Trans-Cinnamaldehyde Exhibits Synergy with Conventional Antibiotic Against Methicillin-Resistant Staphylococcus Aureus

International Journal of Molecular Sciences

Article Trans-Cinnamaldehyde Exhibits Synergy with Conventional Antibiotic against Methicillin-Resistant Staphylococcus aureus

Shu Wang, Ok-Hwa Kang * and Dong-Yeul Kwon *

Department of Oriental Pharmacy, College of Pharmacy and Wonkwang Oriental Medicines Research Institute, Wonkwang University, Iksan 54538, Jeonbuk, Korea; [email protected] * Correspondence: [email protected] (O.-H.K.); [email protected] (D.-Y.K.); Tel.: +82-63-850-6802 (O.-H.K.)

Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen worldwide and has acquired multiple resistance to a wide range of antibiotics. Hence, there is a press- ing need to explore novel strategies to overcome the increase in antimicrobial resistance. The present study aims to investigate the efficacy and mechanism of plant-derived antimicrobials, trans- cinnamaldehyde (TCA) in decreasing MRSA’s resistance to eight conventional antibiotics. A checkerboard dilution test and time–kill curve assay are used to determine the synergistic effects of TCA combined with the antibiotics. The results indicated that TCA increased the antibacterial activity of the antibiotics by 2-16-fold. To study the mechanism of the synergism, we analyzed the mecA transcription gene and the penicillin-binding protein 2a level of MRSA treated with TCA by quantita- tive RT-PCR or Western blot assay. The gene transcription and the protein level were significantly inhibited. Additionally, it was verified that TCA can significantly inhibit the biofilm, which is

highly resistant to antibiotics. The expression of the biofilm regulatory gene hld of MRSA after TCA treatment was also significantly downregulated. These findings suggest that TCA maybe is an

Citation: Wang, S.; Kang, O.-H.; exceptionally potent modulator of antibiotics. Kwon, D.-Y. Trans-Cinnamaldehyde Exhibits Synergy with Conventional Keywords: MRSA; trans-cinnamaldehyde; β-lactam antibiotics; synergy; mecA; PBP2a; bioﬁlm Antibiotic against Methicillin- Resistant Staphylococcus aureus. Int. J. Mol. Sci. 2021, 22, 2752. https://doi.org/10.3390/ijms22052752 1. Introduction Gram-positive bacterium methicillin-resistant Staphylococcus aureus (MRSA) is a promi- Academic Editor: Diego A. Moreno nent human pathogen worldwide [1], which is a major cause of antibiotic-resistant healthcare- and Antonio J. Ruiz-Alcaraz associated infections. In recent years, community-acquired strains of infection have appeared and have begun to spread among healthy persons [2]. MRSA resists most antibiotics, owing Received: 28 January 2021 to the strain that produces the penicillin-binding protein 2a (PBP2a). The PBP2a is a modi- Accepted: 5 March 2021 Published: 9 March 2021 fied penicillin-binding protein encoded by the mecA gene, demonstrating reduced affinity to β-lactam antibiotics. MecA is regulated by genes mecI and mecR1 located on the bacterial

Publisher’s Note: MDPI stays neutral chromosome [3]. In the past, the antibiotics commonly used to treat MRSA were vancomycin with regard to jurisdictional claims in and oral linezolid. However, moderate resistance to vancomycin or linezolid has emerged [4], published maps and institutional afﬁl- posing an urgent demand for novel antibacterial substances or therapeutic options. iations. Natural products have been a rich source of compounds with structural and chemical diversity for drug discovery [5]. Since plant antibacterial agents contain different functional groups, their antibacterial activity can be attributed to multiple mechanisms. Therefore, compared with antibiotics, bacteria are relatively less likely to develop resistance to plant- derived antibacterial substances [6]. Cinnamon is a traditional spice, which is widely used Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. around the world [7]. It has also been used since ancient times as a food preservative by act- This article is an open access article ing against foodborne pathogens and spoilage bacteria [8]. Cinnamon extract components distributed under the terms and have demonstrated a range of medical properties, cinnamic acid possesses antibacterial conditions of the Creative Commons activity based on directed self-assembly [9]; procyanidin C1 acts as a potential insulin Attribution (CC BY) license (https:// sensitizer that targets adipocytes [10]; trans-cinnamaldehyde (TCA) mitigates intestinal creativecommons.org/licenses/by/ inﬂammation [11]. TCA, the main component of cinnamon, is an aromatic aldehyde ex- 4.0/). isting in cinnamon bark extract. It is a food-grade molecule and is listed as a recognized

