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Received 23 Feb 2016 | Accepted 20 Sep 2016 | Published 3 Nov 2016

Wa Siala1, Soa Kucharkov2,3, Annabel Braem4, Jef Vleugels4, Paul M. Tulkens1, Marie-Paule Mingeot-Leclercq1, Patrick Van Dijck 2,3 & Franoise Van Bambeke1

Biolms play a major role in Staphylococcus aureus pathogenicity but respond poorly to antibiotics. Here, we show that the antifungal caspofungin improves the activity of uoroquinolones (moxioxacin, delaoxacin) against S. aureus biolms grown in vitro (96-well plates or catheters) and in vivo (murine model of implanted catheters). The degree of synergy among different clinical isolates is inversely proportional to the expression level of ica operon, the products of which synthesize poly-N-acetyl-glucosamine polymers, a major constituent of biolm matrix. In vitro, caspofungin inhibits the activity of IcaA, which shares homology with

b-1-3-glucan synthase (caspofungins pharmacological target in fungi). This inhibition destructures the matrix, reduces the concentration and polymerization of exopolysaccharides in biolms, and increases uoroquinolone penetration inside biolms. Our study identies a bacterial target for caspofungin and indicates that IcaA inhibitors could potentially be useful in the treatment of biolm-related infections.

1 Pharmacologie cellulaire et molculaire, Louvain Drug Research Institute, Universit catholique de Louvain, 1200 Brussels, Belgium. 2 Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KULeuven, 3000 Leuven, Belgium. 3 Department of Molecular Microbiology, VIB, KULeuven, 3000 Leuven, Belgium. 4 Department of Materials Engineering, KULeuven, 3000 Leuven, Belgium. Correspondence and requests for materials should be addressed to F.V.B. (email: mailto:[email protected]

Web End [email protected] ).

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DOI: 10.1038/ncomms13286 OPEN

The antifungal caspofungin increases uoroquinolone activity against Staphylococcus aureus biolms by inhibiting N-acetylglucosamine transferase

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13286

Staphylococcus aureus is one of the most prevalent human pathogens in the Western world, being capable of causing a wide spectrum of community- or hospital-acquired

infections. S. aureus healthcare-associated infections are related to the capacity of this bacterium to form biolms1. These consist of complex communities of microorganisms encased in a glycocalyx composed of DNA, proteins and polysaccharides. Biolms not only contribute to bacterial colonization of surfaces but also represent a reservoir for continuing bacterial dissemination within the body. Thus, staphylococcal biolms are considered as a main reason for persistence and/or recurrence of infections like endocarditis, osteomyelitis or those associated with indwelling medical devices2,3. These infections are also prone to treatment failure4, ascribed to poor bacterial response to immune defenses and antibiotics57. Unresponsiveness to antibiotics is related to the facts that (i) biolm matrix opposes a barrier to the access of host defenses and antibiotics to embedded bacteria, and (ii) bacteria within biolms adopt a dormant lifestyle poorly responsive to antibiotic action8. Antibiotic combination has been considered as a valuable strategy to act on staphylococcal biolms9,10, but this approach does not address the main pharmacokinetic issue posed by biolms, consisting in insufcient drug penetration within the structure.

In strains of S. aureus expressing the ica operon, a major constituent of the biolm matrix is poly-N-acetyl-glucosamine (PNAG) polymer, also referred to as polysaccharide intercellular adhesin (PIA)5,11,12. The gene products of the icaADBC locus include IcaA (transmembrane N-acetyl-glucosamine transferase synthesizing short PNAG polymers13), IcaD (protein increasing the biosynthetic efciency of IcaA and playing a predominant role in the synthesis of oligomers longer than 20 residues13), IcaB (extracellular N-deacetylase enabling PNAG xation at the bacterial cell surface and biolm formation1,14), and IcaC (putative transmembrane protein initially considered as involved in the polymerization of short chain polymers13 but more recently, being recognized as a O-succinyltransferase catalyzing the O-modication of PNAG during biosynthesis15). Expression of icaA and subsequent PNAG production have been associated with the capacity of S. aureus to produce biolm in vitro, including for clinical isolates collected from device-related infections1618. The expression of the icaADBC

locus in S. aureus depends on the genetic background of the strain and is upregulated in vivo19. Moreover, PNAG-enriched biolms are effectively dispersed by the glycoside hydrolase dispersin B, positioning this polysaccharide as an attractive target for adjunctive therapy20,21. Yet, the applicability of dispersin B itself in the clinics is limited to the eld of wound or catheter-related infections by its proteic nature22,23.

Several alternative, non-protein-based strategies have thus been proposed to improve antibiotic activity against staphylococcal biolms24. Small molecules like quinolines25, 2-aminobenzimidazoles26 or norspermidine and guanidine or biguanide biomimetics27 have proven effective in vitro but have never been tested in vivo, so that their druggability is unknown.Moreover, their mechanism of action is only partially elucidated, making a successful lead optimization and development of more potent analogues uncertain.

We set out to identify, amongst already approved drugs, compounds that would act on extracellular matrix to increase antibiotic activity against staphylococcal biolms. This was thought to facilitate the potential future clinical exploitation of the results. On the basis of the importance of polysaccharidic compounds in the matrix of staphylococcal biolms, we selected for this study caspofungin, an approved antifungal echinochandin28, which acts on Candida and Aspergillus species by inhibiting b-1-3-glucan synthase29. We used clinical isolates of

S. aureus previously demonstrated to be recalcitrant to the action of antibiotics when grown as biolms30. We compared two uoroquinolone antibiotics, namely, (a) moxioxacin, considered as the most potent anti-Gram-positive uoroquinolone among those available on the market31, but which is only modestly active against biolms32, and (b) delaoxacin, an even more potent anti-Gram-positive uoroquinolone currently in phase III of clinical development33, which also showed more promising activity than moxioxacin against biolms30.

We demonstrate that caspofungin markedly improves the activity of both uoroquinolones in in vitro and in vivo models of biolms. This synergy is due to the capacity of caspofungin to inhibit the enzymatic activity of IcaA, which shares homology with the fungal b-1-3-glucan synthase. Thus, we establish a bacterial target for this class of antifungal compounds and document a therapeutic potential of pharmacological inhibitors of IcaA.

Table 1 | Activity of moxioxacin and delaoxacin alone or combined with caspofungin against planktonic bacteria and 24- h-biolms.

Strain* MICw (mg l 1) Fluoroquinolone concentrations (mg l 1) needed to reduce bacterial viability in biolms of 50%z

MXFw DFXw CASw MXF DFXAlone CASy Alone CAS y

ATCC33591 (MRSA) 0.032 0.004 80 1.25 0.1 0.125 0.125 2011S027 (MSSA) 0.125 0.004 80 0.9 0.7 0.5 0.125 Surv2003/1083 (MSSA) 0.125 0.004 160 420|| 17 420|| 2 2009S025 (MRSA) 0.125 0.125 80 420 1.9 4 1

Surv2005/104 (MRSA) 2 0.125 160 420 18 420 2 Surv2005/179 (MRSA) 2 0.016 80 420 3.8 420 4 2009S028 (MRSA) 2 0.016 80 420 3.7 8 0.5

Surv2003/651 (MRSA) 2 0.125 160 420 420 420 8 Xen36 (MSSA) (bioluminescent strainderived from S. aureus ATCC 49525)

1 0.016 80 420 10.4 420 1

*All clinical isolates belong to the epidemic CC5 or CC8 clonal complexes; see Siala et al.30 for origin and description; MSSA, methicillin susceptible S. aureus; MRSA, methicillin-resistant S. aureus.

wMIC, minimal inhibitory concentration; MXF, moxioxacin; DFX, delaoxacin; CAS, caspofungin. zCalculated using the Hill function tted to the data of concentrationresponse experiments similar to those presented in Supplementary Fig. 3 for selected strains.

yUsed at 40 mg l 1.

|| Effect not reached at 20 mg l 1.

