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Cl against Staphylococcus aureus and Pseudomonas aeruginosa forming biofilms on prosthetic material
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Photodynamic antibacterial and antibiofilm activity of RLP068/Cl against Staphylococcus aureus and Pseudomonas aeruginosa forming biofilms on prosthetic material Christian Vassena a,1 , Simone Fenu b,1 , Francesco Giuliani c , Lia Fantetti c , Gabrio Roncucci c , Giulio Simonutti b , Carlo Luca Romanò d , Raffaele De Francesco b , Lorenzo Drago a,e,∗ a
Laboratory of Clinical Chemistry and Microbiology, IRCCS Galeazzi Orthopaedic Institute, Milan, Italy Virology Program, National Institute of Molecular Genetics (INGM), Milan, Italy c Molteni Therapeutics s.r.l., via I. Barontini 8, 50018 Scandicci, Florence, Italy d Center for Reconstructive Surgery of Osteoarticular Infections (C.R.I.O.), IRCCS Galeazzi Orthopaedic Institute, Milan, Italy e Laboratory of Technical Sciences for Laboratory Medicine, Department of Biomedical Science for Health, University of Milan, Milan, Italy b
a r t i c l e
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Article history: Received 10 January 2014 Received in revised form 26 March 2014 Accepted 26 March 2014 Keywords: Antibiofilm photodynamic activity Prosthetic joint infections Confocal laser scanning microscopy Bacterial biofilm
a b s t r a c t Prosthetic joint infections (PJIs) are becoming a growing public health concern in developed countries as more people undergo arthroplasty for bone fixation or joint replacement. Because a wide range of bacterial strains responsible for PJIs can produce biofilms on prosthetic implants and because the biofilm structure confers elevated bacterial resistance to antibiotic therapy, new drugs and therapies are needed to improve the clinical outcome of treatment of PJIs. Antimicrobial photodynamic therapy (APDT), a non-antibiotic broad-spectrum antimicrobial treatment, is also active against multidrug-resistant microorganisms such as meticillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. APDT uses a photosensitiser that targets bacterial cells following exposure to visible light. APDT with RLP068/Cl, a novel photosensitiser, was studied by confocal laser scanning microscopy (CLSM) to evaluate the disruption of MRSA and P. aeruginosa biofilms on prosthetic material. Quantitative CLSM studies showed a reduction in biofilm biomass (biofilm disruption) and a decrease in viable cell numbers, as determined by standard plate counting, in the S. aureus and P. aeruginosa biofilms exposed to APDT with the photosensitiser RLP068/Cl. APDT with RLP068/Cl may be a useful approach to the treatment of PJI-associated biofilms. © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
Introduction Total joint arthroplasties have significantly increased in recent years. Prosthetic joint infection (PJI) is one of the major complications of total joint arthroplasty, usually requiring revision surgery and prolonged courses of intravenous or oral antimicrobial therapy [1]. Despite its relatively low incidence (1–2%), the healthcare costs for treating PJIs are staggeringly high [2], with an increase from US$320 million to $566 million between 2001 and 2009, and
∗ Corresponding author. Present address: Laboratory of Clinical Chemistry and Microbiology, IRCCS Galeazzi Orthopaedic Institute, via R. Galeazzi 4, 20161 Milan, Italy. Tel.: +39 02 6621 4839; fax: +39 02 6621 4774. E-mail address: [email protected] (L. Drago). 1 These two authors contributed equally to this work.
they are expected to rise further to over $1.62 billion by 2020 in the USA alone [3]. Bacterial biofilms are a common cause of persistent infections and are often responsible for PJIs [4,5]. A biofilm is a matrixenclosed microbial population involving cell-to-cell adhesion between micro-organisms and an artificial surface [6]. A common feature shared by biofilm-related infections is their intrinsic resistance to host immunity as well as conventional antimicrobial agents and biocides [7]. Biofilms can tolerate levels of antibiotics 10–1000 times higher than the minimum inhibitory concentration of the corresponding planktonic form. Today, with fewer new antimicrobials coming through the drug development pipeline [8], there is an urgent unsatisfied medical need for alternative drugs and/or therapies. One novel approach to inactivate bacterial biofilms on prosthetic implants is with the use of antimicrobial photodynamic therapy (APDT). By acting on the micro-organisms embedded in the biofilm matrix, the matrix itself and the other
http://dx.doi.org/10.1016/j.ijantimicag.2014.03.012 0924-8579/© 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
Please cite this article in press as: Vassena C, et al. Photodynamic antibacterial and antibiofilm activity of RLP068/Cl against Staphylococcus aureus and Pseudomonas aeruginosa forming biofilms on prosthetic material. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.012
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Fig. 1. (A) Structure of the tetracationic Zn(II) phthalocyanine chloride RLP068/Cl photosensitiser. (B) Visible absorption spectrum of RLP068/Cl.
components stabilising its structure, APDT disrupts the biofilm and reduces its re-growth. APDT is a bimodal strategy that combines light, a chemical substance (or photosensitiser) activated on exposure to light waves within its absorption spectrum, and oxygen to obtain a cytotoxic effect through the production of in situ reactive oxygen species (ROS). In turn, ROS are formed by two reaction pathways. In type 1 reactions, electron transfer occurs from the photosensitiser triplet state to biological substrates resulting in the formation of radical species that react with oxygen to produce ROS such as hydrogen peroxide (H2 O2 ), hydroxyl radical (HO*) and superoxide anion (O2 − ). In type 2 reactions, energy is transferred from the photosensitiser triplet state directly to oxygen to form singlet oxygen (1 O2 ). RLP068/Cl, a recently developed photosensitiser used in the present study, is thought to act mainly through the type 2 pathway [9]. Because the photosensitiser can reach target micro-organisms without affecting surrounding tissues or cells when activated under a suitable illumination protocol [10], the main advantage of APDT is its high target specificity with few undesired side effects, i.e. the drug is inactive in the dark and becomes active only when exposed to light [11]. Since some photosensitisers bind rapidly and selectively to microbial cells, APDT holds promise as an anti-infective approach [12], which is now becoming a therapeutic experimental reality. RLP068/Cl is a cationic Zn(II) phthalocyanine derivative (molecular weight >1300 Da) (Fig. 1) believed to mainly target the bacterial cell wall and membrane both through hydrophobic and electrostatic interactions. Upon activation by light, RLP068/Cl disrupts the cell’s external components through various oxidative reactions initiated by photosensitisation and enters the wall/membranes of bacteria and fungi through a self-promoting uptake process [13]. As this process is unaffected by mechanisms of bacterial resistance to antibiotics, both susceptible and multidrug-resistant bacterial strains may be suitable targets for APDT [14,15]. The multi-target nature and broad spectrum of action of photosensitised inactivation make this approach particularly interesting for treating biofilm-associated PJIs [16]. However, although APDT has long been studied in planktonic cultures, little is known about its potential use against bacteria organised in biofilms. Although the ability of APDT to eradicate bacterial biofilms growing in dental plaques and on oral implants has been variously investigated [17–19], to the best of our knowledge its effect on biofilms developed on prosthetic materials has been far less studied. To fill this gap, here we examined the photodynamic effects of RLP068/Cl on the viability and
architecture of meticillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa biofilms developed on prosthetic material. Materials and methods Bacterial strains The studied micro-organisms were an MRSA strain (SAUMRBP2) and a P. aeruginosa strain (PAE2). Both strains were isolated at the Microbiology Laboratory of the IRCCS Galeazzi Orthopaedic Institute (Milan, Italy) from patients referred to the Center for Reconstructive Surgery of Osteoarticular Infections (C.R.I.O.) of the institute for revision of septic knee implants. The strains were selected for their high capacity to produce in vitro biofilm on prosthetic materials. Bacterial strains were grown overnight in tryptic soy broth (TSB) (bioMérieux, Marcy l’Étoile, France) at 37 ◦ C under aerobic conditions unless otherwise specified. Biofilm growth Sterilised sandblasted titanium discs (25 mm diameter, 5 mm thickness; batch J04051) (Adler Ortho, Milan, Italy) were used as the substrate for biofilm formation. Briefly, discs were placed into six-well flat-bottomed sterile polystyrene microplates (Jet Biofil, China) containing 5 mL of TSB. Overnight cultures of S. aureus and P. aeruginosa were re-suspended to a final concentration of 1.0 × 108 CFU/mL in fresh TSB. Aliquots (200 L) of the diluted bacterial suspension were inoculated in each microplate well containing a titanium disc and were incubated at 37 ◦ C aerobically. Exhausted growth medium containing the non-adherent bacteria was removed 24 h later and was replaced with 5 mL of fresh medium. To obtain mature biofilms, the plates were incubated for a further 48 h. Prior to treatment, the remaining non-adherent bacteria were removed by washing three times with sterile saline solution. Photosensitiser RLP068/Cl, a tetracationic Zn(II) phthalocyanine chloride (Molteni Therapeutics, Florence, Italy), was dissolved in sterile
Please cite this article in press as: Vassena C, et al. Photodynamic antibacterial and antibiofilm activity of RLP068/Cl against Staphylococcus aureus and Pseudomonas aeruginosa forming biofilms on prosthetic material. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.012
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Milli-Q water to give a 1.0 mM stock solution and was stored at −20 ◦ C in the dark until use [20]. Light source Illumination was carried out using a diode laser centred at 689 ± 3 nm (Ceralas PDT 689 nm/3 W; CeramOptec GmbH, Bonn, Germany) equipped with an optical fibre with a final light diffuser (Frontal light diffuser, Model FD; Medlight S.A., Ecublens, Switzerland) positioned at a suitable distance from the biofilm sample to provide a light spot ca. 5 cm in diameter. The fluence rate at the spot level was experimentally determined to be 120 mW/cm2 (Nova laser power/energy metre; Ophir Optronics Ltd., Jerusalem, Israel). A light dose of 60 J/cm2 was administered based on previous experiments in which this value was found to be both effective and clinically safe [21,22]. The maximum wavelength of RLP068/Cl was 690 nm, which overlapped with that of the laser emission of 689 ± 3 nm.
