Received: 31 August 2017 Revised: 19 December 2017 Accepted: 10 January 2018 DOI: 10.1002/ptr.6042 RESEARCH ARTICLE Bioactive compounds from the African medicinal plant Cleistochlamys kirkii as resistance modifiers in bacteria Annamária Kincses1 Ana Margarida | Borisz Varga1 Madureira3 | | Ákos Csonka1,2 3 Maria‐José U. Ferreira Shirley Sancha3 | | | Silva Mulhovo4 Gabriella Spengler | 1 1 Department of Medical Microbiology and Immunobiology, Faculty of Medicine, University of Szeged, Dóm tér 10, Szeged 6720, Hungary 2 Department of Traumatology, Faculty of Medicine, University of Szeged, Semmelweis utca 6, Szeged 6725, Hungary 3 Research Institute for Medicines (iMed. Ulisboa), Faculty of Pharmacy, University of Lisbon, Avenue Professor Gama Pinto, Lisbon 1649‐003, Portugal 4 Mozambican and Ethnoscience Study Center (CEMEC), Faculty of Mathematics and Natural Sciences, Pedagogic University, Lhanguene Campus, Av. de Moçambique, Maputo 21402161, Mozambique Correspondence Gabriella Spengler, Department of Medical Microbiology and Immunobiology, Faculty of Medicine, University of Szeged, Dóm tér 10, Szeged 6720, Hungary. Email: spengler.gabriella@med.u‐szeged.hu Funding information Portuguese Foundation for Science and Technology (FCT), Grant/Award Numbers: UID/ DTP/04138/2013 and PTDC/QEQ‐MED/ 0905/2012; European Social Fund, Grant/ Award Number: TÁMOP 4.2.4. A/2‐11‐1‐ 2012‐0001; ÚNKP‐17‐3 New National Excellence Program of the Ministry of Human Capacities of Hungary; János Bolyai Research Scholarship of the Hungarian Academy of Sciences; University of Szeged, Hungary, Grant/ Award Number: GINOP‐2.3.2‐15‐2016‐ 00012; European Union and the State of Hungary 1 | Cleistochlamys kirkii (Benth) Oliv. (Annonaceae) is a medicinal plant traditionally used in Mozambique to treat infectious diseases. The aim of this study was to find resistance modifiers in C. kirkii for Gram‐positive and Gram‐negative model bacterial strains. One of the most important resistance mechanisms in bacteria is the efflux pump‐related multidrug resistance. Therefore, polycarpol (1), three C‐benzylated flavanones (2–4), and acetylmelodorinol (5) were evaluated for their multidrug resistance‐reverting activity on methicillin‐susceptible and methicillin‐resistant Staphylococcus aureus and Escherichia coli AG100 and AG100 A strains overexpressing and lacking the AcrAB–TolC efflux pump system. The combined effects of antibiotics and compounds (2 and 4) were also assessed by using the checkerboard microdilution method in both S. aureus strains. The relative gene expression of the efflux pump genes was determined by real‐time reverse transcriptase quantitative polymerase chain reaction. The inhibition of quorum sensing was also investigated. The combined effect of the antibiotics and compound 2 or 4 on the methicillin‐ sensitive S. aureus resulted in synergism. The most active compounds 2 and 4 increased the expression of the efflux pump genes. These results suggested that C. kirkii constituents could be effective adjuvants in the antibiotic treatment of infections. KEY W ORDS C‐benzylated flavanones, Cleistochlamys kirkii, efflux pump, Escherichia coli AG100, methicillin‐ resistant Staphylococcus aureus, quorum sensing I N T RO D U CT I O N extrusion of toxic substances into the environment (Aparna, Dineshkumar, Mohanalakshmi, Velmurugan, & Hopper, 2014; Webber The increasing resistance of bacterial isolates is a high concern in the & Piddock, 2003). therapy of infectious diseases. The widespread and inappropriate use In Staphylococcus aureus, the most studied MDR pump is the chro- of antibiotics has contributed to the selection of resistant bacteria. Mul- mosomally encoded NorA, which is a member of the major facilitator tidrug‐resistant (MDR) bacteria show resistance against a broad range of superfamily. NorA can transport hydrophilic compounds, quaternary antimicrobials, and one of the most important resistance mechanisms is ammonium compounds, and dyes. The MepA chromosomally encoded the presence of multidrug‐resistant efflux pumps (EPs). These membrane efflux transporter described in S. aureus belongs to the multidrug and proteins have physiological functions, and they are involved in the toxic compound extrusion family. MepA can recognize fluoroquinolones, Phytotherapy Research. 