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ResultsCaspofungin-uoroquinolone activity on biolms in vitro. In a rst set of experiments, we examined the activity of moxioxacin and delaoxacin alone or combined with caspofungin at a xed concentration (40 mg l 1) against S. aureus mature biolms grown in 96-well plates. The laboratory strain ATCC33591 and seven clinical strains, previously described as clinical isolates forming biolms in vitro30, were used in parallel (see Table 1 for minimal inhibitory concentrations (MICs)). We rst checked that caspofungin did not affect the bacterial viability or biomass in biolms when used alone at the xed concentration selected (Supplementary Fig. 1). Figure 1 illustrates typical results for 4

biolms exposed during 48 h to uoroquinolones alone or combined with caspofungin (data for the other four strains under study are shown in Supplementary Fig. 2). In a rst step, we examined the effect of increasing concentrations of uoroquinolones on bacterial viability in biolms, as assessed in parallel by the measure of residual resorun uorescence (bacterial metabolic activity; left axis) and of colony-forming units (CFUs) (viable bacteria; right axis). Moxioxacin alone (Fig. 1ad) was poorly active on these biolms, reaching a bactericidal effect (3 log10 decrease in CFUs) only against

ATCC33591 biolm at the highest concentration tested (shown in Supplementary Fig. 2). Delaoxacin alone (Fig. 1il) reached a

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Figure 1 | Effect of uoroquinolones alone or combined with caspofungin on biolms from four selected strains. Biolms were incubated during 48 h in the absence or in the presence of the drugs (moxioxacin (ah black) or delaoxacin (ip blue) at increasing concentrations and used alone (ad and il) or combined (eh and mp) with 40 mg l 1 caspofungin). The ordinate shows resorun uorescence (closed bars; left scale; expressed in percentage of the value measured in control conditions (no uoroquinolone added)) and CFU (open bars; right scale; expressed in log10 units). Data are the means.d. of four replicates. Statistical analysis: multiple t-tests comparing data for uoroquinolone alone or combined with caspofungin in the same conditions: ***Po0.001; **Po0.01; *Po0.05.

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13286

bactericidal effect against the ATCC33591 (Supplementary Fig. 2) and 2011S027 but not against the other strains. In sharp contrast, when uoroquinolones were combined with caspofungin, a bactericidal effect was observed against 3 out of 4 strains for moxioxacin (Fig. 1eh) and against all strains for delaoxacin (Fig. 1mp), for which this effect was also reached at lower concentrations (r4 mg l 1). A similar improvement of activity was observed when combining caspofungin with uoroquinolones against biolms from the other strains under study (Supplementary Fig. 2).

Because the same type of results was obtained when determining residual viability based on CFU counts or resorun uorescence, we used the latter technique to obtain full concentrationresponse curves, which allowed us to determine and compare the relative potencies of the drugs (that is, the concentrations needed to reach a specied effect) against these biolms. We also evaluated in the same conditions the effect of uoroquinolones alone or combined with caspofungin on biolm biomass using crystal violet staining (Supplementary Fig. 3 for an illustration for four selected strains). The Hill function tted to the data of these concentrationresponse curves was used to calculate the concentrations of each uoroquinolone (used alone or combined with 40 mg l 1 caspofungin) needed to reduce bacterial viability of 25, 50 or 75% within biolms compared with control, and the corresponding potencies are shown graphically in Fig. 2 for the eight strains investigated. Moxioxacin (Fig. 2a) alone only reduced viability of 25% in six strains and of 50% in two strains. In contrast, its combination with caspofungin (40 mg l 1) achieved 75% reduction of viability for seven strains, with only 2003/651 remaining unaffected by this treatment (Fig. 2b). For delaoxacin alone (Fig. 2c), a 50 and 75% reduction of viability was obtained for four and three strains, respectively, in the range of concentrations investigated, and the corresponding potencies were increased (lower values) when combined with caspofungin (Fig. 2d). For two strains, however, a 75% reduction in viability could not be achieved in the range of concentrations tested even in combination with caspofungin (maximum reduction observed: 65% for 2005/179 and 68% for 2009S028, respectively). Considering then drug effects on biomass, a reduction in crystal violet staining was observed (although to a lesser extent than for viability) for all strains when caspofungin was combined with delaoxacin but only for two of them (2011S027 and 2005/179) when it was combined with moxioxacin.

Using the same experimental design, we also tested the effect of caspofungin on the activity of three other widely used antistaphylococal agents, namely vancomycin, daptomycin and linezolid (Supplementary Table 1). Synergy with caspofungin was observed with no strain when combined with vancomycin, for only one strain when combined with daptomycin, and for only four out of eight strains when combined with linezolid.

The activity of uoroquinolones and caspofungin combined at xed concentrations (10 and 40 mg l 1, respectively) was then examined in a second in vitro model consisting in biolms formed inside polyurethane catheter pieces with the seven clinical strains examined so far and with the bioluminescent strain Xen36 (Fig. 3). When tested alone, caspofungin and moxioxacin were ineffective in this model while delaoxacin signicantly reduced bacterial counts for all strains except 2003/651 (with residual counts remaining, however, Z4.5 log10 CFUs for four strains).

When used in combination, a marked synergy between each uoroquinolone and caspofungin was observed. Thus, moxioxacin gained considerable activity against all strains except 2003/651 and delaoxacin activity was improved against ve strains, including 2003/651. While the extent of synergy widely differed between strains (with reduction in CFU varying

for moxioxacin between 1.9 and 7.6 log10 for strains 2003/651 and 2011S027, respectively), it was more marked for strains showing more adhesion to the catheters (2011S027 and 2003/1083).

Caspofungin-uoroquinolone activity on biolms in vivo. In a next step, we assessed whether uoroquinolones, caspofungin, or their combination could act in vivo on S. aureus biolms present on catheters. Biolms were rst made in vitro and the infected catheters implanted under the skin of BALB/c mice. Biolms were then allowed to develop in vivo for 24 h, after which animals were treated twice daily with either 40 mg kg 1 of uoroquinolone alone or once daily with 4 mg kg 1 of caspofungin alone or with

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Figure 2 | Relative potencies of uoroquinolones alone or combined with caspofungin on biolms. Comparison of relative potencies of uoroquinolones alone (a moxioxacin; c delaoxacin) or combined with a xed concentration (40 mg l 1) of caspofungin (b and d) against biolms. The ordinate shows the concentrations of uoroquinolones needed to reach 25, 50 or 75% reductions in viability as assessed by measuring residual resorun uorescence. Active concentrations were calculated based on the equation of sigmoid concentrationresponse curves obtained for each strain in experiments similar to those illustrated in Supplementary Fig. 3. Each symbol corresponds to a specic strain, as indicated on the top of the graphs. A lower active concentrations corresponds to a higher potency. The horizontal dotted lines separate values for which calculated concentrations were above the actual maximal uoroquinolone concentrations tested(20 mg l 1).

