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Coumarins reduce biofilm formation and the virulence of Escherichia coli O157:H7 Jin-Hyung Lee a,1 , Yong-Guy Kim a,1 , Hyun Seob Cho a , Shi Yong Ryu b , Moo Hwan Cho a , Jintae Lee a,∗ a b
School of Chemical Engineering, Yeungnam University, Gyeongsan, 712-749, Republic of Korea Korea Research Institute of Chemical Technology, Daejeon, 305-606, Republic of Korea
a r t i c l e
i n f o
Article history: Received 24 October 2013 Received in revised form 13 February 2014 Accepted 6 April 2014 Keywords: Biofilm formation Coumarin Escherichia coli O157:H7 Esculetin Umbelliferone Virulence
a b s t r a c t E. coli O157:H7 is the most common cause of hemorrhagic colitis, and no effective therapy exists for E. coli O157:H7 infection. Biofilm formation is closely related to E. coli O157:H7 infection and constitutes a mechanism of antimicrobial resistance. Hence, the antibiofilm or antivirulence approach provides an alternative to antibiotic strategies. Coumarin and its derivatives have a broad range of biological effects, and in this study, the antibiofilm activities of nine coumarins were investigated against E. coli O157:H7. Coumarin or umbelliferone at 50 g/ml was found to inhibit biofilm E. coli O157:H7 formation by more than 80% without affecting bacterial growth. Transcriptional analysis showed that coumarins repressed curli genes and motility genes in E. coli O157:H7, and these findings were in-line with observed reductions in fimbriae production, swarming motility, and biofilm formation. In addition, esculetin repressed Shigalike toxin gene stx2 in E. coli O157:H7 and attenuated its virulence in vivo in the nematode Caenorhabditis elegans. These findings show that coumarins have potential use in antivirulence strategies against persistent E. coli O157:H7 infection. © 2014 Elsevier GmbH. All rights reserved.
Introduction Shiga-like toxigenic E. coli O157:H7 has caused a large number of foodborne outbreaks worldwide. E. coli O157:H7 contamination on various surfaces causes approximately 73,000 infections annually in the U.S., which cost around $405 million in 2003 (Frenzen et al. 2005). However, no effective therapy has been devised to treat E. coli O157:H7 infection, because antibiotics and anti-inflammatory drugs increase the risk of developing hemolyticuremic syndrome (Tarr et al. 2005). E. coli O157:H7 colonizes the large intestine, where it forms attaching and effacing lesions that cause bloody diarrhea (Nataro and Kaper 1998). Of the several virulence factors, Shiga-like toxins Stx1 and Stx2 are considered to be the major virulence factors responsible for these clinical symptoms (Nataro and Kaper 1998). E. coli O157:H7 is able to form biofilms on various biotic and abiotic surfaces, such as, on plants, stainless steel, glass, and polymers
∗ Corresponding author. Tel.: +82 538102533. E-mail address: [email protected] (J. Lee). 1 These authors contributed equally to this work.
(Patel et al. 2011; Ryu and Beuchat 2005). Biofilm cells are difficult to eradicate because of their inherent tolerance to physical and chemical antimicrobial treatments, and thus, pathogenic biofilms can pose serious problems to human health (Costerton et al. 1999). Plants are rich sources of bioactive molecules and thus, are being explored for novel biofilm inhibitors. For example, essential oils (Pérez-Conesa et al. 2006), citrus flavonoids (Lee et al. 2011b; Vikram et al. 2010a), grapefruit limonoids (Vikram et al. 2010b), plant-derived indole derivatives (Lee et al. 2011a), and resveratrol and its dimer viniferin (Cho et al. 2013; Lee et al. 2013a) have been all reported to inhibit E. coli O157:H7 biofilm formation. In the present study, we initially examined the antibiofilm abilities of 560 plant secondary metabolites against E. coli O157:H7, and found that coladonin (a sesquiterpene coumarin) inhibited E. coli O157:H7 biofilm formation. In the subsequent investigation, the antibiofilm activities of eight other coumarins, namely, coumarin, coumarin-3-carboxylic acid, dephnetin, ellagic acid, esculetin, 4hydroxycoumarin, scopoletin, and umbelliferone (Fig. 1A) were measured. Furthermore, an in vivo Caenorhabditis elegans model was used to study the effects of coumarins on E. coli O157:H7 virulence.
http://dx.doi.org/10.1016/j.phymed.2014.04.008 0944-7113/© 2014 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Lee, J.-H., et al., Coumarins reduce biofilm formation and the virulence of Escherichia coli O157:H7. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.04.008
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Fig. 1. Effects of coumarins on biofilm formation of E. coli O157:H7. Biofilm inhibition (%) was quantified after treatment with coumarins (50 g/ml) (A) at 37 ◦ C for 24 h in 96-well plates. Chemical structures are shown. Dose-dependent reductions in E. coli O157:H7 biofilm formation by coladonin, coumarin, or umbelliferone (B). Biofilm formation of E. coli O157:H7/pCM18 tagged with green fluorescent protein in 96-well plates in the presence or absence of coumarins (50 g/ml) (C). The scale bar represents 50 m.