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A 488 nm laser line was used to excite SYTO9, whilst fluorescent emission was detected from 500 nm to 540 nm. PI was excited with a 561 nm laser line, and its fluorescent emission was detected from 600 nm to 695 nm. To minimise the possibility of false co-localisation between fluorescent stains owing to bacterial movement, we opted for a simultaneous acquisition mode of the two channels in which the laser beam scanned the visual field at a frequency of 700 Hz. Moreover, using a third laser line (633 nm) in reflection mode, it was possible to determine the reflecting surfaces of both the titanium disc (starting acquisition point) and the coverslip (ending acquisition point) with high accuracy. Images from five randomly selected areas were acquired for each disc, for each of which sequential optical sections of 2 m were collected in sequence along the z-axis over the complete thickness of the sample. The resulting stacks of images were analysed, quantified and rendered into three-dimensional (3D) mode using Volocity 3D Image Analysis Software (Perkin Elmer, Waltham, MA). Statistical analysis
Photodynamic inactivation studies To investigate the efficacy of APDT with RLP068/Cl on the viability of bacterial cells embedded in the biofilm grown on the prosthetic material, titanium discs were incubated in the dark for 20 min with 5 mL of 50 M RLP068/Cl solution and were then illuminated with a final light dose of 60 J/cm2 . Controls were carried out without treatment (growth control). The illuminated and non-illuminated discs were then placed in sterile containers together with 10 mL of sterile saline solution and were sonicated at room temperature in an ultrasonic bath (WVR, Milan, Italy) for 5 min at a frequency of 30 kHz and a power of 300 W. Serial 10-fold dilutions in sterile saline of the sonicated fluids were plated on tryptic soy agar plates (bioMérieux). Following incubation at 37 ◦ C for 16–18 h, the number of grown colonies was counted. The efficacy of RLP068/Cl on the bacterial biofilm was evaluated by a spectrophotometric method as described in Christensen et al. [23]. Briefly, biofilms grown on the titanium discs were air-dried and were stained by disc immersion in a 5% crystal violet (CV) solution for 15 min and then, after several washings, were air-dried again. Biofilm biomass was estimated by elution of the biofilm-bound CV with 3 mL of ethanol (96%) and then measuring the absorbance of 100 L of eluted dye solution at 595 nm by means of a microplate photometer (MultiskanTM FC; Thermo Scientific; Milan, Italy). All experiments were carried out in triplicate. Confocal laser scanning microscopy (CLSM) studies Microbial biofilm was grown on titanium discs and was treated as described above. Controls were carried out without treatment (growth control), without illumination (photosensitiser alone, dark control) as well as with illumination but without RLP068/Cl (light control). A FilmTracerTM LIVE/DEAD® Biofilm Viability Kit (Molecular Probes, Life Technologies Ltd., Paisley, UK) was used to determine the viability of bacteria within the biofilm and the structural damage after photodynamic treatment: dead cells emitted red fluorescence, whereas cells emitting green fluorescence were considered viable. All samples (both the illuminated and non-illuminated) were stained for 15 min in the dark at room temperature with an appropriate volume of a mixture of the two dyes SYTO9 and propidium iodide (PI) (3 L of each component in 1 mL of saline solution). Stained biofilms were examined with a confocal microscope (Leica TCS SP5; Leica Microsystems CMS GmbH, Mannheim, Germany) using a 20× dry objective (HC PL FLUOTAR 20.0 × 0.50 DRY) plus a 2× electronic zoom. The specimens were coverslipped to minimise air contact and to maintain sample moisture constant.
Results of the CV assays and biomass volume variations obtained from the CLSM image analysis are presented as the mean ± standard deviation (S.D.). Results of the CV assay were analysed using a nonpaired Student’s t-test, whilst the variations in biofilm biomass volumes obtained from CLSM were analysed by non-parametric one-way analysis of variance (ANOVA) followed by the Tukey’s HSD test. Results Antimicrobial activity The antimicrobial activity of RLP068/Cl following illumination was preliminarily evaluated by plate counts. The decrease in cell viability was estimated to be ca. 1.5 log10 CFU/mL both for S. aureus and P. aeruginosa (Fig. 2). Crystal violet assay Antibiofilm activity, defined as the ability to disrupt the biofilm structure, was preliminarily determined using the CV method as described in Christensen et al. [23]. Quantification of the whole biomass present on the titanium discs after treatment showed significant differences between the illuminated and non-illuminated samples. Compared with the untreated samples, the amount of biofilm in the APDT-treated samples was significantly lower (P < 0.001) (Fig. 3). Confocal laser scanning microscopy analysis The FilmTracerTM LIVE/DEAD® Biofilm Viability Kit staining properties, together with the instrumental set-up used, allowed the biomass volume and the ratio between live and dead cells in the matrix to be quantified. Biomass volumes (expressed as the mean ± S.D. of the five acquired areas for each disc both for S. aureus and P. aeruginosa) are shown in Figs. 4 and 5, respectively. As shown in Fig. 4, there was a statistically significant reduction in the biomass volume of the S. aureus sample treated with RLP068/Cl compared with the growth (P < 0.01), light (P < 0.01) and dark controls (P < 0.05). No differences were observed between the dark and growth controls (P > 0.05), whereas significant differences were found between the light and growth controls (P < 0.05). Fig. 5 shows a statistically relevant reduction (P < 0.05) in the biomass volume of the P. aeruginosa sample treated with RLP068/Cl
Please cite this article in press as: Vassena C, et al. Photodynamic antibacterial and antibiofilm activity of RLP068/Cl against Staphylococcus aureus and Pseudomonas aeruginosa forming biofilms on prosthetic material. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.012
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Fig. 2. Antimicrobial activity of RLP068/Cl. Efficacy of killing meticillin-resistant Staphylococcus aureus (SAU-MRBP2) and Pseudomonas aeruginosa (PAE2) cells within the biofilm after 20 min incubation in the dark with 50 M RLP068/Cl, without illumination (white bars) and with illumination (black bars). Colony count from single sample (n = 1).
