CHEMBIOCHEM FULL PAPERS DOI: 10.1002/cbic.201400023

Pseudomonas aeruginosa Biofilm Growth Inhibition on Medical Plastic Materials by Immobilized Esterases and Acylase Johannes Martin Kisch,[a] Christian Utpatel,[b] Lutz Hilterhaus,[a] Wolfgang R. Streit,[b] and Andreas Liese*[a] Biofilms are matrix-encapsulated cell aggregates that cause problems in technical and health-related areas; for example, 65 % of all human infections are biofilm associated. This is mainly due to their ameliorated resistance against antimicrobials and immune systems. Pseudomonas aeruginosa, a biofilmforming organism, is commonly responsible for nosocomial infections. Biofilm development is partly mediated by signal molecules, such as acyl-homoserine lactones (AHLs) in Gram-negative bacteria. We applied horse liver esterase, porcine kidney

acylase, and porcine liver esterase; these can hydrolyze AHLs, thereby inhibiting biofilm formation. As biofilm infections are often related to foreign material introduced into the human body, we immobilized the enzymes on medical plastic materials. Biofilm formation was quantified by Crystal Violet staining and confocal laser scanning microscopy, revealing up to 97 % (on silicone), 54 % (on polyvinyl chloride), and 77 % (on polyurethane) reduced biomass after 68 h growth.

Introduction The term “biofilm” refers to a growth state in which microorganisms are attached to surfaces, interfaces, or each other and encapsulated in a matrix, mainly consisting of polysaccharides, proteins, DNA, and RNA.[1] Biofilms occur practically everywhere and are believed to be the most prominent manifestation of bacteria. Bacteria benefit from the formation of biofilms. They are better protected against environmental changes, other microorganisms, immune systems, and antimicrobial agents such as antibiotics.[2, 3] Although bacterial biofilms can be beneficial (e.g., for cleaning bodies of water) and are being used in waste-water treatment plants[4] and as biocatalysts,[5] they create problems in other areas. Biofouling occurs in heat exchangers, ship hulls, pipe systems, and washing machines, thereby causing efficiency losses and product contamination.[6] Additionally, biofilms cause huge clinical problems. It is believed that 65–80 % of all human infections are biofilm associated[7, 8] and cause up to 550 000 deaths in the US every year,[9] almost as high as the number of projected cancer deaths in 2014 (585 720).[10] Examples of biofilm infection include colitis, conjunctivitis, and urethritis. Infections related to intravascular and urinal catheters are the two most common causes of nosocomial bloodstream infections, with biofilm formation being central to the pathogenesis.[11] Pseudomonas aeruginosa is a biofilm-forming mi[a] J. M. Kisch, Dr. L. Hilterhaus, Prof. A. Liese Institute of Technical Biocatalysis, Hamburg University of Technology Denickestr. 15, 21071 Hamburg (Germany) E-mail: [email protected] [b] C. Utpatel, Prof. W. R. Streit Abteilung fr Mikrobiologie und Biotechnologie Biozentrum Klein Flottbek, Universitt Hamburg Ohnhorststraße 18, 22609 Hamburg (Germany)

 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

crobe and one of the most common organisms responsible for nosocomial infections.[12] Several approaches to prevent bacterial biofilm formation on surfaces are being investigated: reduction of bacterial adhesion by altering surface properties (e.g., hydrophobicity or roughness) or by polymer coatings (e.g., polyethylene glycol);[13] release strategies with agents[14] such as silver nanoparticles,[15, 16] antibiotics,[17, 18] furanones,[19] or nitric oxide;[20] bactericidal coatings[21] like poly(alkylammonium) compositions;[22] or interference with bacterial cell–cell communication.[23, 24] In this study, enzymes were applied to degrade bacterial signal molecules. The development of a biofilm involves the attachment of cells to a surface, formation of a microcolony, maturation of the biofilm, and dissolution of parts of the biofilm to colonize new sites (Figure 1, top).[25] This process, along with other functions such as the secretion of virulence factors, competence, sporulation, swarming, and bioluminescence, is regulated by quorum sensing (QS), which is the ability to coordinate behavior between cells by producing and sensing small, diffusible molecules.[26] Acyl-homoserine lactones (AHLs) form one major class of signal molecules in Gram-negative bacteria. Others are quinolones and furanosyl borate diesters (also Gram-positive). P. aeruginosa QS consists of three inter-related systems. The Las system (signal molecule: N-3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL)) is the primal system, induced at a threshold cell density. 3OC12-HSL gives positive feedback to the Las system and induces the production of N-butyryl-homoserine lactone through the Rhl system as well as of 2-heptyl-3-hydroxy-4-quinolone, the “Pseudomonas quinolone signal” (PQS). The latter gives positive feedback to Las and Rhl systems.[27] ChemBioChem 2014, 15, 1911 – 1919

1911

CHEMBIOCHEM FULL PAPERS

www.chembiochem.org

Figure 1. Biofilm development (top) and inhibition approach (bottom).

