Pathogens and Disease ISSN 2049-632X

RESEARCH ARTICLE

Protease production by Staphylococcus epidermidis and its effect on Staphylococcus aureus biofilms Ilse Vandecandelaere1, Pieter Depuydt2, Hans J. Nelis1 & Tom Coenye1 1 Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium 2 Department of Intensive Care, Ghent University Hospital, Ghent, Belgium

This work establishes a role for S. epidermidis extracellular proteases in inhibition of S. aureus biofilm formation. It is demonstrated that secretion of S. epidermis proteases inhibits development of S. aureus biofilms in both in vitro and in vivo C. elegans infection model.

Keywords nosocomial pathogens; extracellular protease production; biofilms. Correspondence Ilse Vandecandelaere, Laboratory of Pharmaceutical Microbiology, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium. Tel.: 0032 9 264 80 93 fax: 0032 9 264 81 95 e-mail: [email protected] Received 22 November 2013; revised 3 January 2014; accepted 4 January 2014. Final version published online 5 February 2014. doi:10.1111/2049-632X.12133

Abstract Due to the resistance of Staphylococcus aureus to several antibiotics, treatment of S. aureus infections is often difficult. As an alternative to conventional antibiotics, the field of bacterial interference is investigated. Staphylococcus epidermidis produces a serine protease (Esp) which inhibits S. aureus biofilm formation and which degrades S. aureus biofilms. In this study, we investigated the protease production of 114 S. epidermidis isolates, obtained from biofilms on endotracheal tubes (ET). Most of the S. epidermidis isolates secreted a mixture of serine, cysteine and metalloproteases. We found a link between high protease production by S. epidermidis and the absence of S. aureus in ET biofilms obtained from the same patient. Treating S. aureus biofilms with the supernatant (SN) of the most active protease producing S. epidermidis isolates resulted in a significant biomass decrease compared to untreated controls, while the number of metabolically active cells was not affected. The effect on the biofilm biomass was mainly due to serine proteases. Staphylococcus aureus biofilms treated with the SN of protease producing S. epidermidis were thinner with almost no extracellular matrix. An increased survival of Caenorhabditis elegans, infected with S. aureus Mu50, was observed when the SN of protease positive S. epidermidis was added.

Editor: Ake Forsberg

Introduction Staphylococcus aureus is one of the most important causes of community-acquired and health care-associated infections worldwide (Mediavilla et al., 2012). It is a versatile pathogen causing various diseases ranging from skin and soft tissue infections to life-threatening conditions such as pneumonia, bloodstream infections and endocarditis (Sifri et al., 2003). Staphylococcus aureus is able to produce a broad array of virulence factors such as adhesins, enzymes and toxins (Sifri et al., 2003; Becker & von Eiff, 2012). Also, various strategies to evade the host immune response are present in S. aureus (Becker & von Eiff, 2012). The success of S. aureus as a pathogen can partially be attributed to its ability to acquire mobile genetic elements (Lindsay, 2010). For instance, methicillin resistance is easily transferred between different lineages of S. aureus and even between different staphylococcal species (Bloemendaal et al., 2010;

Li et al., 2013). Worldwide, prevalence of methicillin resistant S. aureus (MRSA) has steadily increased over the last three decades (Sifri et al., 2003). In one-third of the European countries, more than 25% of the health care-associated infections are due to MRSA (with the highest abundance in Southern Europe) (Control ECfDPa, 2010) and antibiotic resistance impedes the treatment of S. aureus infections (Moellering, 2012). A primary reservoir of S. aureus is the nasal cavity; up to one-third of the human population is a persistent carrier (Iwase et al., 2010; Becker & von Eiff, 2012). Another important representative of staphylococci in the nares is Staphylococcus epidermidis (Otto, 2012). This commensal skin bacterium is also recognized as an opportunistic pathogen. The majority of infections caused by S. epidermidis are associated with the presence of indwelling medical devices, such as central vascular catheters and prosthetic graft materials (Ziebuhr et al., 2006; Otto, 2012).

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Effect of S. epidermidis proteases on S. aureus biofilms

These infections are related to the ability of S. epidermidis to form biofilms on prostheses (Otto, 2012). In contrast to S. aureus, S. epidermidis does not produce many virulence factors and is one of the most abundant colonizers of the human skin. It plays an important role in maintaining a healthy skin flora, providing so-called colonization resistance (Stecher & Hardt, 2008). To do this, it is believed that S. epidermidis competes with potentially harmful bacteria (Otto, 2012). For instance, it has been suggested that S. epidermidis inhibits the Agr quorum sensing response of S. aureus in a pheromone dependent way (Otto et al., 2001). Another mechanism was reported by Iwase et al. (2010); they described the production of an extracellular serine protease (Esp) by S. epidermidis which inhibits S. aureus biofilm formation. Esp, isolated by Moon et al. (2001), can degrade human fibrogen and complement protein C5 (Dubin et al., 2001; Moon et al., 2001). Pro-Esp, also known as SspA or GluSE, contains a 66-residue propeptide (N-terminal end) and a mature 216-residue Esp peptide (with 59.1% sequence similarity to S. aureus V8 protease) (Ohara-Nemoto et al., 2002). Esp destroys specific proteins of the S. aureus biofilm matrix and of the S. aureus cell wall. At least 75 proteins are degraded, including 11 biofilm formation- and colonization-associated proteins such as extracellular adherence protein, the extracellular matrix protein-binding protein and protein A. Also, Esp selectively destroys several receptors of S. aureus which bind human extracellular proteins (Sugimoto et al., 2013). Importantly, Esp is exclusively produced by S. epidermidis (Ikeda et al., 2004). In the present study, we investigated the production of extracellular proteases by 114 S. epidermidis isolates. These strains were isolated from biofilms on endotracheal tubes (ET) of mechanically ventilated patients (Vandecandelaere et al., 2012). We evaluated the effect of the supernatant (SN) of these S. epidermidis isolates on S. aureus biofilms, using biomass and viability staining, as well as confocal laser scanning microscopy (CLSM). In addition, specific protease inhibitors were used to determine which proteases were produced by S. epidermidis. Finally, we tested the in vivo effect of S. epidermidis SN using a Caenorhabditis elegans infection model.

