Research ArticleExperimental Studies

Inhibition of Quorum Sensing and Efflux Pump System by Trifluoromethyl Ketone Proton Pump Inhibitors

ZOLTÁN G. VARGA, ANA ARMADA, PEDRO CERCA, LEONARD AMARAL, MIOR A.A. MIOR AHMAD SUBKI, MICHAEL A. SAVKA, ERNŐ SZEGEDI, MASAMI KAWASE, NOBORU MOTOHASHI and JOSEPH MOLNÁR
In Vivo March 2012, 26 (2) 277-285;

Abstract

Background: One major microbiological problem is the widespread antibiotic resistance. There is an urgent need for new antibiotics and ways to treat multi-drug-resistant infections. Inhibition of bacterial quorum sensing (QS) systems could be an effective alternative in a smuch as they regulate a broad spectrum of cell functions, including, virulence factor production, biofilm organisation and motility. Influx and efflux bacterial systems involved in quorum sensing (QS) are known to depend on the proton motive force (PMF). Thus, a new series of 12 trifluoromethyl ketones (TFs) known to inhibit the PMF, was investigated for effects on the efflux pump of a QS responding bacterium, for its subsequent effect on the response to a QS signal and its direct inhibition of the response to a QS signal. Materials and Methods: Chromobacterium violaceum 026 (CV026) was used as the indicator strain to evaluate the QS inhibitory effect of TFs. This strain responds to the presence of short carbon chain acyl-homoserine lactones (AHLs) by the development of a purple pigment. Effect on the QS response of CV026 to externally added AHLs was evaluated. In addition, the specific activity of the TFs on the efflux pump system of the CV026 strain and a wild-type Escherichia coli strain was assessed with the aid of the automated real-time ethidium bromide method. Results: From the 12 compounds, 6 proved to be effective inhibitors of the QS response by CV026, as well as inhibit the efflux pumps of CV026 and Escherichia coli. Conclusion: Our results show that TFs have QS inhibitory properties that are mediated through their inhibition of efflux pumps that extrude the noxious QS signal before it reaches its intended target. Because the TFs also inhibit the efflux pump of a pathogenic bacterium, the method used for the evaluation of the TFs in the current study has clinical relevance and may be exploited for the prevention of QS responses of infecting bacteria.

The antimicrobial and antimotility effects of 30 trifluoromethyl ketones (TFs) on various bacterial species have already been studied (1-5). Some of these TFs only inhibit the growth of various Gram-positive bacteria, while others exhibit antimicrobial activity against Gram-negative bacteria and yeasts. The combination of certain derivatives of TFs with promethazine results in a synergistic antibacterial effect (1).

TFs at subinhibitory concentrations inhibit the motility of Proteus vulgaris. Detailed analysis demonstrated that the proportion of mobile bacterial cells is reduced (2), suggesting that the action of TFs at concentrations that have no effect on the viability of the studied bacteria involves the inhibition of an energy source upon which flagellae depend for their action, namely, the proton motive force (PMF) (2).

Quorum sensing (QS) signal systems regulate a broad spectrum of cell functions in bacterial populations (6-8). Among the functions that depend or interact with QS is the secretion of biofilms. Bacteria whose main efflux pump has been deactivated or deleted are deficient in the secretion of QS signals (9) as well as production of biofilm (10). Efflux pumps of the resistance nodulation division (RND) family of transporters, depend upon the energy provided by the PMF for effective function (11, 12). These efflux pumps extrude noxious agents that penetrate the outer cell wall of the organism prior to reaching their intended targets (13). QS signals that are produced by one species for the inhibition of growth of a competing bacterial species can be considered noxious agents; consequently one would expect that compounds shown to inhibit access to energy provided by the PMF would have a negative effect on the main efflux pump of the responding competitor and hence the response to a QS signal such as N-acyl homoserine lactone (AHL) would be negatively affected. Since TFs have been shown to affect access to energy provided by the PMF (5) we have been studying the effects of a series of 12 systematically synthesized TFs (1-12) (Table I) on the QS signal system. This study evaluated the effect of 12 TFs on the expression of the AHL-mediated signal in a system containing the environmental member Sphingomonas sp. Ezf 10-17 strain, as producer of AHL and the Chromobacterium violaceum CV026 as the responding sensor to AHL. Since the effects of the TFs on the QS system by the former system do not distinguish an effect on the producer versus the responder, the effects of the TFs on the responder were directly assessed by combinations of pure commercial N-Hexanoyl-DL-homoserine lactone AHLs and each TF. In addition, we have studied the effects of the TFs on the activity of the efflux pumps of the QS-responding CV026 strain, as well as on the efflux pump system of Escherichia coli, in order to determine the role that an efflux pump system might play in the response to a QS signal.