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Figure 3 | Effect of caspofungin uoroquinolones used alone or in combination on biolms grown in catheters in vitro. Biolms grown in catheters for clinical isolates (ag) and the bioluminescent strain Xen36 (h) during 24 h were exposed to caspofungin (CAS; 40 mg l 1), uoroquinolones alone(10 mg l 1) or combined with 40 mg l 1 CAS. In each graph, the left panel shows data obtained with moxioxacin (MXF; black) and the right panel, data obtained with delaoxacin (DFX; blue) and matching controls for each strain. The graphs show the number of CFU (in log10 units) recovered from catheters after 48 h of incubation in the absence (control (CTRL)) or in the presence of the drugs. Data are meanss.d. for three catheters. Statistical analysis: bars with different letters show data that signicantly different from one another (Po0.01; one- way ANOVA with Tukey post-hoc test).

their combination for 7 days. These doses and schedules were selected as mimicking human exposure in clinical practice taking into consideration the shorter half-life of moxioxacin in mice compared with humans3437. For delaoxacin, in the absence of published humanized pharmacokinetic data, we selected as a starting point a dose equivalent to that of moxioxacin, taking also into account its short half-live and high protein binding in mice38. In a rst experiment, we followed on a daily basis mice with Xen36-infected catheters using bioluminescence imaging (Fig. 4a). In untreated mice, the intensity of the bioluminescence signal increased almost linearly from day 1 to 4 and reached thereafter a plateau (Fig. 4b,c). No difference in signal intensity was observed between untreated mice and those treated with caspofungin alone. Moxioxacin alone was also ineffective over the whole treatment duration (Fig. 4b), but delaoxacin caused a marked decrease in bioluminescence signal as from day 1 (Fig. 4c). When moxioxacin was combined with caspofungin, the bioluminescence signal was signicantly lower from day 4 as compared with mice treated by moxioxacin alone or caspofungin alone (Fig. 4b). When delaoxacin was combined with caspofungin, no signicant difference was observed with animals treated by delaoxacin alone if considering the mean values for all catheters (Fig. 4c). Yet, the establishment of the infection was slower in part of the mice and full eradication was achieved in one of the mice treated by the combination (compare Fig. 4e and Fig. 4d).

The experiment was extended in the same conditions to catheters infected by two clinical isolates against which the combinations were respectively markedly (2011S027) or marginally (2003/651) more effective in vitro than for uoroquinolones alone (Fig. 3). CFUs remaining on catheters were counted at day7. As shown in Fig. 5af, caspofungin alone was ineffective against all biolms. Fluoroquinolones alone caused a limited (B1 log10 for moxioxacin) or a marked (4.57 log10 for delaoxacin) decrease in the number of CFUs recovered from the catheters infected by 2011S027 and Xen36 but not by 2003/651.

In contrast, the number of CFUs recovered from the catheters was signicantly lower in animals treated with the combination of moxioxacin and caspofungin (mean reduction of 2.1, 1.6 and0.4 log10 CFUs for strains 2011S027, Xen36 and 2003/651, respectively (Fig. 5ac)) or with the combination of delaoxacin and caspofungin for strain 2003/651 (reduction of 0.5 log10 CFUs

(Fig. 5f)). Against the two other strains, combining caspofungin with delaoxacin allowed to achieve total (2011S027) or partial (Xen36) sterilization but the difference was not signicant with the already impressive effect reached in mice treated with delaoxacin alone (Fig. 5d,e). To better apprehend the potential of combining delaoxacin with caspofungin, we therefore performed a doseresponse study, using the highly responsive strain 2011S027 (Fig. 5g). Mice were treated with delaoxacin at increasing doses of 10, 20 or 40 mg kg 1 twice daily alone or combined with caspofungin 4 mg kg 1 once daily for 7 days and catheter-associated CFUs were determined at the end of this treatment. Delaoxacin activity was clearly dose-dependent over this range, and the synergy with caspofungin was best seen at the lowest dose (10 mg kg 1) of delaoxacin, which was suboptimal in monotherapy.

Electron microscopy studies of in vivo biolms. Catheters with biolms made by the clinical isolate 2011S027 and recovered from mice after in vivo treatment with moxioxacin alone, caspofungin alone or their combination, that is, focusing on conditions in which the effect of the combination was most evident, were examined by scanning electron microscopy (Fig. 6). A massive biolm matrix with only a few visible bacterial cells was observed on the surface of catheters extracted from control (saline-treated) (Fig. 6a), caspofungin- (Fig. 6b) or moxioxacin-treated animals (Fig. 6c). Some cracks (possibly due to the drying process) did, however, appear on caspofungin-treated biolms. More strikingly, catheters extracted from animals treated with the combination of moxioxacin and caspofungin (Fig. 6d) showed a

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13286

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Figure 4 | Activity of uoroquinolones alone or combined with caspofungin against the bioluminescent strain Xen36 in vivo. Bioluminescentsignal emitted from catheters infected by Xen36, implanted at day 0 in the back of mice treated 24 h after implantation and for the next 7 days with caspofungin (CAS) (4 mg kg 1 of body weight once daily), moxioxacin (MXF) or delaoxacin (DFX) (40 mg kg 1 of body weight twice daily) or with a uoroquinolone and caspofungin (each injected separately and according to its own schedule; (CAS MXF) or (CAS DFX)). All drugs were given by

intraperitoneal injection. Control: animals implanted with infected catheters and treated by normal saline (0.9% NaCl) twice daily. Uninfected: animals implanted with non-infected catheters and left untreated (used for detection of background signal). (a) Representative bioluminescence images for one mouse per group: intensity of the transcutaneous photon emission represented as a pseudocolor image. (b,c) Quantitative analysis per uoroquinolone (moxioxacin and matching controls (b); delaoxacin and matching controls (c)): in vivo bioluminescence signals expressed in photons per second (p s 1), with data expressed as meanss.e.m. Statistical analysis: two-way ANOVA, Tukey post-hoc test. *Po0.001 when comparing combinations versus uoroquinolones alone. (d,e) Quantitative analysis comparing both uoroquinolones when given alone (d) or in combination with caspofungin (e).

The data are shown as individual values with the corresponding means represented by a horizontal coloured line. Images are those of four mice treated during 7 days by the combinations.

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destroyed biolm structure, with less visible matrix and an abundance of bacterial cells in patches spread on the surface.

Caspofungin effect on uoroquinolone penetration in biolms. Previous studies have documented a correlation between the activity of antibiotics against biolm-encased bacteria and their capacity to penetrate the biolm30,39,40. Taking advantage of the intrinsic uorescence of uoroquinolones, we examined their

penetration within biolms grown on glass cover slips using confocal laser scanning microscopy. Figure 7 shows the concentration of uoroquinolones in the depth of biolms produced by four clinical isolates and exposed to 20 mg l 1 of antibiotics alone or combined with 40 mg l 1 caspofungin, together with the corresponding microscopic images. While uoroquinolone penetration was important for the biolm produced by strain 2011S027 (Fig. 7a,e), it was minimal for the three other strains. Combination with caspofungin largely increased uoroquinolone penetration not only in biolm produced by 2011S027 but also in those produced by 2003/1083 and 2005/179 (Fig. 7ac, eg). It also increased delaoxacin (Fig. 7h) but not moxioxacin (Fig. 7d) penetration in biolms produced by 2003/651.

Caspofungin effects on biolm matrix properties. Because poly-b(1-6)-N-acetylglucosamine is a major matrix component11, we examined whether the enhancement of uoroquinolone penetration and the destructuration of biolm matrix induced by caspofungin could be related to a modication in the concentration or polymerization degree of this exopolysaccharide. To this effect, the abundance of poly-b(1-6)-

N-acetylglucosamine in biolms was compared in control conditions and after incubation with caspofungin, for the same four clinical isolates and the reference strain ATCC33591, using an anti-PNAG antiserum (Fig. 8a). Caspofungin markedly decreased the signal for all strains, except 2003/651, suggesting it could interfere with the metabolism of this polysaccharide.