Materials and methods Isolates One hundred and fourteen S. epidermidis isolates were obtained from ET biofilms, originating from mechanically ventilated patients (Ghent University Hospital, Ghent, Belgium). These isolates were not obtained as part of standard patient care. The study was approved by the “Ethical Committee of Ghent University Hospital” (registration number: B6702010156). A written consent was signed by the patient or his/her relatives. More than 300 isolates were obtained from 32 ET biofilms, and identification procedures were described previously (Vandecandelaere et al., 2012). One hundred and fourteen of these isolates were identified as S. epidermidis: 33 S. epidermidis ET isolates (ET stands for 322

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endotracheal tube, representing a first set of tubes) and 81 S. epidermidis EF isolates (EF stands for endotracheal tube follow-up, representing a second collection of tubes). In addition, S. aureus subspecies aureus Mu50 (ATCC, Manassas, VA) and eight S. aureus isolates (EF-077, EF-087, EF-161, EF-207, ET-058, ET-181, ET-190 and ET-199) obtained from the same set of ET biofilms were also included in this study. Pure cultures of staphylococci were grown on Mueller-Hinton agar (MHA; Lab M Limited, Bury, UK) or in Mueller-Hinton broth (MHB; Lab M Limited) at 37 °C for 24 h. Vibrio anguillarum LMG 4411 (BCCM/LMG, Ghent, Belgium) was used as a positive control in the azocasein assay, and pure cultures were grown on Marine agar (MA; BD, Franklin Lakes, NJ) or in Marine broth (MB; BD) at 30 °C for 24 h. Determination of the extracellular protease production Pure cultures of S. epidermidis isolates (24 h old) were inoculated in 10 mL sterile MHB and incubated for 48 h at 37 °C. Vibrio anguillarum LMG 4411 (24 h old) was inoculated in 10 mL sterile MB and incubated for 48 h at 30 °C. After incubation, 1 mL was transferred to 9 mL sterile 0.9% NaCl and serial dilutions were made (10 1 to 10 9). One millilitre of the dilutions was plated on MHA, and after 24 h of incubation at 37 °C, the number of colony-forming units per mL (CFU mL 1) was determined. These CFU mL 1 were used to normalize the results of the protease assay. The residual 9 mL of the S. epidermidis culture was centrifuged at 2800 g for 20 min. Afterwards, SN was transferred to a sterile recipient and subsequently filtered (0.22 lm pore size). The resulting SN was used in the azocasein assay. Four hundred microlitres of azocasein (5 mg mL 1 in 0.1 M Tris-HCl buffer pH 8) (Sigma, Bornem, Belgium) was transferred to a sterile tube and 400 lL of S. epidermidis SN was added. Vibrio anguillarum LMG 4411 was included as a positive control, and centrifuged/filtered MHB and MB were used as negative controls. The tubes, containing SN and azocasein, were incubated for 3 h at 37 °C. The reaction was stopped by adding 100 lL 10% trichloroacetic acid (Sigma), and the tubes were centrifuged at 14000 g for 15 min. Each well of a 96-well flat-bottomed microtiter plate (SPL Life Sciences, Gyeonggi-Do, Korea) was filled with 100 lL 525 mM NaOH (Sigma). After centrifugation of the tubes, 100 lL of the SN was carefully transferred to the microtiter plate. The absorption at 420 nm was measured with a microtiter plate reader (EnVision, Perkin Elmer, Waltham, MA). We included six replicates in each experiment, and the experiments were repeated three times. The net absorbances (three 9 six values) of the S. epidermidis SN and positive control were calculated and normalized to the number of CFU per mL. Effect of S. epidermidis SN on S. aureus biofilms Starting from a S. aureus overnight culture, a dilution containing 107–108 CFU mL 1 was made. Seventy-two wells (all rows except the top and bottom row) of a round-bottomed 96-well microtiter plate were inoculated with 100 lL of the dilution and 24 wells (first and last row)

