- Email: [email protected]
Structural effects on persister control by brominated furanones
Bioorganic & Medicinal Chemistry Letters 23 (2013) 6559–6562
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Structural effects on persister control by brominated furanones Jiachuan Pan a,b, Dacheng Ren a,b,c,d,⇑ a
Department of Biomedical and Chemical Engineering, Syracuse University, Syracuse, NY 13244, United States Syracuse Biomaterials Institute, Syracuse University, Syracuse, NY 13244, United States c Department of Civil and Environmental Engineering, Syracuse University, Syracuse, NY 13244, United States d Department of Biology, Syracuse University, Syracuse, NY 13244, United States b
a r t i c l e
i n f o
Article history: Received 25 September 2013 Revised 29 October 2013 Accepted 30 October 2013 Available online 9 November 2013 Keywords: Antibiotic tolerance Persister Quorum sensing Pseudomonas aeruginosa Brominated furanone Structural effect
a b s t r a c t Bacterial persister cells are a small population of dormant cells that are tolerant to essentially all antibiotics. Recently, we reported that a quorum sensing (QS) inhibitor, (Z)-4-bromo-5-(bromomethylene)-3methylfuran-2(5H)-one (BF8), can revert antibiotic tolerance of Pseudomonas aeruginosa persister cells. To better understand this phenomenon, several synthetic brominated furanones with similar structures were compared for their activities in persister control and inhibition of acyl-homoserine lactone (AHL) mediated QS. The results show that some other furanones in addition to BF8 are also AHL QS inhibitors and can revert antibiotic tolerance of P. aeruginosa PAO1 persister cells. However, not all QS inhibiting BFs can revert persistence at growth non-inhibitory concentrations, suggesting that QS inhibition itself is not sufficient for persister control. Ó 2013 Elsevier Ltd. All rights reserved.
Persister cells are phenotypic variants that can be found in virtually any bacterial culture and are tolerant to different antibiotics.1 It is believed that this subpopulation can survive from aggressive antibiotic treatments, leading to chronic infections with reoccurring symptoms. For example, bacterial strains with highlevel persistence have been isolated from patients with tuberculosis2 and cystic fibrosis associated infections.3 Since antibiotics are not effective against persister cells, it is important to develop better control methods. Recently, we reported that (Z)-4-bromo-5-(bromomethylene)3-methylfuran-2(5H)-one (BF8, Fig. 1A) at 2 lg/mL can sensitize Pseudomonas aeruginosa PAO1 persister cells to antibiotics and BF8 alone at this concentration does not affect the viability of these persister cells.4 In addition, BF8 was found to reduce persistence during the growth of P. aeruginosa PAO14 and the mucoid strain P. aeruginosa PDO300.5 BF8 is a known inhibitor of quorum sensing (QS),4,6,7 a bacterial system of gene regulation in response to cell population density, which regulates different phenotypes8 such as biofilm formation9 and bioluminescence.10 For example, at 10 lg/mL, BF8 was found to inhibit acyl-homoserine lactone (AHL)-mediated QS in Vibrio harveyi BB886 without affecting the viability of this reporter strain.4 Some other BF compounds have also been shown as inhibitors of AHL and autoinducer-2 (AI-2) mediated QS;6,7 and consistently, some BFs have been shown to ⇑ Corresponding author. Tel.: +1 315 443 4409; fax: +1 315 443 9175. E-mail address: [email protected] (D. Ren). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.10.070
interact with the regulator R protein in AHL-mediated QS systems11 and covalently modify LuxS, the enzyme for AI-2 production.12 It is worth noticing that, although this QS inhibitor was found to control P. aeruginosa persister cells,4 the role of QS in persister control is still elusive. The QS signal 3-oxo-C12-homoserine lactone (HSL) has been shown to promote persister formation in exponential cultures of P. aeruginosa.13 Interestingly, we found that 3-oxo-C12-HSL can also sensitize PAO1 persisters (isolated from stationary cultures) to ciprofloxacin when treated in 0.85% NaCl.4 To better understand if QS inhibition plays a role in persister control and to design better antagonists, we conducted this study to compare a group of BFs with similar structures (Fig. 1B) for their effects on P. aeruginosa persistence and AHL-mediated QS. Two non-brominated furanones (NFs) were also involved as controls to understand if the presence of Br is important. Effects of BFs on AHL-mediated QS: To characterize the effects of BFs on AHL-mediated QS, the method described by Surette et al.14 was followed with slight modifications (see Supplementary data for more details). In this assay, the reporter strain V. harveyi BB886 (BAA-1118 from ATCC) produces bioluminescence in response to extracellular AHL.14 Thus, by measuring the relative bioluminescence (luminescence normalized by the number of live V. harveyi BB886 cells) after incubation in the presence or absence of each BF or NF, the effects of BFs and NFs on AHL-mediated QS can be determined. The BF molecules were synthesized as described previously15 and the two NF compounds were purchased from Sigma (St. Louis, MO, USA). Concentrations of 0, 0.1, 0.5, 1,
6560
J. Pan, D. Ren / Bioorg. Med. Chem. Lett. 23 (2013) 6559–6562
Figure 1. Structures of brominated furanones and non-brominated furanones. (A) Structure of BF8. (B) Structures of BFs and NFs used in this study.
Figure 2. Effects of BFs and NFs on AHL-mediated quorum sensing. V. harveyi BB886 was challenged with BFs and NFs at different concentrations. The number of viable V. harveyi BB886 cells after BF treatment, the level of bioluminescence, and the relative bioluminescence (bioluminescence normalized by CFU) are shown.
J. Pan, D. Ren / Bioorg. Med. Chem. Lett. 23 (2013) 6559–6562
10, 30 and 60 lg/mL were tested for each furanone compound. As shown in Figure 2A, addition of BF9 up to 10 lg/mL reduced bioluminescence of the QS reporter V. harveyi BB886 dose-dependently with no effects on the viability of this reporter. For example, the relative bioluminescence was reduced by 99.96 ± 0.69% when BF9 was tested at 10 lg/mL. At higher concentrations, for example, 30 lg/mL, the viability of V. harveyi BB886 was reduced by BF9 and no bioluminescence was detected. BF10, BF11, and BF12 also exhibited similar activities: inhibiting QS without affecting the viability of the reporter strain when the concentration was below a threshold (Fig. 2B-D). For example, at 0.5 lg/mL, BF10 and BF11 reduced the relative bioluminescence by 55.9 ± 0.3% and 91.2 ± 4.3%, respectively (T test, p <0.05 for both conditions). In comparison, the two NFs (NF1: citraconic anhydride; NF2: 4methoxy-2(5H)-furanone), BF13 and BF14 did not significantly repress QS at concentrations that were not inhibitory to the viability of the reporter V. harveyi BB886. For example, NF2 did not affect the viability and QS of V. harveyi BB886 at concentrations up to 60 lg/mL (Fig. 2H). These results indicate that the presence of Br is important to QS control by BFs and the structure of BFs also
6561
has significant impact on such activities. This finding is consistent with some other BFs reported previously.11,16–18 Effects of BFs on P. aeruginosa PAO1 persister cells: To test the effects of BFs on persistence, the persister cells of P. aerugionsa PAO1 were isolated and challenged with BFs and NFs by following a protocol described previously.4,5 All BFs and NFs were tested at 2 lg/ mL with a treatment time of 2 h. The effects on viability were evaluated by plating cells on LB agar plates right after treatment. Meanwhile, a portion of each sample was further treated with 200 lg/mL ciprofloxacin for 3.5 h to determine the number of cells that remained as persisters. More details about this experiment are described in the Supplementary data. The BFs tested in this study exhibited different activities in restoring the susceptibility of P. aeruginosa PAO1 persister cells to the representative antibiotic ciprofloxacin. As shown in Figure 3, BF9 and BF11 exhibited significant activities to sensitize P. aeruginosa PAO1 persister cells to ciprofloxacin at concentrations that did not affect the viability of persister cells in the absence of antibiotic. For example, after treatment with 2 lg/mL BF9 and BF11, 67.01 ± 16.13% and 99.99 ± 0.02% PAO1 persisters were killed by
Figure 3. Effects of BFs and NFs on isolated PAO1 persister cells. After treatment for 2 h, the number of surviving persisters was determined by counting CFU. Meanwhile, a portion of each sample was further challenged with 200 lg/mL ciprofloxacin for 3.5 h to determine the number of cells that remained as persisters by counting CFU.
6562
J. Pan, D. Ren / Bioorg. Med. Chem. Lett. 23 (2013) 6559–6562
200 lg/mL ciprofloxacin, respectively (T test, p <0.05 for both). In comparison, treatment with 2 lg/mL BF9 or BF11 alone did not affect the viability of P. aeruginosa PAO1 persister cells. Also, 200 lg/mL ciprofloxacin itself does not kill persister cells (used to isolated persisters). In comparison, BF14 slightly reduced the viability of PAO1 persister cells but significantly enhanced persister killing by ciprofloxacin. For example, at 2 lg/mL, BF14 reduced the viability of persister cells by 64.6 ± 1.9%, but rendered 99.99 ± 0.01% persister cells sensitive to ciprofloxacin (Fig. 3F). BF10, BF12 and BF13 exhibited stronger killing of P. aeruginosa PAO1 persister cells in the absence of ciprofloxacin (Fig. 3B, D and E). The non-brominated furanones NF1 and NF2 did not exhibit notable effects (Fig. 3G and H). In a previous study, we demonstrated that some compounds from this group of synthetic BFs are inhibitors of Escherichia coli biofilm formation19 and AI-2-mediated QS.6 Here we show that BF9 and BF11 are also potent inhibitors of AHL-mediated QS and can sensitize P. aeruginosa PAO1 persister cells to ciprofloxacin, at concentrations that do not affect the viability of the QS reporter V. harveyi BB886 and the persister cells of P. aeruginosa AO1. In comparison, BF10 and BF12 are also QS inhibitors, but cannot sensitize P. aeruginosa PAO1 persister cells to ciprofloxacin at the concentrations that are not cidal to these persister cells. Although the mechanism of persister control by BFs is still unknown, the differences in QS and persister control by BFs suggest that QS inhibition itself is not sufficient to revert persistence and these BF compounds also have other targets in addition to QS in P. aeruginosa. Further study to identify the targets of BFs and the nature of interaction (e.g., reversible binding or covalent modification) will help improve infection control by eliminating persister cells. Acknowledgments We thank the U.S. National Science Foundation (CAREER1055644 and EFRI-1137186) for partial support of this work. We
are grateful to Professor Thomas K. Wood at Pennsylvania State University for sharing the strain of P. aeruginosa PAO1. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013. 10.070. References and notes 1. Lewis, K. Annu. Rev. Microbiol. 2010, 64, 357. 2. Ojha, A. K.; Baughn, A. D.; Sambandan, D.; Hsu, T.; Trivelli, X.; Guerardel, Y.; Alahari, A.; Kremer, L.; Jacobs, W. R. J.; Hatfull, G. F. Mol. Microbiol. 2008, 69, 164. 3. Mulcahy, L. R.; Burns, J. L.; Lory, S.; Lewis, K. J. Bacteriol. 2010, 192, 6191. 4. Pan, J.; Bahar, A. A.; Syed, H.; Ren, D. PLoS ONE 2012, 7, e45778. 5. Pan, J.; Song, F.; Ren, D. Bioorg. Med. Chem. Lett. 2013, 23, 4648. 6. Hou, S.; Duo, M.; Han, Y.; Luk, Y.-Y.; Ren, D. Inhibiting Microbial Biofilm Formation by Brominated Furanones; Minneapolis, Minnesota: ASM International, 2009. 7. Pan, J.; Ren, D. Expert. Opin. Ther. Pat. 2009, 19, 1581. 8. Miller, M. B.; Bassler, B. L. Annu. Rev. Microbiol. 2001, 55, 165. 9. Hammer, B. K.; Bassler, B. L. Mol. Microbiol. 2003, 50, 101. 10. Winson, M. K.; Swift, S.; Fish, L.; Throup, J. P.; Jørgensen, F.; Chhabra, S. R.; Bycroft, B. W.; Williams, P.; Stewart, G. S. A. B. FEMS Microbiol. Lett. 1998, 163, 185. 11. Defoirdt, T.; Miyamoto, C. M.; Wood, T. K.; Meighen, E. A.; Sorgeloos, P.; Verstraete, W.; Bossier, P. Environ. Microbiol. 2007, 9, 2486. 12. Zang, T.; Lee, B. W. K.; Cannon, L. M.; Ritter, K. A.; Dai, S.; Ren, D.; Wood, T. K.; Zhou, Z. S. Bioorg. Med. Chem. Lett. 2009, 19, 6200. 13. Moker, N.; Dean, C. R.; Tao, J. J. Bacteriol. 2010, 192, 1946. 14. Surette, M. G.; Miller, M. B.; Bassler, B. L. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1639. 15. Han, Y.; Hou, S.; Simon, K. A.; Ren, D.; Luk, Y. Y. Bioorg. Med. Chem. Lett. 2008, 18, 1006. 16. Ren, D.; Bedzyk, L. A.; Ye, R. W.; Thomas, S. M.; Wood, T. K. Biotechnol. Bioeng. 2004, 88, 630. 17. Hentzer, M.; Wu, H.; Andersen, J. B.; Riedel, K.; Rasmussen, T. B.; Bagge, N.; Kumar, N.; Schembri, M. A.; Song, Z.; Kristoffersen, P. EMBO J. 2003, 22, 3803. 18. Martinelli, D.; Grossmann, G.; Séquin, U.; Brandl, H.; Bachofen, R. BMC Microbiol. 2004, 4, 25. 19. Pan, J.; Xie, X.; Tian, W.; Bahar, A. A.; Lin, N.; Song, F.; An, J.; Ren, D. Appl. Microbiol. Biotechnol. 2013, 1.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Structural effects on persister control by brominated furanones Jiachuan Pan a,b, Dacheng Ren a,b,c,d,⇑ a
Department of Biomedical and Chemical Engineering, Syracuse University, Syracuse, NY 13244, United States Syracuse Biomaterials Institute, Syracuse University, Syracuse, NY 13244, United States c Department of Civil and Environmental Engineering, Syracuse University, Syracuse, NY 13244, United States d Department of Biology, Syracuse University, Syracuse, NY 13244, United States b
a r t i c l e
i n f o
Article history: Received 25 September 2013 Revised 29 October 2013 Accepted 30 October 2013 Available online 9 November 2013 Keywords: Antibiotic tolerance Persister Quorum sensing Pseudomonas aeruginosa Brominated furanone Structural effect
a b s t r a c t Bacterial persister cells are a small population of dormant cells that are tolerant to essentially all antibiotics. Recently, we reported that a quorum sensing (QS) inhibitor, (Z)-4-bromo-5-(bromomethylene)-3methylfuran-2(5H)-one (BF8), can revert antibiotic tolerance of Pseudomonas aeruginosa persister cells. To better understand this phenomenon, several synthetic brominated furanones with similar structures were compared for their activities in persister control and inhibition of acyl-homoserine lactone (AHL) mediated QS. The results show that some other furanones in addition to BF8 are also AHL QS inhibitors and can revert antibiotic tolerance of P. aeruginosa PAO1 persister cells. However, not all QS inhibiting BFs can revert persistence at growth non-inhibitory concentrations, suggesting that QS inhibition itself is not sufficient for persister control. Ó 2013 Elsevier Ltd. All rights reserved.
Persister cells are phenotypic variants that can be found in virtually any bacterial culture and are tolerant to different antibiotics.1 It is believed that this subpopulation can survive from aggressive antibiotic treatments, leading to chronic infections with reoccurring symptoms. For example, bacterial strains with highlevel persistence have been isolated from patients with tuberculosis2 and cystic fibrosis associated infections.3 Since antibiotics are not effective against persister cells, it is important to develop better control methods. Recently, we reported that (Z)-4-bromo-5-(bromomethylene)3-methylfuran-2(5H)-one (BF8, Fig. 1A) at 2 lg/mL can sensitize Pseudomonas aeruginosa PAO1 persister cells to antibiotics and BF8 alone at this concentration does not affect the viability of these persister cells.4 In addition, BF8 was found to reduce persistence during the growth of P. aeruginosa PAO14 and the mucoid strain P. aeruginosa PDO300.5 BF8 is a known inhibitor of quorum sensing (QS),4,6,7 a bacterial system of gene regulation in response to cell population density, which regulates different phenotypes8 such as biofilm formation9 and bioluminescence.10 For example, at 10 lg/mL, BF8 was found to inhibit acyl-homoserine lactone (AHL)-mediated QS in Vibrio harveyi BB886 without affecting the viability of this reporter strain.4 Some other BF compounds have also been shown as inhibitors of AHL and autoinducer-2 (AI-2) mediated QS;6,7 and consistently, some BFs have been shown to ⇑ Corresponding author. Tel.: +1 315 443 4409; fax: +1 315 443 9175. E-mail address: [email protected] (D. Ren). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.10.070
interact with the regulator R protein in AHL-mediated QS systems11 and covalently modify LuxS, the enzyme for AI-2 production.12 It is worth noticing that, although this QS inhibitor was found to control P. aeruginosa persister cells,4 the role of QS in persister control is still elusive. The QS signal 3-oxo-C12-homoserine lactone (HSL) has been shown to promote persister formation in exponential cultures of P. aeruginosa.13 Interestingly, we found that 3-oxo-C12-HSL can also sensitize PAO1 persisters (isolated from stationary cultures) to ciprofloxacin when treated in 0.85% NaCl.4 To better understand if QS inhibition plays a role in persister control and to design better antagonists, we conducted this study to compare a group of BFs with similar structures (Fig. 1B) for their effects on P. aeruginosa persistence and AHL-mediated QS. Two non-brominated furanones (NFs) were also involved as controls to understand if the presence of Br is important. Effects of BFs on AHL-mediated QS: To characterize the effects of BFs on AHL-mediated QS, the method described by Surette et al.14 was followed with slight modifications (see Supplementary data for more details). In this assay, the reporter strain V. harveyi BB886 (BAA-1118 from ATCC) produces bioluminescence in response to extracellular AHL.14 Thus, by measuring the relative bioluminescence (luminescence normalized by the number of live V. harveyi BB886 cells) after incubation in the presence or absence of each BF or NF, the effects of BFs and NFs on AHL-mediated QS can be determined. The BF molecules were synthesized as described previously15 and the two NF compounds were purchased from Sigma (St. Louis, MO, USA). Concentrations of 0, 0.1, 0.5, 1,
6560
J. Pan, D. Ren / Bioorg. Med. Chem. Lett. 23 (2013) 6559–6562
Figure 1. Structures of brominated furanones and non-brominated furanones. (A) Structure of BF8. (B) Structures of BFs and NFs used in this study.
Figure 2. Effects of BFs and NFs on AHL-mediated quorum sensing. V. harveyi BB886 was challenged with BFs and NFs at different concentrations. The number of viable V. harveyi BB886 cells after BF treatment, the level of bioluminescence, and the relative bioluminescence (bioluminescence normalized by CFU) are shown.
J. Pan, D. Ren / Bioorg. Med. Chem. Lett. 23 (2013) 6559–6562
10, 30 and 60 lg/mL were tested for each furanone compound. As shown in Figure 2A, addition of BF9 up to 10 lg/mL reduced bioluminescence of the QS reporter V. harveyi BB886 dose-dependently with no effects on the viability of this reporter. For example, the relative bioluminescence was reduced by 99.96 ± 0.69% when BF9 was tested at 10 lg/mL. At higher concentrations, for example, 30 lg/mL, the viability of V. harveyi BB886 was reduced by BF9 and no bioluminescence was detected. BF10, BF11, and BF12 also exhibited similar activities: inhibiting QS without affecting the viability of the reporter strain when the concentration was below a threshold (Fig. 2B-D). For example, at 0.5 lg/mL, BF10 and BF11 reduced the relative bioluminescence by 55.9 ± 0.3% and 91.2 ± 4.3%, respectively (T test, p <0.05 for both conditions). In comparison, the two NFs (NF1: citraconic anhydride; NF2: 4methoxy-2(5H)-furanone), BF13 and BF14 did not significantly repress QS at concentrations that were not inhibitory to the viability of the reporter V. harveyi BB886. For example, NF2 did not affect the viability and QS of V. harveyi BB886 at concentrations up to 60 lg/mL (Fig. 2H). These results indicate that the presence of Br is important to QS control by BFs and the structure of BFs also
6561
has significant impact on such activities. This finding is consistent with some other BFs reported previously.11,16–18 Effects of BFs on P. aeruginosa PAO1 persister cells: To test the effects of BFs on persistence, the persister cells of P. aerugionsa PAO1 were isolated and challenged with BFs and NFs by following a protocol described previously.4,5 All BFs and NFs were tested at 2 lg/ mL with a treatment time of 2 h. The effects on viability were evaluated by plating cells on LB agar plates right after treatment. Meanwhile, a portion of each sample was further treated with 200 lg/mL ciprofloxacin for 3.5 h to determine the number of cells that remained as persisters. More details about this experiment are described in the Supplementary data. The BFs tested in this study exhibited different activities in restoring the susceptibility of P. aeruginosa PAO1 persister cells to the representative antibiotic ciprofloxacin. As shown in Figure 3, BF9 and BF11 exhibited significant activities to sensitize P. aeruginosa PAO1 persister cells to ciprofloxacin at concentrations that did not affect the viability of persister cells in the absence of antibiotic. For example, after treatment with 2 lg/mL BF9 and BF11, 67.01 ± 16.13% and 99.99 ± 0.02% PAO1 persisters were killed by
Figure 3. Effects of BFs and NFs on isolated PAO1 persister cells. After treatment for 2 h, the number of surviving persisters was determined by counting CFU. Meanwhile, a portion of each sample was further challenged with 200 lg/mL ciprofloxacin for 3.5 h to determine the number of cells that remained as persisters by counting CFU.
6562
J. Pan, D. Ren / Bioorg. Med. Chem. Lett. 23 (2013) 6559–6562
200 lg/mL ciprofloxacin, respectively (T test, p <0.05 for both). In comparison, treatment with 2 lg/mL BF9 or BF11 alone did not affect the viability of P. aeruginosa PAO1 persister cells. Also, 200 lg/mL ciprofloxacin itself does not kill persister cells (used to isolated persisters). In comparison, BF14 slightly reduced the viability of PAO1 persister cells but significantly enhanced persister killing by ciprofloxacin. For example, at 2 lg/mL, BF14 reduced the viability of persister cells by 64.6 ± 1.9%, but rendered 99.99 ± 0.01% persister cells sensitive to ciprofloxacin (Fig. 3F). BF10, BF12 and BF13 exhibited stronger killing of P. aeruginosa PAO1 persister cells in the absence of ciprofloxacin (Fig. 3B, D and E). The non-brominated furanones NF1 and NF2 did not exhibit notable effects (Fig. 3G and H). In a previous study, we demonstrated that some compounds from this group of synthetic BFs are inhibitors of Escherichia coli biofilm formation19 and AI-2-mediated QS.6 Here we show that BF9 and BF11 are also potent inhibitors of AHL-mediated QS and can sensitize P. aeruginosa PAO1 persister cells to ciprofloxacin, at concentrations that do not affect the viability of the QS reporter V. harveyi BB886 and the persister cells of P. aeruginosa AO1. In comparison, BF10 and BF12 are also QS inhibitors, but cannot sensitize P. aeruginosa PAO1 persister cells to ciprofloxacin at the concentrations that are not cidal to these persister cells. Although the mechanism of persister control by BFs is still unknown, the differences in QS and persister control by BFs suggest that QS inhibition itself is not sufficient to revert persistence and these BF compounds also have other targets in addition to QS in P. aeruginosa. Further study to identify the targets of BFs and the nature of interaction (e.g., reversible binding or covalent modification) will help improve infection control by eliminating persister cells. Acknowledgments We thank the U.S. National Science Foundation (CAREER1055644 and EFRI-1137186) for partial support of this work. We
are grateful to Professor Thomas K. Wood at Pennsylvania State University for sharing the strain of P. aeruginosa PAO1. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013. 10.070. References and notes 1. Lewis, K. Annu. Rev. Microbiol. 2010, 64, 357. 2. Ojha, A. K.; Baughn, A. D.; Sambandan, D.; Hsu, T.; Trivelli, X.; Guerardel, Y.; Alahari, A.; Kremer, L.; Jacobs, W. R. J.; Hatfull, G. F. Mol. Microbiol. 2008, 69, 164. 3. Mulcahy, L. R.; Burns, J. L.; Lory, S.; Lewis, K. J. Bacteriol. 2010, 192, 6191. 4. Pan, J.; Bahar, A. A.; Syed, H.; Ren, D. PLoS ONE 2012, 7, e45778. 5. Pan, J.; Song, F.; Ren, D. Bioorg. Med. Chem. Lett. 2013, 23, 4648. 6. Hou, S.; Duo, M.; Han, Y.; Luk, Y.-Y.; Ren, D. Inhibiting Microbial Biofilm Formation by Brominated Furanones; Minneapolis, Minnesota: ASM International, 2009. 7. Pan, J.; Ren, D. Expert. Opin. Ther. Pat. 2009, 19, 1581. 8. Miller, M. B.; Bassler, B. L. Annu. Rev. Microbiol. 2001, 55, 165. 9. Hammer, B. K.; Bassler, B. L. Mol. Microbiol. 2003, 50, 101. 10. Winson, M. K.; Swift, S.; Fish, L.; Throup, J. P.; Jørgensen, F.; Chhabra, S. R.; Bycroft, B. W.; Williams, P.; Stewart, G. S. A. B. FEMS Microbiol. Lett. 1998, 163, 185. 11. Defoirdt, T.; Miyamoto, C. M.; Wood, T. K.; Meighen, E. A.; Sorgeloos, P.; Verstraete, W.; Bossier, P. Environ. Microbiol. 2007, 9, 2486. 12. Zang, T.; Lee, B. W. K.; Cannon, L. M.; Ritter, K. A.; Dai, S.; Ren, D.; Wood, T. K.; Zhou, Z. S. Bioorg. Med. Chem. Lett. 2009, 19, 6200. 13. Moker, N.; Dean, C. R.; Tao, J. J. Bacteriol. 2010, 192, 1946. 14. Surette, M. G.; Miller, M. B.; Bassler, B. L. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1639. 15. Han, Y.; Hou, S.; Simon, K. A.; Ren, D.; Luk, Y. Y. Bioorg. Med. Chem. Lett. 2008, 18, 1006. 16. Ren, D.; Bedzyk, L. A.; Ye, R. W.; Thomas, S. M.; Wood, T. K. Biotechnol. Bioeng. 2004, 88, 630. 17. Hentzer, M.; Wu, H.; Andersen, J. B.; Riedel, K.; Rasmussen, T. B.; Bagge, N.; Kumar, N.; Schembri, M. A.; Song, Z.; Kristoffersen, P. EMBO J. 2003, 22, 3803. 18. Martinelli, D.; Grossmann, G.; Séquin, U.; Brandl, H.; Bachofen, R. BMC Microbiol. 2004, 4, 25. 19. Pan, J.; Xie, X.; Tian, W.; Bahar, A. A.; Lin, N.; Song, F.; An, J.; Ren, D. Appl. Microbiol. Biotechnol. 2013, 1.