Evaluation of the efficacy of disinfection procedures againstBurkholderia cenocepacia biofilms

Elke Peeters, Hans J. Nelis, Tom Coenye*

Laboratory for Pharmaceutical Microbiology, Ghent University

Running title: Burkholderia biofilms and disinfectants

*Corresponding author:

Tom Coenye

Laboratory for Pharmaceutical Microbiology

Ghent University

Harelbekestraat 72, B-9000 Ghent, Belgium

Tel.: +32 9 2648141

Fax: +32 9 2648195

Email address:

Summary

In the present study we evaluated the efficacy of various procedures recommended for the disinfection of respiratory equipment and other materials in cystic fibrosis, using both planktonic and sessile Burkholderia cenocepacia cells. A modified European Suspension Test was performed to determine the effects of the disinfection procedures (DPs) on planktonic cells. The ability of the treatments to kill sessile cells and to remove biofilm biomass was evaluated using two resazurin-based viability assays and a crystal violet staining on biofilms grown and treated in 96-well microtiter plates. The effect of chlorhexidine and hydrogen peroxide treatments on the viability of sessile B. cenocepacia cells was significantly 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 insufficient reductions in the number of viablesessile cells. There was no correlation between the ability of the DPsto removebiofilm biomass and their potential to kill biofilm cells. In conclusion, our study indicates that testing of the efficacy of disinfectants should be performed on both planktonic and sessile cells, with particular attention to their effects on cellular viability.

Keywords: B. cepacia complex, disinfectants, biofilm, resistance

1

Introduction

Biofilms are microbial communities of surface-attached sessile cells embedded in a matrix of self-produced extracellular polymeric substances.1 These sessile cells often exhibit an increased resistance to antimicrobial agents due to restricted penetration of antimicrobials into the biofilm, decreased growth rate, expression of various resistance genes and/or the presence of “persister cells” in the biofilm.2,3 This makes biofilm-related infections very challenging to treat.4

Burkholderia cepacia complex bacteria are opportunistic pathogens that can cause severe respiratory tract infections in cystic fibrosis (CF) patients.5 Because these organisms are resistant to many antimicrobial agents, treatment of infected patients tends to be problematic.6In addition, most B. cepacia complexstrains readily form biofilms in various in vitro model systems7,8,9 and there is growing evidence that antibiotic resistance is even higher in those biofilms than in planktonic cells.8,9 Attempts to eradicate B. cepacia complex bacteria from the lungs of CF patients are often unsuccessful, making prevention of acquisition of these organisms essential.10

B. cepacia complex bacteria are transmitted from patienttopatient through close contact or aerosol transfer and through environmental (including nosocomial) acquisitions.11In order to prevent transmission, multiple infection control guidelines (ICG) have been issued. Important aspects of these ICG pertain to 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 Despite the general consensus on the need for ICG, there are considerable variations between different national and international ICG, also in terms of disinfection procedures (DPs). In addition, these DPs 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 efficacy, both against planktonic and sessile Burkholderia cenocepacia cells, of seven disinfectants (DIs) as well ashot water for the disinfection of equipment and environmental surfaces,as part of preventive measures used by CF patients.

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 (Gent, Belgium) and cultured on Nutrient Agar (NA, Oxoid, Hampshire, UK) at 37°C. Both strains belong to the B. cepacia complex test panel16 and readily form biofilms in vitro.7

Disinfection procedures

The following DPs were tested: acetic acid (Sigma, Bornem, Belgium) (concentration: 1.25%; contact time:15, 30 and 60 min), Dettol (Reckitt Benckiser, Brussel, Belgium)(5.0%; 5, 15 and 30 min), ethanol (Certa, Braine-l’Alleud, 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), cetrimide (Certa, Braine-l’Alleud, Belgium)(0.15%; 15 min), chlorhexidine (Fagron, Waregem, Belgium)(0.015% and 0.05%; 15 min), sodium hypochlorite (Forever, Courcelles, Belgium)(0.05%, 0.1% and 0.3%; 5 min). The main disinfecting components of HAC are cetrimide(15%) and chlorhexidine(1.5%). Dettol contains chloroxylenol as disinfecting constituent. We also tested hot water (70°C; 15, 30 and 60 min). Concentrations and contact times were based on various ICG for cleaning and disinfection of respiratory equipment and other materials used by CF patients. All DI solutions were prepared using water of standard hardness (WSH) and were filter sterilized before use (Puradisk FP 30; Whatman, Middlesex, UK).

