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Thermoresponsive oligomers reduce Escherichia coli O157:H7 biofouling and virulence a

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Jin-Hyung Lee , Yong-Guy Kim , Hyun Seob Cho , Jintae Kim , Seong-Cheol Kim , Moo Hwan a

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Cho & Jintae Lee a

School of Chemical Engineering, Yeungnam University, Gyeongsan, Republic of Korea

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Department of Nano, Medical and Polymer Materials, Yeungnam University, Gyeongsan, Republic of Korea Published online: 15 Apr 2014.

Click for updates To cite this article: Jin-Hyung Lee, Yong-Guy Kim, Hyun Seob Cho, Jintae Kim, Seong-Cheol Kim, Moo Hwan Cho & Jintae Lee (2014) Thermoresponsive oligomers reduce Escherichia coli O157:H7 biofouling and virulence, Biofouling: The Journal of Bioadhesion and Biofilm Research, 30:5, 627-637, DOI: 10.1080/08927014.2014.907402 To link to this article: http://dx.doi.org/10.1080/08927014.2014.907402

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Biofouling, 2014 Vol. 30, No. 5, 627–637, http://dx.doi.org/10.1080/08927014.2014.907402

Thermoresponsive oligomers reduce Escherichia coli O157:H7 biofouling and virulence Jin-Hyung Leea,1, Yong-Guy Kima,1, Hyun Seob Choa, Jintae Kimb, Seong-Cheol Kimb*, Moo Hwan Choa and Jintae Leea* a School of Chemical Engineering, Yeungnam University, Gyeongsan, Republic of Korea; bDepartment of Nano, Medical and Polymer Materials, Yeungnam University, Gyeongsan, Republic of Korea

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(Received 29 November 2013; accepted 10 March 2014) Thermoresponsive polymers have potential biomedical applications for drug delivery and tissue engineering. Here, two thermoresponsive oligomers were synthesized, viz. oligo(N-isopropylacrylamide) (ONIPAM) and oligo(N-vinylcaprolactam) (OVCL), and their anti-biofouling abilities investigated against enterohemorrhagic E. coli O157:H7, which produces Shiga-like toxins and forms biofilms. Biofilm formation (biofouling) is closely related to E. coli O157:H7 infection and constitutes a major mechanism of antimicrobial resistance. The synthetic OVCL (MW 679) and three commercial OVCLs (up to MW 54,000) at 30 μg ml−1 were found to inhibit biofouling by E. coli O157:H7 at 37 °C by more than 80% without adversely affecting bacterial growth. The anti-biofouling activity of ONIPAM was weaker than that of OVCL. However, at 25 °C, ONIPAM and OVCL did not affect E. coli O157:H7 biofouling. Transcriptional analysis showed that OVCL temperature-dependently downregulated curli genes in E. coli O157:H7, and this finding was in line with observed reductions in fimbriae production and biofouling. In addition, OVCL downregulated the Shiga-like toxin genes stx1 and stx2 in E. coli O157:H7 and attenuated its in vivo virulence in the nematode Caenorhabditis elegans. These results suggest that OVCL has potential use in antivirulence strategies against persistent E. coli O157:H7 infection. Keywords: biofilm; biofouling; Escherichia coli O157:H7; N-vinylcaprolactam; thermoresponsive polymer; virulence

Introduction Thermoresponsive polymers are a class of ‘smart materials’ with the ability to respond to changes in temperature, and their potential usage in biomedical applications, including drug delivery and tissue engineering, is being widely investigated (Vihola et al. 2005; Ward & Georgiou 2011). Thermoresponsive polymers exhibit lower critical solution temperatures (LCSTs) and are soluble in water below their LCSTs, but insoluble above (Vihola et al. 2005). Poly(N-isopropylacrylamide) (PNIPAM) has been widely studied, but recently another thermoresponsive polymer, poly(N-vinylcaprolactam) (PVCL), which is more biocompatible than PNIPAM, has attracted research attention (Imaz & Forcada 2010). Importantly, PNIPAM and PVCL both have LCST values of ~32 °C and, thus are soluble at room temperature and phase separate at body temperature. Shiga-like toxigenic Escherichia coli O157:H7 has caused a large number of foodborne outbreaks worldwide, but no effective treatment has been devised to treat E. coli O157:H7 infections, primarily because antibiotics and anti-inflammatory drugs increase the risk of developing hemolytic-uremic 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 & Kaper 1998). Of the several virulence factors produced by E. coli O157:H7, the Shiga-like toxins Stx1 and Stx2 are considered to be the major virulence factors responsible for its clinical symptoms (Nataro & Kaper 1998). Furthermore, E. coli O157:H7 kills the nematode Caenorhabditis elegans by producing Shiga-like toxins (Kim et al. 2006; Chou et al. 2013), which allows C. elegans to be used to assess E. coli O157:H7 virulence. In addition, E. coli O157:H7 is able to form biofilms on biotic and abiotic surfaces, such as stainless steel, glass, and polymers (Ryu & Beuchat 2005; Rivas et al. 2007; Patel et al. 2011), and these biofilms are difficult to eradicate because of their inherent tolerance to physical and chemical antimicrobial treatments. Accordingly, pathogenic biofilms pose serious problems to human health (Costerton et al. 1999). Recently, several polymers have been studied to determine whether they inhibit biofouling (biofilm formation) on different surfaces (Rana & Matsuura 2010; Yu et al. 2011). In the present study, two thermoresponsive oligomers (ONIPAM and OVCL) were synthesized and their antibiofouling abilities investigated against E. coli O157:H7. In a subsequent investigation, attempts were made to determine the molecular mechanism responsible for the inhibition of biofouling by OVCL using transcriptional

*Corresponding authors. Email: [email protected] (S-C Kim), [email protected] (J Lee) 1 These authors contributed equally to this work. © 2014 Taylor & Francis

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and phenotypic assays. In addition, an in vivo C. elegans model was used to study the effects of OVCL on the virulence of E. coli O157:H7.

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Materials and methods Materials N-Isopropylacrylamide (NIPAM), N-vinylcaprolactam (VCL), and azobisisobutyronitrile (AIBN) were purchased from Sigma-Aldrich (St Louis, MO). The other chemicals used to produce ONIPAM and OVCL were purchased from Duksan Pure Chemicals (Ansan, Korea). NIPAM was recrystallized from hexane/toluene (1:1 v/v), and VCL was purified by triple recrystallization at reduced temperature using hexane/diethyl ether (2:1 v/v). AIBN was purified by crystallization from methanol and dried under vacuum. Four commercial thermoresponsive OVCLs with different molecular weights (MW 1,300, 32,000, 54,000, and 240,000) were purchased from Polymer Source, Inc. (Dorval, Canada).

Analysis of oligomers The molecular structures and weights of ONIPAM and OVCL were characterized by 1H-NMR (300 MHz, VNS300, Bruker, Germany) using CDCl3 containing a small amount of tetramethylsilane reference as the solvent. Matrix-assisted laser desorption ionization time-offlight (MALDI-TOF) analysis was performed using a Voyager DE-STR (Applied Biosystems, Foster City, CA) at an operating voltage of 20 kV and using a 337 nm N2 laser in reflection mode. Samples were prepared by dissolving ONIPAM or OVCL (10 mg ml−1) in a mixture of acetonitrile:deionized water:formic acid (700:300:1 all μl) containing α-cyano-4-hydroxycinnamic acid (10 mg ml−1). Thermal transition of oligomers was monitored by measuring the intensity of visible light at 550 nm using UV/VIS/NIR (cary5000, Agilent, Korea) spectrometer with a resolution of 0.05 nm.

Bacterial growth assay Serotype E. coli O157:H7 [ATCC43895, EDL933 strain (Strockbine et al. 1986)] was used throughout. E. coli OP50 was used the control strain as well as the food source for C. elegans (Murphy et al. 2003). Experiments were conducted at 25 °C or 37 °C in Luria–Bertani (LB) medium. For cell-growth assessments, optical densities were measured at 600 nm using a spectrophotometer (Optizen 2120UV, Mecasys, Korea). Each experiment was performed using at least two independent cultures.

Biofouling assay A static biofouling assay was performed in 96-well polystyrene plates (SPL Life Sciences, Korea), as previously described (Lee, Cho et al. 2011). To quantify biofouling, biofilms were stained with crystal violet and eluted in 95% ethanol, and absorbance was measured at 570 nm (OD570). The results are the average of at least twelve replicate wells. 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 OVCL or its monomer. Static biofouling was visualized by confocal laser microscopy (Nikon Eclipse Ti, Tokyo) using an excitation wavelength of 488 nm (Ar laser), an emission wavelength of 500–550 nm, and a 20× objective. Color confocal images were produced using NISElements 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 of LB medium in 250 ml shake flasks at a starting OD600 of 0.05 and then cultured at 25 °C or 37 °C for 8 h without agitation in the presence or absence of OVCL (50 μg ml−1). RNase inhibitor (RNAlater, Ambion, TX) was added to prevent RNA degradation. Total RNA was isolated using a Qiagen RNeasy mini Kit (Valencia, CA). qRT-PCR was used to determine the transcription levels of curli genes (csgA and csgB), Shiga-like toxin genes (stx1 and stx2), quorum-sensing genes (luxR, luxS, and tnaA), and motility genes (flhD, motB, and qseB) in E. coli O157:H7 treated with or without ONIPAM or OVCL (50 μg ml−1). Gene-specific primers were used, and rrsG was used as a housekeeping control (Table 1). The qRT-PCR method used has been described previously (Lee, Regmi et al. 2011). qRT-PCR was performed using an SYBR Green master mix (Applied Biosystems) and an ABI StepOne Real-Time PCR System (Applied Biosystems) on two independent cultures. Scanning electron microscope (SEM)-based fimbriae assay SEM was used to observe fimbriae production, as previously described (Lee, Regmi et al. 2011). Briefly, E. coli O157:H7 cells were inoculated on a nylon filter (0.5 × 0.5 mm square) at an initial OD600 of 0.05. Cells

Biofouling Table 1. RT-PCR.

Sequences of the primers used for quantitative

Gene Primer

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rrsG

Forward 5′-TAT TGC ACA ATG GGC GCA AG-3′ Reverse 5′-ACT TAA CAA ACC GCC TGC GT-3′ csgA Forward 5′-AGA TGT TGG TCA GGG CTC AG-3′ Reverse 5′-CGT TGT TAC CAA AGC CAA CC-3′ csgB Forward 5′-ATC AGG CAG CCA TAA TTG GT-3′ Reverse 5′-CCA TAA GCA CCT TGC GAA AT-3′ stx1 Forward 5′-GTC ACA GTA ACA AAC CGT AAC A3′ Reverse 5′-TCG TTG ACT ACT TCT TAT CTG-3′ stx2 Forward 5′-GTT CCG GAA TGC AAA TCA GT-3′ Reverse 5′-CGG CGT CAT CGT ATA CAC AG-3′ luxR Forward 5′-CGC CCT TCA GTG GTG TTT AT-3′ Reverse 5′-CGT CGA GAG ATT CCG GTT TA-3′ luxS Forward 5′-CAT ACC CTG GAG CAC CTG TT-3′ Reverse 5′-TGA TCC TGC ACT TTC AGC AC-3′ tnaA Forward 5′-TGA AGA AGT TGG TCC GAA TAA CGT G-3′ Reverse 5′-CTT TGT ATT CTG CTT CAC GCT GCT T-3′ flhD Forward 5′-TGC ATA CCT CCG AGT TGC TG-3′ Reverse 5′-GCG TGT TGA GAG CAT GAT GC-3′ motB Forward 5′-CAG GGG GAA GTG AAT AAG CA-3′ Reverse 5′-TTC TAA ACA TCG GGC GAT TC-3′ qseB Forward 5′-GGC GAA CCC TTA ACA CTG AA-3′ Reverse 5′-CCA TGC ACG GTA CGA ATA AA-3′

on nylon filters were incubated in the presence of OVCL (50 μg ml−1) at 25 °C or 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×. C. elegans life span assay The C. elegans killing assay was performed as described previously (Lee, Cho, Kim et al. 2013). In brief, E. coli O157:H7 was cultured with or without OVCL (50 μg ml−1) at 37 °C for 24 h, and the overnight culture (10 μl) of E. coli O157:H7 was spread onto NGM plates. Also, E. coli OP50 was used as a control strain compared with the virulent strain, E. coli O157:H7. L4/young adult fer-15;fem-1 (Murphy et al. 2003) worms (n = 60) were infected by placing them on the lawns. Nematodes were incubated at 25 °C 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 Preparation and characterization of the thermoresponsive oligomers In order to increase the interaction between cells and the repeating unit of an oligomer, two thermoresponsive

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oligomers, namely, OVCL and ONIPAM were prepared. Briefly, 30 mmol of each monomer and 3 mmol of AIBN were dissolved in 25 ml of toluene. AIBN initiator (10 mol %) was added to reduce the MWs of the polymers produced. Air in the reaction vessels was degassed through a three-cycle freezing-and-thawing process (Lau & Wu 1999). The reaction flasks were placed into a preheated oil bath at 70 °C. After polymerization for 2 days, the polymer solutions were purified using hexane and diethyl ether. The average number of repeating units calculated from the ratio between the methyl protons of a fragment of AIBN appearing at 2.11 ppm and that of the methylene, 3.22 ppm, in the ε position of caprolactam, was ~ 5.4 (Figure 1A). The average number of repeating units (n) predicted from the major peak in the MALDITOF spectrum of OVCL, 679 Da, was ~4.9, which corresponded well with the value obtained by 1H-NMR (Figure 1B). The degree of polymerization of NIPAM was also calculated. The n value, 6.7, of ONIPAM was calculated from 1H-NMR data using the methine proton at 4.0 ppm (Figure 1C). The MALDI-TOF spectra of ONIPAM show two distinct peaks at 666 and 877 Da of intensity approximately a half of that at 666 Da (Figure 1D). The approximate n value of ONIPAM was calculated considering the intensities of the two major peaks in its spectrum and found to be 6.5 (Xia et al. 2005). Thermal analysis of OVCL and ONIPAM in a thermostatic water bath showed that the LCST of each oligomer was 32 and 33 °C, respectively (Figure 1E). In addition, the intensity of visible light at 550 nm passing through the cuvette was monitored using a UV-Vis spectrometer as a proof for the phase transition of two oligomer solutions (Figure 1F). Temperature-dependent anti-biofouling activities of ONIPAM and OVCL To investigate the anti-biofouling activities of OVCL and ONIPAM as a function of temperature, E. coli O157:H7 biofouling was measured at 25 °C or 37 °C in the presence of ONIPAM or OVCL (0, 30, or 100 μg ml−1). Biofouling was reduced by twofold and by 125-fold by 100 μg ml−1 of ONIPAM or OVCL, respectively, at 37 °C (Figure 2A), but neither oligomer was effective at decreasing biofouling at 25 °C (Figure 2B). The anti-biofouling activity of ONIPAM was weaker than that of OVCL (Figure 2A). To confirm the above result, the anti-biofouling activities of four commercial OVCLs with different molecular weights (MW 1,300, 32,000, 54,000, and 240,000) were investigated at 25 °C or 37 °C. It was found that three OVCLs (MW 1,300, 32,000, and 54,000) markedly inhibited E. coli O157:H7 biofouling at only 37 °C (Figure 2C), but not at 25 °C (Figure 2D). Interestingly,

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Figure 1. Characterization of ONIPAM and OVCL. 1H-NMR spectra of OVCL and ONIPAM are shown in (A) and (C), respectively. MALDI-TOF spectra of OVCL and ONIPAM are shown in (B) and (D), respectively. Numbers of repeating units (n) were calculated by dividing the weights of major peaks by the molecular weight of the monomer. Oligomer structures are shown as inserts. Thermal transition of OVCL and ONIPAM was monitored by measuring the intensity of visible light at 550 nm passing through the cuvette (E). Phase transition of two oligomer solutions was shown as before thermal transition and after thermal transition (F).

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Biofouling

Figure 2. Effects of ONIPAM and OVCL on biofilm formation by E. coli O157:H7 at different temperatures. Biofilm formation (OD570) was quantified after treatment with OVCL MW 679 (0, 30, or 100 μg ml−1) at 37 °C (A) or 25 °C (B) for 24 h in 96-well plates. Anti-biofouling activities of four commercial OVCLs with different molecular weights (MW 1,300, 32,000, 54,000, and 240,000) were investigated at 37 °C (C) or 25 °C (D).