Fig. 3. Cristal violet (CV) assay. Absorbance value at 595 nm for meticillin-resistant Staphylococcus aureus (SAU-MRBP2) and Pseudomonas aeruginosa (PAE2) strains after 20 min incubation in the dark with 50 M RLP068/Cl, without illumination (white bars) and with illumination (black bars). Mean absorbance data (±standard deviation) of experiments carried out in triplicate (n = 3). ***P < 0.001.
Please cite this article in press as: Vassena C, et al. Photodynamic antibacterial and antibiofilm activity of RLP068/Cl against Staphylococcus aureus and Pseudomonas aeruginosa forming biofilms on prosthetic material. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.012
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Fig. 4. Biomass volume of Staphylococcus aureus samples. RLP068/Cl antimicrobial photodynamic therapy significantly reduced S. aureus (SAU-MRBP2) biomass volume upon illumination. Quantification was performed using Leica Application Suite Advanced Fluorescence (LAS-AF) software to obtain the mean biofilm thickness multiplied by the total surface of portions acquired (n = 5). **P < 0.05; ***P < 0.01.
compared with the growth control. No statistically significant differences were found between the controls. The 3D renderings (Figs. 6 and 7) confirmed a decrease in the biofilm volume both of the S. aureus and P. aeruginosa samples treated with RLP068/Cl compared with the controls (growth, dark and light). Fig. 6 shows an example of 3D reconstruction of one of
the acquired portions of an S. aureus biofilm sample grown on a titanium disc. The RLP068/Cl APDT-treated sample (Fig. 6B) clearly shows a reduction in biofilm volume compared with the relative controls (Fig. 6A, C, and D). Fig. 7 shows an example of 3D reconstruction of one of the acquired portions of a P. aeruginosa biofilm sample grown on a
Fig. 5. Biomass volume of Pseudomonas aeruginosa samples. RLP068/Cl antimicrobial photodynamic therapy significantly reduced P. aeruginosa (PAE2) biomass volume upon illumination. Quantification was performed using Leica Application Suite Advanced Fluorescence (LAS-AF) software to obtain the mean biofilm thickness multiplied by the total surface of portions acquired (n = 5). **P < 0.05.
Please cite this article in press as: Vassena C, et al. Photodynamic antibacterial and antibiofilm activity of RLP068/Cl against Staphylococcus aureus and Pseudomonas aeruginosa forming biofilms on prosthetic material. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.012
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Fig. 6. Three-dimensional (3D) image of Staphylococcus aureus samples. 3D reconstruction by confocal laser scanning microscopy of antimicrobial photodynamic therapytreated S. aureus biofilms. Biofilms were grown for 72 h and were then treated with 50 M RLP068/Cl solution for 20 min in the dark and were subsequently illuminated with a total light dose of 60 J/cm2 . Biofilms were stained with FilmTracerTM LIVE/DEAD® Biofilm Viability Kit (Molecular Probes, Life Technologies Ltd., Paisley, UK): SYTO9 (green) represents viable cells; propidium iodide (red) represents dead cells. (A) Bacteria without treatment (growth control); (B) RLP068/Cl-treated bacteria exposed to 60 J/cm2 light dose; (C) bacteria treated with RLP068/Cl in the dark (dark control); and (D) bacteria untreated but illuminated (light control).
Fig. 7. Three-dimensional (3D) image of Pseudomonas aeruginosa samples. 3D reconstruction by confocal laser scanning microscopy of antimicrobial photodynamic therapytreated P. aeruginosa biofilms. Biofilms were grown for 72 h and were then treated with 50 M RLP068/Cl solution for 20 min in the dark and were subsequently illuminated with a total light dose of 60 J/cm2 . Biofilms were stained with FilmTracerTM LIVE/DEAD® Biofilm Viability Kit (Molecular Probes, Life Technologies Ltd., Paisley, UK): SYTO9 (green) represents viable cells; propidium iodide (red) represents dead cells. (A) Bacteria without treatment (growth control); (B) RLP068/Cl-treated bacteria exposed to 60 J/cm2 light dose; (C) bacteria treated with RLP068/Cl in the dark (dark control); and (D) bacteria untreated but illuminated (light control).
Please cite this article in press as: Vassena C, et al. Photodynamic antibacterial and antibiofilm activity of RLP068/Cl against Staphylococcus aureus and Pseudomonas aeruginosa forming biofilms on prosthetic material. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.012
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Fig. 8. Percentage composition of the analysed Staphylococcus aureus samples. Pie charts showing the proportion of meticillin-resistant S. aureus (SAU-MRBP2) biomass components obtained from confocal laser scanning microscopy image analysis using Volocity 3D Image Analysis Software (Perkin Elmer, Waltham, MA): biofilm (blue area); live cells within the biofilm (green area); dead cells within the biofilm (red area); and amount of biofilm ‘lost’ compared with the growth control upon antimicrobial photodynamic therapy for the RLP068/Cl-treated sample and the dark and light controls (grey area).
titanium disc. Also here, the RLP068/Cl APDT-treated sample (Fig. 7B) clearly shows a reduction in biofilm volume compared with the controls (Fig. 7A, C, and D). Quantification of the bacterial cells included in the biomatrix was carried out by measuring the volume of fluorescence expressed by the two specific dyes (SYTO9 and PI) of the staining kit. As expected, there was a substantial difference in the biofilm matrix composition and structure for the two bacterial species [24]. Since the characteristics of the P. aeruginosa matrix made it difficult to quantify the volume occupied by the bacterial cells (live and dead) within the matrix, the experimental evaluation was limited to determination of the biomass volume alone. Conversely, it was possible to quantify both the live and dead cells of S. aureus and to reconstruct the percentage composition of the samples analysed. As shown in Fig. 8, the cells (live or dead) present within the matrix occupied ca. 5% of the total biomass in all of the samples. In the control samples (growth, dark and light), the live cells accounted for 4.7%, 4.8% and 4.9% of the total biomass, respectively, whereas in the RLP068/Cl APDT-treated samples the live cells made up 2.4% and the dead cells made up 2.3% of the total biomass.