Efficacy of DPs against planktonic cells

To assess the effect of the DIs and hot water on planktonic cells, a modified European Suspension Test (EST) was used.17The inactivation solution prescribed in the EST was replaced by a commercially available neutralizer (Dey-Engley Neutralizing Broth; DENB; BD, Sparks, MD, USA) or, in case of testing hot water, by cold WSH (4°C). No differentiation was made between clean and dirty conditions. Prior to the actual analysis, verification of the effectiveness of the chosen neutralizing agent and its non-toxicity against the challenge micro-organisms were determined as described in the EST protocol. The efficacy of all DPs was evaluated after various contact times and all tests were performed in duplicate.

Biofilm formation in microtiter plates

Starting from an overnight culture in Tryptone Soya Broth (TSB, Oxoid), a suspension containing approximately 108 CFU/ml was made in TSB. For each test condition, 12 wells of a round-bottomed polystyrene 96-well microtiter plate (TPP, Trasadingen, Switzerland) were inoculated with 100 µl of this suspension. Twelve wells, filled 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 µl 0.9 % (w/v) NaCl. Subsequently, 100 µl of fresh TSB was added to each well and the plates were further incubated for 20 h. After 4 h adhesion and 20 h biofilm formation, the supernatant was again removed and the wells were rinsed using 100 l 0.9 % (w/v) NaCl.

Treatment of biofilms and neutralization procedures

To assess the effect of each DP on the biofilms, 120 µl of DI or hot water was added to all wells. Every experiment included 12 control wells, in which biofilms were treated with 120 µl of WSH. After the prescribed contact time, the DI was neutralized. To this end, two different neutralization procedures were used. In the first procedure, 80 µl 2.5x-concentrated DENB was added to each well. For the neutralization of hot water, DENB was replaced by cold WSH (4°C). In the second neutralization procedure, the DI was first removed and 200 µl DENB was subsequently added to all wells. Finally, wells were again rinsed using 0.9 % (w/v) NaCl.

Prior to the actual analysis, the neutralization procedures were evaluated to verify their effectiveness for each DI. To evaluate the first neutralization procedure, 80 µl 2.5x-concentrated DENB was added to biofilm containing wells, followed by the addition of 120 µl DI. After a 5 min neutralization time, supernatants were removed and biofilms were washed using 100 µl 0.9 % (w/v) NaCl. Afterwards, 100 µl fresh TSB was added to all wells and growth was evaluated by visual inspection after 24 h of incubation. The first procedure was effective in neutralizing 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 µl of a DENB-DI mixture (containing 180µl DENB and 20µl DI) to the biofilm containing wells. The evaluation ofbiofilm growth after 24 h incubationrevealed that these DIs were sufficientlyneutralized using this second neutralization procedure.

Crystal violet (CV) and resazurin assays

The effects of the treatments were evaluated using three procedures. CV staining was used to assess the effect of the treatment on biofilm 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 µl 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 CV assay,100 µl 99% methanol was added (15 min) for fixation of the biofilms, after which supernatants were removed and the plateswere air-dried. Then, 100 µl of a CV solution (0.1%, Pro-Lab Diagnostics, Richmond Hill, ON, Canada) was added to all wells. After 20 min at room temperature, the excess CV was removed by washing the plates under running tap water. Finally, bound CV was released by adding 150 µl of 33% acetic acid (Sigma). The absorbance was measured at 590 nm using a multilabel microtiter plate reader (Wallac Victor2; Perkin Elmer Life and Analytical Sciences, Boston, MA, USA).

In the resazurin assays, a commercially available resazurin solution (CellTiter-Blue, CTB; Promega, Madison, WI, USA) was used. Stock solutions were stored at –20°C. One hundred µl 0.9 % (w/v) NaCl was added to all wells after rinsing followed by the addition of 20 l CTB solution. Fluorescence was measured after 60 min using a multilabel microtiter plate reader (ex: 560 nm and em: 590 nm).

Statistical analysis

UNIANOVA statistics, Scheffe’s tests and independent samples T-tests were performed using SPSS 15.0 software (SPSS, Chicago, IL, USA).

Results

Effect of DPs on planktonic cells

The survival of planktonic cells after various treatments was evaluated using amodified EST. All treatments resulted in a reduction in the number of microbial cells of at least 99.999% after the prescribed contact time. However, an average reduction of only 98.8% was observed 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 in an average reduction of planktonic B. cenocepacia LMG 18828 cells of 99.99%.Evaluation of shorter treatments also revealed insufficient 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).

Effect of DPs on total biofilm biomass

The effects of the different treatments on the reduction of total biofilm biomass showed that large reductions (more than 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 biofilm biomass compared to the untreated biofilms (Figure 1). Except for ethanol, an increase in concentration of the DIor in theduration of treatment did result in a more efficient removal of biofilm biomass.