Pathogens and Disease (2014), 70, 321–331, © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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were filled with sterile MHB (negative control). After 4 h of incubation at 37 °C, the nonadherent cells were removed and the wells were rinsed with 100 lL physiological saline (PS). Subsequently, the inoculated wells were either filled with 100 lL of S. epidermidis SN (six wells per SN) or with 100 lL sterile filtered MHB (positive control; six wells). The microtiter plate was further incubated for 20 h at 37 °C. Prior to staining and plating, the biofilms were rinsed with 100 lL PS. Two different methods were used: a crystal violet staining (CV; ProLab Diagnostics, Neston, UK) to determine the amount of total biomass and a resazurin staining (Cell Titer BlueTM, CTB; Promega, Leiden, The Netherlands) to determine the number of metabolically active biofilm cells. Six replicates were included in each experiment, and the experiments were repeated three times. For the CV staining, biofilms were first fixed by adding 100 lL 99% methanol (Sigma). After 15 min, the methanol was removed and the plate was air-dried. Afterwards, 100 lL of a 0.1% CV solution was added to the wells and the plate was incubated for 20 min at room temperature. Then, the CV solution was removed, and the plate was extensively washed under running tap water. Finally, 150 lL 33% acetic acid (Sigma) was added to release bound CV. Plates were vigorously mixed (450 r.p.m., 20 min) to obtain complete dissolution of CV. Absorbance was measured at 590 nm (Peeters et al., 2008). The resazurin staining relies on the reduction in resazurin (by metabolically active cells) to fluorescent resofurin. A total of 120 lL of a 1/6 dilution of commercial CTB in PS was added to each well. Fluorescence (kex: 560 nm and kem: 590 nm) was measured after 30 min (Peeters et al., 2008). To determine the number of CFU in the biofilms, 100 lL PS was added to each well and the biofilms were sonicated (5 min) and vortexed (800 r.p.m., 5 min). The resulting cell suspensions were collected in sterile tubes (one tube per biofilm condition). This was repeated once, and afterwards, it was visually checked whether the biofilm was completely removed from the wells. Tenfold dilutions in PS were made (10 1 to 10 9), and 1 mL was plated on MHA using the pour plate method. Plates were incubated at 37 °C for at least 24 h, and afterwards, the number of CFU per biofilm was calculated. For CLSM, biofilms were grown in a 96-well microtiter plate with glass bottom. After 4 h at 37 °C, biofilms were rinsed and treated with the SN of protease positive S. epidermidis (ET-024 or ET-086) or with the SN of protease negative S. epidermidis (ET-167). After 20 h of incubation at 37 °C, biofilms were rinsed with 100 lL PS per well and stained using the LIVE/DEAD BacLight bacterial viability staining kit (Invitrogen, Merelbeke, Belgium) according to the manufacturer’s instructions. In brief, 3 lL Syto (3.34 mM in DMSO) (kex: 482 nm, kem: 500 nm) and 3 lL propidium iodide (20 mM in DMSO) (kex: 490 nm, kem: 635 nm) were mixed in 1 mL PS. One hundred microlitres of this solution was added to each well of the microtiter plate, and the biofilm was incubated for 10–15 min at room temperature. The staining solution was removed and images were taken immediately. The confocal laser scanning microscrope was equipped with

Effect of S. epidermidis proteases on S. aureus biofilms

two lasers, that is 488 nm, bandpass 50 (green fluorescence) and 633 nm, 660 LP filter (red fluorescence) (CVI Melles Griot, Albuquerque, NM). Images (z-stacks) were taken using a Nikon C1si confocal laser scanning microscope system equipped with a Plan Apo VC 60 9 1.4 NA oil immersion objective lens (Nikon, Brussels, Belgium). Images were viewed using the NIS-Elements Viewer 4.0 (Nikon). Confirmation of the role of proteases in the antibiofilm effect of S. epidermidis SN The SN of six isolates was further investigated using specific inhibitors. Aprotinin, a serine protease inhibitor (Yin et al., 2010; Park et al., 2012b) (5 mg mL 1 in sterile water, Sigma), was added to the azocasein assay mixture. Similarily, iodoacetamide (100 mM, Sigma) and EDTA (100 mM, Sigma) were added in the azocasein assay as cysteine and metalloprotease inhibitors (Jankiewicz & Bielawski, 2002; Keller et al., 2004; Yin et al., 2010; Hassanein et al., 2011), respectively. The assay consisted of 400 lL azocasein, 500 lL S. epidermidis SN and 300 lL inhibitor. Combinations of iodoacetamide (200 mM) and EDTA (200 mM), aprotinin (10 mg mL 1) and EDTA (200 mM) or aprotinin (10 mg mL 1) and iodoacetamide (200 mM) were used to determine the residual activity of serine, cysteine and metalloproteases, respectively (400 lL azocasein, 500 lL S. epidermidis SN and 150 lL of each inhibitor). The residual protease activity was expressed as a percentage compared to the protease activity measured without any inhibitors present, set to 100%. Using specific protease inhibitors, we also investigated whether the effect of S. epidermidis SN on S. aureus Mu50 biofilms was due to the presence of proteases in the SN. To this end, protease inhibitors were added to S. aureus Mu50 biofilms, treated with the SN of selected protease positive S. epidermidis isolates. After 4 h of incubation, the biofilms were rinsed and treated with 50 lL SN, 25 lL inhibitor (aprotinin: 5 mg mL 1; iodoacetamide: 100 mM; EDTA: 100 mM) and 25 lL MHB. Control S. aureus Mu50 biofilms were treated with 50 lL SN, 25 lL PS and 25 lL MHB. After 20 h of incubation at 37 °C, biofilms were rinsed and stained with CV. The effect of SN and/or inhibitors on the total biomass of S. aureus Mu50 biofilms was expressed as a percentage of A590 nm, compared to the appropriate control experiment (for example, the combined effect of S. epidermidis SN and aprotinin on S. aureus Mu50 biomass was compared to the effect of aprotinin on S. aureus Mu50 biomass). Evaluating the in vivo effect of S. epidermidis SN using a C. elegans infection model Caenorhabditis elegans strain N2 Dglp-4 Dsek-1 was used. This strain is incapable of producing progeny at 25 °C (Dglp-4) (Beanan & Strome, 1992) and exhibits an enhanced sensitivity to various pathogens (Dsek-1) (Kim et al., 2002). Worms were grown as described by Brenner (1974). In brief, nematodes were grown on nematode growth medium (precultured with E. coli OP50) and main-

