Thiophenone and furanone in control of Escherichia coli O103:H2 virulence.

Pathogens and Disease ISSN 2049-632X

RESEARCH ARTICLE

Thiophenone and furanone in control of Escherichia coli O103:H2 virulence €nn-Stensrud1 & Anne A. Scheie1 Ingun L. Witsø1, Tore Benneche2, Lene K. Vestby3, Live L. Nesse3, Jessica Lo 1 Department of Oral Biology, University of Oslo, Norway 2 Department of Chemistry, University of Oslo, Norway 3 National Veterinary Institute, Oslo, Norway

In this work, the authors show that two quorum-sensing inhibitors, furanone and thiophenone, can prevent biofilm formation by E. coli O103:H2 in vitro possibly by downregulating motility.

Keywords biofilm; quorum sensing; Escherichia coli; virulence; thiophenone. Correspondence Ingun L. Witsø, Department of Oral Biology, Faculty of Dentistry, University of Oslo, Sognsvannsveien 10, 0372 Oslo, Norway. Tel.: +47 22840256 fax: +47 22840302 e-mail: [email protected] Received 22 November 2013; revised 20 December 2013; accepted 23 December 2013. Final version published online 3 February 2014. doi:10.1111/2049-632X.12128

Abstract Escherichia coli are a mutual and foodborne pathogen, causing severe intestinal infections typically characterized by diarrhoea and vomiting. Biofilms are often a common source of pathogenic and nonpathogenic bacteria. Quorum sensing is a phenomenon where bacteria communicate and initiate the regulation of several virulence factors and biofilm formation. Thus, quorum sensing has been a new target in the fight against bacterial biofilms. In this study, we investigated the effect of two quorum-sensing inhibitors for preventing in vitro biofilm formation in wild-type E. coli O103:H2. Furanone F202 originates from the red algae Delisea pulchra, and thiophenone TF101 is a sulphur analogue of furanone. We also investigated the effect of thiophenone and furanone on virulence factors controlled by quorum sensing. Both TF101 and F202 interfered with biofilm formation, although TF101 was more effective. TF101 reduced motility presumably by interfering with flagella production, visualized by microscopic techniques. The expressions of flhd, which are involved in flagella synthesis, were affected by thiophenone. This is the first study exploring the effect of thiophenone on E. coli biofilm formation and virulence factors.

Editor: Ake Forsberg

Introduction Biofilms are sessile bacterial populations, where bacterial cells adhere to a surface and are enclosed in a matrix (Costerton et al., 1995). Bacteria may form biofilm on a wide variety of surfaces, including living tissues, medical devices, industrial piping or natural aquatic systems. One major challenging feature of bacterial biofilms is the tolerance to antibacterial agents and components of the host defence system (Ciofu & Tolker-Nielsen, 2011). Biofilm bacteria are less susceptible to antibiotics than planktonic bacteria. Their ability to resist antimicrobial therapy makes biofilm a major challenge in treatment of biofilm-related infectious diseases. Thus, new and alternative treatments are needed in the fight against severe bacterial biofilm infections. Escherichia coli are gram-negative commensals that typically colonize the gastrointestinal tracts and live in symbiosis with its host (Kaper et al., 2004). However, there

are several clones that have acquired specific virulence traits, which confer an increased ability to adapt to new niches and cause intra- and extraintestinal infections. Enteropathogenic E. coli (EPEC), for example, is associated with infections with symptoms like diarrhoea, emesis/ vomiting and fever (Nataro & Kaper, 1998). EPECs are identified as isolates positive for the intimine gene (aea) but lacking verotoxin genes. This E. coli pathotype is divided into two subgroups: typical EPEC and atypical EPEC (aEPEC). aEPEC have been isolated from both humans and animals, whilste typical EPEC have only been isolated from humans (Trabulsi et al., 2002; Carneiro et al., 2006). aEPEC isolated from animals have been shown to be potentially pathogenic to humans as these strains possess many features necessary to cause illness (Monaghan et al., 2013). Bacteria can sense the concentration of, and communicate with, other bacteria in the same environment through the secretion and detection of small molecules called

Pathogens and Disease (2014), 70, 297–306, © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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Thiophenone and furanone in control of E. coli virulence