View this table:
Table I.

Inhibitory effects of the (TFs) on (QS) and the growth of producing and QS sensor bacterial strains, after 24 hours of incubation. The extent of the colourless zone indicates the inhibitory effect of the given compound on QS signal transmission.

Figure 1.
Figure 1.

Structures of trifluoromethyl ketones investigated in this study.

Materials and Methods

Chemicals. Eleven TFs (1-9, 11 and 12) were synthesized by reaction of the corresponding 2-methylbenzazoles with trifluoroacetic or chlorodifluoroacetic anhydride (14). Compound 10 was prepared by treatment of 2-lithiomethylbenzoxazole with ethyl acetate (15, 16). N-Hexanoyl-DL-homoserine lactone was purchased from Sigma (Budapest, Hungary). TFs were dissolved in, dimethyl sulfoxide (DMSO).

The structures of the TFs are presented in Figure 1. Ethidium bromide and thioridazine were purchased from Sigma (Madrid, Spain).

Bacterial strains. Chromobacterium violaceum is a common bacterium which lives in soil and water. When it attains a high cell density, it produces a purple pigment called violacein (17). The C. violaceum CV026 used as a sensor strain to study the effect of TFs on QS is a Tn5 mutant that cannot synthesize AHLs; it produces the purple pigment only in the presence of externally added inducers (18). This strain has been used to detect a wide range of short-chain AHL molecules and QS inhibitors (19-21). Ezf 10-17 was isolated from a grapevine crown gall tumor. This strain induced pigment production by CV026 and proved to be efficient to study QS interactions (22). Escherichia coli wild-type AG100 [argE3 thi-1 rpsL xyl mtl (gal-uvrB) supE44] was employed for the determination of effects of TFs on the activity of the intrinsic efflux pump of this organism (13).

Medium. A modified LB agar containing yeast extract 5 g, trypton 10 g, NaCl 10 g, K2HPO4 1 g, MgSO4•7H2O 0.3 g and FeNaEDTA 36 mg in 1.0 liter of distilled water was used to study the effect of TFs of QS. Ezf 10-17 was grown on potato dextrose agar (PDA) to prepare signal compounds. E. coli was cultured on Mueller-Hinton broth (MHB) and colonies isolated on Mueller-Hinton agar (MHA) purchased in powder form from Sigma (Madrid, Spain).

Taxonomic identification of Ezf10-17 and analysis of its AHL production. The V3 region of 16S rDNA from Ezf 10-17 was amplified using the forward primer (5’-ACTCCTACGGGAGGCA GCAG-3’) and reverse primer (5’-ATTACCGCGGCTGCTGG-3’) and sequenced. Sequence data were compared and analysed by BLAST against the published 16S V3 sequences available in the database.

The AHLs from the liquid culture of Ezf 10-17 were extracted and concentrated by using acidified ethyl acetate liquid-liquid extraction. The purified AHLs were analysed using thin layer chromatography (TLC) overlaid with C. violaceum CV026 biosensor strain.