A critical property of poly-b(1-6)-N-acetylglucosamine as a matrix constituent is its degree of polymerization, since hydrolysis of polymers leads to biolm dispersal20. The inuence of caspofungin on the degree of PNAG polymerization was therefore evaluated in biolms from the same strains after 24 h of culture in the absence or in the presence of 40 mg l 1 caspofungin. To this effect, PNAG were puried from these biolms and submitted to a treatment by dispersin B to release N-acetylglucosamine monomers and allow for their quantication20 (Fig. 8b). In control conditions, more monomers were generated by dispersin B for 2005/179 and 2003/651 than for the other strains, suggesting a higher degree

Moxifloxacin Delafloxacin

CTRL CAS DFX DFX+CAS

a d

2011S027

Xen36

2003/651

2011S027

A A,B B C

Log 10CFUs/catheter piece

9.58.57.56.55.54.53.52.5

b e

9 8 7 6 5 4 3 2 1 0

9.58.57.56.55.54.53.52.5

CTRL CAS MXF MXF+CAS

10 A A B

A A B C

A A B B

Log 10CFUs/catheter piece

Log 10 CFUs/catheter piece

Log 10CFUs/catheter piece

c f

A A A B A A A B

Figure 5 | Activity of uoroquinolones alone or combined with caspofungin on S. aureus biolms in vivo. (af) Biolms were formed by the clinical isolate 2011S027 (a,d), by the bioluminescent strain Xen36 (b,e) or by the clinical isolate 2003/651 (c,f) in the mouse subcutaneous biolm model. Animals were treated for 7 days with caspofungin(4 mg kg 1 of body weight) once daily, uoroquinolones (40 mg kg 1 of body weight) twice daily, or with a uoroquinolone and caspofungin (each injected separately and according to its own schedule). CAS: caspofungin; MXF: moxioxacin; DFX: delaoxacin. Control animals (CTRL) were injected with normal saline (0.9% NaCl) twice daily. (ac) Experiments with moxioxacin; (df) experiments with delaoxacin. (g) Doseresponse for the activity of delaoxacin alone or combined with caspofungin on biolms from the clinical isolate 2011S027 in the mouse subcutaneous biolm model. Animals were treated for 7 days with caspofungin (CAS; 4 mg kg 1 of body weight) once daily, delaoxacin (DFX; 10, 20 or40 mg kg 1 of body weight) twice daily, or with delaoxacin at each of these doses combined with caspofungin 4 mg kg 1 once daily. Data are presented as the number of CFU (in log10 units) recovered from each individual catheter, with the mean value (and s.e.m. in g) shown in green or red colour for all catheters (ve catheters per animal; three or four animals per group; one experiment with clinical isolates and three experiments with strain Xen36). Statistical analysis (one-way ANOVA; Tukey post-hoc test): groups with different letters are signicantly different from one another (Po0.05).

4 CTRL CAS MXF CAS+MXF

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CAS

DFX (10)

CAS+DFX (10)

CAS+DFX (20)

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DFX (20)

DFX (40)

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13286

a b

Figure 6 | Scanning electron microscopy images of S. aureus 2011S027 biolms developed in vivo inside polyurethane catheters pieces. Fragments were retrieved from animals treated with sterile saline (a; control group), 4 mg kg 1 caspofungin once daily (b), 40 mg kg 1 moxioxacin twice daily (c) or their combination (d). Scale bars, 5 mm.

of PNAG polymerization in the corresponding biolms. In caspofungin-treated biolms, the concentration of monomers was reduced to 5060% in all strains except 2003/651, which remained unaffected. This strongly suggests that caspofungin impairs N-acetylglucosamine polymerization in biolms formed from strains that are susceptible to its effects.

Biolm matrix also contains other constituents like extra-cellular DNA and proteins. Their concentration was therefore also evaluated in biolms that were incubated during 48 h with 40 mg l 1 caspofungin versus controls, but no effect was observed, indicating that the action of caspofungin was not unspecic (Supplementary Fig. 4).

Inhibition of N-acetylglucosamine transferase by caspofungin. Poly-b(1-6)-N-acetylglucosamine is synthesized by enzymes encoded by the ica operon comprising four genes (icaA, icaD icaB and icaC). Among them, icaA encodes a membrane-located N-acetylglucosamine transferase that catalyzes the addition of new N-acetylglucosamine monomers to the growing polymer (Fig. 9a)13. The mode of action of caspofungin as antifungal agent is to prevent the incorporation of uridine diphosphate (UDP)-glucose into b-1-3-glucan by inhibiting the fungal b-1-3-glucan synthase29. A BLAST and clustalW analysis revealed conserved regions between the sequence of S. aureus icaA and that of the genes encoding b-1-3-glucan synthase from diverse fungal species (Supplementary Fig. 5). Interestingly, these regions correspond to conserved amino acids described as catalytic residues (Asp134; Asp227; Arg276) in IcaA13. On the basis of the data presented in Fig. 8, which strongly suggest an effect of caspofungin on N-acetylglucosamine incorporation in growing PNAG polymers, we investigated whether caspofungin could inhibit IcaA activity. We rst compared the enzymatic activity in protein extracts prepared from strain ATCC33591 or its DicaA mutant (Fig. 9b)

and found that the enzymatic activity of the extract from the wild-type strain was 5.62 IU mg 1 against only 0.02 IU mg 1 for the mutant extract. Notably, the enzymatic activity of the wild-type extract was markedly inhibited in the presence of 40 mg l 1 caspofungin (residual activity: 0.10 IU mg 1). This led us to conclude that caspofungin can inhibit bacterial

N-acetylglucosamine transferase activity.

We therefore examined the effect of increasing concentrations of caspofungin on UDP release by extracts from ATCC33591 and 2003/651. These two strains were selected because they express icaA, respectively, to the lowest and the highest level among the studied strains (see Supplementary Table 2; this table also shows the expression levels of the other genes of the ica operon). In parallel, we also sequenced icaA in ATCC33591 and 2003/651, looking for potential mutations that could explain differences in caspofungin inhibition towards IcaA activity from these two strains. As illustrated in Supplementary Fig. 6, only six silent mutations were found in 2003/651, which did not alter the amino acid sequence but could contribute to explain the high expression level of icaA in this strain by an exchange in the used codon. It has indeed been observed in several species that gene expression levels tend to correlate with the codon usage41 and that rare codons increase 4 to 20-fold gene expression levels42,43 possibly by making easier ribosome trafcking throughout the coding sequence44.

As illustrated in Fig. 9c, a clear concentration-effect for the inhibition of IcaA activity by caspofungin was obtained for both strains, with, however, a major difference in EC50 values(1.6 versus 9 mg l 1, respectively). Considering then the eight strains under investigation, we looked for a possible correlation between (a) the concentrations of uoroquinolones needed to achieve a specic reduction in viability within biolms when combined with caspofungin and (b) the level of icaA expression. We selected as target effect a reduction in viability of 25% for moxioxacin and of 50% for delaoxacin, because the latter was more active than the former when used alone. A highly signicant correlation (Pearsons correlation coefcient 40.84) between these two parameters was observed (Fig. 9d,e).

DiscussionBecause of the difculties of eradicating bacterial infections with current antibiotic treatments once biolms are formed, discovering innovative strategies that specically enhance antibiotic efcacy in this setting contributes to ll a therapeutic gap and therefore answers a clear unmet medical need24,45,46. Our work contributes to this effort by demonstrating the adjuvant properties of caspofungin towards uoroquinolone activity

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MXF MXF+CAS DFX

MXF

CAS+MXF

e DFX+CAS

Moxifloxacin Delafloxacin

MXF conc. in biofilm (mg l1 )

14 12 10

8 6 4 2 0

0 5 10 15 20 0.0 5.0 10.0

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CAS+DFX

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14 12 10

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0 2.5

Biofilm depth (m) Biofilm depth (m)

Figure 7 | Effect of caspofungin on uoroquinolone penetration within biolms. (ad) Moxioxacin (MXF); (eh) delaoxacin (DFX). The graphs compare the concentration of uoroquinolone in biolms incubated with 20 mg l 1 of uoroquinolone alone (MXF or DFX) or combined with40 mg l 1 caspofungin (MXF CAS or DFX CAS). The horizontal dotted line corresponds to the uoroquinolone MIC for the corresponding strain.

