 100 400

All data represent the mean of over 24 replicates, involving three

biological replicates for each strain.



Kl. pneumoniae, and Ps. aeruginosa biofilm development

was impeded by ~60%.

Discussion

Healthcare professionals, to support the care and treat-

ment of patients, increasingly use medical devices such as

catheters, shunts, orthopaedic implants and wound dress-

ings. While medical devices offer regained structure and

function to the body, they are a persistent source of

infection (Darouiche 2001; Van and Michiels 2005). Such

infections not only present profound economic burdens

for society but infections associated with the insertion of

a medical device lead to significant levels of morbidity

and are sometimes fatal (Polonio et al. 2001). Bacterial

communities known as biofilms play a central role in

device-associated infections (Costerton 2007; Hatt and

Rather 2008). Preventing biofilm formation is key to pro-

longing the lifetime of any medical device and reducing

infection-related complications.

The ability of many conventional antimicrobials to

inhibit biofilm formation has been assessed and some

success has been found with the use of heavy metals, par-

ticularly silver. Hydrogel-coated latex catheters impreg-

nated with silver on both the outer surface and lumen of

the catheter have shown some effectiveness, however,

only in the short-term (Bologna et al. 1999; Verleyen

et al. 1999), possibly because the main challenge of the

urinary catheter is the hugely mixed population of resis-

tant micro-organisms that form a biofilm and cause

infection, including bacteria, which display heavy metal

resistance (Woods et al. 2009). Therefore, silver may not

be an ideal inhibitor in the long term, as it may select for

organisms with silver resistance. Furthermore, there is

conflicting evidence in the literature as to the efficacy of

silver in the clinical environment (Johnson et al. 2006).

Here, we have studied the antimicrobial and biofilm

inhibitory properties of a proprietary hygiene product,

ByotrolTM G32, which is a commercial formulation based

upon a mixture of poly(hexylmethylbiguanide) chloride

(PHMB), didecyldimethylammonium chloride (DDQ)

and dimethyl benzyl ammonium chloride (BAC). PHMB

is a polymeric cationic antimicrobial agent, which for

many years has been used in the domestic, food and

medical industries (Gilbert and Moore 2005; Kim et al.

2011). BAC and DDQ are quaternary ammonium com-

pounds that have also been well studied as antimicrobial

agents and used in a wide variety of settings (Ioannou

et al. 2007). Although PHMB, DDQ and BAC all display

impressive antimicrobial activity, we demonstrate that

these constituents are synergistic in the ByotrolTM G32

formulation and present a much greater degree of

antimicrobial activity.

The microtitre plate biofilm formation assay demon-

strates a correlation between the MIC results for the inhi-

bition of planktonic cells and the inhibition of biofilm

forming cells, that is, the fewer the planktonic cells, the

fewer the number of viable cells that are able to form a

biofilm. However, the results from the microtitre plate

biofilm formation assay validates the activity of ByotrolTM

G32 against planktonic cells and therefore as an inhibitor

of early biofilm formation. Ps. aeruginosa is inherently

resistant to many antimicrobial agents due to surface fac-

tors such as its outer membrane impermeability and

active drug efflux mechanisms (Drenkard 2003; Trott

et al. 2007). The MIC for the planktonic cells of Ps. aeru-

ginosa is lower than the biofilm inhibition concentration,

which may be a result of fewer planktonic cells being

killed; therefore, perhaps a greater number of cells were

able to proliferate as a biofilm.

By comparison, ByotrolTM is least effective against

Ps. aeruginosa. This may be explained by the suggestion

that biocides, which have a broad spectrum of activity

4·0

3·0

2·0

1·0

0·0Control0·3 0·6 3 5 15 60 100 200 400 1000

Control0·3 0·6 3 5 15 60 100 200 400 1000

Control0·3 0·6 3 5 15 60 100 200 400 1000

Control 0·3 0·6 3 5 15 60 100 200 400 1000

0·4

0·3

0·2

0·1

0·0

0·8

0·6

0·4

0·2

0·0

3·02·52·01·51·00·50·0

E. coli K12

E. coli

K. pneumoniae

P. aeruginosa

OD

595

nm


Figure 4 Minimum Biofilm Eradication Concentration assay to deter-

mine the biofilm eradication activity of ByotrolTM G32. The biofilms

were grown statically on a lid with protruding polystyrene pegs for

18 h and treated with ByotrolTM G32 for 18 h. The lid carrying pegs

were re-incubated in fresh media for a further 18 h. The extent of

remaining biofilm was determined by crystal violet staining. All data

represent the mean of 24 replicates, involving three biological repli-

cates for each strain. Error bars indicate the standard error of the

mean.



and nonspecific targets, may cause nonspecific resistance

mechanisms, for example in Ps. aeruginosa, the hyperex-

pression of multidrug efflux pumps (Gilbert and McBain

2003). There may also be changes to the outer mem-

brane, which reduces the permeability of the membrane

to the biocide (McDonnell and Russell 1999; Gilbert

2005). However, the higher MIC of ByotrolTM for

Ps. aeruginosa compared to that of the other organisms

tested is likely to be due to increased tolerance rather

than resistance (Gilbert and McBain 2003).