Int. J. Mol. Sci. 2021, 22, 2752. https://doi.org/10.3390/ijms22052752 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 2752 2 of 9

safe agent by the Food and Drug Administration [12]. In recent years, there are various studies on TCA as a natural antibacterial substance, with antibacterial activity against Listeria monocytogenes, Cronobacter spp., and MRSA [13]. In this study, we evaluated the synergistic antibacterial activity of TCA combined with eight conventional antibiotics and investigated the mechanism at the molecular level.

2. Results 2.1. Synergistic Antibacterial Activity A broth microdilution assay was used to determine the minimal inhibitory concentration (MIC) of TCA, and the value against all strains (reference strain ATCC 33591 and two clinical isolates) was 250 µg/mL. The results of checkerboard dilution are indicated in Table1. For all studied strains, the fractional inhibitory concentration index (FICI) of TCA combinations with conventional antibiotics (including ampicillin, oxacillin, gentamicin, vancomycin, amoxicillin, ceftazidine, amikacin, and cefoxitin) were 0.19–0.1, showing synergy, partial synergy, and additive effect. TCA combined with amikacin had the best synergism, with a mean FICI of 0.27. TCA combined with gentamicin, vancomycin, and amoxicillin had a signiﬁcant synergistic effect. The remaining combinations, including those with the antibiotics, ampicillin, oxacillin, ceftazidine, and cefoxitin showed relatively less signiﬁcant interactions with TCA against MRSA, although the MIC value of antibiotics also reduced by at least 2-fold against all three strains.

Table 1. The MIC value of conventional antibiotic and synergistic effect of TCA combined with an antibiotic.

ATCC 33591 DPS-1 DPS-3 Antibiotics MIC MIC MIC Fold FICI Fold FICI Fold FICI (µg/mL) (µg/mL) (µg/mL) Ampicillin 62.5 2 1 31.3 4 0.75 62.5 4 0.75 Oxacillin 62.5 4 0.75 500 2 1 500 8 0.25 Gentamicin 3.9 4 0.75 125 4 0.37 250 4 0.5 Vancomycin 250 8 0.25 250 2 1 500 2 1 Amoxicillin 62.5 8 0.63 125 4 0.5 125 8 0.25 Ceftazidine 125 2 1 125 2 1 250 4 0.75 Amikacin 31.2 4 0.38 31.2 8 0.25 62.5 16 0.19 Cefoxitin 31.2 4 0.75 62.5 8 0.62 250 4 0.5 TCA, trans-cinnamaldehyde; MIC, minimal inhibitory concentration; Fold, Fold reduction of the MIC of antibiotics. FICI, The fractional inhibitory concentrations index.

2.2. Time–Kill Assay According to the results of the checkerboard assays, three antibiotics with excellent synergistic effect with TCA were selected to further explore the synergistic effect by a time–kill assay. The use of antibiotics with 1/2 MIC alone had no effect on the growth of bacteria after 24 h. In all combination groups, bacterial growth was detected to be inhibited after 4 h (Figure1a–c). TCA (1/2 MIC) combined with three antibiotics (1/2 MIC), amikacin, gentamicin, and oxacillin in the treatment of S. aureus, ATCC 33591, DPS-1, and DPS-3, displayed significantly synergistic interactions, respectively, and more than 3 log10 reductions in colony count after 24 h. Besides, a combination of TCA (1/2 MIC) and gentamicin (1/2 MIC) exhibited bactericidal activity against MRSA (DPS-1) after overnight incubation. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 9

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 9 Int. J. Mol. Sci. 2021, 22, 2752 3 of 9

