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Bioresponsive polymers for the detection of bacterial contaminations in platelet concentrates
New Biotechnology Volume 31, Number 2 March 2014
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Research Paper
Bioresponsive polymers for the detection of bacterial contaminations in platelet concentrates Clemens Gamerith1, Andrea Heinzle1, Konstantin P. Schneider1, Elisabeth Hulla-Gumbsch1, Ulrike Gewessler1, Laurent Ducoroy2, Michael Gehrer3, Thomas Wagner4, Eva Sigl1 and Georg M. Guebitz1,5 1
Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria Macopharma, Chausse´e Fernand Forest 200, 59200 Tourcoing, France 3 Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Universita¨tsplatz 4, 8010 Graz, Austria 4 Department of Blood Group Serology and Transfusion Medicine, Medical University of Graz, Auenbruggerplatz 3, 8036 Graz, Austria 5 Institute of Environmental Biotechnology, University of Natural Resources and Life Sciences, Vienna, Konrad Lorenz Strasse 20, 3430 Tulln, Austria 2
Bacterial contamination of platelet concentrates (PCs) can lead to fatal transfusion transmitted diseases and is the most abundant infectious risk in transfusion medicine. The storage conditions of PCs provide a good environment for bacterial growth. The detection of these contaminations at an early stage is therefore important to avoid the transfusion of contaminated samples. In this study, bioresponsive polymer (BRP) systems were used for the detection of microorganisms in PCs. The backbone of the polymer consisted of labelled protein (casein), which was demonstrated to be degraded by pure proteases as models and by extracellular enzymes released by contaminating microorganisms. The concomitant colour change was easily visible to the naked eye. To enhance stability, the protein was cross-linked with glycidyl methacrylate (GMA). The cross-linked polymer was easier to handle but was less sensitive than the non-cross-linked material. A contamination of a PC with 10 CFU/mL S. aureus was detectable after 24 hours. The visible colour reaction was quantified as a DE value according to the CIELab concept. A DE value of 21.8 was already reached after 24 hours. Hence, this simple but effective system could prevent transfusion of a contaminated PC.
Introduction Bacterial contamination of blood products represents a considerable risk in transfusion medicine whereby the most abundant infectious risk proceeds from bacterially contaminated platelet concentrates (PCs) [1]. The storage conditions of PCs (20–248C) provide a good environment for bacterial growth. Approximately 1 in 2000–3000 platelet products are bacterially contaminated [2,3]. The risk of transfusion-transmitted bacterial infections is lowered by different detection systems which are used for the screening of bacterial contamination in PCs (BacT/ALERT, eBDS, PGD) [4–8]. These systems often require special equipment and Corresponding author: Sigl, E. ([email protected])
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expertise. The procedures can be time consuming and may also lead to false positive signals [9]. Moreover, none of these detection methods measure parameters directly in PCs. An integrated sensor device could give a faster response, which could lead to a lower detection limit. Although different methods are used nowadays, transfusion-transmitted bacterial infections still occur and represent a serious risk to patients [10–12]. Propionibacterium acnes and different Staphylococcus species are the most frequent pathogens detected in contaminated PCs [2,13]. Transfusions of PCs contaminated with P. acnes rarely lead to transfusion reactions. Hence, these contaminations are considered to be less relevant in comparison to different Staphylococcus contaminations [14]. Therefore, Staphylococcus spp. were chosen as
1871-6784/$ - see front matter ß 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.nbt.2013.11.001
model pathogens in this study for detection with bioresponsive systems. The use of in situ bioresponsive systems can provide a cheap and easy to use alternative to established equipment-based systems. Bioresponsive devices not only are designed to indicate the presence of contaminating bacteria via simple colour changes but could also release antimicrobial molecules. Common systems of (bio) responsive polymers are designed to be sensitive to pH or temperature changes in different environments [15,16]. A more selective system is obtained with devices responding to enzymes as triggers. The contaminating pathogens release extracellular enzymes which can be used to trigger different reactions. These triggers can provoke a controlled release of different substances ranging from small dyes to large and complex molecules such as antibiotics. Previous studies conducted by our group and by others have shown that bioresponsive polymers (BRPs) based on enzyme triggered reactions are an appropriate method to detect both contaminating organisms [17–19] and wound infection [20–23]. This technology can be used to detect contaminations at an early stage. Bacteria in contaminated PCs, such as S. epidermidis, S. aureus and P. acnes, produce extracellular proteases which could serve as biomarkers for their detection [24– 26]. Therefore, in this study BRPs based on modified and labelled proteins were constructed for the detection of proteases secreted by potential contaminants/pathogens. The stability of the biopolymers was enhanced by cross-linking through insertion of methacrylic groups. Biotransformation in the presence of contaminating organisms with concomitant colour changes was assessed as an indicator of bacterial contamination.
Materials and methods Chemicals and enzymes Casein from bovine milk, Casein Hydrolysate, Reactive Black 5, glycidyl methacrylate (GMA), sodium persulphate, tetramethylethylendiamine (TEMED), tryptic soy broth, sodium sulphate, sodium carbonate, hydrochloric acid (HCl), neomycin or trisuphate salt hydrate and protease from Aspergillus oryzae were all supplied by Sigma–Aldrich (St. Louis, USA). Storage solution for platelets (SSP+-buffer) as well as PC bags (small and large) was obtained from MacoPharma (Tourcoing, France). Columbia-IIIagar plates containing 5% sheep blood were purchased from Becton Dickinson (Heidelberg, Germany). S. aureus (American type culture collection (ATCC) 29213) was received from the Institute of Hygiene, Microbiology and Environmental Medicine (Medical University Graz, Austria) culture collection. PCs from the Department of Blood Group Serology and Transfusion Medicine (Medical University Graz, Austria) were used.
