Journal of Hospital Infection (2008) 70, 361e368

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www.elsevierhealth.com/journals/jhin

Evaluation of the efcacy of disinfection procedures against Burkholderia cenocepacia biolms

E. Peeters, H.J. Nelis, T. Coenye*
Laboratory for Pharmaceutical Microbiology, Ghent University, Belgium
Received 11 December 2007; accepted 29 August 2008 Available online 1 November 2008

KEYWORDS
Biolm; B. cepacia complex; Disinfectants; Resistance

Summary In the present study we evaluated the efcacy of various procedures recommended for the disinfection of respiratory equipment and other materials in cystic brosis, using both planktonic and sessile Burkholderia cenocepacia cells. A modied European Suspension Test was performed to determine the effects of the disinfection procedures on planktonic cells. The ability of the treatments to kill sessile cells and to remove biolm biomass was evaluated using two resazurin-based viability assays and a crystal violet staining on biolms grown and treated in 96well microtitre plates. The effect of chlorhexidine and hydrogen peroxide treatments on the viability of sessile B. cenocepacia cells was clearly reduced compared to the effects on planktonic cells. Treatments with low concentrations of sodium hypochlorite (0.05%, 5 min) and acetic acid (1.25%, 15 min) also resulted in insufcient reductions in the number of viable sessile cells. There was no relation between the ability of the disinfectants to remove biolm biomass and their potential to kill biolm cells. In conclusion, our study indicates that testing of the efcacy of disinfectants should be performed on both planktonic and sessile cells, with particular attention to their effects on cellular viability. 2008 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.

Introduction
* Corresponding author. Address: T. Coenye, Laboratory for Pharmaceutical Microbiology, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium. Tel.: 32 9 2648141; fax: 32 9 2648195. E-mail address: Tom.Coenye@Ugent.be

Biolms are microbial communities of surfaceattached sessile cells embedded in a matrix of self-produced extracellular polymeric substances.1 These sessile cells often show increased resistance

0195-6701/$ - see front matter 2008 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jhin.2008.08.015

362 to antimicrobial agents due to restricted penetration of antimicrobials into the biolm, decreased growth rate, expression of various resistance genes and/or the presence of persister cells in the biolm.2,3 This makes biolm-related infections very challenging to treat.4 Burkholderia cepacia complex bacteria are opportunistic pathogens that can cause severe respiratory tract infections in cystic brosis (CF) patients.5 Since these organisms are resistant to many antimicrobial agents, treatment of infected patients tends to be difcult.6 In addition, most B. cepacia complex strains readily form biolms in various in vitro model systems and there is growing evidence that antibiotic resistance is even higher in those biolms than in planktonic cells.7e9 Attempts to eradicate B. cepacia complex bacteria from the lungs of CF patients are often unsuccessful, making it important to prevent acquisition of these organisms.10 B. cepacia complex bacteria are transmitted from patient to patient through close contact or aerosol transfer as well as through environmental acquisition.11 In order to prevent transmission, multiple infection control guidelines have been issued. Important aspects of these guidelines concern the cleaning and disinfection of respiratory equipment.12 This is essential to prevent infections in CF patients as bacteria present in these devices can be aerosolised during use, thus being a potential source of infection.13,14 In addition, several guidelines, also provide protocols for cleaning and disinfection of environmental surfaces. Despite the general consensus on the need for infection control guidelines, there are considerable variations between different national and international guidelines, also in terms of disinfection procedures. In addition, these disinfection procedures are mostly based on susceptibilities of planktonic cells and do not take into account the increased resistance of sessile cells.15 In the present study we investigated the efcacy both against planktonic and sessile Burkholderia cenocepacia cells, of eight disinfectants and hot water for the disinfection of equipment and environmental surfaces as part of preventive measures used by CF patients.