autoinducers. When the concentration of autoinducers reaches a specific threshold (a quorum), they initiate the regulation of several virulence factors and biofilm formation (Miller & Bassler, 2001; Xue et al., 2009). This specific phenomenon is known as quorum sensing. Autoinducer-2 (AI-2), a product of the luxS gene, is suggested to serve as inter- and intraspecies signalling molecule (Bassler et al., 1997; Surette et al., 1999). The substrate of LuxS is S-Ribosylhomocysteine, which is cleaved to yield homocystein and 4, 5-dihydroxypentan-2, 3-dione (DPD). DPD cyclizes spontaneously and forms AI-2 (Schauder et al., 2001). Previous studies have also shown that AI-2 regulates several genes in E. coli, including genes involved in flagella synthesis, motility and other virulence factors (Sperandio et al., 2001; Ren et al., 2004). In E. coli, different characteristics such as motility and swarming are associated with biofilm formation (Pratt & Kolter, 1998; O’Toole et al., 2000). Motility is believed to be a critical feature for biofilm formation. Bacterial motility is mediated by rotating flagella, which enables the bacteria to move in a coordinated fashion. According to Pratt & Kolter (1998), flagella perform four exclusive roles; they enable the bacteria to swim towards nutrients associated with a surface or towards signals; they are required for the bacteria to initially reach a surface; flagella enable the bacteria to spread along a surface; and lastly, the flagella provide physical adherence to an abiotic surface. Swarming is an organized movement of a group of bacteria, mediated by flagella. A critical cell density is necessary to initiate the swarming process. Therefore, swarming is believed to be coupled to quorum sensing (Eberl et al., 1996; Sperandio et al., 2002; Verstraeten et al., 2008). Quorum-sensing E. coli regulators B and C (QseBC) belong to a two-component regulatory system involved in motility and regulation of flagella. Transcription of qseBC is activated by AI-2 (Sperandio et al., 2002). In E. coli, over 50 genes are involved in the expression of flagella (Chilcott & Hughes, 2000), and QseBC is involved in regulation of the flagella assembly, namely by regulating the master regulator FlhDC (Clarke & Sperandio, 2005). The flhDC operon encodes the transcriptional activators that control the expression of the middle and late genes of the flagella regulon, for example fliA (Liu & Matsumura, 1994; Chilcott & Hughes, 2000; Patrick & Kearns, 2012). fliA encodes an alternative sigma subunit of RNA polymerase (r28) and confers specificity to the flagellar promoters of the late genes (Ohnishi et al., 1990). MotA, encoded by the motA gene, is a subunit in the proton motive force generator, involved in the late flagella assembly (Kutsukake et al., 1990). Escherichia coli strains possess adherence factors that allow them to adhere and colonize a substratum (Kaper et al., 2004), and bacterial attachment is involved in the first step of biofilm formation. Type 1 fimbriae that mediate mannose-sensitive agglutination have shown to play a role in biofilm formation of E. coli (Pratt & Kolter, 1998). In addition to expression of fimbriae, bacterial cell surface hydrophobicity is also a determinant factor in bacterial adhesion to surfaces (van der Mei et al., 1995). 298

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Interfering with bacterial communication could be a new approach to control bacterial infections. Several chemical compounds have been identified as quorum-sensing inhibitors (QSI) (Vattem et al., 2007; Lee et al., 2011; Jakobsen et al., 2012), for example halogenated furanones. These furanones were initially isolated from the red algae Delisea pulchra and were discovered due to their ability to inhibit bacterial growth and colonization (Kjelleberg & Steinberg, 2001). A series of brominated furanones have been synthesized and found to interfere with bacterial communication, inhibiting AI-2-regulated features such as biofilm formation, swarming and virulence (Ren et al., 2001; €nn-Stensrud et al., 2007; Benneche et al., 2008; He Lo et al., 2012). We have synthesized sulphur analogues of furanone (Benneche et al., 2006) that have shown to be even more effective in inhibiting biofilm formation in Staph€nn-Stensrud et al., ylococcus epidermidis than furanone (Lo 2010, 2012). Previous studies in gram-positive and gram-negative strains have revealed an effect of both F202 and TF101 in biofilm formation in vitro at concentrations which did not have any impact on the planktonic €nn-Stensrud et al., 2007, 2012; Vestby et al., growth (Lo 2010; Defoirdt et al., 2012). TF101 and F202 have previously shown not to be toxic to eukaryotic cells at low €nn-Stensrud et al., 2012). concentrations (Lo The aim of this study was to investigate and compare the effect of the sulphur analogue thiophenone 101 (TF101) and furanone 202 (F202; Fig. 1) in wild-type E. coli O103:H2 strains and to study the effect on phenotypes believed to be regulated by quorum sensing.

Material and methods Bacterial strains and media Three E. coli O103:H2 isolates from sheep were used in this study (Table 1). They were all verified at the National Reference Laboratory at the Norwegian Veterinary Institute. The strains were stored at 80 °C in Luria–Bertani (LB) broth (Difco) supplemented with 15% glycerol and recovered on LB agar plates (bactotrypton 10 g L 1, yeast extract 5 g L 1, agar 15 g L 1) at 37 °C overnight. The bacterial cultures were transferred into LB broth and incubated with shaking overnight at 37 °C to obtain a working culture.

(a)

(b)

Fig. 1 Structure of (Z)-5-(bromomethylene) furan-2(5H)-one (1a, F202) and (Z)-5-(bromomethylene)-thiophene-2(5H)-one (1b, TF101).


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Table 1 Bacterial strains used in this study Strain ID number

Genus

Phenotype

Reference

2006-22-1153 (1153) 2006-22-1242 (1242) 2007-60-10705 (10705) MM32

E. coli O103:H2 E. coli O103:H2 E. coli O103:H2 V. Harveyi

Stx1 , stx2 , eae+

JAF548 pAKluxI

V. harveyi

Sekse et al. (2013) Sekse et al. (2013) Sekse et al. (2013) Miller et al. (2004) Defoirdt et al. (2012)

Stx1 , stx2 , eae Stx1 , stx2 , eae luxN::Cm, luxS::Tn5Kan Luminescence independent of the quorumsensing system

Thiophenone and furanone were dissolved in absolute ethanol to a concentration of 10 mM and stored at 80 °C. The freezing stock was further diluted in LB media to a working solution, without precipitation. Effect of TF101 and F202 on phenotypic features Biofilm formation Overnight cultures were diluted 1 : 200 in fresh LB without NaCl (LBwo/NaCl: bactotrypton 10 g L 1, yeast extract 5 g L 1). The strains were incubated at 37 °C for 5 h and diluted to OD600 nm = 1.0. Samples of 200 lL were transferred to flat-bottom, 96-well polystyrene microtiter plates. Furanone F202 or thiophenone TF101 was added to the wells at final concentrations of 5, 10 or 50 lM. The plates were incubated for 48 h at 20 °C (Nesse et al., 2013). Biofilm quantity was assessed after removing the planktonic cells by inverting the plates and washing the wells twice with 0.9% NaCl. Adherent cells were stained with 0.1% safranine solution for 30 min, followed by washing at least three times with 0.9% NaCl. The safranine stain was released with 30% glacial acetic acid, and OD530 nm was measured (Synergy HT Multi-Detection Microtiterplate Reader, Biotek, VT). The assay was performed in six parallels, and the experiment was repeated twice. The biofilm mass was calculated as % of control. Planktonic growth To assure that the effect on biofilm formation was not due to a growth inhibiting effect, the effect of F202 and TF101 on planktonic growth was assessed. Cultures of each E. coli strain supplemented with F202 or T101 at concentrations of 5, 10 or 50 lM were incubated in LB wo/NaCl with shaking at 20 and 37 °C. OD600 nm was measured every second hour until stationary phase. Cells in LB wo/NaCl medium without F202 or TF101 were similarly assayed as negative controls. The experiment was performed in triplicates and repeated at least twice. Scanning electron microscopy Scanning electron microscopy (SEM) was used to visually confirm the effect of F202 and TF101 on biofilm formation.

Biofilm was allowed to form on polystyrene coverslips (Nunc Thermanox Coverslips, Thermo Scientific, Rochester, NY) immersed into separate culture wells. After incubation (20 °C, 48 h), the planktonic cells were removed by washing with 0.9% NaCl, and the adherent cells were fixed with 2.5% glutaraldehyde in 0.1 M Sørensen phosphate buffer and stored at 4 °C until examined by SEM (model XL 30 ESEM, €nn-Stensrud et al., Philips, Eindhoven) as described (Lo 2007). Swarming motility The effect of TF101 and F202 on swarming motility was studied in semi-solid agar plates with 0.5% agar (10 g L 1 tryptone, 5 g L 1 yeast extract, 5 g L 1 NaCl, 10 g L 1 glucose, 5 g L 1 agar with F202 or TF101 added, 10 lM final concentration). Overnight cultures, diluted to OD600 nm = 1.0, were spot-inoculated (1 lL) on the agar plates. After incubation at 37 °C for 10 h, the halo diameter (mm) of the swarming motility was measured and compared with control on agar without TF101 or F202. Expression of flagella SEM was used to study the effect of TF101 or F202 on expression of flagella. Escherichia coli 1242 was used as a representative strain. Cell cultures supplemented with 10 lM F202 or TF101 were incubated with shaking at 37 °C for 5 h. Cell cultures without furanone or thiophenone were assayed as negative control. 10 lL of the cell suspension was added to a polystyrene coverslip and let to dry at room temperature to allow the cells to attach to the surface. The cells were fixed and treated for SEM as described above. Expression of flagella was also studied by atomic force microscopy (AFM). Escherichia coli strain 1242 was incubated at 37 °C for 5 h in LB wo/NaCl with or without 10 lM TF101 or F202. The cells were washed twice in 0.9% NaCl solution and mixed with 100 mM Tris-MgCl2. Ten microlitres aliquots were transferred to freshly cleaved mica. The samples were incubated for 10 min at room temperature, followed by washing with 10 9 100 lL milliQ- H2O. Hereafter, the samples were dried by applying a soft jet stream of N2. AFM was performed in intermittent contact mode in air using the NanoWizard instrument from JPK (Berlin, Germany). The scanning probes were NSC35/AIBS purchased from MicroMash (Estonia). Real-time PCR Escherichia coli strain 1242 was grown planktonically for 2.5 h at 37 °C to OD600 nm 0.5. TF101, F202 or (S)-4, 5-dihydroxy-2, 3-pentanedione (DPD; OMM Scientific Inc., TX) was added to final concentrations of 10 lM, and the cultures were incubated for another 60 min. Total RNA was isolated from harvested E. coli cells using the High Pure RNA isolation kit (Roche Applied Science, Mannheim, Germany) as described by the manufacturer. DNase was used during the RNA extraction to remove remaining DNA, and the RNA was stored at 80 °C. cDNA was synthetized using MMLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit (Epicentreâ Biotechnologies, Ilumina Inc.,


