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thiazolidinedione derivatives on Propionibacterium acnes biofilm formation in vitro and to assess their effect on the susceptibility of P. acnes biofilms towards.
Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Thiazolidinedione derivatives as novel agents against Propionibacterium acnes biofilms G. Brackman1, K. Forier2,3, A. A. A. Al Quntar4,5, E. De Canck1, C. D. Enk6, M. Srebnik4, K. Braeckmans2,3 and T. Coenye1 1 2 3 4 5 6

Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Ghent, Belgium Center for Nano- and Biophotonics, Ghent University, Gent, Belgium Institute of Drug Research, Hebrew University, Jerusalem, Israel Department of Material Engineering, Al Quds University, Jerusalem, Palestinian Authority Department of Dermatology, Hadassah-Hebrew University Medical School, Jerusalem, Israel

Keywords antimicrobials, biofilm eradication, biofilm inhibition, Propionibacterium acnes, Thiazolidinedione. Correspondence Gilles Brackman, Laboratory of Pharmaceutical Microbiology (LPM), Universiteit Gent Harelbekestraat 72, B-9000 Gent, Belgium. E-mail: [email protected] 2013/1749: received 27 August 2013, revised 22 October 2013 and accepted 24 October 2013 doi:10.1111/jam.12378

Abstract Aims: The aim of the present study was to determine the effect of two thiazolidinedione derivatives on Propionibacterium acnes biofilm formation in vitro and to assess their effect on the susceptibility of P. acnes biofilms towards antimicrobials. Methods and Results: The compounds were shown to have a moderate to strong antibiofilm activity when used in subinhibitory concentrations. These compounds do not affect P. acnes attachment but lead to increased dispersal of biofilm cells. This dispersal results in an increased killing of the P. acnes biofilm cells by conventional antimicrobials. Conclusion: The antibiofilm effect and the effect on biofilm susceptibility of the thiazolidinedione-derived quorum sensing inhibitors were clearly demonstrated. Significance and Impact of the Study: Propionibacterium acnes infections are difficult to treat due to the presence of biofilms at the infection site and the associated resistance towards conventional antimicrobials. Our results indicate that these thiazolidinedione derivatives can be promising leads used for the treatment of P. acnes infections and as anti-acne drugs.

Introduction Propionibacterium acnes is an aerotolerant, anaerobic Gram-positive commensal of the human skin. However, P. acnes can cause a wide variety of opportunistic infections including chronic skin wounds, biomaterial-related and intravascular infections, and P. acnes also plays a role in acne vulgaris (Brook 2008; Perry and Lambert 2011). There is growing evidence that P. acnes forms biofilms at the infection site. Alexeyev and Jahns (2012) demonstrated the presence of P. acnes biofilms in biopsies of sebaceous follicles obtained from acne patients by staining these with specific antibodies against co-haemolysin CAMP factor, a putative virulence determinant. In addition P. acnes has been shown to develop biofilms in orthopaedic and neurosurgical infection sites (Rammage 492

et al. 2003; Bayston et al. 2007). This biofilm lifestyle contributes to the pathogenicity and the difficulty to treat P. acnes infections. For example, P. acnes biofilm cells are more resistant to antimicrobial agents and produce more lipase compared to planktonic cells (Coenye et al. 2007; Humphrey 2012). Thus, 1 in 5 acne patients treated with erythromycin or clindamycin had antibiotic-resistant strains on their skin (Crawford et al.1979; Ross et al. 2001). In addition, P. acnes biofilm cells produce more autoinducer-2 (AI-2) compared to planktonic cells (Coenye et al. 2007). AI-2 is a mixture of signalling molecules used by a wide variety of bacteria to coordinate virulence in response to population density by a communication mechanism known as quorum sensing (QS) (Miller and Bassler 2001). Although P. acnes produces AI-2, to date no receptor for AI-2 or signal transduction pathway has