E. Peeters et al. (Oxoid, Basingstoke, UK) at 37  C. Both strains belong to the B. cepacia complex test panel and readily form biolms in vitro.7,16

Disinfection procedures
The following disinfection procedures were tested: acetic acid (Sigma, Bornem, Belgium) (concentration: 1.25%; contact time: 15, 30 and 60 min), Dettol (Reckitt Benckiser, Brussels, Belgium) (5.0%; 5, 15 and 30 min), ethanol (Certa, Braine-lAlleud, Belgium) [70% (v/v); 2, 5 and 10 min], Hospital antiseptic concentrate (HAC; Regent Medical, Manchester, UK) (1.0%; 15 min), hydrogen peroxide (Acros, Geel, Belgium) (0.3%, 0.5%, 1.0% and 3.0%; 30 min), and sodium hypochlorite (Forever, Courcelles, Belgium) (0.05%, 0.1% and 0.3%; 5 min). We also tested hot water (70  C; 15, 30 and 60 min). As the main disinfecting components of HAC are cetrimide (15%) and chlorhexidine (1.5%), tests on these individual disinfectants were also included: cetrimide (Certa) (0.15%; 15 min), chlorhexidine (Fagron, Waregem, Belgium) (0.015% and 0.05%; 15 min). Dettol contains chloroxylenol as disinfecting constituent. Concentrations and contact times were based on various infection control guidelines for cleaning and disinfection of respiratory equipment and other materials used by CF patients. All disinfectant solutions were prepared using water of standard hardness (WSH) and were lter-sterilised before use (Puradisk FP 30; Whatman, Middlesex, UK).

Efcacy of disinfection procedures against planktonic cells

To assess the effect of the disinfectants and hot water on planktonic cells, a modied European Suspension Test (EST) was used.17 The EST is a standard quantitative suspension test used to assess the bactericidal activity of a disinfectant. Basically, a bacterial suspension is added to a disinfectant solution and the fraction of surviving micro-organisms is determined after a prescribed contact time at a given temperature. The procedure was slightly altered as the inactivation solution prescribed in the EST was replaced by a commercially available neutraliser (DeyeEngley Neutralising Broth; DENB; BD, Sparks, MD, USA) or, in case of testing hot water, by cold WSH (4  C). Prior to the actual analysis, verication of the effectiveness of the chosen neutralising agent and its non-toxicity against the challenge microorganisms were determined as described in the EST protocol. The efcacy of all disinfection procedures was evaluated after various contact times and all tests were performed in duplicate.

Methods
Bacterial strains
B. cenocepacia (formerly B. cepacia genomovar III) LMG 16656 and B. cenocepacia LMG 18828 were obtained from the BCCM/LMG Bacteria Collection (Ghent, Belgium) and cultured on Nutrient Agar

Bacterial biolms and disinfectants

363 that these disinfectants were sufciently neutralised using this second neutralisation procedure.

Biolm formation in microtitre plates

Starting from an overnight culture in Tryptone Soya Broth (TSB, Oxoid), a suspension containing w108 cfu/mL was made in TSB. For each test condition, 12 wells of a round-bottomed polystyrene 96-well microtitre plate (TPP, Trasadingen, Switzerland) were inoculated with 100 mL of this suspension. Twelve wells, lled with sterile medium, served as blanks. Following 4 h of adhesion, the supernatant (containing non-adhered cells) was removed from each well and plates were rinsed using 100 mL 0.9% (w/v) NaCl. Subsequently, 100 mL of fresh TSB was added to each well and the plates were further incubated for 20 h. After 4 h adhesion and 20 h biolm formation, the supernatant was again removed and the wells were rinsed using 100 mL 0.9% (w/v) NaCl.

Crystal violet and resazurin assays

The effects of the treatments were evaluated using three procedures. Crystal violet staining was used to assess the effect of the treatment on biolm biomass removal (only performed on B. cenocepacia LMG 18828) and a resazurin viability staining was applied immediately after treatment to determine the reduction in number of viable cells. In addition, regrowth of sessile cells was evaluated by adding 100 mL of fresh TSB to each well, and after an additional growth period of 24 h a resazurin viability staining was performed. Each experiment was performed in triplicate. In the crystal violet assay, 100 mL 99% methanol was added (15 min) for xation of the biolms, after which supernatants were removed and the plates were air-dried. Then 100 mL of a crystal violet solution (0.1%, Pro-Lab Diagnostics, Richmond Hill, ON, Canada) was added to all wells. After 20 min at room temperature, the excess crystal violet was removed by washing the plates under running tap water. Finally, the bound crystal violet was released by adding 150 mL of 33% acetic acid (Sigma). The absorbance was measured at 590 nm using a multilabel microtitre plate reader (Wallac Victor; Perkin Elmer Life and Analytical Sciences, Boston, MA, USA). In the resazurin assays, a commercially available resazurin solution (CellTiter-Blue; Promega, Madison, WI, USA) was used. Stock solutions were stored at 20  C. A volume of 100 mL 0.9% (w/v) NaCl was added to all wells after rinsing, followed by the addition of 20 mL CellTiter-Blue solution. Fluorescence was measured after 60 min using a multilabel microtitre plate reader (lex: 560 nm; lem: 590 nm).