299


Madison, WI) as described by the manufacturer. Expression of flhD, fliA and motA was studied using primer pairs as described in Table 2. Expression of rpoA was used to normalize the data (Table 2). The assay was carried out using Stratagene Mx3005 P Multiplex Quantitative PCR systems (Stratagene, La Jolla, CA) using the Thermo Scientific Maxima SYBR Green/ROX qPCR Master Mix (Fermentas, Germany). The gradient thermocycling programme was set for 40 cycles at 95 °C for 15 s, 59 °C for 30 s and 72 °C for 30 s, with an initial cycle at 95 °C for 10 min. The data were collected and compared using MXPRO software. ATP measurements As reduced biofilm formation and motility also could be ascribed to an effect of TF101 or F202 on ATP generation, we assayed ATP in the cell cultures using the BacTiter-Glo microbial cell viability assay (Promega, Madison, WI). Overnight cultures were diluted 1 : 1000 in LB WO/NaCl with or without TF101 or F202 and grown for 5 h at 37 °C. Hundred microlitres of the bacterial suspensions were mixed with 100 lL of the BacTiter-Glo reagent in black-walled, optical-bottom 96-well plates (Nunc, Denmark). Luminescence was measured (Synergy HT Multi-Detection Microtiterplate Reader). LB wo/NaCl was used to measure the background luminescence Expression of fimbriae Bacteria expressing Type 1 fimbriae are able to agglutinate yeast cells due to high mannose-binding. Expression of Type 1 fimbriae was performedaccording to Hancock et al. (2011), with slight modifications. Briefly, 10 mL of LB media were inoculated with 10 lL of overnight culture and incubated at 37 °C for 5 h. One-hour prior to examination, TF101 or F202 was added to the cultures at 10 lM concentrations. Bacterial culture without TF101 or F202 was used as control. Aliquots of the bacterial suspension (10 lL) were mixed with yeast cells (Saccharomyces cerevisiae, 0.01 g mL 1), and agglutination was investigated and scored by the time it took for the yeast cells to agglutinate (+++ = 0–10 s, ++ = 10–60 s, + = > 60 s). Bacterial adherence to hydrocarbons (BATH) As adhesion may depend also on hydrophobicity of the bacteria, the effect of T101 and F202 on cell hydrophobicity was measured as adhesion to hydrocarbons. Second, overnight cultures were prepared for each strain. After 24 h, F202 or TF101 was added to the cultures to a final Table 2 Primers used in this study ILW007_fliA ILW007_fliA ILW008_motA ILW008_motA ILW003_flhD ILW003_flhD ILW004_rpoA ILW004_rpoA

300

Forward Reverse Forward Reverse Forward Reverse Forward Reverse

5′-tgctcgacaccaataacagc-3′ 5′-gcagttgttgtagcgggttt-3′ 5′-atgcagtgcgtcaaagtcac-3′ 5′-gcacatgctcttccagttca-3′ 5′-ggttaagctggcagaaacca-3′ 5′-gatgccggtatgaatttgct-3′ 5′-caaccattctggctgaacaa-3′ 5′-gcggacagtcaattccagat-3′

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concentration of 10 lM and then the cultures were incubated for another 24 h. The cultures were centrifuged at 600 g for 10 min and washed twice with PBS (NaCl 8 g L 1, KCl 0.2 g L 1, Na2HPO4 1.44 g L 1 and KH2PO4 0.24 g L 1, adjusted to pH 7.2). The bacterial pellets were resuspended in PBS to OD600 nm of 0.6–0.8 (A0). 500 lL of hexadecane was added to the suspension, mixed thoroughly by vortexing for 1 min (426 g) and incubated at room temperature for 30 min. OD600 nm of the aqueous phase was measured (A). The percentage of adhered bacteria to hexadecane was calculated by the equation: % adhesion to hexadecane = [(A0 A)/A0] 9 100. Interference with AI-2 communication Bioluminescence AI-2 production by E. coli was assessed as the ability of cell-free culture supernatants to induce bioluminescence in the reporter strain Vibrio harveyi MM32 (ATCC BAA-1121), a non-AI-2 producing quorum-sensing mutant with only the LuxP AI-2 receptor. Interference with AI-2 communication was assessed as the ability of TF101 or F202 to interfere with bioluminescence induced by E. coli cell-free culture supernatants in the reporter strains V. harveyi MM32 (ATCC BAA-1121) and JAF548 pAKlux1, the latter being a constitutively bioluminescent mutant (Defoirdt et al., 2012). Cell-free supernatants were prepared by centrifugation of mid-log phase cultures of each E. coli strain (10 000 g, 10 min, 4 °C) followed by sterile filtration (0.2 lm). Vibrio harveyi mutants MM32 and JAF548 pAKlux1 were grown overnight in LB medium containing 35 g L 1 of sea salt (Sigma–Aldrich, St Louis, MO), diluted to an OD600 nm of c. 0.5 and distributed in a 96-well microtiterplate. Cell-free supernatants were added (1 : 10) to the wells containing V. harveyi MM32 or JAF548 pAKlux1. TF101 or F202 (5 or 10 lM) was added, and the cultures were further incubated at 30 °C with shaking. 30 min after thiophenone or furanone addition, luminescence was measured with Synergy HT Multi-Detection Microtiterplate Reader, and the results compared with nonexposed controls. To study whether TF101 or F202 affected AI-2 production, E. coli cultures in mid-exponential phase were pretreated (45 min) with 10 lM TF101 or F202, and cell-free supernatants were prepared as previously described. The ability of the supernatants to induce bioluminescence in V. harveyi MM32 was measured. Statistics All experiments were performed as at least two independent experiments with at least three parallels in each, using freshly prepared reagents unless otherwise stated. One-way ANOVA followed by the Student–Newman–Keuls method was used for the multiple comparisons of biofilm formation. For the motility analysis, One-way ANOVA followed by the Holm– Sidak method was used. For all statistical analysis, the level of statistical significance was set at P < 0.05. The data were analysed by EXCELâ 2010 (Microsoftâ, Redmond, WA) and SIGMAPLOT version 12.5