2018;1–8. wileyonlinelibrary.com/journal/ptr Copyright © 2018 John Wiley & Sons, Ltd. 1 2 KINCSES ET AL. glycylcyclines, dyes, and quaternary ammonium compounds (Costa, of several compounds with different scaffolds. Among them, Viveiros, Amaral, & Couto, 2013). C‐benzylated flavanones, namely, dichamanetin and the α,β‐unsatu- The discovery of plant‐derived antimicrobials has drawn particular rated lactone (−)‐cleistenolide, have shown relevant antibacterial activ- attention (Balogh et al., 2014; Hintz, Matthews, & Di, 2015). Numerous ity against Gram‐positive bacteria, including drug‐resistant strains phytochemicals have minimal toxic activity, and they could be used in (Pereira et al., 2016). Moreover, in the combination with β‐lactam anti- order to overcome drug resistance in bacteria by blocking multidrug biotics and vancomycin, polycarpol bearing a triterpenic scaffold exhib- EPs (Stavri, Piddock, & Gibbons, 2007). It is important to note that EP ited a strong synergistic effect against Gram‐positive resistant strains. inhibitors (EPIs) from plant sources can inhibit the activity of bacterial In this study, polycarpol (1) and C‐benzylated flavanones 2–4 and a efflux systems; furthermore, they can potentiate the efficacy of antibi- heptane derivative (5) were further investigated for their ability as both otics as well. It has been described that reserpine, isolated from the roots EP and QS inhibitors of the efflux systems of Gram‐negative and of Rauwolfia vomitoria, inhibits the Bmr EP of Bacillus subtilis (Klyachko, Gram‐positive bacteria. Furthermore, the ability of compounds 2 and Schuldiner, & Neyfakh, 1997). Several Berberis species producing berber- 4 to potentiate the effect of tetracycline and the fluoroquinolone anti- ine were also found to synthesize 5′‐methoxyhydnocarpin, an inhibitor biotic ciprofloxacin on methicillin‐susceptible and methicillin‐resistant of the NorA MDR pump of S. aureus (Stermitz, Lorenz, Tawara, Zenewicz, S. aureus (MRSA) strains was also described. & Lewis, 2000). Cucurbitane‐type triterpenoids, isolated from the aerial parts of the African medicinal plant Momordica balsamina, have shown activity against the EP systems of Gram‐positive bacteria (Ramalhete, 2 MATERIALS AND METHODS | Da Cruz, et al., 2011a; Ramalhete, Lopes, et al., 2011b; Ramalhete, Spengler, et al., 2011c). 2.1 | Compounds tested The bacterial quorum sensing (QS) is a cell‐to‐cell communication Triterpene polycarpol (1), C‐benzylated flavanones chamanetin (2), system that is based on chemical signals, namely, autoinducers (AIs). In isochamanetin (3), dichamanetin (4), and the heptane derivative Gram‐negative bacteria, the most common AI is the N‐acyl homoserine acetylmelodorinol (5; Figure 1) were isolated from the methanol lactone (AHL; Varga et al., 2011). EPs and QS signals play an important extract of the root barks of C. kirkii, as it has previously been described role in the development of bacterial virulence. The QS system and the (Pereira et al., 2016). The purity of all the compounds was higher than AIs are able to influence the expression of transporter genes. These 95% based on high‐performance liquid chromatography analysis and EPs have the ability to transport AI molecules to the external environ- nuclear magnetic resonance spectroscopy. The compounds were dis- ment of bacteria, thus facilitating cell‐to‐cell communication (Spengler, solved in dimethyl sulfoxide (DMSO). Kincses, Gajdács, & Amaral, 2017). Cleistochlamys kirkii (Benth) Oliv. (Annonaceae) is an African medicinal plant traditionally used in Mozambique for the treatment of wound infections, tuberculosis, and rheumatism (Verzár & Petri, 1987). Previously, in a research for antimicrobial compounds from African 2.2 | Reagents and media Promethazine (PMZ; EGIS), ethidium bromide (EB), verapamil, tetracycline‐hydrochloride (TET), ciprofloxacin‐hydrochloride (CIP), and medicinal plants (Cabral et al., 2015; Kolaczkowski et al., 2010; Luria–Bertani (LB) broth, and LB agar were purchased from Sigma‐ Madureira, Ramalhete, Mulhovo, Duarte, & Ferreira, 2012; Pereira Aldrich Chemie GmbH (Steinheim, Germany). The modified LB medium et al., 2016; Ramalhete et al., 2010), bioassay‐guided fractionation of (LB*) was prepared from yeast extract 5 g/L, tryptone 10 g/L, NaCl the methanol extract of the root barks of C. kirkii led to the isolation 10 g/L, K2HPO4 1 g/L, MgSO4 × 7H2O 0.3 g/L, and FeNaEDTA FIGURE 1 1–5 Chemical structures of compounds KINCSES 3 ET AL. 36 mg/L. In case of modified LB* agar, the LB* medium was supple- synergism, an additive effect (or no interaction), and antagonism, mented with agar 20 g/L (Difco). pH was adjusted to 7.2. Tryptic soy respectively (Chou & Martin, 2005). broth (TSB), tryptic soy agar, and Mueller–Hinton (MH) broth were purchased from Scharlau Chemie S. A. (Barcelona, Spain). 2.3 | Bacterial strains Compounds were evaluated against the Gram‐negative wild‐type 2.6 | Real‐time accumulation assay by Roche LightCycler real‐time thermocycler The activity of compounds on the real‐time accumulation of EB was assessed by the automated EB method (Viveiros et al., 2008) using a Escherichia coli K‐12 AG100 strain [argE3 thi‐1 rpsL xyl mtl LightCycler real‐time thermocycler (LightCycler 1.5, Roche, Indianapo- Δ(gal‐uvrB) supE44], expressing the AcrAB–TolC EP at its basal level lis, IN, USA). Briefly, an aliquot of an overnight culture of the S. aureus and its AcrAB–TolC‐deleted mutant E. coli AG100 A strain. These strain in TSB medium was transferred to fresh TSB medium, and it was strains were kindly provided by Prof. Dr. Hiroshi Nikaido (Department incubated until it reached an optical density (OD) of 0.6 at 600 nm. In of Molecular and Cell Biology and Chemistry, University of California, case of E. coli, the medium used in the assay was LB broth; the prepa- Berkeley, CA, USA). ration of the inoculum was similar to the one of S. aureus. The cells The compounds were further evaluated against two Gram‐positive were washed with phosphate‐buffered saline (PBS; pH 7.4) and centri- strains, S. aureus American Type Culture Collection (ATCC) 25923, fuged at 13,000 × g for 3 min, the pellets were resuspended in PBS used as the methicillin‐susceptible reference strain, and the methicillin (pH 7.4), and the OD was adjusted to 0.6 at 600 nm. The compounds and ofloxacin‐resistant S. aureus 272123 clinical isolate, which was were added individually at different concentrations at MIC/2, MIC/3, kindly provided by Prof. Dr. Leonard Amaral (Institute of Hygiene MIC/4, or MIC/5 (in double concentrated form) to the EB solution in and Tropical Medicine, Lisbon, Portugal). PBS. The final concentration of EB was based on the MIC and the fluo- For QS tests, the following strains were used: Chromobacterium rescent signal produced by this amount of EB. In case of S. aureus violaceum 026 (CV026) as sensor strain and Enterobacter cloaceae strains, the concentration of EB was 0.5 μg/ml, for E. coli AG100 31298 as AHL producer strain (a clinical isolate from a wound). When 1 μg/ml, and in case of E. coli AG100 A, it was 0.25 μg/ml. Then, C. violaceum reaches a high cell density, it produces a purple pigment, 10 μl of the EB solution containing the compound was transferred into namely, violacein (Ballantine, Beer, Crutchley, Dodd, & Palmer, 1958). standard glass capillary tubes of 20 μl maximum volume (Roche), and 2.4 Determination of minimum inhibitory concentrations by microdilution method the capillaries. The capillaries containing the samples were placed into 10 μl of bacterial suspension (OD of 0.6 at 600 nm) was added to | The minimum inhibitory concentrations (MICs) of compounds were determined according to the Clinical and Laboratory Standard Institute guidelines (2017) in three independent assays. The solvent DMSO had no antibacterial effect. 2.5 | the carousel (Roche), and the fluorescence was monitored at the FL‐2 channel in every minute on a real‐time basis. From the real‐time data, the activity of the compound, namely, the relative final fluorescence index (RFI) of the last time point (minute 30) of the EB accumulation assay, was calculated according to the following formula: Interaction between antibiotics and compounds RFI ¼ The combined effect of compounds and antibiotics on the growth inhibition of S. aureus was evaluated by the checkerboard method. Twofold serial dilutions of antibiotics were prepared in MH broth on the horizontal rows of microtiter plate and then cross‐diluted vertically by twofold serial dilutions of the compounds (Wolfart et al., 2006). For this assay, only the compounds with well‐defined MIC values could be used. Consequently, the combination assays were carried out on methicillin‐susceptible and MRSA strains. The dilutions of the antibiotics (TET or CIP) were made in a horizontal direction in 100 μl, and the dilutions of compounds were made vertically in the microtiter plate RFtreated −RF untreated RF untreated where RFtreated is the relative fluorescence at the last time point of EB retention curve in the presence of an inhibitor and RFuntreated is the relative fluorescence at the last time point of the EB retention curve of the untreated control having the solvent control (DMSO). Verapamil was applied as positive control on Gram‐positive strains, and PMZ was used on Gram‐negative strains. 2.7 | Assay for quorum sensing inhibition in 50 μl. After the dilution of an overnight culture, bacterial cells were LB* was used for these experiments. The sensor strain C. violaceum resuspended in MH medium containing 1 × 104 cells and distributed 026 and the AHL producer strains E. cloaceae 31298 were inoculated into each well. The plates were incubated for 18 hr at 37 °C. The cell as parallel lines and incubated at room temperature (20 °C) for growth rate was determined after MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐ 24–48 hr. QS inhibition was monitored by the agar diffusion method. 2,5‐diphenyltetrazolium bromide) staining, as described elsewhere Filter paper discs (7.0 mm in diameter) were impregnated with 10 μl (Wolfart et al., 2006). The combination index (CI) values at 90% growth of stock solutions (10 mM) of the compounds in DMSO. The discs inhibition (ED90) were determined by using CompuSyn software to were placed between the parallel lines of the sensor and the AHL plot 4 or 5 data points for each ratio (www.combosyn.com, ComboSyn, producer strains on the surface of the nutrient agar. The plates were Inc., Paramus, NJ, USA). CI values were calculated by means of the incubated at room temperature for another 24–48 hr, and the interac- median‐effect equation, where CI < 1, CI = 1, and CI > 1 represent tions between the strains and compounds were evaluated for the 4 KINCSES reduction in the size of the zone of pigment production and the zone ET AL. Hercules, CA, USA), and the manufacturer's recommendations of the of growth inhibition of the affected strains, in millimeters. PMZ was SensiFAST™ SYBR No‐ROX One‐Step Kit (Bioline GmbH, Luckenwalde, applied as positive control (Varga et al., 2011). Germany) were strictly adhered to. Briefly, each well of the 96‐well microtiter plates in a final volume of 20 μl contained 10 μl of the 2× SensiFAST™ SYBR No‐ROX One‐Step Mix, 0.2 μl reverse transcriptase, 2.8 | Expression analyses of genes by real‐time reverse transcriptase quantitative polymerase chain reaction S. aureus ATCC 25923 and S. aureus 272123 strains were cultured in TSB broth and were incubated overnight at 37 °C with shaking. On 0.4 μl RiboSafe RNase Inhibitor, 5.4 μl diethyl pyrocarbonate‐treated water, 500 nM of each primer, and approximately 20 ng of the total RNA in RNAase‐free water. Thermal cycling was initiated with a denaturation step of 5 min at 95 °C, followed by 40 cycles, each of 10 s at 95 °C, 30 s at 57 °C, and 20 s at 72 °C. the day of RNA isolation, the bacterial suspensions (OD of 0.6 at 600 nm) were transferred to 10‐ml tubes in 3‐ml aliquots, and 5 μM of compound 2 or 0.5 μM of compound 4 was added to the tubes, which were incubated at 37 °C. After 4 hr of culturing, the tubes were centrifuged at 12,000 × g for 2 min. Pellets were suspended in 100 μl 3 3.1 RESULTS | | In vitro antibacterial activity of compounds Tris–EDTA buffer containing 1 mg/ml lysozyme by vigorous vortexing, Compounds (1–5) were assessed for their antibacterial activity against and they were incubated at 37 °C for 10 min. The total RNA was iso- methicillin‐susceptible S. aureus ATCC 25923, and the methicillin‐ and lated in an RNase‐free environment using the NucleoSpin RNA kit ofloxacin‐resistant S. aureus 272123 clinical isolate. Wild‐type E. coli (Macherey Nagel, Düren, Germany) according to the manufacturer's K‐12 AG100 strain, and E. coli AG100 A strain, overexpressing and instructions. Purified RNA was stored in RNase‐free water in nucle- lacking the AcrAB–TolC EP system, respectively, were used as Gram‐ ase‐free collection tubes and was maintained at −20 °C until quantifi- negative models. In addition, the antibacterial activity of the cation was performed. The concentration of the extracted RNA compounds was tested on QS strains C. violaceum and E. cloaceae. templates was assessed by spectrophotometry at 260 nm. Expression Concerning the antibacterial effect of the compounds, chamanetin of the EP genes norA and mepA was studied by reverse transcription (2) and dichamanetin (4) had a potent antibacterial effect on the of the total RNA. The data obtained for gene targets were normalized S. aureus strains. MIC value of compound 2 was 12.5 μM on reference against the S. aureus 16S ribosomal RNA measured in the same sample. S. aureus; however, the MIC of the methicillin‐ and ofloxacin‐resistant The primers (Couto, Costa, Viveiros, Martins, & Amaral, 2008) used in strain was 25 μM. Compound 4 was the most effective flavanone the assay were the following: because its MIC value on S. aureus ATCC 25923 was 0.8 μM; further- 1. Sequence (5′‐3′) of norA (246 bp) TCGTCTTAGCGTTCGG more, on the methicillin‐resistant strain, it was 1.56 μM. The compounds had no antibacterial effect on the Gram‐negative TTTA (Fw) TCCAGTAACCATCGGC E. coli AG100, AG100 A, C. violaceum, and E. cloaceae strains. AATA (Rv) 2. Sequence (5′‐3′) of mepA (198 bp) TGCTGCTGCTCTGTTC TTTA (Fw) GCGAAGTTTCCATAAT 3. Sequence (5′‐3′) of 16S rRNS (492 bp) 3.2 | Combination effects of chamanetin (2) and dichamanetin (4) with antibiotics GTGC (Rv) The type of interaction between the antibacterial C‐benzylated flava- AGAGTTTGATCMTGGC nones 2 and 4 and tetracycline and the fluoroquinolone antibiotic TCAG (Fw) ciprofloxacin was evaluated on methicillin‐susceptible (ATCC 25923) GWATTACCGCG and MRSA strains by the checkerboard assay. The results are GCKGCTG (Rv) presented in Tables 1a and 1b as CI values. CI values <1 indicate a synergistic interaction between the compound and the antibiotic Real‐time quantification of the RNA templates by real‐time one‐ (Chou & Martin, 2005). As it can be observed, the combined effect of step reverse transcriptase quantitative polymerase chain reaction was TET and compound 2 or 4 on S. aureus ATCC 25923 resulted in syner- performed in a CFX96 Touch real‐time PCR detection system (Bio‐Rad, gism. The most effective ratio of antibiotic and compound was 1:20 TABLE 1a Combination assays on Staphylococcus aureus ATCC 25923 strain Staphylococcus aureus ATCC 25923 Combination Best ratio CI at ED90 SD (+/−) Interaction Synergism Tetracycline + chamanetin (2) 1:20 0.63786 0.13419 Tetracycline + dichamanetin (4) 1:1 0.42093 0.10354 Synergism Ciprofloxacin + chamanetin (2) 1.3:12.5 0.81577 0.23974 Slight synergism Ciprofloxacin + dichamanetin (4) 1.3:1 0.68615 0.27953 Synergism Note. Starting concentration of tetracycline: 5.2 μM; ciprofloxacin: 2.6 μM; and compound 2: 25 μM and compound 4: 2 μM. ATCC = American Type Culture Collection; CI = combination index. KINCSES 5 ET AL. TABLE 1b Combination assays on MRSA 272123 strain Staphylococcus aureus 272123 Combination Best ratio CI at ED90 SD (+/−) Interaction Tetracycline + chamanetin (2) 6:25 1.4595 0.29390 Antagonism Tetracycline + dichamanetin (4) 100:1 0.59402 0.34521 Synergism Ciprofloxacin + chamanetin (2) 13:8 0.85030 0.21648 Slight synergism Ciprofloxacin + dichamanetin (4) 32:1 1.35064 0.24946 Moderate antagonism Note. Starting concentration of tetracycline: 100 μM; ciprofloxacin: 64 μM; and compound 2: 50 μM and compound 4: 3 μM. Ratio: antibiotic and tested compound (μM). CI < 1, CI = 1, and CI > 1 represent synergism, an additive effect (or no interaction), and antagonism, respectively. CI = combination index; MRSA = methicillin‐resistant Staphylococcus aureus. and 1:1, respectively. Similarly, CIP also acted synergistically with compounds 2 and 4 were selected for gene expression studies. In the compounds 2 and 4, being the most active ratio of antibiotic and com- real‐time quantitative RT‐PCR assay, the genes of NorA and MepA pound 1.3:12.5 and 1.3:1, respectively (Table 1a). transporters were investigated. As shown in Figure 2bA, compound 2 Against the MRSA strain, compound 2 showed antagonistic effect at 5 μM significantly up‐regulated the expression of norA and mepA with TET and slight synergism with CIP. The interactions of compound genes after 4 hr of exposure in the MRSA strain. Compound 4 at 4 with TET and CIP on the MRSA strain were synergism and moderate 0.5 μM also significantly up‐regulated both EP genes after 4 hr of antagonism, respectively (Table 1b). exposure in the MRSA as presented in Figure 2bB. In the S. aureus ATCC strain, the expression level of the mepA gene was not influ- 3.3 | Efflux pump‐inhibiting activity The EB accumulation assay provides information about the intracellu- enced; nevertheless, the norA gene was significantly up‐regulated by compounds 2 (19.84‐fold increase) at 5 μM and 4 (2.39‐fold increase) at 0.5 μM (data not shown). lar accumulation of the general EP substrate EB. A potential EPI increases the fluorescence level of EB because of its accumulation within the bacterial cell. The EP‐inhibiting activity of the compounds 4 | DISCUSSION was compared on the basis of the RFI of the real‐time accumulation curves in Gram‐positive and Gram‐negative strains (Figure 2a). In case Natural compounds isolated from plants represent a valuable source of of real‐time EB accumulation by the LightCycler thermocycler, the new antibacterial agents. When analyzing the results, the potent anti- amount of EB accumulated by cells is higher if the difference between bacterial activity found for dichamanetin (4) and also the significant RFtreated and RFuntreated is greater; therefore, the degree of inhibition of activity of chamanetin (2) corroborated our previous studies on this the EP system by the compound becomes greater. set of rare C‐benzylated flavanones (Pereira et al., 2016). The strongest As shown in Figure 2a, compounds 2, 3, and 5 had EP‐inhibiting activity of dichamanetin (4), when compared with chamanetin (2), activity compared with verapamil (RFI: 0.29) on the S. aureus ATCC seems to be due to its higher lipophilic character resulting from the 25923 strain, and the most active compound was compound 2. How- extra benzyl group at C‐6. In contrast to chamanetin (2), isochamanetin ever, compounds 1–5 had no EPI activity on the MRSA strain at the (3), differing only in the position of the benzyl group, was