The tridimensional images were obtained using confocal laser scanning microscopy for the corresponding biolms stained either with 0.5 mM of 5-cyano-2,3-ditolyl tetrazolium chloride (red signal: MXF experiment) or with LIVE/DEAD (green signal: living bacteria; red: dead bacteria). The moxioxacin uorescence signal appears as green (preventing us from using LIVE/DEAD staining of the corresponding biolm) and the delaoxacin signal, in blue. Scale bars, 20 mm.

against S. aureus biolms. This synergy is observed not only in two in vitro models (biolms growing in 96-well plates or on catheters), but also in vivo, using a series of clinical isolates that were previously demonstrated as poorly susceptible to these and to other antibiotics when growing as biolms in vitro30.

Caspofungin is described as an antifungal agent with no intrinsic antibacterial activity. We present here three pieces of convergent experimental evidence that caspofungin increases the activity of uoroquinolone by destructuring S. aureus biolm matrix through an inhibition of the bacterial N-acetylglucosamine transferase (IcaA). First, caspofungin inhibits in vitro the

enzymatic activity of IcaA in the range of concentrations at which it also increases uoroquinolone activity on biolms. Second, caspofungin decreases the concentration and the degree of polymerization of poly-b(1-6)-N-acetylglucosamine in biolms. Third, while the degree of synergy between caspofungin and uoroquinolones markedly differs among the two antibiotics tested as well as among strains, it correlates with the respective level of expression of icaA in these strains. Although unexpected, inhibition of bacterial IcaA by caspofungin can be rationalized by the fact that it is a homologue of b-1-3-glucan synthase, the fungal target of caspofungin. While caspofungin proved effective

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Control + CAS

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+ CAS

*** B

***

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GlcNAc (g ml1 )

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Strains

Figure 8 | Effect of caspofungin on poly-b(1-6)-N-acetylglucosamine in biolms. Biolms were incubated during 24 h in the absence (control) or in the presence ( CAS) of 40 mg l 1 caspofungin. (a) Immunoblot analysis

of PNAG puried from biolms. (b) Determination of N-acetyl-glucosamine (GlcNAc) concentration after treatment of puried PNAG during 1 h with0.1% Dispersin B. The released GlcNAc monomers were detected by uorescence (lexc 545 nm; lem 604 nm). All data are means.d. of triplicates. Statistical analysis (two-way ANOVA; Tukey post-hoc test): groups with different letters (caps: control; small letters: CAS) are

signicantly different from one another (Po0.05); *** (Po0.001): comparison of control to CAS for each individual strain.

to inhibit poly-b(1-6)-N-acetylglucosamine polymerization in bacterial membrane extracts and to disperse it within biolms, it only exerted modest effects on the biomass or on the three-dimensional structure of biolms in the absence of moxioxacin. This suggests that bacterial killing (by an antibiotic) is required to observe an effective disruption of the biolm. This hypothesis is coherent with the observation that bacterial death within biolms is associated with a facilitation of biolm dispersal, related to the creation of voids within the matrix47,48. The main role of caspofungin, therefore, would be to increase the ability of uoroquinolones to penetrate more deeply into the biolm by decreasing the amount and/or the degree of polymerization of N-acetyl-glucosamine polymer present in the matrix network. We previously demonstrated that the bacterial killing exerted by delaoxacin towards bacteria present in biolms formed by the same strains is strictly dependent on its capacity to penetrate the matrix30. We complement this observation here by showing that its killing activity is much lower against those strains that express icaA to high levels, in close correlation with its decreased penetration in the biolm. We also extend this observation to another uoroquinolone, moxioxacin. This is coherent with the fact that, beside their contribution in bacterial adhesion and aggregation, exopolysaccharides are also critical for the maintenance of the biolm architecture and viscoelastic

properties49,50, playing, therefore, a key role in limiting antibiotic penetration. Supporting this specic role for exopolysaccharides, planktonic cultures of S. aureus that spontaneously form aggregates because of a production of these polymers have been also shown to be refractory to antibiotic activity, but to regain susceptibility on disruption of the aggregates by sonication51. Thus, caspofungin can be considered as a dispersal agent capable of improving antibiotic activity against biolms in a similar way as enzymes such as dispersin B, proteinase K or DNase I22,52,53. However, we show here that its association with a uoroquinolone antibiotic is essential for maximal efcacy. The key advantage of combining a dispersal agent and a bactericidal antibiotic is, indeed, that the latter also acts on planktonic bacteria, which may avoid the spreading of bacterial clumps released from the matrix on dispersal and the subsequent reestablishment of a biolm elsewhere.

Our work has two main limitations. First, synergy was mainly demonstrated for uoroquinolones and was not observed or only in a limited fashion for two other well established anti-staphylococcal antibiotics. For vancomycin and daptomycin, this may partly be due to their large molecular mass (1,449 and 1,620 g mol 1, respectively) versus uoroquinolones (B400 g mol 1), that may hamper their ability to diffuse into the biolm30 even if disrupted by caspofungin. For linezolid, for which the molecular mass (337 g mol 1) is lower than that of uoroquinolones, this could be due to its bacteriostatic effect against S. aureus, thus limiting its overall activity. Beside antibiotic properties, we cannot exclude that other matrix properties can contribute to prevent antibiotic action. Yet, we did not nd any relationship between antibiotic loss of activity and the biolm content in other major constituents like proteins or extracellular DNA. Likewise, we did not observe signicant differences among strains in the expression levels of icaC, which encodes an O-succinyltransferase that is thought to inuence biolm accumulation15. A second limitation of our study is that we only examined biolms formed from S. aureus, whereas those formed by related species such as Staphylococcus epidermidis are also clinically relevant. Interestingly, this organism also expresses the ica locus and produces a poly-b(1-6)-N-acetylglucosamine-rich matrix54,55.

In spite of these limitations, and although remaining focused on in vitro and animal demonstrations, our nding may have important clinical implications. First, the synergistic effects observed in vivo were obtained while using caspofungin and uoroquinolones at doses that are clinically relevant, suggesting they could be also observed in humans. Second, S. aureus strains expressing the ica locus are highly prevalent in biolm-related infections. A recent study showed that 85.6% of strains collected from human or bovine infections expressed ica genes, among which 95.4% were biolm producers18. Likewise, in a collection of strains causing catheter-related urinary infections, 88.6% were biolm-producers and all of them expressed the ica locus16. In this context, an Argentinian study showed that 35% of strains collected in nasal swabs from hospitalized patients or from staff are ica-positive and that all were slime producers56. Conversely, ica-negative strains producing slime have been rarely described inS. aureus infection57, underlining the interest of detecting icaA expression in clinical isolates prior using inhibitors of IcaA as adjuvant therapy. Third, we document the promising activity of delaoxacin, a uoroquinolone in clinical development, against staphylococcal biolms, especially when combined with caspofungin against strains expressing icaA to high levels. Fourth, and perhaps more importantly, no usable pharmacological inhibitor of IcaA has been described so far, and we have identied here an agent that is already approved for clinical use in

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1 )

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1 2 4 8 16 32

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icaA relative expression

Figure 9 | Ica A (N-acetylglucosamine transferase) activity and its inhibition by caspofungin. (a) Reaction catalyzed by IcaA. (b) Activity of IcaA in membrane protein extracts from strain ATCC33591 and its DicaA mutant; increasing concentrations of extracts (1.2580 mg ml 1) were incubated with 40 mM UDP-GlcNAc and 400 mM N-acetylglucosamine without (ATCC33591 and DicaA) or with 40 mg l 1 caspofungin (ATCC33591 CAS) during