Figure 1. Time–kill curves of the synergistic effect of TCA in combination with three conventional antibiotics at sub-inhibitory concentrations against methicillin-resistant Staphy- lococcus aureus (MRSA). (a) Time–kill curves of combination between TCA and amikacin against reference strain ATCC 33591. (b) Time–kill curves of combination between TCA and gentamicin against clinical isolates DPS-1. (c) Time–kill curves of combination be- Figure 1. Time–kill curves of the synergistic effect of TCA in combination with three conventional antibiotics at sub- tween TCA and oxacillin against clinical isolates DPS-3. TCA: trans-cinnamaldehyde; AK; inhibitory concentrationsFigure 1. Time–kill against methicillin-resistantcurves of the synergisticStaphylococcus effect of aureus TCA(MRSA). in combination (a) Time–kill with curves three of con- combination CFU, colony-forming units; MIC, minimum inhibitory concentration. between TCAventional and amikacin antibiotics against at reference sub-inhibitory strain ATCC concentrations 33591. (b) Time–kill against curves methicillin-resistant of combination between Staphy- TCA and gentamicin against clinical isolates DPS-1. (c) Time–kill curves of combination between TCA and oxacillin against clinical lococcus aureus2.3. TCA(MRSA). Represses (a) Time–kill the Transcription curves of of combinationMecA, MecR1, between and MecI TCA in S. and aureus amikacin isolates DPS-3.against TCA: trans-cinnamaldehyde;reference strain ATCC AK; 33591. CFU, colony-forming(b) Time–kill curves units; MIC, of combination minimum inhibitory between concentration. TCA and gentamicin2.3.The against TCA transcriptional Represses clinical theisolates Transcriptionlevels DPS-1. of mecA of(c, MecA,)mecR1, Time–kill MecR1, and curvesmecI and wereMecI of combinationinhibited in S. aureus in S.be- aureus upon tween TCAthe and treatment oxacillinThe transcriptional withagainst 1/8 clinical MIC levelsconcentrations isolates of DPS-3.mecA ,of TCA:mecR1, TCA, trans-cinnamaldehyde; and themecI transcriptionwere inhibited AK;levels in ofS. three aureus CFU, colony-forminggenesupon were the units;affected treatment MIC, by minimumthe with treatment 1/8 MICinhibitory with concentrations gra concentration.ded subinhibitory of TCA, andconcentrations the transcription (Figure levels 2). In ofthe three presence genes of were 1/2 MIC affected of TC byA, the the treatment transcriptional with graded levels subinhibitoryof mecA, mecR1, concentrations and mecI 2.3. TCA Represseswere(Figure decreased the2 Transcription). In theby 1.8-fold, presence of MecA, 2.3-fold, of 1/2 MecR1, MIC and and of2.5-fold, TCA, MecI therespectively.in S. transcriptional aureus levels of mecA, mecR1, and mecI were decreased by 1.8-fold, 2.3-fold, and 2.5-fold, respectively. The transcriptional levels of mecA, mecR1, and mecI were inhibited in S. aureus upon the treatment with 1/8 MIC concentrations of TCA, and the transcription levels of three genes were affected by the treatment with graded subinhibitory concentrations (Figure 2). In the presence of 1/2 MIC of TCA, the transcriptional levels of mecA, mecR1, and mecI were decreased by 1.8-fold, 2.3-fold, and 2.5-fold, respectively.

Figure 2. The expression of mecA, mecR1, and mecI in MRSA (ATCC 33591) cultures in the presence Figureof sub-concentrations 2. The expression of TCA.of mecA The, relativemecR1, geneand expressionmecI in MRSA of mecA (ATCC, mecR1 33591), and mecI cultureswas reduced in the in presencea dose-dependent of sub-concentrations manner. The dataof TC wereA. The presented relative as gene the mean expression± standard of mecA deviation, mecR1 of the, and three mecIindependent was reduced experiments. in a dose-dependent ** represents p manner.< 0.01. The data were presented as the mean ± standard2.4. Expression deviation of PBP2aof the three in MRSA independent Treated with experiments. TCA ** represents p < 0.01. Western blot was used to evaluate the expression level of PBP2A in TCA treated 2.4.or Expression untreated of MRSAPBP2a strainsin MRSA (Figure Treated3). with The TCA PBP2a levels were signiﬁcantly reduced af- Figure 2. The expression of mecA, mecR1, and mecI in MRSA (ATCC 33591) cultures in the ter treatment with TCA of sub-concentrations (1/8 MIC, 1/4 MIC, and 1/2 MIC), and presence of sub-concentrations of TCA. The relative gene expression of mecA, mecR1, and TCAWestern caused blot a concentration-dependent was used to evaluate the decreaseexpression in level the expression of PBP2A ofin PBP2a.TCA treated Moreover, or mecI was reduced in a dose-dependent manner. The data were presented as the mean ± untreatedthe PBP2a MRSA production strains (Figure of S. aureus 3). Thestrains PBP2a was levels almost were undetectable significantly after reduced exposure after to standard deviationtreatment1/2 MIC of with the of TCA.three TCA The inofdependent sub-concentrations expression experiments. levels of (1/8 PBP2A ** MIC, represents in MRSA1/4 MIC, pdecreased < 0.01.and 1/2 afterMIC), TCA and addition, TCA corresponding to the low expression of mecA transcription after primer extension. 2.4. Expression of PBP2a in MRSA Treated with TCA