Discussion PJIs caused by biofilm-producing bacteria are a feared complication of arthroplasty for bone fixation or joint replacement. Even with routine precautions, such as pre-operative antimicrobial prophylaxis and surgical environment protection, the risk of intra-operative infection is 1% and 2% for hip and knee replacement, respectively [2]. Orthopaedic implant-related infections are usually managed with complete device removal, long-term antimicrobial therapy with conventional drugs and, eventually, re-implantation [1]. Novel approaches to PJI eradication rely on the synergistic effect gained from the use of combined strategies based on the action of biofilm-disrupting agents coupled with antibiotics [25], although an effective antibiofilm treatment specifically for PJIs is not yet available. In this scenario, APDT is an innovative option for treating PJIs in the clinical setting. Building on the promising results from in vitro tests on Grampositive and Gram-negative bacteria in planktonic form [14,15], as well as in the treatment of chronic wounds where bacteria are predominantly present as biofilm [21], in this study we tested the antimicrobial and antibiofilm efficacy of APDT with RLP068/Cl
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against clinical isolates of S. aureus and P. aeruginosa forming biofilms on prosthetic material. The RLP068/Cl concentration used for these tests (50 M) reduced free-floating bacteria by a factor of almost 7 log10 [14,15]. Following RLP068/Cl treatment and illumination of the biofilm samples, bacterial inactivation was less than that observed in the corresponding planktonic forms. These findings were expected since bacteria organised as biofilm are not only less susceptible to antibiotics but are also more resistant to environmental stress. Therefore, a long-lasting therapy with elevated drug dosage is needed which, however, raises the risk of increased resistance to currently used antimicrobial agents [26]. On the other hand, the short incubation time of the photosensitiser with the microorganisms and the typically multi-target nature of photosensitised inactivation processes prevent the possible expression of protective factors (e.g. biosynthesis of stress proteins), thus minimising the risk of emergence of resistant strains [15]. The antibiofilm activity of RLP068/Cl APDT was preliminarily investigated using the CV assay. CV is a basic dye that readily binds to negatively charged molecules (and thus to acidic polysaccharides in the extracellular matrix) [27]. In our experience, owing to the non-specific nature of the dye, this method yields only semiquantitative data for staining both living and dead cells and biofilm matrix. Moreover, the ethanol used to solubilise the dye does not guarantee complete extraction from the biomass. Nevertheless, the current results showed an estimated decrease of 45% and 38% in S. aureus and P. aeruginosa whole biomass, respectively. CLSM is one of the most sensitive and specific techniques for biofilm structure visualisation and analysis. To further investigate the changes in the spatial structure of the biofilm induced by RLP068/Cl APDT treatment, samples of mature biofilm grown on sandblasted titanium discs were analysed after staining with the FilmTracerTM LIVE/DEAD® Biofilm Viability Kit. It was possible to quantify the volume occupied by the live and dead bacterial cells within the biomass only for S. aureus. As demonstrated by fluorescence quantification (Volocity software) and observable from the 3D renderings, RLP068/Cl APDT significantly reduced the biomass volume of both micro-organisms compared with the relative growth control. Moreover, the light control showed a decrease in biomass volume of S. aureus probably due to aspecific physical phenomena. As expected, albeit not statistically significant, a mild effect on biomass volume was observable in the dark control, likely due to the non-photodynamic antibiofilm activity of the molecule. As confirmation of the antibiofilm effect of RLP068/Cl APDT exerted by the photodynamic action mechanism, a greater decrease in biomass volume was observed in the treated samples in which there was appreciable antibacterial action (up to 50%), not observable in either the dark or light controls (Fig. 8). Unfortunately, it was not possible to determine the ratio between live and dead cells in the P. aeruginosa biofilm. In particular, it was difficult to estimate the amount of PI bonded to the dead cells, which precluded reliable quantification of the number of viable cells. A decrease in biomass volume of P. aeruginosa after APDT treatment was also observed, even if statistical significance can be reported only for the growth control. This may be ascribed to the nature of the P. aeruginosa biofilm, especially the difference in matrix composition [24] compared with biofilm formed by S. aureus. These results, obtained under experimental in vitro conditions, suggest that APDT with RLP068/Cl exerts antibiofilm and antimicrobial activities already after a single treatment against bacterial biofilm produced by a clinical strain of MRSA. Although activity against P. aeruginosa biofilm was observed, further experimentation and refinement of technical settings are needed to improve the capability to study the biofilm produced by this microbe. Microbial biofilms can quickly re-form unless completely eradicated. This peculiarity poses a major challenge to treating
infections. A future area of focus will involve a larger number of micro-organisms in order to determine the long-lasting effectiveness as a function of the frequency of APDT treatments. This could be done by evaluating antibiofilm and antibacterial efficacy mediated by repeated in vitro APDT treatment. Repeated treatments over time will likely amplify the effects observed after a single treatment by increasing the reduction of biomass volume and decreasing the number of surviving cells. If this hypothesis is confirmed, further animal studies will be required to establish the optimum parameters (number of APDT treatments, time between treatments) for an effective and safe protocol to translate from laboratory science to clinical use for treating PJIs. The aim of this study was to obtain ‘proof of concept’ for the application to PJIs of RLP068/Cl APDT, already successfully tested in a phase 2 clinical trial for the treatment of diabetic foot ulcer infections. This study holds particular interest as there are currently no alternative effective treatments that exert both antibacterial and antibiofilm activity. Whilst substances with antibiofilm properties, such as N-acetyl cysteine or d amino acids, are able to trigger biofilm disassembly, they do not exhibit a pronounced antibacterial activity [28,29]. Conversely, many antibiotics possess antibacterial activity but are not active against biofilms. Combined treatment that disrupts microbial biofilm and possesses antibacterial activity [30,31], as might be achieved with the APTD approach, could represent an innovative therapeutic technology. Acknowledgments The authors are grateful to Elena De Vecchi (Laboratory of Clinical Chemistry and Microbiology, IRCCS Galeazzi Orthopaedic Institute, Milan, Italy) for helpful scientific discussions. The authors also thank Riccardo Rossi [Computational Biology & Data Analysis, Istituto Nazionale Genetica Molecolare (INGM), Milan, Italy] for assistance with the statistical analysis. Funding: This study was supported by IRCCS Galeazzi Orthopaedic Institute (Milan, Italy) with a grant for current research [no. L-4063]. Competing interests: FG, LF and GR are Molteni Therapeutics employees, which is the owner of RLP068/Cl patents. All other authors declare no competing interests. Ethical approval: Not required. References [1] Darouiche RO. Treatment of infections associated with surgical implants. N Engl J Med 2004;350:1422–9. [2] Sia IG, Berbari EF, Karchmer AW. Prosthetic joint infections. Infect Dis Clin North Am 2005;19:885–91. [3] Kurtz SM, Lau E, Watson H, Schmier JK, Parvizi J. Economic burden of periprosthetic joint infection in the United States. J Arthroplasty 2012;27(Suppl):61–5. [4] Zimmerli W, Trampuz A, Ochsner PE. Prosthetic-joint infections. N Engl J Med 2014;351:1645–54. [5] Gallo J, Kolár M, Novotny´ R, Riháková P, Tichá V. Pathogenesis of prosthesisrelated infection. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2003;147:27–35. [6] Ellington JK, Reilly SS, Ramp WK, Smeltzer MS, Kellam JF, Hudson MC. Mechanisms of Staphylococcus aureus invasion of cultured osteoblasts. Microb Pathog 1999;26:317–23. [7] Hall-Stoodley L, Stoodley P. Evolving concepts in biofilm infections. Cell Microbiol 2009;11:1034–43. [8] Talbot GH, Bradley J, Edwards Jr JE, Gilbert D, Scheld M, Bartlett JG, et al. Bad bugs need drugs: an update on the development pipeline from the Antimicrobial Availability Task Force of the Infectious Diseases Society of America. Clin Infect Dis 2006;42:657–68. [9] Demidova TN, Hamblin MR. Photodynamic therapy targeted to pathogens. Int J Immunopathol Pharmacol 2004;17:245–54. [10] Soncin M, Fabbris C, Busetti A, Dei D, Nistri D, Roncucci G, et al. Approaches to selectivity in the Zn(II)-phthalocyanine-photosensitized inactivation of wildtype and antibiotic-resistant Staphylococcus aureus. Photochem Photobiol Sci 2002;1:815–9.