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tained at 15 °C. Worms were bleached by adding 1 mL 5% sodium hypochlorite (Sigma) and 0.5 mL 4 M NaOH (Sigma). The resulting eggs were incubated for 3–4 days at 25 °C to obtain L4 stage worms (Brenner, 1974). The effect of S. epidermidis SN was evaluated as follows. In a 24-well flat-bottomed microtiter plate, approximately 30 L4 stage worms were added per well in growth medium containing 95% M9 buffer (3 g KH2PO4, 6 g Na2HPO4, 1 mL 1 M MgSO4 per litre), 5% brain heart infusion broth (Lab M Limited) and 0.1 v/v % of a 5 mg mL 1 cholesterol solution (Sigma). Worms were infected by adding 250 lL of a S. aureus Mu50 cell suspension (107–108 CFU mL 1), and 250 lL S. epidermidis SN was added. The effect of the SN of two protease positive and one protease negative S. epidermidis strains on infected C. elegans was evaluated. The total volume per well was 1 mL. The plates were incubated at 25 °C and scored for live and dead worms every 24 h (up to 72 h). Worms were considered dead if they were straightened and if no movement was observed. Data analysis Statistical analysis was performed using the Mann–Whitney U-test (SPSS, version 19.0; SPSS Inc., Chicago, IL), and statistical significance was defined as a P value < 0.05.

Results and discussion

compared to the positive control (Vibrio anguillarum LMG 4411) (Fig. 1, Supporting information, Fig. S1). The protease activity of more than half the isolates (n = 62) was lower than 10% of the activity of the positive control, and only for 10 isolates, the protease activity was > 20% of the activity of the positive control. Staphylococcus epidermidis isolates ET-024, ET-028b, ET-084 and ET-086 exhibited the highest protease production (Fig. 1, Fig. S1). We subsequently investigated whether there was a relation between the production of proteases by S. epidermidis and the presence of S. aureus in ET biofilms from which these S. epidermidis isolates were recovered. From seven biofilms, both S. epidermidis (n = 29) and S. aureus (n = 40) were isolated (Table 1, Table S1). When S. aureus was isolated from a biofilm in which S. epidermidis was also present, we observed a significantly lower protease production; we compared the normalized absorbance values of SN of S. epidermidis co-isolated with S. aureus (0.002232  0.000417) with the normalized average absorbance of SN of S. epidermidis, present in ET biofilms without S. aureus (0.004376  0.000334) (P < 0.05) (Fig. 1, Fig. S1, Table S1). These results suggest that there is a link between protease production of S. epidermidis and the absence of S. aureus in an ET biofilm obtained from the same patient. Our data are in line with the results of Iwase et al. (2010), who found that S. epidermidis has an inhibitory effect on S. aureus biofilms.

Protease production of S. epidermidis and link with the presence of S. aureus

Effect of SN of protease positive S. epidermidis on S. aureus biofilms

Of the 114 S. epidermidis isolates investigated, 112 produced extracellular proteases, albeit that the levels were low

Staphylococcus aureus Mu50 biofilms were treated with the SN of 53 selected S. epidermidis isolates. As revealed by

0.020 0.018 0.016

Absorbance per cfu/ml

0.014 0.012 0.010 0.008 0.006 0.004 0.002

EF-030 EF-037 EF-045 EF-054 EF-062 EF-064 EF-101 EF-106 EF-119 EF-123 EF-124 EF-125 EF-131 EF-152 EF-163 EF-170 EF-174 EF-176 EF-177 EF-180 EF-186 EF-192 EF-201 EF-208 EF-282 EF-283 EF-291 EF-294 EF-295 EF-333 EF-340 EF-344 EF-345 EF-348 EF-350 ET-013 ET-014 ET-016 ET-020 ET-024 ET-028b ET-037 ET-041 ET-046 ET-048 ET-051 ET-052 ET-059 ET-060 ET-066 ET-073 ET-076 ET-081 ET-084 ET-086 ET-096 ET-107 ET-124 ET-130 ET-150 ET-167

0.000

Selected S. epidermidis isolates

Fig. 1 Protease production by selected Staphylococcus epidermidis isolates; a complete overview is shown in Fig. S1. Bars and error bars represent average absorbance values at 420 nm normalized to CFU mL 1 and the standard error mean, respectively. Black bars: Staphylococcus aureus was isolated from the same ET biofilm from which S. epidermidis was recovered.