120 min; activity was evaluated by the amount of UDP liberated in the reaction medium; all data are meanss.d. of triplicates. (c) Inhibition of IcaA activity in membrane protein extracts from strain ATCC33591 or the clinical isolate 2003/651 (both at 80 mg ml 1) exposed to increasing concentrations of caspofungin (CAS) during 120 min; all data are meanss.d. of triplicates; (d,e): correlation between the icaA relative expression and the relative potency of the uoroquinolonecaspofungin combinations against biolms. Each symbol corresponds to a specic strain. Fluoroquinolone potency is expressed as the concentration needed to reduce of 25% (C25; for moxioxacin (d)) or of 50% (C50; for delaoxacin (e)) the bacterial viability within biolms exposed to the uoroquinolone combined with 40 mg l 1 caspofungin (data from Fig. 2). The graph shows the actual data (symbols; bivariate t) surrounded by the bivariate normal ellipse for 95% condence interval (r is the Pearsons correlation coefcient).

fungal infections. Although caspofungin is a low-afnity inhibitor of IcaA, our results warrant further studies to validate such inhibitors as potential adjuvants for antibiolm antibiotherapy.

We fully realize that administering caspofungin to patients who are not infected by fungi could affect the fungal ora. We hope that our work will stimulate research for development of more potent IcaA inhibitors acting specically on the staphylococcal enzyme and devoid of antifungal activity.

Methods

Materials. Microbiological standards or solutions for injection were obtained from the following sources: moxioxacin HCl (powder potency: 90.9%, from Bayer HealthCare; Leverkusen, Germany; a solution for injection was prepared in NaCl0.9%); B.5.delaoxacin (powder potency: 95.7%) and its intravenous formulation, from Melinta Therapeutics (New Haven, CT, USA); caspofungin diacetate (powder potency: 90.1%), from Sigma-Aldrich (St Louis, MO, USA) and Cancidas, from

MSD (Brussels, Belgium). The other antibiotics were used as powder or solution for injection approved for human use in Belgium and complying with the provisions of the European Pharmacopoeia (vancomycin as Vancomycine Mylan, Mylan Inc, Canonsburg, PA, USA; linezolid as Zyvoxid, Pzer Inc. (New York, NY, USA); daptomycin as Cubicin, Novartis (Horsham, UK)). Media for bacterial culture were from Becton Dickinson Company (Franklin Lakes, NJ, USA).

Bacterial strains. The S. aureus ATCC33591 (methicillin-resistant) strain was used as reference strain. Seven clinical strains isolated from various human anatomical sites but all belonging to the pandemic clonal complexes CC5 or CC8 ofS. aureus were selected from the collection of the Belgian Reference Centre forS. aureus (Hpital Erasme, Universit libre de Bruxelles, Brussels) (Table 1). The bioluminescent strain Xen36 (Caliper Life Sciences, Hopkinton, MA, USA) was originally derived from S. aureus ATCC49525 and expresses a stable copy of a modied Photorhabdus luminescens luxABCDE operon58,59. MICs were determined by microdilution assay according to the recommendations of the Clinical & Laboratory Standards Institute60 (Table 1).

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In vitro biolm model. Mature biolms were obtained by growing bacterial strains for 24 h in 96-well tissue culture plates (VWR (Radnor, PA, USA); European cat. number 734-2327) in Trypticase Soy Broth supplemented with 2% NaCl and 1% glucose, with a starting inoculum adjusted to an optical density at 620 nm (OD620nm) of 0.005 in a volume of 200 ml, as previously described32. Biolms were

then exposed for 48 h to antibiotics (concentration range: 0.12520 mg l 1) alone or in combination with caspofungin (40 mg l 1). Bacterial viability in the biolm was measured using the redox indicator resazurin, as previously described30,32, or by CFU counting. To this effect, biolms were washed twice with phosphate buffer saline (PBS), sonicated (Branson 5510 Ultrasonics bath) for 10 min in 1 ml PBS and diluted aliquots were plated on tryptic soy agar (TSA) plates to allow CFU counting after overnight incubation. Biolm biomass was determined using crystal violet staining, following a published procedure30,32. In brief, at the end of the incubation period, the medium was removed and wells were washed with PBS, xed at 60 C for 1 h and stained by a 2.3% crystal violet solution prepared in ethanol 20% (Sigma-Aldrich). After elimination of the dye in excess under running water, crystal violet xed to the biolm was resolubilized by addition of 33% glacial acetic acid and incubation at room temperature for 1 h. Absorbance was read at 570 nm.

Biolm grown on catheters in vitro. Biolms were studied inside triple-lumen polyurethane central venous catheters (Certox duo/trio; B. Braun Melsungen AG, Melsungen, Germany) as described earlier61. Briey, 1 cm long catheters were incubated overnight in 100% fetal bovine serum (Sigma-Aldrich). Catheters were incubated 90 min at 37 C in trypticase soy broth supplemented with 2% NaCl and 1% glucose with bacterial strains at an initial density of 0.005 to allow adhesion, then transferred to new medium and incubated during 24 h. Biolms were then exposed to 10 mg l 1 uoroquinolone, 40 mg l 1 caspofungin or their combination for 48 h. Catheter pieces were washed with PBS, sonicated and diluted before plating on TSA and CFU counting, as described in the previous paragraph.

Murine subcutaneous biolm model. Female pathogen-free 20 g 8-week old BALB/c mice (Janvier Labs, Saint Berthevin, France) were kept individually in ventilated cages and provided with food and water ad libitum. All animal experiments were performed in accordance with the regulations and approval of the Ethical Committee of KULeuven (project number P125/2011). Animals were immunosuppressed by adding 0.4 mg l 1 dexamethasone (Organon Laboratories

Limited, Cambridge, UK) in their drinking water 24 h before the catheter implant and during the whole experiment. Biolms were studied using clinical isolates (2011S027 or 2003/651) or the bioluminescent strain (Xen36). Serum-coated catheters were incubated with the bacteria during the period of adhesion (90 min at 37 C) as described above. Afterwards, catheters were washed twice with PBS and subsequently implanted subcutaneously in the back of mice as described hereunder. First, general anaesthesia was achieved by intraperitoneal injection of a mixture of 45 mg kg 1 ketamine (Ketamine1000; Pzer, Puurs, Belgium) and 0.6 mg kg 1 medetomidine (Domitor; Pzer) and local anaesthesia by application of a 2%

xylocaine (AstraZeneca BV, Zoetermeer, Netherlands) on the skin. The lower back of the mice was then shaved and disinfected with 0.5% chlorhexidine in 70% alcohol. A 10 mm incision was made longitudinally and ve catheter fragments were implanted per mouse. Biolms were allowed to mature in vivo for 24 h before treatment. Fluoroquinolones and caspofungin were administered intraperitoneally. Whereas caspofungin was administered once daily (4 mg kg 1 of body weight), uoroquinolones (up to 40 mg kg 1 of body weight) were injected twice daily for 7 days. A control group of animals was injected twice daily with saline only. After 7 days of treatment, animals were killed by cervical dislocation and catheters were removed, washed twice with PBS (to remove non-device associated bacteria) and sonicated. The number of viable bacteria recovered from the biolms was then quantied by CFU counting after plating and overnight growth at 37 C.

In vivo bioluminescent imaging. Biolms made with the bioluminescent strain Xen36 were prepared and animal treated exactly as described above. Mice were imaged every day using an In Vivo Imaging System (IVIS Spectrum, Perkin-Elmer, Waltham, MA, USA). During the imaging, mice were anaesthetized using a gas mixture of isourane in oxygen (1.52%) and placed by groups of four animals in the apparatus. Frames were acquired with a eld of view of 23 cm. Consecutive scans with acquisition time of 5 min (binning 2) were acquired until maximal signal intensity was reached. The signal was quantied by using Living Image software (version 4.0, Perkin-Elmer) and reported as photon ux per second(p s 1) for a rectangular region of interest placed over each ve catheters)62.