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[21] Simonetti O, Cirioni O, Orlando F, Alongi C, Lucarini G, Silvestri C, et al. Effectiveness of antimicrobial photodynamic therapy with a single treatment of RLP068/Cl in an experimental model of Staphylococcus aureus wound infection. Br J Dermatol 2012;164:987–95. [22] Mannucci E, Genovese S, Monami M, Navalesi G, Dotta F, Anichini R, et al. Photodynamic topical antimicrobial therapy for infected foot ulcers in patients with diabetes: a randomized, double-blind, placebo-controlled study – D.A.N.T.E (Diabetic ulcer Antimicrobial New Topical treatment Evaluation) study. Acta Diabetol 2013 [Epub ahead of print]. [23] Christensen GD, Simpson WA, Younger JJ, Baddour LM, Barrett FF, Melton DM, et al. Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiol 1985;22:996–1006. [24] Sutherland IW. Biofilm exopolysaccharides: a strong and sticky framework. Microbiology 2001;147:3–9. [25] Arciola CR, Montanaro L, Costerton JW. New trends in diagnosis and control strategies for implant infections. Int J Artif Organs 2011;34:727–36. [26] Davies D. Understanding biofilm resistance to antimicrobial agents. Nat Rev Drug Discov 2003;2:114–22. [27] Li X, Yan Z, Xu J. Quantitative variation of biofilms among strains in natural populations of Candida albicans. Microbiology 2003;149:353–62. [28] Drago L, De Vecchi E, Mattina R, Romanò CL. Activity of N-acetyl-l-cysteine against biofilm of Staphylococcus aureus and Pseudomonas aeruginosa on orthopedic prosthetic materials. Int J Artif Organs 2013;36:39–46. [29] Kolodkin-Gal I, Romero D, Cao S, Clardy J, Kolter R, Losick R. d-Amino acids trigger biofilm disassembly. Science 2010;328:627–9. [30] Leite B, Gomes F, Teixeira P, Souza C, Pizzolitto E, Oliveira R. Combined effect of linezolid and N-acetylcysteine against Staphylococcus epidermidis biofilms. Enferm Infecc Microbiol Clin 2013;31:655–9. [31] Lu Q, Yu J, Bao L, Ran T, Zhong H. Effects of combined treatment with ambroxol and ciprofloxacin on catheter-associated Pseudomonas aeruginosa biofilms in a rat model. Chemotherapy 2013;59:51–6.
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International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Photodynamic antibacterial and antibiofilm activity of RLP068/Cl against Staphylococcus aureus and Pseudomonas aeruginosa forming biofilms on prosthetic material Christian Vassena a,1 , Simone Fenu b,1 , Francesco Giuliani c , Lia Fantetti c , Gabrio Roncucci c , Giulio Simonutti b , Carlo Luca Romanò d , Raffaele De Francesco b , Lorenzo Drago a,e,∗ a
Laboratory of Clinical Chemistry and Microbiology, IRCCS Galeazzi Orthopaedic Institute, Milan, Italy Virology Program, National Institute of Molecular Genetics (INGM), Milan, Italy c Molteni Therapeutics s.r.l., via I. Barontini 8, 50018 Scandicci, Florence, Italy d Center for Reconstructive Surgery of Osteoarticular Infections (C.R.I.O.), IRCCS Galeazzi Orthopaedic Institute, Milan, Italy e Laboratory of Technical Sciences for Laboratory Medicine, Department of Biomedical Science for Health, University of Milan, Milan, Italy b
a r t i c l e
i n f o
Article history: Received 10 January 2014 Received in revised form 26 March 2014 Accepted 26 March 2014 Keywords: Antibiofilm photodynamic activity Prosthetic joint infections Confocal laser scanning microscopy Bacterial biofilm
a b s t r a c t Prosthetic joint infections (PJIs) are becoming a growing public health concern in developed countries as more people undergo arthroplasty for bone fixation or joint replacement. Because a wide range of bacterial strains responsible for PJIs can produce biofilms on prosthetic implants and because the biofilm structure confers elevated bacterial resistance to antibiotic therapy, new drugs and therapies are needed to improve the clinical outcome of treatment of PJIs. Antimicrobial photodynamic therapy (APDT), a non-antibiotic broad-spectrum antimicrobial treatment, is also active against multidrug-resistant microorganisms such as meticillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. APDT uses a photosensitiser that targets bacterial cells following exposure to visible light. APDT with RLP068/Cl, a novel photosensitiser, was studied by confocal laser scanning microscopy (CLSM) to evaluate the disruption of MRSA and P. aeruginosa biofilms on prosthetic material. Quantitative CLSM studies showed a reduction in biofilm biomass (biofilm disruption) and a decrease in viable cell numbers, as determined by standard plate counting, in the S. aureus and P. aeruginosa biofilms exposed to APDT with the photosensitiser RLP068/Cl. APDT with RLP068/Cl may be a useful approach to the treatment of PJI-associated biofilms. © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
Introduction Total joint arthroplasties have significantly increased in recent years. Prosthetic joint infection (PJI) is one of the major complications of total joint arthroplasty, usually requiring revision surgery and prolonged courses of intravenous or oral antimicrobial therapy [1]. Despite its relatively low incidence (1–2%), the healthcare costs for treating PJIs are staggeringly high [2], with an increase from US$320 million to $566 million between 2001 and 2009, and
∗ Corresponding author. Present address: Laboratory of Clinical Chemistry and Microbiology, IRCCS Galeazzi Orthopaedic Institute, via R. Galeazzi 4, 20161 Milan, Italy. Tel.: +39 02 6621 4839; fax: +39 02 6621 4774. E-mail address: [email protected] (L. Drago). 1 These two authors contributed equally to this work.
they are expected to rise further to over $1.62 billion by 2020 in the USA alone [3]. Bacterial biofilms are a common cause of persistent infections and are often responsible for PJIs [4,5]. A biofilm is a matrixenclosed microbial population involving cell-to-cell adhesion between micro-organisms and an artificial surface [6]. A common feature shared by biofilm-related infections is their intrinsic resistance to host immunity as well as conventional antimicrobial agents and biocides [7]. Biofilms can tolerate levels of antibiotics 10–1000 times higher than the minimum inhibitory concentration of the corresponding planktonic form. Today, with fewer new antimicrobials coming through the drug development pipeline [8], there is an urgent unsatisfied medical need for alternative drugs and/or therapies. One novel approach to inactivate bacterial biofilms on prosthetic implants is with the use of antimicrobial photodynamic therapy (APDT). By acting on the micro-organisms embedded in the biofilm matrix, the matrix itself and the other
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Fig. 1. (A) Structure of the tetracationic Zn(II) phthalocyanine chloride RLP068/Cl photosensitiser. (B) Visible absorption spectrum of RLP068/Cl.