Evaluation of the effect of TFs on the response by CV026 to AHL. Isolated colonies of the AHL-responding CV026 were plated directly as a 6 to 7 cm line on the surface of the modified LB agar. One microlitre of solution containing 5 or 10 ng of N-Hexanoyl-DL-homoserine lactone AHLs was mixed with 10 μl of the stock (2 mg/ml or 0.4 mg/ml) solution of the potential QS inhibitor TF compound. Filter paper discs (7.0 mm in diameter) were impregnated with 11 μl of the mixture of different concentrations of each TF and AHL, and the disks were placed on the inoculated line of the CV026 sensor strain (18). For sets of assays that aimed to evaluate the interaction between AHL and each TF, blank filter disks were separately impregnated with 11 μl of AHL alone, with 11 μl each of the TFs alone, and with 11 μl of DMSO, the latter serving as an absolute control. The plates were incubated at room temperature (ca. 20°C) for 24-48 hs.

Minimum inhibitory concentration (MIC) of each TF on CV026 and E. coli AG100. The MIC of TFs was determined by the broth dilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines (23).

Assessment of the effects of each TF on the activity of the efflux pump systems of CV026 and Escherichia coli AG100. The activity of the TFs on the real-time accumulation of ethidium bromide (EB) was assessed by the automated EB method, previously described in detail (24), using the Rotor-Gene 3000™ thermocycler with real-time analysis software (Corbett Research, Sydney, NSW, Australia). Briefly, E. coli AG100 was cultured in MHB medium until the culture reached an optical density (OD) of 0.6 at 600 nm, the culture was then centrifuged at 13,000 rpm for 3 min, the pellets were re-suspended in phosphate-buffered saline (PBS; pH 7.4) with a final concentration of glucose of 0.4% and the OD adjusted to 0.6 at 600 nm. Aliquots of 45 μl of the cell suspension were distributed to 0.2 ml tubes. The TFs were individually added at concentrations equal to half their MIC against the strain in 5 μl volumes of their stock solutions, and finally 45 μl of EB to yield a final concentration of 1 mg/l (Sigma-Aldrich) in PBS, with and without glucose, were added. Note that the selection of a concentration of each TF at half its MIC is due to the empirical fact that at this concentration there is no significant effect on the viability of the organism (24, 25). It is also important to note that prior to the experiments described, the maximum concentration of EB which was within the capacity of the bacterium to extrude, was determined at least three times. For the wild-type E. coli AG100 reference and the CV026 strains employed in the study, these concentrations of EB were determined to be 1 and 0.5 mg/l, respectively (13, 24, 25). The tubes were placed into a Rotor-Gene 3000TM thermocycler and the fluorescence monitored on a real-time basis. From the real-time data, the activity of the TF, namely the relative final fluorescence (RFF) of the last time point (minute 30) of the EB accumulation assay was calculated according to the formula: Embedded Image Where RFtreated is the relative fluorescence at the last time point of the EB retention curve in the presence of an inhibitor, and RFuntreated is the relative fluorescence at the last time point of the EB retention curve of the untreated control. The greater the difference between RFtreated and RFuntreated, the greater the degree of EB accumulated and, therefore, the greater the degree of inhibition of the efflux pump system of the bacterium promoted by the agent at that concentration.

The RFF was then divided by the concentration of the TF that corresponded to half its MIC. This yielded a measure of the effect of each TF at a milligram level (specific activity) and therefore afforded comparison of each TF for activity against the efflux pump systems of the CV026 and E. coli AG100 strains. The experiments were repeated three times and the specific activity values presented are the average of three independent assays. This method of analysis has been previously presented (26). Thioridazine (TZ) an efflux pump inhibitor (12), was used as as a positive control.

Results

In our previous studies, the unidentified grapevine tumor isolate Ezf 10-17 proved to be an inducer of violacein production by C. violaceum CV026. Thus, this pair of inducer/sensor strains was successfully used to study potential QS inhibitors (21) (Figure 2). To identify Ezf 10-17, we sequenced the V3 region of 16S rDNA gene from its genome. Comparing the sequence data to those found in databases, this strain proved to be a member of the Sphingomonadaceae family.

Signal production of Ezf 10-17 was analysed by TLC overlaid with C. violaceum CV026. As compared to the standard AHLs, Ezf 10-17 produces a strong signal that co-migrates with 3-oxo-C6 AHL. Additionally, weaker signals which seem to be identical with C6 AHL, 3-oxo-C8 AHL and C8 AHL were also observed (Figure 3). These data support our earlier observations on the suitability of Ezf 10-17 in QS assays with C. violaceum CV026 (21).

