Accepted Manuscript Title: Phototoxic effect of hypericin alone and in combination with acetylcysteine on Staphylococcus aureus biofilms Author: Nasim Kashef Shima Karami Gholamreza Esmaeeli Djavid PII: DOI: Reference:
S1572-1000(15)00036-8 http://dx.doi.org/doi:10.1016/j.pdpdt.2015.04.001 PDPDT 640
To appear in:
Photodiagnosis and Photodynamic Therapy
Received date: Revised date: Accepted date:
14-9-2014 2-4-2015 6-4-2015
Please cite this article as: Kashef N, Karami S, Djavid GE, Phototoxic effect of hypericin alone and in combination with acetylcysteine on Staphylococcus aureus biofilms, Photodiagnosis and Photodynamic Therapy (2015), http://dx.doi.org/10.1016/j.pdpdt.2015.04.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highlights 1- We evaluated the in vitro bactericidal effect of HYP-PDI on clinical Staphylococcus aureus biofilms.
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2- In vitro bactericidal effect of HYP-PDI on S. aureus biofilms treated with acetylcysteine was also studied.
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3- HYP-PDI did not result in a reduction in viable count for each of the strains when grown in biofilms.
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4- HYP-PDI applied on biofilms treated with acetylcysteine was able to disrupt pre-formed biofilms.
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5- Acetylcysteine may enhance the efficacy of PDI by disrupting the biofilm structure.
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Title:
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Phototoxic effect of hypericin alone and in combination with acetylcysteine on Staphylococcus aureus biofilms
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Authors: Nasim Kashef1, Shima Karami2, Gholamreza Esmaeeli Djavid3 1
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Assistant Professor of Medical Bacteriology, Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran Tel: +98-21-61113558 Fax: +98-21-66492992 E-mail: [email protected] 2
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MSc, Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran Tel: +98-21-61113558 Fax: +98-21-66492992 E-mail: [email protected] 3
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MD, Iranian Research Center for Medical Lasers, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran Tel: +98-21-66480877 Fax: +98-21-66952040 E-mail: [email protected]
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Corresponding author: Mailing address: Nasim Kashef, Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran Tel: +98-21-61113558 Fax: +98-21-66492992 E-mail: [email protected] 2
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Abstract Background: Resistance of bacteria against antibiotics and antimicrobials is arising worldwide
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and there is an urgent need for strategies that are capable of inactivating biofilm-state pathogens
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with less potential of developing resistance in pathogens. A promising approach could be
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photodynamic inactivation (PDI) which uses light in combination with a photosensitizer to
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induce a phototoxic reaction. In this study, we evaluated the in vitro phototoxic effect of
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hypericin (HYP) alone and in combination with acetylcysteine (AC) on Staphylococcus aureus
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biofilms. AC, a mucolytic agent, reduces the production of extracellular polysaccharide matrix
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while promoting the disruption of mature biofilm.
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Methods: In vitro phototoxic effect of HYP alone (0.5 µg/ml, light dose: 16 J/cm2), and in
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combination with AC (10 mg/ml) on ten clinical S. aureus isolates and S. aureus (ATCC 25923)
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biofilms was studied. Effect of HYP concentration (0.5 µg/ml) and light dose (8 J/cm2) on PDI
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of all eleven S. aureus strains in planktonic forms was also investigated.
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Results: HYP-PDI did not result in a reduction in viable count for each of the strains when
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grown in biofilms. However, HYP-PDI applied on biofilms treated with AC was able to disrupt
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pre-formed biofilms (viable count reduction ranging from 5.2 to 6.3 log10-unit in comparison to
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controls in all tested strains). A 6.5 log killing was obtained for S. aureus (ATCC 25923)
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planktonic cells treated with 0.5 µg/ml at 8 J/cm2. For this set of PDI parameters, ten clinical S.
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aureus isolates showed 5.5-6.7 log killing.
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Conclusion: HYP-PDI in combination with AC had significant ability to eradicate the pre-
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formed mature biofilms of S. aureus strains.
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Key words: photodynamic inactivation, hypericin, Staphylococcus aureus, acetylcysteine
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Introduction
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In many environments bacteria exist as a complex, multispecies surface-associated community
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termed biofilm. Organisms within biofilms are embedded in a self-produced matrix of
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extracellular polymeric substance (EPS) composed of polysaccharides, proteins, lipids, and
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extracellular DNA. Several advantages exist for bacteria that live in a biofilm phenotype
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including structural stability, firm adherence to biotic or abiotic surfaces, increased virulence,
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and resistance to both antimicrobial therapy and the host immune response (1-3).
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Staphylococcus aureus is an important human pathogen associated with numerous skin diseases
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including wound infections. The difficulty in eradicating S. aureus colonization with
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conventional antibiotic therapy may be due to the presence of biofilm (4). EPS itself may slow
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drug-diffusion by its higher viscosity or can even act as a barrier (5). Furthermore, development
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of a biofilm leads to an enormous genetic diversity of its cells, which provides insurance for the
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cells for better adaptation to rapid alteration of environment conditions (6). Also, persister cells
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are being formed, when bacteria grow as a biofilm. These are in a dormant, non-dividing state
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and provide tolerance toward antimicrobial agents (7).
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S. aureus biofilms have a negative effect on wound healing as evidenced by their ability to
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induce keratinocyte apoptosis in vitro and inhibit re-epithelialization in an in vivo animal wound
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model (8,9). In addition, biofilms may also increase the inflammatory response characteristic of
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chronic wounds, thus promoting tissue damage and further contributing to the non-healing
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phenotype (10).
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With increasing the resistance of pathogenic bacteria including S. aureus to antimicrobial agents,
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there is a need for the development of alternative antibacterial strategies; photodynamic
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inactivation (PDI) could be the method of choice. PDI involves killing of organisms by light in
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the presence of a photosensitizing agent (11). The local combination of light and the
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photosensitive drug in the frame of PDI avoids systemic effects on bacterial flora and by this
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minimizes side effects. Reports of successful inactivation of multi-drug-resistant strains of S.
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aureus declare PDI to be the method of choice for treatment of such local infections (12,13).
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Several studies have been reported PDI mediated by hypericin (HYP), a powerful photosensitizer
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(14-20 ). HYP is a phenanthroperylene quinine pigment naturally occuring in Hypericum plants.
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It is characterized by a high quantum yield for formation of reactive oxygen species and very
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slow photobleaching (21).
