Susceptibility of MRSA biofilms to denture-cleansing agents

R E S E A R C H L E T T E R

SusceptibilityofMRSAbio¢lms todenture-cleansingagentsDiana Lee1, Julie Howlett1, Jonathan Pratten1, Nicola Mordan1, Ailbhe McDonald1, Michael Wilson1 &Derren Ready2

1UCL Eastman Dental Institute, London, UK; and 2Microbial Diseases, Eastman Dental Hospital, UCLH NHS Foundation Trust, London, UK

Correspondence: D. Ready, Microbial

Diseases, Eastman Dental Hospital, UCLH

NHS Foundation Trust, 256 Gray’s Inn Road,

London WC1X 8LD, UK. Tel.: 144 20 7915

1050; fax: 144 20 7915 1127; e-mail:

[email protected]

Received 18 July 2008; accepted 21 November

2008.

First published online 22 December 2008.

DOI:10.1111/j.1574-6968.2008.01463.x

Editor: William Wade

Keywords

MRSA; denture; disinfection; oral cavity;

biofilm; antimicrobial susceptibility.

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial

pathogen, which is responsible for considerable morbidity and mortality in

the United Kingdom. The major reservoir of this organism is thought to be the

anterior nares, but there is increasing evidence that this pathogen is present in the

oral cavity, particularly in denture wearers. The purpose of this study was to

determine whether MRSA, grown as biofilms on denture acrylic resin, could be

eradicated using commercially available agents. EMRSA-15 or EMRSA-16 was

grown in a model system on the surface of denture acrylic resin for 4, 24 or 120 h

before the samples were exposed to a range of disinfectants for time intervals of 1, 5

and 10 min. All of the agents reduced the number of cultivable MRSA bacteria

present on the acrylic resin surface at 4 h, with 2% sodium hypochlorite (NaOCl)

eliminating MRSA below the level of detection after an exposure of 1 min.

However, the established MRSA biofilms (24 and 120 h) were more resistant to

killing by the agents, although 2% NaOCl was still able to eradicate all ages of

MRSA biofilms within 1 min of exposure.

Introduction

Staphylococcus aureus, particularly methicillin-resistant

strains of the organism (MRSA), are important nosocomial

pathogens. MRSA infection occurs mainly in people aged

65 or older (Maudsley et al., 2004) and poses a serious

challenge to healthcare providers. Although the importance

of methicillin-sensitive S. aureus (MSSA) and MRSA as

pathogens has long been recognized, the significance of their

presence within the oral cavity has only been highlighted in

the literature in the last decade (Rossi et al., 1997; Smith

et al., 2001, 2003a, b; Maeda et al., 2007). A number of oral

infections are caused at least in part by S. aureus, for

example, angular cheilitis, parotitis and staphylococcal mu-

cositis (Smith et al., 2003a, b), and it has been confirmed

that MSSA and MRSA can be isolated from the oral cavity

(Smith et al., 2001, 2003a, b). Furthermore, there has been

an increase in the number of reports suggesting that

S. aureus from the oral cavity can cause infection at distant

sites (Smith et al., 2001, 2003a, b).

The major reservoir of S. aureus is thought to be the anterior

nares, but there is increasing evidence that the organism is

present in the oral cavity, particularly in denture wearers

(Ayliffe et al., 1998; Smith et al., 2001). Staphylococcus aureus

has been isolated from 24% to 36% of healthy oral cavities,

but the incidence has been reported to rise to 48% in denture-

wearing patients (Dahlen et al., 1982; Ohman et al., 1995). In

one UK study carried out over a 3-year period, 5% of oral

specimens containing S. aureus were shown to be MRSA

strains (Smith et al., 2003b). In a separate study, MRSA was

recovered from 10% of unselected denture-wearing patients

in Japan (Tawara et al., 1996) and from 19% of the mouths

of an elderly institutionalized group (Owen, 1994).

