DRAGANA D. BOŽIĆ¹, MARINA T. MILENKOVIĆ¹, BRANKA M. IVKOVIĆ², ANDERS RHOD LARSEN³ and IVANA B. ĆIRKOVIĆ⁴

¹ Department of Microbiology and Immunology, University of Belgrade-Faculty of Pharmacy, Belgrade, Serbia
² Department of Pharmaceutical Chemistry, University of Belgrade-Faculty of Pharmacy, Belgrade, Serbia
³ Department of Microbiological Surveillance and Research, Statens Serum Institute, Copenhagen, Denmark
⁴ Department of Bacteriology, Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Belgrade, Serbia

*Corresponding author: D.D. Božić, Department of Microbiology and Immunology, University of  Belgrade-Faculty of Pharmacy, Belgrade, Serbia; e-mail: dragana.vucicevic@pharmacy.bg.ac.rs.

Submitted 25 July 2014, revised 3 April 2015, accepted 20 May 2015
DOI: 10.5604/17331331.1185237

Abstract

Pathogenicity of methicillin-resistant Staphylococcus aureus (MRSA) is associated with a broad spectrum of virulence factors, amongst which is α-hemolysin. The aim of this study was to investigate the effect of three newly-synthesized chalcones (1,3- Bis-(2-hydroxy-phenyl)-propenone, 3-(3-Hydroxy-phenyl)-1-(2-hydroxy-phenyl)-propenone and 3-(4-Hydroxy-phenyl)-1-(2-hydroxy-phenyl)-propenone) on α-hemolysin production of clinical isolates of MRSA. Subinhibitory concentrations of the tested compounds reduced hemolytic activity of MRSA strains, with almost complete abolishment of hemolysis at concentrations in the range of 1/2-1/4 x MIC (25-12.5 μg/ml). In conclusion, newly-synthesized chalcones tested in this study showed potent inhibitory activity on α-hemolysin production of multiresistant and genetically diverse MRSA strains.

Key words: α-hemolysin,  chalcones, MRSA

Pathogenicity of methicillin-resistant Staphylococcus aureus (MRSA) is directly associated with a broad spectrum of virulence factors, amongst which is α-hemolysin (i.e. HIa or α-toxin). It is generally considered that α-hemolysin plays a central role in the pathogenesis of staphylococcal infections, especially in pulmonary infections caused by these bacteria (Bubeck Wardenburg et al., 2007; Burlak et al., 2007; Montgomery et al., 2008). Approximately one half of staphylococcal necrotizing pneumonia cases affecting previously healthy adults and children are caused by community-associated MRSA strains (Ragle and Bubeck Wardenburg, 2009). Besides direct lysis of the pulmonary cells, α-hemolysin also activates alveolar macrophages or monocytes, induces massive polymorphonuclear leukocyte influx into lung parenchyma with subsequent degranulation and destruction of microvascular endothelium and adjacent tissues, and induces platelet-neutrophil co-aggregation leading to the host response to toxin that contribute to the severity of lung destruction (Parimon et al., 2013). Such complex pathogenicity and occurrence of multiresistant strains urge the development of antimicrobial compounds that selectively target virulence factors. In past years, several studies investigated the inhibitory activity of various compounds against extracellular virulence factors of S. aureus (Escaich, 2008; Cegelski et al., 2008). Previously we have reported antimicrobial activity of three newly-synthesized chalcones against clinical isolates of MRSA, and inhibitory effect on the expression of virulence factors related to the early step of bacterial invasion-adherence (i.e. biofilm formation, glycocalyx production and adherence to human fibronectin) (Božić et al., 2014). Therefore, the aim of this study was to investigate the effect of 1,3- Bis-(2-hydroxy-phenyl)-propenone (O-OH), 3-(3-Hydroxy-phenyl)-1-(2-hydroxy-phenyl)-propenone (M-OH) and 3-(4-Hydroxy-phenyl)-1-(2-hydroxy-phenyl)-propenone (P-OH) on α-hemolysin production of clinical isolates of multiresistant and genetically diverse strains of MRSA.