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been identified in this organism. However, the presence of yet unknown receptors of AI-2 cannot be excluded. Several QS inhibitors (QSI) specifically targeting AI-2 QS systems have been developed (Ni et al. 2009). Recently, thiazolidinedione derivatives were observed to be potent QSI of the AI-2 QS system (Brackman et al. 2013). When used at 100 lmol l 1, (Z)-5-octylidenethiazolidine-2,4-dione (TZD8) and (Z)-5-decylidenethiazolidine-2,4-dione (TZD10) (Fig. 1), consisting of a thiazolidinedione backbone and alkyl side chain containing eight and ten carbons, respectively, completely blocked AI-2 QS in a Vibrio harveyi biosensor (Brackman et al. 2013). Although several QSI affect biofilm formation and susceptibility (Brackman et al. 2011), the antibiofilm properties of these highly potent QSI have not yet been investigated. In view of the role of P. acnes biofilms in skin infections, the difficulty to eradicate these biofilms and the potential role of biofilms in the development of microbial resistance in acne treatment, we investigated the effect of two thiazolidinedionederived QSI on P. acnes biofilm formation in vitro. The compounds are applied in subminimal inhibitory concentrations (sub-MICs), that is, concentrations that do not inhibit the growth of the bacteria. We further evaluated whether these QSI could increase the susceptibility of P. acnes biofilms towards antimicrobials such as minocycline, erythromycin, clindamycin, oxytetracycline and benzoylperoxide, which are commonly used as anti-acne treatment (James et al. 2008). Materials and methods Strains and culture conditions The following P. acnes strains were used: LMG 16711 (isolated from human facial acne in the UK), LMG 16712

O

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(isolated from human acne) and LMG 16715 (isolated from human blood). All strains were obtained from the BCCM/LMG Bacteria Collection (Gent, Belgium) and were cultured on reinforced clostridial medium (RCM) (Oxoid, Erembodegem-Aalst, Belgium). Plates were incubated at 37°C under anaerobic conditions using the AnaeroGen compact (Oxoid). Chemicals The following antimicrobials were used: minocycline (Mc), erythromycin (Em), clindamycin (Cm), oxytetracycline (Otc) and benzoylperoxide (BPO) (all from SigmaAldrich, Bornem, Belgium). The antimicrobials were directly dissolved in RCM (5000 mg l 1 Mc, 10 000 mg l 1 Cm, 5000 mg l 1 Otc), in ethanol (final concentration of 5000 mg l 1 erythromycin) or in acetone (final concentration of 25 000 mg l 1 BPO). The QSI (Z)-5octylidenethiazolidine-2,4-dione (TZD-8) and (Z)-5-decylidenethiazolidine-2,4-dione (TZD-10) (Fig. 1) were synthesized as described previously (Brackman et al. 2013) and dissolved in DMSO (Sigma-Aldrich). Determination of the minimal inhibitory concentration Minimal inhibitory concentrations of QSI and the antimicrobials were determined in triplicate according to the EUCAST broth microdilution protocol, using flat-bottom 96-well microtitre plates (TPP, Trasadingen, Switzerland) (Coenye et al. 2012). The inoculum was standardized to approximately 5 9 105 CFU ml 1. The plates were incubated anaerobically at 37°C for 20 h, and the optical density at 590 nm was determined using a multilabel microtitre plate reader (Envision; PerkinElmer LAS, Waltham, MA). The concentrations tested ranged from 1000 to 05 lmol l 1 for the QSI, 4000 mg to 0008 mg L 1 for the antibiotics and 25 000 mg to 10 mg l 1 for BPO. The lowest concentration for which a similar optical density was observed in the inoculated as in the blank wells was recorded as the MIC. For each antibiotic, the MIC was determined in the presence and absence of TZD8 or TZD10 (both at 100 lmol l 1). If the MIC determined in both conditions differed, checkerboard testing was performed to determine whether the interaction is synergistic (fractional inhibitory concentration index [FICI] ≤ 05) or indifferent (FICI > 05–4) (Odds 2003). Biofilm inhibitory and eradicating activity of the QSI Propionibacterium acnes biofilms were formed as previously described (Coenye et al. 2012). In brief, 48-h-old P. acnes cultures in RCM were centrifuged, resuspended in double-concentrated RCM (2 9 RCM) and diluted to