Scanning electron microscopy. Mounted samples were sputter-coated with AuPd and viewed using a scanning electron microscope operated at standard high vacuum settings at a 10-mm working distance and 10-keV accelerating voltage (FEI XL30-FEG microscope, Philips Nederland B.V., Eindhoven, the Netherlands).

Confocal laser scanning microscopy for visualization of biolms. Biolm samples were imaged using a Cell Observer s.d. confocal uorescent microscope (Carl Zeiss AG, Oberkochen, Germany) using spinning disc technology (Yokogawa Electric Corporation, Tokyo, Japan) and controlled by the AxioVision software

(AxioVs40 V 4.8.2.0; Zeiss). Optimal confocal settings (camera exposure time, CSU disk speed) were determined in preliminary experiments. Image stacks of each sample were acquired at a resolution of 700 500 pixels and recorded using

Z-Stack module for acquisition of image series from different focus planes and used to construct three-dimensional images with AxioVision software.

Fluoroquinolone penetration within biolms. Twenty-four hour biolms were grown on cover slips and incubated for 1 h with 20 mg l 1 uoroquinolone alone or in combination with 40 mg l 1 caspofungin. Biolms were washed twice with 1 ml PBS and stained for 30 min in the dark with LIVE/DEAD bacterial viability kit (L-7007; Thermo Fisher Scientic, Waltham, MA, USA) or with 0.5 mM 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) (RedoxSensor vitality kit; Invitrogen, Carlsbad, CA, USA), as described previously30. CTC is a colourless, non-uorescent and membrane permeable compound, which is readily reduced via electron transport activity to uorescent, insoluble CTC-formazan that accumulates inside bacteria63. Stained biolms were then washed with 1 ml PBS buffer. Excitation/emission wavelengths were set as follows: 415 nm/500550 nm for moxioxacin; 395 nm/450 nm for delaoxacin, 488 nm/570620 nm for CTC-formazan signal, 488 nm/500550 nm for Syto 9 and 561 nm/570620 nm for propidium iodide (LIVE/DEAD staining). Fluoroquinolone concentrations within biolms were then calculated using calibration curves built using uoroquinolone solutions (concentrations ranging from 5 to 50 mg l 1) examined in the microscope using the same settings as for samples30.

PNAG purication and immunoblot analysis. PNAG was extracted as previously described64. Briey, biolms were resuspended in PBS, centrifuged and resuspended in 0.5 M EDTA, and incubated at 100 C for 5 min and at 85 C for 30 min. After a new centrifugation, the supernatant was rst dialyzed against deionized water for 18 h and then against a buffer (50 mM TrisHCl pH 8; 20 mM MgCl2) for 18 h (cut-off of dialysis membrane: 2 kDa). The crude polysaccharide preparation was treated with 100 mg ml 1 a-amylase, 500 mg ml 1 lysozyme,250 mg ml 1 DNase I and 100 mg ml 1 RNase A at 37 C for 2 h, then by 2 mg ml 1 proteinase K for 16 h at 55 C in the presence of 1 mM CaCl2 and 0.5%

sodium dodecyl sulfate. The samples were then incubated at 85 C for 1 h to inactivate proteinase K and dialyzed against deionized water for 18 h. Polysaccharide preparations were then lyophilized and dissolved in 200 ml PBS.

A 20 ml aliquot was spotted onto a PVDF membrane, which was air-dried and blocked with 0.5% milk in TBS buffer (150 mM NaCl and 10 mM TrisHCl(pH 7.4)) overnight at 4 C. The membrane was then incubated overnight at 4 C with PNAG antiserum (1:4,000)65 (kindly provided Dr Gerald B. Pier; Brigham and Womens Hospital, Boston, MA, USA), washed and probed with 1:5,000 goat anti-rabbit HRP for 2 h. Spots were visualized with the SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientic) and analyzed using the FUSION-CAP Software (Analis, Belgium).

GlcNAc determination by MorganElson assay with uorimetric detection.

Puried PNAG (125 ml) was incubated with 0.1% dispersin B (Symbiose, Belgium) for 1 h at 37 C to cleave them in N-acetylglucosamine (GlcNAc) monomers (note that the dialysis steps performed during the purication procedure allowed to eliminate the pre-formed monomeric forms). These were quantied by the MorganElson reaction66. The sample was added by 25 ml of tetraborate reagent (K2B4O7 4H2O 0.8 M; 3 min at 100 C), then by 0.75 ml of

p-dimethylaminobenzaldehyde reagent (prepared as described67; 10 min incubation at 37 C). The released reducing terminal GlcNAc in the supernatant were quantied in the supernate (recovered after 30 s centrifugation at 8,000g) by uorimetry (lexc: 545 nm; lem: 604 nm)68 based on a calibration curve constructed using GlcNAc standards (Sigma-Aldrich).

Assay of proteins and DNA in biolms. Biolms were grown in 6-wells plates during 24 h after which they were incubated during 48 h in the presence of40 mg l 1 caspofungin or in control conditions. Extracellular DNA (eDNA) was quantied as previously described69. In brief, biolms were washed twice and chilled at 4 C for 1 h, after which 500 ml of 0.5 M EDTA was added to each well.

After centrifugation (5 min, 12,000g), supernatants were discarded, and biolms were resuspended in eDNA extraction solution (50 mM Tris HCl, 10 mM EDTA,

500 mM NaCl, pH 8) and transferred into chilled tubes. After centrifugation(5 min; 4 C; 18,000g), 100 ml of supernatant was transferred to a tube containing 300 ml of TE buffer (10 mM Tris HCl, 1 mM EDTA, pH 8), and extracted once

with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) and once with chloroform/isoamyl alcohol (24:1). The aqueous phase of each sample was then mixed with three volumes of ice-cold 100% (vol/vol) ethanol and 1/10 volume of 3 M sodium acetate (pH 5.2) and stored at 20 C. The next day, the ethanol-

precipitated DNA was collected by centrifugation (20 min; 4 C; 18,000 g), washed with ice-cold 70% (vol/vol) ethanol, air-dried and dissolved in 20 ml TE buffer. DNA concentration was then determined with NanoDrop Ultravioletvis spectrophotometer (Thermo Fisher Scientic). Total proteins were quantied following a described procedure70. Biolm cells were pelleted by centrifugation at 5,500g for 10 min. Proteins present in the supernatant were precipitated by trichloroacetic acid (TCA). Biolm cells were gently resuspended in 5 ml PBS

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(pH 10) containing complete Protease Inhibitor Cocktail (Roche Life Science, Penzberg, Germany) (according to manufacturers instruction). Cells were incubated at 4 C with gentle rotation for 1 h and debris pelleted by centrifugation at 5,500g for 10 min. Proteins for biolms supernatants and biolms cells were then assayed using Bradford method.

Determination of the N-acetylglucosamine transferase activity in vitro.

Protein extracts from membranes of ATCC33591 and of its DicaA mutant (a kind gift from Prof. Friedrich Gtz, Universitat Tbingen, Germany) were prepared as follows. Fifty ml of overnight cultures were collected by centrifugation, and cell pellets were resuspended in 500 ml of buffer A (50 mM TrisHCl, pH 7.5, 10 mM

MgCl2, 4 mM dithiothreitol). Cells were disrupted by 3 1 min vortexing in Corex

tubes with glass beads (Sigma-Aldrich) (diameter of 0.30.60 mm; two times the weight of the cell pellet). DNase I (20 mg ml 1) was added before breaking the cells.