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automated separator (Compomat G3, NPBI, Amsterdam, the Netherlands). Subsequently, according to a randomisation scheme, buffy coats from four different donors and one bag containing 300 mL of additive solution (PAS3; composition, 116 mM sodium chloride, 30 mM sodium acetate, 2.94 g/l sodium citrate, pH 7.2) were connected by use of a sterile connection device (TSCD, Terumo Corp., Tokyo, Japan) and were pooled in one container. Subsequently a 1 l polyolefin bag (Baxter) and a white-blood-cell-reduction filter (Autostop BC, Pall, Cornwall, UK) were connected, and the pool was centrifuged at 500 g for 8 min at 228C. The supernatant was expressed immediately into the storage bag by means of a plasma extractor. The buffy coat pool was stored for up to five days at 228C on a flat-bed agitator under constant agitation at 60 rpm [27].
Growth kinetics of S. aureus in PCs Three different PCs (A, B and C, 300 mL) were each divided under sterile conditions into four smaller bags (75 mL) obtained from MacoPharma. For each of the three PCs three different inoculation concentrations were used (3 2, 40 10, 470 60 CFU/mL as well as one negative control). An overnight culture (ONC) of S. aureus was cultivated in 40 mL tryptic soy broth at 378C in a baffled 300 mL Erlenmeyer flask under aerobic conditions and shaking at 100 rpm on a Multitron II shaker (Laurel, USA). The different dilutions of the ONC for the inoculation of the PCs were performed with sterile SSP+-buffer (storage solution for platelets+) to avoid further nutrients supply in the PC. The inoculation was performed using sterile syringes. After the injection, the syringe was purged three times with PC. Further, the spiked samples were plated on Columbia-III-agar plates. The following volumes were plated: negative control and 470 CFU/mL: 100 mL three times each; 3 and 40 CFU/mL: 1 mL three times each, each mL divided into 333 mL aliquots for plating. After 24 hours of incubation the grown colonies were counted. The spiked PCs were stored at room temperature (228C) and at gentle agitation at 30 rpm. After 24 hours, the CFU/mL were determined again using 100 mL aliquots.
Oxygen measurements The oxygen consumption measurement was carried out according to a method previously described by Greimel et al. [28]. Briefly, an optical oxygen meter (Firesting O2, Pyro Science, Aachen, Germany) was used for the online measurements in PC bags. The sensor spots for the O2 measurement were integrated into the bags under sterile conditions, before the PC was transferred. One PC was divided into four smaller bags (each 40 mL). Two of them were used as negative control and the other two were contaminated with 10 CFU/mL (S. aureus). The PCs were stored at room temperature (228C) with gentle agitation (60 rpm).
PC preparation Preparation of blood cells from whole blood donations
Production of BRPs
Buffy coat-derived PCs were prepared from 450 mL 10% whole blood obtained from healthy volunteer blood donors according to the Austrian regulations for blood donation and after informed consent. Whole blood was collected into triple bags containing 63 mL CPD in the primary bag (MacoPharma, Tourcoing, France) and centrifuged at 4000 g for 10 min at 208C within 12 hours. Red blood cells (RBCs) and plasma were separated from the buffy coat fraction and transferred into satellite containers by use of an
For the production of BRP, casein was labelled with a reactive dye. 1 g Reactive Black 5 was dissolved in 50 mL ddH2O (dd: double distilled). 50 mL additive solution (5 g of sodium sulphate + 2 g of sodium carbonate dissolved in 50 mL ddH2O) was added and incubated with 5 g of non-water-soluble casein from bovine milk for 10 min at 258C under shaking conditions. A similar method with reactive orange 16 was recently published [29]. After incubation for 45 min at 658C the reaction mixture was washed with www.elsevier.com/locate/nbt
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ddH2O. The washing procedure was repeated until the supernatant was clear. In a final step the dyed casein was frozen in liquid nitrogen and dried via lyophilisation overnight (Labconco Freezone 4.5 freeze dryer, Kansas City, USA). The BRP was stored under exclusion of light. Sterilisation was performed using ethanol (70%) and an end-over-end rotator for 24 hours. The ethanol was removed after 24 hours and the sterilised BRP was washed with sterile SSP+-buffer several times.
Production of cross-linked BRPs (clBRPs) Research Paper
For functionalisation of casein, a procedure for the modification of polysaccharides with GMA, which was recently published [17,19,30], was slightly modified. Briefly, 15 g of hydrolysed casein from bovine milk were dissolved in 85 mL ddH2O. After adding 7.5 mL GMA and 0.5 mL 10 M HCl the reaction was stirred for 48 hours at 358C. The samples were stored until further use at 48C. Polymerisation of the modified protein was performed based on a modified method previously described [17,19]. 15 g of caseinmethacrylate were mixed with 2 g of BRP and put into a glass container (3 cm 5 cm). After adding 20 mg sodium persulphate the polymerisation process was started by adding 8 mL of TEMED. After finishing the polymerisation the polymer was cut into 1 cm 1 cm pieces and was washed with ddH2O for a couple of times. The obtained clBRPs were treated in two different ways. In both approaches (A and B), the samples were dried in an oven at 608C over night. After the drying process, only samples B were additionally steam sterilised at 1218C for 20 min.