Materials and methods Bacterial strains, plant compounds, and growth assay Shiga-like toxigenic E. coli O157:H7 (ATCC43895, EDL933 strain, Strockbine et al. 1986) was used throughout the study. All experiments were conducted at 37 ◦ C in Luria-Bertani (LB) medium. 560 plant-derived chemicals (Supplementary Table 1) were obtained from the Natural Product Library in the Korea Chemical Bank (http://www.chembank.org, Daejeon, Republic of Korea). These chemicals were purified from a variety of plants and included terpenoids, flavonoids, polyphenols, saponins, and others (Cha et al. 2011; Cho et al. 2002; Choi et al. 2010; Koh et al. 2009). All chemicals were dissolved in dimethyl sulfoxide (DMSO). Seven coumarins
(coumarin, coumarin-3-carboxylic acid, ellagic acid, esculetin, 4hydroxycoumarin, scopoletin, and umbelliferone) were purchased from Sigma–Aldrich (St. Louis, USA) and dephnetin was obtained from TCI Tokyo Chemical Industry (Tokyo, Japan). For cell growth measurements, optical densities were measured at 600 nm using a spectrophotometer (Optizen 2120UV, Mecasys, KOREA). Each experiment was performed using at least two independent cultures. Biofilm assay and antibiofilm screening A static biofilm formation assay was performed in 96-well polystyrene plates (SPL Life Sciences, Korea), as previously reported (Lee et al. 2011a). Biofilms were stained with crystal violet and
Please cite this article in press as: Lee, J.-H., et al., Coumarins reduce biofilm formation and the virulence of Escherichia coli O157:H7. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.04.008
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dissolved in 95% ethanol, and absorbances were measured at 570 nm (OD570 ) to quantify biofilm formation. For initial antibiofilm screening, E. coli O157:H7 was cultured with plant secondary metabolites at 10 g/ml in LB medium for 24 h. Cultures were performed twice in quadruplicate. For more detailed analysis, the results of at least twelve replicate wells were averaged.
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and scored as alive or dead on a daily basis by gently touching them with a platinum wire. Worms that crawled onto the walls of culture plates were eliminated from the analysis. Three independent experiments were conducted.
Results and discussion Confocal laser microscopy E. coli O157:H7/pCM18 tagged with green fluorescent protein was cultured in 96-well plates (SPL Life Sciences, Korea) with or without coumarins. Static biofilm formation was visualized by confocal laser microscopy (Nikon eclipse Ti, Tokyo) using an excitation wavelength 488 nm (Ar laser), emission wavelengths of 500–550 nm, and a 20× objective. Color confocal images were produced using NIS-Elements C version 3.2 (Nikon eclipse). For each experiment, at least 10 random positions in each of four independent cultures were chosen for microscopic analysis. RNA isolation and quantitative real-time RT-PCR For transcriptional analysis, E. coli O157:H7 was inoculated into 25 ml LB medium in 250 ml shake flasks at a starting OD600 of 0.05, and then cultured at 37 ◦ C for 5 h at 250 rpm shaking in the presence or absence of coumarins (50 g/ml). RNase inhibitor (RNAlater, Ambion, TX, USA) was added to prevent RNA degradation. Total RNA was isolated using a Qiagen RNeasy mini Kit (Valencia, CA, USA). qRT-PCR was used to investigate the transcription levels of curli genes (csgA and csgB), motility genes (flhD and motB), Shigalike toxin genes (stx1 and stx2), and quorum-sensing genes (lsrA and luxS) in E. coli O157:H7 treated with or without coumarins (50 g/ml). Gene specific primers were used and rrsG was used as a housekeeping control (Supplementary Table 2). The qRT-PCR method used has been previously described (Lee et al. 2011a). qRT-PCR was performed using a SYBR Green master mix (Applied Biosystems, Foster City, USA) and an ABI StepOne Real-Time PCR System (Applied Biosystems) on two independent cultures. Fimbriae assay using SEM SEM was used to observe fimbriae production, as previously described (Lee et al. 2011a). Briefly, EHEC cells were inoculated on a nylon filter (0.5 mm × 0.5 mm square) at an initial OD600 of 0.05. Cells and nylon filters were incubated together in the presence of coumarins (50 g/ml) at 37 ◦ C for 24 h without shaking to form biofilms. After critical-point drying, specimens were examined using an SEM (S-4100; Hitachi, Japan) at a voltage of 15 kV and a magnification of 10,000×. Motility assay Agar (0.3%) containing 1% tryptone and 0.25% NaCl was used to assess swimming motility (Sperandio et al. 2002), and LB medium supplemented with 0.8% glucose and 0.5% agar to assess swarming motility (Ling et al. 2010). Coumarins were added to motility agar and DMSO was added as a control. The sizes of swimming halos were measured after 20 h. Each experiment was performed using at least three independent cultures. C. elegans killing assay The C. elegans killing assay was performed as described previously (Lee et al. 2013b). In brief, 10 l of an overnight E. coli O157:H7 culture was spread onto NGM plates, and L4/young adult fer-15;fem-1 (Murphy et al. 2003) worms (n = 60) were infected by placing them on the lawns. The nematodes were incubated at 25 ◦ C