Recently, an additional P. aeruginosa QS signal molecule, IQS, from Sulfolobus solfataricus was immobilized on nanoaluminawas described; this is under the control of the Las system and functionalized cellulose membrane discs; when these were regulates the Rhl system and PQS production.[28] IQS producplaced in P. aeruginosa PAO1 cultures, virulence factor production is also induced under phosphate-limited conditions, linktion was reduced.[34] In this study PKA, HLE, and PLE were immobilized on silicone ing QS to environmental influences. (SIL), polyvinyl chloride (PVC), and polyurethane (PU) surfaces; Quorum quenching (QQ) interferes with the QS systems at these inhibited P. aeruginosa PAO1 biofilm growth by up to the stage of either signal-molecule production, accumulation, 98 %. These plastic materials (Medicoplast, Illingen, Germany) or detection, and is one promising approach to inhibit bacteriare used for catheter production. Surfaces were amino-funcal virulence and biofilm formation.[29] In recent years, several AHL-degrading QQ enzymes have been derived from metagetionalized by layer-by-layer (LBL) coating of polyethyleneimine nomic screenings and from bacterial isolates.[23] Among these are lactonases that hydrolyze the lactone ring, acylases that hydrolyze the amide bond, and oxidoreductases that reduce keto groups at the third carbon atom of the acyl chain (Scheme 1).[30] Porcine kidney acylase (PKA) has already been proposed as a QQ enzyme,[31] while porcine liver esterase (PLE) and horse liver esterase (HLE) have not yet been reported to hydrolyze AHLs or inhibit biofilm formation. Immobilization of quorum quenching enzymes has been investigated. PKA was immobilized on magnetic carriers in a membrane bioreactor, decreasing the drop in transmembrane pressure.[32] It was also immobilized on polyamide nanofiltration membranes and reduced biofilm formation.[33] Finally, a lactonase Scheme 1. Enzymatic degradation of AHLs.  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemBioChem 2014, 15, 1911 – 1919

1912

CHEMBIOCHEM FULL PAPERS (PEI) and polystyrene sulfonate (PSS) by electrostatic forces. Subsequently, enzymes were covalently immobilized with a glutardialdehyde (GDA) linker (Figure 1, bottom). Tubes of all three materials were tested for biofilm growth inhibition in “tube flow” assays, and SIL plates were assayed in a flow cell with confocal scanning laser microscopy (CLSM).

www.chembiochem.org homo-serine lactone, and 3OC12-HSL were also shown to be hydrolyzed by PLE, with 24.5 % higher conversion of 3OC12-HSL compared to C12-HSL (data not shown).

LBL polymer coating of surface materials

Results AHL degradation by esterases and acylase AHL degradation by HLE and PLE was shown by following the concentrations of N-butyryl-l-homoserine lactone (C4-HSL), Nhexanoyl-l-homoserine lactone (C6-HSL), N-octanoyl-l-homoserine lactone (C8-HSL), and N-dodecanoyl-l-homoserine lactone (C12-HSL) in pH 7 potassium phosphate buffer (KPi) at 30 8C over time. Ethyl acetate (EtOAc) extracts were measured by GC/MS with bovine serum albumin (BSA) as the control. AHLs undergo autohydrolysis in aqueous media, which explains the declining AHL concentrations in the BSA controls. However, AHL concentrations in HLE and PLE experiments were significantly lower due to enzymatic degradation (Figure 2), except for C4-HSL and C6-HSL in HLE experiments (Figure 2 A and B). The corresponding p values were below 5.8  104. PLE showed 170 % higher conversion after 8 h compared to HLE for all four AHLs and 21.5 % higher for C12-HSL. The highest conversion for all four AHLs under these conditions was 119 nmol per mg of enzyme in four hours. Additional experiments were carried out for PLE with LC/MS/ MS analysis to verify the suggested lactone ring hydrolysis. N3-oxo-hexanoyl-l-homoserine lactone, N-3-oxo-octanoyl-l-

LBL polymer coating was carried out by Surflay Nanotec (Berlin, Germany). Multiple layers of alternately charged polyelectrolytes (PEI, MW 25 000 and PSS, MW 70 000) were bound to the corresponding surface through electrostatic forces by using established coating techniques as described elsewhere.[35] CLSM analysis of rhodamine-labeled PEI coatings was carried out to verify the coating process. The thickness of the coating was about 1 nm. Figure 3 shows the results of the CLSM analysis for SIL, PVC, and PU tubes. The tubes were cut radially and lengthwise, and the coatings were analyzed on the inside and outside. CLSM excitation voltages were adjusted to elicit similar emission levels, thus a lower voltage represent more rhodamine-labeled PEI on the surface. SIL offered the best coating characteristics, followed by PU and PVC.

Figure 3. CLSM analysis of rhodamine-labeled PEI coatings (1 mm picture width); left to right: SIL inside 610 V, SIL outside 650 V, PVC inside 830 V, PVC outside 870 V, PU inside 780 V, PU outside 820 V.

Analysis of enzyme immobilization Covalent enzyme immobilization with GDA linker (see the Experimental Section) was verified by the activity of immobilized esterase, as tested by a para-nitrophenyl acetate (pNP-A) assay. The activity of PLE immobilized on the inside of PEI-coated SIL tubes decreased over time (Figure 4): initial activity 1.7 U m2 ; after 48 days’ storage at 4 8C, 0.9 U m2, corresponding to a half-life of 53 days. Biofilm tube system

Figure 2. AHL concentrations over time in 100 mm KPi buffer, pH 7, 30 8C, with 1 mg mL1 enzyme (&: HLE, or control (^: BSA), 50 mm AHL; measured by GC/MS after EtOAc extraction.