Enzyme triggered colour reaction of clBRPs and BRPs The clBRPs (1 cm 1 cm) obtained were transferred into 20 mL glass containers and 10 mL of SSP+-buffer and different protease concentrations (protease from A. oryzae; 0, 0.03, 0.1, 0.3, and 2.5 U) were added. The colour change during the incubation was monitored at 590 nm (Platereader infinite M200, Tecan, Ma¨nnedorf, Switzerland). In a next step, the colour change of clBRPs upon incubation with S. aureus was assessed. One PC was spiked with S. aureus (1 103 CFU/mL) and a second one served as a control. The PCs were incubated at room temperature at gentle agitation (30 rpm) for 48 hours. Thereafter, growth of the bacteria was stopped through the addition of neomycin (25 mg/mL). 2 mL of each PC (control and S. aureus) presumably containing enzymes secreted by S. aureus were then added to cuvettes containing clBRPs (1 cm 1 cm) and their colour change was measured after 48 hours of incubation at 30 rpm with a Colorlite sph 850 (Colorlite GmbH, Katlenburg-Lindau, Germany) spectrophotometer. BRPs were compared to clBRPs in terms of reaction time. 20 mg of BRPs were suspended in 5 mL SSP+-buffer. Different protease concentrations (protease from A. oryzae; 0, 0.01, 0.03, 0.06, 0.125, 0.3, 0.6, 1.25, 0.3, 0.6, 1.25, 2.5, 5 and 10 U) were added. The samples were incubated at room temperature (228C) under shaking conditions. For the absorbance measurements 100 mL of each sample were transferred into a 96-well plate. Absorbance at 590 nm was measured after 20, 60 and 180 min. A proof of concept was also performed directly in PC. 40 mL PC were transferred into sterile 250 mL flasks. 80 mg of the BRPs were added as substrate for the trigger enzymes. One sample was spiked with S. aureus (10 CFU/mL) whereas the control was not inoculated. For the measurement of the colour change, 2 mL of each 152
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flask were transferred into a cuvette and a Colorlite sph 850 was used for the colour measurement after 0, 24 and 48 hours.
Results and discussion Bacterial growth in PC bags In a first step, growth of S. aureus in PCs was investigated. CFU analysis together with oxygen consumption in PC bags after 24 hours indicated a considerably fast growth of this bacterium in PCs (Fig. 1). Upon inoculation with 10 CFU/mL, the oxygen concentration in samples (S. aureus 1 and 2) showed a rapid decrease indicating bacterial contamination. After 30 hours of incubation, only less than 10% of the air saturation was left which correlated with the growth of the organisms. Figure 1 clearly illustrates that initial CFU/mL values of 3 increased to values around 1.5 105 CFU/mL within 24 hours. Initial concentrations of 40 CFU/mL led to 2 106 CFU/mL after 24 hours incubation. These results are comparable with the studies of Brecher et al. [31]. By contrast, the oxygen concentration of the two control samples (controls 1 and 2) only slightly decreased over the first 30 hours, probably due to the additional air which was inadvertently entrapped in the bags and hence led to an oxygen equilibrium between the liquid and the aqueous phase. The oxygen consumption of platelets could be an alternative explanation for the slow decrease at the beginning. The well established BacT/ALERT system for detection of bacterial contaminations is designed to take platelet samples at least 24 hours after collection or later. However, the growth kinetics of S. aureus demonstrate that under the storage conditions of the PCs bacterial growth can be fast enough to reach crucial values already before 24 hours.
BRP design for detection of microbial contaminations Two different types of BRPs (BRP and clBRP) were designed and tested for the detection of contaminating pathogens directly in
FIGURE 1
Growth of S. aureus in PC bags. (a) Oxygen concentration of two contaminated samples inoculated with S. aureus (10 CFU/mL) and two control PC bags. (b) Bacterial counts of samples x, y and z after incubation for 24 hours at 228C and gentle agitation inoculated with 3 2, 40 10 and 470 60 CFU/mL, respectively.
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FIGURE 2
Schematic representation of the BRP system integrated into a PC bag. The operating biomatrix consists of cross-linked casein which is hydrolysed by proteases secreted by contaminating pathogens. This degradation process of the biomatrix can be observed via release of a dye (Reactive Black 5).
PCs. Thereby extracellular proteases secreted by relevant pathogens like S. aureus trigger the hydrolysis of the BRPs consequently leading to a colour change. A schematic representation of this BRPbased detection system is shown in Fig. 2. The most frequent pathogens (P. acnes, S. aureus and S. epidermidis) are able to produce extracellular proteases [24–26]. Hence, in this study an operating biomatrix was constructed consisting of modified model proteins. The constructed BRP consisted of a water-insoluble protein (casein) which was modified with a reactive dye (Reactive Black 5). Hydrolysis of this BRP leads to a visible colour reaction. For the clBRP in a first step, water-soluble hydrolysed casein was modified with GMA. On the one hand, the epoxy group of the GMA enables cross-linking with amines, hydroxyl groups or carboxylic acids on the casein. On the other hand, the acrylic groups were used for the cross-linking with other vinyl monomers [32]. The stained BRP, which was embedded into the clBRP, was used for the visualisation of the degradation process. The radical polymerisation process was started by the addition of sodium persulphate and TEMED as
described by Reis et al. [33]. The main benefit of the cross linked polymer was an enhanced usability as well as stability [34] and can therefore improve hydrogel properties which are widely used in medical applications [35,36].