The suggestion that prosthetic devices in the oral cavity,

including dentures, may encourage colonization of MRSA

and indeed, act as a source of cross-infection or recoloniza-

tion, is of growing concern (Rossi et al., 1997; Smith et al.,

2001, 2003a, b; Maeda et al., 2007). This may be especially

problematic in institutional settings, including hospitals,

where denture cleaning is often undertaken by carers,

potentiating horizontal transmission to other vulnerable

hosts. Additionally, there is a risk of cross-infection to and

from the dental and medical team in any setting. A recent

survey of MRSA prevalence in intensive care units showed

that 16% of patients were either colonized or infected with

MRSA (Hails et al., 2003).

FEMS Microbiol Lett 291 (2009) 241–246 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Denture cleaning and disinfection procedures rely on a

range of antimicrobial agents; however their ability to

eradicate MRSA, growing as a biofilm i.e. the natural state

of the organism in denture-associated plaque, has not been

fully assessed. The aim of this work was to determine

whether current procedures for chemical decontamination

of dentures are sufficient to eradicate planktonic, early

attached cells, and established and mature MRSA biofilms

present on denture acrylic resin.

Materials and methods

Minimum inhibitory concentration (MIC) ofdenture cleansers

Susceptibility testing was performed by a broth dilution

method (CLSI, 2006). Antimicrobial agents were double

diluted in Mueller Hinton broth. A standardized bacterial

inoculum was prepared of either EMRSA-15 (NCTC 13142)

or EMRSA-16 (NCTC 13143), added to the diluted anti-

microbial agents and incubated for 18 h at 37 1C. The agents

tested were 2% sodium hypochlorite (NaOCl) (Miltons

Sterilising Fluid, Milton Pharmaceutical Ltd, Nantes,

France); 2% w/v aldehyde-free, oxygen-releasing disinfec-

tant solution recommended for disinfection of prosthetic

material and dental impressions (Performs-ID, Schulke &

Mayr UK Ltd, Sheffield, UK) and a 1.5% w/v alkaline

peroxide denture cleanser solution [Steradent Active plus,

Reckitt Benckiser (UK) Ltd, Swindon, UK]. Miltons Ster-

ilising Fluid was used as supplied by the manufacturer;

Performs-ID and Steradent Active plus were prepared as

per the manufacturer’s instruction in sterile water. The MIC

was defined as the lowest concentration of the agent that

inhibited visible growth.

Testing of denture cleansers against sessileorganisms

Biofilms were grown on denture acrylic resin in a Constant

Depth Film Fermenter (CDFF), which is an artificial mouth

model able to generate large numbers of identical biofilms.

A 10-mL volume from an overnight culture of EMRSA-15 or

EMRSA-16 was added to 500 mL of nutrient broth, mixed

and pumped into the fermenter over an 8-h period to

provide the bacterial inoculum. The inoculation vessel was

disconnected and a medium reservoir containing sterile

nutrient broth was connected and delivered via a peristaltic

pump at a rate of 0.5 mL min�1. Five millimeter denture

acrylic resin discs were prepared by firstly making a mould by

pouring addition-cured silicone (Shera Duo-Sil H, Addition-

cure Silicone duplicating system, Shera GmbH & Co. KG,

Lemforde, Germany) over blank Perspex rods with a dia-

meter of 5 mm. A self-curing acrylic resin was then prepared

by mixing the polymer powder (Rapid Repair Polymer,

shade pink, Chaperlin & Jacobs Ltd, Sutton, UK) and

monomer liquid (Rapid Repair Monomer, Chaperlin &

Jacobs Ltd) according to the manufacturer’s instructions.

The mixture was packed into the mould and the whole setup

then placed into a pressure pot for a curing cycle of 15 min

at room temperature. The prepared acrylic resin rod (5 mm

in diameter) was removed from the mould and cut into

sections with a depth of 8.0–10.0 mm, the samples were

polished to standardize the surface area. All samples were

then soaked in plain water for two 24-h cycles before use.