 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

~:

PLE)

To grow and measure P. aeruginosa biofilms on medical plastic materials, a tube system was designed with Crystal Violet (CV) staining analysis, by using

ChemBioChem 2014, 15, 1911 – 1919

1913

CHEMBIOCHEM FULL PAPERS

www.chembiochem.org

Figure 4. Activity of polymer immobilized on the inside of a PEI coated SIL tube (i.d. 3 mm, length 8 mm), 100 mm KPi buffer, pH 7.5, 30 8C, 2 mm pNPA. ^: experimental data; c: y = 1.67  e0.0131 x.

a modified protocol of Peeters et al.[36] The tube system consisted of a medium reservoir, a peristaltic pump, growth and inoculation tubes, a waste container, and connections. The system was optimized for robustness regarding changes in inoculation procedure, attachment time, and CV staining analysis. Inoculation with a ten times higher or ten times lower cell concentration, attachment times between 10 and 90 min, and variations in the CV staining procedure had no significant influence on measured OD590 values (data not shown). The setup is described in the Experimental Section. Figure 5 shows the extent of P. aeruginosa biofilm growth in eight SIL tubes in parallel. The tubes were disassembled and analyzed at six time points to obtain growth curves. (Three tubes were analyzed at 70 h: standard deviation, 6.2 %, thus illustrating the versatility and reproducibility of the system). The biomass increase over time is in accordance with the biofilm development scheme (Figure 1). It can be assumed that cells attached to the tube wall and formed microcolonies up to 48 h, followed by rapid, QS-regulated biofilm growth.

Figure 5. P. aeruginosa biofilm SIL tube experiment with CV staining analysis for 60 min attachment time, 77 h growth time, 0.1 mL min1, and RT: indicated time points are the CV staining analysis results of single tubes. After 70 h three tubes were analyzed (standard deviation 6.2 %). A = absorbance units.

To investigate temperature dependence of the inhibitory effect, P. aeruginosa biofilm growth inhibition in SIL tubes with immobilized PLE was monitored at room temperature (RT = 20 8C) and at 35 8C. Control biofilm growth at RT was 37.5 % lower than at 35 8C (Figure 7). Biofilm growth was inhibited by 84.4 % at RT and by 81.8 % at 35 8C, compared to the corresponding controls. The time dependence of the inhibitory effect was evaluated in SIL tubes with immobilized PLE. Biofilm growth was measured over 28 days (Figure 8). The lowest growth inhibition was 53.5 % after five days, and the highest was 87.1 % after 68 h (mean, 64.3 %). CLSM imaging To further evaluate the inhibitory effect, CLSM images were acquired of P. aeruginosa biofilms grown on SIL with and without immobilized PLE. A flow cell was constructed (see the Experi-

Biofilm growth inhibition Biofilm growth inhibition experiments were carried out in SIL, PVC, and PU tubes, with the the described polymer coating and HLE, PKA, and PLE immobilization on the inner surface. Experiments were carried out in duplicate, except for the time-resolved experiments (Figures 5 and 8, below); untreated tubes were used as controls. Figure 6 A shows that neither the polymer coating, nor bound GDA, nor immobilized BSA had any influence on biofilm growth. Biofilm growth inhibition rates of immobilized HLE, PKA and PLE in polymer-coated SIL, PV, and PU tubes (Figure 6 B–D ) were 95.3, 87.4, and 97.6% in SIL tubes, 53.9, 43, and 48.7% in PVC tubes, and 77.1, 73.8, and 71.4% in PU tubes, respectively.

Figure 6. P. aeruginosa biofilm tube experiments with CV staining analysis for 60 min attachment time, 68 h growth time, 0.1 mL min1, and RT: A) untreated SIL tubes, with PEI coating (PEI), with polymer coating and GDA linker, with polymer coating, GDA linker and immobilized BSA; B) SIL tubes with immobilized HLE, PKA, or PLE; C) PVC tubes with immobilized HLE, PKA, or PLE; D) PU tubes with immobilized HLE, PKA, or PLE.

 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemBioChem 2014, 15, 1911 – 1919

1914

CHEMBIOCHEM FULL PAPERS

www.chembiochem.org Discussion

Figure 7. P. aeruginosa biofilm tube experiment with CV staining analysis for 60 min attachment time, 68 h growth time, 0.1 mL min1, RT, and 35 8C: SIL tubes without or with immobilized PLE, at room temperature and at 35 8C.

Figure 8. P. aeruginosa biofilm SIL tube experiment with CV staining analysis for 60 min attachment time, 28 days’ growth time, 0.1 mL min1, and RT. Indicated time points are the CV staining analysis results of single control tubes (^) and tubes with immobilized PLE (&).

mental Section), and SIL plates (15  10  2 mm) with immobilized PLE were placed in the flow cell, height adjusted to provide 1 mm space between the plates and the cover slip lid, and monitored for P. aeruginosa biofilm growth. Figure 9 shows a representative image of a biofilm grown on a SIL control plate and on a SIL plate with immobilized PLE in one chamber. Biofilm growth after 68 h on the SIL plate with immobilized PLE was significantly reduced. The corresponding pvalues are 1  105 (area) and 3.2  105 (volume). The sequence of the three SIL plates in the flow direction in this experiment was control!PLE!control. Images were recorded: four from the first control plate, eight from the plate with immobilized PLE, and four from the second control plate. Imaris software (Bitplane AG, Zrich, Switzerland) was used to calculate surfaces and volumes from the 16 images. The inhibition effects were 85.2 and 93.7 % (biofilm surface area and volume, respectively).

