Antimicrobial Effects of Violacein Against Planktonic Cells and Bioﬁlms of Staphylococcus Aureus

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Article Antimicrobial Effects of Violacein against Planktonic Cells and Bioﬁlms of Staphylococcus aureus

Andressa H. M. Batista 1,* ID , Anne C. D. Moreira 1, Rafael M. de Carvalho 1, Gleilton W. P. Sales 1 ID , Patrícia C. N. Nogueira 2, Thalles B. Grangeiro 3 ID , Suelen C. Medeiros 3, Edilberto R. Silveira 2 and Nádia A. P. Nogueira 1

1 Clinical Department of Toxicological Analysis, Faculty of Pharmacy, Federal University of Ceará, Fortaleza 60356000, Brazil; [email protected] (A.C.D.M.); [email protected] (R.M.d.C.); [email protected] (G.W.P.S.); [email protected] (N.A.P.N.) 2 Organic Chemistry Department, Federal University of Ceará, Fortaleza 60356000, Brazil; [email protected] (P.C.N.N.); [email protected] (E.R.S.) 3 Biology Department, Federal University of Ceará, Fortaleza 60356000, Brazil; [email protected] (T.B.G.); [email protected] (S.C.M.) * Correspondence: [email protected]; Tel.: +55-85-3366-8267

Received: 7 August 2017; Accepted: 9 September 2017; Published: 25 September 2017

Abstract: Violacein is an indole compound, produced by Chromobacterium violaceum, a bacteria present in tropical and subtropical areas. Among its numerous biological activities, its antimicrobial potential stands out. This study aims to determine the antimicrobial activity of VIO on S. aureus in planktonic culture and biofilms. VIO showed excellent antimicrobial activity in inhibiting and killing S. aureus in planktonic cultures and biofilm formation. The minimum bactericidal concentration (5 µg/mL) of VIO caused the death of S. aureus after 3–4 h of exposure and the minimum inhibitory concentration (1.25 µg/mL) of VIO inhibited bacterial growth within the first 8 h of contact. Biofilm formation was also strongly inhibited by VIO (1.25 µg/mL), in contrast to the higher resistance verified for S. aureus in mature biofilm (40 µg/mL). The high bacterial metabolic activity favored VIO activity; however, the good activity observed during phases of reduced metabolism indicates that VIO action involves more than one mechanism. Thus, VIO is a promising molecule for the development of an antimicrobial drug for the eradication of S. aureus infections.

Keywords: violacein; antimicrobial activity; bioﬁlms; Staphylococcus aureus

1. Introduction Staphylococcus aureus, one of the main etiological agents of acquired infections in the community and the environment [1], has a remarkable ability to adapt and an enormous ability to rapidly develop resistance to countless antibiotics [2]. Its ability to produce numerous virulence factors contributes to its pathogenicity and ability to cause a variety of infectious diseases [3]. Biofilms are heterogeneous bacterial communities, irreversibly bound to a complex matrix consisting of DNA, proteins, and polysaccharides [3], which have an altered phenotype regarding growth rate and gene transcription [4]. Microbial growth in biofilm plays an important role during infection by providing different defense mechanisms to the microorganism. The biofilm matrix can prevent the access of immune cells, such as macrophages [5], as well as promote increased tolerance of microorganisms to antimicrobial agents [6]. The ability of S. aureus to form biofilms on medical devices, such as catheters and prostheses, increases its virulence and contributes to treatment failure [4,7]. Compared to its planktonic state, S. aureus in biofilms shows a significant difference in gene expression and in its physiology [8].

Molecules 2017, 22, 1534; doi:10.3390/molecules22101534 www.mdpi.com/journal/molecules Molecules 2017, 22, 1534 2 of 10 Molecules 2017, 22, 1534 2 of 10

Thus,Thus, due due to to the difficulty difﬁculty for antimicrobial agents to promote inhibition and eradication of S. aureus biofilm,bioﬁlm, the search for new therapies is extremely important for the successful treatment of of infectiousinfectious diseases. diseases. ViolaceinViolacein (VIO) is a purple bisindole metabolite, natural violet pigment produced by several Gram-negativeGram-negative bacteria, bacteria, including including the thespecies species ChromobacteriumChromobacterium violaceum violaceum. VIO is. a VIObioactive is a secondary bioactive metabolite,secondary metabolite,formed by the formed condensation by the condensation of two tryptoph of twoan molecules tryptophan through molecules the action through of proteins the action [9,10] of (Figureproteins 1). [9 ,10] (Figure1).

O H 20 N 11 19 10 14 12 18 21 HO 5 17 4 13 6 3 22 15 23 7 1 2 O 16 N 9 N 8 H H

FigureFigure 1. 1. StructureStructure of of violacein violacein from from the NMR.

VIOVIO has has attracted attracted much much attention attention in in the the scientific scientific community community due due to to its its several several pharmacological pharmacological properties, such such as antitumor, antibacterial, antivi antiviral,ral, antiparasitic, antioxidant antioxidant action, action, as as well well as fungicidefungicide and and leishmanicidal leishmanicidal properties [10–13]. [10–13]. AnAn excellent excellent antibacterial antibacterial activity activity of of VIO VIO has has been been demonstrated demonstrated in invitro vitro assaysassays [13], [13 as], well as well as itsas itssynergistic synergistic potential potential in inassociation association with with gentamicin, gentamicin, cefadroxil, cefadroxil, azithromycin, azithromycin, and and kanamycin, kanamycin, suggestingsuggesting the possibility possibility of using using it it as as an an altern alternativeative therapy therapy [14], [14], when when ad administeredministered in in combination combination with otherother antibiotics,antibiotics, VIO VIO is is more more effective effective than than the usethe ofuse antibiotics of antibiotics alone [alone10]. Nanoparticles [10]. Nanoparticles loaded loadedwith VIO with were VIO two were to fivetwo timesto five more times effective more effective than VIO than alone VIO against alone strainsagainst of strains methicillin-resistant of methicillin- resistantS. aureus S.(MRSA) aureus [(MRSA)15]. [15]. GivenGiven the the concern about about the the emergence emergence of of antibiotic antibiotic-resistant-resistant strains, strains, there there is is a a great great need need for for searchingsearching and and investigating investigating new new molecules, molecules, as as well well as asunderstanding understanding their their mechanism mechanism of action. of action. In thisIn this context, context, VIO VIO with with its its excellent excellent antimicrobial antimicrobial activity activity especially especially on on S.S. aureus aureus, ,has has emerged emerged as as a a promising molecule.molecule. However,However, it it is is necessary necessary to to elucidate elucidate its its antimicrobial antimicrobial mechanism mechanism of action of action so that so thatVIO VIO can becomecan become a new a drugnew drug for the for treatment the treatment of microbial of microbial infections infections [13,15 [13,15].]. The aim The of aim this of study this studywas to was determine to determine the antistaphylococcal the antistaphylococcal activity activi of VIOty of in VIO planktonic in planktonic cultures cultures and biofilms. and biofilms.

2.2. Results Results

2.1.2.1. Structure Structure Characterization Characterization 1 TheThe 1H-NMRH-NMR spectrum spectrum showed showed 13 13 proton proton signals, signals, four four singlets singlets at at δδ 11.88,11.88, 10.72, 10.72, 10.60, 10.60, and and 9.33; 9.33; sixsix doublets doublets at at δ 8.93 ( J = 8.0 8.0 Hz, H-19), 8.06 ( JJ = 3.1 3.1 Hz, Hz, 2 2 H), H), 7.55 ( JJ = 2.0 2.0 Hz, Hz, H-13), H-13), 7.35 7.35 (J (J == 8.7 8.7 Hz, Hz, H-8),H-8), 7.23 ( JJ = 2.0 2.0 Hz, Hz, H-5), H-5), and and 6.83 6.83 ( (JJ == 8.0 8.0 Hz, Hz, H-22); H-22); two two triplets triplets of of doublet doublet at at δδ 7.207.20 ( (JJ == 8.0; 8.0; 1.0 1.0 Hz, Hz, 21-H)21-H) and 6.95 ( JJ == 8.0, 8.0, 1.0 1.0 Hz, Hz, H-20); H-20); and, and, finally, ﬁnally, a double doublet at δ 6.79 (J = 8.7; 2.0 Hz, H-7). TheThe singlets at at δ 11.88 (NH-1), 10.72 (NH-10) and 10.60 (NH-15) were attributed to hydrogen atomsatoms bonded bonded to nitrogen nitrogen atoms, while the singlet singlet at at δδ 9.339.33 was was associated associated to to the the proton proton of of a a hydroxyl, hydroxyl, 13 suggestedsuggested as an oxygenate oxygenatedd carbon substituent at δ 152.9. The 13C-NMRC-NMR spectrum spectrum showed showed 20 20 carbon carbon signals,signals, in in agreement agreement with the deduced molecular formula, all related to sp2 carbons (Table(Table1 1).).

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Table 1. 1H- and 13C-NMR spectrals data of VIO.

HSQC HMBC 2 3 δC δH (Mult, J/Hz) JCH JCH 2 129.7 8.06 (d 3.1) 3 105.8 — H-2 NH-1, H-13, H-5 4 125.6 — NH-1, H-2, H-8 5 104.6 7.23 d (2.0) OH, H-7 6 152.9 — OH, H-5, H-7 H-8 7 113.1 6.79 dd (8.7, 2.0) OH, H-5 8 113.4 7.35 d (8.7) 9 131.6 — NH-1 H-2, H-5, H-7 11 171.7 — NH-10 H-13 12 137.0 — H-13 NH-10 13 97.0 7.55 d (2.0) NH-10 14 147.7 — NH-10, H-13 16 170.3 — NH-15 17 118.7 — NH-15, H-19 18 122.4 — NH-15, H-20, H-22 19 126.4 8.93 d (8.0) H-20 H-21 20 120.9 6.95 td (8.0, 1.0) H-22 21 129.4 7.20 td (8.0, 1.0) H-20 H-19 22 109.1 6.83 d (8.0) H-20 23 141.8 — NH-15 H-19, H-21 NH-1 11.88 (s) NH-10 10.72 (s) NH-15 10.60 (s) OH 9.33 (s)

DMSO-d6, 500 e 75 MHz.

All these data, together with the comparison with the NMR data in the literature [16], allowed the identiﬁcation of the compound as (3E)-[3-(1,2-dihydro-5-(5-hydroxy-1H-indol- 3-yl)-2-oxo-3H-pyrrol-3-ylidene)-1,3-dihydro]-2H-indol-2-one, known as VIO. The Supplementary Materials are available online. NMR are available as Supporting Information.

2.2. Antimicrobial Activity of VIO The values of MIC and MBC ranged from 1.25 to 20 µg/mL and 5 to 40 µg/mL, respectively. Interestingly, VIO showed excellent activity inhibiting bioﬁlm growth, with MBIC equal to MIC, indicating that its action occurs during bioﬁlm formation, when the cells show intense metabolism (Table2).

Table 2. VIO antimicrobial activity on planktonic cultures and bioﬁlms of S. aureus.

VIO µg/mL Microbial Strain MIC MBC MBIC S. aureus ATCC 6538P (OSSA) 1.25 5 1.25 S. aureus ATCC 14458 (OSSA) 2.5 10 2.5 S. aureus ATCC 3359 (ORSA) 20 40 20 S. aureus CCBH 5330 (ORSA) 5 20 5 MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; MBIC: minimum bioﬁlm inhibitory concentration; ATCC: American type culture collection; CCBH: culture collection of hospital-acquired bacteria. OSSA: oxacillin-susceptible strain, ORSA: oxacillin-resistant strain. Assays were performed in triplicate.

The results shown in Figure1 exhibiting the viability of planktonic cultures of S. aureus exposed to MIC, 2× MIC, and 4× MIC for 2, 4, 6, 8, 10, 12, and 24 h at 37 ◦C. VIO showed antimicrobial activity, Molecules 20172017,, 2222,, 15341534 44 ofof 1010

MoleculesThe2017 results, 22, 1534 shown in Figure 1 exhibiting the viability of planktonic cultures of S. aureus exposedexposed4 of 10 toto MIC,MIC, 2×2× MIC,MIC, andand 4×4× MICMIC forfor 2,2, 4,4, 6,6, 8,8, 10,10, 12,12, andand 2424 hh atat 3737 °C.°C. VIOVIO showedshowed antimicrobialantimicrobial activity,activity, depending on concentration, against cells of S. aureus ATCC 6538P, completely preventing bacterial depending on concentration, against against cells cells of of S.S. aureus aureus ATCC 6538P, completely preventing bacterial metabolism after 3–4 h of exposure to 5 μg/mL (Figure 2). metabolism after 3–4 h of exposure to 5 µμg/mL (Figure (Figure 22).).

p<0.05 14

12 * 10 * * Control 10 * * * MIC * * MIC 8 CFU/mL * * 2xMIC CFU/mL * * 2xMIC 10 10 * 4xMIC log * log 6 * * * * * * 4

2 * * * ** * 0 4 8 12 16 20 24 Time (h) Figure 2. Effects of VIO on the viability of S. aureus ATCC 6538P. VIO (MIC = 1.25 μg/mL); Control: Figure 2. Effects of VIO on the viability of S. aureus ATCC 6538P.6538P. VIOVIO (MIC(MIC == 1.251.25 µμg/mL);g/mL); Control: without VIO. Detection limit was 1022 colony forming units per milliliter (CFU/mL) and the assays without VIO.VIO. DetectionDetection limit limit was was 10 210colony colony forming forming units units per per milliliter milliliter (CFU/mL) (CFU/mL) and theand assays the assays were were performed in triplicate. Data are expressed as mean ±SEM of three experiments. * p < 0.05 wereperformed performed in triplicate. in triplicate. Data are Data expressed are expr asessed mean as± SEMmean of ±SEM three experiments.of three experiments. * p < 0.05 compared* p < 0.05 compared with the control. withcompared the control. with the control.

InIn thethe beginningbeginning ofof thethe exponentialexponential growthgrowth phasephase (4(4 h),h), whenwhen cellscells areare youngyoung andand havehave anan intenseintense In the beginning of the exponential growth phase (4 h), when cells are young and have an metabolic activity, an initial reduction of 3 log1010 CFU/mLCFU/mL waswas observedobserved inin thethe microbialmicrobial populationpopulation intense metabolic activity, an initial reduction of 3 log CFU/mL was observed in the microbial (Figure(Figure 3).3). AtAt thethe endend ofof thethe exponentialexponential growthgrowth phase10 (8 h) and stationary growth phase, VIO population (Figure3). At the end of the exponential growth phase (8 h) and stationary growth phase, inhibitoryinhibitory actionaction cancan alsoalso bebe observed,observed, butbut toto aa lowerlower intensityintensity (1–2(1–2 loglog1010 CFU/mL).CFU/mL). VIO inhibitory action can also be observed, but to a lower intensity (1–2 log10 CFU/mL).

p<0.05 18 16 Control 14 14 * * T=0 12 12 * T=4h * 10 * T=8h CFU/mL *

CFU/mL * 10 10 T=24h 8 T=24h log log * * 6 * 4

0 4 8 0 4 8 1212 2424 2828 3232 4848 Time (h)

Figure 3. VIO effect on S. aureus ATCC 6538P viability at the phases of exponential and stationary Figure 3. VIOVIO effecteffect onon S. aureus ATCCATCC 6538P6538P viabilityviability atat thethe phasesphases ofof exponentialexponential andand stationarystationary growth. VIO (MIC = 1.25 µg/mL) was added at the start of incubation (t = 0), and after 4, 8, and 24 h. growth. VIO (MIC = 1.25 μg/mL) was added at the start of incubation (tt == 0),0), andand afterafter 4,4, 8,8, andand 2424 h.h. Microbial growth was assessed after 4 and 24 h of exposure. Control: without VIO. Detection limit was Microbial growth was assessed after 4 and 24 h of exposure. Control: without VIO. Detection limit 102 colony-forming units per milliliter (CFU/mL) and assays were performed in triplicate. * p < 0.05 was 1022 colony-formingcolony-forming unitsunits perper millilitermilliliter (CFU/mL)(CFU/mL) andand assaysassays werewere performedperformed inin triplicate.triplicate. ** p << 0.050.05 compared with the control. comparedcompared withwith thethe control.control.

Molecules 2017, 22, 1534 5 of 10 MoleculesMolecules 20172017, ,2222, ,1534 1534 55 of of 10 10 2.3. Antibiofilm Activity 2.3. Antibiofilm Activity 2.3. AntibiofilmVIO effect Activityon mature biofilm viability was the concentration of 40 μg/mL. Our results show total VIO effect on mature biofilm viability was the concentration of 40 μg/mL. Our results show total inhibition of bacterial metabolism and loss of S. aureus cell viability in mature biofilm after 150 min inhibitionVIO effectof bacterial on mature metabolism biofilm viabilityand loss wasof S. the aureus concentration cell viability of 40in µmatureg/mL. Ourbiofilm results after show 150 min total of exposure to MBEC (Figure 4). ofinhibition exposure of to bacterial MBEC (Figure metabolism 4). and loss of S. aureus cell viability in mature biofilm after 150 min of exposure to MBEC (Figure4). p<0.05 p<0.0510 10 8 *** 8 *** 6 6 CFU/ml 10 CFU/ml 4 MBEC 10 MBEC log 4 log 2 2 *** *** ** * *** *** 0 *** ** * *** 0 0 0 0 ol 6 8 40 2 10 0 0 10 XA ntrol 6 8 40 2 o 1 10 OXA Cntr o O C Concentration(μg/ml) μ Concentration( g/ml)

Figure 4. Effect of different concentrations of VIO on the viability of biofilm formed by S. aureus ATCC FigureFigure 4. 4. EffectEffect of of different different concentrations concentrations of of VIO VIO on on the the viability viability of of biofilm biofilm formed formed by by S.S. aureus aureus ATCCATCC 6538P following a 24 h exposure. MBEC: minimum biofilm eradication concentration; Control: 6538P6538P following aa 2424 h exposure.h exposure. MBEC: MBEC: minimum minimum biofilm biofilm eradication eradication concentration; concentration; Control: Control: without without VIO; OXA: oxacillinµ 20 μg/mL. Detection limit2 was 102 colony forming units per milliliter withoutVIO; OXA: VIO; oxacillin OXA: oxacillin 20 g/mL. 20 Detectionμg/mL. Detection limit was limit 10 colonywas 10 forming2 colony unitsforming per milliliterunits per (CFU/mL) milliliter (CFU/mL) and the assays were performed in triplicate. Data are expressed± as mean ± SEM of three (CFU/mL)and the assays and the were assays performed were performed in triplicate. in Data tripli arecate. expressed Data are as expressed mean SEM as mean of three ± SEM experiments. of three ***experiments.p < 0.05 compared *** p < 0.05 with compared the control. with the control. experiments. *** p < 0.05 compared with the control. A better effect was obtained when VIO concentration was 2× MBEC, with a significantly greater AA better better effect effect was was obtained obtained when when VIO VIO concentrat concentrationion was was 2× 2× MBEC,MBEC, with with a asignificantly significantly greater greater reduction of cell viability (3 log10 CFU/mL) being observed when compared to the MBEC activity reductionreduction of of cell cell viability viability (3 (3 log log1010 CFU/mL)CFU/mL) being being observed observed when when compared compared to to the the MBEC MBEC activity activity (<1 log10 CFU/mL) after 30 min of exposure and total cell inactivity between 90 and 120 min. The VIO (<1(<1 log log10 CFU/mL)CFU/mL) after after 30 30 min min of of exposure exposure and and total total cell cell inactivity inactivity between between 90 90 and and 120 120 min. min. The The VIO VIO subinhibitory10 concentration (0.5× MBEC) inhibited cell growth in biofilm when compared to the subinhibitorysubinhibitory concentration concentration (0.5× (0.5× MBEC)MBEC) inhibited inhibited cell cell growth growth in in biofilm biofilm when when compared compared to to the the control (p < 0.05), but was not able to reduce the number of viable cells (Figure 5). controlcontrol ( (pp << 0.05), 0.05), but but was was not not able able to to reduce reduce the the number number of of viable viable cells cells (Figure (Figure 5).5).

p<0.05 p<0.05 10 10

8 Control 8 Control * * 0.5 x MBEC * * 0.5 x MBEC 6 * * MBEC CFU/mL 6 * * MBEC 10 CFU/mL * * 2 x MBEC

10 *

log * 2 x MBEC

log * 4 * * 4 * * 2 * * * 2 0 30 60 90 120* *150 0 30 60 90 120 150 Time (min) Time (min) Staphylococcus aureus Figure 5.5. Time–kill curves for Staphylococcus aureus ATCC 6538P biofilm bioﬁlm after treatment with VIO. FigureMBEC: 5. minimum Time–kill bioﬁlmcurves for eradication Staphylococcus concentration aureus ATCC (40 µ g/mL);6538P biofilm Control: after without treatment VIO. with Detection VIO. MBEC: minimum biofilm eradication concentration (40 μg/mL); Control: without VIO. Detection limit MBEC:limit was minimum 102 colony-forming biofilm eradication units per concentration milliliter (CFU/mL) (40 μg/mL); and Control: assays without were performed VIO. Detection in triplicate. limit was 102 colony-forming units per milliliter (CFU/mL) and assays were performed in triplicate. Data wasData 10 are2 colony-forming expressed as mean units± perSEM milliliter of three (CFU/mL) experiments. and *assaysp < 0.05 were compared performed with in the triplicate. control. Data are expressed as mean ±SEM of three experiments. * p < 0.05 compared with the control. are expressed as mean ±SEM of three experiments. * p < 0.05 compared with the control.

Molecules 2017, 22, 1534 6 of 10

3. Discussion Many biological activities have been identified for VIO and among them we can highlight the anti-inflammatory, anti-diarrheal, anti-ulcer, anti-tumor, antimalarial, antimicrobial, and antimycobacterial activities [10,11,13,17–19]. Our results show VIO antimicrobial activity on S. aureus strains, both resistant and susceptible to oxacillin, in suspension and in biofilm. All tested strains were susceptible to VIO and the OSSA strains showed greater susceptibility than ORSA strains. S. aureus strains are often responsible for hospital infections, increasing the costs to the health system and increasing mortality and morbidity rates. Furthermore, the resistance of S. aureus to antimicrobial agents used in the treatment of infectious diseases hinders or even prevents treatment success, which is enhanced by its biofilm growth [20]. In order to consider VIO a molecule with potential for the development of a new chemotherapy drug for the treatment of staphylococcal infections, it is important to establish the kinetics of its antimicrobial action on cells in planktonic cultures and in biofilm. For that purpose, we performed the time–kill assay, which determines the time of action of a compound antimicrobial activity. The used strain was S. aureus ATCC 6538P, the most sensitive among the tested strains and the only one that had its growth in mature biofilm eradicated by the action of VIO at the tested concentration range (MBEC = 40 µg/mL). The S. aureus ATCC 6538P strain was also used in other assays of this study. The VIO MIC (1.25 µg/mL) was able to inhibit the growth of S. aureus for up to 8 h, a period of intense metabolic activity. After this period, exponential growth was observed until 24 h of incubation, when growth was once again inhibited. The recovery of microbial growth after 8 h may be related to possible decomposition of VIO, as well as the reduction in cell activity levels, suggesting that VIO mechanism of action involves macromolecule synthesis. The lower sensitivity of S. aureus in mature biofilm, verified in our study, confirms the importance of the active microbial metabolism for VIO action, as the bacteria inside the biofilm have reduced metabolic activity [21]. The influence of cell metabolism on VIO activity is also confirmed by the VIO ability to inhibit biofilm formation. Considering the occurrence of possible VIO decomposition after 8 h of incubation, as well as the possibility that VIO inhibitory action be favored by the high cell metabolism rate, one can infer that cell growth inhibition observed after 24 h, when cell division is minimal, may be associated with the high nutritional depletion and metabolite concentration. VIO showed inhibitory activity in all stages of S. aureus growth. In these assays, the substance is added at different stages of growth and its activity is assessed after 4 and 24 h. The fact that VIO was added to each of the different stages of bacterial growth might have prevented its decomposition and favored its action, even at the low bacterial metabolism stages. Thus, an initial decrease in the microbial population (up to 4 h) was demonstrated for all phases. This was not observed when VIO was added only at the beginning of incubation, in the time–kill assay, which reinforces the idea that VIO may have degraded after 4–8 h, in microbial culture, under the experimental conditions. The ability of S. aureus to form biofilms is a major virulence factor and contributes to the success of the infectious process [20]. The greater resistance of S. aureus in the mature biofilm matrix, when compared to that of its cells in planktonic culture, can be attributed to the ability of the bacterial biofilm to limit the access and dissemination of antimicrobial agents in its interior. In the biofilm, the cells are protected from antimicrobial agents, environmental conditions, and from the host’s immune response, and can display an increase of up to 1000 times the antimicrobial resistance rate [22]. In this study, we demonstrate that VIO has an excellent antimicrobial activity against S. aureus in intensive metabolism (exponential growth phase), possibly showing that its mechanism of action involves the synthesis of macromolecules. However, we also verified VIO antimicrobial action against S. aureus at all growth stages, although at a lower intensity, suggesting the existence of more than one antimicrobial mechanism of action for VIO. Molecules 2017, 22, 1534 7 of 10

4. Materials and Methods

4.1. Violacein VIO extracted from the strain Chromobacterium violaceum ATCC 12472 was puriﬁed and characterized by nuclear magnetic resonance (NMR) [23].

4.2. Bacterial Strains This study used four reference strains: S. aureus ATCC 6538P and S. aureus ATCC 14458 (oxacillin-susceptible strains-OSSA), S. aureus ATCC 33591 and S. aureus CCBH 5330 (oxacillin-resistant strains-ORSA).

4.3. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) The MIC was determined using the microdilution method in culture broth [24]. Aliquots of the microbial suspensions (106 CFU/mL) in Brain–Heart Infusion Broth (BHI) (Merck Millipore Corporation) were added to the wells containing 95 µL of BHI and 100 µL of VIO (0.019–40 µg/mL), in 0.1% Dimethyl Sulfoxide (DMSO). The microplates were incubated for 24 h/37 ◦C. The MIC was considered as the lowest VIO concentration capable of completely inhibiting microbial growth. As experiment controls, sterile BHI broth, amikacin, and 0.1% DMSO were used. The MBC was determined by counting viable cells [25]. Brieﬂy, 5 µL of inoculum were collected and plated. The MBC was regarded as the lowest VIO concentration capable of determining the death of 99.9% of S. aureus cells of the initial inoculum. The assays were performed in triplicate.

4.4. Time–Kill Assay Aliquots of 20 µL of VIO (MIC, 2× MIC, 4× MIC) were added to the microplate wells containing 100 µL of BHI broth and 80 µL of bacterial suspension (106 CFU/mL). The microplates were incubated at 37 ◦C and 10 µL aliquots were removed, diluted in 0.85% sterile saline solution and plated for colony counting, at predetermined time intervals (0, 2, 4, 6, 8, 10, 12, and 24 h) [26]. Microbial cultures with no exposure to VIO were used as experimental control. The assays were performed in triplicate and the results were expressed in log10 CFU/mL.

4.5. Determination of VIO Effect on S. aureus at Different Stages of Microbial Metabolism A microbial suspension was exposed to VIO MIC. VIO was added at the start of the incubation period (t = 0) and after 4, 8, and 24 h and microbial growth was determined after 4 and 24 h of exposure. Cultures of S. aureus, without VIO addition and submitted to the same experimental conditions were used as control [27]. The experiment was performed in triplicate and the results expressed as log10 CFU/mL.

4.6. Determination of Minimum Biofilm Inhibitory Concentration (MBIC) The effect of VIO on biofilm formation was assessed by determining the MBIC [28]. The biofilm was obtained and its mass quantified using the crystal violet technique [29]. The strains were cultivated in Tryptic Soy Broth (TSB) with 1% (p/v) of glucose for 24 h/37 ◦C. Aliquots of 100 µL of the culture and 100 µL of VIO (0.019 to 80 µg/mL) were added to microplates and incubated for 24 h/37 ◦C. The wells were washed and 200 µL of 99% methanol was added for 15 min. After, aliquots of 200 µL of crystal violet solution (CV) 2% (v/v) were added for 15 min. The CV uptake by the bacterial cells was released by adding 160 µL of 33% acetic acid and optical density reading was performed at 570 (OD570) on a microplate reader (Model Synergy HT, Biotek, Winooski, VT, USA). MBIC was regarded as the lowest concentration of VIO capable of inhibiting the formation of bacterial biofilm and the experiments were performed in triplicate. Molecules 2017, 22, 1534 8 of 10

4.7. Determination of Minimum Biofilm Eradication Concentration (MBEC) MBEC was determined to evaluate VIO effect on mature biofilm viability [30]. The S. aureus ATCC 6538P strains were grown in TSB supplemented with 1% (p/v) of glucose (24 h/37 ◦C). Aliquots of 100 µL (106 CFU/mL) were transferred into microplates and incubated for 72 h/37 ◦C. 50 µL of TSB and 50 µL of VIO (10–160 µg/mL) were added to the wells containing biofilm. The microplates were incubated for 24 h/37 ◦C and the viability of the remaining biofilms was measured by counting the colonies. Biofilms not treated with VIO were used as controls. MBEC was considered the lowest VIO concentration able to prevent microbial growth. The experiments were performed in triplicate and the results were expressed in log10 CFU/mL.

4.8. Biofilm Time–Kill Assay VIO aliquots of 20 µL (0.5, 1, and 2× MBEC) were added to microplates containing 100 µL of TSB and mature biofilm of S. aureus ATCC 6538P, formed in 72 h as previously described. The microplates were incubated at 37 ◦C, and after 24 h the remaining biofilm viability was measured by counting the colonies at the time of VIO addition and at predetermined time intervals (30, 60, 90, 120, and 150 min) after VIO addition. Aliquots of the biofilm serial dilutions were plated and the counting of colonies was performed after 24 h/37 ◦C. Biofilms not treated with VIO were used as controls. The experiments were performed in triplicate and the results were expressed in log10 CFU/mL [30].

4.9. Statistical Analysis Statistical analyses were performed using one-way ANOVA with post hoc Bonferroni test. The data were performed in triplicate. The charts are shown as mean ± standard deviation. Data were considered signiﬁcant when p < 0.05.

5. Conclusions VIO is a promising molecule for the development of an antimicrobial drug for the eradication of S. aureus bioﬁlm infections. However, while its action is enhanced by the active cell metabolism, it is also present in cells with reduced metabolism.

Supplementary Materials: The following are available online. 1H- and 13C-NMR of VIO. Acknowledgments: We thank Coordination for Enhancement of Higher Education Personnel (CAPES) for their financial support. Author Contributions: A.H.M.B. assisted of growing C. violaceum, in the production of violacein and in the implementation of microbiological experiments and wrote the article; A.C.D.M. and G.W.P.S. assisted in the implementation of microbiological experiments; R.M.d.C. assisted in growing C. violaceum and in the production violacein; T.B.G. and S.C.M. provided the strain Chromobacterium violaceum ATCC 12472, assisted of growing C. violaceum and in the production violacein; E.R.S. and P.C.N.N. assisted in the purification and characterization of VIO; N.A.P.N. assisted in the implementation of microbiological experiments and contributed to the coordination of the study and wrote the article. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are not available from the authors.

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