Western blot was used to evaluate the expression level of PBP2A in TCA treated or untreated MRSA strains (Figure 3). The PBP2a levels were significantly reduced after treatment with TCA of sub-concentrations (1/8 MIC, 1/4 MIC, and 1/2 MIC), and TCA Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 9

caused a concentration-dependent decrease in the expression of PBP2a. Moreover, the PBP2a production of S. aureus strains was almost undetectable after exposure to 1/2 MIC Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 9 of TCA. The expression levels of PBP2A in MRSA decreased after TCA addition, corresponding to the low expression of mecA transcription after primer extension.

caused a concentration-dependent decrease in the expression of PBP2a. Moreover, the PBP2a production of S. aureus strains was almost undetectable after exposure to 1/2 MIC of TCA. The expression levels of PBP2A in MRSA decreased after TCA addition, corre- Int. J. Mol. Sci. 2021, 22, 2752 sponding to the low expression of mecA transcription after primer extension. 4 of 9

Figure 3. Effect of TCA at sub-concentrations on the expression of penicillin-binding protein 2a (PBP2a) in MRSA (ATCC 33591). Lane 1, the control was treated without TCA; Lane 2-4, PBP2a production after treatment with TCA at 1/8 MIC, 1/4 MIC, and 1/2 MIC, respectively. Figure 3. Effect of TCA at sub-concentrations on the expression of penicillin-binding protein 2a (PBP2a) in MRSA (ATCC 33591). Lane 1, the control was treated without TCA; Lane 2-4, PBP2a 2.5. BiofilmFigure Inhibitory 3. Effect Assay of andTCA the at Transcription sub-concentrations of hld in on S. theaureus expression of penicillin-binding protein 2aproduction (PBP2a) afterin MRSA treatment (ATCC with 33591). TCA at 1/8Lane MIC, 1, the 1/4 MIC,control and was 1/2 treated MIC, respectively. without TCA; The Lanebiofilm 2-4,2.5. formation PBP2a Biofilm production Inhibitory of the two Assay afterstrains and treatment (mean the Transcription of wi 1 thisolates TCA of ATCCat hld 1/8 in MIC, S.33591 aureus 1/4 and MIC, 1 stand- and 1/2 MIC, ard isolaterespectively. DPS-1) in the presence of sub-concentration of TCA was inhibitory signifi- The biofilm formation of the two strains (mean of 1 isolates ATCC 33591 and 1 standard cantly, and TCA caused a concentration-dependent inhibitory effect on the biofilm for- isolate DPS-1) in the presence of sub-concentration of TCA was inhibitory significantly, mation (Figure2.5. Biofilm 4a). TCAInhibitory also Assaysignificantly and the down-regulatedTranscription of hld the in expression S. aureus of the biofilm and TCA caused a concentration-dependent inhibitory effect on the biofilm formation regulatory gene(Figure hld of 4MRSA.a). TCA In also the significantlypresence of 1/2 down-regulated MIC of TCA, the transcriptional expression of the levels biofilm regulatory The biofilm formation of the two strains (mean of 1 isolates ATCC 33591 and 1 stand- of hld were reducedgene hld byof 5.5-fold MRSA. or In 5-fold the presence on ATCC of 1/2 33591 MIC or of DPS-1, TCA, therespectively transcriptional (Figure levels of hld were ard isolate DPS-1) in the presence of sub-concentration of TCA was inhibitory signifi- 4b). reduced by 5.5-fold or 5-fold on ATCC 33591 or DPS-1, respectively (Figure4b). cantly, and TCA caused a concentration-dependent inhibitory effect on the biofilm formation (Figure 4a). TCA also significantly down-regulated the expression of the biofilm regulatory gene hld of MRSA. In the presence of 1/2 MIC of TCA, the transcriptional levels of hld were reduced by 5.5-fold or 5-fold on ATCC 33591 or DPS-1, respectively (Figure 4b).