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Figure 3. Dose-dependent effects of OVCL on E. coli O157:H7 biofilm formation and cell growth. OVCL dose-dependently reduced E. coli O157:H7 biofilm formation at 37 °C (A). Planktonic cell growth of E. coli O157:H7 in the presence of OVCL MW 679 (0, 50, or 1,000 μg ml−1) was measured at 600 nm in 250 ml flasks agitated at 250 rpm at 37 °C (B) or 25 °C (C). Biofilm formation by E. coli O157:H7/pCM18 tagged with the green fluorescent protein in 96-well plates in the presence or absence of VCL monomer or OVCL (50 μg ml−1) at 37 °C (D). The scale bars represent 50 μm.

the longest OVCL (MW 240,000) showed much weaker activity than the other three OVCLs (MW 1,300, 32,000, and 54,000) at 37 °C (Figure 2C). Also, the synthesized OVCL (the smallest MW 679) at 100 μg ml−1 showed the highest activity (Figure 2A). Therefore, the most potent OVCL (MW 679) was subjected to further study. Since E. coli O157:H7 forms more biofilm at 25 °C than 37 °C after incubation for 24 h (Figure 2A and B), the anti-biofouling activity of OVCL was assayed at 25 °C after incubation for 12 h when the biofilm formation of E. coli O157:H7 was one third of the biofilm formation after 24 h incubation. However, there was no anti-biofouling effect of OVCL at 25 °C after 12 h incubation (data not shown), so OVCL was found to inhibit E. coli O157:H7 biofouling in a temperature-dependent manner.

Inhibition of E. coli O157:H7 biofouling by OVCL without affecting cell growth In a more detailed study, OVCL dose-dependently inhibited E. coli O157:H7 biofouling at 37 °C, whereas its monomer did not (Figure 3A). Since bacteria form biofilms on the bottom as well as on the sides of 96-well plates, confocal laser microscopy and E. coli O157:H7/ pCM18 tagging a green fluorescent protein were used to observe well-bottom biofilms. Microscopic observations matched the quantitative biofouling data (Figure 3D). The cell growth of E. coli O157:H7 was measured because anti-biofouling activity was required with no bactericidal activity. It was found that OVCL at concentrations up to 1,000 μg ml−1 did not inhibit E. coli O157:H7 growth at 37 °C (Figure 3B) and 25 °C (Figure 3C).

Biofouling Transcriptional changes in biofouling-related genes in E. coli O157:H7 cells by OVCL and ONIPAM

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To investigate the molecular mechanism responsible for the inhibition of biofouling, qRT-PCR was used to examine the differential expression of selected biofoulingrelated genes (Lee, Cho, Joo et al. 2013) and toxin genes in E. coli O157:H7 cells treated with or without OVCL. The gene expression of the csg operon involved in curli formation was found to be markedly downregulated by OVCL at 37 °C (Figure 4A), suggesting that OVCL inhibits fimbriae production. Also, OVCL downregulated two Shiga-like toxin genes (stx1 and stx2) more than twofold (Figure 4A). The expression of quorum sensing genes and motility genes was less influenced by OVCL, but at 25 °C, OVCL did not influence the expression of biofoulingrelated genes (Figure 4B), and OIPAM did not appreciably change gene expression at 37 °C (Figure 4C). Reduction of fimbriae formation in E. coli O157:H7 by OVCL Since fimbriae are important for biofilm formation by E. coli O157:H7 (Ryu & Beuchat 2005; Uhlich et al. 2006; Rendón et al. 2007; Lee, Regmi et al. 2011; Lee, Cho, Joo et al. 2013), and the gene expression of the csg operon was found to be downregulated by OVCL (Figure 4), fimbriae production was investigated by SEM. In line with the gene expression data, OVCL at 50 μg ml−1 clearly reduced fimbriae production, whereas its monomer did not at 37 °C (Figure 5A). Also, few biofilm cells attached to nylon filters in the presence of OVCL (Figure 5A), and there was no effect of OVCL on fimbriae production at 25 °C (Figure 5B). Therefore, downregulation of curli genes and fimbriae production is a possible mechanism for the anti-biofouling activity of OVCL. Reduction of E. coli O157:H7 virulence by OVCL Since E. coli O157:H7 kills the nematode C. elegans by producing Shiga-like toxins (Kim et al. 2006; Chou et al. 2013), the effects of OVCL on the survival of infected C. elegans were assayed. It was found that OVCL prolonged the survival of C. elegans in the presence of E. coli O157:H7 in a dose-dependent manner, indicating that OVCL reduces the virulence of E. coli O157:H7 (Figure 6A). Also, this result was in line with the observed downregulation of Shiga-like toxin stx1 and stx2 genes by OVCL at 37 °C (Figure 4A). Additionally, survival of C. elegans was investigated with E. coli OP50, which is a common food source for the nematode. As expected, there was no harmful effect of OVCL on the nematode (Figure 6B). Compared with the result with non-toxic E. coli OP50, the addition of OVCL (200 μg ml−1) to the E. coli O157:H7 almost

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abolished the virulence of E. coli O157:H7 to the level of E. coli OP50 (Figures 6A and 6B.) Discussion Various polymers have been shown to have antimicrobial and anti-biofouling activities (Rana & Matsuura 2010; Yu et al. 2011). The present study reports for the first time that thermoresponsive OVCL exhibits anti-biofouling activity and anti-virulence activity against enterohemorrhagic E. coli O157:H7 without inhibiting planktonic cell growth. It is important to note that PVCL is biocompatible (Vihola et al. 2005; Imaz & Forcada 2010), as many antibiotics are toxic and exhibit drug resistance. Hence, this anti-biofouling approach provides an alternative to antibiotic-based strategies. It was found that OVCL at concentrations up to 1,000 μg ml−1 did not affect the cell growth of E. coli O157:H7 (Figure 3B and C). Previously, it was reported that PVCL in concentrations up to 10 mg ml−1 was not harmful to animal cell cultures, whereas PNIPAM induced cellular cytotoxicity at 37 °C (Vihola et al. 2005). Therefore, low toxicity is one of the advantages of treatment with OVCL or PVCL. The molecular mechanism responsible for the anti-biofouling effect by OVCL at 37 °C was found to involve the downregulation of curli genes (Figure 4A) and resultant reductions in fimbriae production (Figure 5A). In addition, OVCL downregulated the expression of two Shiga-like toxin genes (Figure 4A) and attenuated E. coli O157:H7 virulence in vivo in the authors’ C. elegans model (Figure 6). These findings indicate that OVCL has potential use in anti-virulence strategies against E. coli O157:H7 infections. Hydrophobicity plays a role in biofilm formation, and it is usually presumed that biofouling propensity increases with substratum hydrophobicity (Rana & Matsuura 2010). Although OVCL is more hydrophobic than its monomer at 37 °C, the presence of OVCL inhibited E. coli O157:H7 biofouling in this study. How OVCL influences the expression of curli genes and Shiga-like toxins is intriguing. Although OVCL reduced fimbriae production (Figure 5), it has been reported that hydrophobicity and fimbriae production are not clearly related in E. coli O157:H7 (Goulter et al. 2010). Further investigation is required to understand how OVCL reaches, transports, or interacts at some target sites and whether the effect of OVCL is functional to other bacteria. Antifouling bioactive surfaces are of great importance for various biomedical and environmental applications (Cheng et al. 2009; Yu et al. 2011). Practically, OVCL can be immobilized on solid surfaces to prevent

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Figure 4. Transcriptional profiles of E. coli O157:H7 cells treated with or without thermoresponsive oligomers. E. coli O157:H7 was cultivated in LB medium with or without OVCL MW 679 (50 μg ml−1) for 8 h without agitation at 37 °C (A) or 25 °C (B) and with ONIPAM (50 μg ml−1) at 37 °C (C). Transcriptional profiles were measured by qRT-PCR. Relative gene expressions represent transcriptional levels after treatment with OVCL vs untreated controls (value 1.0). The experiment was performed in duplicate.

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Figure 5. Effect of OVCL 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 OVCL MW 679 (50 μg ml−1) for 24 h at 37 °C (A) or 25 °C (B). The scale bars represent 3 μm.

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Figure 6. Effects of OVCL 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 (A) and E. coli OP50 as a control (B) in the presence of OVCL MW 679 (0, 50, or 200 μg ml−1). The experiment was performed in triplicate (n = 90).

biofouling. Hence, it would be interesting to apply different molar masses of OVCL onto the surfaces of catheters, dental materials, and implants, for example. The findings of the present study suggest that OVCL should be considered a potential anti-virulence agent and treatment for E. coli O157:H7 infections. Acknowledgements 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 2010-0021871 to J-H. Lee and J. Lee, respectively).

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Thermoresponsive oligomers reduce Escherichia coli O157:H7 biofouling and virulence.

Thermoresponsive polymers have potential biomedical applications for drug delivery and tissue engineering. Here, two thermoresponsive oligomers were s...
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