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In our pervious study, we evaluated the in vitro bactericidal effect of HYP-PDI on different
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bacterial species, assessing its photocytotoxicity to primary human fibroblasts to determine
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possible side effects (20). Our results showed that HYP had a high phototoxicity to S. aureus,
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Enterococcus faecalis and Escherichia coli at extremely low drug concentrations and did not
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induce significant cytotoxic effects on human fibroblasts in culture. However, planktonic
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bacterial cultures as employed in that study represent the first step in the evaluation of a
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photoactive compound for its use in PDI. So, the purpose of the present study was to evaluate the
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in vitro phototoxic effect of HYP alone and in combination with acetylcysteine (AC) on
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Staphylococcus aureus biofilms. AC, also known as N-acetylcysteine, is a mucolytic agent that
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disrupts disulfide bonds in mucus and, for this reason; it reduces the viscosity of secretions (22).
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AC decreases biofilm formation by a variety of bacteria (22-24) and reduces the production of
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extracellular polysaccharide matrix (25) while promoting the disruption of mature biofilm
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(23,24).
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Materials and methods
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Bacterial species and culture conditions
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A total of ten clinical S. aureus recovered from acute and chronic wounds were used in this
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study. In addition, S. aureus (ATCC 25923) was also included. The in vitro antimicrobial
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susceptibility of clinical isolates was determined according to CLSI guidelines (26). Antibiotic
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discs were purchased from Padtan Teb Company, Iran. Eight clinical isolates were resistant to
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the following antibiotics: amoxicillin—clavulanate (20/10 µg), oxacillin (1 µg), ciprofloxacin (5
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µg), gentamicin (10 µg), and amikacin (30 µg). S. aureus (UTMC 1484) was resistant to
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ciprofloxacin, amoxicillin—clavulanate and gentamicin and sensitive to oxacillin and amikacin.
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S. aureus (UTMC 1474) was resistant to ciprofloxacin, amoxicillin—clavulanate and sensitive to
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oxacillin, gentamicin and amikacin.
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Organisms were maintained by weekly subculture on nutrient agar (Merck, Germany). All
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organisms were grown aerobically in nutrient agar plates at 37 °C for 18-24 h. Then a suspension
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of each organism was prepared in sterile phosphate-buffered saline (PBS, pH 7.4) to reach the
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turbidity of McFarland standards. No. 2 (a concentration of 6×108 CFU/ml).
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Photosensitizer (PS) and light source
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HYP (Tocris Bioscience, UK, purity > 98%) was dissolved as a stock at 100 µg/ml in dimethyl
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sulphoxide (DMSO). Stock solution was kept at 4°C in the dark and further diluted in sterile PBS
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(pH 7.4) when needed.
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The light source consisted of a light-emitting diode (LED) array. The samples were exposed to
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LEDs (wavelength of 590 nm, measured power of 10 mW).
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Photodynamic inactivation of Staphylococcus aureus planktonic cells
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Bacterial suspensions were incubated with 0.5 µg/ml HYP in the dark at 37 ºC for 5 minutes.
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The HYP-treated cells were centrifuged (6000 rpm for 15 minutes), and washed twice with
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sterile PBS (pH 7.4). Aliquots of 200 µl treated cells were placed in a 96-well microtiter plate
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and irradiated with the light source (5 min, 8 J/cm2). The plates were kept covered during the
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illumination in order to maintain the sterility of the culture. After that, 100 µl of cell suspension
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was spread on nutrient agar in 10-fold serial dilutions. Colonies were counted after incubation
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for 24 h at 37 ºC. All experiments were repeated three times (20).
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Every experiment was accompanied by three control samples: a negative control (no PS, no
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illumination), a light control (no PS, illumination same as PDI samples) and a dark control (with
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PS, no illumination).
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Assessment of Biofilm formation
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S. aureus strains were grown overnight in tryptic soya broth (TSB, Merck, Germany)
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supplemented with 0.2% glucose at 37 ºC and then diluted 1:50 in fresh TSB. For each test strain
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200 µl of inoculums was added to 6 wells of a 96-well flat-bottomed sterile polystyrene
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microplate. A volume of 200 µl TSB was added to the remaining wells and the plate incubated
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for 24 h at 37 ºC. Then, the plate contents emptied out and washed two times with PBS (pH 7.4)
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to remove the planktonic cells. The cells were fixed with 95% ethanol for 10 min and stained
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with 0.4% crystal violet for 15 min. After several washing, the wells were air dried. For a
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quantitative estimation of biofilms, crystal violet was solubilized with 10% glacial acetic acid
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and absorbance of the solubilized dye was determined at 490 nm in a microplate reader (27).
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Photodynamic inactivation of Staphylococcus aureus biofilms
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Bacterial suspensions (6×108 CFU/ml) were diluted at 1:100, in TSB containing 0.2% glucose.
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Aliquots (100 µl) of the diluted bacterial suspensions and 100 µl of fresh TSB containing 0.2%
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glucose were inoculated into 96-well flat-bottomed sterile polystyrene microplates (SPL, Korea),
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and incubated for 18 h at 37°C. Following incubation, the plate contents washed once with fresh
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PBS (pH 7.4) to remove the planktonic cells. To investigate the effect of PDI, 200 µl of 0.5
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µg/ml HYP was added to each well and the plates were incubated in the dark for 5 min at room
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temperature. Wells used as controls were incubated with PBS only. Following incubation, HYP
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was carefully removed from the microwells and the biofilms were washed once with fresh PBS.
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HYP-treated biofilms were irradiated with light source for 10 minutes (16 J/cm2). The biofilms
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were scraped carefully, and then vortexed for 30 s to homogenize the samples. Treated and
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untreated samples were serially diluted, plated on the nutrient agar plates, and incubated for 24 h
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at 37°C in the dark. For each set of measurements, controls consisting of biofilms treated with
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HYP but not exposed to light, exposed to light only, and treated with neither HYP nor light were
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included (27).
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Photodynamic inactivation of Staphylococcus aureus biofilms treated with acetylcysteine
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AC (Hexal, Germany) was dissolved in water to make a 10 mg/ml solution. Biofilm formation
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was performed as described above. After removing the planktonic cells, 200 µl of 10 mg/ml AC
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was added to each well and the plates were incubated in the dark for 10 min at 37 ºC. Following
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incubation, AC was carefully removed from the microwells and HYP diluted in AC solution (at
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final concentration of 0.5 µg/ml) was added to each well and the plates were incubated in the
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dark for 5 min at 37 ºC. Following incubation, HYP was carefully removed from the microwells
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and the biofilms were washed once with fresh PBS. HYP-treated biofilms were irradiated with
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light source for 10 minutes (16 J/cm2). The biofilms were scraped carefully, and then vortexed
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for 30 s to homogenize the samples. Treated and untreated samples were serially diluted, plated
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on the nutrient agar plates, and incubated for 24 h at 37°C in the dark.
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Statistical analysis
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Values were expressed as log10 means ± standard deviation. Comparisons between means of
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groups were analyzed using the One-Way ANOVA and Post Hoc Tukey tests. P 0.05).
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The PDI was applied to all 10 clinical S. aureus isolates, and S. aureus (ATCC 25923) strains. In
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the case of clinical S. aureus strains, the obtained results for HYP-PDI included viable count
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reduction ranging from 5.5 to 6.7 log10-unit. A 6.5 log killing was obtained for S. aureus (ATCC
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25923) treated with 0.5 µg/ml at 8 J/cm2 (figure 1).
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Biofilm formation ability of Staphylococcus aureus strains
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Table 1 shows the biofilm producing ability of ten clinical S. aureus isolates and S. aureus
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(ATCC 25923). According to our results, all 11 S. aureus strains were strong biofilm-producing
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organisms (OD490 > 0.6). For instance, the biofilm-positive strain S. aureus (UTMC 1442) gave
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an OD490 reading of 2.88 ± 0.45, which is a factor of 4.2 greater than that formed by the biofilm-
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positive strain S. aureus (UTMC 1451).
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Photodynamic inactivation of Staphylococcus aureus biofilms
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Our preliminary experiments showed that the treatment of biofilms with HYP (at final
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concentrations of 0.5 µg/ml) in the dark did not exhibit any toxicity against the bacterial cells.
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Similarly, the exposure of cells to light (16 J/cm2) alone did not cause any change in cell survival
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compared to the survival rates of untreated controls (data not shown).
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Unlike to the findings for planktonically grown cultures, HYP-PDI did not result in a reduction
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in viable count for each of the strains when grown in biofilms (figure 2). It was shown that
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bacteria growing as biofims were more resistant to applied PDI compared with planktonic forms.
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Photodynamic inactivation of Staphylococcus aureus biofilms treated with acetylcysteine
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When exposed to AC (10 mg/ml solution) only, there was no statistically significant decrease in
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log10 viable count, in comparison with the untreated control, for each 11 biofilm-grown strains (p
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> 0.05).
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HYP-PDI was found to have significant effects (p < 0.001) on pre-formed biofilms treated with
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AC (figure 3). HYP-PDI did not reduce viable cell counts in the pre-formed biofilms of all tested
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strains compared to the controls (p > 0.05, 0.01 log decrease in viable cells), while, HYP-PDI
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applied on biofilms treated with AC showed the highest ability to disrupt pre-formed biofilms
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(viable count reduction ranging from 5.2 to 6.3 log10-unit in comparison to controls in all clinical
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S. aureus strains). A 5.7 log killing was obtained for S. aureus (ATCC 25923) treated with 0.5
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µg/ml at 16 J/cm2.
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Discussion
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One of the major goals in the field of modern Clinical Microbiology is to attempt to develop new
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strategies capable of reducing the incidence of biofilm infections and of effectively curing
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chronic conditions related to the establishment of these difficult-to-eradicate bacterial structures
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(28). Therefore, in the present study, we evaluated the PDI efficacies on ten clinical S. aureus
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and S. aureus (ATCC 25923) by incubating the bacterial cultures with HYP followed by yellow
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light irradiation and assessed the phototoxic effects of HYP toward pre-formed biofilms of those
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strains. HYP used in this study was very effective in inactivation of all tested S. aureus
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planktonic cells upon illumination; at concentration of 0.5 µg/ml a decrease in bacterial count of
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5.5-6.7 log units was achieved. Our previous study also showed a 6.3 log killing for S. aureus
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(ATCC 25923) treated with 1 µg/ml at 48 J/cm2 (20). Similar to our results, Yow et al. study
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showed that the combination of HYP (8 µM) and light irradiation (30 J/cm2) could induce
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significant killing of methicillin-sensitive and -resistant S. aureus cells (> 6 log reduction) (16).
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In the present study, HYP failed to inactivate S. aureus biofilm cells upon illumination. It was
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shown that bacteria growing as biofilms were more resistant to applied PDI compared with
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planktonic forms. As reported by others (29,30), the dye concentration and the light dose used
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for the photoinactivation of biofilms were considerably higher than those required to inactivate S.
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aureus suspensions. The differences in the photodynamic efficacies of the drug could be due to
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several reasons. In fact, cells growing in biofilm differ from their planktonic counterparts in a
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number of aspects, such as the cell wall composition, rate of growth, and presence of
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polysaccharide intercellular adhesin (PIA), which may block the uptake of the PS and
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penetration of light and thereby reduce the photosensitizing process (31,32). According to Gad et
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al. study, abundant production of PIA has been suggested to obstruct the diffusion of the PS
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through the matrix, thus reducing the susceptibility of biofilms to photosensitization (33).
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To improve the efficiency of PDI using HYP on bacteria growing as biofilms, we used AC, a
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mucolytic agent. AC is widely used in medical practice via inhalation, oral and intravenous
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routes (34-36), and it has an excellent safety profile (37). In addition, AC has been used to treat
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aspergilloma by local installation (38). Its efficacy in chronic obstructive pulmonary disease and
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nephropathy has been reported (39). AC, which is a glutathione precursor, provides
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neuroprotection by preventing oxidative damage (39, 40). AC is also used in the treatment of
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chronic bronchitis (41).
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AC, as an antibiofilm agent, has been used in several studies alone or in combination with
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antimicrobials (42-47). Mansouri et al. studied in vitro antimicrobial activities of central venous
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catheters impregnated with AC and levofloxacin (ACLEV) against a range of important clinical
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pathogens. ACLEV catheters produced the most active and durable antimicrobial effect against
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both Gram-positive and Gram-negative isolates (42). In Leite et al. study, the combined effect of
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rifampicin with AC, in the control of Staphylococcus epidermidis biofilms was assessed.
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Biofilms of two S. epidermidis strains (9142 and 1457) were treated with AC (4 and 40 mg/mL)
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and 10 mg/L (peak serum) of rifampicin alone and in combination. AC at 40 mg/L alone or in
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combination with rifampicin significantly reduced (4 log10) the number of biofilm cells (46). El-
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Feky et al. also evaluated the effect of ciprofloxacin and AC, alone and in combination, on
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biofilm production and pre-formed mature biofilms of important clinical pathogens related to
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ureteral stent surfaces. Ciprofloxacin and AC (2 and 4 mg/ml) inhibited biofilm production by ≥
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60% in all tested microorganisms. Disruption of pre-formed biofilms of all tested
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microorganisms was found to be ≥ 78% in the presence of ciprofloxacin and ≥ 62% in the
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presence of AC (2 and 4 mg/ml), compared to controls. Ciprofloxacin/AC showed the highest
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inhibitory effect on biofilm production (94-100%) and the highest disruptive effect on the pre-
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formed biofilms (86-100%) in comparison to controls. AC was found to increase the therapeutic
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efficacy of ciprofloxacin by degrading the extracellular polysaccharide matrix of biofilms (47).
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In our study, the applied HYP-PDI on pre-formed biofilms treated with AC consistently
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decreased (5.2 to 6.3 log10) viable biofilm-associated bacteria relative to the controls. Since the
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susceptibility of biofilm-associated clinical S. aureus isolates, and S. aureus (ATCC 25923) to
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HYP-PDI was enhanced in biofilms treated with AC, it is possible that an antibiofilm/PDI
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combination would be synergistic. By degrading the extracellular polysaccharide matrix of
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biofilm (23,25), it is possible that AC may have made the biofilm-associated bacteria more
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susceptible to phototoxic effect of HYP, although we did not specifically test this hypothesis.
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To the best of our knowledge, there are no published reports on the combined use of AC and PDI
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for the inactivation of biofilms. PDI is proposed as an alternative antimicrobial method for the
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inactivation of biofilm-related diseases owing to several advantages over the conventional
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antibacterial treatment. However, the efficacy of PDI for biofilm treatment depends mainly on
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the penetration of the PS and light to the deeper layers. Our study shows that pretreatment of
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biofilms with AC may enhance the efficacy of PDI by disrupting the biofilm structure and
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thereby enhancing the PS and light penetration. Thus, the use of combined AC and PDI has the
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advantage of treating and eradicating even thick biofilms and avoiding the disadvantage of using
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antibiotics, with the consequent generation of antimicrobial resistance. Therefore, an
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experimental approach involving a combination of AC and PDI in treating biofilm-associated
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infections may be worth exploring.
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Acknowledgment
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Support was provided by the College of Science, University of Tehran and Iranian Research
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Center for Medical Lasers, Academic Center for Education, Culture and Research.
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aureus. Photochem Photobiol Sci 2010; 9:365-9.
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19. Hager B, Strauss WS, Falk H. Cationic hypericin derivatives as novel agents with
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photobactericidal activity: synthesis and photodynamic inactivation of Propionibacterium acnes.
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Photochem Photobiol 2009; 85:1201-6.
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20. Kashef N, Borghei YS, Djavid GE. Photodynamic effect of hypericin on the microorganisms
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and primary human fibroblasts. Photodiagnosis Photodyn Ther 2013; 10: 150-155.
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21. de Melo Wde C, Lee AN, Perussi JR, Hamblin MR. Electroporation enhances antimicrobial
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photodynamic therapy mediated by the hydrophobic photosensitizer, hypericin. Photodiagnosis
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Photodyn Ther 2013; 10:647-50.
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22. Perez-Giraldo C, Rodriguez-Benito A, Moran FJ, Hurtado C, Blanco MT, Gomez-Garcia
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AC. Influence of N-acetylcysteine on the formation of biofilm by Staphylococcus epidermidis. J
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Antimicrob Chemother 1997; 39: 643-6.
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23. Marchese A, Bozzolasco MGualco L, Debbia EA, Schito GC, Schito AM. Effect of
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fosfomycin alone and in combination with N-acetylcysteine on E. coli biofilms. Int J Antimicrob
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Agents 2003; 22 (Suppl. 2): 95–100.
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24. Schwandt LQ, Van Weissenbruch R, Stokroos I, Van der Mei HC, Busscher HJ, Albers FW.
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2004. Prevention of biofilm formation by dairy products and N-acetylcysteine on voice
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prostheses in an artificial throat. Acta Otolaryngol 2004; 124:726–731.
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25. Olofsson AC, Hermansson M, Elwing H. N-acetyl-L-cysteine affects growth, extracellular
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polysaccharide production, and bacterial biofilm formation on solid surfaces. Appl Environ
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Clinical and Laboratory Standards Institute; 2012.
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27. Sharma M, Visai L, Bragheri F, Cristiani I, Gupta PK, Speziale P. Toluidine blue-mediated
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photodynamic effects on staphylococcal biofilms. Antimicrob Agents Chemother 2008; 52:299-
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305.
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28. Stewart PS. New ways to stop biofilm infections. Lancet 2003; 361:97.
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29. Demidova TN, Hamblin MR. Effect of cell-photosensitizer binding and cell density on
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microbial photoinactivation. Antimicrob Agents Chemother 2005; 49:2329–2335.
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30. Usacheva MN, Teichert MC, Biel MA. Comparison of the methylene blue and toluidine blue
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photobactericidal efficacy against gram-positive and gram-negative microorganisms. Lasers Surg
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Med 2001; 29:165–173.
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31. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent
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infections. Science 1999; 284:1318–1322.
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32. Zanin IC, Lobo MM, Rodrigues LK, Pimenta LA, Ho¨fling JF, Goncalves RB.
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Photosensitization of in vitro biofilms by toluidine blue O combined with light-emitting diode.
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Eur J Oral Sci 2006; 114:64–69.
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33.Gad F, Zahra T, Hasan T, Hamblin MR. Effects of growth phase and extracellular slime on
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photodynamic inactivation of gram-positive pathogenic bacteria. Antimicrob Agents Chemother
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2004; 48:2173–2178.
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34. Marzullo L. 2005. An update of N-acetylcysteine treatment for acute acetaminophen toxicity
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in children. Curr Opin Pediatr 2005; 17:239–245.
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35. Oldemeyer JB, Biddle WP, Wurdeman RL, Mooss AN, Cichowski E, Hilleman DE.
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Acetylcysteine in the prevention of contrast induced nephropathy after coronary angiography.
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36. Yip L, Dart RC, Hurlbut KM. Intravenous administration of oral N-acetylcysteine. Crit Care
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37. Kao LW, Kirk MA, Furbee RB, Mehta NH, Skinner JR, Brizendine EJ. What is the rate of
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suspected acetaminophen poisoning? Ann Emerg Med 2003; 42:741–750.
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38. Kauffman CA. Quandary about treatment of aspergillomas persists. Lancet 1996; 347: 1640.
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pharmacology and clinical utility. Expert Opin Biol Ther 2008; 8: 1955-62.
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40. Berk M, Copolov DL, Dean O, Lu K, Jeavons S, Schapkaitz I, et al. N-acetylcysteine for
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depressive symptoms in bipolar disorder -- a double-blind randomized placebo-controlled trial.
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Biol Psychiatry 2008; 64: 468-75.
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41. Stey C, Steurer J, Bachmann S, Medici TC, Tramer MR. The effect of oral N-acetylcysteine
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in chronic bronchitis: a quantitative systematic review. Eur Respir J 2000; 16: 253-62.
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42. Mansouri MD, Hull RA, Stager CE, Cadle RM, Darouiche RO. In vitro activity and
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durability of a combination of an antibiofilm and an antibiotic against vascular catheter
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colonization. Antimicrob Agents Chemother. 2013; 57(1):621-5.
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43. Drago L, De Vecchi E, Mattina R, Romanò CL. Activity of N-acetyl-L-cysteine against
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44. Quah SY, Wu S, Lui JN, Sum CP, Tan KS. N-acetylcysteine inhibits growth and eradicates
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biofilm of Enterococcus faecalis. J Endod. 2012; 38(1):81-5.
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45. Aslam S, Darouiche RO. Role of antibiofilm-antimicrobial agents in controlling device-
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related infections. Int J Artif Organs. 2011; 34(9):752-8.
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46. Leite B, Gomes F, Teixeira P, Souza C, Pizzolitto E, Oliveira R. Staphylococcus epidermidis
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biofilms control by N-acetylcysteine and rifampicin. Am J Ther. 2013; 20(4):322-8.
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47. El-Feky MA, El-Rehewy MS, Hassan MA, Abolella HA, Abd El-Baky RM, Gad GF. Effect
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of ciprofloxacin and N-acetylcysteine on bacterial adherence and biofilm formation on ureteral
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stent surfaces. Pol J Microbiol. 2009; 58(3):261-7.
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Tables
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Table 1. Biofilm formation ability of ten drug-resistant S. aureus isolates and S. aureus (ATCC
3
25923). Absorbance of crystal violet (for each test strain in 6 wells) was determined at 490 nm
4
and reported as Mean ± SD.
5 Mean ± SD (OD490)
S. aureus strains
ATCC 25923
1.44 ± 0.29
UTMC 1451
0.69 ± 0.22
UTMC 1485
1.11 ± 0.34
UTMC 1439
1.57 ± 0.66
UTMC 1442
2.88 ± 0.45
UTMC 1446
2.06 ± 0.37
UTMC 1455
1.61 ± 0.56
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UTMC 1461
1.38 ± 0.43 0.78 ± 0.20
UTMC1474
0.73 ± 0.19
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UTMC 1484
1.02 ± 0.33
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Mean ± SD (OD490)
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S. aureus strains
UTMC 1478
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Figures
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Figure 1. Photodynamic inactivation of Staphylococcus aureus planktonic cells [6×108 CFU/ml
10
were sensitized with hypericin (final concentration: 0.5 µg/ml) and after washing excess dye,
11
exposed to 8 J/cm2 light dose].
21
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Figure 2. Photodynamic inactivation of Staphylococcus aureus biofilms (pre-formed biofilms
11
were sensitized with 0.5 µg/ml hypericin and after washing excess dye, exposed to 16 J/cm2 light
12
dose).
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Figure 3. Photodynamic inactivation of Staphylococcus aureus biofilms treated with
2
acetylcysteine (pre-formed biofilms were treated with 10 mg/ml AC, sensitized with 0.5 µg/ml
3
hypericin and after washing excess dye, exposed to 16 J/cm2 light dose).
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S1572-1000(15)00036-8 http://dx.doi.org/doi:10.1016/j.pdpdt.2015.04.001 PDPDT 640
To appear in:
Photodiagnosis and Photodynamic Therapy
Received date: Revised date: Accepted date:
14-9-2014 2-4-2015 6-4-2015
Please cite this article as: Kashef N, Karami S, Djavid GE, Phototoxic effect of hypericin alone and in combination with acetylcysteine on Staphylococcus aureus biofilms, Photodiagnosis and Photodynamic Therapy (2015), http://dx.doi.org/10.1016/j.pdpdt.2015.04.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Highlights 1- We evaluated the in vitro bactericidal effect of HYP-PDI on clinical Staphylococcus aureus biofilms.
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2- In vitro bactericidal effect of HYP-PDI on S. aureus biofilms treated with acetylcysteine was also studied.
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3- HYP-PDI did not result in a reduction in viable count for each of the strains when grown in biofilms.
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4- HYP-PDI applied on biofilms treated with acetylcysteine was able to disrupt pre-formed biofilms.
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5- Acetylcysteine may enhance the efficacy of PDI by disrupting the biofilm structure.
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Title:
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Phototoxic effect of hypericin alone and in combination with acetylcysteine on Staphylococcus aureus biofilms
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Authors: Nasim Kashef1, Shima Karami2, Gholamreza Esmaeeli Djavid3 1
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Assistant Professor of Medical Bacteriology, Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran Tel: +98-21-61113558 Fax: +98-21-66492992 E-mail: [email protected] 2
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MSc, Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran Tel: +98-21-61113558 Fax: +98-21-66492992 E-mail: [email protected] 3
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MD, Iranian Research Center for Medical Lasers, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran Tel: +98-21-66480877 Fax: +98-21-66952040 E-mail: [email protected]
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Corresponding author: Mailing address: Nasim Kashef, Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran Tel: +98-21-61113558 Fax: +98-21-66492992 E-mail: [email protected] 2
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Abstract Background: Resistance of bacteria against antibiotics and antimicrobials is arising worldwide
2
and there is an urgent need for strategies that are capable of inactivating biofilm-state pathogens
3
with less potential of developing resistance in pathogens. A promising approach could be
4
photodynamic inactivation (PDI) which uses light in combination with a photosensitizer to
5
induce a phototoxic reaction. In this study, we evaluated the in vitro phototoxic effect of
6
hypericin (HYP) alone and in combination with acetylcysteine (AC) on Staphylococcus aureus
7
biofilms. AC, a mucolytic agent, reduces the production of extracellular polysaccharide matrix
8
while promoting the disruption of mature biofilm.
9
Methods: In vitro phototoxic effect of HYP alone (0.5 µg/ml, light dose: 16 J/cm2), and in
10
combination with AC (10 mg/ml) on ten clinical S. aureus isolates and S. aureus (ATCC 25923)
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biofilms was studied. Effect of HYP concentration (0.5 µg/ml) and light dose (8 J/cm2) on PDI
12
of all eleven S. aureus strains in planktonic forms was also investigated.
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Results: HYP-PDI did not result in a reduction in viable count for each of the strains when
14
grown in biofilms. However, HYP-PDI applied on biofilms treated with AC was able to disrupt
15
pre-formed biofilms (viable count reduction ranging from 5.2 to 6.3 log10-unit in comparison to
16
controls in all tested strains). A 6.5 log killing was obtained for S. aureus (ATCC 25923)
17
planktonic cells treated with 0.5 µg/ml at 8 J/cm2. For this set of PDI parameters, ten clinical S.
18
aureus isolates showed 5.5-6.7 log killing.
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Conclusion: HYP-PDI in combination with AC had significant ability to eradicate the pre-
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formed mature biofilms of S. aureus strains.
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Key words: photodynamic inactivation, hypericin, Staphylococcus aureus, acetylcysteine
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Introduction
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In many environments bacteria exist as a complex, multispecies surface-associated community
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termed biofilm. Organisms within biofilms are embedded in a self-produced matrix of
4
extracellular polymeric substance (EPS) composed of polysaccharides, proteins, lipids, and
5
extracellular DNA. Several advantages exist for bacteria that live in a biofilm phenotype
6
including structural stability, firm adherence to biotic or abiotic surfaces, increased virulence,
7
and resistance to both antimicrobial therapy and the host immune response (1-3).
8
Staphylococcus aureus is an important human pathogen associated with numerous skin diseases
9
including wound infections. The difficulty in eradicating S. aureus colonization with
10
conventional antibiotic therapy may be due to the presence of biofilm (4). EPS itself may slow
11
drug-diffusion by its higher viscosity or can even act as a barrier (5). Furthermore, development
12
of a biofilm leads to an enormous genetic diversity of its cells, which provides insurance for the
13
cells for better adaptation to rapid alteration of environment conditions (6). Also, persister cells
14
are being formed, when bacteria grow as a biofilm. These are in a dormant, non-dividing state
15
and provide tolerance toward antimicrobial agents (7).
16
S. aureus biofilms have a negative effect on wound healing as evidenced by their ability to
17
induce keratinocyte apoptosis in vitro and inhibit re-epithelialization in an in vivo animal wound
18
model (8,9). In addition, biofilms may also increase the inflammatory response characteristic of
19
chronic wounds, thus promoting tissue damage and further contributing to the non-healing
20
phenotype (10).
21
With increasing the resistance of pathogenic bacteria including S. aureus to antimicrobial agents,
22
there is a need for the development of alternative antibacterial strategies; photodynamic
23
inactivation (PDI) could be the method of choice. PDI involves killing of organisms by light in
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the presence of a photosensitizing agent (11). The local combination of light and the
2
photosensitive drug in the frame of PDI avoids systemic effects on bacterial flora and by this
3
minimizes side effects. Reports of successful inactivation of multi-drug-resistant strains of S.
4
aureus declare PDI to be the method of choice for treatment of such local infections (12,13).
5
Several studies have been reported PDI mediated by hypericin (HYP), a powerful photosensitizer
6
(14-20 ). HYP is a phenanthroperylene quinine pigment naturally occuring in Hypericum plants.
7
It is characterized by a high quantum yield for formation of reactive oxygen species and very
8
slow photobleaching (21).
9
In our pervious study, we evaluated the in vitro bactericidal effect of HYP-PDI on different
10
bacterial species, assessing its photocytotoxicity to primary human fibroblasts to determine
11
possible side effects (20). Our results showed that HYP had a high phototoxicity to S. aureus,
12
Enterococcus faecalis and Escherichia coli at extremely low drug concentrations and did not
13
induce significant cytotoxic effects on human fibroblasts in culture. However, planktonic
14
bacterial cultures as employed in that study represent the first step in the evaluation of a
15
photoactive compound for its use in PDI. So, the purpose of the present study was to evaluate the
16
in vitro phototoxic effect of HYP alone and in combination with acetylcysteine (AC) on
17
Staphylococcus aureus biofilms. AC, also known as N-acetylcysteine, is a mucolytic agent that
18
disrupts disulfide bonds in mucus and, for this reason; it reduces the viscosity of secretions (22).
19
AC decreases biofilm formation by a variety of bacteria (22-24) and reduces the production of
20
extracellular polysaccharide matrix (25) while promoting the disruption of mature biofilm
21
(23,24).
22
Materials and methods
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Bacterial species and culture conditions
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A total of ten clinical S. aureus recovered from acute and chronic wounds were used in this
2
study. In addition, S. aureus (ATCC 25923) was also included. The in vitro antimicrobial
3
susceptibility of clinical isolates was determined according to CLSI guidelines (26). Antibiotic
4
discs were purchased from Padtan Teb Company, Iran. Eight clinical isolates were resistant to
5
the following antibiotics: amoxicillin—clavulanate (20/10 µg), oxacillin (1 µg), ciprofloxacin (5
6
µg), gentamicin (10 µg), and amikacin (30 µg). S. aureus (UTMC 1484) was resistant to
7
ciprofloxacin, amoxicillin—clavulanate and gentamicin and sensitive to oxacillin and amikacin.
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S. aureus (UTMC 1474) was resistant to ciprofloxacin, amoxicillin—clavulanate and sensitive to
9
oxacillin, gentamicin and amikacin.
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Organisms were maintained by weekly subculture on nutrient agar (Merck, Germany). All
11
organisms were grown aerobically in nutrient agar plates at 37 °C for 18-24 h. Then a suspension
12
of each organism was prepared in sterile phosphate-buffered saline (PBS, pH 7.4) to reach the
13
turbidity of McFarland standards. No. 2 (a concentration of 6×108 CFU/ml).
14
Photosensitizer (PS) and light source
15
HYP (Tocris Bioscience, UK, purity > 98%) was dissolved as a stock at 100 µg/ml in dimethyl
16
sulphoxide (DMSO). Stock solution was kept at 4°C in the dark and further diluted in sterile PBS
17
(pH 7.4) when needed.
18
The light source consisted of a light-emitting diode (LED) array. The samples were exposed to
19
LEDs (wavelength of 590 nm, measured power of 10 mW).
20
Photodynamic inactivation of Staphylococcus aureus planktonic cells
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Bacterial suspensions were incubated with 0.5 µg/ml HYP in the dark at 37 ºC for 5 minutes.
2
The HYP-treated cells were centrifuged (6000 rpm for 15 minutes), and washed twice with
3
sterile PBS (pH 7.4). Aliquots of 200 µl treated cells were placed in a 96-well microtiter plate
4
and irradiated with the light source (5 min, 8 J/cm2). The plates were kept covered during the
5
illumination in order to maintain the sterility of the culture. After that, 100 µl of cell suspension
6
was spread on nutrient agar in 10-fold serial dilutions. Colonies were counted after incubation
7
for 24 h at 37 ºC. All experiments were repeated three times (20).
8
Every experiment was accompanied by three control samples: a negative control (no PS, no
9
illumination), a light control (no PS, illumination same as PDI samples) and a dark control (with
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PS, no illumination).
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Assessment of Biofilm formation
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S. aureus strains were grown overnight in tryptic soya broth (TSB, Merck, Germany)
13
supplemented with 0.2% glucose at 37 ºC and then diluted 1:50 in fresh TSB. For each test strain
14
200 µl of inoculums was added to 6 wells of a 96-well flat-bottomed sterile polystyrene
15
microplate. A volume of 200 µl TSB was added to the remaining wells and the plate incubated
16
for 24 h at 37 ºC. Then, the plate contents emptied out and washed two times with PBS (pH 7.4)
17
to remove the planktonic cells. The cells were fixed with 95% ethanol for 10 min and stained
18
with 0.4% crystal violet for 15 min. After several washing, the wells were air dried. For a
19
quantitative estimation of biofilms, crystal violet was solubilized with 10% glacial acetic acid
20
and absorbance of the solubilized dye was determined at 490 nm in a microplate reader (27).