components stabilising its structure, APDT disrupts the biofilm and reduces its re-growth. APDT is a bimodal strategy that combines light, a chemical substance (or photosensitiser) activated on exposure to light waves within its absorption spectrum, and oxygen to obtain a cytotoxic effect through the production of in situ reactive oxygen species (ROS). In turn, ROS are formed by two reaction pathways. In type 1 reactions, electron transfer occurs from the photosensitiser triplet state to biological substrates resulting in the formation of radical species that react with oxygen to produce ROS such as hydrogen peroxide (H2 O2 ), hydroxyl radical (HO*) and superoxide anion (O2 − ). In type 2 reactions, energy is transferred from the photosensitiser triplet state directly to oxygen to form singlet oxygen (1 O2 ). RLP068/Cl, a recently developed photosensitiser used in the present study, is thought to act mainly through the type 2 pathway [9]. Because the photosensitiser can reach target micro-organisms without affecting surrounding tissues or cells when activated under a suitable illumination protocol [10], the main advantage of APDT is its high target specificity with few undesired side effects, i.e. the drug is inactive in the dark and becomes active only when exposed to light [11]. Since some photosensitisers bind rapidly and selectively to microbial cells, APDT holds promise as an anti-infective approach [12], which is now becoming a therapeutic experimental reality. RLP068/Cl is a cationic Zn(II) phthalocyanine derivative (molecular weight >1300 Da) (Fig. 1) believed to mainly target the bacterial cell wall and membrane both through hydrophobic and electrostatic interactions. Upon activation by light, RLP068/Cl disrupts the cell’s external components through various oxidative reactions initiated by photosensitisation and enters the wall/membranes of bacteria and fungi through a self-promoting uptake process [13]. As this process is unaffected by mechanisms of bacterial resistance to antibiotics, both susceptible and multidrug-resistant bacterial strains may be suitable targets for APDT [14,15]. The multi-target nature and broad spectrum of action of photosensitised inactivation make this approach particularly interesting for treating biofilm-associated PJIs [16]. However, although APDT has long been studied in planktonic cultures, little is known about its potential use against bacteria organised in biofilms. Although the ability of APDT to eradicate bacterial biofilms growing in dental plaques and on oral implants has been variously investigated [17–19], to the best of our knowledge its effect on biofilms developed on prosthetic materials has been far less studied. To fill this gap, here we examined the photodynamic effects of RLP068/Cl on the viability and
architecture of meticillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa biofilms developed on prosthetic material. Materials and methods Bacterial strains The studied micro-organisms were an MRSA strain (SAUMRBP2) and a P. aeruginosa strain (PAE2). Both strains were isolated at the Microbiology Laboratory of the IRCCS Galeazzi Orthopaedic Institute (Milan, Italy) from patients referred to the Center for Reconstructive Surgery of Osteoarticular Infections (C.R.I.O.) of the institute for revision of septic knee implants. The strains were selected for their high capacity to produce in vitro biofilm on prosthetic materials. Bacterial strains were grown overnight in tryptic soy broth (TSB) (bioMérieux, Marcy l’Étoile, France) at 37 ◦ C under aerobic conditions unless otherwise specified. Biofilm growth Sterilised sandblasted titanium discs (25 mm diameter, 5 mm thickness; batch J04051) (Adler Ortho, Milan, Italy) were used as the substrate for biofilm formation. Briefly, discs were placed into six-well flat-bottomed sterile polystyrene microplates (Jet Biofil, China) containing 5 mL of TSB. Overnight cultures of S. aureus and P. aeruginosa were re-suspended to a final concentration of 1.0 × 108 CFU/mL in fresh TSB. Aliquots (200 L) of the diluted bacterial suspension were inoculated in each microplate well containing a titanium disc and were incubated at 37 ◦ C aerobically. Exhausted growth medium containing the non-adherent bacteria was removed 24 h later and was replaced with 5 mL of fresh medium. To obtain mature biofilms, the plates were incubated for a further 48 h. Prior to treatment, the remaining non-adherent bacteria were removed by washing three times with sterile saline solution. Photosensitiser RLP068/Cl, a tetracationic Zn(II) phthalocyanine chloride (Molteni Therapeutics, Florence, Italy), was dissolved in sterile
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Milli-Q water to give a 1.0 mM stock solution and was stored at −20 ◦ C in the dark until use [20]. Light source Illumination was carried out using a diode laser centred at 689 ± 3 nm (Ceralas PDT 689 nm/3 W; CeramOptec GmbH, Bonn, Germany) equipped with an optical fibre with a final light diffuser (Frontal light diffuser, Model FD; Medlight S.A., Ecublens, Switzerland) positioned at a suitable distance from the biofilm sample to provide a light spot ca. 5 cm in diameter. The fluence rate at the spot level was experimentally determined to be 120 mW/cm2 (Nova laser power/energy metre; Ophir Optronics Ltd., Jerusalem, Israel). A light dose of 60 J/cm2 was administered based on previous experiments in which this value was found to be both effective and clinically safe [21,22]. The maximum wavelength of RLP068/Cl was 690 nm, which overlapped with that of the laser emission of 689 ± 3 nm.
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A 488 nm laser line was used to excite SYTO9, whilst fluorescent emission was detected from 500 nm to 540 nm. PI was excited with a 561 nm laser line, and its fluorescent emission was detected from 600 nm to 695 nm. To minimise the possibility of false co-localisation between fluorescent stains owing to bacterial movement, we opted for a simultaneous acquisition mode of the two channels in which the laser beam scanned the visual field at a frequency of 700 Hz. Moreover, using a third laser line (633 nm) in reflection mode, it was possible to determine the reflecting surfaces of both the titanium disc (starting acquisition point) and the coverslip (ending acquisition point) with high accuracy. Images from five randomly selected areas were acquired for each disc, for each of which sequential optical sections of 2 m were collected in sequence along the z-axis over the complete thickness of the sample. The resulting stacks of images were analysed, quantified and rendered into three-dimensional (3D) mode using Volocity 3D Image Analysis Software (Perkin Elmer, Waltham, MA). Statistical analysis
Photodynamic inactivation studies To investigate the efficacy of APDT with RLP068/Cl on the viability of bacterial cells embedded in the biofilm grown on the prosthetic material, titanium discs were incubated in the dark for 20 min with 5 mL of 50 M RLP068/Cl solution and were then illuminated with a final light dose of 60 J/cm2 . Controls were carried out without treatment (growth control). The illuminated and non-illuminated discs were then placed in sterile containers together with 10 mL of sterile saline solution and were sonicated at room temperature in an ultrasonic bath (WVR, Milan, Italy) for 5 min at a frequency of 30 kHz and a power of 300 W. Serial 10-fold dilutions in sterile saline of the sonicated fluids were plated on tryptic soy agar plates (bioMérieux). Following incubation at 37 ◦ C for 16–18 h, the number of grown colonies was counted. The efficacy of RLP068/Cl on the bacterial biofilm was evaluated by a spectrophotometric method as described in Christensen et al. [23]. Briefly, biofilms grown on the titanium discs were air-dried and were stained by disc immersion in a 5% crystal violet (CV) solution for 15 min and then, after several washings, were air-dried again. Biofilm biomass was estimated by elution of the biofilm-bound CV with 3 mL of ethanol (96%) and then measuring the absorbance of 100 L of eluted dye solution at 595 nm by means of a microplate photometer (MultiskanTM FC; Thermo Scientific; Milan, Italy). All experiments were carried out in triplicate. Confocal laser scanning microscopy (CLSM) studies Microbial biofilm was grown on titanium discs and was treated as described above. Controls were carried out without treatment (growth control), without illumination (photosensitiser alone, dark control) as well as with illumination but without RLP068/Cl (light control). A FilmTracerTM LIVE/DEAD® Biofilm Viability Kit (Molecular Probes, Life Technologies Ltd., Paisley, UK) was used to determine the viability of bacteria within the biofilm and the structural damage after photodynamic treatment: dead cells emitted red fluorescence, whereas cells emitting green fluorescence were considered viable. All samples (both the illuminated and non-illuminated) were stained for 15 min in the dark at room temperature with an appropriate volume of a mixture of the two dyes SYTO9 and propidium iodide (PI) (3 L of each component in 1 mL of saline solution). Stained biofilms were examined with a confocal microscope (Leica TCS SP5; Leica Microsystems CMS GmbH, Mannheim, Germany) using a 20× dry objective (HC PL FLUOTAR 20.0 × 0.50 DRY) plus a 2× electronic zoom. The specimens were coverslipped to minimise air contact and to maintain sample moisture constant.