I.L. Witsø et al.

Results Effect of TF101 and F202 on phenotypic features Biofilm formation and growth Both TF101 and F202 reduced biofilm formation in microtiter plates at the concentrations tested in all three E. coli strains (P ≤ 0.05; Fig. 2a). At 50 lM, TF101 was more efficient than F202 in reducing biofilm formation in all strains (P < 0.05). TF101 was also more efficient than F202 in reducing biofilm formation in strains 1242 and 10705, at 10 lM (P ≤ 0.05). These two strains formed more biofilm compared with strain 1153. The reduction in biofilm was confirmed by SEM (Fig. 2b). The effect on planktonic growth was studied by OD600 nm measurements. There was no effect of F202 or TF101 on growth at concentrations 5 lM or 10 lM, whereas 50 lM retarded growth slightly during exponential phase (Fig. 2c and Supporting Information, Fig. S1). This effect seemed to cease when the cells reached stationary phase. Due to the growth retarding effect of 50 lM TF101 and F202, 10 lM was selected as the highest concentration used in further experiments. Motility At 10 lM, TF101 gave a significant decrease in swarming motility (P < 0.05) compared with the control (Fig. 3a and b). F202 had no effect on motility at 10 lM. TF101 reduced motility in all the three strains more efficiently compared with F202 (P ≤ 0.05). Figure 3a shows representative pictures of swarming motility phenotype with and without TF101 or F202. Expression of flagella and fimbria As seen in the SEM images, expression of flagella was reduced upon exposure to TF101 (Fig. 3c), and F202 did not affect the number of flagella which appeared similar to the control. Type 1 fimbriae expression was confirmed by yeast cell agglutination. The control sample and the sample with F202 showed positive yeast agglutination within 10 s, whilst addition of TF101 gave a negative agglutination result, indicating an inhibitory effect of TF101 on expression of fimbriae (Table 3). The results from the phenotypic assays were confirmed by atomic force microscopy showing reduced numbers of both fimbriae and flagella after treatment with TF101 (Fig. 3d). Effect of TF101 and F202 on gene expression The relative expression of flhD, fliA and motA is shown in Fig. 4. Addition of DPD gave an increase in the expression of all three genes (P < 0.05). TF101 gave a significant change in gene expression of flhD compared with F202. Neither TF101 nor F202 affected expression of fliA or motA ATP measurements The BacTiter-Glo microbial assay measures ATP as an indicator of metabolic activity and cell viability. The relative ATP values are shown in Table 3. Overall, there was no statistically significant effect of 10 lM TF101 on ATP levels


in the three strains. Strain 1242 and 10705 showed higher ATP levels compared with strain 1153. The former strains were the two most biofilm-producing strains. Conversely, in strain 1242, addition of F202 gave reduced ATP levels compared with the control (P < 0.05, Table 3). Hydrophobicity In an attempt to understand the mechanism of action of thiophenone and furanone, their effect on bacterial hydrophobicity was studied using BATH. Neither TF101 nor F202 affected the hydrophobicity in the three E. coli strains (Table 3). Thiophenone and furanone interfere with AI-2 communication Supernatants from all three strains induced bioluminescence in V. harveyi MM32. By adding TF101 or F202 to the MM32 and E. coli supernatant mixture, the bioluminescence was significantly reduced compared with the control (P < 0.05; Fig. 5a). When TF101 was added, the bioluminescence was decreased by over 40% at the lowest concentration (5 lM). The reduction in bioluminescence of F202 was around 50% at the lowest concentration (5 lM). The decrease in bioluminescence was in a concentration-dependent manner. In E. coli 10705, there was a significant difference in bioluminescence interference between TF101 and F202, with F202 being more efficient. In the two other strains, there was no significant difference between TF101 and F202. JAF548 pAKlux1 produces bioluminescence independently of the quorum-sensing system. Both TF101 and F202 seemed to affect bioluminescence to varying degrees in JAF548 pAKlux1 (Fig. 5b). All concentrations of F202 significantly reduced the bioluminescence compared with the control (P < 0.05), whilst TF101 reduced bioluminescence significantly only at 10 lM. F202 reduced bioluminescence in JAF548 more than TF101 at both concentrations tested (P < 0.05). To study whether TF101 or F202 affected AI-2 production, the same protocol was followed comparing supernatants from cultures pre-exposed to TF101 or F202 with supernatants from unexposed cells. The supernatants induced bioluminescence in MM32 (Fig. 5c) with no difference between the samples pre-exposed with TF101 or F202 and the unexposed controls (P > 0.05).

Discussion In this study, the effect of thiophenone on E. coli strains was tested for the first time. Further, the structural analogues of thiophenone and furanone were compared for effect on selected phenotypic and virulence traits in E. coli. To our knowledge, this is the first study exploring the effect of thiophenone on physiology and gene expression in gram-negative bacteria. The present results clearly showed interference with E. coli biofilm formation by both TF101 and F202 at nongrowth inhibitory concentrations. TF101 was more efficient in controlling biofilm formation at lower


301


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(a)

(b) 1a

1b

1c

2a

2b

2c

3a

3b

3c

(c)

302

Fig. 2 The effect of 5, 10 or 50 lM of TF101 or F202 on phenotypic features in three Escherichia coli O103:H2 strains. (a) All concentrations tested gave a significant reduction in biofilm formation compared with the control sample. The results are mean relative values and standard errors from two independent experiments with six parallels in each. *Significant different from control. **Significant different from control and the same concentration of F202 (P < 0.05). (b) Scanning electron micrographs of E. coli biofilm formation on coverslips supplemented with 10 lM TF101 (1B–3B) or 10 lM F202 (1C–3C). The controls are shown in the left panel (1A – E. coli 1153, 2A – E. coli 1242, 3A – E. coli 10705). (c) TF101 (a) and F202 (b) effect on planktonic growth at 20 °C. Growth of E. coli 1153, E. coli 1242 and E. coli 10705 was measured as plaktonic growth at OD600 nm for 24 h with different concentrations of TF101 and F202. The data represent mean value (n = 6), OD600 nm, optical density at 600 nm.


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(a)

(b)

(c)

Fig. 3 Motility in Escherichia coli O103:H2. (a) Swarming motility in three E. coli O103: H2 strains with and without 10 lM TF101 or F202. (b) Graphical presentation of motility in E. coli exposed to 10 lM TF101 or 10 lM F202. The data represent mean values from three different experiments. **Significantly different from control and F202. (c) The effect of TF101 and F202 on expression of flagella in planktonic cells. The flagella are designated with an arrow. (d) AFM used to study extracellular structures on E. coli exposed to TF101 or F202 (scale 10 lM).

(d)

Table 3 TF101 and F202 effect on phenotypic and metabolic activity E. coli 1153 Control ATP levels (relative values)† Agglutination‡ Hydrofobicity %§,¶

10 lM TF101

E. coli 1242 10 lM F202

Control

10 lM TF101

1.00

0.78

0.94

1.00

1.03

53.43

+ 54.40

52.08

74.30

+ 74. 51

E. coli 10705 10 lM F202 0.76 (*)

81.8

Control

10 lM TF101

10 lM F202

1.00

0.94

0.99

71.62

+ 72.85

74.43

The results represent mean values (n ≤ 6). † F202 gave a decrease in ATP levels in E. coli 1242 (P < 0.05). In the other strains, no difference was observed in ATP levels. (*Significant different from control). ‡ Addition of TF101 to the culture inhibited yeast agglutination, indicating an effect on expression of fimbriae [(+) effect of TF101 or F202, ( ) no effect of TF101 or F202]. § TF101 and F202 did not influence the bacteria’s ability to adhere to hydrocarbons (hydrofobicity). ¶ [(A0 A)/A0] 9 100.

concentrations compared with F202. This is in line with a €nn-Stensrud et al., previous study in S. epidermidis (Lo 2012). In previous studies, thiophenone TF101 and furanone F202 have also been shown to interfere with biolumi€nn-Stensrud et al., 2010, 2012; nescence in V. harveyi (Lo Defoirdt et al., 2012). F202 has also been shown to interfere with biofilm formation in gram-negative Salmonella enterica strains and in E. coli O103:H2 (Vestby et al., 2010, 2013). It has been indicated that they both act as QSI (Ren et al., 2001; Defoirdt et al., 2012). The mechanisms behind the biofilm inhibitory effect of thiophenones and furanones are, however, still discussed and unclear. Studies in V. harveyi have shown that brominated furanone and thiophenone TF101 prevent binding of the quorum-sensing master regulator protein LuxR to its target promoter sequences (Defoirdt et al., 2007, 2012).

Quorum-sensing controls a wide range of bacterial characteristics, including motility and adhesion (Antunes et al., 2010). Both of these factors are involved in biofilm formation. By studying gene expression in air–liquid interphase biofilm in E. coli K-12 wild type, Ren et al. (2004) found that furanone repressed biofilm formation and expression of genes involved in motility and biofilm formation. They found that furanone acted by blocking AI-2 signalling post-transcriptionally, notably by binding covalently to AI-2. In this study, furanone did not interfere with motility or expression of flagella at 10 lM, whilst thiophenone was shown to affect both motility and expression of flagella (Fig. 3). This may be seen in context with the biofilm results, which showed that thiophenone was more effective at lower concentrations compared with furanone. Biofilm formation and motility were studied under different experimental conditions, which might make them difficult to compare.


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Fig. 4 Effect of DPD, F202 or TF101 on expression of genes involved in flagella assembly. Relative expression of flhD, fliA and motA in cells exposed to 10 lM DPD, F202 or TF101 compared with nontreated cells (denoted as 0.0). The data represent mean values from two independent assays with a total of 6 parallels. *P < 0.05.

However, this is an in vitro study, where the limits of the methods need to be taken into account. To rotate, the flagella depend on energy. According to the ATP measurements, TF101 did not interfere with ATP generating metabolic reactions, whilst the SEM images used to study expression of flagella showed that TF101 in contrast to F202 affected expression of flagella. Hence, reduction in motility by TF101 was most likely due to reduced expression of flagella and independent of interference with ATP production. Several genes associated with flagella assembly in E. coli have been identified (Chilcott & Hughes, 2000). Thus, it was interesting to study whether some of these genes were affected in response to TF101 or F202. All three genes were upregulated after addition of DPD. This is in line with a

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previous microarray study (Ren et al., 2004) showing upregulation by AI-2 of several genes and downregulation by furanone of the same genes. We observed no significant effect on expression of flhD, fliA or motA after treatment with F202. Strain background and experimental conditions might explain the contradictive results. One important factor is also that Ren et al. (2004) studied bacteria in biofilm, whilst our gene expression study was preformed on bacteria in planktonic state in exponential phase. Additionally, they used a structurally different furanone at a higher concentration. Our results correlate very well with our motility results, showing that swarming motility was not reduced by F202. In a previous study, we observed that 50 lM furanone reduced motility in the same strains without affecting expression of flagella (Vestby et al., 2013). This may suggest that furanone acts on biological mechanisms other than quorum sensing, for example flagellar function. However, furanone seems less effective than TF101, and higher concentrations might be needed to observe an effect. We found that TF101 repressed expression of flhD. fliA showed a slight, statistically nonsignificant decrease in expression, whilst expression of motA did not show any change in response to TF101. Motility and transcription of flagellar genes are very complex and sensitive to several environmental conditions (Chilcott & Hughes, 2000), which may account for the discrepancy of the difference in expression of the three genes. They are also expressed at different time points during growth. fliA and motA are expressed later than flhD, and that may encounter the larger effect of TF101 on flhD gene expression compared with the two other genes at this specific time point. Previous studies have shown that TF101 interferes with €nn-Stensrud et al., quorum sensing (Defoirdt et al., 2012; Lo

(a)

(b)

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(c)

Fig. 5 Bioluminescence in Vibrio harveyi MM32 induced by Escherichia coli. (a) Bioluminescence in the reporter strain V. harveyi MM32 induced by E. coli 1153, 1242 and 10705, with and without TF101 or F202. (b) Bioluminescence in constitutive bioluminescent mutant V. harveyi JAF548 pAKlux1 with and without TF101 and F202. (c) Bioluminescence in reporter strain V. harveyi MM32 induced by E. coli supernatants pretreated with 10 lM TF101 and 10 lM F202, respectively. Black bars represent control without any treatment, dark grey represents F202 and light grey represents TF101. The results are mean values from two independent experiments with 6 parallels. *Significant from control. **Significant from control and F202 (P < 0.05).


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2012). QseBC is also involved in regulation of the flagella genes and is regulated by quorum sensing (Sperandio et al., 2002). Whether QseBC is involved in the reduction of biofilm and motility in this study is not known. However, QseBC could be a new target in the approach to understand more of the mechanisms of thiophenone and furanone. Adhesion is an important step in biofilm formation. Escherichia coli cells treated with TF101 showed decreased ability to agglutinate yeast cells, indicating decreased expression of Type 1 fimbriae, which are important adhesion mediators in E. coli biofilm. The same result was not observed when the cells were treated with F202. Thus, furanone does not seem to interfere with expression of Type 1 fimbriae at the concentration tested. Hydrophobicity also plays an important role in bacterial adhesion and is linked to the expression of fimbriae, especially Type 1 fimbriae in E. coli. One would thus expect that inhibition of Type 1 expression, as seen in bacteria exposed to TF101, would show reduction in hydrophobicity. This was, however, not observed in the present study. F202 and TF101 did not affect hydrophobicity of the studied strains. Previous studies with hydrophobicity in E. coli have given conflicting results as to the hydrophobic properties. Growth media, origin of the strain and methods used to determine hydrophobicity could explain contradictory results (Goulter et al., 2009). The sensitivity of the hydrocarbon adherence method may be insufficient; thus, complementary testing is needed to elucidate the impact of hydrophobicity in adhesion of E. coli and the effect of QSI. In accordance with previous studies, we would expect thiophenone and furanone to affect quorum sensing via AI-2 €nn-Stensrud et al., 2007, 2012). Using V. harveyi MM32 (Lo reporter strain, we were able to show that the present E. coli strains produced AI-2, indicating that all three strains are able to communicate through AI-2. The reduction in bioluminescence after addition of TF101 and F202 supports the notion that TF101 and F202 may act through interference with AI-2 interspecies communication possibly by competing for its receptor. JAF548 pAKlux1 produces bioluminescence that is independent of quorum sensing. The strain was included to assess possible quorum-sensing-independent effects. Our results showed that both TF101 and F202 interfered with the bioluminescence in JAF548 pAKlux1, with F202 showing the greatest reduction in bioluminescence in both JAF548 pAKlux1 and MM32. This may indicate that besides interference with quorum-sensing communication, TF101 and F202 might affect E. coli by mechanisms undetected by the growth and ATP assays. Interestingly, previous microarray studies have shown that brominated furanone induces stress responses in E. coli K-12 (Ren et al., 2004). Taken together, we cannot exclude the possibility that TF101 and F202, in addition to interference with quorum sensing, induce stress responses, with the greatest response after exposure to F202. To assess the effect of TF101 and F202 on AI-2 production, bacterial cells were exposed to TF101 and F202 prior to supernatant preparation. Bioluminescence assay with V. harveyi MM32 was used to quantify differences in light production. Our results showed that bioluminescence in MM32 was not reduced in samples


pretreated with TF101 or F202, indicating that TF101 and F202 most likely do not interfere with LuxS synthase. In conclusion, the present study showed that both furanone and thiophenone reduced biofilm formation in E. coli O103:H2, in vitro. As thiophenone was the most effective substance in reducing biofilm formation and motility at nongrowth inhibitory concentrations, this molecule has caught attention for further studies. The present results open for further investigations to find QSI in the fight against infections caused by E. coli. Further molecular studies are needed to understand and learn more about the mechanisms underlying the biofilm reducing effect and the role of thiophenone as a quorum-sensing inhibitor. References Antunes LC, Ferreira RB, Buckner MM & Finlay BB (2010) Quorum sensing in bacterial virulence. Microbiology 156: 2271–2282. Bassler BL, Greenberg EP & Stevens AM (1997) Cross-species induction of luminescence in the quorum-sensing bacterium Vibrio harveyi. J Bacteriol 179: 4043–4045. €nn J & Scheie AA (2006) Synthesis of (E)- and Benneche T, Lo (Z)-5-(bromomethylene)furan-2(5H)-one by bromodecarboxylation of (E)-2-(5-oxofuran-2(5H)-ylidene)acetic acid. Synth Commun 36: 1401–1404. € nn- Stensrud J (2008) Benneche T, Hussain Z, Scheie AA & Lo Synthesis of 5-(bromomethylene)furan-2(5H)-ones and 3-(bromomethylene)isobenzofuran-1(3H)-ones as inhibitors of microbial quorum sensing. New J Chem 32: 1567–1572. Carneiro LA, Lins MC, Garcia FR, Silva AP, Mauller PM, Alves GB, Rosa AC, Andrade JR, Freitas-Almeida AC & Queiroz ML (2006) Phenotypic and genotypic characterisation of Escherichia coli strains serogrouped as enteropathogenic E. coli (EPEC) isolated from pasteurised milk. Int J Food Microbiol 108: 15–21. Chilcott GS & Hughes KT (2000) Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar Typhimurium and Escherichia coli. Microbiol Mol Biol Rev 64: 694–708. Ciofu O & Tolker-Nielsen T (2011) Antibiotic Tolerance and Resistance in Biofilms. Biofilm Infections (Bjarnsholt T, Jensen PØ, Moser C & Høiby N, eds), pp. 215–229. Springer, New York, NY. Clarke MB & Sperandio V (2005) Transcriptional regulation of flhDC by QseBC and sigma (FliA) in enterohaemorrhagic Escherichia coli. Mol Microbiol 57: 1734–1749. Costerton JW, Lewandowski Z, Caldwell DE, Korber DR & Lappin-Scott HM (1995) Microbial biofilms. Annu Rev Microbiol 49: 711–745. Defoirdt T, Miyamoto CM, Wood TK, Meighen EA, Sorgeloos P, Verstraete W & Bossier P (2007) The natural furanone (5Z)-4-bro mo-5-(bromomethylene)-3-butyl-2(5H)-furanone disrupts quorum sensing-regulated gene expression in Vibrio harveyi by decreasing the DNA-binding activity of the transcriptional regulator protein luxR. Environ Microbiol 9: 2486–2495. Defoirdt T, Benneche T, Brackman G, Coenye T, Sorgeloos P & Scheie AA (2012) A quorum sensing-disrupting brominated thiophenone with a promising therapeutic potential to treat luminescent vibriosis. PLoS One 7: e41788. Eberl L, Winson MK, Sternberg C, Stewart GS, Christiansen G, Chhabra SR, Bycroft B, Williams P, Molin S & Givskov M (1996) Involvement of N-acyl-L-hormoserine lactone autoinducers in controlling the multicellular behaviour of Serratia liquefaciens. Mol Microbiol 20: 127–136. Goulter RM, Gentle IR & Dykes GA (2009) Issues in determining factors influencing bacterial attachment: a review using the


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Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. TF101 (a) and F202 (b) effect on planktonic growth at 37 °C.


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