In order for an agent to be correctly evaluated for effects on a QS system whose intensity of colour is dependent upon the growth of the producer of the QS signal and the growth of the responding bacterium, the concentration of the agent that is to provide meaningful interpretation, must be one that does not affect the viability of either bacterium. As summarized in Table I, various TFs have antimicrobial activities against the producer and responding bacteria. Consequently, the amounts of TFs selected for the evaluation of effects on the QS system were at or below those that had no effect on the growth of either species. Because of space limitation, the range of effects of the TFs on the QS system cannot be pictorially presented. Rather, the effects are presented in Figure 4 and Table II. Briefly, TF 5 had the least inhibitory effect (deep colour associated with the responder CV026) and TF 3 had the greatest inhibitory effect on the response of the CV026. The effect of the TFs on the QS system is clearly inhibitory. Whether the effect is due to the TF inhibiting the release of the QS signal or to the inhibition of the response of the responding species cannot be distinguished from the above evaluation.

Figure 2.
Figure 2.

The quorum sensing response of CV026 to the presence of the (AHL) producer Ezf 10-17. A: CV026 responder strain alone. B: CV026 responder strain (right) with Ezf 10-17 AHL producer strain (left). The deep colour associated with the responder strain CV026 was found only when CV026 was cultured in parallel with the AHL producer Ezf 10-17.

The direct effects of each TF on the QS response by CV026 was determined with the use of disks impregnated with combinations of a constant amount of the AHL and differing amounts of TF. As evident from the example provided in Figure 5, whereas the disks with TF alone (Figure 5A) do not produce the purple colour associated with CV026, the presence of pure AHL in the disk led to the production of the deep purple colour associated with CV026 (Figure 5B). The presence of the TF that inhibited the production of colour in the QS assay described, when in combination with the AHL, inhibits the production of the purple colour by the responding CV026 (Figure 5C). These results clearly show that the TF has a powerful inhibitory effect on the QS responding strain. However, the question of whether the same TF can inhibit the secretion of the QS signal by the producer species remains unanswered. The activity of each TF at half its MIC on the efflux pump system of the CV026 is exemplified by Figure 6. The data presented on those figures suggest that all of the TFs have activity against the efflux pump of CV026. However, because the concentrations of TFs used in the assay were half of their MICs, and the MICs of the TFs against the strain differed significantly, the activities presented in Figure 6 do not permit a comparison of the activities between TFs. However, calculation of the specific activity of each TF by the formula for RFF presented in the Materials and Methods section affords a comparison, and these data are presented in Table III. Briefly, the activity of the positive control thioridazine was 1.02. Comparison of the activity of each relative to the positive control, TFs 2 and 3 have the greatest activity against the efflux pump system of CV026; TFs 1, 4, 5 and 9 have significant activity; and, TFs 7, 8, 10, 11 and 12 have no activity. The demonstrations that the TFs inhibit the response of an environmental strain to a QS signal and the same TFs inhibit the efflux pump of the environmental responding strain, by themselves, do not support clinical interest in the TFs for possible use in the therapy of a bacterial infection. Therefore, in order to establish the needed support for the claim that indeed, the TFs have clinical value, the TFs were examined for activity against the efflux pump system of E. coli, a pathogenic bacterium. Since the number of graphs needed to depict each effect is large, an example of the data obtained is presented in Figure 7 for TF 4. As evident from the figure, the presence of TF 4 promotes an increase of fluorescence due to the accumulation of EB whereas in the absence of the compound, no significant increase of fluorescence takes place (the curve is rather flat). The effects of the TFs on the activity of the efflux pump is summarised in Table IV. This Table provides the concentration of the positive control thioridazine and each of the TFs that corresponds to half their MIC. As noted in Table IV, TFs 2 and 3 have the highest activity against the efflux pump system of E. coli. TFs 1, 4, 5 and 9 are also very active since their inhibitory activity exceeds that of thioridazine, the efflux pump inhibitor that serves as positive control.