Treatment of biolms and neutralisation procedures

To assess the effect of each disinfection procedure on the biolms, 120 mL of disinfectant or hot water was added to all wells. Every experiment included 12 control wells, in which biolms were treated with 120 mL of WSH. After the prescribed contact time, the disinfectant was neutralised. To this end, two different neutralisation procedures were used. In the rst procedure, 80 mL 2.5 concentrated DENB was added to each well. For the neutralisation of hot water, DENB was replaced by cold WSH (4  C). In the second neutralisation procedure, the disinfectant was rst removed and 200 mL DENB was subsequently added to all wells. Wells were again rinsed using 0.9% (w/v) NaCl. Prior to the actual analysis, the neutralisation procedures were evaluated to verify their effectiveness for each disinfectant. To evaluate the rst neutralisation procedure, 80 mL 2.5-concentrated DENB was added to biolm-containing wells, followed by the addition of 120 mL disinfectant. After a 5 min neutralisation time, supernatants were removed and biolms were washed using 100 mL 0.9% (w/v) NaCl. Afterwards, 100 mL fresh TSB was added to all wells and growth was evaluated by visual inspection after 24 h of incubation. The rst procedure was effective in neutralising cetrimide (0.15%), chlorhexidine (0.015% and 0.05%), HAC (1%), H2O2 (0.3%, 0.5% and 1.0%) and NaOCl (0.05%, 0.1% and 0.3%). For all other treatments a second procedure was tested, applying 200 mL of a DENBedisinfectant mixture (containing 180 mL DENB and 20 mL disinfectant) to the biolm-containing wells. The evaluation of biolm growth after 24 h incubation revealed

Statistical analysis
Univariate analysis of variance statistics, Scheffes tests and independent samples t-tests were performed using SPSS 15.0 software (SPSS, Chicago, IL, USA).

Results
Effect of disinfection procedures on planktonic cells
The survival of planktonic cells after various treatments was evaluated using a modied EST. All treatments resulted in a reduction in the number of microbial cells of at least 99.999% after the

364 prescribed contact time. There was an average reduction of only 98.8%, however, when treating B. cenocepacia LMG 16656 planktonic cultures with chlorhexidine (0.05%; 15 min). Additionally, treatments with a low concentration of H2O2 (0.3%; 30 min) yielded an average reduction of planktonic B. cenocepacia LMG 18828 cells of 99.99%. Evaluation of shorter treatments also revealed insufcient disinfection (<99.999%) for H2O2 (0.5%, 5 min), hot water (70  C, 1 min) and acetic acid (1.25%, 1 min) in at least one of both strains (data not shown).

E. Peeters et al. treatments. In contrast, regrowth was observed for biolms of both strains after chlorhexidine treatments and after H2O2 treatments (except 3.0% for B. cenocepacia LMG 18828) (Figure 2). In addition, slight regrowth was also observed after treating B. cenocepacia LMG 18828 biolms with a low concentration of NaOCl (0.05%; 5 min). Regrowth of the latter biolms also occurred in the majority of wells treated with 1.25% acetic acid (15 min) but the extent of regrowth was highly variable (data not shown).

Effect of disinfection procedures on total biolm biomass

The effects of the different treatments on the reduction of total biolm biomass showed that large reductions (>50%) were only obtained with Dettol, hot water (70  C; 30 and 60 min), H2O2 (3.0%; 30 min) and NaOCl. Treatments with low concentrations of H2O2 (0.3%; 30 min) or chlorhexidine (0.015%; 15 min) resulted in negligible reductions of the biolm biomass compared to the untreated biolms (Figure 1). Except for ethanol, an increase in concentration of the disinfectant or in the duration of treatment did result in a more efcient removal of biolm biomass.