inactive at concentrations applied in the assay. the concentration tested. Thus, besides the importance of lipophilicity, Concerning the inhibitory activity on Gram‐negatives, triterpene polycarpol (1) and acetylmelodorinol (5) compared with the PMZ the presence of a benzyl moiety at C‐8 appears to play a decisive role in the antibacterial activity of this type of compounds. (RFI: 0.15) could inhibit the AcrAB–TolC system of E. coli AG100. Com- In the combination assays, chamanetin (2) and dichamanetin (4) pound 1 proved to be the most effective EPI (Figure 2a). On the basis had synergistic activity with tetracycline and ciprofloxacin on the of the real‐time accumulation data, compounds 1–5 had no effect on S. aureus ATCC 25923 strain. Furthermore, on the MRSA strain, the E. coli AG100 A strain lacking the AcrAB–TolC pump. dichamanetin (4) and chamanetin (2), combined with tetracycline and ciprofloxacin, respectively, also showed synergism, which indicates 3.4 | Anti‐quorum sensing activity The QS inhibition activity of compounds was defined measuring the that these compounds could be potential adjuvants in the therapy. Besides having an antibacterial effect, both compounds (2 and 4) could inhibit the activity of EPs, they were QS inhibitors, and they sig- colorless zone around the disc on C. violaceum as described previously nificantly increased the expression of EP genes norA and mepA after (Varga et al., 2011). Compounds 1, 2, 4, and 5 were able to inhibit 4 hr of exposure in the MRSA strain. Furthermore, the NorA and MepA effectively the QS between CV026 and E. cloaceae compared with pumps of the MRSA strain could not be inhibited because of the over- the positive control PMZ (Table 2). expression of these pumps. The change in gene expression could be due to the stress response against compounds 2 and 4 because these 3.5 | Relative expression of efflux pump genes compounds as potential noxious agents for S. aureus had to be extruded from the cytoplasm as soon as possible. This stress response In order to evaluate the effect of compounds on the relative expres- can be the explanation for the up‐regulation of the EP genes after 4 hr. sion of EP genes in both S. aureus strains, the most effective The difference between MRSA and ATCC is due to the overexpression 6 KINCSES ET AL. The minimum inhibitory concentration (MIC) and efflux pump modulating effect of compounds 1–5. (a) Relative final fluorescence index (RFI) for the effect of compounds 1–5 on the AcrAB–TolC‐ expressing Escherichia coli AG100 and Staphylococcus aureus American Type Culture Collection (ATCC) 25923 strains. (b) Relative gene expression levels of norA and mepA genes in the presence of (A) chamanetin (2) and (B) dichamanetin (4) in methicillin resistant Staphylococcus aureus (MRSA) 272123, after 4‐hr exposure. The line denotes the threshold value, which was set at a twofold increase in transcripts FIGURE 2 of the mepA gene in the resistant strain and it has low expression level in Inhibitory effects of compounds 1–5 on QS signal transmission TABLE 2 Compound QS inhibition zone in mm 1 51 2 50 3 — 4 53 5 52 Promethazine 46 Note. Ten microliters of 10 mM stock solution was added into the filter paper discs (10 μM per disc), and the colorless zone around the disc measured on Chromobacterium violaceum was measured after incubation for 24–48 hr at room temperature. QS = quorum sensing. the ATCC strain. Compounds 2 and 4 increased the expression of mepA gene in the MRSA, but in the ATCC strain, these compounds did not influence the expression level of mepA. It has been demonstrated by other studies that the overexpression of EPs confers a fitness cost for the organism, for example, a resistant isolate overexpressing EPs shows reduced production of virulence determinants. Usually the overexpression of pump genes is often related to global effects on bacterial physiology influencing virulence (Ledda et al., 2017). In