21
Photodynamic inactivation of Staphylococcus aureus biofilms
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Bacterial suspensions (6×108 CFU/ml) were diluted at 1:100, in TSB containing 0.2% glucose.
2
Aliquots (100 µl) of the diluted bacterial suspensions and 100 µl of fresh TSB containing 0.2%
3
glucose were inoculated into 96-well flat-bottomed sterile polystyrene microplates (SPL, Korea),
4
and incubated for 18 h at 37°C. Following incubation, the plate contents washed once with fresh
5
PBS (pH 7.4) to remove the planktonic cells. To investigate the effect of PDI, 200 µl of 0.5
6
µg/ml HYP was added to each well and the plates were incubated in the dark for 5 min at room
7
temperature. Wells used as controls were incubated with PBS only. Following incubation, HYP
8
was carefully removed from the microwells and the biofilms were washed once with fresh PBS.
9
HYP-treated biofilms were irradiated with light source for 10 minutes (16 J/cm2). The biofilms
10
were scraped carefully, and then vortexed for 30 s to homogenize the samples. Treated and
11
untreated samples were serially diluted, plated on the nutrient agar plates, and incubated for 24 h
12
at 37°C in the dark. For each set of measurements, controls consisting of biofilms treated with
13
HYP but not exposed to light, exposed to light only, and treated with neither HYP nor light were
14
included (27).
15
Photodynamic inactivation of Staphylococcus aureus biofilms treated with acetylcysteine
16
AC (Hexal, Germany) was dissolved in water to make a 10 mg/ml solution. Biofilm formation
17
was performed as described above. After removing the planktonic cells, 200 µl of 10 mg/ml AC
18
was added to each well and the plates were incubated in the dark for 10 min at 37 ºC. Following
19
incubation, AC was carefully removed from the microwells and HYP diluted in AC solution (at
20
final concentration of 0.5 µg/ml) was added to each well and the plates were incubated in the
21
dark for 5 min at 37 ºC. Following incubation, HYP was carefully removed from the microwells
22
and the biofilms were washed once with fresh PBS. HYP-treated biofilms were irradiated with
23
light source for 10 minutes (16 J/cm2). The biofilms were scraped carefully, and then vortexed
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for 30 s to homogenize the samples. Treated and untreated samples were serially diluted, plated
2
on the nutrient agar plates, and incubated for 24 h at 37°C in the dark.
3
Statistical analysis
4
Values were expressed as log10 means ± standard deviation. Comparisons between means of
5
groups were analyzed using the One-Way ANOVA and Post Hoc Tukey tests. P 0.05).
13
The PDI was applied to all 10 clinical S. aureus isolates, and S. aureus (ATCC 25923) strains. In
14
the case of clinical S. aureus strains, the obtained results for HYP-PDI included viable count
15
reduction ranging from 5.5 to 6.7 log10-unit. A 6.5 log killing was obtained for S. aureus (ATCC
16
25923) treated with 0.5 µg/ml at 8 J/cm2 (figure 1).
17
Biofilm formation ability of Staphylococcus aureus strains
18
Table 1 shows the biofilm producing ability of ten clinical S. aureus isolates and S. aureus
19
(ATCC 25923). According to our results, all 11 S. aureus strains were strong biofilm-producing
20
organisms (OD490 > 0.6). For instance, the biofilm-positive strain S. aureus (UTMC 1442) gave
21
an OD490 reading of 2.88 ± 0.45, which is a factor of 4.2 greater than that formed by the biofilm-
22
positive strain S. aureus (UTMC 1451).
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Photodynamic inactivation of Staphylococcus aureus biofilms
2
Our preliminary experiments showed that the treatment of biofilms with HYP (at final
3
concentrations of 0.5 µg/ml) in the dark did not exhibit any toxicity against the bacterial cells.
4
Similarly, the exposure of cells to light (16 J/cm2) alone did not cause any change in cell survival
5
compared to the survival rates of untreated controls (data not shown).
6
Unlike to the findings for planktonically grown cultures, HYP-PDI did not result in a reduction
7
in viable count for each of the strains when grown in biofilms (figure 2). It was shown that
8
bacteria growing as biofims were more resistant to applied PDI compared with planktonic forms.
9
Photodynamic inactivation of Staphylococcus aureus biofilms treated with acetylcysteine
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When exposed to AC (10 mg/ml solution) only, there was no statistically significant decrease in
11
log10 viable count, in comparison with the untreated control, for each 11 biofilm-grown strains (p
12
> 0.05).
13
HYP-PDI was found to have significant effects (p < 0.001) on pre-formed biofilms treated with
14
AC (figure 3). HYP-PDI did not reduce viable cell counts in the pre-formed biofilms of all tested
15
strains compared to the controls (p > 0.05, 0.01 log decrease in viable cells), while, HYP-PDI
16
applied on biofilms treated with AC showed the highest ability to disrupt pre-formed biofilms
17
(viable count reduction ranging from 5.2 to 6.3 log10-unit in comparison to controls in all clinical
18
S. aureus strains). A 5.7 log killing was obtained for S. aureus (ATCC 25923) treated with 0.5
19
µg/ml at 16 J/cm2.
20
Discussion
21
One of the major goals in the field of modern Clinical Microbiology is to attempt to develop new
22
strategies capable of reducing the incidence of biofilm infections and of effectively curing
23
chronic conditions related to the establishment of these difficult-to-eradicate bacterial structures
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(28). Therefore, in the present study, we evaluated the PDI efficacies on ten clinical S. aureus
2
and S. aureus (ATCC 25923) by incubating the bacterial cultures with HYP followed by yellow
3
light irradiation and assessed the phototoxic effects of HYP toward pre-formed biofilms of those
4
strains. HYP used in this study was very effective in inactivation of all tested S. aureus
5
planktonic cells upon illumination; at concentration of 0.5 µg/ml a decrease in bacterial count of
6
5.5-6.7 log units was achieved. Our previous study also showed a 6.3 log killing for S. aureus
7
(ATCC 25923) treated with 1 µg/ml at 48 J/cm2 (20). Similar to our results, Yow et al. study
8
showed that the combination of HYP (8 µM) and light irradiation (30 J/cm2) could induce
9
significant killing of methicillin-sensitive and -resistant S. aureus cells (> 6 log reduction) (16).