Figure 4. (a) Inhibition of TCA at sub-inhibitory concentrations on MRSA (ATCC 33591 and DPS-1) biofilm. (b) The expressionFigure of hld in 4. MRSA (a) Inhibition (ATCC 33591 of TCA and DPS-1) at sub-inhibitory cultures in the concentrations presence of TCA on at MRSA various (ATCC concentrations. 33591 The data were presentedand asDPS-1) the mean biofilm.± standard (b) The deviation expression of the of three hld in independent MRSA (ATCC experiments. 33591 and ** represents DPS-1) culturesp < 0.01. in the presence3. of Discussion TCA at various concentrations. The data were presented as the mean ± standard deviation of the three independent experiments. ** represents p < 0.01. With the slow development of novel drugs, a combination of established drugs that restore therapeutic effects through synergistic interactions is a promising method to over- 3. DiscussionFigure 4. (a) Inhibition of TCA at sub-inhibitory concentrations on MRSA (ATCC 33591 and DPS-1)come thebiofilm. increasing (b) The antibiotic expression resistance of hld [in3]. MRSA In the (ATCC present 33591 study, and the synergisticDPS-1) cultures anti-MRSA With the slow development of novel drugs, a combination of established drugs that in the effectspresence and of mechanism TCA at various of the concentrat natural antibacterialions. The data agent were TCA presented and eight as the conventional mean ± an- restore therapeutictibiotics effects including throughβ -lactamsynergistic antibiotics interactions were is evaluated a promising for the method first time. to over- The study pointed standard deviation of the three independent experiments. ** represents p < 0.01. come the increasingout that antibiotic TCA had resistance a potent potential[3]. In the to present reverse study, the sensitivity the synergistic of conventional anti- antibiotics MRSA effects andto MRSA. mechanism The resultsof the natural of checkerboard antibacterial dilution agent indicatedTCA and thateight TCA conven- had synergistic or 3. Discussion tional antibioticspartial including synergistic β-lactam effects antibiotics with all thewere eight evaluated conventional for the antibiotics first time. tested, The especially in study pointedWith combinationout thatthe slowTCA with developmenthad amikacin, a potent ofpote which novelntial reduced drugs, to reverse thea combination MIC the ofsensitivity amikacin of established of against conven- MRSA drugs bythat 16-fold tional antibioticsrestoreand therapeutic tothe MRSA. FICI valueTheeffects results was through as of low ch synergisticeckerboard as 0.19. According interactions dilution to indicated the is a standard promising that ofTCA method the had Clinical to over- and Labo- come ratorythe increasing Standards antibiotic Institute resistance (CLSI), the [3]. clinical In the MRSA present strain study, DPS-3 the is synergistic an oxacillin-resistant anti- MRSAstrain. effects The and MIC mechanism of oxacillin of isthe reduced natural by antibacterial 8-fold when agent combined TCA withand TCAeight againstconven- MRSA. tionalTCA antibiotics significantly including improved β-lactam the antibiotics antibacterial were sensitivity evaluated of oxacillin.for the first However, time. The the MIC study ofpointed oxacillin outis that only TCA reduced had a bypotent 2-fold pote whenntial theto reverse TCA combined the sensitivity with oxacillinof conven- against tional clinicalantibiotics DPS-1. to MRSA. This may The be results due to of the ch differentialeckerboard expression dilution indicated of the mecA thatgene TCA in had different clinical strains. Time–kill assay was employed to further evaluate the synergy effect of TCA combined with conventional antibiotics, and three results with excellent synergy effect were listed in the present study. The results demonstrated that compared with treatment with antibiotics or TCA alone at sub-MIC concentration, the two-drug combination groups exhibited a significant synergy effect and more than 3 log10 reduction in colony count after 24 h, Int. J. Mol. Sci. 2021, 22, 2752 5 of 9