Table I
Genotyping and resistance profiles of MRSA strains

CC type	spa type	N° of strains	SCCmec type	agr  type	PVL	Resistance profile (> 50% of strains)
CC5	t041	9	1	2	–	AM, GEN, KAN, TOB, STR, LIN, CLIN, ER, CLA, AZ, SPIRA, TET, DOX, CHL
	t041	1	1	1	–	AM, GEN, KAN, TOB, STR, LIN, CLIN, ER, CLA, AZ, SPIRA, CHL
	t003	1	2	2	–	KAN, TOB, LIN, CLIN, ER, CLA, AZ, SPIRA
CC8	t030	2	3	1	–	AM, GEN, KAN, TOB, STR, ER, CLA, AZ, TET, DOX, MIN, CHL
	t030	1	3	2	–	AM, GEN, KAN, TOB, STR, LIN, CLIN, ER, CLA, AZ, SPIRA, TET, DOX, MIN
	t969	1	3	1	–	AM, GEN, KAN, TOB, STR, ER, CLA, AZ, TET, DOX
CC45	t015	1	1	2	–	AM, GEN, KAN, TOB, STR, LIN, CLIN, ER, CLA, AZ, SPIRA, CHL
	t583	1	4	1	–	AM, GEN, KAN, TOB, STR, ER, CLA, AZ
CC80	t044	1	4	3	+	KAN, STR, TET
CC152	t595	1	5	1	+	GEN, KAN, TOB, LIN, TET
	t6371	1	5	1	–	GEN, KAN, TOB, ER, CLA, AZ

AM-amikacin, GEN-gentamicin, KAN-kanamycin, TOB-tobramycin, STR-streptomycin, LIN-lincomycin, CLIN-clindamycin, ER-erythromycin, CLA-clarithromycin, AZ-azithromycin, SPIRA-spiramycin, TET-tetracycline, DOX-doxycycline, MIN-minocycline, CHL-chloramphenicol.

Antibacterial activity of chalcones was tested against 20 clinical isolates of MRSA isolated from blood (4), wound (6), sputum (3), endotracheal tube (2), abdominal drain (1), nose (1), skin (1), urine (1) and external auditory canal (1) and one laboratory control strain of methicillin-resistant S. aureus ATCC 43300 (KWIK-STIK^TM, Microbiologics, USA). Identification of the isolates and methicillin resistance were determined by VITEK 2 test cards GP and AST-P580 (bioMérieux, France) and confirmed by PCR for nuc (Brakstad et al., 1992) and mecA (Bignardi et al., 1996) genes. Genotyping (SCCmec, agr, pvl and spa typing) of MRSA was performed according to previously described protocols (Boye et al., 2007; Lina et al., 2003; Harmsen et al., 2003). The spa types were clustered into spa clonal complexes (CCs) using the algorithm based upon repeat pattern (BURP) with Ridom Staph-Type 1.4 software (http://www.ridom.de). The  multiresistance profile of MRSA strains was determined by VITEK 2 test card AST-P580 and further supplemented with disc diffusion test according to CLSI guidelines (CLSI, 2007). Chalcones tested in this study (Fig. 1) were obtained from the Department of Pharmaceutical Chemistry, University of Belgrade-Faculty of Pharmacy, Belgrade, Serbia. Compounds were prepared and characterized as previously described (Božić et al., 2014) and their antimicrobial activity was determined by broth microdilution test according to CLSI guidelines (CLSI 2007). Hemolytic activities of MRSA culture supernatants were determined according to the method of Rowe and Welch (1994). The data obtained in this study were analyzed in SPSS statistical program (PASW statistics for Windows, Version 18.0, Chicago: SPSS Inc. USA) using methods of descriptive statistics, Chi square test and Mann-Whitney U test.

Clinical isolates of MRSA were genetically heterogeneous and expressed multiresistance phenotype (Table I). MRSA strains were classified into SCCmec type I (55.5%), II (5.0%), III (20.0%), IV (10.0%) and V (10.0%) and agr type I (35.0%), II (60.0%) and III (5.0%).  Strains belonged to 10 spa types and were clustered into 5 spa CCs, with most frequent CC5 (55.0%) and CC8 (20.0%).