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Effect of S. epidermidis proteases on S. aureus biofilms

% Total biomass compared to an untreated control

CV staining, most treatments resulted in a moderate (< 20%) but significant reduction (P < 0.05) of total biofilm biomass compared to S. aureus Mu50 biofilms treated with centrifuged/filtered MHB (Fig. S2). However, S. aureus Mu50 biofilms treated with the SN of protease positive S. epidermidis ET-024 ( 52.2%), ET-028b ( 52.9%), ET-066 ( 51.0%), ET-073 ( 49.5%), ET-084 ( 44.2%) and ET-086 ( 44.4%) showed a more pronounced biomass reduction. Staphylococcus aureus strains, isolated from the same set of ET biofilms, were also included in the experiments. Biofilms from four S. aureus isolates (ET-058, ET-181, ET-190 and ET-199), were treated with the SN of six selected protease positive S. epidermidis isolates (ET-024, ET-028b, ET-060, ET-066, ET-084 and ET-086). In addition, biofilms from four other S. aureus isolates (EF-077, EF-087, EF-161 and EF-207) were treated with the SN of 6 other protease positive S. epidermidis isolates, that is EF-034, EF-045, EF-108, EF-125, EF-171 and EF-344. Our results show that there was an effect of the SN of most of these S. epidermidis isolates on the biomass of S. aureus biofilms (Fig. 2). A reduction in biomass (range: 3.2–87.6%) was observed. Remarkably, treatment with the SN of protease positive S. epidermidis isolates had a large effect (P < 0.05) on the biomass of S. aureus ET-181, EF-161 and EF-207 as it resulted in a biomass reduction of approximately 80% (Fig. 2). The effect of S. epidermidis SN was highly S. aureus strain dependent (Table 1). For instance, treatment of S. aureus ET-058, ET-181, ET-190 and ET-199 biofilms with the SN of S. epidermidis ET-024 resulted in a biomass decrease of 23.7%, 72.3%, 40.1% and 42.3% (P < 0.05), respectively (Fig. 2).

The number of metabolically active cells in S. aureus Mu50 biofilms treated with the SN of 53 selected protease positive S. epidermidis was determined by a resazurin-based viability staining. Only treatment with the SN of protease positive S. epidermidis ET-024, ET-028b and ET-084 resulted in a significant (although small) reduction in the number of metabolically active cells in S. aureus Mu50 biofilms (< 20% compared to negative controls) (Fig. S3). Treatment with the SN of the other protease positive S. epidermidis had almost no effect on the number of metabolically active cells in S. aureus Mu50 biofilms (Fig. S3). Although there was some variation, the same trend was observed when biofilms of S. aureus isolates, obtained from the same set of ET biofilms, were treated with the SN of 12 selected protease positive S. epidermidis isolates (ET-024, ET-028b, ET-060, ET-066, ET-084, ET-086, EF-034, EF-045, EF-108, EF-125, EF-171 and EF-344) (data not shown). In addition, planktonic growth of S. aureus Mu50 was not affected by SN of protease positive S. epidermidis ET-024 and ET-086 (Fig. S4). Compared to the results of the biomass staining, the effect on the number of viable cells in S. aureus biofilms was almost negligible. Staphylococcus aureus Mu50 biofilms were treated with the SN of 33 selected protease positive S. epidermidis isolates, and plate counting was performed. Most treatments had no significant effect on the number of CFU per biofilm. However, treatment of S. aureus Mu50 biofilms with SN of ET-024, ET-028b, ET-076, ET-084 and ET-086 resulted in approximately 10% less CFU per biofilm (P < 0.05) compared to negative controls (biofilms treated with centrifuged/filtered MHB) (Fig. S5).

120

100

80

60

40

20

SN ET-024 SN ET-028b SN ET-060 SN ET-066 SN ET-084 SN ET-086 SN ET-167 Control SN ET-024 SN ET-028b SN ET-060 SN ET-066 SN ET-084 SN ET-086 SN ET-167 Control SN ET-024 SN ET-028b SN ET-060 SN ET-066 SN ET-084 SN ET-086 SN ET-167 Control SN ET-024 SN ET-028b SN ET-060 SN ET-066 SN ET-084 SN ET-086 SN ET-167 Control SN EF-034 SN EF-045 SN EF-108 SN EF-125 SN EF-171 SN EF-344 SN EF-350 Control SN EF-034 SN EF-045 SN EF-108 SN EF-125 SN EF-171 SN EF-344 SN EF-350 Control SN EF-034 SN EF-045 SN EF-108 SN EF-125 SN EF-171 SN EF-344 SN EF-350 Control SN EF-034 SN EF-045 SN EF-108 SN EF-125 SN EF-171 SN EF-344 SN EF-350 Control

0

S. aureus ET-058

S. aureus ET-181

S. aureus ET-190

S. aureus ET-199

S. aureus EF-077

S. aureus EF-087

S. aureus EF-161

S. aureus EF-207

Fig. 2 CV staining of biofilms of Staphylococcus aureus isolates, obtained from ET biofilms, treated with the SN of selected Staphylococcus epidermidis isolates. Staphylococcus aureus ET-058, S. aureus ET-181, S. aureus ET-190 and S. aureus ET-199 were treated with the SN of 6 protease positive S. epidermidis (ET-024, ET-028b, ET-060, ET-066, ET-084 and ET-086) and SN of 1 protease negative S. epidermidis (ET-167). Four S. aureus EF isolates (EF-077, EF-087, EF-161 and EF-207) were treated with the SN of 6 protease positive S. epidermidis (EF-034, EF-045, EF-108, EF-125, EF-171 and EF-344) and SN of 1 protease negative S. epidermidis (EF-350). Average absorbance values at 590 nm of treated S. aureus biofilms are expressed as the percentage compared to negative controls (treated with centrifuged/filtered MHB). The error bars represent the standard error mean.