respectively. TCA combined with gentamicin in sub-MIC can completely inhibit the growth of MRSA even after 24 h. In addition, TCA significantly inhibited MRSA growth after 4 h, which may mean that combination therapy inhibits the bacteria faster than the traditional single antibiotic therapy. We supported that the synergy mechanism between TCA and antibiotics was mul- tifaceted. First, the results of qRT-PCR and Western blot revealed that TCA inhibited mecA and PBP2a in a dose-dependent manner at sub-concentrations, suggesting that the synergistic effect of TCA and β-lactam antibiotics was caused by the fact that TCA down- regulated the transcription and translation of antibiotic resistance gene mecA. Significantly, synergy was also presented when TCA was combined with non-beta lactam antibiotics. We speculated that was because TCA can destroy the biofilm and cell membrane of MRSA. The results of the biofilm inhibition assay indicated that TCA can significantly inhibit the biofilm formation and the expression of the biofilm regulatory gene hld [14]. The biofilm of MRSA has serious clinical implications, making it difficult to eradicate and more tolerant to antibiotic therapy [15]. When the formation of biofilm was inhibited, the resistance of MRSA to antibiotics can be reduced, which may be one of the reasons that TCA combined with antibiotics has a synergistic effect. In addition, it has been reported that TCA has a hydrophobic structure that can change the permeability of cell membranes and destroy the integrity of cell membranes and cell walls [16]. We speculated that the efficiency of antibiotics entering into cells increased as TCA destroyed the cell membrane, implying that TCA improved the sensitivity of antibiotics to bacteria. TCA is an essential oil derived from cinnamon bark, with certain limitations, such as low water solubility and low stability. Recent studies have demonstrated that the encapsu- lation of essential oil by nanomaterials can increase chemical stability and solubility [17], equipping TCA with promising clinical significance as an antibiotic reversal agent. In conclusion, our results indicated that TCA exhibited a potent in vitro ability to restore antibiotic sensitivity against MRSA at sub-MIC, revealing the promising potential of TCA to overcome the antibiotic resistance crisis and be developed to a useful botanical synergist of antibiotics for the control of MRSA. However, the study has certain limitations. First, the S. aureus used in the study only one reference strain and two clinical isolates, which were confined to a selected population from a single hospital in Korea with a specific endemic [4]. Moreover, it may limit the external validity of the test results. Second, TCA is a food-grade compound. Nevertheless, there may be potential side effects when it is used in combination with antibiotics. Their clinical usage needs to be determined using in vivo efficacy studies. In addition to discovering novel antibacterial treatments, implementing interventions to control the spread of MRSA in the community and improve compliance with basic infection control measures also have crucial significance for further community-associated MRSA transmission and infections [18].

4. Materials and Methods 4.1. Reagents TCA, crystal violet, ampicillin, oxacillin, gentamicin, vancomycin, amoxicillin, ceftazidine, amikacin, and cefoxitin were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Mueller-Hinton agar, Mueller-Hinton broth, and skim milk were obtained from Difco Laboratories (Baltimore, MD, USA). Primary antibodies to PBP2a were obtained from DiNonA Inc. (Seoul, Korea). Anti-mouse IgG secondary antibody was obtained from Thermo Fisher Scientiﬁc Inc. (Carlsbad, CA, USA). The chemiluminescent ECL assay kit was obtained from ATTO Corp. (Tokyo, Japan). E.Z.N.A. Bacterial RNA Kit was purchased from Omega Bio-Tek (Norcross, GA, USA). The sequences of primers used in this study were obtained from Bioneer (Daejeon, Korea) (Table2). SMART ™ bacterial protein extraction solution was obtained from Intron BioTechnology, Inc. (Seongnam, Korea). QuantiTect Reverse Transcription Kit was obtained from (Dusseldorf, Germany). Power SYBR Green PCR Master Mix was obtained from Life Technologies LTD (Warrington, UK). Int. J. Mol. Sci. 2021, 22, 2752 6 of 9

Table 2. Primers used in qRT-PCR.