To prevent the formation of bacterial biofilms on surfaces, two main aspects have to be considered. Firstly, an agent that interferes with biofilm development has to be found. Secondly, the active agent has to be brought or linked to the surface on which biofilm development is to be hindered. Adding the agent to the medium has limited efficiency, because the agent diffuses and is not confined to the area of biofilm formation. Often this is not an option. Reducing bacterial adhesion by surface modification or polymer coating is a promising approach, but might still be limited because of the diverse substances and adhesion mechanisms in complex, biological environments. Bactericidal coatings often require direct contact with bacteria, but this can be hindered by adhered cell debris or other substances. Active agent release strategies have shown good results in vitro and in vivo, but have the disadvantage of distributing the agents, with possible side effects such as damaging healthy tissue and causing resistance and selection pressure (antibiotics).[14] Interference with bacterial QS at the stages of signal molecule synthesis, accumulation, or detection[24] can inhibit bacterial biofilm formation and virulence.[23] Degradation of signal molecules by immobilized enzymes might still be effective even if covered with cells or other substances, as signal molecules can diffuse towards the enzymes. Selection pressure is not as concerning as it is for antibiotics, but mutations in QS systems have been described.[37, 38] Other drawbacks are enzyme deactivation or dissolution, and the diversity of bacterial signal molecules. A lot of research has been carried out to find enzymes that can interfere with QS by degrading signal molecules. Strain isolates have been the main sources of such QQ enzymes. Possible degradation mechanisms for AHLs are hydrolysis of the lactone ring, amide bond hydrolysis, and reduction of keto groups at the third carbon atom of the fatty acid chain (Scheme 1). Commercial enzymes often offer better stability, have known properties, and offer ease of access compared to enzymes from metagenomic screenings or bacterial isolates. We discovered that commercially available esterases HLE and PLE were able to reduce the concentration of several AHLs by lactone ring hydrolysis, including C4-HSL and 3OC12-HSL, which are associated with QS in P. aeruginosa. Additionally, we used

Figure 9. P. aeruginosa biofilm experiment with CLSM analysis for 60 min attachment time, 68 h growth time, 0.225 mL min 1, and 35 8C. SIL plates in flow cell, A) control and B) with immobilized PLE; C) Mean areas and volumes (from eight images each) calculated with Imaris software, normalized to corresponding control.

 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemBioChem 2014, 15, 1911 – 1919

1915

CHEMBIOCHEM FULL PAPERS PKA, which has already been described to interfere with AHL QS. Although the activities for all three enzymes were low (~ 119 nmol per mg of enzyme conversion in 4 h), AHL concentrations in biofilms are in the nanomolar range,[39] so low conversions can still have an impact. Comparison of the determined activities with activities of previously described QQ enzymes (such as AiiA[40, 41] or AiiB)[42] is difficult, as kinetic parameters are usually determined from AHL starting concentrations around 10 mm. This would require extrapolation of kcat and Km values over more than two orders of magnitude. Wang et al. also specified minimum AHL concentrations for AiiA at which conversions could be detected. These were 20–400 times higher than the concentrations used in this study (> 10 mm for C4-HSL and 1 mm for 3OC12-HSL). As stated in the introduction, QQ enzyme immobilization has been described previously. Yeon et al. immobilized PKA on magnetic carriers by LBL coating and adsorption or covalent binding.[32] The immobilized compound reduced transmembrane pressure drop as well as protein and extracellular polymeric substances (EPS) content of the filter cake. Kim et al. adsorbed PKA-chitosan aggregates on polyamide membranes with subsequent GDA crosslinking.[33] Activity and stability of the immobilizates were determined with N-acetyl-l-methionine. The membrane was assayed for biofilm growth in a flow cell by in situ fluorescence microscopy and ex situ CLSM. The CLSM results indicated less biofilm growth and less EPS production. Transmembrane flux was also increased. Ng et al. adsorbed a phosphotriesterase-like lactonase from S. solfataricus on alumina-functionalized cellulose membrane discs by electrostatic forces.[34] The discs were added to P. aeruginosa PAO1 liquid cultures, and they reduced casein-hydrolyzing activity as well as elastase and pyocyanin production (measured photometrically). The distinct features of our approach are PEI/PSS LBL coating of polymers used for catheter production followed by covalent enzyme immobilization with GDA linker, the novel use of HLE and PLE as QQ enzymes, CLSM biofilm formation analysis in a flow cell, and the use of a tube flow assay (Figure 10). This allowed us to carry out up to 16 experiments in parallel, to investigate different materials, temperatures, and time dependencies, to compare one described and two new QQ enzymes, and to analyze biofilm formation in situ. Covalent enzyme immobilization is known for low amounts of enzyme leakage,[43] although this still has to be verified for this application of QQ enzymes. Dissolution of the polymer coating also has to be addressed. Surflay Nanotec claims good chemical and physical resistance for their coatings. Determination of immobilization yields is difficult because the inner surface of each growth tube is 7.54 cm2 and the amount of protein on this surface is about 95 ng (estimated in previous experiments with porous glass particles by the same immobilization method). Rhodamine-labeled PEI coating was analyzed by CLSM (Figure 3). The amount of PEI on the different materials seemed to have an influence on the observed biofilm inhibition by QQ enzymes. This suggests that more PEI on a surface leads to more enzyme activity, which leads to a higher biofilm growth inhibition (Figure 6).  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chembiochem.org

Figure 10. Biofilm tube system setup.