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an OD600 nm = 02. Fifty microlitre of the diluted bacterial suspension was transferred to the wells of a roundbottom 96-well microtitre plate (TPP). Negative controls received 50 ll MilliQ. Positive controls received 50 ll of the QSI. Bacteria were allowed to adhere and grow without agitation for 4 h at 37 °C under anaerobic conditions. After 4 h, medium was removed, and the adhered cells were washed with sterile physiological saline (09% NaCl; PS). After this washing step, negative control wells were filled with 50 ll 2 9 RCM and 50 ll MilliQ. Other wells were filled with 50 ll 2 9 MH and 50 ll of the test compounds, and the plate was incubated for 20 h at 37°C. To evaluate the effect on mature biofilms, control biofilms were formed in the absence of test compounds, as described above. After 24 h of biofilm formation, the medium was removed, the wells were rinsed with PS, and fresh medium containing the compounds was added to the wells. The plates were then incubated for an additional 24 h at 37°C. After biofilm formation or treatment of mature biofilms, the biomass and the number of metabolically active cells were quantified by crystal violet (CV) and resazurin-based viability staining (Cell-titer Blue, Promega, Leiden, the Netherlands), respectively, as described previously (Peeters et al. 2008). The control signal for CV staining corresponded to an absorbance value at 590 nm wavelength of 1481  0212, 0776  0114 and 1176  0199 for P. acnes LMG 16711, LMG 16712 and LMG 16715, respectively. The control fluorescence signal (excitation and emission filters of 535 and 590 nm) for resazurin-based viability staining corresponded to 166 433  16 262, 183 604  20 649 and 144 486  21 145 for P. acnes LMG 16711, LMG 16712 and LMG 16715, respectively. Each compound was tested on ten wells in each assay, and each assay was repeated at least three times (n ≥ 30). Effect of QSI on adherence and detachment of Propionibacterium acnes For quantification of the numbers of adhering and detached cells, P. acnes LMG 16711, LMG 16712 and LMG 16715 were allowed to form biofilms on silicone discs (Q7-4735; Dow Corning) in a 24-well plate in the presence and absence of test compounds (100 lmol l 1). In brief, 500 ll OD600 02 P. acnes was transferred to the wells of a 24-well plate (TPP) containing a sterile silicone disc. Subsequently, 500 ll of the test compound was added. To control wells, we added 500 ll control solution. Bacteria were allowed to adhere and grow without agitation anaerobically for 4 h at 37°C. After 4 h, growth medium was removed, discs were rinsed with 09% (w/v) NaCl and 500 ll RCM and 500 ll QSI in RCM was added. The plate was further incubated for 24 h at 37°C. 494

The number of culturable sessile cells present on the silicone disc and the total number of cells present in the surrounding medium and the rinsing solution were determined after 4 h and 24 h by plating as described previously (Brackman et al. 2009). In brief, each disc was transferred to 10 ml 09% (w/v) NaCl. The tubes were subjected three times to 30 s of sonication (Branson 3510, 42 kHz, 100 W; Branson Ultrasonics Corp., Danbury, CT) and 30 s of vortex mixing to remove the biofilm cells from the discs. No cells were killed during sonication (Bjerkan et al. 2009). Using this procedure, all cells were removed from the discs, and clumps of cells were sufficiently broken apart. Combining vortexing and sonication was previously shown to be superior to scraping, vortexing or sonication alone to break biofilm clumps (Rieger et al. 2009). The number of sessile P. acnes LMG 16711, LMG 16712 and LMG 16715 cells were quantified by plating on RCM agar. All plates were incubated at 37°C for 24 h, and the number of CFUs per disc was calculated by counting colonies on the plates. To quantify the number of bacteria released from the biofilms on the silicone discs and the number of cells adhering to the disc, the number of cells in the medium and rinsing solution after 4 and 24 h was determined by plating. Each compound was tested on three discs in each assay, and each assay was repeated at least three times (n ≥ 9). Effect of QSI on the antimicrobial susceptibility of Propionibacterium acnes biofilms Propionibacterium acnes biofilms were formed as described above. To evaluate the effect of QSI on biofilm susceptibility, biofilms were formed as described above in the presence (pretreatment) or absence of QSI (100 lmol l 1). After biofilm formation, the biofilms were rinsed with PS, and fresh RCM was added with and without antibiotics alone or in combination with QSI (combined treatment). Antibiotic concentrations were 5000 mg l 1 for Mc, 10 000 mg l 1 for Cm, 5000 mg l 1 for Otc, 5000 mg l 1 for erythromycin and 25 000 mg l 1 for BPO. Subsequently, plates were incubated anaerobically for 24 h at 37°C. After 24 h of treatment, plates were rinsed with PS, and the number of living biofilm cells was quantified using a resazurin-based viability stain as described previously (Peeters et al. 2008). Confocal laser scanning microscopy For Confocal laser scanning microscopy (CLSM) analysis, 100 ll of OD600 02 bacterial suspension was transferred to the wells of a black 96-well plate with a glass bottom (Greiner Bio-One, Wemmel, Belgium). Biofilm formation