Primer Sequence (50-30) 16S F: ACTCCTACGGGAGGCAGCAG R: ATTACCGCGGCTGCTGG mecA F: CAATGCCAAAATCTCAGGTAAAGTG R: AACCATCGTTACGGATTGCTTC mecR1 F: GTGCTCGTCTCCACGTTAATTCCA R: GACTAACCGAAGAAGTCGTGTCAG mecI F: CGTTATAAGTGTACGAATGGTTTTTG R: TCATCTGCAGAATGGGAAGTT hld F: ATTTGTTCACTGTGTCGATAATCC R: GGAGTGATTTCAATGGCACAAG

4.2. Susceptibility Testing of TCA with Conventional Antibiotics The MIC values of TCA were determined either alone or in combination with conventional antibiotics (ampicillin, oxacillin, gentamicin, vancomycin, amoxicillin, ceftazidine, amikacin, and cefoxitin) according to the guidelines of the Clinical and Laboratory Stan- dards Institute (CLSI) by microdilution and checkerboard assays [19]. S. aureus strains were cultured on MHA at 37 ◦C for 24 h. Serial dilutions of TCA with antibiotics were mixed in Mueller Hinton broth (MHB). The MRSA inocula were adjusted to 0.5 McFarland standard in MHB. The bacterial concentration of the final inoculum was 1.5 × 105 CFU/well. Each MIC value was determined after a 24 h incubation period at 37 ◦C and defined as the lowest concentration that completely inhibited the growth of the bacteria. In vitro interaction between the drugs was quantified by determining the fractional inhibitory concentration index (FICI), as follows: ∑FIC: FICA + FICB = MICA + B/MICA alone + MICB + A/MICB alone. The combination was considered as synergy for FICI ≤ 0.5, partial synergy for 0.5 < FICI ≤ 0.75, additive effect for 0.75 < FICI ≤ 1, indifferent for 1 < FICI ≤ 4 and antagonism for FICI > 4. At the same time, the fold reduction of the MIC of antibiotics against MRSA alone to combined with TCA also was calculated, which is abbreviated as Fold in Table1. All tests were performed in triplicate.

4.3. Bacterial Strains and Conditions S. aureus ATCC 33591 (American Type Culture Collection, Manassas, VA, USA) was used as a reference strain. Two clinical isolates of MRSA DPS-1 and DPS-3 were isolated from patients at the Wonkwang University Hospital (Jeonbuk, Korea). Bacteria were maintained on Mueller–Hinton agar (MHA) plates. Liquid cultures for S. aureus strains were cultured in Mueller–Hinton broth (MHB) at 37 ◦C.

4.4. Time–Kill Assays The synergistic antimicrobial effect was further determined by a time–kill assay, which used 1/2 MIC of TCA and 1/2 MIC of three conventional antibiotics (amikacin, gentamicin, and oxacillin) against the growth S. aureus (reference strain ATCC33591 and clinical isolates DPS-1, DPS-3) according to the previous method [10]. Brieﬂy, bacteria cultures incubated in MHA at 37 ◦C for 24 h were diluted with sterilized MHB to 1.5 × 105 CFU/mL, and the diluted cultures were incubated at 37 ◦C for 24 h. Then, aliquots (100 µL) of the culture were removed after an incubation period at (0, 4, 8, 16, and 24 h), and serial 10-fold dilutions were prepared in saline as needed. The numbers of viable cells were counted on a drug-free MHA plate after 24 h incubation. Three independent experiments were carried out and the data presented as the mean ± SD. Finally, the graphics as log10 CFU/mL versus time were plotted to describe the results of time–kill assays, and the combinations decrease of CFU/mL by ≥2 log10 was considered as a synergistic effect [20]. Int. J. Mol. Sci. 2021, 22, 2752 7 of 9

