R E S EA RCH AR T I C L E

Catheter lock technique: in vitro efficacy of ethanol foreradication of methicillin-resistant staphylococcal biofilm

compared with other agents

Abhijit Chaudhury1, Jayaprada Rangineni2 & Venkatramana B2

1Department of Clinical Microbiology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India; and 2Department of

Microbiology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India

Correspondence: Abhijit Chaudhury,

Alipiri Road, Tirupati, Andhra Pradesh

517507, India. Tel.: +91 9885096261;

fax: +91 8772286803; e-mail:

abhijitch2001@yahoo.co.in

Received 28 September 2011; revised 21

February 2012; accepted 21 February 2012.

DOI: 10.1111/j.1574-695X.2012.00950.x

Editor: Niels Høiby

Keywords

biofilm; CVC; Staphylococcus.

Abstract

Biofilm formation in central venous catheters (CVC) is a prerequisite for cathe-

ter-related bloodstream infection (CRBSI). The catheter lock technique has

been used to treat biofilm infection, but the ideal agent, concentration and the

minimum exposure time necessary to eradicate the biofilms are not clearly

known. In this study, biofilm-producing strains of staphylococci were used to

find out the minimum biofilm eradication concentration of ethanol compared

with three other conventional antibacterial agents. Eight representative methi-

cillin-resistant staphylococci, from colonized CVCs, were studied. The biofilms

were exposed to 1, 5 and 10 mg mL�1 of gentamicin, ciprofloxacin and vanco-

mycin. The ethanol concentrations used were 20%, 40% and 80%. Biofilms

were examined for the presence of live organisms after exposure to these agents

from 30 min to 24 h. The three antibiotics were unable to eradicate the bio-

films even after 24 h, while ethanol at 40% concentration could do so for all

the isolates in 1 h. Our study highlights the efficacy and rationale of using

40% ethanol for a short period as catheter lock solution to eradicate biofilms

and thus to prevent CRBSI, instead of using high concentrations of antibiotics

for extended periods.

Introduction

Central venous catheters (CVCs) are increasingly being

used, sometimes for prolonged periods in multiple speci-

alities. Apart from its use in haemodialysis patients, they

are also used to monitor haemodynamic status, adminis-

tration of parenteral nutrition and for various therapies

in oncology, neonatal intensive care units and other criti-

cal care settings (Mermel et al., 2001). Data from CDC,

Atlanta, attribute 12–25% mortality among critically ill

patients because of CVC-related infections (O’Grady

et al., 2011). The infection begins as intraluminal or

external surface colonization of catheters by various

organisms, and subsequent biofilm formation. Dislodge-

ment of these organisms from the biofilm and subsequent

entry into the bloodstream initiates the classical catheter-

related bloodstream infection (CRBSI). Coagulase-nega-

tive staphylococci (particularly Staphylococcus epidermidis)

and Staphylococcus aureus remain the most common

organisms involved in these infections (Raad & Hanna,

2002).

Various modalities have been tried to prevent or to

eradicate biofilms in CVCs. Systemic antibiotics have

been extensively used, but its response has been subopti-

mal (Benjamin et al., 2001; Allon, 2004), because these

agents cannot eradicate bacteria embedded in the biofilms

(Donlan & Costerton, 2002). The antibiotic lock tech-

nique (ALT) is an attractive alternative where the catheter

lumen is just filled (without spillage into circulation) with

an antibiotic solution at high concentration and allowed

to remain there for a period of time (Messing et al.,

1988). The effectiveness of ALT has been studied in vari-

ous trials, but there is still no consensus clinical guideline

for its use (O’Grady et al., 2011). Concerns have also

been raised about development of antibiotic resistance,

allergic reactions and toxicity. In recent years, the use of

ethanol has been reported to be extremely effective as a

catheter lock solution (Metcalf et al., 2004; Onland et al.,

FEMS Immunol Med Microbiol && (2012) 1–4 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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2006). As regards the various agents used as lock solu-

tions, there is still a large gap in our knowledge regarding

the optimum agent, its concentration and ideal exposure

time necessary to eradicate a microbial biofilm in CVC

(Berrington & Gould, 2001). Our study was performed to

address this issue in an in vitro mature biofilm model

using three conventional antibiotics and also ethanol at

various concentrations.

Materials and methods

Bacterial isolates

A total of 100 consecutive CVC tips from femoral or jug-

ular veins of patients undergoing haemodialysis and sub-

mitted to the microbiology department were analysed.

Roll-plate technique of the catheter tips was followed by

vortexing in phosphate-buffered saline (PBS) and subse-

quent plating on sheep blood agar. The latter procedure

was carried out to recover the organisms present in the

biofilm in the intraluminal surface of the catheter. The

presence of more than 15 colonies by roll-plate technique

and/or more than 103 CFU mL�1 of the vortex specimen

were taken as significant.