Unbroken cells and glass beads were sedimented (10 min, 2,000g), and the supernatant was saved. The procedure was repeated twice and all supernatants were combined. Membranes were sedimented from the crude extract by centrifugation (40 min, 20,000g), resuspended in buffer A, extracted with 2% (w/v) Triton X-100 (in buffer A) for 2 h with gentle shaking, sedimented again, washed once with buffer A and resuspended in 1 ml of buffer A. Protein concentrations were determined by the Lowrys method.

To determine N-acetylglucosamine transferase activity, increasing concentrations (1.2580 mg ml 1) of membrane extracts were incubated for 2 h at 37 C in 250 mM MES-NaOH buffer pH 6.25 containing 40 mM UDP-GlcNAc, 20 mM MnCl2, 400 mM N-acetylglucosamine, 1% (w/v) Triton X-100, in a total volume of 50 ml. The reaction was stopped by addition of 50 ml of Milli-Q water and boiling for 2 min. The mixture was then centrifuged at 20,000g for 5 min to remove denatured proteins and then supernatant was collected. The N-acetylglucosamine transferase activity was evaluated by measuring the amount of UDP liberated in the reaction using the Transcreener UDP2 FP Kit (BellBrook Labs, Madison,WI, USA), according to the manufacturers recommendations. Fluorescence polarization was measured using a Perkin-Elmer LS55 uorimeter (Perkin-Elmer). UDP concentration was then calculated based on a titration curve established as described in Supplementary Fig. 7.

RNA isolation from biolms and quantitative real-time PCR. RNAs were isolated from 24 h-old biolms. Biolms formed in 6-well polystyrene plates were washed thrice with sterile distilled water. Bacterial cells were detached by rapid scraping and resuspended in cold sterile distilled water. Suspensions were immediately incubated with 1 ml of RNA protect (Qiagen GmbH, Hilden, Germany), vortexed for 5 s and incubated for 5 min at room temperature, and pelleted by centrifugation at 10,000g for 10 min. Cell pellets were resuspended in 100 ml of 4 C sterile RNase-free distilled water (Qiagen). Total RNA was isolated using RNeasy mini kit (Qiagen). The RNA quality and quantity was checked by agarose gel electrophoresis and by measuring the absorbance at 260 and 280 nm using a NanoDropM spectrophotometer. Puried RNA was immediately converted to cDNA using transcription rst strand cDNA synthesis kit (Roche Applied Science) with random hexamer primers according to the manufacturers instructions. Quantitative PCR reactions were performed in triplicates in 96-well plates using 2 ml of cDNA, 10 ml of SYBR Green Master Mix, 0.5 ml of 100 mM of each primer, and 7 ml of sterile RNase-free water. The following primers were used: icaA gene forward (50-CGAGAAAAAGAATATGGCTG-30) and reverse (50-ACCATGTTGC GTAACCACCT-30); 16s rRNA gene forward (50-CGAAGGCGACTTTCTGG TCT-30) reverse (50-TACTCCCCAGGCGGAGTGCT-30). The reaction was started with an initial denaturation at 95 C for 5 min, followed by 40 amplication cycles of 95 C for 20 s, 60 C for 20 s and 72 C for 20 s. The X-fold change of transcription level was calculated using a relative standard curve method as described previously71.

Sequencing icaA gene. cDNA from the ATCC33591 strain and from the 2003/651 isolate were amplied via PCR using Phusion High-Fidelity DNA Polymerase following manufacturers protocol (Thermo Fischer Scientic). The primers used for DNA amplication were icaAfwd:50-GTTATCAATAATCTTATC

CTT-30 and icaArev: 50-AGTTTCAAATATATCTAAAAT-30. The PCR product (1611pb) was sequenced at Beckman Coulter Genomics facilities (Beckman Coulter Genomics, Takeley, Essex, UK) following Sanger protocol. The primers used for DNA sequencing were icaAfwd; icaArev and seqicaAREV: 50-CCAATGTTTCT

GGAACCAACA-30.

Data analyses and statistical analyses. Curve-tting analyses of concentration-effect relationships were made with GraphPad Prism version 4.03 or version 7.01 (GraphPad Software, San Diego, CA, USA) and correlations with JMP Pro version12.1.0 (SAS Institute, Marlow, Buckinghamshire, UK). Statistical analyses were made with GraphPad Instat version 3.06 (GraphPad Software) or GraphPad version 7.01.

Data availability. The authors declare that the data supporting the ndings of this study are available within the article and its Supplementary Information les.

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Acknowledgements

W.S. was a postdoctoral fellow of the programme Prospective Research for Brussels from

Innoviris (Brussels Institute for Research and Innovation); S.K. is supported by a post

doctoral grant of the Fonds Wetenschappelijk Onderzoek, F.V.B. is Matre de Recherches

of the Fonds de la Recherche Scientique. This work was supported by the Interuniversity

Attraction Poles Programme initiated by the Belgian Science Policy Ofce (programme

IAP P7/28); the Fonds de la Recherche Scientique (Grants 3.4.588.10F, 3.4530.12 and

T.0134.13) and Innoviris. We thank Pr. Friedrich Gtz (Universitat Tbingen, Germany)

for the kind gift of the DicaA mutant of the ATCC33591 strain, Pr. Gerald B. Pier

(Brigham and Womens Hospital, Boston, MA), for the kind gift of the anti-PNAG

antiserum, Tinne Buelens and Uwe Himmelreich (MoSAIC facility, KULeuven), for their

help during bioluminescence imaging and Celia Lobo Romero and Cindy Colombo

(KULeuven) for technical assistance during in vivo experimental procedures.

Author contributions

W.S. performed the in vitro experiments; S.K., the in vivo experiments, A.B. and J.V., the

electron microscopy. W.S., S.K. and F.V.B. designed the studies. P.V.D. and F.V.B.

supervised the work with the help of M.-P.M.-L. and P.M.T. W.S., S.K. and F.V.B. wrote the

manuscript. P.M.T., M.-P.M.-L. and P.V.D. contributed to the writing of the manuscript.

Additional information

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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Competing nancial interests: F.V.B. and P.M.T. have received research grants from

Melinta Therapeutics for projects unrelated to the present work. The remaining authors

declare no competing nancial interests.

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Biofilms play a major role in Staphylococcus aureus pathogenicity but respond poorly to antibiotics. Here, we show that the antifungal caspofungin improves the activity of fluoroquinolones (moxifloxacin, delafloxacin) against S. aureus biofilms grown in vitro (96-well plates or catheters) and in vivo (murine model of implanted catheters). The degree of synergy among different clinical isolates is inversely proportional to the expression level of ica operon, the products of which synthesize poly-N-acetyl-glucosamine polymers, a major constituent of biofilm matrix. In vitro, caspofungin inhibits the activity of IcaA, which shares homology with β-1-3-glucan synthase (caspofungin's pharmacological target in fungi). This inhibition destructures the matrix, reduces the concentration and polymerization of exopolysaccharides in biofilms, and increases fluoroquinolone penetration inside biofilms. Our study identifies a bacterial target for caspofungin and indicates that IcaA inhibitors could potentially be useful in the treatment of biofilm-related infections.

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Title

The antifungal caspofungin increases fluoroquinolone activity against Staphylococcus aureus biofilms by inhibiting N-acetylglucosamine transferase

Author

Siala, Wafi; Kucharíková, Sona; Braem, Annabel; Vleugels, Jef; Tulkens, Paul M; Mingeot-leclercq, Marie-paule; Van Dijck, Patrick; Van Bambeke, Françoise

Pages

13286

Publication year

2016

Publication date

Nov 2016

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms13286

ProQuest document ID

1835681428

The antifungal caspofungin increases fluoroquinolone activity against Staphylococcus aureus biofilms by inhibiting N-acetylglucosamine transferase

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