Tested chalcones exerted inhibitory activity against MRSA, with the order of potency of chalcones (average MIC±SD) as following: O-OH (MIC=37.5 ±13.2 μg/ml) > M-OH (MIC=97.5 ±27.5 μg/ml) > P-OH (MIC=110.8 ±21.1 μg/ml). The most significant dose-dependent inhibition of hemolysis was observed in MRSA supernatants cultivated with O-OH chalcone (Fig. 2). A 91.6-99.7% reduction of hemolysis was detected in all MRSA strains cultivated with ½ x MIC of O-OH. Hemolytic activity of MRSA strains cultivated with 1/2 x MIC (25.0 µg/ml) and 1/4 x MIC (12.5 µg/ml) of O-OH was in the range of 3.8-8.2% of the positive control (p < 0.001). Mild increase of the hemolytic activity was detected after cultivation with 1/8 x MIC (6.2 µg/ml) up to 19% (p < 0.01), and 1/16 x MIC (3.1 µg/ml) up to 34.6% (p < 0.01). The effect of M-OH and P-OH was tested in the two-fold higher concentrations than O-OH chalcone (50.0-6.2 µg/ml), corresponding the same range of MIC’s (1/2 x MIC – 1/16 x MIC). Hemolytic activity of MRSA strains cultivated with 1/2 x MIC and 1/4 x MIC of M-OH was in the range of 7.1-18.8% of the positive control (p < 0.01; p < 0.001), and cultivation with 1/8 x MIC lead to the increase of the hemolysis up to 40.7% of the positive control (p < 0.05). The lowest applied concentration of M-OH (1/16 x MIC, 6.2 µg/ml) did not reduce the hemolytic activity of MRSA (p > 0.05). Statistically significant inhibition of hemolytic activity of MRSA strains cultivated with P-OH chalcone occurred only in concentrations of 1/2 x MIC (7.7%; p < 0.01) and 1/4 x MIC (43.8%; p < 0.05) (Fig. 2). Chalcones themselves did not perform any hemolytic activity or induced hemolysis of rabbit erythrocytes in the same range of MIC’s (1/2 x MIC – 1/16 x MIC; p > 0.05).

Antistaphylococcal activity of various chalcones is strongly correlated with their chemical characteristics. It is generally considered that lipophilicity of the ring A (Nowakowska, 2007) and presence of free hydroxyl group/s at various positions of the ring B are necessary for antistaphylococcal activity of these compounds (Sato et al., 1996; Alcaraz et al., 2000; Kromann et al., 2004; Talia et al., 2011). Three compounds investigated in our study carrying free hydroxyl group at various positions of the B ring exerted strong anti-MRSA activity, with 1,3- Bis-(2-hydroxy-phenyl)-propenone (O-OH) bearing the hydroxyl group at the position 2 of ring B as the most active one. These results are in accordance with the findings of other investigators (Alcaraz et al., 2000; Kromann et al., 2004). Subinhibitory concentrations of the tested compounds also inhibited hemolytic activity of MRSA strains in a dose dependent manner, with almost complete abolishment of hemolysis at concentrations in the range of 1/2 – 1/4 MIC (25-12.5 μg/ml). Inhibition of hemolytic activity of α-hemolysin was dose dependent. Chalcone with free hydroxyl group at the position 2 of ring B was the most active one, since the lowest applied concentration of this compound (1/16 x MIC, 3.1 µg/ml) exerted statistically significant inhibition of hemolysis. Higher concentrations of other tested compounds exerted similar inhibitory effect, with partial or complete recovery of hemolytic activity of α-hemolysin when applied in the lowest concentrations (1/16 x MIC, 6.2 µg/ml). Previously it has been reported that subinhibitory concentrations of chalcones with similar chemical structure isolated from natural sources, like licochalcone A and E, inhibit α-hemolysin production (Qiu et al., 2010a; Zhou et al., 2012). Licochalcone A possess anti-MRSA activity in the range of MIC’s from 2.0-8.0 µg/ml (Fukai et al., 2002; Qiu et al., 2010a), and inhibitory effect on α-hemolysin production in the range of 1/2 – 1/8 x MIC (Qiu et al., 2010a). Licochalcone E was found to be the most active licochalcone against S. aureus, since the inhibitory effect was detected in the range of MIC’s from 1.0-4.0 µg/ml, and inhibition of α-hemolysin production in the range of 1/4 – 1/16 x MIC (Zhou et al., 2012).