Figure 3.
Figure 3.

Detection of N-acyl-homoserine lactones (AHL) from Ezf 10-17. Pure, commercial AHLs (lanes 1-4) and purified AHL produced by Ezf 10-17 (lane 5) isolated by thin layer chromatography. The plate overlaid with the responder CV026. The application of varying concentrations of pure commerical AHLs provide standards of the degree of response (intensity of colour). EZF 10-17 AHL extracts were prepared with acidified ethyl acetate of re-suspended 4-day old cultures of EzF 10-17 grown on potato dextrose agar.

Figure 4.
Figure 4.

The length and intensity of the purple colouration induced by 10 ng AHL/disc on Chromobacterium violaceum 026, after 24-hour incubation in the presence of TFs applied at 20 μg/disc. The effective inhibitors reduce the length and intensity of the purple colouration. The scale indicates an increasing intensity of colouration. A lower colour intensity means higher inhibition of QS. −, No coloration; +, white-purple; ++, palepurple; +++, purple; ++++, darkpurple.

Figure 5.
Figure 5.

The effect of (TFs) on the response of CV026 (AHL). A: Control disc with TF4 (20 μg) alone (no colour).B: Control disc with AHL (10 ng) alone (deep purple colouration). C: Disc with TF4 (20 μg/disc) and AHL (10 ng) (very light purple colouration).

View this table:
Table II.

Effects of (TFs) on (AHL)-mediated quorum sensing.

View this table:
Table III.

The related final fluorescence (RFF) and specific activity (SA) of each (TF) on the efflux pump system of CV026. Samples consisted of saline plus 1 mg/l of ethidium bromide, 0.4% glucose, and without and with half (MIC) of positive control and TFs. Assessment of fluorescence took place at 37°C for 30 minutes. Data in bold identifies TFs that express very high inhibitory activity against the efflux pump system of CV026.

View this table:
Table IV.

The effects of (TFs) on the efflux pump system of Escherichia coli AG 100.

Figure 6.
Figure 6.

The effect of half the minimum inhibitory concentration of (TFs) 1, 4, 5, 9 (A), and 6, 7, 8, 10, 11, 12 (B) as compared to the positive control thioridazine (TZ).

Figure 7.
Figure 7.

The effect of TF 4 on the activity of the efflux pump system of Escherichia. coli AG100. The concentration of the TF 4 that correspond to half minimum inhibitory concentration was 30 mg/l. Note that the control does not accumulate ethidium bromide during the 30 minutes of the assay.