Initially, 560 purified phytochemicals were screened to identify new antibiofilm compounds against E. coli O157:H7. To minimize antimicrobial effects of compounds, all compounds at 10 g/ml were used for the initial screening. Of the 560 purified chemicals tested, four inhibited E. coli O157:H7 biofilm formation by more than 70%. These four chemicals were coladonin, ginkgolic acid, vitisin A, and vitisin B. Because coladonin is a sesquiterpene coumarin and various derivatives of coumarin are commercially available, coumarins and coladonin were focused on further study. To identify more potent antibiofilm compounds than coladonin, eight coumarin derivatives, namely, coumarin, coumarin-3-carboxylic acid, dephnetin, ellagic acid, esculetin, 4hydroxycoumarin, scopoletin, and umbelliferone at 50 g/ml were further investigated (Fig. 1A). Of the coumarins tested, coumarin and umbelliferone most significantly dose-dependently reduced biofilm formation after 24 h (Fig. 1B). Specifically, coumarin and umbelliferone at 50 g/ml inhibited E. coli O157:H7 biofilm formation by more than 80 and 90%, respectively. However, coladonin at higher concentrations (20, 50, or 100 g/ml) did not show a better performance than at 10 g/ml. Because E. coli O157:H7 forms biofilms on the bottoms and sides of 96-well plates, confocal laser microscopy was used to observe the bottom biofilms. Microscopic observations confirmed that coumarin and umbelliferone dramatically inhibited the formation of bottom biofilms (Fig. 1C) and total biofilms (Fig. 1B). The cell growth of E. coli O157:H7 was investigated to identify non-cytotoxic compounds with antibiofilm activity. The presence of coumarin, esculetin, and umbelliferone at concentrations up to 50 g/ml did not diminish E. coli O157:H7 cell growth, but at 200 g/ml they had inhibitory effects (Fig. 2). It is important to note that reduced biofilm formation by coumarins was due to its antibiofilm activity and not to its antimicrobial activity. Unlike antibiotics that aim to inhibit cell growth, biofilm inhibitors that do not inhibit bacterial growth may reduce the risk of drug resistance (Clatworthy et al. 2007). To investigate the molecular mechanism responsible for biofilm inhibition, qRT-PCR was used to determine differential expression of biofilm-related genes in E. coli O157:H7 cells treated with or without coumarin, umbelliferone, or esculetin. Most notably, the gene expression of the csg operon (csgA and csgB), which is involved in curli formation, was markedly repressed by coumarin, umbelliferone, and esculetin (Fig. 3). In addition, these three coumarins significantly repressed the gene expression of motility genes (flhD and motB). However, they changed the expression patterns of Shiga-like toxin genes and quorum sensing genes in different ways. In particular, esculetin repressed Shiga-like toxin II gene (stx2), whereas coumarin and umbelliferone did not. Since fimbriae are important for E. coli O157:H7 biofilm formation (Rendón et al. 2007; Ryu and Beuchat 2005), and the gene expression of the csg operon was found to be repressed by all three coumarins, fimbriae production was investigated by SEM. In-line with the gene expression data, coumarin and umbelliferone clearly reduced fimbriae production (Fig. 4). Furthermore, few biofilm cells attached to nylon membranes in the presence of coumarin or umbelliferone, which suggested, biofilm inhibition by these two coumarins was caused at least in part by fimbriae reduction. Because motility plays a role in E. coli biofilm formation (Pratt and Kolter 1998; Ren et al. 2001), the impacts of coumarins on the
Please cite this article in press as: Lee, J.-H., et al., Coumarins reduce biofilm formation and the virulence of Escherichia coli O157:H7. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.04.008
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Fig. 3. Transcriptional profiles of E. coli O157:H7 cells treated with or without coumarins. E. coli O157:H7 was cultivated in LB medium with or without coumarins (50 g/ml) for 5 h at 250 rpm shaking. Transcriptional profiles were measured by qRT-PCR. Relative gene expressions represent transcriptional levels after treatment with coumarins versus untreated controls (value 1.0). The experiment was performed in duplicate.