Results of the CV assays and biomass volume variations obtained from the CLSM image analysis are presented as the mean ± standard deviation (S.D.). Results of the CV assay were analysed using a nonpaired Student’s t-test, whilst the variations in biofilm biomass volumes obtained from CLSM were analysed by non-parametric one-way analysis of variance (ANOVA) followed by the Tukey’s HSD test. Results Antimicrobial activity The antimicrobial activity of RLP068/Cl following illumination was preliminarily evaluated by plate counts. The decrease in cell viability was estimated to be ca. 1.5 log10 CFU/mL both for S. aureus and P. aeruginosa (Fig. 2). Crystal violet assay Antibiofilm activity, defined as the ability to disrupt the biofilm structure, was preliminarily determined using the CV method as described in Christensen et al. [23]. Quantification of the whole biomass present on the titanium discs after treatment showed significant differences between the illuminated and non-illuminated samples. Compared with the untreated samples, the amount of biofilm in the APDT-treated samples was significantly lower (P < 0.001) (Fig. 3). Confocal laser scanning microscopy analysis The FilmTracerTM LIVE/DEAD® Biofilm Viability Kit staining properties, together with the instrumental set-up used, allowed the biomass volume and the ratio between live and dead cells in the matrix to be quantified. Biomass volumes (expressed as the mean ± S.D. of the five acquired areas for each disc both for S. aureus and P. aeruginosa) are shown in Figs. 4 and 5, respectively. As shown in Fig. 4, there was a statistically significant reduction in the biomass volume of the S. aureus sample treated with RLP068/Cl compared with the growth (P < 0.01), light (P < 0.01) and dark controls (P < 0.05). No differences were observed between the dark and growth controls (P > 0.05), whereas significant differences were found between the light and growth controls (P < 0.05). Fig. 5 shows a statistically relevant reduction (P < 0.05) in the biomass volume of the P. aeruginosa sample treated with RLP068/Cl
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Fig. 2. Antimicrobial activity of RLP068/Cl. Efficacy of killing meticillin-resistant Staphylococcus aureus (SAU-MRBP2) and Pseudomonas aeruginosa (PAE2) cells within the biofilm after 20 min incubation in the dark with 50 M RLP068/Cl, without illumination (white bars) and with illumination (black bars). Colony count from single sample (n = 1).
Fig. 3. Cristal violet (CV) assay. Absorbance value at 595 nm for meticillin-resistant Staphylococcus aureus (SAU-MRBP2) and Pseudomonas aeruginosa (PAE2) strains after 20 min incubation in the dark with 50 M RLP068/Cl, without illumination (white bars) and with illumination (black bars). Mean absorbance data (±standard deviation) of experiments carried out in triplicate (n = 3). ***P < 0.001.
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Fig. 4. Biomass volume of Staphylococcus aureus samples. RLP068/Cl antimicrobial photodynamic therapy significantly reduced S. aureus (SAU-MRBP2) biomass volume upon illumination. Quantification was performed using Leica Application Suite Advanced Fluorescence (LAS-AF) software to obtain the mean biofilm thickness multiplied by the total surface of portions acquired (n = 5). **P < 0.05; ***P < 0.01.
compared with the growth control. No statistically significant differences were found between the controls. The 3D renderings (Figs. 6 and 7) confirmed a decrease in the biofilm volume both of the S. aureus and P. aeruginosa samples treated with RLP068/Cl compared with the controls (growth, dark and light). Fig. 6 shows an example of 3D reconstruction of one of
the acquired portions of an S. aureus biofilm sample grown on a titanium disc. The RLP068/Cl APDT-treated sample (Fig. 6B) clearly shows a reduction in biofilm volume compared with the relative controls (Fig. 6A, C, and D). Fig. 7 shows an example of 3D reconstruction of one of the acquired portions of a P. aeruginosa biofilm sample grown on a
Fig. 5. Biomass volume of Pseudomonas aeruginosa samples. RLP068/Cl antimicrobial photodynamic therapy significantly reduced P. aeruginosa (PAE2) biomass volume upon illumination. Quantification was performed using Leica Application Suite Advanced Fluorescence (LAS-AF) software to obtain the mean biofilm thickness multiplied by the total surface of portions acquired (n = 5). **P < 0.05.
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Fig. 6. Three-dimensional (3D) image of Staphylococcus aureus samples. 3D reconstruction by confocal laser scanning microscopy of antimicrobial photodynamic therapytreated S. aureus biofilms. Biofilms were grown for 72 h and were then treated with 50 M RLP068/Cl solution for 20 min in the dark and were subsequently illuminated with a total light dose of 60 J/cm2 . Biofilms were stained with FilmTracerTM LIVE/DEAD® Biofilm Viability Kit (Molecular Probes, Life Technologies Ltd., Paisley, UK): SYTO9 (green) represents viable cells; propidium iodide (red) represents dead cells. (A) Bacteria without treatment (growth control); (B) RLP068/Cl-treated bacteria exposed to 60 J/cm2 light dose; (C) bacteria treated with RLP068/Cl in the dark (dark control); and (D) bacteria untreated but illuminated (light control).
Fig. 7. Three-dimensional (3D) image of Pseudomonas aeruginosa samples. 3D reconstruction by confocal laser scanning microscopy of antimicrobial photodynamic therapytreated P. aeruginosa biofilms. Biofilms were grown for 72 h and were then treated with 50 M RLP068/Cl solution for 20 min in the dark and were subsequently illuminated with a total light dose of 60 J/cm2 . Biofilms were stained with FilmTracerTM LIVE/DEAD® Biofilm Viability Kit (Molecular Probes, Life Technologies Ltd., Paisley, UK): SYTO9 (green) represents viable cells; propidium iodide (red) represents dead cells. (A) Bacteria without treatment (growth control); (B) RLP068/Cl-treated bacteria exposed to 60 J/cm2 light dose; (C) bacteria treated with RLP068/Cl in the dark (dark control); and (D) bacteria untreated but illuminated (light control).
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Fig. 8. Percentage composition of the analysed Staphylococcus aureus samples. Pie charts showing the proportion of meticillin-resistant S. aureus (SAU-MRBP2) biomass components obtained from confocal laser scanning microscopy image analysis using Volocity 3D Image Analysis Software (Perkin Elmer, Waltham, MA): biofilm (blue area); live cells within the biofilm (green area); dead cells within the biofilm (red area); and amount of biofilm ‘lost’ compared with the growth control upon antimicrobial photodynamic therapy for the RLP068/Cl-treated sample and the dark and light controls (grey area).
titanium disc. Also here, the RLP068/Cl APDT-treated sample (Fig. 7B) clearly shows a reduction in biofilm volume compared with the controls (Fig. 7A, C, and D). Quantification of the bacterial cells included in the biomatrix was carried out by measuring the volume of fluorescence expressed by the two specific dyes (SYTO9 and PI) of the staining kit. As expected, there was a substantial difference in the biofilm matrix composition and structure for the two bacterial species [24]. Since the characteristics of the P. aeruginosa matrix made it difficult to quantify the volume occupied by the bacterial cells (live and dead) within the matrix, the experimental evaluation was limited to determination of the biomass volume alone. Conversely, it was possible to quantify both the live and dead cells of S. aureus and to reconstruct the percentage composition of the samples analysed. As shown in Fig. 8, the cells (live or dead) present within the matrix occupied ca. 5% of the total biomass in all of the samples. In the control samples (growth, dark and light), the live cells accounted for 4.7%, 4.8% and 4.9% of the total biomass, respectively, whereas in the RLP068/Cl APDT-treated samples the live cells made up 2.4% and the dead cells made up 2.3% of the total biomass.