Discs were aseptically removed from the fermenter at 4,

24 and 120 h. Triplicate samples were placed into 1 mL of

each test agent or sterile phosphate-buffered saline and kept

for 1, 5 or 10 min before being removed, placed into 1 mL of

neutralizing buffer (Becton Dickinson, Detroit, MI) and

then vortexed for 1 min to disrupt the biofilm. The samples

were serially diluted and bacterial survival was determined

by spread plating in duplicate onto Columbia Blood Agar

plates (Oxoid Ltd, Basingstoke, UK) containing 5% defibri-

nated horse blood (E & O Laboratories, Bonnybridge, UK).

After 24-h incubation, colonies were counted, allowing the

number and percentage of isolates killed by the different

regimes to be calculated.

Scanning electron microscopy (SEM)

To carry out SEM, samples were fixed in 3% glutaraldehyde

in 0.1 M sodium cacodylate buffer at 4 1C overnight. The

specimens were dehydrated in a graded series of alcohol

(20%, 50%, 70%, 90% and 100%� three 10-min applica-

tion time). The samples were then transferred into hexam-

ethyldisilazane (TABB Laboratories Ltd, Reading, UK) for

1–2 min before drying on filter paper in a fume cupboard.

Once dry, the specimens were placed on aluminium stereo-

scan stubs with adhesive carbon tags (Agar Scientific,

Stansted, UK). The samples were coated with a metallic

layer of gold/palladium in a Polaron E5000 sputter coater

(Quorum technology Ltd, Newhaven, UK) and viewed in a

Cambridge Stereoscan 90B Electron Microscope (Zeiss,

Cambridge, UK).

Statistical analysis

Analysis of the viable counts before and after exposure to the

different agents was carried out using the Mann–Whitney

U-test. Data were analysed using SPSS software, and the 5%

level of significance was used throughout these analyses.

Results

MIC of EMRSA-15 and EMRSA-16

All three agents inhibited planktonic EMRSA-15 and EMR-

SA-16. NaOCl was the most effective with an MIC of 0.03%

FEMS Microbiol Lett 291 (2009) 241–246c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

242 D. Lee et al.

(1 : 64 dilution) when tested against EMRSA-15 or EMRSA-

16. The MIC of Performs-ID when tested against either

strain of MRSA was 0.12% (1 : 16 dilution). Steradent was

also able to inhibit the growth of both MRSA strains,

although EMRSA-16 appeared to be less susceptible to this

agent with a 0.38% w/v (1 : 4 dilution) being required to

inhibit growth of this bacterium compared with EMRSA-15,

which was inhibited at a concentration of 0.09% (1 : 16

dilution).

EMRSA-16 and EMRSA-16 biofilms

The number of bacteria present on the surface of the denture

acrylic resin increased over the 120-h time period (Fig. 1).

At 4 h, an average of 3.5� 104 CFU mm�2 of EMRSA-15 and

7.6� 103 CFU mm�2 of EMRSA-16 were recovered from

the denture acrylic resin disks. This number increased after

24 h with 4.1� 105 CFU mm�2 of EMRSA-15 and 9.7�105 CFU mm�2 of EMRSA-16 attached to the denture acrylic

resin surface. The average total viable count of the mature

biofilm at 120 h had increased further to 1.1� 107 CFU mm�2

for EMRSA-15 and 2.4� 107 CFU mm�2 for EMRSA-16.

Susceptibility of EMRSA-15 and EMRSA-16biofilms

Four-hour sessile EMRSA-15 or EMRSA-16 were rapidly

killed by Miltons at all three time intervals (Figs 2 and 3). A

10-min exposure time to Steradent led to elimination of 4-h

sessile EMRSA-15 and EMRSA-16 below the level of detec-

tion (2.7 CFU mm�2). Exposure to Steradent for 1 min was

the least effective regime and achieved a 38.6% and 91.1%

reduction in the number of viable EMRSA-15 and EMRSA-

16 cells, respectively. A 10-min exposure time to Performs-

ID was sufficient to eliminate EMRSA-16 below the level of

detection; however, total elimination of EMRSA-15 was

not achieved with 7.1� 102 CFU mm�2 surviving these

conditions.