Discussion
We examined the antimicrobial effectiveness of various disinfection procedures against planktonic and sessile B. cenocepacia cells. Our results demonstrate that most disinfection procedures were capable of achieving a 99.999% reduction in the number of planktonic cells. The use of chlorhexidine or a low concentration of H2O2, however, did not always result in meaningful reductions (reductions in number of planktonic cells <99.999%). In addition, the use of shorter treatments drastically reduced the effectiveness of most disinfectants and of hot water, indicating the need for a strict adherence to the duration of treatment. Most disinfection procedures were also found to be effective for the killing of sessile B. cenocepacia cells, as shown by the absence of signicant uorescence in the resazurin viability assays (Table I; independent samples t-test, P > 0.01). However, the effects of chlorhexidine and H2O2 on biolms were reduced compared to identical treatments on planktonic cells. Increased resistance of sessile Gram-negative bacteria, including B. cepacia complex, against chlorhexidine has been described previously.18e20 Our results conrm the poor efcacy of chlorhexidine against these biolms. It is further shown that B. cenocepacia biolms are highly resistant to H2O2. When applying H2O2 to the biolms of both strains, strong effervescence was observed. Studies on the penetration of H2O2 into Pseudomonas aeruginosa biolms suggest that H2O2 is neutralised in the surface layers of the biolm at a faster rate than it can diffuse into the biolm interior. Besides this reactionediffusion interaction, additional protective mechanisms might also contribute towards increased resistance of these biolms.21 Presence of catalases/peroxidases in B. cenocepacia has been described previously and the combination of the expression of these enzymes and biolmspecic factors may lead to the failure of H2O2 treatments.22e24

Effect of disinfection procedures on viable sessile cells

The reductions in number of viable cells immediately after treatments were evaluated using a resazurin assay. Most treatments resulted in the absence of a signicant uorescence signal (Table I; independent samples t-test, P > 0.01). However, treatments with chlorhexidine and H2O2 failed to eradicate a substantial part of the sessile B. cenocepacia LMG 16656 cells. Similarly, limited reductions in the uorescence signals were also observed when treating B. cenocepacia LMG 18828 biolms with chlorhexidine (0.015%; 15 min) or H2O2 (0.3%; 30 min) (Figure 2).

Effect of disinfection procedures on biolm regrowth

Possible regrowth of sessile cells after various treatments was demonstrated using a resazurin assay after a regrowth period of 24 h. In general, the results obtained in the regrowth assays conrm those obtained in the resazurin assays performed immediately after treatment (Table I). Most treatments resulted in an absence of regrowth of sessile cells, indicating that all sessile cells present in the biolms were killed by these

Bacterial biolms and disinfectants

120

100

Relative absorbance (590 mm; )

80

60

40

20

0 Disinfectant Concentration (%) Contact time (min)

AA 1.25 15 30 60 5

Det 5 15 30 2

Eth 70 5 10 15

HW 70C 30 60 0.3

H2O2 0.5 30 1.0 3.0

HAC 1.0 15

Cet

Chx 0.05

NaOCI 0.1 5 0.3

0.15 0.015 0.05 15 15

Disinfection procedure
Figure 1 Effect of various treatments on the removal of B. cenocepacia LMG 18828 biolm biomass. Average relative absorbance signals (%) obtained with the crystal violet assay on B. cenocepacia LMG 18828 biolms, showing the effects of the various treatments on the reductions in biolm biomass. Biolms were treated with acetic acid (AA), Dettol (Det), ethanol (Eth), hot water (HW), H2O2, Hospital antiseptic concentrate (HAC), cetrimide (Cet), chlorhexidine (Chx) or NaOCl. Error bars indicate standard deviations of three experiments.

365

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E. Peeters et al.

Table I Mean (SD) relative uorescence signals obtained with the resazurin assay performed immediately after treatment and after a regrowth period of 24 h for biolms of B. cenocepacia LMG 18828 and LMG 16656a Product Concentration Contact (%) time (min) B. cenocepacia LMG 16656 B. cenocepacia LMG 18828

Resazurin assay Resazurin assay Resazurin assay Resazurin assay immediately after after 24 h of immediately after after 24 h of regrowth (%) treatment (%) regrowth (%) treatment (%) e e e e e e e e e e e e 83.6 (5.6) 57.5 (7.7) 31.4 (10.8) 4.9 (0.8) e e 104.8 (2.3) 79.4 (6.8) e e e e e e e e e e e e e e e 68.7 (13.1) 37.9 (1.2) 19.9 (10.2) 15.8 (1.6) e e 102.0 (1.7) 80.5 (9.8) e e e e e e e e e e e e e e e 18.1 (3.1) e e e e e 22.4 (5.7) e 11.2 (0.5) e e 5.7 (5.2) e e e e e e e e e e e 34.4 (9.6) 16.8 (4.0) 3.9 (1.6) e e e 8.2 (1.5) 4.4 (0.9) 9.7 (1.9) e e