The AHL hydrolysis activity of HLE and PLE was low, so the activity of the immobilized enzymes could not be measured with an AHL assay. pNP-acetate was used to determine the activity of immobilized PLE, and this was shown to have the highest activity at 1.7 U m2 with a half-life of 53 days. Ad- or absorption of pNP and pNP-acetate on or into the tube was detected by comparing measurements in tubes and in cuvettes; controls without enzyme resulted in higher absorption (OD410) of cuvette samples compared to tube samples. The biofilm tube system is an easy and rapid method to measure biofilm growth under multiple conditions. The setup is similar to that with a classical flow cell, but with a growth tube (i.d. 3 mm) in place of the flow cell. It would also be possible to incorporate glass tubes, thus allowing microscopy analysis. The results were reproducible (~ 10 % standard deviation). It was proven that neither the PEI coating, GDA linker, nor immobilized BSA had any influence on P. aeruginosa biofilm growth. There was significant biofilm growth inhibition on SIL, PVC, and PU with immobilized QQ enzymes (Figure 6). The highest p-values for each material were 1.9  103 for SIL (PKA), 7.7  103 for PVC (PKA), and 9.4  103 for PU (PLE). PLE showed 170 % higher conversion than HLE (hydrolysis of C4-HSL, C6-HSL, and C8-HSL and C12-HSL combined), the conversion was only 21.5 % higher for C12-HSL (Figure 2 D). Although the conversion results were obtained with C12-HSL, 3OC12-HSL was also shown to be hydrolyzed by PLE with similar conversion (24.5 % higher conversion compared to C12-HSL). P. aeruginosa biofilm growth inhibition on SIL, PVC, and PU after 68 h (Figure 6 B, C) was similar for HLE and PLE. As the Las system (3OC12-HSL as signal molecule) is superior to P. aeruginosa QS, it can be hypothesized that activity towards 3OC12-HSL degradation is crucial for QQ in P. aeruginosa. However, several factors have to be considered when comparing enzyme activity assays in buffer and biofilm growth inhibition by immobilized enzymes, including PEI coating and EPS matrix effects, activity ChemBioChem 2014, 15, 1911 – 1919

1916

CHEMBIOCHEM FULL PAPERS changes caused by the immobilization procedure, competing substrates, and substrate concentration. One drawback of the tube system is that biofilms grown in plastic tubes cannot be assayed with a microscope without opening the tube. Additionally, CV staining only gives total biomass information. Therefore, a flow-cell assay was implemented with the possibility to insert plates of different materials and to analyze biofilm growth on the surface of the plates by CLSM. Careful adjustment of the height of plastic plates (to ~ 1 mm below the cover slip lid) on the top of the flow cell is crucial to ensure similar flow characteristics for all plates. To further minimize such influences, plates with immobilized PLE were placed between two control plates. Four pictures were taken of each control plate and eight of the plate with immobilized PLE. The results were consistent with those obtained with the SIL tubes. However, time-resolved analysis might yield still more insight into the inhibition effects. Our data clearly suggest that immobilization of commercial enzymes HLE, PKA, and PLE results in reduced biofilm formation. As it is highly likely that immobilized esterases and acylase also hydrolyze metabolites other than P. aeruginosa AHLs, it might be difficult to prove that reduced biofilm formation is indeed a direct result of QQ. It is perhaps noteworthy that the role of QS during biofilm formation has been critically discussed within this research framework, as LasI and RhlI mutants are not necessarily biofilm negative (unpublished data).[44, 45] Thus, the reduced biofilm formation could also be a result of reduced motility. Biofilm growth characteristics can still strengthen the suggested QQ. In the 68 h biofilm tube experiments, the bottoms of the tubes with immobilized enzymes were regularly colonized by bacteria, whereas the sides and the top remained biofilm free. This suggests that cells that attached to the bottom of the tubes during inoculation phase were growing but not spreading over the entire tube surface. Also, in the flow cell, P. aeruginosa did not spread across the whole SIL plate surface. Swarming of P. aeruginosa is QS regulated, thus the observed reduced motility suggests a link with QQ.[46] Biofilm growth is temperature dependent, but the inhibitory effect is comparable between room temperature and 35 8C. The effect was shown to persist over 28 days on SIL with immobilized PLE. This proves that even if surfaces are covered with cells (as was the case, in contrast to the 68 h experiments), the immobilized enzymes retained their function. This is especially important when focusing on applications in complex biological environments.

Conclusions In this study it was shown, that P. aeruginosa biofilm growth can be significantly inhibited on different clinically relevant plastic materials by immobilization of commercially available enzymes HLA, PKE, and PLE. Polymer coating made it possible to apply the method to different materials. Presumably P. aeruginosa biofilm growth inhibition is affected by both the amount of adsorbed PEI on a surface and the enzymatic activity of 3OC12-HSL degradation. PLE immobilizates on SIL are  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chembiochem.org stable (half-life, 53 days) under storage conditions, and growth inhibition is maintained over 28 days of P. aeruginosa biofilm growth. CLSM analysis confirmed the results obtained with CV staining. Further research should address quantitative analysis of the PEI coating and enzymatic activity on the surface, time-resolved CLSM biofilm imaging on plastic materials as well as AHL profiles and transcriptome/proteome analyses of inhibited and control biofilms. Multispecies biofilms are the cause of most human infections.[47] Thus, bacterial strains that use signal molecules other than AHLs, such as autoinducer-2, should also be inhibited, for example, by co-immobilization of other QQ enzymes. Mixed-species biofilms and culture media that mimic natural media (e.g., urine, serum, and blood) should be tested, as well as the possible reinforcing effects of QQ enzymes when combined with, for example, agents that reduce bacterial adhesion or releasing antibiotics.