The 24-h established EMRSA-15 and EMRSA-16 biofilms

were rapidly killed by Milton, with bacterial numbers below

the level of detection even after an exposure period of only

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

Contro

l

Milto

n 1

min

Stera

dent

1 m

in

Perfo

rm 1

min

Milto

n 5

min

Stera

dent

5 m

in

Perfo

rm 5

min

Milto

n 10

min

Stera

dent

10

min

Perfo

rm 1

0 m

in

Agent / exposure time

CF

U m

m−2

Fig. 2. Survival of a 4-, 24- and 120-h EMRSA-15

after exposure to Miltons, Performs-ID and

Steradent for 1, 5 and 10 min. White bars represent

4-h sessile biofilms, grey bars represent 24-h

established biofilms and black bars represent 120-h

mature biofilms. Error bars represent SDs.

Fig. 1. SEM images of MRSA deposition and biofilm development on

denture acrylic resin after 4 h (a), 24 h (b) and 120 h (c).


243Susceptibility of MRSA denture biofilms

1 min (Figs 2 and 3). A 1-min exposure to Steradent was the

least effective regime when tested against EMRSA-15 with

only 16.9% of the viable cells killed; however, a 10-min

exposure to Steradent did result in a significant reduction in

the viable numbers of EMRSA-15 (99.7% kill; Po 0.05) and

EMRSA-16 (98.4% kill; Po 0.05). Exposure to Performs-

ID for 1 min significantly reduced the viable numbers of the

established 24 h EMRSA-16 biofilms from 9.7� 105 to

2.2� 103 CFU mm�2 (99.8% kill; Po 0.05) and EMRSA-15

biofilms from 4.1� 105 to 2.0� 104 CFU mm�2 (95.1% kill;

Po 0.05).

The results obtained using the mature 120-h EMRSA-15

or EMRSA-16 biofilms showed that these two pathogens

were again rapidly killed by Miltons with a 1-min exposure

time being sufficient to eliminate these bacteria below the

level of detection (Figs 2 and 3). Mature EMRSA-15 or

EMRSA-16 biofilms could not be eradicated below the level

of detection with a 10-min exposure to either Performs-ID

or Steradent, as demonstrated by a 100% survival rate for

EMRSA-16 after exposure to Steradent for this time period.

A 10-min exposure to Performs-ID, however, did lead to a

significant reduction in the total viable numbers of mature

120 h biofilms of EMRSA-15 (4 99.99% kill; Po 0.05) and

EMRSA-16 (96.83% kill; Po 0.05).

Discussion

The two epidemic strains, EMRSA-15 and EMRSA-16, are

the dominant forms of MRSA in the United Kingdom. The

possibility that dentures may act as a reservoir of MRSA is

becoming increasingly acknowledged (Rossi et al., 1997;

Maeda et al., 2007) and, therefore, the aim of this study was

to establish whether current disinfection cleansers are effec-

tive in killing MRSA when growing as biofilms on denture

acrylic resin.

Four phases of MRSA growth were used in this study:

planktonic, sessile (4 h), established biofilms (24 h) and

mature biofilms (120 h). Studying biofilm development

and perturbation requires the use of a reproducible model

system. The CDFF has previously been used to produce

in vitro denture plaque biofilms with similar compositions

to those seen in vivo (Lamfon et al., 2005). Furthermore,

using this model, longitudinal analysis can be carried out.

Indeed, it has previously been shown that the susceptibility

of biofilms to antimicrobial agents changes throughout

biofilm development (Lamfon et al., 2004).

Our results suggest that the only agent that was able to

consistently eradicate EMRSA-15 and EMRSA-16 when

grown in all four phases was Miltons Sterilising Fluid. This

agent eliminated bacterial numbers below the level of

detection even with an exposure time of only 1 min.