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and treatment was conducted as described above. Afterwards, the wells were rinsed with PS and filled with 100 ll of a staining solution (3 ll of SYTO9 and 3 ll of propidium iodide in 1 ml of PS). The plates were incubated for 15 min at room temperature. Fluorescence and transmission images of the biofilms were obtained with a Nikon C1 confocal laser scanning module attached to a motorized Nikon TE2000-E inverted microscope (Nikon Benelux, Brussels, Belgium) equipped with a Plan Apo VC 60 9 14 NA oil immersion objective. The SYTO9 fluorophore was excited with the Argon ion 488-nm laser line, and emission light was collected using a 500- to 550-nm band-pass filter. Propidium iodide was excited by 561-nm laser light, and emission light was collected with a 660-nm long-pass filter. Dual colour confocal Z-stacks were recorded of the biofilm samples. Tests were performed on at least two wells for each situation, and representative images are shown. Statistics The normal distribution of the data was checked using the Shapiro–Wilk test. The data were analysed using a one-way ANOVA Dunnet statistical analysis. Statistical analysis and linear regression analysis were carried out using SPSS software, version 19.0 (SPSS, Chicago, IL, USA). Results MIC of antibiotics and QSI alone or in combination on Propionibacterium acnes Minimal inhibitory concentrations were determined for all antimicrobials and QSI alone and in combination. A MIC above 1000 lmol l 1 was observed for TZD8 for all three P. acnes strains (Table 1). A MIC of 250 lmol l 1 for TZD10 was observed for P. acnes LMG 16711 and LMG 16712, while a MIC of 1000 lmol l 1 was observed for P. acnes LMG 16715. For all further experiments, TZD8 and TZD10 were used in a concentration of 100 lmol l 1. This concentration was chosen as it is well below the MIC for the P. acnes strains and as a similar concentration of TZD8 and TZD10 was previously shown to completely block QS in V. harveyi (Brackman et al. 2013). A MIC of 1 mg l 1, 05 mg l 1 and 0125 mg l 1 was found for all three P. acnes strains for erythromycin, minocycline and clindamycin, respectively. MICs of oxytetracycline and benzoylperoxide ranged between 64– 128 mg l 1 and 6250–12 500 mg l 1, respectively. None of the QSI altered the susceptibility of planktonic P. acnes cells towards erythromycin, minocycline, clindamycin and oxytetracycline. Although minor differences in MIC

Effect of thiazolidinedione on biofilm

Table 1 Minimal inhibitory concentrations of different antibiotics (mg l 1) and QS inhibitors (lmol l 1) for planktonically grown cells MIC value for the different compounds alone or in combination Propionibacterium acnes Compounds

LMG16711

LMG16712

LMG16715

TZD8 TZD10 Em Em-TZD8* Em-TZD10* Mc Mc-TZD8* Mc-TZD10 Cm Cm-TZD8* Cm-TZD10* Otc Otc-TZD8* Otc-TZD10* BPO BPO-TZD8* BPO-TZD10*

1000 250 1 1 1 05 05 05 0125 0125 0125 64 64 64 6300 6300 3200

1000 250 1 1 1 05 05 05 0125 0125 0125 128 128 128 6300 3200 3200

1000 1000 1 1 1 05 05 05 0125 0125 0125 64 64 64 12 500 6300 6300

*MIC of the antibiotic when used in combination with TZD8 or TZD10 (both at 100 lmol l 1).