Experimental Section AHL activity assay: C4-HSL, C6-HSL, C7-HSL, C8-HSL, and C12-HSL were purchased from Cayman Chemical Company (Ann Arbor, MI). A stock of C7-HSL (110 mm) was prepared in EtOAc and a stock solution containing each of the other AHLs (5 mm) was prepared in DMSO. HLE (EC 3.1.1.1), PKA (EC 3.5.1.14), and PLE (EC 3.1.1.1) were purchased from Sigma–Aldrich. A stock of each enzyme (10 mg mL1) was prepared in KPi (pH 7, 100 mm). Enzyme stock (100 mL) was added to KPi (890 mL) in a GC vial, and this was placed in an MHR 11 thermoshaker (HLC BioTech, Bovenden, Germany; 200 rpm, 30 8C). After 15 min AHL stock (10 mL) was added (final, 50 mm) to start the reaction. The solution was mixed by inverting, a sample (100 mL) was taken, and the GC vial was placed back in the thermoshaker. EtOAc (300 mL) was added to the sample, the vial was vortexed twice for 5 s, and the phases were allowed to separate. Supernatant (100 mL) was transferred to a GC vial inlay and C7-HSL stock (10 mL) was used as internal standard. Samples were taken at 0, 4, and 8 h, and all experiments were carried out in triplicate. GC/MS: Agilent multimode injector G3440A #150: injection volume 1 mL, splitless; 75 8C for 0.2 min, 600 8C min1, 250 8C for 3 min, total helium flow 54 mL min1, septum purge: 3 mL min1; oven: Agilent GC 7890A, column: Agilent 19091S-433 (30 m, 250  0.25 mm), helium flow 1 mL min1, 75 8C for 2 min, 10 8C min1, 290 8C for 10 min, AUX temperature 290 8C. MSD: Agilent 5975C, solvent delay 5 min, EMV mode relative 0 = 1200 V, scan mode SIM m/z 143 and 156, 4.53 cycles per second, MS source temperature 230 8C, MS quad temperature 150 8C. AHL retention times: 13 min (C4-HSL), 15.5 min (C6-HSL), 16.6 min (C7-HSL), 17.7 min (C8-HSL), and 21.5 min (C12-HSL). Layer-by-layer polymer coating: LBL coating with PEI and PSS was carried out by Surflay Nanotec with proprietary modifications to the general coating method described by Decher and Schlenoff.[35] Confocal laser scanning microscopy of rhodamine-labeled PEI coating was carried out by Surflay Nanotec. Enzyme immobilization: Tubes/plates were autoclaved, washed with H2O (3 ), filled/covered with GDA (5 %), incubated at 4 8C for 1 h, then washed with H2O (4 ) and KPi (pH 7, 1  100 mm). Enzymes were dissolved in KPi (1 mg mL1). The tubes/plates were filled/covered with enzyme solution and incubated at 4 8C overnight. Immobilizates were washed with KPi four times to remove ChemBioChem 2014, 15, 1911 – 1919

1917

CHEMBIOCHEM FULL PAPERS

www.chembiochem.org

unbound enzyme. For biofilm assays, all solutions were filter sterilized (pore size, 0.45 mm).

sured. Relative OD values (relative to the highest control of each experiment) are shown in the graphs.

para-Nitrophenol assay: A stock solution (200 mm) of para-nitrophenyl acetate (Sigma–Aldrich) was prepared in DMSO and stored at 20 8C. Aliquots (100 mL) were added to KPi (900 mL, pH 7.5, 100 mm) to create the reaction stock, and aliquots from this (100 mL, final 2 mm) were added to KPi (900 mL) in a cuvette. OD410 was measured with a UV-160A photometer (Shimadzu, Kyoto, Japan), and the reaction mixture was placed in tubes (i.d. 3 mm, length 81 mm) with immobilized enzymes. The reaction mixture was removed from the tube and transferred to a fresh cuvette, OD410 was measured, and the mixture was placed back in the tube. Samples were taken at 0, 15, and 30 min; experiments were carried out in duplicate. Activities were corrected by control experiments (tubes with immobilized BSA). The tubes were stored in KPi at 4 8C between measurements.

Biofilm cultivation in flow cell reactor: A glass frame (inner dimensions: width: 15 mm, length: 50 mm, height: 4 mm) with tube connections was constructed by a glassblower. The flow cell was closed from one side by gluing a microscope slide (Carl Roth) to the frame using solvent-free SIL glue E43 (DETAKTA, Norderstedt, Germany), connected as for the growth tubes in the tube system, and autoclaved. SIL plates (15  10  2 mm, Medicoplast) were placed in the flow cell aseptically, and adjusted to 1 mm below the upper end of the glass frame with small cover slips. The flow cell was sealed from the top with a cover slip and silicone glue. The system was rinsed with medium for at least one hour and inoculated with LB overnight culture (4 mL, OD590 of 0.01) of P. aeruginosa PAO1 tagged with red fluorescent protein (DsRed)[48] through the inoculation tube. After one hour attachment, the flow was set to 0.225 mL min1 (flow velocity, 1.5 cm min1), with a residence time in the flow cell of 7.33 min (Reynolds number, 0.35).

Biofilm cultivation in tube system: See Figure 10. The tube system consisted of a 5 L medium bottle (Schott, Mainz, Germany) to which an inlet and an outlet were added by a glassblower. The inlet was connected to a sterile filter, and the outlet was connected to an SIL tube (i.d. 8 mm), which was connected to a second tube (i.d. 3 mm) with a reducer and then to eight tubes (3 mm) through Y connectors. These were connected to SIL pump tubes (i.d. 1.52 mm; IDEX Health & Science, Wertheim, Germany). The pump tubes were placed in an IPC pump (IDEX) and connected to the inoculation tubes (i.d. 3 mm), which were connected to the growth tubes (i.d. 3 mm, length 81 mm); these were connected to waste tubes. All reducers, connectors, and tubes (except pump tubes and growth tubes) were purchased from Carl Roth. Pump tubes were bought from IDEX, and growth tubes were bought from Medicoplast (Illingen, Germany). Minimal medium contained gluconic acid sodium salt (10 mm), KNO3 (10 mm), NaH2PO4 (1.25 mm), and K2HPO4 (2.8 mm), and was adjusted to pH 7.5 with KOH or NaOH. This was placed in the medium bottle, and the whole system was autoclaved. Presterilized MgSO4 was added aseptically (final, 1 mm). Before the inoculation, the system was run for at least 1 h to equilibrate and remove gas bubbles. P. aeruginosa PAO1 was grown in lysogeny broth (LB) at 35 8C on a KS-10 shaker (Edmund Bhler, Hechingen, Germany; 200 rpm). Overnight culture was diluted to an OD590 of 0.01 in LB, and samples (0.6 mL) were injected into the growth tube with a syringe and needle (penetrating the inoculation tube and moving the tip of the needle through the connector into the growth tube). After 1 h of attachment time, the flow rate was set to 0.1 mL min1 (flow velocity, 1.41 cm min1), with a residence time of 5.73 min in the growth tubes (Reynolds number, 0.97). All experiments were carried out in duplicate, except the time-resolved experiments (Figures 5 and 8), which were carried out once. Crystal Violet staining: The tube connection to the medium bottle was clamped and disconnected, thus allowing the medium in the tube system to be replaced by air. The growth tubes were disassembled from the system, connected to a syringe, filled with methanol, and incubated for 20 min. The methanol was removed, and the tube was allowed to dry for at least one hour. CV working solution (0.1 %, w/v) in H2O was prepared from a stock solution in ethanol (1.5 %, w/v). The tubes were filled with CV working solution and incubated for 40 min. The tubes were emptied, washed four times with NaCl (0.9 %, w/v in H2O), placed in a 15 mL falcon tube containing acetic acid (5 mL, 33 %), vortexed, and OD590 was mea-

 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

CLSM biofilm imaging and data treatment: Image acquisition was performed with an eclipse 80i C1 confocal laser-scanning microscope (Nikon Instruments Inc., Melville, US). Images were obtained through a Plan Flour objective (20  /0.5 DIC M/N2; Nikon), with excitation from a helium/neon laser (543 nm) and detection at 605 nm. Additional settings were: 1.68 ms pixel dwell time, 512 (x)  512 (y) steps, 1.5 mm (z) step size,  100 gain, and 60 mm pinhole. Three-dimensional image reconstructions, surface area and volume quantification of biofilms was done using IMARIS software (version x64 7.3.1; Bitplane AG, Zrich, Switzerland). Statistics: p-values were calculated by using Welch’s t-test[49] with the p-value calculator from Social Science Statistics (http:// www.socscistatistics.com/pvalues/tdistribution.aspx).

Acknowledgements We thank Dr. Babu Halan for guidance with the flow cell and CLSM analysis, Dr. Uwe Jandt for assistance with the CLSM, Prof. Leo Eberl for providing the P. aeruginosa PAO1 tagged with red fluorescent protein DsRed, as well as the Federal Ministry for Education and Research (BMBF), project number 0315587D, for financial support. Keywords: biofilms · immobilization · lactones · polymer coating · quorum quenching [1] P. Stoodley, K. Sauer, D. G. Davies, J. W. Costerton, Annu. Rev. Microbiol. 2002, 56, 187 – 209. [2] L. Hall-Stoodley, J. W. Costerton, P. Stoodley, Nat. Rev. Microbiol. 2004, 2, 95 – 108. [3] N. Høiby, T. Bjarnsholt, M. Givskov, S. Molin, O. Ciofu, Int. J. Antimicrob. Agents 2010, 35, 322 – 332. [4] Z. Lewandowski, J. P. Boltz in Treatise on Water Science, Vol 4: WaterQuality Engineering (Ed.: P. A. Wilderer), Elsevier, Oxford, 2011, pp. 529 – 570. [5] B. Halan, K. Buehler, A. Schmid, Trends Biotechnol. 2012, 30, 453 – 465. [6] Springer Series on Biofilms (Eds.: H.-C. Flemming, P. Murthy, R. Venkatesan, K. Cooksey), Springer, Berlin, 2009. [7] A. Resch, R. Rosenstein, C. Nerz, F. Gçtz, Appl. Environ. Microbiol. 2005, 71, 2663 – 2676. [8] D. Davies, Nat. Rev. Drug Discovery 2003, 2, 114 – 122. [9] R. D. Wolcott, D. D. Rhoads, M. E. Bennett, B. M. Wolcott, L. Gogokhia, J. W. Costerton, S. E. Dowd, J. Wound Care 2010, 19, 45 – 53.

ChemBioChem 2014, 15, 1911 – 1919

1918

CHEMBIOCHEM FULL PAPERS [10] R. Siegel, J. Ma, Z. Zou, A. Jemal, Ca-Cancer J. Clin. 2014, 64, 9 – 29. [11] B. W. Trautner, R. O. Darouiche, Arch. Intern. Med. 2004, 164, 842 – 850. [12] A report from the NNIS System, Am. J. Infect. Control 2004, 32, 470 – 485. [13] I. Banerjee, R. C. Pangule, R. S. Kane, Adv. Mater. 2011, 23, 690 – 718. [14] E. M. Hetrick, M. H. Schoenfisch, Chem. Soc. Rev. 2006, 35, 780 – 789. [15] D. Roe, B. Karandikar, N. Bonn-Savage, B. Gibbins, J.-B. Roullet, J. Antimicrob. Chemother. 2008, 61, 869 – 876. [16] C. Aymonier, U. Schlotterbeck, L. Antonietti, P. Zacharias, R. Thomann, J. C. Tiller, S. Mecking, Chem. Commun. 2002, 3018 – 3019. [17] M. E. Skindersoe, M. Alhede, R. Phipps, L. Yang, P. O. Jensen, T. B. Rasmussen, T. Bjarnsholt, T. Tolker-Nielsen, N. Høiby, M. Givskov, Antimicrob. Agents Chemother. 2008, 52, 3648 – 3663. [18] I. Raad, R. Darouiche, R. Hachem, M. Sacilowski, G. P. Bodey, Antimicrob. Agents Chemother. 1995, 39, 2397 – 2400. [19] J. Lçnn-Stensrud, M. A. Landin, T. Benneche, F. C. Petersen, A. A. Scheie, J. Antimicrob. Chemother. 2009, 63, 309 – 316. [20] E. M. Hetrick, J. H. Shin, H. S. Paul, M. H. Schoenfisch, Biomaterials 2009, 30, 2782 – 2789. [21] M. Chen, Q. Yu, H. Sun, Int. J. Mol. Sci. 2013, 14, 18488 – 18501. [22] P. Kurt, L. Wood, D. E. Ohman, K. J. Wynne, Langmuir 2007, 23, 4719 – 4723. [23] B. LaSarre, M. J. Federle, Microbiol. Mol. Biol. Rev. 2013, 77, 73 – 111. [24] W. Wang, T. Morohoshi, T. Ikeda, L. Chen, Acta Biochim. Biophys. Sin. 2008, 40, 1023 – 1028. [25] B. A. Annous, P. M. Fratamico, J. L. Smith, J. Food Sci. 2009, 74, R24 – R37. [26] N. A. Whitehead, A. M. Barnard, H. Slater, N. J. Simpson, G. P. Salmond, FEMS Microbiol. Rev. 2001, 25, 365 – 404. [27] S. L. McKnight, B. H. Iglewski, E. C. Pesci, J. Bacteriol. 2000, 182, 2702 – 2708. [28] J. Lee, J. Wu, Y. Deng, J. Wang, C. Wang, J. Wang, C. Chang, Y. Dong, P. Williams, L.-H. Zhang, Nat. Chem. Biol. 2013, 9, 339 – 343. [29] N. Amara, B. P. Krom, G. F. Kaufmann, M. M. Meijler, Chem. Rev. 2011, 111, 195 – 208. [30] R. Czajkowski, S. Jafra, Acta Biochim. Pol. 2009, 56, 1 – 16. [31] F. Xu, T. Byun, H.-J. Dussen, K. R. Duke, J. Biotechnol. 2003, 101, 89 – 96. [32] K.-M. Yeon, C.-H. Lee, J. Kim, Environ. Sci. Technol. 2009, 43, 7403 – 7409. [33] J.-H. Kim, D.-C. Choi, K.-M. Yeon, S.-R. Kim, C.-H. Lee, Environ. Sci. Technol. 2011, 45, 1601 – 1607.

 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chembiochem.org [34] F. S. W. Ng, D. M. Wright, S. Y. K. Seah, Appl. Environ. Microbiol. 2011, 77, 1181 – 1186. [35] Multilayer Thin Films: Polymer and Polyelectrolyte Multilayer Systems, (Eds.: G. Decher, J. B. Schlenoff), Wiley-VCH, Weinheim, 2002. [36] E. Peeters, H. J. Nelis, T. Coenye, J. Microbiol. Methods 2008, 72, 157 – 165. [37] T. Kçhler, A. Buckling, C. van Delden, Proc. Natl. Acad. Sci. USA 2009, 106, 6339 – 6344. [38] D. A. D’Argenio, M. Wu, L. R. Hoffman, H. D. Kulasekara, E. Dziel, E. E. Smith, H. Nguyen, R. K. Ernst, T. J. L. Freeman, D. H. Spencer, M. Brittnacher, H. S. Hayden, S. Selgrade, M. Klausen, D. R. Goodlett, J. L. Burns, B. W. Ramsey, S. I. Miller, Mol. Microbiol. 2007, 64, 512 – 533. [39] J. P. Pearson, K. M. Gray, L. Passador, K. D. Tucker, A. Eberhard, B. H. Iglewski, E. P. Greenberg, Proc. Natl. Acad. Sci. USA 1994, 91, 197 – 201. [40] Y.-H. Dong, J.-L. Xu, X.-Z. Li, L.-H. Zhang, Proc. Natl. Acad. Sci. USA 2000, 97, 3526 – 3531. [41] L.-H. Wang, L.-X. Weng, Y.-H. Dong, L.-H. Zhang J. Biol. Chem. 2004, 279, 13645 – 13651. [42] D. Liu, P. W. Thomas, J. Momb, Q. Q. Hoang, G. A. Petsko, D. Ringe, W. Fast, Biochemistry 2007, 46, 11789 – 11799. [43] B. Brena, P. Gonzlez-Pombo, F. Batista-Viera in Methods in Molecular Biology, Vol. 1051: Immobilization of Enzymes and Cells (Ed.: J. M. Guisan), Humana, Totowa, 2013, pp. 15 – 31. [44] A. Heydorn, B. Ersbøll, J. Kato, M. Hentzer, M. R. Parsek, T. Tolker-Nielsen, M. Givskov, S. Molin, Appl. Environ. Microbiol. 2002, 68, 2008 – 2017. [45] C. T. O’Loughlin, L. C. Miller, A. Siryaporn, K. Drescher, M. F. Semmelhack, B. L. Bassler, Proc. Natl. Acad. Sci. USA 2013, 110, 17981 – 17986. [46] J. D. Shrout, D. L. Chopp, C. L. Just, M. Hentzer, M. Givskov, M. R. Parsek, Mol. Microbiol. 2006, 62, 1264 – 1277. [47] R. Wolcott, S. Dowd, Plast. Reconstr. Surg. 2011, 127, 28S – 35S. [48] K. Riedel, M. Hentzer, O. Geisenberger, B. Huber, A. Steidle, H. Wu, N. Høiby, M. Givskov, S. Molin, L. Eberl, Microbiology 2001, 147, 3249 – 3262. [49] B. L. Welch, Biometrika 1947, 34, 28 – 35.

Received: January 9, 2014 Published online on July 15, 2014

ChemBioChem 2014, 15, 1911 – 1919

1919