Previous studies have shown that NaOCl not only kills

viable bacterial cells but also is able to remove the biofilm

from the surface (Clegg et al., 2006). Furthermore, in

addition to its antimicrobial activity, NaOCl also may

remove stains and dissolve mucin and other organic sub-

stances (Jagger & Harrison, 1995). However, as NaOCl has

previously been shown to cause bleaching of denture acrylic

resin and corrosion of the metal parts of dentures, dentists

may be apprehensive to use it for this application; however,

the short immersion times, seen in this study, required to

eliminate MRSA biofilms may avoid or minimize this

complication (Jagger & Harrison, 1995).

Bacteria in the planktonic phase of growth are routinely

used to determine the susceptibility to antimicrobial agents.

The MIC data for Performs-ID and Steradent demon-

strated that planktonic EMRSA-15 and EMRSA-16 were

susceptible to these two agents. However, the biofilm data

showed that these two agents had less ability to reduce the

24- and 120-h biofilms, to viable counts below the level of

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08C

FU

mm

−2

Contro

l

Milto

n 1

min

Stera

dent

1 m

in

Perfo

rm 1

min

Milto

n 5

min

Stera

dent

5 m

in

Perfo

rm 5

min

Milto

n 10

min

Stera

dent

10

min

Perfo

rm 1

0 m

in

Agent / exposure time

Fig. 3. Survival of a 4-, 24- and 120-h EMRSA-16

after exposure to Miltons, Performs-ID and

Steradent for 1, 5 and 10 min. White bars

represent 4-h sessile biofilms, grey bars represent

24-h established biofilms and black bars represent

120-h mature biofilms. Error bars represent SDs.


244 D. Lee et al.

detection. Biofilm communities are known to be far more

resistant to antimicrobial agents than their planktonic

counterparts and for many years it has been obvious that

standardized tests such as MIC are no longer appropriate on

their own (Ready et al., 2002). This study was carried out

using monospecies biofilms of MRSA, and as MRSA on

denture materials in vivo would be present in multispecies

biofilms, their susceptibility profile may be further altered.

The ability of antimicrobial agents to eradicate the carriage

of multispecies biofilms harbouring EMRSA requires

further investigation.

Performs-ID significantly reduced EMRSA-15 and

EMRA-16 at all four phases of growth. Performs-ID is a

product that is currently used in both dental clinics and

hospital environments and, as it is not NaOCl based, its use

avoids complications such as bleaching. Although this

product did not eliminate MRSA below the level of detec-

tion at each time point, a 10-min exposure with this product

significantly reduced the microbial burden of the established

120-h MRSA biofilms. Steradent also demonstrated a high

level of anti-MRSA activity, especially against 4-h early-

attached cells, with MRSA eliminated below the level of

detection after a 10-min exposure time.

If in-patients are identified as MRSA carriers in a hospital

or nursing home setting, then disinfection for time periods

4 10 min would be possible. Therefore the ability to eradi-

cate mature MRSA denture biofilms after extended period of

exposure (including overnight soaking) to antimicrobial

agents, combined with the potential beneficial antibiofilm

activity of other antimicrobial agents used for denture disin-

fection such as chlorhexidine, glutaraldehyde, acetic acid,

hydrogen peroxide and sodium perborate (Buergers et al.,

2008; Da Silva et al., 2008) requires further investigation.

This study has demonstrated that planktonic MRSA are

rapidly killed using these three antimicrobial agents, which

correlates with the findings from a study by Maeda et al.

(2007). However, established (120 h) MRSA biofilms are

difficult to eradicate unless 2% NaOCl is used. Currently,

eradication of this organism from patients with high MRSA

carriage rates (elderly patients either hospitalized or in

nursing homes) may be significantly hindered if one possi-

ble source of reinfection, the patient’s own dentures, is either

ignored or inadequately disinfected.

Acknowledgement

This work was undertaken at UCLH/UCL, which received a

proportion of funding from the Department of Health’s

NIHR Biomedical Research Centres Funding Scheme, UK.

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