towards P. acnes were observed for some combinations of benzoylperoxide and QSI in comparison with benzoylperoxide alone, these differences were within the acceptable margin of error and were not considered relevant. The FICI were higher than 05 and lower than 4 for all combinations, indicating that there was no interaction and that the observed interactions are indifferent (data not shown). Effect QSI on Propionibacterium acnes biofilm formation and on mature biofilms There was a significant decrease in biofilm formation relative to the control when the P. acnes strains were grown in the presence of the QSI (Fig. 2a). The use of TZD10 or TZD8 (both at 100 lmol l 1) resulted in a reduction in biofilm biomass of more than 40% in P. acnes LMG 16711 and LMG 16712 and more than 35% in P. acnes LMG 16715. Based on the data obtained with CTB staining, all compounds significantly affected the number of metabolically active cells in all strains tested (Fig. 2a). Similarly, biofilm biomass was reduced by more than 20% when 24-h-old biofilms were treated with the QSI (Fig. 2b). A decrease in the number of metabolically active cells was also observed when mature biofilms were treated with TZD10 or TZD8 (Fig. 2b).

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Figure 2 Antibiofilm activity of TZD8 and TZD10 against P. acnes LMG 16711, LMG 16712 and LMG 16715 biofilms. (a) The biofilm inhibitory activity was assessed by growing P. acnes biofilms in the absence (CTRL) and presence of TZD8 or TZD10. (b) The biofilm eradicating activity was assessed by treating a 24-h-old biofilm with TZD8 or TZD10. The biomass was quantified by CV staining (black bars), while cell viability was quantified by CTB staining (grey bars), CV signals (absorbance values at 590 nm) and CTB signals (excitation and emission filters of 535 and 590 nm) are presented as a percentage compared to a control biofilm formed in the absence of compounds (set to 100%). All treatments were significantly different from the control (P < 005).

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Effect of QSI on sessile cell numbers and detachment of Propionibacterium acnes biofilms As all compounds are tested in sub-MIC concentrations, a reduction in biofilm biomass and in number of metabolically active cells cannot be explained by growth inhibitory effects. This suggests that the compounds reduce initial attachment or increase detachment at later stages of biofilm development. This was investigated by determining the number of CFUs present on the discs (after 4 h and 24 h of biofilm formation) and by determining the number of CFUs present in the surrounding growth medium and in the rinsing solution of a P. acnes LMG 16711 biofilm. After 4 h of adhesion, the number of cells present on the disc and in the growth medium did not differ significantly between untreated biofilms and biofilms treated with QSI (data not shown). In contrast, the number of cells present in the growth medium and on the discs significantly differed for 24-h-old biofilms formed in the presence of QSI (Table 2). The medium surrounding the control biofilm only contained 167 (115) 9103 cells, while this was 973 (373) 9 104 and 450 (183) 9105 for biofilms formed in the presence of TZD8 and TZD10, respectively. In addition, significantly more cells were removed during rinsing. Similarly, the number of cells present on the disc, in the growth medium and in the rinsing solution was significantly different for 24-hold biofilm treated with the QSI compared to the untreated biofilm (Table 2). The growth medium surrounding the control biofilm contained only 787 (083) 9105 cells, while this was 333 (033) 9 106 and 236 (046) 9106 for biofilms formed in the presence of TZD8 or TZD10, respectively. Between five to ten times more CFU/biofilm were removed from biofilms formed in the presence of TZD8 and TZD10 during the rinsing step, compared to the control biofilm. Finally, significantly less cells were present on the discs of 24-h-old biofilm treated with the QSI compared to the control

disc. In contrast, total cell numbers did not differ significantly. Effect of QSI on the antimicrobial susceptibility of Propionibacterium acnes biofilms We subsequently assessed whether the effect of the QSI on biofilms would also result in an increased susceptibility of these biofilms towards commonly used antimicrobial agents. For this reason, biofilms pretreated with QSI during formation were subsequently treated with an antimicrobial agent commonly used to treat acne. In addition, 24-h-old biofilms were simultaneously treated with a combination of an antimicrobial and a QSI. The selected concentrations represent actual therapeutic concentrations. To determine the fraction of surviving cells after treatment, we used a resazurin-based viability staining. All antibiotics resulted in a significant (P < 005) reduction in the number of metabolically active cells in all three P. acnes strains (Fig. 3). However, some clear differences were observed between the different antimicrobials. The use of erythromycin (5000 mg l 1), clindamycin (10 000 mg l 1) and benzoylperoxide (25 000 mg l 1) resulted in only moderate reductions (90%). Pretreatment with QSI significantly increased biofilm susceptibility towards treatment. For example, only 41  28%, 02  03%, 175  33%, 20  06% and 229  38% P. acnes LMG 16711 biofilm cells survived treatment with erythromycin, minocycline, clindamycin, oxytetracycline and benzoylperoxide, respectively, when biofilm were formed in the presence of TZD10 (Fig. 3a). Similar results were also observed for pretreatment with TZD8 and for the other P. acnes strains (Fig. 3a). In addition, combined treatment of a 24-h-old biofilm with a QSI and an antimicrobial agents resulted in significantly higher killing compared to treatment with the antimicrobial agent alone (Fig. 3b). For example, only 82  23%,

Table 2 Number of cells present in the growth medium (M), rinsing solution (RS) and on the discs of a P. acnes LMG 16711 biofilm grown in the presence of QSI and mature biofilm treated with QSI. Number of cells are presented as CFU ml 1  standard deviation Compound

M

Numbers of cells in biofilms formed in the presence of QSI Control 167 (115) 9 103 TZD8 973 (373) 9 104* TZD10 450 (183) 9 105* Numbers of cells in 24-h-old biofilms treated for additional 24 h with QSI Control 787 (083) 9 105 TZD8 333 (033) 9 106* TZD10 236 (046) 9 106*

RS

Disc

793 (310) 9 102 120 (054) 9 105* 477 (182) 9 104*

347 (010) 9 106 665 (309) 9 105* 531 (145) 9 105*

136 (040) 9 105 193 (069) 9 106* 117 (038) 9 106*

437 (104) 9 106 668 (248) 9 105* 663 (479) 9 105*

*Cell numbers are significantly different from the control (P < 005; Mann–Whitney U-test).

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Figure 3 Effect of TZD8 and TZD10 on biofilm susceptibility. (a) Propionibacterium acnes LMG 16711 (black bars), LMG 16712 (dark grey bars) and LMG 16712 (light grey bars) control biofilms and biofilms receiving pretreatment with TZD8 or TZD10 were subsequently treated with an antimicrobial agent. (b) Propionibacterium acnes LMG 16711 (black bars), LMG 16712 (dark grey bars) and LMG 16712 (light grey bars) biofilms received no treatment (CTRL) or a combined treatment of TZD8 or TZD10 with an antimicrobial agent. Cell viability was quantified by CTB staining and are presented as a percentage fluorescence signal (excitation and emission filters of 535 and 590 nm) compared to a control biofilm formed in the absence of compounds (set to 100%) (log scale).

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18  05%, 144  35%, 32  105% and 73  67% P. acnes LMG 16711 biofilm cells survived treatment with TZD10 in combination with erythromycin, minocycline, clindamycin, oxytetracycline or benzoylperoxide, respectively (Fig. 3b). Similar results were also obtained for combined treatment with TZD8 and an antimicrobial agent and for the other P. acnes strains (Fig. 3b).

of a 24-h-old biofilm with TZD10 significantly affected biofilm thickness. Biofilms treated with TZD10 were also less structured and contained fewer cells. In addition, significantly more biofilm cells are killed when a P. acnes LMG 16711 biofilm formed in the presence of TZD10 is treated with erythromycin or when a 24-h-old biofilm is treated with a combination of erythromycin and TZD10.

Effect of QSI on biofilm morphology

Discussion

Consistent with the results obtained by plating and by resazurin staining, clear differences in biofilm morphology were observed between the different treatments. Treatment of a 24-h-old biofilm with erythromycin alone resulted in a moderate killing of the biofilm cells and a limited reduction in biofilm thickness compared to an untreated control biofilm (Fig. 4). In contrast, pretreatment with TZD10 during biofilm formation or treatment

Propionibacterium acnes infections are difficult to treat due to the presence of biofilms at the infection site and the associated resistance towards conventional antimicrobials. For this reason, novel agents targeting P. acnes biofilm formation are needed. QS is a process that could be targeted by these novel agents. Different QSI have been shown to affect biofilm formation and susceptibility in species other than P. acnes (Brackman et al. 2011). In

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Figure 4 Representative confocal images of P. acnes LMG16711 biofilms. (a) Biofilm receiving no treatment, (b) biofilm receiving a treatment with erythromycin, (c) biofilm grown in the presence of TZD10 receiving no antibiotic treatment or (d) biofilm grown in the presence of TZD10 receiving a treatment with erythromycin, (e) Propionibacterium acnes LMG 16711 24-h-old biofilms treated with TZD10 and (f) 24-h-old biofilm treated with TZD10 in combination with erythromycin.

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addition, in view of the role of P. acnes biofilms in skin infections, the difficulty to eradicate these biofilms and the potential role of biofilms in the development of microbial resistance in acne treatment, we investigated the effect of two thiazolidinedione-derived QSI on P. acnes biofilm formation in vitro. The antimicrobial agents included in this study were selected because they are often used in the treatment of acne. Although the thiazolidinediones did not affect the susceptibility of planktonic P. acnes strains towards conventional antiacne drugs, a clear antibiofilm effect was observed. As the thiazolidinedione derivatives are tested in sub-MIC concentrations, the observed reduction in biofilm biomass and in number of metabolically active cells cannot be explained by growth inhibitory effect on the cells. This suggests that the compounds reduce initial attachment or increase detachment at later stages of biofilm development. This was investigated by determining the number of CFUs present on the discs (after 4 h and 24 h of biofilms formation) and by determining the number of CFUs present in the surrounding growth medium and in the rinsing solution of a P. acnes LMG 16711 biofilm. Indeed, although no effect was observed during the adhesion step, significantly different cell numbers were present in the growth medium and on the discs of 24-h-old biofilms formed in the presence of QSI. In addition, between five to ten times more CFU/biofilm were removed from biofilms formed in the presence of TZD8 and TZD10 during the rinsing step, compared to the control biofilm. These data suggest that TZD8 and TZD10 do not exert their effect during initial stages of attachment, but rather affect later stages of biofilm development. We subsequently assessed whether the effect of the thiazolidinedione derivatives on biofilms would also result in an increased susceptibility of these biofilms towards commonly used antimicrobial agents used in actual therapeutic concentrations. Although the antibiotics alone only moderately affected the biofilms, combined treatment clearly increased biofilm susceptibility. These data confirm that biofilm dispersal due to the QSI can result in an increased killing of the biofilm cells by antimicrobial agents. Whether these QSI indeed exert their effect by blocking a QS-dependent biofilm mechanism or by actively inducing a dispersal mechanism remains to be investigated. Propionibacterium acnes produces AI-2, but to date, no AI-2 receptor or signal transduction pathway has been identified in this organism. While an increased production of AI-2 as well as an upregulation of virulence activity is observed in vitro in biofilms of P. acnes compared to their planktonic counterparts, evidence for a direct causal relationship between both phenomena is lacking (Coenye et al. 2007). In addition, whether these 500

compounds also interfere with other cellular processes besides biofilm formation and whether these compounds would also exert similar effects in vivo remains to be investigated. Although this effect of QSI on P. acnes has not been described before, similar effects for QSI have been described for other bacteria. Baicalin hydrate affected biofilm maturation and increased instability in Burkholderia cenocepacia biofilms (Brackman et al. 2009), while a furanone-like compound was shown to affect Pseudomonas aeruginosa biofilm architecture and enhance detachment from the biofilm (Hentzer et al. 2002). Similarly, the QSI RIP was shown to affect biofilm formation in Staphylococcus aureus (Kiran et al. 2008). Altogether, these data indicate that although the mechanism(s) by which they exert their activity is presently unknown, these compounds can be promising leads for the development of novel drugs to treat P. acnes infections. Acknowledgements We dedicate this article to the memory of our friend and colleague Professor M. Srebnik who passed away in December 2011. This work was supported by Interuniversity Attraction Poles Programme initiated by the Belgian Science Policy Office, by the ‘Special Research fund of Ghent University’ (BOF09/GOA/011) and by the IWTSBO Programme. Conflict of interest No conflict of interest declared. References Alexeyev, O.A. and Jahns, A.C. (2012) Sampling and detection of skin Propionibacterium acnes: current status. Anaerobe 18, 479–483. Bayston, R., Ashraf, W., Barker-Davies, R., Tucker, E., Clement, R., Clayton, J., Freeman, B.J. and Nuradeen, B. (2007) Biofilm formation by Propionibacterium acnes on biomaterials in vitro and in vivo: impact on diagnosis and treatment. J Biomed Mater Res A 81A, 705–709. Bjerkan, G., Witso, E. and Bergh, K. (2009) Sonication is superior to scraping for retrieval of bacteria in biofilm on titanium and steel surfaces in vitro. Acta Orthopedica 80, 245–250. Brackman, G., Hillaert, U., Van Calenbergh, S., Nelis, H.J. and Coenye, T. (2009) Use of quorum sensing inhibitors to interfere with biofilm formation and development in Burkholderia multivorans and Burkholderia cenocepacia. Res Microbiol 160, 144–151.