Intervention at the early stages of biofilm development

and inhibiting planktonic cells before they are able to

attach to a surface is most desirable. For this reason, the

inhibition of viable planktonic bacteria to attach to the

surface was evaluated at 18 h with the microtitre plate

biofilm formation assay and 8 h for the pretreatment of a

glass surface, as these time points reflect the early stages

of biofilm formation.

Pretreatment of a surface with ByotrolTM G32 in this

study provided the preliminary data as to the efficacy of

ByotrolTM as an antimicrobial coating. AFM clearly shows

the deposition of a nonstructured thin film over the con-

centration range for ByotrolTM G32. Glass has a negative

zeta potential at pH 7 (Gu and Li 2000); therefore, we

propose that as PHMB is a positively charged polyelectro-

lyte, it strongly adheres to the surface through opposite

charge interaction (Borkovec and Papastavrou 2008) and

serves to enhance the weaker adhesion of both BAQ and

DDQ through the formation of a composite film.

To place these findings in a clinical context, the stabil-

ity and lifetime of this coating under the flow of urine

would also be an important factor in the success of Byo-

trolTM as a catheter coating. The more stable the coating,

the longer the lifetime of the catheter. That being the

case, any long-term toxicity associated with ByotrolTM

against uroepithelial cells would also need to be assessed.

It should be noted that PHMB and BAC, the compounds

demonstrating the highest antimicrobial activity in the

ByotrolTM formulation, are already widely used in the

environmental and clinical setting, including wound care

100

0 0

nm

20µM 10

020

µ M 10

0 20µM

100 20µM

10

1009080706050403020100

10090807060504030201000 1 2 3 4

Film

dep

th (

nm)

Film

dep

th (

nm)

Line distance (µm)0 1 2 3 4

Line distance (µm)

4·0

3·5

3·0

2·5

2·0

1·5

1·0

0·5

0·0U

E. coli K12

E. coli

K. pneumoniae

P. aeruginosa

C U C U C U C

(a)

(b)

(c)

Figure 5 The effects of ByotrolTM G32-coated

glass surfaces on biofilm development. (a) A

glass surface was coated with 5 lg ml�1 and

1 mg ml�1 ByotrolTM G32 for 18 h. A scratch

was made across the surface to indicate the

depth of the coatings (b) The film depth was

determined by measuring the distance across

the scratches in three distinct areas. ( )

Line1; ( ) Line2 and ( ) Line3. (c) Biofilms

of Escherichia coli K12 (black bars), and

clinical isolates of E. coli (grey bars), Klebsiella

pneumoniae (white bars) and Pseudomonas

aeruginosa (hashed bars), were grown

statically for 8 h at 37°C on uncoated glass

surfaces (Control) and glass surfaces coated

with 5 lg ml�1 and 1 mg ml�1 ByotrolTM

G32. The extent of biofilm formation was

determined by solubilization of crystal violet

used to stain adhered cells. The absorbance

values of solubilized stain represent the

mean of six biological replicates for each

strain. Error bars indicate the standard error

of the mean.



dressings and ophthalmic solutions. In a clinical review,

PHMB was stated to have good clinical safety with no

known toxic risks (Gray et al. 2010), and even with long-

term use, BAC was stated to pose no clinical risk (Marple

et al. 2004). Therefore, although cytotoxicity testing

would need to be performed prior to a ByotrolTM-coated

catheter entering the clinical setting, it is probable that

ByotrolTM should be safe to use.

In conclusion, ByotrolTM G32 has the ability to reduce

bacterial growth as an antimicrobial in suspension, as a

coating, and is also able to disrupt an existing biofilm. It

may therefore hold great promise not only as an antimi-

crobial coating for medical devices but also as a sanitizing

agent for the removal of established biofilms.

Acknowledgements

We would like to thank Age UK for funding this research

and Byotrol Technology Ltd. for sample provision. The

microscopes used in this study were part of The Bioimag-

ing Facility at the University of Manchester, purchased

with grants from BBSRC, the Wellcome Trust and the

University of Manchester Strategic Fund.

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The antibiofilm effects of Byotrol™ G32