Precise mechanism of antimicrobial activity of chalcones and inhibition of α-hemolysin synthesis has not been clarified yet. Several different mechanisms could be involved, like direct enzyme inhibition, inhibition of DNA or RNA synthesis (Mori et al., 1987; Ohemeng et al., 1993) or interference with energy metabolism by inhibition of NADH-cytochrome c reductase, reducing the energy required for active uptake of various metabolites and biosynthesis of macromolecules (Haraguchi et al., 1998; Cushnie and Lamb, 2011). Licochalcone A (Qiu et al., 2010a) or thymol (Qiu et al., 2010b) perform inhibitory effect on α-hemolysin synthesis through inhibition of agrA transcription, one of the components of the agr global regulatory system of S. aureus. Besides influence on agr global regulatory system, licochalcone E also directly inhibits expression of hla-gene encoding α-hemolysin synthesis (Zhou et al., 2012). Accordingly, it is possible that chalcones also inhibit production of α-hemolysin through mechanisms proposed by Qui et al. and Zhou et al. Further studies are necessary to elucidate the precise mechanisms of inhibitory effect of tested chalcones on hemolytic activity of α-hemolysin.

Acknowledgments
Funding: The work of Dragana D. Božić and Ivana B. Ćirković was supported by the Ministry of Education, Science and Technological Development, Republic of Serbia (project grant no. 175039).

Literature

Alcaraz L.E., S.E. Blanco, O.N. Puig, F. Tomas and F.H. Ferretti. 2000. Antibacterial activity of flavonoids against methicillin-resistant Staphylococcus aureus strains. J. Theor. Biol. 205:231-240.

Bignardi G.E., N. Woodford, A. Chapman, A.P. Johnson and D.C.E. Speller. 1996. Detection of the mec-A and phenotypic detection of resistance in Staphylococcus aureus isolates with borderline or low-level methicillin resistance. J. Antimicrob. Chemoth. 37:53-63.

Boye K., M.D. Bartels, I.S. Andersen, J.A. Møller and H. Westh. 2007. A new multiplex PCR for easy screening of methicillin-resistant Staphylococcus aureus SCCmec types I-V. Clin. Microbiol. Infect. 13:725-727.

Božić D.D., M. Milenković, B. Ivković and I. Ćirković. 2014. Newly-synthesized chalcones-inhibition of adherence and biofilm formation of methicillin-resistant Staphylococcus aureus. Braz. J. Microbiol. 45:263-270.

Brakstad O.G., K. Aabakk and J.A. Maeland. 1992. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J. Clin. Microbiol. 30:1654-1660.

Bubeck Wardenburg J., T. Bae, M. Otto, F.R. DeLeo and O. Schneewind. 2007. Poring over pores: alpha-hemolysin and Panton-Valentine leukocidin in Staphylococcus aureus pneumonia. Nat. Med. 13:1405-1406.

Burlak C., C.H. Hammer, M.A. Robinson, A.R. Whitney, M.J. McGavin, B.N. Kreiswirth and F.R. DeLeo. 2007. Global analysis of community associated methicillin-resistant Staphylococcus aureus exoproteins reveals molecules produced in vitro and during infection. Cell. Microbiol. 9:1172-1190.

Cegelski L., G.R. Marshall, G.R. Eldridge and S.J. Hultgren. 2008. The biology and future prospects of antivirulence therapies. Nat. Rev. Microbiol. 6:17-27.

Clinical Laboratory Standards Institute (CLSI). 2007. Performance standards for antimicrobial susceptibility testing, 17th Informational Supplement. Approved Standard. CLSI document M100-S17. Wayne, PA, USA.

Cushnie T.P. and A.J. Lamb. 2011. Recent advances in understanding the antibacterial properties of flavonoids. Int. J Antimicrob. Agents 38:99-107.

Escaich S. 2008. Antivirulence as a new antibacterial approach for chemotherapy. Curr. Opin. Chem. Biol. 12:400-408.