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In the present study, HYP failed to inactivate S. aureus biofilm cells upon illumination. It was
11
shown that bacteria growing as biofilms were more resistant to applied PDI compared with
12
planktonic forms. As reported by others (29,30), the dye concentration and the light dose used
13
for the photoinactivation of biofilms were considerably higher than those required to inactivate S.
14
aureus suspensions. The differences in the photodynamic efficacies of the drug could be due to
15
several reasons. In fact, cells growing in biofilm differ from their planktonic counterparts in a
16
number of aspects, such as the cell wall composition, rate of growth, and presence of
17
polysaccharide intercellular adhesin (PIA), which may block the uptake of the PS and
18
penetration of light and thereby reduce the photosensitizing process (31,32). According to Gad et
19
al. study, abundant production of PIA has been suggested to obstruct the diffusion of the PS
20
through the matrix, thus reducing the susceptibility of biofilms to photosensitization (33).
21
To improve the efficiency of PDI using HYP on bacteria growing as biofilms, we used AC, a
22
mucolytic agent. AC is widely used in medical practice via inhalation, oral and intravenous
23
routes (34-36), and it has an excellent safety profile (37). In addition, AC has been used to treat
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aspergilloma by local installation (38). Its efficacy in chronic obstructive pulmonary disease and
2
nephropathy has been reported (39). AC, which is a glutathione precursor, provides
3
neuroprotection by preventing oxidative damage (39, 40). AC is also used in the treatment of
4
chronic bronchitis (41).
5
AC, as an antibiofilm agent, has been used in several studies alone or in combination with
6
antimicrobials (42-47). Mansouri et al. studied in vitro antimicrobial activities of central venous
7
catheters impregnated with AC and levofloxacin (ACLEV) against a range of important clinical
8
pathogens. ACLEV catheters produced the most active and durable antimicrobial effect against
9
both Gram-positive and Gram-negative isolates (42). In Leite et al. study, the combined effect of
10
rifampicin with AC, in the control of Staphylococcus epidermidis biofilms was assessed.
11
Biofilms of two S. epidermidis strains (9142 and 1457) were treated with AC (4 and 40 mg/mL)
12
and 10 mg/L (peak serum) of rifampicin alone and in combination. AC at 40 mg/L alone or in
13
combination with rifampicin significantly reduced (4 log10) the number of biofilm cells (46). El-
14
Feky et al. also evaluated the effect of ciprofloxacin and AC, alone and in combination, on
15
biofilm production and pre-formed mature biofilms of important clinical pathogens related to
16
ureteral stent surfaces. Ciprofloxacin and AC (2 and 4 mg/ml) inhibited biofilm production by ≥
17
60% in all tested microorganisms. Disruption of pre-formed biofilms of all tested
18
microorganisms was found to be ≥ 78% in the presence of ciprofloxacin and ≥ 62% in the
19
presence of AC (2 and 4 mg/ml), compared to controls. Ciprofloxacin/AC showed the highest
20
inhibitory effect on biofilm production (94-100%) and the highest disruptive effect on the pre-
21
formed biofilms (86-100%) in comparison to controls. AC was found to increase the therapeutic
22
efficacy of ciprofloxacin by degrading the extracellular polysaccharide matrix of biofilms (47).
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In our study, the applied HYP-PDI on pre-formed biofilms treated with AC consistently
2
decreased (5.2 to 6.3 log10) viable biofilm-associated bacteria relative to the controls. Since the
3
susceptibility of biofilm-associated clinical S. aureus isolates, and S. aureus (ATCC 25923) to
4
HYP-PDI was enhanced in biofilms treated with AC, it is possible that an antibiofilm/PDI
5
combination would be synergistic. By degrading the extracellular polysaccharide matrix of
6
biofilm (23,25), it is possible that AC may have made the biofilm-associated bacteria more
7
susceptible to phototoxic effect of HYP, although we did not specifically test this hypothesis.
8
To the best of our knowledge, there are no published reports on the combined use of AC and PDI
9
for the inactivation of biofilms. PDI is proposed as an alternative antimicrobial method for the
10
inactivation of biofilm-related diseases owing to several advantages over the conventional
11
antibacterial treatment. However, the efficacy of PDI for biofilm treatment depends mainly on
12
the penetration of the PS and light to the deeper layers. Our study shows that pretreatment of
13
biofilms with AC may enhance the efficacy of PDI by disrupting the biofilm structure and
14
thereby enhancing the PS and light penetration. Thus, the use of combined AC and PDI has the
15
advantage of treating and eradicating even thick biofilms and avoiding the disadvantage of using
16
antibiotics, with the consequent generation of antimicrobial resistance. Therefore, an
17
experimental approach involving a combination of AC and PDI in treating biofilm-associated
18
infections may be worth exploring.
19
Acknowledgment
20
Support was provided by the College of Science, University of Tehran and Iranian Research
21
Center for Medical Lasers, Academic Center for Education, Culture and Research.
22
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Tables
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Table 1. Biofilm formation ability of ten drug-resistant S. aureus isolates and S. aureus (ATCC
3
25923). Absorbance of crystal violet (for each test strain in 6 wells) was determined at 490 nm
4
and reported as Mean ± SD.
5 Mean ± SD (OD490)
S. aureus strains
ATCC 25923
1.44 ± 0.29
UTMC 1451
0.69 ± 0.22
UTMC 1485
1.11 ± 0.34
UTMC 1439
1.57 ± 0.66
UTMC 1442
2.88 ± 0.45
UTMC 1446
2.06 ± 0.37
UTMC 1455
1.61 ± 0.56
an M
UTMC 1461
1.38 ± 0.43 0.78 ± 0.20
UTMC1474
0.73 ± 0.19
ed
UTMC 1484
1.02 ± 0.33
pt Ac ce
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Mean ± SD (OD490)
us
S. aureus strains
UTMC 1478
6
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Figures
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Figure 1. Photodynamic inactivation of Staphylococcus aureus planktonic cells [6×108 CFU/ml
10
were sensitized with hypericin (final concentration: 0.5 µg/ml) and after washing excess dye,
11
exposed to 8 J/cm2 light dose].
21
Page 21 of 23
1 2
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Figure 2. Photodynamic inactivation of Staphylococcus aureus biofilms (pre-formed biofilms
11
were sensitized with 0.5 µg/ml hypericin and after washing excess dye, exposed to 16 J/cm2 light
12
dose).
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Figure 3. Photodynamic inactivation of Staphylococcus aureus biofilms treated with
2
acetylcysteine (pre-formed biofilms were treated with 10 mg/ml AC, sensitized with 0.5 µg/ml
3
hypericin and after washing excess dye, exposed to 16 J/cm2 light dose).
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8 9 10
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Page 23 of 23