Discussion PJIs caused by biofilm-producing bacteria are a feared complication of arthroplasty for bone fixation or joint replacement. Even with routine precautions, such as pre-operative antimicrobial prophylaxis and surgical environment protection, the risk of intra-operative infection is 1% and 2% for hip and knee replacement, respectively [2]. Orthopaedic implant-related infections are usually managed with complete device removal, long-term antimicrobial therapy with conventional drugs and, eventually, re-implantation [1]. Novel approaches to PJI eradication rely on the synergistic effect gained from the use of combined strategies based on the action of biofilm-disrupting agents coupled with antibiotics [25], although an effective antibiofilm treatment specifically for PJIs is not yet available. In this scenario, APDT is an innovative option for treating PJIs in the clinical setting. Building on the promising results from in vitro tests on Grampositive and Gram-negative bacteria in planktonic form [14,15], as well as in the treatment of chronic wounds where bacteria are predominantly present as biofilm [21], in this study we tested the antimicrobial and antibiofilm efficacy of APDT with RLP068/Cl
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against clinical isolates of S. aureus and P. aeruginosa forming biofilms on prosthetic material. The RLP068/Cl concentration used for these tests (50 M) reduced free-floating bacteria by a factor of almost 7 log10 [14,15]. Following RLP068/Cl treatment and illumination of the biofilm samples, bacterial inactivation was less than that observed in the corresponding planktonic forms. These findings were expected since bacteria organised as biofilm are not only less susceptible to antibiotics but are also more resistant to environmental stress. Therefore, a long-lasting therapy with elevated drug dosage is needed which, however, raises the risk of increased resistance to currently used antimicrobial agents [26]. On the other hand, the short incubation time of the photosensitiser with the microorganisms and the typically multi-target nature of photosensitised inactivation processes prevent the possible expression of protective factors (e.g. biosynthesis of stress proteins), thus minimising the risk of emergence of resistant strains [15]. The antibiofilm activity of RLP068/Cl APDT was preliminarily investigated using the CV assay. CV is a basic dye that readily binds to negatively charged molecules (and thus to acidic polysaccharides in the extracellular matrix) [27]. In our experience, owing to the non-specific nature of the dye, this method yields only semiquantitative data for staining both living and dead cells and biofilm matrix. Moreover, the ethanol used to solubilise the dye does not guarantee complete extraction from the biomass. Nevertheless, the current results showed an estimated decrease of 45% and 38% in S. aureus and P. aeruginosa whole biomass, respectively. CLSM is one of the most sensitive and specific techniques for biofilm structure visualisation and analysis. To further investigate the changes in the spatial structure of the biofilm induced by RLP068/Cl APDT treatment, samples of mature biofilm grown on sandblasted titanium discs were analysed after staining with the FilmTracerTM LIVE/DEAD® Biofilm Viability Kit. It was possible to quantify the volume occupied by the live and dead bacterial cells within the biomass only for S. aureus. As demonstrated by fluorescence quantification (Volocity software) and observable from the 3D renderings, RLP068/Cl APDT significantly reduced the biomass volume of both micro-organisms compared with the relative growth control. Moreover, the light control showed a decrease in biomass volume of S. aureus probably due to aspecific physical phenomena. As expected, albeit not statistically significant, a mild effect on biomass volume was observable in the dark control, likely due to the non-photodynamic antibiofilm activity of the molecule. As confirmation of the antibiofilm effect of RLP068/Cl APDT exerted by the photodynamic action mechanism, a greater decrease in biomass volume was observed in the treated samples in which there was appreciable antibacterial action (up to 50%), not observable in either the dark or light controls (Fig. 8). Unfortunately, it was not possible to determine the ratio between live and dead cells in the P. aeruginosa biofilm. In particular, it was difficult to estimate the amount of PI bonded to the dead cells, which precluded reliable quantification of the number of viable cells. A decrease in biomass volume of P. aeruginosa after APDT treatment was also observed, even if statistical significance can be reported only for the growth control. This may be ascribed to the nature of the P. aeruginosa biofilm, especially the difference in matrix composition [24] compared with biofilm formed by S. aureus. These results, obtained under experimental in vitro conditions, suggest that APDT with RLP068/Cl exerts antibiofilm and antimicrobial activities already after a single treatment against bacterial biofilm produced by a clinical strain of MRSA. Although activity against P. aeruginosa biofilm was observed, further experimentation and refinement of technical settings are needed to improve the capability to study the biofilm produced by this microbe. Microbial biofilms can quickly re-form unless completely eradicated. This peculiarity poses a major challenge to treating
infections. A future area of focus will involve a larger number of micro-organisms in order to determine the long-lasting effectiveness as a function of the frequency of APDT treatments. This could be done by evaluating antibiofilm and antibacterial efficacy mediated by repeated in vitro APDT treatment. Repeated treatments over time will likely amplify the effects observed after a single treatment by increasing the reduction of biomass volume and decreasing the number of surviving cells. If this hypothesis is confirmed, further animal studies will be required to establish the optimum parameters (number of APDT treatments, time between treatments) for an effective and safe protocol to translate from laboratory science to clinical use for treating PJIs. The aim of this study was to obtain ‘proof of concept’ for the application to PJIs of RLP068/Cl APDT, already successfully tested in a phase 2 clinical trial for the treatment of diabetic foot ulcer infections. This study holds particular interest as there are currently no alternative effective treatments that exert both antibacterial and antibiofilm activity. Whilst substances with antibiofilm properties, such as N-acetyl cysteine or d amino acids, are able to trigger biofilm disassembly, they do not exhibit a pronounced antibacterial activity [28,29]. Conversely, many antibiotics possess antibacterial activity but are not active against biofilms. Combined treatment that disrupts microbial biofilm and possesses antibacterial activity [30,31], as might be achieved with the APTD approach, could represent an innovative therapeutic technology. Acknowledgments The authors are grateful to Elena De Vecchi (Laboratory of Clinical Chemistry and Microbiology, IRCCS Galeazzi Orthopaedic Institute, Milan, Italy) for helpful scientific discussions. The authors also thank Riccardo Rossi [Computational Biology & Data Analysis, Istituto Nazionale Genetica Molecolare (INGM), Milan, Italy] for assistance with the statistical analysis. Funding: This study was supported by IRCCS Galeazzi Orthopaedic Institute (Milan, Italy) with a grant for current research [no. L-4063]. Competing interests: FG, LF and GR are Molteni Therapeutics employees, which is the owner of RLP068/Cl patents. All other authors declare no competing interests. Ethical approval: Not required. References [1] Darouiche RO. Treatment of infections associated with surgical implants. N Engl J Med 2004;350:1422–9. [2] Sia IG, Berbari EF, Karchmer AW. Prosthetic joint infections. Infect Dis Clin North Am 2005;19:885–91. [3] Kurtz SM, Lau E, Watson H, Schmier JK, Parvizi J. Economic burden of periprosthetic joint infection in the United States. J Arthroplasty 2012;27(Suppl):61–5. [4] Zimmerli W, Trampuz A, Ochsner PE. Prosthetic-joint infections. N Engl J Med 2014;351:1645–54. [5] Gallo J, Kolár M, Novotny´ R, Riháková P, Tichá V. Pathogenesis of prosthesisrelated infection. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2003;147:27–35. [6] Ellington JK, Reilly SS, Ramp WK, Smeltzer MS, Kellam JF, Hudson MC. Mechanisms of Staphylococcus aureus invasion of cultured osteoblasts. Microb Pathog 1999;26:317–23. [7] Hall-Stoodley L, Stoodley P. Evolving concepts in biofilm infections. Cell Microbiol 2009;11:1034–43. [8] Talbot GH, Bradley J, Edwards Jr JE, Gilbert D, Scheld M, Bartlett JG, et al. Bad bugs need drugs: an update on the development pipeline from the Antimicrobial Availability Task Force of the Infectious Diseases Society of America. Clin Infect Dis 2006;42:657–68. [9] Demidova TN, Hamblin MR. Photodynamic therapy targeted to pathogens. Int J Immunopathol Pharmacol 2004;17:245–54. [10] Soncin M, Fabbris C, Busetti A, Dei D, Nistri D, Roncucci G, et al. Approaches to selectivity in the Zn(II)-phthalocyanine-photosensitized inactivation of wildtype and antibiotic-resistant Staphylococcus aureus. Photochem Photobiol Sci 2002;1:815–9.
Please cite this article in press as: Vassena C, et al. Photodynamic antibacterial and antibiofilm activity of RLP068/Cl against Staphylococcus aureus and Pseudomonas aeruginosa forming biofilms on prosthetic material. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.012
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Please cite this article in press as: Vassena C, et al. Photodynamic antibacterial and antibiofilm activity of RLP068/Cl against Staphylococcus aureus and Pseudomonas aeruginosa forming biofilms on prosthetic material. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.012