Fukai T., A. Marumo, K. Kaitou, T. Kanda, S. Terada and T. Nomura. 2002.  Antimicrobial activity of licorice flavonoids against methicillin-resistant Staphylococcus aureus. Fitoterapia 73:536-539.

Haraguchi H., K. Tanimoto, Y. Tamura, K. Miyutani and T. Kinoshita. 1998. Mode of antibacterial action of retrochalcones from Glycyrrhiza infiltrata. Phytochemistry 48:125-129.

Harmsen D., H. Claus, W. Witte, J. Rothganger, D. Turnwald and U. Vogel. 2003. Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management. J. Clin. Microbiol. 41:5442-5448.

Kromann H., M. Larsen, T. Boesen, K. Schønning and S.F. Nielsen. 2004. Synthesis of prenylated benzaldehydes and their use in the synthesis of analogues of licochalcone A. Eur. J. Med. Chem. 39:993-1000.

Lina G., F. Boutite, A. Tristan, M. Bes, J. Etienne and F. Vandenesch. 2003. Bacterial competition for human nasal cavity colonization: role of staphylococcal agr alleles. Appl. Environ. Microbiol. 69:18-23.

Montgomery C.P., S. Boyle-Vavra, P.V. Adem, J.C. Lee, A.N. Husain, J. Clasen and R.S. Daum. 2008. Comparison of virulence in community-associated methicillin-resistant Staphylococcus aureus pulsotypes USA300 and USA400 in a rat model of pneumonia. J. Infect. Dis. 198:561-570.

Mori A., C. Nishino, N. Enoki and S. Tawata. 1987. Antibacterial activity and mode of action of plant flavonoids against Proteus vulgaris and Staphylococcus aureus. Phytochemistry 26:2231-2234.

Nowakowska Z. 2007. A review of anti-infective and anti-inflammatory chalcones. Eur. J. Med. Chem. 42:125-137.

Ohemeng K.A., C.F. Schwender, K.P. Fu and J.F. Barrett. 1993. DNA gyrase inhibitory and antibacterial activity of some flavones (1). Bioorg. Med. Chem. Lett. 3:225-230.

Parimon T., Z. Li and D.D. Bolz. 2013. Staphylococcus aureus Alpha-hemolysin Promotes Platelet-Neutrophil Aggregate Formation.  J. Infect. Dis. 208(5):761-70.

Qiu J., Y. Jiang, L. Xia, H. Xiang, H. Feng, S. Pu, N. Huang, L. Yu and X. Deng. 2010a. Subinhibitory concentrations of licochalcone A decrease alpha-toxin production in both methicillin-sensitive and methicillin-resistant Staphylococcus aureus isolates. Lett. Appl. Microbiol. 50:223-229.

Qiu J., D. Wang, H. Xiang, H. Feng, Y. Jiang, L. Xia, J. Dong, J. Lu, L. Yu and X. Deng.  2010b. Subinhibitory concentrations of thymol reduce enterotoxins A and B and alpha-hemolysin production in Staphylococcus aureus isolates. PLoS One. 5:e9736.

Ragle E.B. and J. Bubeck Wardenburg. 2009. Anti-Alpha-hemolysin monoclonal antibodies mediate protection against Staphylococcus aureus pneumonia. Infect. Immun. 77:2712-2718.

Rowe G.E. and R.A. Welch. 1994. Assays of hemolytic toxins. Methods. Enzymol. 235:657-667.

Sato M., H. Tsuchiya, T. Miyazaki, S. Fujiwara, R. Yamaguchi, H. Kureshiro and M. Iinuma. 1996. Antibacterial activity of hydroxychalcone against methicillin-resistant Staphylococcus aureus. Int. J. Antimicrob. Agents 6:227-231.

Talia J.M., N.B. Debattista and N.B. Pappano. 2011. New antimicrobial combinations: substituted chalcones-oxacillin against methicillin resistant Staphylococcus aureus. Braz. J. Microbiol. 42:470-475.

Zhou T., D. Xumin and J. Qiu. 2012. Antimicrobial activity of Licochalcone E against Staphylococcus aureus and its impact on the production of staphylococcal alpha-toxin. J. Microbiol. Biotechnol. 22:800-805.