4.5. Western Blot Analysis Western blot analysis was carried out to evaluate the effect of TCA on the expression of PBP2a according to the methods described previously [21]. S. aureus strains (ATCC 33591) were grown in MHB for 24 h and treated with sub-concentrations of TCA for 30 min. Cell protein extracts were harvested by centrifugation at 3000× g for 10 min and the protein concentration was determined by a Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). The supernatants were subjected to SDS-PAGE and electroblotted onto Amersham HybondTM-P membranes (GE Healthcare, Piscataway, NJ, USA). The membranes were blocked by 5% skim milk and probed with monoclonal mouse anti-PBP2a primary antibody (diluted 1:1000, DiNonA, Seoul, Korea) overnight at 4 ◦C and re-probed with anti-mouse IgG secondary antibody (diluted 1:2000, Enzo Life Sciences, Ann Arbor, MI, USA) at room temperature for 2 h. Then, the membranes were supplemented with ECL Prime Western Blotting Detection reagent (GE Healthcare Life Sciences, Incheon, Korea), and an ImageQuant LAS-4000 mini chemical luminescent imager (GE Healthcare Life Sciences) was used to visualize the bands [22].

4.6. qRT-PCR S. aureus ATCC 33591 in MHB was treated with sub-inhibitory concentrations (1/8 MIC, 1/4 MIC, and 1/2 MIC) of TCA for 0.5 h with untreated as a control. Easy-RED BYF RNA was prepared with the Easy-RedByFrNA extraction kit according to the manufacturer’s instructions (iNtRON Biotechnology, Seongnam, Korea). RNA was reverse transcribed into cDNA and was performed by a cDNA synthesis kit (iNtRON Biotechnol- ogy) for ﬁrst-strand cDNA synthesis, in accordance with the manufacturer’s instructions, in order to synthesize the RNA template for qRT-PCR. Primers used are presented in Table1. A total 20 µL volume was used in the qRT-PCR: 2 µL sample cDNA and 2 µL of primer mix (10 µM), 10 µL of 2 × SYBR premix (Life Technologies, Carlsbad, CA, USA), and 6 µL of nuclease-free water. The PCR was performed by the StepOnePlus real-time PCR system (Applied Biosystems, Courtaboeuf, France).

4.7. Biofilm Inhibition Assay The effect of the TCA on the biofilm formation of S. aureus (ATCC 33591 and DPS-1) had been identified by biofilm inhibition assay as previously described [9]. One hundred microliters of overnight cultures (0.5 MacFarland bacterial culture) were added and treated with various concentrations of TCA (1/16 MIC, 1/8 MIC, and 1/2 MIC) to each well of 96-well microtiter plates. After incubation for 24 h at 37 ◦C, the planktonic cells were removed, washed with PBS for 3 times in each well, stained with 1% (w/v) crystal violet for 10 min, and then washed again with PBS. The stained biofilms were solubilized in 100 µL of absolute ethanol and the optical density (OD) values were measured at 600 nm. All biofilm assays were performed in triplicates, and the negative control was the bacteria in their respective media without any drug. Finally, the percentage of inhibition of biofilm was calculated by the following formula. Percentage of inhibition = 100 − [(OD 600 nm of the treated wells)/(mean OD 600 nm of the negative control wells) × 100)].

4.8. Statistical Analysis Analyses were performed in triplicate and data were presented as the mean ± standard deviation. The results were statistically analyzed by an independent Scheffe’st-test (SPSS software version 22.0; IBM SPSS, Armonk, NY, USA). A p-value of less than 0.05 was considered to be statistically signiﬁcant.

Author Contributions: Conceptualization, D.-Y.K. and S.W.; data curation, S.W.; formal analysis, S.W.; funding acquisition, D.-Y.K.; investigation, S.W.; project administration, D.-Y.K. and O.-H.K.; resource, O.-H.K.; supervision, D.-Y.K. and O.-H.K.; validation, D.-Y.K. and O.-H.K.; visualization, S.W.; writing—original draft, S.W.; writing—review and editing, D.-Y.K. and O.-H.K. All authors have read and agreed to the published version of the manuscript. Int. J. Mol. Sci. 2021, 22, 2752 8 of 9

Funding: This study was supported by the Basic Science Research Program through the National Re- search Foundation (NRF) of Korea funded by the Ministry of Education (NRF-2016R1D1A1B03934552) and by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (NRF-2017R1A2B1009979), and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2020R1F1A1054227). Institutional Review Board Statement: This research has been reviewed by the IACUC and it has been approved(Approved Number WKU17-04). Informed Consent Statement: Patient consent was received and performed according to the guidelines of the Wonkwang University Institutional Review Board IRB. Data Availability Statement: Personal information is included, so it was conducted for research only. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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