Suspected staphylococcal colonies were picked up for

further identification. Gram stain followed by tube coagu-

lase test was carried out with these colonies. Coagulase-

negative isolates were further characterized to species level

by using a battery of tests as described earlier (Banner-

man & Peacock, 2007). Staphylococcus aureus and S. epi-

dermidis strains were further tested. Methicillin sensitivity

testing was carried out using oxacillin 1 lg discs (Hi

Media, India) and reconfirmed with Cefoxitin 30 lg discs

(Hi Media). The antibiogram was determined by standard

Kirby Bauer’s technique (CLSI, 2010). Strains sensitive to

gentamicin, ciprofloxacin and vancomycin but resistant

to oxacillin were used for the biofilm experiment.

Biofilm-forming potential of staphylococcal

isolates

Quantitative biofilm experiment was carried out by the

method described earlier (Rachid et al., 2000). An 1 : 100

dilution of overnight broth culture of the organism was

performed in brain heart infusion broth (Hi Media) with

1% glucose supplementation that has been found to be

potent inducer for slime production. Two hundred

micro-litres of the diluted broth culture were transferred

to 96-well polystyrene flat-bottomed ELISA plates (Tar-

sons, India). After overnight incubation at 37 °C, plateswere washed three times with PBS and stained with crys-

tal violet solution. The plates were read at 490 nm in the

ELISA reader, and optical density of > 0.120 was consid-

ered biofilm positive.

Antibiotic and ethanol activity on biofilm

Eight representative strains comprising four each of MRSA

and MRSE, all of which were strong biofilm producers

(OD: 0.408–0.798), were used to find out whether a high

concentration of selected antibiotics or ethanol at varying

concentrations could disperse and eradicate formed

mature biofilms. Preformed 24-h biofilms were exposed

to the antibiotics as follows: gentamicin, ciprofloxacin,

vancomycin at concentrations of 1, 5 and 10 mg mL�1

and ethanol at 20%, 40% and 80% strengths. Individual

strains to be tested were exposed separately to 200 lL of

the above agents at the particular concentration for peri-

ods of 30 min, 1 h, 3 h, 6 h and 24 h. After the specified

contact time period, the wells were thoroughly washed

three times with PBS, and the biofilm material was har-

vested by scraping the wells using sterile cotton swabs.

Harvested biofilm was homogenized and plated on Trypti-

case soy agar with 5% defibrinated sheep blood. Any

growth on blood agar after 24-h exposure was considered

as noneradicaiton of the biofilm organisms.

Results and discussion

Antibiotic catheter lock solution has been found to pre-

vent or reduce CRBSI rates significantly and resultant

catheter removal rates compared with heparin lock used

alone (Yahav et al., 2008). However, the guidelines from

the Infectious Diseases Society of America and CDC,

Atlanta, mention the use of antibiotic lock as prophylaxis

for catheter-related infections in selected patient popula-

tions only, but includes its use as a therapeutic option for

intraluminal infection when the device is not removed

(Mermel et al., 2001; O’Grady et al., 2011). In our study,

the three antibiotics used were unable to eliminate the

mature staphylococcal biofilms even after prolonged

exposure of up to 24 h and at very high pharmacological

concentrations of 1000, 5000 and 10 000 lg mL�1 as evi-

denced by the growth of harvested biofilm organisms on

blood agar (Table 1). ALT can be used in two situations –first, as a preventive measure to decrease the incidence

of CRBSI and secondly, in a situation where bloodstream

infection has already set in. In the latter situation, salvage

of the colonized catheter should be attempted as first

option. Our study was aimed at finding an effective agent

that could eradicate a fully formed biofilm. The three

antibiotics studied were not effective in this aspect after

maximum exposure time of 24 h. In agreement with an

earlier study (Qu et al., 2009), we found that gentamicin

ª 2012 Federation of European Microbiological Societies FEMS Immunol Med Microbiol && (2012) 1–4Published by Blackwell Publishing Ltd. All rights reserved

2 A. Chaudhury et al.

and vancomycin are unsuitable as catheter lock solutions,

although in their study, ciprofloxacin was able to kill the

biofilm embedded cells. In a similar study (Lee et al.,

2006), it was shown that eradication of mature biofilms is

possible with vancomycin and ciprofloxacin by prolonged

exposure of up to 14 days. However, such a prolonged

treatment of the catheter in the presence of overt CRBSI

in real-life situations may not be acceptable.