Effect of DPs 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 significant fluorescence signal (Table I).However, treatments with chlorhexidine and H2O2(0.3%, 0.5% and 1.0%; 30 min)failed to eradicate a substantial part ofthe sessile B. cenocepacia LMG 16656 cells. Similarly, limited reductions in the fluorescence signals were also observed when treating B. cenocepacia LMG 18828 biofilms with chlorhexidine (0.015%; 15 min) or H2O2 (0.3%; 30 min). The average relativefluorescence signals obtained in these experiments are shown in Table I.

Effect of DPs on biofilmregrowth

Possible regrowth of sessile cells after various treatments was demonstrated using a resazurin assay after a regrowth period of 24 h.In general, theresults obtained in the regrowth assays confirm those obtained in the resazurin assaysperformed immediately after treatment(Table I).Most treatments resulted in an absence of regrowth of sessile cells, indicating that all sessile cells present in the biofilms were killed by these treatments. In contrast, regrowth was observed for biofilms of both strains after chlorhexidinetreatments and after H2O2treatments (except 3.0% for B. cenocepacia LMG 18828). In addition, slight regrowth was also observed after treating B. cenocepacia LMG 18828 biofilms with a low concentration of NaOCl (0.05%; 5 min). Regrowth of the latter biofilms 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).

Discussion

The present study is concerned with the antimicrobial effectiveness of various DPs against planktonic and sessile B. cenocepacia cells. Our results demonstrate that most DPs were capable of achieving a 99.999% reduction in the number of planktonic cells. However, the use of chlorhexidine or a low concentration of H2O2did not always result in meaningful reductions. In addition, the use of shorter treatments drastically reduced the effectiveness of most DIs and of hot water, indicating the need for a strict adherence to the duration of treatment.

Most DPs were also found to be effective for the killing of sessile B. cenocepacia cells, as evidenced from the absence of significant fluorescence signals in the resazurin viability assays (Table I). However, the antimicrobial effectiveness of chlorhexidine and H2O2 treatments on biofilms was significantly reduced compared to planktonic cells.Increased resistance of sessile Gram-negative bacteria, including the B. cepacia complex bacteriaagainst chlorhexidine has been described previously.18,19,20 Our results confirm the poor efficacy of chlorhexidine against these biofilms, indicating that its use should not be recommended in ICG for CF patients.

It is further shown that B. cenocepacia biofilms are highly resistant to H2O2. When applying H2O2 to the biofilms of both strains, strong effervescence was observed. Studies on the penetration of H2O2 in P. aeruginosa biofilms suggest that H2O2 is neutralized in the surface layers of the biofilm by catalases at a faster rate than it can diffuse into the biofilm interior. Besides this reaction-diffusion interaction, additional protective mechanisms might also contribute to the increased resistance of these biofilms.21 Presence of catalases/peroxidases in B. cenocepacia has been described previously22,23,24and the combination of the expression of these enzymes and biofilm-specific factors may lead to the failure of H2O2 treatments.

The presence of regrowth after treating B. cenocepacia LMG 18828 biofilms 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 sufficient to eradicate viable sessile cells.

Although acetic acid has insufficient activity against some potential CF pathogens (e.g. S. aureus) and is no longer recommended to disinfect respiratory equipment by the ICG,its use continues to be recommended in some nebulizer manuals. Recent surveys also indicate that diluted vinegar is still used by 10-20% of patients.25,26 Our data confirm that the use of acetic acid for disinfection purposes should be discouraged, as regrowth of B. cenocepacia biofilms can occur following this treatment.

Although most treatments yielded similar results in the resazurin assays performed immediately after treatment and after an additional regrowth period of 24h, 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 24h led to higher fluorescence signals.

A comparison between the results obtained in the CV assays and in both resazurin assaysindicate there is no correlation between the removal of biofilm biomass by the DPs and their potential to kill biofilm cells. Although the CV assay provides useful information on the efficacy of the DPs to remove biofilm remnants, it should not be used to assess the ability of DPs to eradicate viable sessile cells. These results emphasize the importance of viability assays to evaluate the effects of DPs on sessile cells.

In conclusion, our study shows that efficacy testing of DI should be performed both on planktonic and sessile cells. The results obtained with sessile cells should be taken into account when formulating ICG for the cleaning and disinfection of respiratory equipment and environmental surfaces. However, further experiments on a broad range of biofilm-forming B. cepaciacomplex strains may be necessary to further define the optimal DPs.

Acknowledgements

This research was financially supported by the BOF of Ghent University (EP) and FWO-Vlaanderen (TC).

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