Gram‐negatives, compounds 1–5 had no antibacterial activity. Polycarpol (1) was the most effective EPI, inhibiting the AcrAB–TolC transport system in the E. coli AG100 strain. The AcrAB–TolC system belongs to the resistance nodulation division transporters, utilizing the proton motive force to extrude toxic agents out of the bacterial KINCSES 7 ET AL. cell. Since compound 1 had no EPI effect on the AcrAB–TolC pump‐ Gabriella Spengler http://orcid.org/0000-0001-8085-0950 deleted E. coli strain, it may have a direct EPI effect on the AcrAB–TolC transporter system. The effect of polycarpol could be due to its high RE FE RE NC ES lipophilicity (Pereira et al., 2016), which can increase the membrane Aparna, V., Dineshkumar, K., Mohanalakshmi, N., Velmurugan, D., & Hopper, W. (2014). Identification of natural compound inhibitors for multidrug efflux pumps of Escherichia coli and Pseudomonas aeruginosa using in silico high‐throughput virtual screening and in vitro validation. PLoS One, 9, e101840. permeability of bacteria. In addition to flavanones 2 and 4, polycarpol (1) and acetylmelodorinol (5) were also able to inhibit effectively the bacterial communication, suggesting that they could be applied as anti‐QS agents. 5 | Balogh, E. P., Mosolygó, T., Tiricz, H., Szabó, A. M., Karai, A., Kerekes, F., … Burián, K. (2014). Anti‐chlamydial effect of plant peptides. Acta Microbiol Immunol, 61, 229–239. C O N CL U S I O N S To summarize our findings, C. kirkii constituents, chamanetin (2) and dichamanetin (4), inhibited the growth of both S. aureus strains. The three flavanones tested (2–4) increased the accumulation of the EP substrate EB on sensitive S. aureus strain, and compounds 1, 2, 4, and 5 prevented the cell‐to‐cell communication. Polycarpol (1) was an effective inhibitor of the AcrAB–TolC system of E. coli, which might be explained by its interference with membrane permeability and modulation of the function of the pump. Dichamanetin (4) and chamanetin (2) showed synergistic effect on MRSA and sensitive S. aureus strains implying that they could restore and increase the efficacy of antibiotics, especially in MDR infections. It can be assumed that the advantageous properties of C. kirkii constituents can influence the QS system and operation of EPs decreasing virulence and antibiotic resistance (Varga et al., 2011). According to the results, the most active compounds, chamanetin (2) and dichamanetin (4), could be possible resistance modifiers because they possess antibacterial, EPI, and anti‐QS properties; moreover, these compounds might be used as adjuvants in the antibiotic therapy because they may be able to reduce the bacterial virulence. Furthermore, they can potentiate the activity of antibiotics. ACKNOWLEDGEMEN TS This research was supported by the European Union and the State of Hungary and by the Portuguese Foundation for Science and Technology (FCT), Projects PTDC/QEQ‐MED/0905/2012 and UID/DTP/ 04138/2013, and co‐financed by the European Social Fund in the framework of TÁMOP 4.2.4. A/2‐11‐1‐2012‐0001 “National Excellence Program.” This study was supported by the GINOP‐2.3.2‐15‐ 2016‐00012 project (University of Szeged, Hungary), and Gabriella Spengler was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. Annamária Kincses was supported by the ÚNKP‐17‐3 New National Excellence Program of the Ministry of Human Capacities of Hungary. The authors thank the Portuguese Embassy in Mozambique, as well as the Portuguese Office of International Affairs, for plant transport. CONF LICT OF INTE R ES T The authors declare no conflict of interest. ORCID Maria‐José U. Ferreira Ballantine JA, Beer RJS, Crutchley DJ, Dodd GM, Palmer DR. 1958. The synthesis of violacein and related compounds. Proc Chem Soc 232–33. http://orcid.org/0000-0002-8742-1486 Cabral, V., Luo, X., Junqueira, E., Costa, S. S., Mulhovo, S., DuarteA, C. I., … Ferreira, M. J. U. (2015). 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