Ethanol was able to kill the biofilm cells of all the iso-

lates within one hour of exposure at a concentration of

40%. Exposure for a longer period was necessary when a

lower concentration of 20% ethanol was used (Table 2).

Higher concentrations of this agent could eradicate the

biofilms in less than an hour. Our observation is in agree-

ment with earlier studies (Sherertz et al., 2006; Qu et al.,

2009) where it was found that ethanol was a more effec-

tive agent for eradication of biofilm compared with anti-

biotics. Ethanol at these low concentrations has been

shown not to interfere with the biomechanical properties

of polyurethane catheters (Crinch et al., 2005), and it

is also compatible with heparin, which is commonly

employed as anticoagulant in the catheter lock (Ackoun-

dou-N’guessen et al., 2006). Additionally, ethanol appears

to have its own anticoagulant activity that may prevent

catheter thrombus formation, thereby making it unneces-

sary to mix heparin with ethanol as lock solution (Sher-

ertz et al., 2006). Ethanol acts as a broad spectrum

bactericidal and fungicidal agent, and there is no concern

about resistance development (Metcalf et al., 2004). It is

thought that ethanol works as an effective biofilm eradi-

cating agent because of its small molecular size and its

hydrophilic nature that helps it to easily penetrate the

highly hydrated biofilm matrix and exert its killing action

(Qu et al., 2009). Mild side effects in the form of flush-

ing, headache, dizziness and nausea have been noted

(Slobbe et al., 2010), but only when the ethanol lock

solution was flushed into circulation. Withdrawal of the

solution after the desired time can circumvent these sub-

jective side effects.

The methodologies adopted in our study need some

explanation. We have used polystyrene material as a plat-

form for biofilm formation, although catheters are com-

monly made up of polyurethane or Teflon. However, in

our experiment, it was found that all the selected strains

adhered adequately to polystyrene and produced strong

biofilms, which has justified our use of this material for

growing the biofilms. We chose polystyrene as this mate-

rial is widely used to study biofilm formation and also

because of the easier availability of polystyrene microtitre

plates in the clinical microbiology laboratories. One con-

cern about the possible effect of alcohol on the mechani-

cal properties of catheter materials has also been

addressed earlier (Crinch et al., 2005).

Secondly, we have collected the antibiotic-/ethanol-

treated biofilm material by scraping with sterile cotton-

tipped swabs and then plated on the blood agar. In

biofilm studies, sonication is more commonly used to

dissociate biofilms, particularly if grown on pegs. For bio-

films grown at the bottom of microtitre plates, sonication

has a poor efficiency, and there is a large variation in cell

release (Qu et al., 2009). Others have found that sonica-

tion may fail to dissociate the cells completely (Raad

et al., 2007), or else the shocked cells may fail to recover

and grow after direct plating (Zimmerli et al., 1994). Our

method is a variation of the method of scraping/vor-

texing/plating used by earlier workers (Raad et al., 2007

Qu et al., 2009), and it is less labour intensive and with

no chance of cross-contamination that can occur during

sonication of microtitre plates. The third and the last

explanation concerns the determination of end point. The

present study has relied on the complete absence of any

recoverable bacteria after ethanol/antibiotic treatment. In

contrast, other studies have observed the log reduction of

bacterial population after exposure to the inhibitory

agents (Lee et al., 2006; Sherertz et al., 2006). To come

to a definitive conclusion about the efficacy of an agent

in the eradication of biofilms, mere reduction of the cell

population may not be enough. This is because of the fact

that the remaining viable cells or ‘persisters’, however

few, will cause the reaccumulation of the biofilm once the

antimicrobial effect is removed (Lewis, 2005).

In conclusion, the result of our study proves the high

effectiveness of 40% ethanol for one hour over three

common antibiotics in eradicating staphylococcal bio-

films. This may prove to be an ideal agent in salvaging

catheters colonized with staphylococci in the presence

of CRBSI. Randomized placebo control trials in patient

populations are necessary to confirm the true significance

of this study.

Table 1. Effect of antibiotic exposure on mature biofilms

SN Antibiotic No. 30 min 1 h 3 h 6 h 24 h

1 Gentamicin 8 + + + + +

2 Ciprofloxacin 8 + + + + +

3 Vancomycin 8 + + + + +

+, growth positivity.

Table 2. Effect of ethanol exposure on mature biofilms

Sn

Ethanol concentration

(%) 30 min 1 h 3 h 6 h

1 20 8 8 8 5

2 40 5 Nil Nil Nil

3 80 1 Nil Nil Nil

Number of strains growing after the exposure time (n = 8).

FEMS Immunol Med Microbiol && (2012) 1–4 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Ethanol catheter lock 3

Conflict of interest

The authors declare no conflict of interest.

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