swimming and swarming motilities of E. coli O157:H7 were also investigated. Interestingly, coumarin and umbelliferone reduced swarming motility (Fig. 5A) but had no effect on swimming motility (Fig. 5B). Thus, it would appear that swarming motility rather than swimming motility influenced E. coli O157:H7 biofilm reduction in this study. Since E. coli O157:H7 kills the nematode C. elegans by producing Shiga-like toxins (Chou et al. 2013; Kim et al. 2006), the effects of coumarin, esculetin, and umbelliferone on infected C. elegans survival were assayed. It was found that esculetin dose-dependently prolonged C. elegans survival in the presence of E. coli O157:H7, indicating that esculetin reduces the virulence of E. coli O157:H7 (Fig. 6). Furthermore, this result was in-line with the observed repression of Shiga-like toxin stx2 gene by esculetin (Fig. 3). However, neither
coumarin nor umbelliferone affected C. elegans survival or Shigalike toxin gene expression (data not shown). Hence, it would be interesting to investigate how only esculetin down-regulates the stx2 gene and E. coli O157:H7 virulence. More than 1,300 coumarins have been identified in a variety of plants (Hoult and Payá 1996). Furthermore, coumarin and its derivatives have been found to exert anti-coagulant, antitumor, antiviral, anti-inflammatory, antioxidant, and antimicrobial effects (Riveiro et al. 2010). Previously, esculetin and umbelliferone at 5 mM (∼800 g/ml) were reported to inhibit the growth of E. coli O157:H7 (Duncan et al. 1998), and recently, esculetin (6,7dihydroxycoumarin) at up to 128 g/ml was reported to prevent biofilm formation by Staphylococcus aureus without affecting its cell growth (Dürig et al. 2010). Although high doses of coumarin are known to be associated with liver toxicity in rodents (Felter et al. 2006), coumarin is not a genotoxic agent, and it was concluded that exposure to coumarin from food and/or cosmetic products poses no health risk in humans (Lake 1999). In the literature the nonsubstituted coumarin is reported to possess cancerogenic potential (Lake 1999), however, in case of therapeutic application the dosage limit of 0.1 mg coumarin per kg body mass and day is considered harmless as average intake and has to be observed accordingly. We report for the first time that coumarin and umbelliferone at concentrations up to 50 g/ml exhibit high antibiofilm formation activity against enterohemorrhagic E. coli O157:H7 without inhibiting planktonic cell growth. Interestingly, hydroxylation of coumarin markedly affects the anti-biofilm effect (Fig. 1A). Hydroxylation in position 4 dramatically diminishes the activity while hydroxylation in position 7 enhances it. The di-hydroxylation of coumarin in positions 6 and 7 (esculetin) diminishes the activity, a 6-methoxy group (scopoletin) instead of a 6-hydroxyl group does not produce any change in the activity, and the introduction of a carboxyl group in position 3 diminishes the activity respective of coumarin. Also, hydroxylation in position 8 (dephnetin) dramatically diminishes the activity compared to coumarin and
Fig. 4. Effect of coumarins on fimbriae production by E. coli O157:H7. SEM was used to examine fimbriae production by biofilm cells grown on a nylon filter in LB in the presence or absence of coumarins (50 g/ml) for 24 h.
Please cite this article in press as: Lee, J.-H., et al., Coumarins reduce biofilm formation and the virulence of Escherichia coli O157:H7. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.04.008
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Fig. 5. Impacts of coumarins on E. coli O157:H7 motility. Biofilm-inhibiting coumarins (50 g/ml) influenced swarming (A) and swimming (B) motilities. Each experiment was performed using three independent cultures; one representative data set is shown.
Acknowledgements
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All plant secondary metabolites used in this study were kindly provided by the Korean Chemical Bank at the Korean Research Institute of Chemical Technology. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant nos. 2012R1A1A3010534 and 20100021871 to J-H. Lee and J. Lee, respectively). Appendix A. Supplementary data
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Fig. 6. Effects of esculetin on the survival of nematodes infected with E. coli O157:H7. Solid killing assays of C. elegans strains fer-15;fem-1 infected with E. coli O157:H7 in the presence of esculetin (0, 50, and 100 g/ml). Nematodes infected with E. coli O157:H7 in the absence of coumarins were used as controls. The experiment was performed three times (n = 60).
umbelliferone. The introduction of a sesquiterpene in position 7 (coladonin) does not produce any beneficial change, suggesting that the coumarin scheleton is a better biofilm inhibitor than coladonin. In the present study, biofilm inhibition by coumarins was partially elucidated as being caused by the repressions of curli genes and motility genes by coumarin and by umbelliferone, and resultant reductions in fimbriae production (Fig. 4) and swarming motility (Fig. 5). Furthermore, the study shows that esculetin attenuates E. coli O157:H7 virulence in vivo in the nematode C. elegans (Fig. 6). These findings indicate that coumarins have potential use in antivirulence strategies against E. coli O157:H7 infection.