Discussion

The results presented in this study show that some TFs have inhibitory activities against the response of CV026 to a QS signal such as AHL and the efflux pump systems of the CV026 and E. coli strains. Comparison of the efflux pump inhibitory activities of the TFs towards both strains suggests that the inhibition is practically identical in both cases. This suggests that the TFs have clinical value. Comparison of the individual inhibition induced by each TF on the QS response by CV026 to AHLs indicates that TF 3 exerts the greatest inhibition on the QS response. By comparison of the MIC of each effective TF, it is clear that the most effective inhibitors of the efflux pump system of E. coli also have the most potent antibacterial activity. TFs such as TF 7, 10 and 11 are devoid of any significant antibacterial activity (MIC for TF 7 and 10 is 480 mg/l, and for TF 11, it is 240 mg/l) and have little activity against the efflux pump of E. coli. The demonstration of a QS response by the method used in this study requires the growth of the responding organism. If an agent inhibits growth, it pre-empts any response since there are no bacteria present to respond. The application of 20 μg of each TF to discs promoted strong antibacterial effects by TFs with very low MICs. Applying amounts of a TF that have significant antibacterial properties to a disc below the inhibitory concentration exceeds the sensitivity of the system, since the distance between evident growth and the absence of a response (no colour) is masked by the deep purple colour associated with the growing population that is less than a millimetre from the disc. This is why TFs with very high MICs were able to produce evidence of an inhibition of the QS response by CV026, whereas for TFs with low MICs, with the exception of TF 3 (MIC 7.5 mg/l), the antibacterial effect of the TF pre-empted growth. QS is essential for various pathogens. In nature there are many types of organisms which can quench QS signals of other species. For example, Bacillus species produce lactonase enzyme to degrade the AHL signals of Gram-negative species. Eukaryotic cells also deploy QS inhibitors to prevent bacterial infection. QS has great importance in the organisation of biofilms (6), the production of virulence factors (8), and also in the spread of antibiotic resistance. The antibiotic resistance of bacteria in biofilms is several hundred or even thousand-fold higher than the one of free living bacteria. Without the ability to make biofilms or virulence factors production, pathogens lose the ability to cause infections. Therefore, if we reduce or completely block bacterial QS, we can reduce these QS-dependent/related activities as well. Our previous study demonstrated that phenothiazines were able to inhibit the QS system that involved Ezf 10-17, the producer of the signal and CV026, the responder to the signal (27). The phenothiazine thioridazine, an inhibitor of efflux pumps of Gram-negative bacteria (11, 12, 24, 25) also inhibits the response of CV026 to pure AHL (data not shown). Because phenothiazines and TFs that inhibit the efflux pumps of the CV026 and E. coli, as shown in the current study, also inhibit access to energy supplied by the PMF (4, 5), we believe that the response to a QS signal depends upon a functional efflux pump system which extrudes the noxious QS signal before it reaches its intended target. Moreover, because all secretory activity of a bacterium such as the secretion of a QS signal, is controlled (28-30), and the main efflux pump systems of Gram-negative bacteria are the secretory paths of internally produced noxious agents (31-34), the inhibition of the efflux pump system of a QS signal producer will result in obviating the secretion of the QS signal. Therefore, it is our contention that inhibitors of an efflux pump, such as phenothiazines and now also TFs, will inhibit the secretion of the QS signal and the response to the QS signal. The results of the current study clearly show that various TFs have the ability to inhibit the response of C. violaceum 026 to the QS signal AHL and to inhibit the efflux pump of the QS-responding CV026 as well as the one of E. coli. This ability is a direct one since none of the TFs formed a complex with AHL (spectrophotometric data not shown). Since some TFs inhibit the QS response, inhibit an efflux pump system and also have significant antibacterial activity, TFs have a promising future for therapy of problematic infections that rely on an efflux-mediated multidrug-resistant phenotypes and which, due to QS, make their therapy problematic.

Acknowledgements

The Authors are grateful for the support from the Szeged Foundation of Cancer Research and a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan (Kawase, no. 20590114). L. Amaral was supported by BCC grant SFRH/BCC/51099/2010 provided by the Fundação para a Ciência e a Tecnologia (FCT) of Portugal. Mior Ahmad Subki is currently enrolled in the Biotechnology Program at Rochester Institute of Technology (RIT, NY, USA) and was supported by a Undergraduate Research Fellowship from the American Society for Microbiology and the College of Science at RIT. M. A. Savka wishes to thank the School of Biological and Medical Sciences, College of Science, at RIT for the support of this work through Faculty Evaluation and Development (FEAD) 2010 awards. E. Szegedi was supported by Hungarian Scientific Research Fund (OTKA) Grant no. K-83121. The Authors are grateful for the skilful technical assistance of Aniko Váradi Vigyikán.

Footnotes

  • ↵* Cost Action BM0701 of the European Commission/European Science Foundation.

  • Received November 15, 2011.
  • Revision received December 22, 2011.
  • Accepted December 23, 2011.