Colour reaction of the clBRPs in the presence of proteases or S. aureus clBRPs were incubated with different protease concentrations and the colour changes were measured. The detection limit was also determined. SSP+-buffer was used to simulate the real conditions in PC. Figure 3(a) shows the colour change dependence on enzyme activity. The colour change was recorded directly in the supernatant in the reaction vessel. The control sample showed that the clBRP was at least stable for 96 hours. Figure 3(a) demonstrates the colour formation dependence on enzyme activity after 24 hours. Small enzyme concentrations of 0.03 U led to a 50% increase in colour formation after 24 hours. Higher concentrations significantly reduce the detection time. The polymer treated with
FIGURE 3
Colour change of clBRPs in the presence of proteases. (a) Visual colour change of the operating biomatrix supernatant after incubation with five different protease concentrations for 24 hours in SSP+-buffer. Measured absorbance increase example shown for incubation for 24 hours. (b) Visual and quantified colour change of clBRPs in contaminated PC. The clBRP in sample A was dried before use; that in sample B was dried and autoclaved. The PC was incubated with S. aureus (1 103 CFU/mL) for 48 hours. Thereafter, the colour reaction was carried out in cuvettes and colour change was quantified after 48 hours as DE values according to the CIELab concept. www.elsevier.com/locate/nbt
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0.25 U substantially increased colour formation after 3 hours (2.6 higher) and showed a 600% higher value after 24 hours. With this experiment, the protease dependent colour reaction of the BRP was clearly demonstrated and confirmed the potential of enzyme-responsive polymers as recently postulated by Thornton et al. [37] and others. In a next step, the clBRP was tested directly in PCs. BRP A in Fig. 3(b) was dried before use, BRP B was dried and steam sterilised. The BRP was still stable after steam sterilisation, provided it was dried beforehand. One PC was inoculated with 1 103 CFU/mL S. aureus and incubated for 48 hours directly in a PC bag. A second PC (not inoculated) served as control. After 48 hours incubation, antibiotics were added to stop and inhibit bacterial growth during the release experiment. Consequently, any hydrolysis and concomitant colour change of the BRPs can be related to the presence of extracellular proteases. Figure 3(b) shows the colour change of the contaminated PCs after 48 hours incubation. The negative controls showed no significant change in colour, whereas the samples inoculated with S. aureus showed a clear difference when compared to the original sample before incubation. Absorbance measurements directly in PC are not possible due to turbidity. Therefore, the DE values (according to the CIELab concept) were measured for the quantification of the colour change. High DE values of around 35 were obtained for both treatment methods of the polymer (A and B). Hence, enzyme triggered dye liberation was clearly achieved resulting in a visible colour change. A contamination of a PC with S. aureus could be indicated by a clBRP, but the incubation time still has to be shortened. Although proteolytic degradation of casein is well known for numerous proteases [38–40], cross-linking obviously hinders access of the enzyme.
Colour reaction of the BRPs in presence of proteases or S. aureus A decrease of the reaction time was achieved by the use of BRP which were not cross-linked. The samples were centrifuged before the
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absorbance increase was determined after 20, 60 and 180 min, respectively (Fig. 4). Higher enzyme concentrations led to a higher sensitivity of the detection system. A controlled release was measurable after 20 min using enzyme concentrations above 0.5 U/mL. Compared to the clBRP, an eightfold higher signal was measured after 3 hours and 0.12 U/mL trigger enzyme. This enhancement clearly improves the sensitivity of the BRP system. Similarly, when directly incubated in PC, a significant faster response was also obtained. Figure 4(b) illustrates the colour change in the contaminated PC in comparison to the control sample. The polymer was sterilised with ethanol before transfer to the reaction solution. The contaminated sample was inoculated with 10 CFU/mL. The DE value after 24 hours of incubation at room temperature and gentle agitation was 21.8, and after 48 hours considerably higher (49.4). A contamination with 10 CFU/mL could be detected after 24 hours using this BRP system. According to literature, the BacT/ALERT detection system (Biomerieux) has a detection time of around 11 hours for inoculation with 10 CFU/mL (S. aureus) [41–43]. The manufacturer manual recommends at least 24 hours incubation before injection into the detection system which leads to a final detection time of 35 hours. The Pan Genera Detection Immunoassay (Verax Biomedical Incorporated) gives the first positive signal after 48 hours of incubation with 0.13 CFU/mL S. aureus [44]. Nine out of ten samples inoculated with 169 CFU/mL S. aureus were detected after 24 hours using the Pall BDS system (Pall Corporation) [45]. Hence, the detection time of BRP studied here is within the magnitude of the commercial available systems or even lower. The response time could be fast enough to avoid the transfusion of a contaminated PC. Hence, this system could lower the risk of transfusion-transmitted infections. The use of an integrated detection system also avoids the possibility of false positive signals. It has to be noted that for a medical application the model substances (dye, protein) would have to be exchanged according to legal regulations of medical products.
FIGURE 4
Hydrolysis of BRPs. In contrast to the previous experiments, the BRP used in this attempt were not cross-linked. Different enzyme concentrations were used in (a) to visualise the colour change. The dye released using enzyme concentrations above 120 mU/mL can be detected after 20 min. (b) The colour change of contaminated (S. aureus, 10 CFU/mL) and control samples was quantified as DE values according to the CIELab concept. An initial inoculation with 10 CFU/mL S. aureus was detected after 24 hours of incubation. 154
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Conclusion
Acknowledgements
In this study it was demonstrated that protein based BRPs can be used for the detection of pathogens in PCs. Thereby, a visible colour reaction indicates a contamination and gives a clear yes/no signal. The backbone of the polymer was designed to be degradable by extracellular proteases released from contaminating pathogens. The model substances used in this study would have to be changed in future experiments according to legal regulations of medical products while a screening for a larger number of different pathogens should also be conducted. Fluorescent dyes could be used to improve the sensitivity of the detection system.
This study was performed within the Austrian Centre of Industrial Biotechnology ACIB, the COMET K Project MacroFun project and the COST Action 868. This work has been supported by the Federal Ministry of Economy, Family and Youth (BMWFJ), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol and ZIT – Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG.
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Bioresponsive polymers for the detection of bacterial contaminations in platelet concentrates Clemens Gamerith1, Andrea Heinzle1, Konstantin P. Schneider1, Elisabeth Hulla-Gumbsch1, Ulrike Gewessler1, Laurent Ducoroy2, Michael Gehrer3, Thomas Wagner4, Eva Sigl1 and Georg M. Guebitz1,5 1
Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria Macopharma, Chausse´e Fernand Forest 200, 59200 Tourcoing, France 3 Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Universita¨tsplatz 4, 8010 Graz, Austria 4 Department of Blood Group Serology and Transfusion Medicine, Medical University of Graz, Auenbruggerplatz 3, 8036 Graz, Austria 5 Institute of Environmental Biotechnology, University of Natural Resources and Life Sciences, Vienna, Konrad Lorenz Strasse 20, 3430 Tulln, Austria 2
Bacterial contamination of platelet concentrates (PCs) can lead to fatal transfusion transmitted diseases and is the most abundant infectious risk in transfusion medicine. The storage conditions of PCs provide a good environment for bacterial growth. The detection of these contaminations at an early stage is therefore important to avoid the transfusion of contaminated samples. In this study, bioresponsive polymer (BRP) systems were used for the detection of microorganisms in PCs. The backbone of the polymer consisted of labelled protein (casein), which was demonstrated to be degraded by pure proteases as models and by extracellular enzymes released by contaminating microorganisms. The concomitant colour change was easily visible to the naked eye. To enhance stability, the protein was cross-linked with glycidyl methacrylate (GMA). The cross-linked polymer was easier to handle but was less sensitive than the non-cross-linked material. A contamination of a PC with 10 CFU/mL S. aureus was detectable after 24 hours. The visible colour reaction was quantified as a DE value according to the CIELab concept. A DE value of 21.8 was already reached after 24 hours. Hence, this simple but effective system could prevent transfusion of a contaminated PC.
Introduction Bacterial contamination of blood products represents a considerable risk in transfusion medicine whereby the most abundant infectious risk proceeds from bacterially contaminated platelet concentrates (PCs) [1]. The storage conditions of PCs (20–248C) provide a good environment for bacterial growth. Approximately 1 in 2000–3000 platelet products are bacterially contaminated [2,3]. The risk of transfusion-transmitted bacterial infections is lowered by different detection systems which are used for the screening of bacterial contamination in PCs (BacT/ALERT, eBDS, PGD) [4–8]. These systems often require special equipment and Corresponding author: Sigl, E. ([email protected])
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expertise. The procedures can be time consuming and may also lead to false positive signals [9]. Moreover, none of these detection methods measure parameters directly in PCs. An integrated sensor device could give a faster response, which could lead to a lower detection limit. Although different methods are used nowadays, transfusion-transmitted bacterial infections still occur and represent a serious risk to patients [10–12]. Propionibacterium acnes and different Staphylococcus species are the most frequent pathogens detected in contaminated PCs [2,13]. Transfusions of PCs contaminated with P. acnes rarely lead to transfusion reactions. Hence, these contaminations are considered to be less relevant in comparison to different Staphylococcus contaminations [14]. Therefore, Staphylococcus spp. were chosen as
1871-6784/$ - see front matter ß 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.nbt.2013.11.001
model pathogens in this study for detection with bioresponsive systems. The use of in situ bioresponsive systems can provide a cheap and easy to use alternative to established equipment-based systems. Bioresponsive devices not only are designed to indicate the presence of contaminating bacteria via simple colour changes but could also release antimicrobial molecules. Common systems of (bio) responsive polymers are designed to be sensitive to pH or temperature changes in different environments [15,16]. A more selective system is obtained with devices responding to enzymes as triggers. The contaminating pathogens release extracellular enzymes which can be used to trigger different reactions. These triggers can provoke a controlled release of different substances ranging from small dyes to large and complex molecules such as antibiotics. Previous studies conducted by our group and by others have shown that bioresponsive polymers (BRPs) based on enzyme triggered reactions are an appropriate method to detect both contaminating organisms [17–19] and wound infection [20–23]. This technology can be used to detect contaminations at an early stage. Bacteria in contaminated PCs, such as S. epidermidis, S. aureus and P. acnes, produce extracellular proteases which could serve as biomarkers for their detection [24– 26]. Therefore, in this study BRPs based on modified and labelled proteins were constructed for the detection of proteases secreted by potential contaminants/pathogens. The stability of the biopolymers was enhanced by cross-linking through insertion of methacrylic groups. Biotransformation in the presence of contaminating organisms with concomitant colour changes was assessed as an indicator of bacterial contamination.
Materials and methods Chemicals and enzymes Casein from bovine milk, Casein Hydrolysate, Reactive Black 5, glycidyl methacrylate (GMA), sodium persulphate, tetramethylethylendiamine (TEMED), tryptic soy broth, sodium sulphate, sodium carbonate, hydrochloric acid (HCl), neomycin or trisuphate salt hydrate and protease from Aspergillus oryzae were all supplied by Sigma–Aldrich (St. Louis, USA). Storage solution for platelets (SSP+-buffer) as well as PC bags (small and large) was obtained from MacoPharma (Tourcoing, France). Columbia-IIIagar plates containing 5% sheep blood were purchased from Becton Dickinson (Heidelberg, Germany). S. aureus (American type culture collection (ATCC) 29213) was received from the Institute of Hygiene, Microbiology and Environmental Medicine (Medical University Graz, Austria) culture collection. PCs from the Department of Blood Group Serology and Transfusion Medicine (Medical University Graz, Austria) were used.
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automated separator (Compomat G3, NPBI, Amsterdam, the Netherlands). Subsequently, according to a randomisation scheme, buffy coats from four different donors and one bag containing 300 mL of additive solution (PAS3; composition, 116 mM sodium chloride, 30 mM sodium acetate, 2.94 g/l sodium citrate, pH 7.2) were connected by use of a sterile connection device (TSCD, Terumo Corp., Tokyo, Japan) and were pooled in one container. Subsequently a 1 l polyolefin bag (Baxter) and a white-blood-cell-reduction filter (Autostop BC, Pall, Cornwall, UK) were connected, and the pool was centrifuged at 500 g for 8 min at 228C. The supernatant was expressed immediately into the storage bag by means of a plasma extractor. The buffy coat pool was stored for up to five days at 228C on a flat-bed agitator under constant agitation at 60 rpm [27].
Growth kinetics of S. aureus in PCs Three different PCs (A, B and C, 300 mL) were each divided under sterile conditions into four smaller bags (75 mL) obtained from MacoPharma. For each of the three PCs three different inoculation concentrations were used (3 2, 40 10, 470 60 CFU/mL as well as one negative control). An overnight culture (ONC) of S. aureus was cultivated in 40 mL tryptic soy broth at 378C in a baffled 300 mL Erlenmeyer flask under aerobic conditions and shaking at 100 rpm on a Multitron II shaker (Laurel, USA). The different dilutions of the ONC for the inoculation of the PCs were performed with sterile SSP+-buffer (storage solution for platelets+) to avoid further nutrients supply in the PC. The inoculation was performed using sterile syringes. After the injection, the syringe was purged three times with PC. Further, the spiked samples were plated on Columbia-III-agar plates. The following volumes were plated: negative control and 470 CFU/mL: 100 mL three times each; 3 and 40 CFU/mL: 1 mL three times each, each mL divided into 333 mL aliquots for plating. After 24 hours of incubation the grown colonies were counted. The spiked PCs were stored at room temperature (228C) and at gentle agitation at 30 rpm. After 24 hours, the CFU/mL were determined again using 100 mL aliquots.
Oxygen measurements The oxygen consumption measurement was carried out according to a method previously described by Greimel et al. [28]. Briefly, an optical oxygen meter (Firesting O2, Pyro Science, Aachen, Germany) was used for the online measurements in PC bags. The sensor spots for the O2 measurement were integrated into the bags under sterile conditions, before the PC was transferred. One PC was divided into four smaller bags (each 40 mL). Two of them were used as negative control and the other two were contaminated with 10 CFU/mL (S. aureus). The PCs were stored at room temperature (228C) with gentle agitation (60 rpm).
PC preparation Preparation of blood cells from whole blood donations
Production of BRPs
Buffy coat-derived PCs were prepared from 450 mL 10% whole blood obtained from healthy volunteer blood donors according to the Austrian regulations for blood donation and after informed consent. Whole blood was collected into triple bags containing 63 mL CPD in the primary bag (MacoPharma, Tourcoing, France) and centrifuged at 4000 g for 10 min at 208C within 12 hours. Red blood cells (RBCs) and plasma were separated from the buffy coat fraction and transferred into satellite containers by use of an
For the production of BRP, casein was labelled with a reactive dye. 1 g Reactive Black 5 was dissolved in 50 mL ddH2O (dd: double distilled). 50 mL additive solution (5 g of sodium sulphate + 2 g of sodium carbonate dissolved in 50 mL ddH2O) was added and incubated with 5 g of non-water-soluble casein from bovine milk for 10 min at 258C under shaking conditions. A similar method with reactive orange 16 was recently published [29]. After incubation for 45 min at 658C the reaction mixture was washed with www.elsevier.com/locate/nbt
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ddH2O. The washing procedure was repeated until the supernatant was clear. In a final step the dyed casein was frozen in liquid nitrogen and dried via lyophilisation overnight (Labconco Freezone 4.5 freeze dryer, Kansas City, USA). The BRP was stored under exclusion of light. Sterilisation was performed using ethanol (70%) and an end-over-end rotator for 24 hours. The ethanol was removed after 24 hours and the sterilised BRP was washed with sterile SSP+-buffer several times.
Production of cross-linked BRPs (clBRPs) Research Paper
For functionalisation of casein, a procedure for the modification of polysaccharides with GMA, which was recently published [17,19,30], was slightly modified. Briefly, 15 g of hydrolysed casein from bovine milk were dissolved in 85 mL ddH2O. After adding 7.5 mL GMA and 0.5 mL 10 M HCl the reaction was stirred for 48 hours at 358C. The samples were stored until further use at 48C. Polymerisation of the modified protein was performed based on a modified method previously described [17,19]. 15 g of caseinmethacrylate were mixed with 2 g of BRP and put into a glass container (3 cm 5 cm). After adding 20 mg sodium persulphate the polymerisation process was started by adding 8 mL of TEMED. After finishing the polymerisation the polymer was cut into 1 cm 1 cm pieces and was washed with ddH2O for a couple of times. The obtained clBRPs were treated in two different ways. In both approaches (A and B), the samples were dried in an oven at 608C over night. After the drying process, only samples B were additionally steam sterilised at 1218C for 20 min.
Enzyme triggered colour reaction of clBRPs and BRPs The clBRPs (1 cm 1 cm) obtained were transferred into 20 mL glass containers and 10 mL of SSP+-buffer and different protease concentrations (protease from A. oryzae; 0, 0.03, 0.1, 0.3, and 2.5 U) were added. The colour change during the incubation was monitored at 590 nm (Platereader infinite M200, Tecan, Ma¨nnedorf, Switzerland). In a next step, the colour change of clBRPs upon incubation with S. aureus was assessed. One PC was spiked with S. aureus (1 103 CFU/mL) and a second one served as a control. The PCs were incubated at room temperature at gentle agitation (30 rpm) for 48 hours. Thereafter, growth of the bacteria was stopped through the addition of neomycin (25 mg/mL). 2 mL of each PC (control and S. aureus) presumably containing enzymes secreted by S. aureus were then added to cuvettes containing clBRPs (1 cm 1 cm) and their colour change was measured after 48 hours of incubation at 30 rpm with a Colorlite sph 850 (Colorlite GmbH, Katlenburg-Lindau, Germany) spectrophotometer. BRPs were compared to clBRPs in terms of reaction time. 20 mg of BRPs were suspended in 5 mL SSP+-buffer. Different protease concentrations (protease from A. oryzae; 0, 0.01, 0.03, 0.06, 0.125, 0.3, 0.6, 1.25, 0.3, 0.6, 1.25, 2.5, 5 and 10 U) were added. The samples were incubated at room temperature (228C) under shaking conditions. For the absorbance measurements 100 mL of each sample were transferred into a 96-well plate. Absorbance at 590 nm was measured after 20, 60 and 180 min. A proof of concept was also performed directly in PC. 40 mL PC were transferred into sterile 250 mL flasks. 80 mg of the BRPs were added as substrate for the trigger enzymes. One sample was spiked with S. aureus (10 CFU/mL) whereas the control was not inoculated. For the measurement of the colour change, 2 mL of each 152
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flask were transferred into a cuvette and a Colorlite sph 850 was used for the colour measurement after 0, 24 and 48 hours.
Results and discussion Bacterial growth in PC bags In a first step, growth of S. aureus in PCs was investigated. CFU analysis together with oxygen consumption in PC bags after 24 hours indicated a considerably fast growth of this bacterium in PCs (Fig. 1). Upon inoculation with 10 CFU/mL, the oxygen concentration in samples (S. aureus 1 and 2) showed a rapid decrease indicating bacterial contamination. After 30 hours of incubation, only less than 10% of the air saturation was left which correlated with the growth of the organisms. Figure 1 clearly illustrates that initial CFU/mL values of 3 increased to values around 1.5 105 CFU/mL within 24 hours. Initial concentrations of 40 CFU/mL led to 2 106 CFU/mL after 24 hours incubation. These results are comparable with the studies of Brecher et al. [31]. By contrast, the oxygen concentration of the two control samples (controls 1 and 2) only slightly decreased over the first 30 hours, probably due to the additional air which was inadvertently entrapped in the bags and hence led to an oxygen equilibrium between the liquid and the aqueous phase. The oxygen consumption of platelets could be an alternative explanation for the slow decrease at the beginning. The well established BacT/ALERT system for detection of bacterial contaminations is designed to take platelet samples at least 24 hours after collection or later. However, the growth kinetics of S. aureus demonstrate that under the storage conditions of the PCs bacterial growth can be fast enough to reach crucial values already before 24 hours.
BRP design for detection of microbial contaminations Two different types of BRPs (BRP and clBRP) were designed and tested for the detection of contaminating pathogens directly in
FIGURE 1
Growth of S. aureus in PC bags. (a) Oxygen concentration of two contaminated samples inoculated with S. aureus (10 CFU/mL) and two control PC bags. (b) Bacterial counts of samples x, y and z after incubation for 24 hours at 228C and gentle agitation inoculated with 3 2, 40 10 and 470 60 CFU/mL, respectively.
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FIGURE 2
Schematic representation of the BRP system integrated into a PC bag. The operating biomatrix consists of cross-linked casein which is hydrolysed by proteases secreted by contaminating pathogens. This degradation process of the biomatrix can be observed via release of a dye (Reactive Black 5).
PCs. Thereby extracellular proteases secreted by relevant pathogens like S. aureus trigger the hydrolysis of the BRPs consequently leading to a colour change. A schematic representation of this BRPbased detection system is shown in Fig. 2. The most frequent pathogens (P. acnes, S. aureus and S. epidermidis) are able to produce extracellular proteases [24–26]. Hence, in this study an operating biomatrix was constructed consisting of modified model proteins. The constructed BRP consisted of a water-insoluble protein (casein) which was modified with a reactive dye (Reactive Black 5). Hydrolysis of this BRP leads to a visible colour reaction. For the clBRP in a first step, water-soluble hydrolysed casein was modified with GMA. On the one hand, the epoxy group of the GMA enables cross-linking with amines, hydroxyl groups or carboxylic acids on the casein. On the other hand, the acrylic groups were used for the cross-linking with other vinyl monomers [32]. The stained BRP, which was embedded into the clBRP, was used for the visualisation of the degradation process. The radical polymerisation process was started by the addition of sodium persulphate and TEMED as
described by Reis et al. [33]. The main benefit of the cross linked polymer was an enhanced usability as well as stability [34] and can therefore improve hydrogel properties which are widely used in medical applications [35,36].
Colour reaction of the clBRPs in the presence of proteases or S. aureus clBRPs were incubated with different protease concentrations and the colour changes were measured. The detection limit was also determined. SSP+-buffer was used to simulate the real conditions in PC. Figure 3(a) shows the colour change dependence on enzyme activity. The colour change was recorded directly in the supernatant in the reaction vessel. The control sample showed that the clBRP was at least stable for 96 hours. Figure 3(a) demonstrates the colour formation dependence on enzyme activity after 24 hours. Small enzyme concentrations of 0.03 U led to a 50% increase in colour formation after 24 hours. Higher concentrations significantly reduce the detection time. The polymer treated with
FIGURE 3
Colour change of clBRPs in the presence of proteases. (a) Visual colour change of the operating biomatrix supernatant after incubation with five different protease concentrations for 24 hours in SSP+-buffer. Measured absorbance increase example shown for incubation for 24 hours. (b) Visual and quantified colour change of clBRPs in contaminated PC. The clBRP in sample A was dried before use; that in sample B was dried and autoclaved. The PC was incubated with S. aureus (1 103 CFU/mL) for 48 hours. Thereafter, the colour reaction was carried out in cuvettes and colour change was quantified after 48 hours as DE values according to the CIELab concept. www.elsevier.com/locate/nbt
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0.25 U substantially increased colour formation after 3 hours (2.6 higher) and showed a 600% higher value after 24 hours. With this experiment, the protease dependent colour reaction of the BRP was clearly demonstrated and confirmed the potential of enzyme-responsive polymers as recently postulated by Thornton et al. [37] and others. In a next step, the clBRP was tested directly in PCs. BRP A in Fig. 3(b) was dried before use, BRP B was dried and steam sterilised. The BRP was still stable after steam sterilisation, provided it was dried beforehand. One PC was inoculated with 1 103 CFU/mL S. aureus and incubated for 48 hours directly in a PC bag. A second PC (not inoculated) served as control. After 48 hours incubation, antibiotics were added to stop and inhibit bacterial growth during the release experiment. Consequently, any hydrolysis and concomitant colour change of the BRPs can be related to the presence of extracellular proteases. Figure 3(b) shows the colour change of the contaminated PCs after 48 hours incubation. The negative controls showed no significant change in colour, whereas the samples inoculated with S. aureus showed a clear difference when compared to the original sample before incubation. Absorbance measurements directly in PC are not possible due to turbidity. Therefore, the DE values (according to the CIELab concept) were measured for the quantification of the colour change. High DE values of around 35 were obtained for both treatment methods of the polymer (A and B). Hence, enzyme triggered dye liberation was clearly achieved resulting in a visible colour change. A contamination of a PC with S. aureus could be indicated by a clBRP, but the incubation time still has to be shortened. Although proteolytic degradation of casein is well known for numerous proteases [38–40], cross-linking obviously hinders access of the enzyme.
Colour reaction of the BRPs in presence of proteases or S. aureus A decrease of the reaction time was achieved by the use of BRP which were not cross-linked. The samples were centrifuged before the
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absorbance increase was determined after 20, 60 and 180 min, respectively (Fig. 4). Higher enzyme concentrations led to a higher sensitivity of the detection system. A controlled release was measurable after 20 min using enzyme concentrations above 0.5 U/mL. Compared to the clBRP, an eightfold higher signal was measured after 3 hours and 0.12 U/mL trigger enzyme. This enhancement clearly improves the sensitivity of the BRP system. Similarly, when directly incubated in PC, a significant faster response was also obtained. Figure 4(b) illustrates the colour change in the contaminated PC in comparison to the control sample. The polymer was sterilised with ethanol before transfer to the reaction solution. The contaminated sample was inoculated with 10 CFU/mL. The DE value after 24 hours of incubation at room temperature and gentle agitation was 21.8, and after 48 hours considerably higher (49.4). A contamination with 10 CFU/mL could be detected after 24 hours using this BRP system. According to literature, the BacT/ALERT detection system (Biomerieux) has a detection time of around 11 hours for inoculation with 10 CFU/mL (S. aureus) [41–43]. The manufacturer manual recommends at least 24 hours incubation before injection into the detection system which leads to a final detection time of 35 hours. The Pan Genera Detection Immunoassay (Verax Biomedical Incorporated) gives the first positive signal after 48 hours of incubation with 0.13 CFU/mL S. aureus [44]. Nine out of ten samples inoculated with 169 CFU/mL S. aureus were detected after 24 hours using the Pall BDS system (Pall Corporation) [45]. Hence, the detection time of BRP studied here is within the magnitude of the commercial available systems or even lower. The response time could be fast enough to avoid the transfusion of a contaminated PC. Hence, this system could lower the risk of transfusion-transmitted infections. The use of an integrated detection system also avoids the possibility of false positive signals. It has to be noted that for a medical application the model substances (dye, protein) would have to be exchanged according to legal regulations of medical products.
FIGURE 4
Hydrolysis of BRPs. In contrast to the previous experiments, the BRP used in this attempt were not cross-linked. Different enzyme concentrations were used in (a) to visualise the colour change. The dye released using enzyme concentrations above 120 mU/mL can be detected after 20 min. (b) The colour change of contaminated (S. aureus, 10 CFU/mL) and control samples was quantified as DE values according to the CIELab concept. An initial inoculation with 10 CFU/mL S. aureus was detected after 24 hours of incubation. 154
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Conclusion
Acknowledgements
In this study it was demonstrated that protein based BRPs can be used for the detection of pathogens in PCs. Thereby, a visible colour reaction indicates a contamination and gives a clear yes/no signal. The backbone of the polymer was designed to be degradable by extracellular proteases released from contaminating pathogens. The model substances used in this study would have to be changed in future experiments according to legal regulations of medical products while a screening for a larger number of different pathogens should also be conducted. Fluorescent dyes could be used to improve the sensitivity of the detection system.
This study was performed within the Austrian Centre of Industrial Biotechnology ACIB, the COMET K Project MacroFun project and the COST Action 868. This work has been supported by the Federal Ministry of Economy, Family and Youth (BMWFJ), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol and ZIT – Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG.
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