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Brackman, G., Cos, P., Maes, L., Nelis, H.J. and Coenye, T. (2011) Quorum sensing inhibitors increase the susceptibility of bacterial biofilms to antibiotics in vitro and in vivo. Antimicrob Agents Chemother 55, 2655–2661. Brackman, G., Al Quntar, A.A., Enk, C.D., Karalic, I., Nelis, H.J., Van Calenbergh, S., Srebnik, M. and Coenye, T. (2013) Synthesis and evaluation of thiazolidinedione and dioxazaborocane analogues as inhibitors of AI-2 quorum sensing in Vibrio harveyi. Bioorg Med Chem 21, 660–667. Brook, I. (2008) Microbiology and management of joint and bone infections due to anaerobic bacteria. J Orthop Sci 13, 160–169. Coenye, T., Peeters, E. and Nelis, H.J. (2007) Biofilm formation by Propionibacterium acnes is associated with increased resistance to antimicrobial agents and increased production of putative virulence factors. Res Microbiol 158, 386–392. Coenye, T., Brackman, G., Rigole, P., De Witte, E., Honraet, K., Rossel, B. and Nelis, H.J. (2012) Eradication of Propionibacterium acnes biofilms by plant extracts and putative identification of icariin, resveratrol and salidroside as active compounds. Phytomedicine 19, 409–412. Crawford, W.W., Crawford, I.P., Stoughton, R.B. and Cornell, R.C. (1979) Laboratory induction and clinical occurrence of combined clindamycin and erythromycin resistance in Corynebacterium acnes. J Invest Dermatol 72, 187–190. Hentzer, M., Riedel, K., Rasmussen, T.B., Heydorn, A., Andersen, J.B., Parsek, M.R., Rice, S.A., Eberl, L. et al. (2002) Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound. Microbiology 148, 87–102. Humphrey, S. (2012) Antibiotic resistance in acne treatment. Skin therapy let 17, 1–3. James, K.A., Burkhart, C.N. and Morrell, D.S. (2008) Emerging drugs for acne. Expert Opin Emerging Drugs 14, 649–659.

Effect of thiazolidinedione on biofilm

Kiran, M.D., Giacometti, A., Cirioni, O. and Balaban, N. (2008) Suppression of biofilm related, device-associated infections by staphylococcal quorum sensing inhibitors. Int J Artif Organs 31, 761–770. Miller, M.B. and Bassler, B.L. (2001) Quorum sensing in bacteria. Annu Rev Microbiol 55, 165–199. Ni, N., Li, M., Wang, J. and Wang, B. (2009) inhibitors and antagonists of bacterial quorum sensing. Med Res Rev 29, 65–124. Odds, F.C. (2003) Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother 52, 1. Peeters, E., Nelis, H.J. and Coenye, T. (2008) Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. J Microbiol Methods 72, 157–165. Perry, A. and Lambert, P. (2011) Propionibacterium acnes: infection beyond the skin. Expert Rev Anti Infect Ther 9, 1149–1156. Rammage, G., Tunney, M.M., Patrick, S., Gorman, S.P. and Nixon, J.R. (2003) Formation of Propionibacterium acnes biofilms on orthopaedic biomaterials and their susceptibility to antimicrobials. Biomaterials 24, 3221–3227. Rieger, U.M., Pierer, G., Luscher, N.J. and Trampuz, A. (2009) Sonication of removed breast implants for improved detection of subclinical infection. Aesthetic Plast Surg 33, 404–408. Ross, J.L., Snelling, A.M., Eady, E. A., Cove, J.H., Cunliffe, W.J., Leyden, J.J., Collignon, P., Dreno, B. et al. (2001) Phenotypic and genotypic characterization of antibioticresistant Propionibacterium acnes isolated from acne patients attending dermatology clinics in Europe, the USA, Japan, and Australia. Br J Dermatol 144, 339–346.

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