Conflict of interest The authors have no potential conflict of interest to disclose.
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Coumarins reduce biofilm formation and the virulence of Escherichia coli O157:H7 Jin-Hyung Lee a,1 , Yong-Guy Kim a,1 , Hyun Seob Cho a , Shi Yong Ryu b , Moo Hwan Cho a , Jintae Lee a,∗ a b
School of Chemical Engineering, Yeungnam University, Gyeongsan, 712-749, Republic of Korea Korea Research Institute of Chemical Technology, Daejeon, 305-606, Republic of Korea
a r t i c l e
i n f o
Article history: Received 24 October 2013 Received in revised form 13 February 2014 Accepted 6 April 2014 Keywords: Biofilm formation Coumarin Escherichia coli O157:H7 Esculetin Umbelliferone Virulence
a b s t r a c t E. coli O157:H7 is the most common cause of hemorrhagic colitis, and no effective therapy exists for E. coli O157:H7 infection. Biofilm formation is closely related to E. coli O157:H7 infection and constitutes a mechanism of antimicrobial resistance. Hence, the antibiofilm or antivirulence approach provides an alternative to antibiotic strategies. Coumarin and its derivatives have a broad range of biological effects, and in this study, the antibiofilm activities of nine coumarins were investigated against E. coli O157:H7. Coumarin or umbelliferone at 50 g/ml was found to inhibit biofilm E. coli O157:H7 formation by more than 80% without affecting bacterial growth. Transcriptional analysis showed that coumarins repressed curli genes and motility genes in E. coli O157:H7, and these findings were in-line with observed reductions in fimbriae production, swarming motility, and biofilm formation. In addition, esculetin repressed Shigalike toxin gene stx2 in E. coli O157:H7 and attenuated its virulence in vivo in the nematode Caenorhabditis elegans. These findings show that coumarins have potential use in antivirulence strategies against persistent E. coli O157:H7 infection. © 2014 Elsevier GmbH. All rights reserved.
Introduction Shiga-like toxigenic E. coli O157:H7 has caused a large number of foodborne outbreaks worldwide. E. coli O157:H7 contamination on various surfaces causes approximately 73,000 infections annually in the U.S., which cost around $405 million in 2003 (Frenzen et al. 2005). However, no effective therapy has been devised to treat E. coli O157:H7 infection, because antibiotics and anti-inflammatory drugs increase the risk of developing hemolyticuremic syndrome (Tarr et al. 2005). E. coli O157:H7 colonizes the large intestine, where it forms attaching and effacing lesions that cause bloody diarrhea (Nataro and Kaper 1998). Of the several virulence factors, Shiga-like toxins Stx1 and Stx2 are considered to be the major virulence factors responsible for these clinical symptoms (Nataro and Kaper 1998). E. coli O157:H7 is able to form biofilms on various biotic and abiotic surfaces, such as, on plants, stainless steel, glass, and polymers
∗ Corresponding author. Tel.: +82 538102533. E-mail address: [email protected] (J. Lee). 1 These authors contributed equally to this work.
(Patel et al. 2011; Ryu and Beuchat 2005). Biofilm cells are difficult to eradicate because of their inherent tolerance to physical and chemical antimicrobial treatments, and thus, pathogenic biofilms can pose serious problems to human health (Costerton et al. 1999). Plants are rich sources of bioactive molecules and thus, are being explored for novel biofilm inhibitors. For example, essential oils (Pérez-Conesa et al. 2006), citrus flavonoids (Lee et al. 2011b; Vikram et al. 2010a), grapefruit limonoids (Vikram et al. 2010b), plant-derived indole derivatives (Lee et al. 2011a), and resveratrol and its dimer viniferin (Cho et al. 2013; Lee et al. 2013a) have been all reported to inhibit E. coli O157:H7 biofilm formation. In the present study, we initially examined the antibiofilm abilities of 560 plant secondary metabolites against E. coli O157:H7, and found that coladonin (a sesquiterpene coumarin) inhibited E. coli O157:H7 biofilm formation. In the subsequent investigation, the antibiofilm activities of eight other coumarins, namely, coumarin, coumarin-3-carboxylic acid, dephnetin, ellagic acid, esculetin, 4hydroxycoumarin, scopoletin, and umbelliferone (Fig. 1A) were measured. Furthermore, an in vivo Caenorhabditis elegans model was used to study the effects of coumarins on E. coli O157:H7 virulence.
http://dx.doi.org/10.1016/j.phymed.2014.04.008 0944-7113/© 2014 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Lee, J.-H., et al., Coumarins reduce biofilm formation and the virulence of Escherichia coli O157:H7. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.04.008
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Fig. 1. Effects of coumarins on biofilm formation of E. coli O157:H7. Biofilm inhibition (%) was quantified after treatment with coumarins (50 g/ml) (A) at 37 ◦ C for 24 h in 96-well plates. Chemical structures are shown. Dose-dependent reductions in E. coli O157:H7 biofilm formation by coladonin, coumarin, or umbelliferone (B). Biofilm formation of E. coli O157:H7/pCM18 tagged with green fluorescent protein in 96-well plates in the presence or absence of coumarins (50 g/ml) (C). The scale bar represents 50 m.