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Table 1 Selected Staphylococcus aureus isolates, used in the present study

Patient

S. aureus

S. epidermidis

S. epidermidis protease activity

Biofilm formation by S. aureus

1 2 3 4 5 6 7

ET-058 ET-181 ET-190 ET-199 EF-077 EF-087 EF-161

Positive (0.00723) Negative (0.00013) – – – – Positive (0.00168)*

2.256 2.990 2.355 2.063 3.604 3.934 4.124

8

EF-207

ET-059 ET-167 – – – – EF-163, EF-164, EF-167, EF-170, EF-171, EF-172, EF-173 and EF-174 EF-204, EF-205, EF-208, EF-209, EF-210 and EF-211

Positive (0.00101)*

2.957  0.276

Co-isolation with S. epidermidis, protease production of S. epidermidis (the average absorbance per CFU mL to form biofilms (evaluated by CV staining) are also given. *All S. epidermidis isolates within that sample are protease positive. –: S. epidermidis was not isolated from that sample.

CLSM was used to evaluate the effect of the SN of two protease positive S. epidermidis isolates (ET-024 and ET-086) on S. aureus Mu50 and S. aureus ET-058 biofilms. Staphylococcus aureus Mu50 and S. aureus ET-058 biofilms (both were treated with centrifuged/filtered MHB) showed a normal mature biofilm structure with cell clusters (Fig. 3). In contrast, S. aureus Mu50 and S. aureus ET-058 biofilms, treated with the SN of ET-024 and ET-086, were thinner and did not have the typical three-dimensional structure (Fig. 3). For instance, S. aureus Mu50 biofilms treated with ET-024 SN were 6.8 lm thick ( 8.5 lm compared to the controls). Our results show that treatment of S. aureus biofilms with the SN of protease positive S. epidermidis isolates resulted in a decrease of biomass. CV stains the bacterial cells (dead and alive) and the extracellular matrix present in a biofilm, and it appears that treatment of S. aureus biofilms with S. epidermidis SN had a large impact on the extracellular matrix. Targets for Esp include biofilm matrix proteins (e.g. Eap, Emp) and parts of the cell wall (e.g. fibrinogen) of S. aureus (Sugimoto et al., 2013). Degradation of these proteins resulted in an inhibition of S. aureus biofilm formation and a destruction of existing S. aureus biofilms (Iwase et al., 2010; Sugimoto et al., 2013). Thus, it is likely that the effect of S. epidermidis SN is due to proteolytic activity. The observation that treatment with S. epidermidis SN had no or very little effect on the number of metabolically active cells in S. aureus biofilms is not completely unexpected. Artini et al. (2013) treated S. aureus and S. epidermidis biofilms with a commercial serratiopeptidase, and although they observed an effect on biofilm formation and biomass, the bacterial viability (i.e. planktonic growth rate) was not affected. Iwase et al. (2010) and Sugimoto et al. (2013) reported that Esp had no bactericidal effect on S. aureus cells, and Park et al. (2012a) showed that the SN of Pseudomonas aeruginosa PAO1 (containing LasB elastase) inhibited S. aureus biofilm formation and degraded existing S. aureus biofilms without affecting the planktonic growth rate of S. aureus cells. 326

1

      

0.188 0.124 0.128 0.138 0.179 0.163 0.233

is given) and the ability of S. aureus

Effect of SN of protease negative S. epidermidis on S. aureus biofilms No significant biomass reduction was observed when S. aureus Mu50 biofilms or biofilms from S. aureus isolates (obtained from ET biofilms) were treated with the SN of protease negative S. epidermidis strains ET-167 and EF-350, except for S. aureus ET-181 treated with S. epidermidis ET-167 SN (P = 0.01) and S. aureus EF-161 treated with S. epidermidis EF-350 SN (P = 0.006) (Fig. S2, Fig. 3). In addition, no significant effect on the number of metabolically active cells was observed when S. aureus biofilms were treated with the SN of S. epidermidis ET-167 and EF-350 (Fig. S3). Also, we observed no significant difference in the number of CFU per biofilm between treated S. aureus Mu50 biofilms and negative controls (Fig. S5 for S. aureus Mu50 treated with ET-167). CLSM images showed that S. aureus Mu50 and ET-058 biofilms treated with the SN of protease negative S. epidermidis had a normal but thinner structure compared to the controls (Fig. 3). Treatment of S. aureus biofilms with the SN of protease negative S. epidermidis only had a minor effect (P > 0.05) on the biomass and biofilm structure and no effect on the number of metabolically active cells compared to the controls. This suggests that the effect of S. epidermidis SN on S. aureus can largely be attributed to the presence of proteases. Confirmation of the role of proteases in the antibiofilm effect of S. epidermidis SN Six isolates were selected for the experiments, including four isolates that exhibited the highest protease activity (ET-024, ET-028, ET-084 and ET-086) among our entire collection. The SN of these isolates was further characterized using three different protease inhibitors (Fig. 4). Aprotinin is a commonly used serine protease inhibitor (Lo & Hughes, 1996; Park et al., 2012b). Iodoacetamide and

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Fig. 3 Confocal laser scanning microscopy of Staphylococcus aureus Mu50 biofilms (a). (b) and (c) show S. aureus Mu50 biofilms treated with the SN of two protease positive Staphylococcus epidermidis, that is ET-024 and ET-086, respectively. CLSM image of a S. aureus Mu50 biofilm treated with the SN of protease negative S. epidermidis ET-167 is shown in (d). (e–g) CLSM images of untreated S. aureus ET-058 biofilms and S. aureus ET-058 biofilms treated with SN of protease positive ET-024 and ET-086, respectively. (h) shows a S. aureus ET-058 biofilm, treated with the SN of protease negative ET-167.