References

  1. ↵
    1. Kawase M,
    2. Motohashi N,
    3. Sakagami H,
    4. Kanamoto T,
    5. Nakashima H,
    6. Ferenczy L,
    7. Wolfard K,
    8. Miskolci Cs,
    9. Molnár J
    : Antimicrobial activity of trifluoromethyl ketones and their synergism with promethazine. Int J Antimicrobial Agents 18: 161-165, 2001.
    OpenUrlPubMed
  2. ↵
    1. Wolfart K,
    2. Molnar A,
    3. Kawase M,
    4. Motohashi N,
    5. Molnar J
    : Effect of trifluoromethyl ketones on the motility of Proteus vulgaris. Biol Pharm Bull 27: 1462-1464, 2004.
    OpenUrlCrossRefPubMed
    1. Spengler G,
    2. Molnár A,
    3. Klausz G,
    4. Mándy I,
    5. Kawase M,
    6. Motohashi N,
    7. Molnar J
    : The antimotility action of trifluoromethyl ketone on some Gram-negative bacteria. Acta Microbiol Immunol Hung 51: 351-358, 2004.
    OpenUrlPubMed
  4. ↵
    1. Spengler G,
    2. Molnár A,
    3. Klausz G,
    4. Mándy I,
    5. Kawase M,
    6. Motohashi N,
    7. Molnar J
    : Inhibitory action of a new proton pump inhibitor, trifluoroketone derivative against the motility of clarithromycin-susceptible and resistant Helicobacter pylori. Int J Antimicrobial Agents 23: 631-633, 2004.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Molnár A,
    2. Wolfart K,
    3. Kawase M,
    4. Motohashi N,
    5. Molnar J
    : Effect of trifluoromethyl ketone on the motility of proton pump deficient mutant of E. coli strain and its wild-type. In Vivo 18: 505-508, 2004.
    OpenUrlPubMed
  6. ↵
    1. Dickshat JS
    : Quorum sensing and bacterial biofilms. Nat Pro Rep 27: 343-369, 2010.
    OpenUrl
    1. Amara N,
    2. Mashiach R,
    3. Amar D,
    4. Krief P,
    5. Spieser S,
    6. Bottomley M,
    7. Aharoni A,
    8. Meijler M
    : Covalent inhibition of bacterial quorum sensing. J Am Chem Soc 131: 10610-10619, 2009.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Chan WC,
    2. Coyle BJ,
    3. Williams P
    : Virulence regulation and quorum sensing in Staphylococcal infections: Competitive AgrC antagonists as quorum-sensing inhibitors. J Med Chem 47: 4633-4641, 2004.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Pumbwe L,
    2. Skilbeck CA,
    3. Wexler HM
    : Presence of quorum-sensing systems associated with multidrug resistance and biofilm formation in Bacteroides fragilis. Microb Ecol 56: 412-9, 2008.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Kvist M,
    2. Hancock V,
    3. Klemm P
    : Inactivation of efflux pumps abolishes bacterial biofilm formation. Appl Environ Microbiol 74: 7376-7382, 2008.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Amaral L,
    2. Fanning S,
    3. Pagès JM
    : Efflux pumps of gram-negative bacteria: genetic responses to stress and the modulation of their activity by pH, inhibitors, and phenothiazines. Adv Enzymol Relat Areas Mol Biol 77: 61-108, 2011.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Amaral L,
    2. Cerca P,
    3. Spengler G,
    4. Machado L,
    5. Martins A,
    6. Couto I,
    7. Viveiros M,
    8. Fanning S,
    9. Pages JM
    : Ethidium bromide efflux by Salmonella: modulation by metabolic energy, pH, ions and phenothiazines. Int J Antimicrob Agents 38: 140-145, 2011.
    OpenUrlPubMed
  13. ↵
    1. Martins A,
    2. Iversen C,
    3. Rodrigues L,
    4. Spengler G,
    5. Ramos J,
    6. Kern WV,
    7. Couto I,
    8. Viveiros M,
    9. Fanning S,
    10. Pages JM,
    11. Amaral L
    : An AcrAB-mediated multidrug-resistant phenotype is maintained following restoration of wild-type activities by efflux pump genes and their regulators. Int J Antimicrob Agents 34: 602-604, 2009.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Kawase M,
    2. Teshima M,
    3. Saito S,
    4. Tani S
    : Trifluoroacetylation of methylpyridines and other methylazines: A convenient access to trifluoroacetonylazines. Heterocycles 48: 2103-2109, 1998.
    OpenUrl
  15. ↵
    1. Lipshutz BH,
    2. Hungate RW
    : Metalation studies of trisubstituted oxazoles. J Org Chem 46: 1410-1413, 1981.
    OpenUrl
  16. ↵
    1. Babichev FS,
    2. