Anti-Bacterial Activity of Phenolic Compounds Against Streptococcus Pyogenes

medicines

Short Note Anti-Bacterial Activity of Phenolic Compounds against Streptococcus pyogenes

Sabrina Macé 1, Lisbeth Truelstrup Hansen 2,† and H.P. Vasantha Rupasinghe 1,3,*

1 Department of Plant, Food, and Environmental Sciences, Faculty of Agriculture, Dalhousie University, Truro, NS B2N 5E3, Canada; [email protected] 2 Department of Process Engineering and Applied Science, Faculty of Engineering, Dalhousie University, Halifax, NS B3H 4R2, Canada; [email protected] 3 Department of Pathology, Faculty of Medicine, Dalhousie University, Halifax, NS B3H 4H7, Canada * Correspondence: [email protected]; Tel.: +1-902-893-6623 † Current address: National Food Institute, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.

Academic Editor: Eleni Skaltsa Received: 1 April 2017; Accepted: 26 April 2017; Published: 1 May 2017

Abstract: Background: Worldwide, Streptococcus pyogenes is the leading cause of bacterial pharyngitis. To reduce the use of antibiotics, antimicrobial phytochemical-containing remedies, which have long been in use in traditional medicine, may provide new approaches for management of streptococcal pharyngitis. The objective of this study was to assess the inhibitory activities of 25 natural phenolic compounds against three strains of S. pyogenes. Methods: After an initial screening, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the nine most effective phenolic compounds were determined. The effect of four compounds with the lowest MIC and MBC on streptococcal growth and biofilm formation was also studied. Results: 1,2-Naphthoquinone and 5-hydroxy-1,4-naphthoquinone elicited the greatest anti-S. pyogenes activities with MICs ranging from 0.39 to 6.25 µg mL−1 and MBCs of 100 µg mL−1. Both naphthoquinones inhibited the biofilm formation at concentrations ranging from 12.5 to 50 µg mL−1. Biofilm reduction and altered bacterial cell structures were visible in scanning electron microscopy images of naphthoquinone-treated cells. Conclusion: In conclusion, 1,2-naphthoquinone and 5-hydroxy-1,4-naphthoquinone inhibit S. pyogenes and should be further investigated as candidates for the management of streptococcal pharyngitis.

Keywords: pharyngitis; strep throat; bioﬁlm; naphthoquinone; infection; disease; polyphenols

1. Introduction Pharyngitis or bacterial infection of the throat is a common disease, which is often caused by Streptococcus pyogenes [1,2]. Worldwide, S. pyogenes are responsible for an estimated 600 million cases of throat infections per year [3]. β-Lactams, such as penicillin, are the preferred antibiotics in the treatment of S. pyogenes throat infections, with macrolides being used for patients with β-lactam hypersensitivity. However, while resistance to β-lactams has so far not emerged in S. pyogenes, resistances were found for macrolides and some quinolones (fluoroquinolone) [3,4]. Moreover, S. pyogenes forms biofilm, which has been associated with antibiotic treatment failures [5]. Thus, there is an urgent need to find new potent antibacterial and anti-biofilm agents which could find use in alternative and/or complementary therapy for streptococcal pharyngitis. In addition to treatment with prescribed medications, throat lozenges containing chemical anesthetics and antiseptics are being used by patients in the relief of the discomfort associated with the infection. The antimicrobial quaternary ammonium compound, dequalinium chloride, is similarly used in the treatment of common infections of the mouth and throat and incorporated in candy-based

Medicines 2017, 4, 25; doi:10.3390/medicines4020025 www.mdpi.com/journal/medicines Medicines 2017, 4, 25 2 of 9 lozenge formulations [6]. The antiseptic and local anesthetic hexylresorcinol has also been included as an active ingredient in throat lozenges [7]. Natural products derived from plants have been used in traditional medicine since the ancient times and are, now, being widely studied for incorporation into mainstream products. Plant-derived compounds of interest are mostly secondary metabolites, which possess antimicrobial properties against microbial pathogens and spoilers [8]. Recently, a diverse range of phytochemical antibacterial agents has been reported to suppress the growth of S. pyogenes, including polyphenols (i.e., ﬂavonoids) and terpenes [9–12]. A recent review describes the mechanisms of inhibition of different Streptococcus species, including S. pyogenes, by phytochemicals through the prevention of the bacterial adherence of the pharynx, inhibition of glycolytic enzyme and pH drop, reduction of bioﬁlm, and alteration of cell surface hydrophobicity [2]. The objective of the present study was to identify, among 25 different commercially available plant-derived phenolic compounds, the ones with anti-S. pyogenes activities, which could be incorporated in dehydrated honey throat lozenges and similar products.

2. Material and Methods

2.1. Bacterial Strains and Growth Conditions The experiments used the following human S. pyogenes strains: S. pyogenes ATCC ® 19615 ™, S. pyogenes ATCC ® 49399 ™ and a clinical isolate obtained from a patient with pharyngitis (Bacteriology, Queen Elizabeth II Hospital, Halifax, NS, Canada). The strains were routinely cultured in Brain Heart Infusion (BHI) (Oxoid, Thermo Fisher Scientiﬁc, Waltham, MA, USA) media at 37 ◦C under aerobic conditions.

2.2. Compounds Twenty-five phenolic compounds belonging to 12 different classes: three simple phenols (eugenol, thymol, and pyrocatechol), three isoflavones (daidzin, daidzein, and genistein), one chalcone (phloretin), one chalcone glycoside (phlorizin), two flavan-3-ols (epicatechin and epigallocatechin gallate, EGCG), two flavanones (naringenin and hesperidin), two flavones (flavone and naringin), two flavonols (myricetin and quercetin hydrate), one flavonol glucoside (quercetin-3-O-glucoside), one hydroxybenzoates (gallic acid), three hydroxycinnamates (tran-ferulic acid, cinnamaldehyde, and warfarin), two naphthoquinones (1,2-naphthoquinone and 5-hydro-1,4-naphthoquinone), one stilbene (resveratrol), one tannin (tannic acid), and two chemical compounds (used as positive controls), one quaternary ammonium compound (dequalinium chloride), and one substituted phenol (4-hexylresorcinol) were evaluated for their antibacterial properties. The compounds were purchased from Sigma-Aldrich Company (Saint-Louis, MO, USA). Before each experiment, the stock solutions were freshly made to a concentration of 10 mg mL−1 in 100% DMSO.

2.3. Preliminary Screening Using the Micro-Dilution Broth Antibacterial Assay The inhibitory effect of the phenolic compounds (100 µg mL−1) was initially assessed on S. pyogenes ATCC 19615 and ATCC 49399, using the broth micro-dilution assay as described by Clinical and Laboratory Standards Institute [13]. Brieﬂy, in a 96-well micro-plate, 2 µL of the 100 mg mL−1 stock solution was added to 98 µL of BHI broth in order to reach a ﬁnal test compound concentration of 100 µg mL−1. Finally, for each strain, 100 µL of standardized inoculum was added, resulting in −1 an initial bacterial concentration of 5 log10 (CFU mL ). Bacterial inocula in BHI broth (2 µL water) and BHI broth +1% DMSO were also run as a control. The growth of the bacteria was assessed after ◦ TM incubation for 24 h at 37 C by reading the A600 nm using a plate reader (Epoch , Biotek, Winooski, VT, USA). Each test was done in triplicate. In order to determine the most active compounds, a decrease in the A600 nm of 0.1 or more in the presence of the tested compounds in comparison to the control (bacteria inocula in BHI +1% DMSO) was considered to be indicative of an inhibition of the growth. Medicines 2017, 4, 25 3 of 9

2.4. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) MIC broth micro-dilution assay was performed as described in the previous section, except 2-fold dilutions were used to yield final test compound concentrations ranging from 0.19 to 100 µg mL−1, on the three S. pyogenes strains. The MIC was determined as the first concentration where the decrease of A600 nm was significant (p < 0.05) in comparison with the control. To determine MBC, 30 µL from each well, where no growth was detected—i.e., compound concentration ≥MIC—was spread on BHI agar plates and incubated for 24–48 h at 37 ◦C. The lowest compound concentration resulting in no growth on the agar plate represented the MBC. Each sample was tested in triplicate and each experiment was repeated three times.

2.5. Time-Kill Assay The effect of concentrations ranging from 1 to 16 times MIC of the four most active compounds on the growth of S. pyogenes ATCC 19615 was quantiﬁed after 0, 2, 4, 6, 8, 10, and 24 h at 37 ◦C. At each time point, an aliquot (100 µL) was pipetted, serially diluted, and plated on BHI agar followed by −1 enumeration and calculation of the mean log10 CFU mL of the duplicate samples. Each time-kill experiment was repeated in three biologically independent assays (n = 6).

2.6. Inhibition of Biofilm Formation The ability of S. pyogenes to form biofilm in the presence of 1,2-naphthoquinone, 5-hydroxy-1- naphthoquinone, dequalinium chloride, or 4-hexylresorcinol (0.19–100 µg mL−1) was assessed by measuring the metabolic activity of sessile biofilm cells using a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) dye assay. After an exposure period of three days at 37 ◦C, the planktonic bacterial cells were eliminated by carefully removing the BHI medium from the wells. Adapted MTT assay was performed as described in Vybrant® MTT Cell Proliferation Assay Kit Protocol ◦ (Thermo Fisher Scientific, Waltham, MA, USA). After incubation for 10 min at 37 C, the A540 nm was measured and used to calculate the minimum biofilm inhibitory concentration (MBIC), defined as the first concentration where the decrease in A540 nm was significant (p < 0.05) in comparison with the control.

2.7. Scanning Electron Microscopy (SEM) S. pyogenes ATCC 19615 biofilms formed (72 h at 37 ◦C) in the presence of MBIC concentrations of 1,2 naphthoquinone, 5-hydroxy 1,4 naphthoquinone, dequalinium chloride, 4-hexylresorcinol were prepared in microplates. The S. pyogenes biofilms were also grown in BHI broth (water) and BHI broth +1% DMSO as controls. After incubation, a sample fixation method for SEM [14] adapted for microplate was performed. After discarding the last solution, the plate was air dried for 2 h in a fume-hood. The bottom of the wells (location of the biofilms) were then removed with a heated blade and mounted on aluminum mounts using the carbon adhesive and finally sputter-coated with gold and examined using a Hitachi S-4700 SEM (Tokyo, Japan).

2.8. Statistical Analysis During the determination of the MIC and MBIC, statistically signiﬁcant differences (Student t-test (paired, two tailed), p < 0.05) between test compound concentrations and controls were determined using the Student t-test on the A600 nm or A540 nm data, respectively.

3. Results The results of the screening test against two S. pyogenes strains, ATCC 19615 and ATCC 49399 showed that nine compounds exhibited antibacterial activity against at least one strain at the concentration of 100 µg mL−1. Six of them, phlorizin, naringenin, myrcetin 1,2-naphthoquinone, 5-hydroxy-1,4-naphthoquinone, and resveratrol inhibited both strains like the two positive controls. Medicines 2017, 4, 25 4 of 9

Medicines 2017, 4, 25 4 of 8 Flavone and thymol affected the growth of strain ATCC 19615 while cinnamaldehyde inhibited only ATCC 49399. Flavone and thymol affected the growth of strain ATCC 19615 while cinnamaldehyde inhibited only The MIC of those nine phytochemicals (Table1) varied from 0.39 µg mL−1 to >100 µg mL−1 and the ATCC 49399. MBC from 100 µg mL−1 to >100 µg mL−1 with some sensitivity/resistance variations among strains. The MIC of those nine phytochemicals (Table 1) varied from 0.39 µg mL−1 to >100 µg mL−1 and 1,2-Naphthoquinone and 5-hydroxy-1,4-naphthoquinone exhibited the best inhibitory effect with the MBC from 100 µg mL−1 to >100 µg mL−1 with some sensitivity/resistance variations among strains. similar MIC values relative to the positive controls and were selected for further analysis of their effect 1,2-Naphthoquinone and 5-hydroxy-1,4-naphthoquinone exhibited the best inhibitory effect with on bacterial growth and biofilm formation. similar MIC values relative to the positive controls and were selected for further analysis of their The four compounds that higher concentrations increased the lag phase and the bacterial effect on bacterial growth and biofilm formation. −1 log10-reduction (Figure1). The presence of low concentrations (<12.5 µg mL ) of 1,2-naphthoquinone The four compounds that higher concentrations increased the lag phase and the bacterial log10- delayed growth during the first 10 h but after 24 h the bacterial concentration reached around reduction (Figure 1). The presence of low concentrations (<12.5 µg mL−1) of 1,2-naphthoquinone −1 −1 8 log10 CFU mL (Figure1A). A concentration of 25 µg mL displayed a bacteriostatic effect delayed growth during the first 10 h but after 24 h the bacterial concentration reached around 8 log10 for the first 10 h, after which growth resumed to reach 5 log CFU mL−1 after 24 h. At 50 µg mL−1, CFU mL−1 (Figure 1A). A concentration of 25 µg mL−1 displayed10 a bacteriostatic effect for the first 10 −1 the growth was inhibited and a bacterial log10-reduction of more than 3.5 log10 CFU mL observed h, after which growth resumed to reach 5 log10 CFU mL−1 after 24 h. At 50 µg mL−1, the growth was after 24 h. inhibited and a bacterial log10-reduction of more than 3.5 log10 CFU mL−1 observed after 24 h.

FigureFigure 1.1.The The effect effect of of several several concentrations concentrations of theof fourthe four compounds compounds on the on growth the growth of S. pyogenes of S. pyogenesATCC 19615ATCC at 19615 37 ◦C: (atA )37 1,2-naphthoquinone °C: (A) 1,2-naphthoquinone (MIC: 3.125 µg(MIC: mL− 13.125); (B) 5-hydroxy-1,4-naphthoquinoneµg mL−1); (B) 5-hydroxy-1,4- (MIC:naphthoquinone 1.56 µg mL (MIC:−1); ( C1.56) dequalinium µg mL−1); (C chloride) dequalinium (MIC: 0.39 chlorideµg mL (MIC:−1); and0.39 (µgD) mL 4-hexylresorcinol−1); and (D) 4- (MIC:hexylresorcinol 1.56 µg mL (MIC:−1). Arrows1.56 µg mL indicate−1). Arrows that plate indicate count that values plate were coun belowt values the were detection below the threshold detection of −1 −1 1.22threshold log10 CFU of 1.22 mL log10. CFU mL .

−1 AdministrationAdministration of of 1.56 1.56µg µg mL mL−1 5-hydroxy-1,4-naphthoquinone 5-hydroxy-1,4-naphthoquinone caused caused populations populations to remain to remain under under 6 log10 CFU−1 mL−1 for 10 h; however, after 24 h more than 7 log10 CFU− 1mL−1 was reached (Figure 6 log10 CFU mL for 10 h; however, after 24 h more than 7 log10 CFU mL was reached (Figure1B). −1 A1B). bacteriostatic A bacteriostatic effect effect was was similarly similarly observed observed with with 3.125 3.125µ gµg mL mL−1 5-hydroxy-1,4-naphthoquinone where bacterial counts were under 6 log10 CFU mL−1 −after1 24 h. Using concentrations of 6.25 and 12.5 where bacterial counts were under 6 log10 CFU mL after 24 h. Using concentrations of 6.25 and µg mL−1, numbers−1 stayed around 4–5 log10 CFU mL−1 for 10 h− 1and then dropped about 2 log10 CFU 12.5 µg mL , numbers stayed around 4–5 log10 CFU mL for 10 h and then dropped about mL−1. With 25 µg−1 mL−1, the bacterial−1 concentrations underwent a 2 log10-reduction after 10 h and 2 log10 CFU mL . With 25 µg mL , the bacterial concentrations underwent a 2 log10-reduction after reached the limit of detection (>1.22 log10 CFU mL−1) after 24− 1h. 10 h and reached the limit of detection (>1.22 log10 CFU mL ) after 24 h. ForFor thethe dequaliniumdequalinium chloridechloride (Figure(Figure1 1C),C), the the same same trend trend of of inhibition inhibition was was observed observed where where the the −1 lowestlowest testtest concentrations of of 0.39 0.39 µgµg mL mL− permitted1 permitted growth growth and and a twice-higher a twice-higher concentration concentration of 0.78 of −1 −1 0.78µg mLµg mL displayed−1 displayed a bacteriostatic a bacteriostatic effect. effect. After After 2424 h, h,the the concentrations concentrations of of 1.56 and 3.1253.125µ µgg mL mL−1 induced more than 2.5 log10 CFU mL−1 and 6.25 µg mL−1 caused a >4 log10-reduction (below the detection limit).

Medicines 2017, 4, 25 5 of 9

−1 −1 induced more than 2.5 log10 CFU mL and 6.25 µg mL caused a >4 log10-reduction (below the detection limit). The concentrations of 4-hexylresorcinol ranging from 1.56 to 6.25 µg mL−1 caused a growth −1 delay and a small log10-reduction (under 1 log10 CFU mL ) after 24 h. However, the concentration of −1 −1 25 µg mL induced a log10-reduction of more than 3 log10 CFU mL after 24 h (Figure1D). Medicines 2017,, 4,, 2525 5 of 8 Medicines 2017, 4, 25 5 of 8 TheMedicines two 2017 naphthoquinones,, 4,, 2525 as well as the substituted phenol, 4-hexylresorcinol,5 of 8 inhibited Medicines 2017, 4, 25 5 of 8 Medicines 2017, 4, 25 −1 −−11 5 of 8 −1 biofilmMedicines formationThe 2017 concentrations, 4, 25 with MBICs of 4-hexylresorcinol of between ranging 25–50 µfromg mL 1.56 tofor 6.25 1,2-naphthoquinone, µg mL−1 causedcaused aa growthgrowth 12.5–50delaydelay5 of 8 µg mL The concentrations of 4-hexylresorcinol ranging from 1.56 to 6.25 µg mL−1 caused a growth delay The concentrations of 4-hexylresorcinol ranging−1 from 1.56 to 6.25 µg mL−1 caused a growth delay and aThe small concentrations log10-reduction of 4- (underhexylresorcinol 1 log10 CFU ranging mL−1) fromafter 1.5624 h. to However, 6.25 µg mL− the−1 causedconcentration a growth of delay25 µg and aThe small concentrations log10-reduction of 4- (underhexylresorcinol 1 log10 CFU ranging mL−1) fromafter 1.5624 h. to However, 6.25 µg mL the1 −1 concentrationcaused a growth of delay25 µg for 5-hydroxy-1,4-naphthoquinone,and aThe small concentrations log10-reduction of 4- (underhexylresorcinol 1 log and10 CFU fromranging mL− 12.51) fromafter to 1.5624 25h. to However,µ 6.25g mL µg mL the−1for concentrationcaused 4-hexylresorcinol a growth of 25delay µg (Table1). and− 1aThe small concentrations log10-reduction of 4- (underhexylresorcinol 1 log10 CFU ranging mL−1) fromafter 1.5624− 1h. to However, 6.25 µg mL the−−11 causedconcentration a growth of delay25 µg andmL− 1aThe induced small concentrations log a10 log-reduction10-reduction of 4- (underhexylresorcinol of more 1 log than10 CFU ranging3 logmL10− 1CFU) fromafter mL 1.5624− 1h. afterto However, 6.25 24 µg h (Figure mL the causedconcentrationcaused 1D). aa growthgrowth of delay25delay µg andmL− 1a induced small log a10 log-reduction10-reduction (under of more 1 log than10 CFU 3 logmL10− 1CFU) after mL 24− 1h. after However, 24 h (Figure the concentration 1D). of− 25 µg andmL− 1a induced small log a 10log-reduction10-reduction (under of more 1 log than10 CFU 3 log mL10−1 CFU) after mL 24− 1h. after However, 24 h (Figure the concentration 1D). of 125 µg DequaliniumandmL− 1a inducedsmall chloride log a10 10log-reduction10-reduction displayed (under of themore 1 log lowest 10than10 CFU 3 logmL MBIC10−−1 1)CFU after ranging mL 24− 1h. after However, from 24 h (Figure 0.39–0.78 the concentration 1D). µ g mL of 25. Theµg sensitivity andmL − 1a Theinduced small two log naphthoquinonesa log-reduction-reduction10-reduction (under(under ofas morewell 11 loglog asthan theCFUCFU 3 substitu log mLmL10 CFU)) afterafterted mLphenol, 2424− 1h.h. after However,However, 4-hexylresorci 24 h (Figure thethe concentrationconcentration nol,1D). inhibited ofof biofilm 2525 µgµg mL−1 Theinduced two naphthoquinonesa log10-reduction ofas morewell as than the 3 substitu log10 CFUted mLphenol,−1 after 4-hexylresorci 24 h (Figure nol,1D). inhibited biofilm mL−1 Theinduced two naphthoquinones a log10-reduction ofas morewell as than the 3 substitu log10 CFUted mLphenol,−1 after 4-hexylresorci 24 h (Figurenol, 1D). inhibited biofilm to themL inhibition−1 Theinducedinduced two naphthoquinones a ofa loglog biofilm1010-reduction-reduction formation of ofas more morewell as thanthan varied the 33 substitu loglog among1010 −CFUCFU1 ted mL mLphenol, the−1 afterafter strains 4-hexylresorci 2424 hh (Figure(Figure and depended nol, 1D).1D). inhibited on biofilm− the1 compounds. formationThe two with naphthoquinones MBICs of between as well 25–50 as the µg substitu mL−1 tedtedforfor phenol,phenol,1,2-naphthoquinone,1,2-naphthoquinone, 4-hexylresorci4-hexylresorci nol,12.5–5012.5–50 inhibited µgµg mLmL biofilm−1 forfor formationThe two with naphthoquinones MBICs of between as well 25–50 as the µg substitu mL−1 tedfor phenol,1,2-naphthoquinone, 4-hexylresorci nol,12.5–50 inhibited µg mL biofilm−1 for formationThe two with naphthoquinones MBICs of between as well 25–50 as the µg substitu mL−1 tedfor phenol,1,2-naphthoquinone, −4-hexylresorci1 nol,12.5–50 inhibited µg mL biofilm−1 for formation5-hydroxy-1,4-naphthoquinone,The two with naphthoquinones MBICs of between as and well 25–50 fromas the µg 12.5substitu mL to−1 tedtedfor25 phenol,phenol,µg1,2-naphthoquinone, mL −4-hexylresorci4-hexylresorci1 for 4-hexylresorcinol nol,12.5–50 inhibited µg (Table mL biofilm−1 for1). ATCCformation5-hydroxy-1,4-naphthoquinone, 19615 was with more MBICs sensitive of between toand 1,2-naphthoquinone 25–50 from µg12.5 mL to−1 25for µg1,2-naphthoquinone, mL but−1 morefor 4-hexylresorcinol resistant 12.5–50 to 4-hexylresorcinolµg (Table mL−1 for1). than formation5-hydroxy-1,4-naphthoquinone, with MBICs of between and 25–50 from µg12.5 mL to−1 25for µg1,2-naphthoquinone, mL−1 for 4-hexylresorcinol 12.5–50 µg (Table mL−1 for1). formation5-hydroxy-1,4-naphthoquinone, with MBICs of between and 25–50 from µg12.5 mL to−−11 for25 µg1,2-naphthoquinone, mL−1 for 4-hexylresorcinol 12.5–50−1 µg (Table mL−−11 for1). formation5-hydroxy-1,4-naphthoquinone,Dequaliniumformation withwith chloride MBICsMBICs displayed ofof betweenbetween theand lowest25–50 25–50from µgMBICµg12.5 mLmL toranging 25forfor µg1,2-naphthoquinone,1,2-naphthoquinone, from mL 0.39–0.78−1 for 4-hexylresorcinol µg mL 12.5–5012.5–50−1. The sensitivityµgµg (Table mLmL forfor1). to 5-hydroxy-1,4-naphthoquinone,Dequalinium chloride displayed theand lowest from MBIC12.5 toranging 25 µg from mL 0.39–0.78−1 for 4-hexylresorcinol µg mL−1. The sensitivity (Table 1).to the two5-hydroxy-1,4-naphthoquinone,Dequalinium other strains chloride (Table displayed1). theand lowest from MBIC12.5 toranging 25 µg from mL −0.39–0.781 for 4-hexylresorcinol µg mL−1. The sensitivity (Table 1).to 5-hydroxy-1,4-naphthoquinone,Dequalinium chloride displayed andthe lowest from MBIC12.5 toranging 25 µg from mL 0.39–0.78−−11 for 4-hexylresorcinol µg mL−1. The sensitivity (Table 1). to 5-hydroxy-1,4-naphthoquinone,Dequaliniumthe inhibition chlorideof biofilm displayed formation theand varied lowest from among MBIC12.5 the toranging strains25 µg from andmL depended0.39–0.78 forfor 4-hexylresorcinol4-hexylresorcinol µg on mLthe− compounds.1. The sensitivity (Table(Table ATCC 1).1).to theDequalinium inhibition chlorideof biofilm displayed formation the varied lowest among MBIC the ranging strains from and depended0.39–0.78 µgon mLthe− compounds.1. The sensitivity ATCC to Dequaliniumthe inhibition chloride of biofilm displayed formation the varied lowest among MBIC the ranging strains from and 0.39–0.78depended µg on mL the−− 1compounds.1.. TheThe sensitivitysensitivity ATCC toto Dequaliniumthe19615 inhibition was more chlorideof sensitivebiofilm displayed formation to 1,2-naphthoquinone the varied lowest among MBIC butthe ranging strainsmore resistantfrom and − depended0.39–0.781 to 4-hexylresorcinol µg on mLthe compounds... TheThe sensitivitysensitivitythan the ATCC two toto Tablethe19615 inhibition 1.wasMinimum more of sensitivebiofilm inhibitory formation to 1,2-naphthoquinone concentration varied among (MIC,butthe strainsmoreµg resistant mLand depended), to minimum 4-hexylresorcinol on the bactericidal compounds. than the concentrationATCC two the19615the inhibitioninhibition was more ofof sensitivebiofilmbiofilm formationformation to 1,2-naphthoquinone variedvaried amongamong butthethe strainsstrainsmore resistant andand dependeddepended to 4-hexylresorcinol onon thethe compounds.compounds. than the ATCCATCC two other19615 strainswas more (Table−1 sensitive 1) to 1,2-naphthoquinone but more resistant to 4-hexylresorcinol than the two −1 (MBC,1961519615other strainswasµwasgmL moremore (Table )sensitivesensitive of 1) 11 compounds toto 1,2-naphthoquinone1,2-naphthoquinone and minimum butbut more biofilmmore resistantresistant inhibitory toto 4-hexylresorcinol4-hexylresorcinol concentration than (MBIC,than thethe µ twotwog mL ) of other strains (Table 1) otherother strainsstrains (Table(Table 1)1) −1 S. pyogenes. theother fourTable strains most 1. (Table MinimumMinimum active 1) compounds inhibitoryinhibitory concentrationconcentration against three (MIC,(MIC, different µgµg mLmL−1 strains),), minimumminimum of bactericidalbactericidal concentrationconcentration Table 1. Minimum inhibitory concentration (MIC, µg mL−1), minimum bactericidal concentration Table 1. Minimum−1 inhibitory concentration (MIC, µg mL−1), minimum bactericidal concentration−1 Table(MBC, 1.µg Minimum mL−1) of 11 inhibitory compounds concentration and minimum (MIC, biofilm µg mLinhibitory−1), minimum concentration bactericidal (MBIC, concentration µg mL−1) of Table(MBC, 1.µg Minimum mL−1) of 11 inhibitory compounds concentration and minimum (MIC, biofilm µg mLinhibitory−1), minimum concentration bactericidal (MBIC, concentration µg mL−1) of Table(MBC, 1.µg Minimum mL−1) of 11 inhibitory compounds concentration and minimum (MIC, biofilm µg mLinhibitory−1 ), minimum concentration bactericidal (MBIC, concentration µg mL−1) of Table(MBC, 1.µg Minimum mL−1) of 11 inhibitory compounds concentration and minimum (MIC, biofilm µg mL inhibitory−−11), minimum concentration bactericidal (MBIC, concentration µg mL−1) of StreptococcusTable(MBC,the four 1.µg most MinimumMinimum mL −active1) of 11 inhibitorycompoundsinhibitory compounds concentrationconcentration against and minimum three (MIC, (MIC,different biofilm µgµg strains mLmLinhibitory),), ofminimumminimum S. concentration pyogenes. bactericidalbactericidal (MBIC, concentrationconcentration µg mL −1) of the(MBC, four µg most mL −active1) of 11 compounds compounds against and minimum threeATCC different biofilm 19615 strains inhibitory of S. concentrationpyogenes. ATCC 49399 (MBIC, µg mL−1) Aof Clinical Isolate Pyogenes(MBC,(MBC,the Strains four µgµg most mLmL− Chemical−1active1)) ofof 1111 compounds compoundscompounds Structure against andand minimumminimum three different biofilmbiofilm strains iinhibitorynhibitory of S. concentration concentrationpyogenes. (MBIC,(MBIC, µgµg mLmL−−11)) ofof Streptococcusthethe fourfour mostmost activeactive compoundscompounds againstagainst threethree differentdifferent strainsstrains ofof S. pyogenes. CompoundsStreptococcusthe four most active compounds againstMIC threeATCC different MBC 19615 strains MBIC of S. pyogenes. MICATCC 49399MBC MBICA Clinical MIC Isolate MBC MBIC PyogenesStreptococcusthethe Strains fourfour mostmost activeactiveChemical compoundscompounds Structure againstagainst threethreeATCC differentdifferent 19615 strainsstrains ofof S.S. pyogenes.pyogenes.ATCC 49399 A Clinical Isolate PyogenesStreptococcus Strains Chemical Structure ATCC 19615 ATCC 49399 A Clinical Isolate PyogenesStreptococcus Strains Chemical Structure ATCC 19615 ATCC 49399 A Clinical Isolate PyogenesStreptococcusCompounds Strains Chemical Structure MICATCC MBC 19615 MBIC MICATCC MBC 49399 MBIC MICA ClinicalMBC IsolateMBIC PyogenesStreptococcusStreptococcusCompounds Strains Chemical Structure MICATCC MBC 19615 MBIC MICATCC MBC 49399 MBIC MICA ClinicalMBC IsolateMBIC PyogenesCompounds Strains Chemical Structure MICATCCATCC MBC 1961519615 MBIC MICATCCATCC MBC 4939949399 MBIC MICAA ClinicalClinicalMBC IsolateIsolateMBIC PyogenesPyogenesCompounds StrainsStrains ChemicalChemical StructureStructure MIC MBC MBIC MIC MBC MBIC MIC MBC MBIC Compounds MIC MBCa MBIC MIC MBC MBIC MIC MBC MBIC PhlorizinCompoundsCompounds MI100CC MBC MBC >100 MBIC MBICnd MIC MIC 100 MBC MBC >100 MBIC MBIC MIC nd MBC 100MBIC >100 nd a Phlorizin 100 >100 a nd 100 >100 nd 100 >100 nd Phlorizin 100 >100 a nd 100 >100 nd 100 >100 nd Phlorizin 100 >100 a nd 100 >100 nd 100 >100 nd Phlorizin 100 >100 a nd 100 >100 nd 100 >100 nd Phlorizin 100 >100 a nd 100 >100 nd 100 >100 nd Phlorizin 100 >100 a nd 100 >100 nd 100 >100 nd Phlorizin 100 >100 a nd 100 >100 nd 100 >100 nd PhloriPhlorizinzin 100100 >100>100 aa ndnd 100100 >100>100 ndnd 100100 >100>100 ndnd

Naringenin 50 >100 nd 50 >100 nd 50 >100 nd Naringenin 50 >100 nd 50 >100 nd 50 >100 nd Naringenin 50 >100 nd 50 >100 nd 50 >100 nd Naringenin 50 >100 nd 50 >100 nd 50 >100 nd Naringenin 50 >100 nd 50 >100 nd 50 >100 nd Naringenin 5500 >100>100 ndnd 5050 >100>100 ndnd 5050 >100>100 ndnd

FlavoneFlavone 5050 >100 >100 nd nd>100 >100>100 >100nd nd 25 >100 25 nd >100 nd Flavone 50 >100 nd >100 >100 nd 25 >100 nd Flavone 50 >100 nd >100 >100 nd 25 >100 nd Flavone 50 >100 nd >100 >100 nd 25 >100 nd FlavoneFlavone 5050 >100>100 nd nd >100>100 >100>100 ndnd 25 25 >100>100 ndnd

MyricetinMyricetin 5050 >100 >100 nd nd 100 100>100 >100nd 100nd >100 100 nd >100 nd Myricetin 50 >100 nd 100 >100 nd 100 >100 nd Myricetin 50 >100 nd 100 >100 nd 100 >100 nd Myricetin 50 >100 nd 100 >100 nd 100 >100 nd Myricetin 5050 >100>100 nd nd 100 100 >100>100 ndnd 100 100 >100>100 ndnd

Thymol 25 >100 nd >100 >100 nd ≥100 >100 nd ThymolThymol 2525 >100 >100 nd nd>100 >100>100 >100nd ≥ nd100 >100≥100 nd >100 nd Thymol 25 >100 nd >100 >100 nd ≥100 >100 nd Thymol 25 >100 nd >100 >100 nd ≥100 >100 nd ThymolThymol 2525 >100>100 nd nd >100>100 >100>100 ndnd ≥≥100100 >100>100 nd nd ThymolThymol1,2- 2525 >100>100 nd nd >100>100 >100>100 ndnd ≥≥100100 >100>100 nd nd 1,2- 3.125 100 25 6.25 100 50 6.25 100 50 Naphthoquinone1,2- 3.125 100 25 6.25 100 50 6.25 100 50 1,2-NaphthoquinoneNaphthoquinone1,2- 3.1253.125 100 100 25 25 6.25 6.25100 100 50 6.25 50 100 6.25 50 100 50 Naphthoquinone1,2- 3.125 100 25 6.25 100 50 6.25 100 50 Naphthoquinone1,2-1,2- 3.125 100 25 6.25 100 50 6.25 100 50 Naphthoquinone 3.1253.125 100100 25 25 6.25 6.25 100100 50 50 6.25 6.25 100100 50 50 Naphthoquinone 5-Hydroxy-1,4- 5-Hydroxy-1,4- 1.56 100 12.5 1.56 >100 50 0.39 100 12.5 5-Hydroxy-1,naphthoquinone5-Hydroxy-1,4- 1.56 100 12.5 1.56 >100 50 0.39 100 12.5 naphthoquinone5-Hydroxy-1,4- 1.56 100 12.5 1.56 >100 50 0.39 100 12.5 naphthoquinone5-Hydroxy-1,4- 1.561.56 100 100 12.5 12.51.56 1.56>100 >10050 0.39 50 100 0.39 12.5 100 12.5 4-naphthoquinonenaphthoquinone5-Hydroxy-1,4-5-Hydroxy-1,4- 1.56 100 12.5 1.56 >100 50 0.39 100 12.5 naphthoquinone 1.561.56 100100 12.5 12.5 1.561.56 >100>100 5050 0.39 0.39 100100 12.512.5 naphthoquinonenaphthoquinone

Resveratrol 100 >100 nd 50 >100 nd >100 >100 nd Resveratrol 100 >100 nd 50 >100 nd >100 >100 nd ResveratrolResveratrol 100100 >100 >100 nd nd50 50>100 >100nd >100 nd >100 >100 nd >100 nd Resveratrol 100 >100 nd 50 >100 nd >100 >100 nd ResveratrolResveratrol 100100 >100>100 ndnd 5050 >100>100 ndnd >100>100 >100>100 ndnd

Cinnamaldehyde >100 >100 nd 50 >100 nd 25 >100 nd Cinnamaldehyde >100 >100 nd 50 >100 nd 25 >100 nd Cinnamaldehyde >100 >100 nd 50 >100 nd 25 >100 nd CinnamaldehydeCinnamaldehyde >100>100 >100 >100 nd nd50 50>100 >100nd nd25 >100 25 nd >100 nd CinnamaldehydeCinnamaldehyde >100>100 >100>100 ndnd 5050 >100>100 ndnd 2525 >100>100 ndnd Dequalinium Dequalinium 0.39 12.5 0.78 0.78 25 0.78 0.78 12.5 0.39 Dequaliniumchloride 0.39 12.5 0.78 0.78 25 0.78 0.78 12.5 0.39 Dequaliniumchloride 0.39 12.5 0.78 0.78 25 0.78 0.78 12.5 0.39 DequaliniumDequaliniumchloride 0.39 12.5 0.78 0.78 25 0.78 0.78 12.5 0.39 DequaliniumDequaliniumchloride 0.39 12.5 0.78 0.78 25 0.78 0.78 12.5 0.39 chloride 0.390.390.39 12.512.5 12.5 0.78 0.78 0.780.780.78 0.782525 25 0.78 0.78 0.78 0.78 12.512.5 0.78 0.390.39 12.5 0.39 chloridechloridechloride 4-Hexylresorcinol 1.56 50 25 6.25 50 12.5 12.5 50 12.5 4-Hexylresorcinol 1.56 50 25 6.25 50 12.5 12.5 50 12.5 4-Hexylresorcinol 1.56 50 25 6.25 50 12.5 12.5 50 12.5 4-Hexylresorcinol 1.56 50 25 6.25 50 12.5 12.5 50 12.5 4-Hexylresorcinolaa 1.56 50 25 −−116.25 50 12.5 12.5 50 12.5 4-Hexylresorcinol4-Hexylresorcinola MBCMBC waswas higherhigher thanthan thethe maximummaximum concentrationconcentration1.561.56 5050 ofof 100100 µgµg 25 25 mLmL−16.256.25 usedused inin5050 thisthis study;study;12.512.5 nd:nd:12.5 12.5not-determined.not-determined. 5050 12.512.5 4-Hexylresorcinola MBC was higher than the maximum concentration1.56 of 50 100 µg mL 25−1 used 6.25 in this study; 50 nd: 12.5not-determined. 12.5 50 12.5 a MBC was higher than the maximum concentration of 100 µg mL−1 used in this study; nd: not-determined. a MBC was higher than the maximum concentration of 100 µg mL −1 used in this study; nd: not-determined. a MBCSEM was images higher showed than the themaximum visual concentrationreduction of ofS. 100pyogenes µg mL ATCC−1 used in19615 this study;biofilm nd: in not-determined. the presence of aa MBCMBCSEM waswas images higherhigher showed thanthan thethe maximumthemaximum visual concentration concentrationreduction of of ofS. 100100 pyogenes µgµg mLmL −−ATCC11 usedused in in19615 thisthis study; study;biofilm nd:nd: in not-determined.not-determined. the presence of a MBC MBCMBCSEM was was images higherhigher showed thanthan than thethe the themaximummaximum maximum visual concentrationconcentrationreduction concentration of of ofS. 100100 pyogenes of 100µgµg mLmLµg ATCC mL usedused−1 ininused19615 thisthis in study; study;biofilm this nd: study;nd: in not-determined.not-determined. the nd: presence not-determined. of the MBICSEM imagesof the fourshowed compounds the visual by reduction strongly ofredu S. cingpyogenes the concentrationATCC 19615 biofilm of the attachedin thethe presencepresence cells and ofof the MBICSEMSEM imagesimagesof the four showedshowed compounds thethe visualvisual by reductionreduction strongly ofofredu S.S. cingpyogenespyogenes the concentrationATCC 19615 biofilm of the attachedin thethe presencepresence cells and ofof thethe MBICMBIC ofof thethe fourfour compoundscompounds byby stronglystrongly redureducingcing thethe concentrationconcentration ofof thethe attachedattached cellscells andand thethe MBICMBIC ofof thethe fourfour compoundscompounds byby stronglystrongly redureducingcing thethe concentrationconcentration ofof thethe attachedattached cellscells andand Medicines 2017, 4, 25 6 of 9

Medicines 2017, 4, 25 6 of 8 SEM images showed the visual reduction of S. pyogenes ATCC 19615 bioﬁlm in the presence of the MBIC of the four compounds by strongly reducing the concentration of the attached cells and modifying their shape (Figure 2). The presence of 1% DMSO in BHI (Figure 2B) reduced the chain modifying their shape (Figure2). The presence of 1% DMSO in BHI (Figure2B) reduced the chain length of the cocci and biofilm density, and also appeared to shrink the cells slightly in comparison length of the cocci and bioﬁlm density, and also appeared to shrink the cells slightly in comparison to to BHI only treatment (Figure 2A). Figure 2C shows that 25 µg mL−1 of 1,2-naphthoquinone reduced BHI only treatment (Figure2A). Figure2C shows that 25 µg mL−1 of 1,2-naphthoquinone reduced stronglystrongly the the concentration concentration of of the the attached attached cells, leavingleaving onlyonly a a few few long long and and thin thin chains chains comprised comprised of of −1 shrunken,shrunken, irregular irregular cells. InIn thethe presence presence of of 12.5 12.5µg mLµg −mL1 of 5-hydroxy-1,4-naphthoquinone of 5-hydroxy-1,4-naphthoquinone (Figure (Figure2D), 2D),smaller smaller cells cells aggregated aggregated in clusters in clusters of chains. of chains. Figure2 FigureE presents 2E thepresents effect ofthe dequalinium effect of dequalinium chloride chlorideat 0.78 µatg 0.78 mL− 1µg, which mL−1, induced which induced the formation the formation of very small of very groups small of bacterial groups cells of bacterial and completely cells and completelyaltered their altered shape. their The MBICshape. of The 4-hexylresorcinol MBIC of 4-hexylresorcinol (Figure2F) affected (Figure the chain 2F) formationaffected the of thechain formationbacteria andof the only bacteria a few smalland only chains a few containing small chains 2–4 cells containing were observed. 2–4 cells were observed.

FigureFigure 2. 2.ScanningScanning electron electron microscopy microscopy images images of the effecteffectof ofMBIC MBIC concentrations concentrations of of the the compounds compounds onon the the biofilm bioﬁlm formation formation by strain S. S. pyogenes pyogenesATCC ATCC 19615 19615 at 37at ◦37C: °C:S. pyogenes S. pyogenesin BHI in (BHIA) and (A) with and 1%with 1%DMSO DMSO (B ();BS.); S. pyogenes pyogeneswith with 25 25µg µg mL mL−1 −of1 of 1,2 1,2 naphthoquinone naphthoquinone (C );(C 12.5); 12.5µg µg mL mL−1 of−1 of 5-hydroxy-1,4 5-hydroxy-1,4 naphthoquinonenaphthoquinone ( (DD);); 0.78 µµggmL mL−−11 ofofdequalinium dequalinium chloride chloride (E) ( andE) and 25 µ 25g mL µg− 1mLof− 4-hexylresorcinol1 of 4-hexylresorcinol (F). (F).Magniﬁcation Magnification value: value: I, × I,1000; ×1000; II, ×II,5000; ×5000; III, III,×1000; ×1000; IV, IV,×15,000; ×15,000; V, V,×25,000. ×25,000.

4. 4.Discussion Discussion AccordingAccording to to the the literature, literature, the the MICs ofof phenolicphenolic compoundscompounds against against pharyngitis pharyngitis pathogens pathogens − rangerange from from 1.52 1.52 to to100 100 µgµ mLg mL−1 [9–12].1 [9–12 Among]. Among all the all thephenolic phenolic compounds compounds tested tested in this in this study, study, nine −1 compoundsnine compounds showed showed an anti-Streptococcal an anti-Streptococcal activity activity at a concentration at a concentration lower lowerthan 100 than µg 100 mLµ−g1. Gyawali mL . and Ibrahim. (2014) [8] stated that the hydroxyl (-OH) group in phenolic compounds may cause bacterial inhibition and described the importance of double bonds (number and position) in relation to antimicrobial effectiveness. Inferences could be made to the present study as, among the nine active compounds, the most anti-S. pyogenes naphthoquinones possess two carbonyl groups in an aromatic ring as part of their quinone structure. This hypothesis is supported by a recent review

Medicines 2017, 4, 25 7 of 9

Gyawali and Ibrahim. (2014) [8] stated that the hydroxyl (-OH) group in phenolic compounds may cause bacterial inhibition and described the importance of double bonds (number and position) in relation to antimicrobial effectiveness. Inferences could be made to the present study as, among the nine active compounds, the most anti-S. pyogenes naphthoquinones possess two carbonyl groups in an aromatic ring as part of their quinone structure. This hypothesis is supported by a recent review which associated the oxydo-reduction activity of the quinone structure in 2-hydroxy-1,4-naphthoquinone with the generation of reactive oxygen species (ROS) and damage of macromolecules such as DNA, proteins, and lipids [15]. Ndi et al. (2007) [11] isolated another naphthoquinone from Eremophila serrula, which also exhibited antagonism against S. pyogenes strain ATCC 10389 with low MIC (7.8 µg mL−1) and MBC (15.6 µg mL−1). Clinically, most antibacterials are described as potentially being both bactericidal and bacteriostatic. This was also observed in this study as the tested compounds. Even if a bactericidal action is preferred in the context of treatment, achieving a bacteriostatic effect may advantageously inhibit exotoxin production in Staphylococci and Streptococci thereby avoiding induction of the toxic shock syndrome [16]. In comparison with the kinetics of the tested compounds, rhodomyrtone—the potent bioactive compound from Rhodomyrtus tomentosa—appears to be slightly more effective: it exerted a faster bactericidal activity on S. pyogenes (within 5 and 6 h) at lower concentrations (6.5–12.5 µg mL−1)[17]. However, rhodomyrtone (Sigma-Aldrich Co., Saint-Louis, MO, USA) is about 2000–4500 times more expensive than the naphthoquinones tested. To the best of our knowledge, the anti-biofilm potential of naphthoquinones against S. pyogenes has not been explored. The inhibition of biofilm formation is an interesting way to prevent the formation of well-organized attached bacterial biofilms and, thus, pharyngitis. We observed that the naphthoquinones elicited significant anti-biofilm activity against S. pyogenes, with metabolic MBICs in the same range as 4-hexylresorcinol and this was confirmed by SEM, where S. pyogenes ATCC 19,615 cells appeared less aggregated, showed morphological changes, and reduced biofilm formation. These observations are in accordance with the potential of phenolic compounds to damage the membrane structure as well as anti-S. pyogenes properties of phytochemicals such as anti-adhesion, anti-biofilm, and quorum-sensing inhibition [2,8]. The generation of ROS during the bio-reduction of naphthoquinones [15] might be linked to the loss of cell wall integrity observed. In addition, the visible effect of 1% DMSO on the bacteria, which do not appear on the growth (Figure1), could also slightly enhance the antibacterial effect of the phytochemicals. Considering the potential clinical application of our study, additional experiments could be conducted on combination of natural antibacterial agents and currently used antibiotics to enhance the present management practices against S. pyogenes. If the combination demonstrates a synergic and/or a complementary effect (e.g., anti-inflammatory and antitussive), it could become a combined treatment for patient’s pain relief. Moreover, it is also possible to incorporate the efficacious natural compounds in dehydrated honey lozenges. Honey itself has a recognized effect as being systemic antitussive and antimicrobial natural source [18–20]. Interestingly, a recent study reported that honey with a high phenolic content presented the highest antimicrobial activity [20]. Further investigations are required to affirm the synergic effect with honey but also understand the safety of the use of naphthoquinones in throat lozenges before making any recommendations for their use for managing streptococcal pharyngitis.

Acknowledgments: This project was cofounded by the Natural Sciences and Engineering Council (NSERC) of Canada and Island Abbey Foods Ltd, Charlottetown, PEI, Canada. We are grateful to R. Davidson (Queen Elizabeth II Hospital, Halifax, NS, Canada) for providing the clinical isolate of S. pyogenes. Author Contributions: Sabrina Macé and H.P. Vasantha Rupasinghe designed the study in collaboration with Lisbeth Truelstrup Hansen. Sabrina Macé performed the experiments, analyzed the data, and wrote the manuscript. H.P. Vasantha Rupasinghe and Lisbeth Truelstrup Hansen supervised the study and the manuscript writing. Conﬂicts of Interest: The authors declare no conﬂict of interest. Medicines 2017, 4, 25 8 of 9

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