Effect of S. epidermidis proteases on S. aureus biofilms

(a)

(b)

(c)

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% residual protease activity compared to untreated SN

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EDTA are used as cysteine and metalloprotease inhibitors, respectively (Jankiewicz & Bielawski, 2002; Keller et al., 2004; Yin et al., 2010; Hassanein et al., 2011). Adding aprotinin and iodoacetamide to the azocasein assay significantly (P < 0.05) reduced the protease activity, indicating that both serine and cysteine proteases are produced by the S. epidermidis isolates. In contrast, the addition of EDTA to the SN of S. epidermidis ET-024, ET-066 and ET-086 did not result in a significant reduction in the protease activity (Fig. 4), indicating that the amount of metalloproteases produced by these S. epidermidis isolates was negligible. Addition of both aprotinin and iodoacetamide resulted in the largest reduction in protease activity (residual absorbance ranging from 10% to 27% compared to untreated SN) (P < 0.05). These results confirm that metalloproteases did not account for a large portion of protease activity. The combination of iodoacetamide and EDTA had the lowest impact on the protease activity (residual absorbance ranging from 40% to 61% compared to untreated SN), indicating that protease activity by S. epidermidis is mainly due to the production of serine proteases (Fig. 4). Protease inhibitors were also added to S. aureus Mu50 biofilms, which were treated with the SN of selected protease positive S. epidermidis isolates (ET-024, ET-028, ET-060, ET-066, ET-084 and ET-086). When aprotinin was added to treated S. aureus Mu50 bioflms, the effect of the SN on S. aureus Mu50 biofilms was much less pronounced (Fig. 5). We observed a biomass increase of at least 20% compared to S. aureus Mu50 biofilms treated with S. epidermidis SN alone (P < 0.05), indicating that serine proteases have an effect on S. aureus Mu50 biofilms. Adding iodoacetamide and EDTA also resulted in a biomass increase compared to S. aureus Mu50 biofilms treated with S. epidermidis SN alone (Fig. 5). When a combination of aprotinin and iodoacetamide was added to treated S. aureus Mu50 biofilms, we observed that the effect of the SN on the biomass was almost completely abolished (P < 0.05). This demonstrates that metalloproteases only 328

ET-086

Fig. 4 The effect of serine (aprotinin), cysteine (iodoacetamide) and metalloprotease (EDTA) inhibitors on the SN of selected Staphylococcus epidermidis isolates; residual protease activity is expressed as the average percentage compared to the protease activity of the SN without inhibitors. The error bars represent the standard error mean. *: P < 0.05, compared to % protease activity without inhibitor.

play a minor role in the effect of S. epidermidis SN on S. aureus Mu50 biofilms. In contrast, when both iodoacetamide and EDTA were added, no significant increase (P > 0.05) in biomass was observed compared to S. aureus Mu50 biofilms treated with S. epidermidis SN alone. This confirmed that the effect of S. epidermidis SN on S. aureus Mu50 biofilms is largely due to serine proteases. However, our results demonstrated that cysteine proteases also contributed to the effect of S. epidermidis SN on S. aureus Mu50 biofilms (Fig. 5). In summary, the results of these inhibition experiments show that serine proteases are largely responsible for the observed antibiofilm effect against S. aureus biofilms treated with the SN of S. epidermidis isolates. However, the biomass of treated S. aureus Mu50 biofilms to which protease inhibitors were added was still lower compared to untreated biofilms. These data suggest that other components in the S. epidermidis SN also affect S. aureus biofilms. Effect of S. epidermidis SN on the survival rate of infected C. elegans The nematodes were infected with S. aureus Mu50, and the effect of adding SN of two protease positive (ET-024 and ET-028b) and one protease negative (ET-167) S. epidermidis isolates on infected C. elegans was evaluated. The addition of S. epidermidis SN to uninfected nematodes did not significantly affect the survival (Fig. 6). Infecting C. elegans with S. aureus Mu50 resulted in a survival of 56.5  1.9% (after 24 h) or 50.8  1.9% (after 48 h) (Fig. 6). Addition of SN of two protease positive S. epidermidis isolates to infected C. elegans resulted in an increase in survival of 34.2% (ET-024) or 23.5% (ET-028b) after 24 h and of 35.7% (ET-024) or 34.8% (ET-028b) after 48 h (Fig. 6). No C. elegans survived when protease inhibitors (aprotinin, iodoacetamide and EDTA) were added. Biofilm formation contributes to the pathogenicity of S. aureus towards C. elegans (Begun et al., 2007), and it is likely that

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No inhibitor Aprotinin Iodoacetamide EDTA Aprotinin + iodoacetamide Aprotinin + EDTA Iodoacetamide + EDTA

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S. aureus Mu50 biofilms treated with the SN of selected S. epidermidis isolates

120 100 80

*

° 60

°

*

% Survival of C. elegans compared to control

Fig. 5 The effect of serine, cysteine and metalloprotease inhibitors on Staphylococcus aureus Mu50 biofilms treated with the SN of selected Staphylococcus epidermidis isolates. The biomass of treated biofilms is expressed as the average percentage compared to the appropriate control. The error bars represent the standard error mean. *: P < 0.05, compared to % total biomass of S. aureus Mu50 biofilms treated with S. epidermidis SN.