Acetic acid

1.25%

Dettol

5%

Ethanol

70% (v/v)

Hot water

70  C

H2O2

0.3% 0.5% 1.0% 3.0% HAC 1.0% Cetrimide 0.15% Chlorhexidine 0.015% 0.05% NaOCl 0.05% 0.1% 0.3%

15 30 60 5 15 30 2 5 10 15 30 60 30 30 30 30 15 15 15 15 5 5 5

HAC, Hospital antiseptic concentrate. a If the uorescence signals obtained in the treated wells did not differ signicantly from those of the blank wells (i.e. if no surviving cells were observed), the average relative uorescence signal was replaced by e (independent samples t-tests; P > 0.01).

The presence of regrowth after treating B. cenocepacia LMG 18828 biolms with 0.05% NaOCl for 5 min indicates that this treatment should not be used. The use of NaOCl in higher concentrations appears to be sufcient to eradicate viable sessile cells. Although acetic acid has insufcient activity against some potential CF pathogens (e.g. Staphylococcus aureus) and is no longer recommended to disinfect respiratory equipment by the infection control guidelines, its use continues to be recommended in some nebuliser manuals. Recent surveys also indicate that diluted vinegar is still used by 10e20% of patients.25,26 Our data conrm that the use of acetic acid for disinfection purposes should be discouraged, as regrowth of B. cenocepacia biolms can occur following this treatment. Although most procedures yielded similar results in the resazurin assays performed immediately after treatment and after an additional regrowth period of 24 h, there were slight

differences between the outcomes of some treatments (Table I). The latter treatments caused a substantial reduction in the number of viable cells, which resulted in signals below the lower limit of detection in the resazurin assay performed immediately after treatment. However, allowing the residual cells to multiply during a period of 24 h led to higher uorescence signals. A comparison between the results obtained in the crystal violet assays and in both resazurin assays indicates that there is no relationship between the removal of biolm biomass and the potential to kill biolm cells. Although the crystal violet assay provides useful information on the efcacy of the disinfection procedure to remove biolm remnants, it should not be used to assess their ability to eradicate viable sessile cells. These results emphasise the importance of viability assays to evaluate the effects of disinfection procedures on sessile cells. In conclusion, our study shows that efcacy testing of disinfectants should be performed both

Bacterial biolms and disinfectants

110 100 90 80 Relative fluorescence ( ) 70 60 50 40 30 20 10 0 10 Chx 0.015 Chx 0.05 H2O2 0.3 H2O2 0.5 H2O2 1.0 H2O2 3.0 Chx 0.015 Chx 0.05 H2O2 0.3 H2O2 0.5 H2O2 1.0

367

H2O2 3.0

B. cenocepacia LMG 16656

B. cenocepacia LMG 18828

Figure 2 Effect of treatments with chlorhexidine and H2O2 on the number of viable sessile cells present in B. cenocepacia LMG 18828 biolms and LMG 16656 biolms immediately after treatment and after a regrowth period of 24 h. Average relative uorescence signals (%), obtained with the resazurin-based viability assay, show the effects of these treatments on the reductions in the numbers of viable sessile cells in B. cenocepacia LMG 16656 and LMG 18828 biolms treated with either chlorhexidine (Chx) or H2O2. Assays were performed immediately after treatment (black bars) or after a regrowth period of 24 h (grey bars). Error bars indicate standard deviations of three experiments.

on planktonic and sessile cells. The results obtained with sessile cells should be taken into account when formulating infection control guidelines for the cleaning and disinfection of respiratory equipment and environmental surfaces. Further experiments on a broad range of biolm-forming B. cepacia complex strains may be necessary to further dene the optimal disinfection procedures. In addition, it will be interesting to evaluate the effect of these disinfection procedures on mixed species biolms, as these will most likely provide a greater challenge.

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Conict of interest statement None declared. Funding sources This research was nancially supported by the BOF of Ghent University (E.P.) and FWOVlaanderen (T.C.).

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