Materials and methods Bacterial strains, plant compounds, and growth assay Shiga-like toxigenic E. coli O157:H7 (ATCC43895, EDL933 strain, Strockbine et al. 1986) was used throughout the study. All experiments were conducted at 37 ◦ C in Luria-Bertani (LB) medium. 560 plant-derived chemicals (Supplementary Table 1) were obtained from the Natural Product Library in the Korea Chemical Bank (http://www.chembank.org, Daejeon, Republic of Korea). These chemicals were purified from a variety of plants and included terpenoids, flavonoids, polyphenols, saponins, and others (Cha et al. 2011; Cho et al. 2002; Choi et al. 2010; Koh et al. 2009). All chemicals were dissolved in dimethyl sulfoxide (DMSO). Seven coumarins
(coumarin, coumarin-3-carboxylic acid, ellagic acid, esculetin, 4hydroxycoumarin, scopoletin, and umbelliferone) were purchased from Sigma–Aldrich (St. Louis, USA) and dephnetin was obtained from TCI Tokyo Chemical Industry (Tokyo, Japan). For cell growth measurements, optical densities were measured at 600 nm using a spectrophotometer (Optizen 2120UV, Mecasys, KOREA). Each experiment was performed using at least two independent cultures. Biofilm assay and antibiofilm screening A static biofilm formation assay was performed in 96-well polystyrene plates (SPL Life Sciences, Korea), as previously reported (Lee et al. 2011a). Biofilms were stained with crystal violet and
Please cite this article in press as: Lee, J.-H., et al., Coumarins reduce biofilm formation and the virulence of Escherichia coli O157:H7. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.04.008
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dissolved in 95% ethanol, and absorbances were measured at 570 nm (OD570 ) to quantify biofilm formation. For initial antibiofilm screening, E. coli O157:H7 was cultured with plant secondary metabolites at 10 g/ml in LB medium for 24 h. Cultures were performed twice in quadruplicate. For more detailed analysis, the results of at least twelve replicate wells were averaged.
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and scored as alive or dead on a daily basis by gently touching them with a platinum wire. Worms that crawled onto the walls of culture plates were eliminated from the analysis. Three independent experiments were conducted.
Results and discussion Confocal laser microscopy E. coli O157:H7/pCM18 tagged with green fluorescent protein was cultured in 96-well plates (SPL Life Sciences, Korea) with or without coumarins. Static biofilm formation was visualized by confocal laser microscopy (Nikon eclipse Ti, Tokyo) using an excitation wavelength 488 nm (Ar laser), emission wavelengths of 500–550 nm, and a 20× objective. Color confocal images were produced using NIS-Elements C version 3.2 (Nikon eclipse). For each experiment, at least 10 random positions in each of four independent cultures were chosen for microscopic analysis. RNA isolation and quantitative real-time RT-PCR For transcriptional analysis, E. coli O157:H7 was inoculated into 25 ml LB medium in 250 ml shake flasks at a starting OD600 of 0.05, and then cultured at 37 ◦ C for 5 h at 250 rpm shaking in the presence or absence of coumarins (50 g/ml). RNase inhibitor (RNAlater, Ambion, TX, USA) was added to prevent RNA degradation. Total RNA was isolated using a Qiagen RNeasy mini Kit (Valencia, CA, USA). qRT-PCR was used to investigate the transcription levels of curli genes (csgA and csgB), motility genes (flhD and motB), Shigalike toxin genes (stx1 and stx2), and quorum-sensing genes (lsrA and luxS) in E. coli O157:H7 treated with or without coumarins (50 g/ml). Gene specific primers were used and rrsG was used as a housekeeping control (Supplementary Table 2). The qRT-PCR method used has been previously described (Lee et al. 2011a). qRT-PCR was performed using a SYBR Green master mix (Applied Biosystems, Foster City, USA) and an ABI StepOne Real-Time PCR System (Applied Biosystems) on two independent cultures. Fimbriae assay using SEM SEM was used to observe fimbriae production, as previously described (Lee et al. 2011a). Briefly, EHEC cells were inoculated on a nylon filter (0.5 mm × 0.5 mm square) at an initial OD600 of 0.05. Cells and nylon filters were incubated together in the presence of coumarins (50 g/ml) at 37 ◦ C for 24 h without shaking to form biofilms. After critical-point drying, specimens were examined using an SEM (S-4100; Hitachi, Japan) at a voltage of 15 kV and a magnification of 10,000×. Motility assay Agar (0.3%) containing 1% tryptone and 0.25% NaCl was used to assess swimming motility (Sperandio et al. 2002), and LB medium supplemented with 0.8% glucose and 0.5% agar to assess swarming motility (Ling et al. 2010). Coumarins were added to motility agar and DMSO was added as a control. The sizes of swimming halos were measured after 20 h. Each experiment was performed using at least three independent cultures. C. elegans killing assay The C. elegans killing assay was performed as described previously (Lee et al. 2013b). In brief, 10 l of an overnight E. coli O157:H7 culture was spread onto NGM plates, and L4/young adult fer-15;fem-1 (Murphy et al. 2003) worms (n = 60) were infected by placing them on the lawns. The nematodes were incubated at 25 ◦ C