% Total biomass compared to the appropiate control

120

40 20 0 Mu50 Uninfected

SN ET-024 + Mu50 SN ET-028b + Mu50 SN ET-167 + Mu50

Infected

Protease positive S. epidermidis

Protease negative S. epidermidis

Fig. 6 The effect on survival rate of Caenorhabditis elegans infected with Staphylococcus aureus Mu50 treated with SN of protease positive (ET-024 and ET-028b) and protease negative (ET-167) Staphylococcus epidermidis isolates. Bars represent the average percentage survival compared to controls (treated with centrifuged/filtered MHB). The error bars represent the standard error mean. *: the % survival is significantly different compared to uninfected worms. °: the % survival is significantly different compared to infected worms treated with SN of protease positive S. epidermidis.

proteases in the S. epidermidis SN interact with virulence factors secreted by S. aureus Mu50 or that they degrade parts of the extracellular matrix produced by S. aureus Mu50 (Kuroda et al., 2001; Begun et al., 2007; Wang et al., 2007; Qiu et al., 2010). The addition of SN of protease negative S. epidermidis also significantly increased (P < 0.05) the survival of infected C. elegans but to a lesser extent (7.9% after 24 h or 13.9% after 48 h) (Fig. 6). The increase in survival was significantly higher (P < 0.05) if the infected worms were treated with the SN of protease positive S. epidermidis compared to treatment with the SN of protease negative S. epidermidis. Our results suggest that proteases play a role in the increased survival of infected C. elegans but as the addition of SN of protease

negative S. epidermidis also resulted in a small but significant increase in C. elegans survival, it is likely that other components in the SN also have an effect.

Conclusion In the present study, we demonstrated that S. epidermidis isolates produce proteases. We found a link between high protease production of S. epidermidis and the absence of S. aureus in ET biofilms obtained from the same patient. The SN of S. epidermidis isolates, containing proteases, had an effect on the biomass of S. aureus biofilms, but the number of metabolically active cells in S. aureus biofilms was not affected. Using specific protease inhibitors, we were

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able to demonstrate that the effect of S. epidermidis SN on S. aureus biofilms was mainly due to serine protease activity. In addition, adding SN of protease positive S. epidermidis to infected C. elegans increased their survival. Our data suggest that protease production by S. epidermidis isolates provides a competitive advantage in biofilm formation and also plays a role in establishing colonization resistance towards S. aureus.

Authors’ contribution All authors have read and approved the manuscript. I.V.D.C. designed the study, performed the experiments and wrote the manuscript. P.D., H.N. and T.C. designed the study and reviewed the manuscript.

Acknowledgements This research has been funded by FWO-Vlaanderen (project 3G001211) and by the Interuniversity Attraction Poles Program initiated by the Belgian Science Policy Office. The  Zegers, Inge Van den Haute and authors want to thank Chloe Alexander Stabij for their excellent technical assistance. We also want to thank Katrien Fourier for taking the CLSM images. All authors declare that they have no financial or nonfinancial competing interests in relation to this manuscript. References Artini M, Papa R, Scoarughi GL, Galano E, Barbato G, Pucci P & Selan L (2013) Comparison of the action of different proteases on virulence properties related to the staphylococcal surface. J Appl Microbiol 114: 266–277. Beanan MJ & Strome S (1992) Characterization of a germ-line proliferation mutation in C. elegans. Development 116: 755–766. Becker K & von Eiff C (2012) Staphylococcus, Micrococcus, and other catalase-positive cocci. Manual of Clinical Microbiology, Vol. 10 (Versalovic J, ed.), pp. 308–330. ASM press, Washington DC. Begun J, Gaiani JM, Rohde H, Mack D, Calderwood SB, Ausubel FM & Sifri CD (2007) Staphylococcal biofilm exopolysaccharide protects against Caenorhabditis elegans immune defenses. PLoS Pathog 3: e57. Bloemendaal AL, Brouwer EC & Fluit AC (2010) Methicillin resistance transfer from Staphylocccus epidermidis to methicillin-susceptible Staphylococcus aureus in a patient during antibiotic therapy. PLoS ONE 5: e11841. Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77: 71–94. Control ECfDPa (2010) Annual epidemiological report on communicable diseases. pp. ECDC, Stockholm. Dubin G, Chmiel D, Mak P, Rakwalska M, Rzychon M & Dubin A (2001) Molecular cloning and biochemical characterisation of proteases from Staphylococcus epidermidis. Biol Chem 382: 1575–1582. Hassanein WA, Kotb E, Awny NM & El-Zawahry YA (2011) Fibrinolysis and anticoagulant potential of a metallo protease produced by Bacillus subtilis K42. J Biosci 36: 773–779. Ikeda Y, Ohara-Nemoto Y, Kimura S, Ishibashi K & Kikuchi K (2004) PCR-based identification of Staphylococcus epidermidis targeting gseA encoding the glutamic-acid-specific protease. Can J Microbiol 50: 493–498.

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Effect of S. epidermidis proteases on S. aureus biofilms

Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. Protease production of the 114 S. epidermidis isolates and Vibrio anguillarum LMG 4411 (positive control). Fig. S2. Biomass staining of S. aureus Mu50 biofilms treated with the SN of selected S. epidermidis isolates. Fig. S3. Resazurin-based viability staining of S. aureus Mu50 biofilms treated with the SN of selected S. epidermidis isolates. Fig. S4. The effect of SN of S. epidermidis ET-024 and ET-086 on the planktonic growth of S. aureus Mu50. Fig. S5. The number of CFU per biofilm of S. aureus Mu50 biofilms treated with the SN of selected S. epidermidis isolates. Table S1. Isolation of S. epidermidis and S. aureus from ET biofilms.

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