Initially, 560 purified phytochemicals were screened to identify new antibiofilm compounds against E. coli O157:H7. To minimize antimicrobial effects of compounds, all compounds at 10 g/ml were used for the initial screening. Of the 560 purified chemicals tested, four inhibited E. coli O157:H7 biofilm formation by more than 70%. These four chemicals were coladonin, ginkgolic acid, vitisin A, and vitisin B. Because coladonin is a sesquiterpene coumarin and various derivatives of coumarin are commercially available, coumarins and coladonin were focused on further study. To identify more potent antibiofilm compounds than coladonin, eight coumarin derivatives, namely, coumarin, coumarin-3-carboxylic acid, dephnetin, ellagic acid, esculetin, 4hydroxycoumarin, scopoletin, and umbelliferone at 50 g/ml were further investigated (Fig. 1A). Of the coumarins tested, coumarin and umbelliferone most significantly dose-dependently reduced biofilm formation after 24 h (Fig. 1B). Specifically, coumarin and umbelliferone at 50 g/ml inhibited E. coli O157:H7 biofilm formation by more than 80 and 90%, respectively. However, coladonin at higher concentrations (20, 50, or 100 g/ml) did not show a better performance than at 10 g/ml. Because E. coli O157:H7 forms biofilms on the bottoms and sides of 96-well plates, confocal laser microscopy was used to observe the bottom biofilms. Microscopic observations confirmed that coumarin and umbelliferone dramatically inhibited the formation of bottom biofilms (Fig. 1C) and total biofilms (Fig. 1B). The cell growth of E. coli O157:H7 was investigated to identify non-cytotoxic compounds with antibiofilm activity. The presence of coumarin, esculetin, and umbelliferone at concentrations up to 50 g/ml did not diminish E. coli O157:H7 cell growth, but at 200 g/ml they had inhibitory effects (Fig. 2). It is important to note that reduced biofilm formation by coumarins was due to its antibiofilm activity and not to its antimicrobial activity. Unlike antibiotics that aim to inhibit cell growth, biofilm inhibitors that do not inhibit bacterial growth may reduce the risk of drug resistance (Clatworthy et al. 2007). To investigate the molecular mechanism responsible for biofilm inhibition, qRT-PCR was used to determine differential expression of biofilm-related genes in E. coli O157:H7 cells treated with or without coumarin, umbelliferone, or esculetin. Most notably, the gene expression of the csg operon (csgA and csgB), which is involved in curli formation, was markedly repressed by coumarin, umbelliferone, and esculetin (Fig. 3). In addition, these three coumarins significantly repressed the gene expression of motility genes (flhD and motB). However, they changed the expression patterns of Shiga-like toxin genes and quorum sensing genes in different ways. In particular, esculetin repressed Shiga-like toxin II gene (stx2), whereas coumarin and umbelliferone did not. Since fimbriae are important for E. coli O157:H7 biofilm formation (Rendón et al. 2007; Ryu and Beuchat 2005), and the gene expression of the csg operon was found to be repressed by all three coumarins, fimbriae production was investigated by SEM. In-line with the gene expression data, coumarin and umbelliferone clearly reduced fimbriae production (Fig. 4). Furthermore, few biofilm cells attached to nylon membranes in the presence of coumarin or umbelliferone, which suggested, biofilm inhibition by these two coumarins was caused at least in part by fimbriae reduction. Because motility plays a role in E. coli biofilm formation (Pratt and Kolter 1998; Ren et al. 2001), the impacts of coumarins on the
Please cite this article in press as: Lee, J.-H., et al., Coumarins reduce biofilm formation and the virulence of Escherichia coli O157:H7. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.04.008
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Fig. 3. Transcriptional profiles of E. coli O157:H7 cells treated with or without coumarins. E. coli O157:H7 was cultivated in LB medium with or without coumarins (50 g/ml) for 5 h at 250 rpm shaking. Transcriptional profiles were measured by qRT-PCR. Relative gene expressions represent transcriptional levels after treatment with coumarins versus untreated controls (value 1.0). The experiment was performed in duplicate.
swimming and swarming motilities of E. coli O157:H7 were also investigated. Interestingly, coumarin and umbelliferone reduced swarming motility (Fig. 5A) but had no effect on swimming motility (Fig. 5B). Thus, it would appear that swarming motility rather than swimming motility influenced E. coli O157:H7 biofilm reduction in this study. Since E. coli O157:H7 kills the nematode C. elegans by producing Shiga-like toxins (Chou et al. 2013; Kim et al. 2006), the effects of coumarin, esculetin, and umbelliferone on infected C. elegans survival were assayed. It was found that esculetin dose-dependently prolonged C. elegans survival in the presence of E. coli O157:H7, indicating that esculetin reduces the virulence of E. coli O157:H7 (Fig. 6). Furthermore, this result was in-line with the observed repression of Shiga-like toxin stx2 gene by esculetin (Fig. 3). However, neither
coumarin nor umbelliferone affected C. elegans survival or Shigalike toxin gene expression (data not shown). Hence, it would be interesting to investigate how only esculetin down-regulates the stx2 gene and E. coli O157:H7 virulence. More than 1,300 coumarins have been identified in a variety of plants (Hoult and Payá 1996). Furthermore, coumarin and its derivatives have been found to exert anti-coagulant, antitumor, antiviral, anti-inflammatory, antioxidant, and antimicrobial effects (Riveiro et al. 2010). Previously, esculetin and umbelliferone at 5 mM (∼800 g/ml) were reported to inhibit the growth of E. coli O157:H7 (Duncan et al. 1998), and recently, esculetin (6,7dihydroxycoumarin) at up to 128 g/ml was reported to prevent biofilm formation by Staphylococcus aureus without affecting its cell growth (Dürig et al. 2010). Although high doses of coumarin are known to be associated with liver toxicity in rodents (Felter et al. 2006), coumarin is not a genotoxic agent, and it was concluded that exposure to coumarin from food and/or cosmetic products poses no health risk in humans (Lake 1999). In the literature the nonsubstituted coumarin is reported to possess cancerogenic potential (Lake 1999), however, in case of therapeutic application the dosage limit of 0.1 mg coumarin per kg body mass and day is considered harmless as average intake and has to be observed accordingly. We report for the first time that coumarin and umbelliferone at concentrations up to 50 g/ml exhibit high antibiofilm formation activity against enterohemorrhagic E. coli O157:H7 without inhibiting planktonic cell growth. Interestingly, hydroxylation of coumarin markedly affects the anti-biofilm effect (Fig. 1A). Hydroxylation in position 4 dramatically diminishes the activity while hydroxylation in position 7 enhances it. The di-hydroxylation of coumarin in positions 6 and 7 (esculetin) diminishes the activity, a 6-methoxy group (scopoletin) instead of a 6-hydroxyl group does not produce any change in the activity, and the introduction of a carboxyl group in position 3 diminishes the activity respective of coumarin. Also, hydroxylation in position 8 (dephnetin) dramatically diminishes the activity compared to coumarin and
Fig. 4. Effect of coumarins on fimbriae production by E. coli O157:H7. SEM was used to examine fimbriae production by biofilm cells grown on a nylon filter in LB in the presence or absence of coumarins (50 g/ml) for 24 h.
Please cite this article in press as: Lee, J.-H., et al., Coumarins reduce biofilm formation and the virulence of Escherichia coli O157:H7. Phytomedicine (2014), http://dx.doi.org/10.1016/j.phymed.2014.04.008
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Fig. 5. Impacts of coumarins on E. coli O157:H7 motility. Biofilm-inhibiting coumarins (50 g/ml) influenced swarming (A) and swimming (B) motilities. Each experiment was performed using three independent cultures; one representative data set is shown.
Acknowledgements
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All plant secondary metabolites used in this study were kindly provided by the Korean Chemical Bank at the Korean Research Institute of Chemical Technology. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant nos. 2012R1A1A3010534 and 20100021871 to J-H. Lee and J. Lee, respectively). Appendix A. Supplementary data
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umbelliferone. The introduction of a sesquiterpene in position 7 (coladonin) does not produce any beneficial change, suggesting that the coumarin scheleton is a better biofilm inhibitor than coladonin. In the present study, biofilm inhibition by coumarins was partially elucidated as being caused by the repressions of curli genes and motility genes by coumarin and by umbelliferone, and resultant reductions in fimbriae production (Fig. 4) and swarming motility (Fig. 5). Furthermore, the study shows that esculetin attenuates E. coli O157:H7 virulence in vivo in the nematode C. elegans (Fig. 6). These findings indicate that coumarins have potential use in antivirulence strategies against E. coli O157:H7 infection.
Conflict of interest The authors have no potential conflict of interest to disclose.
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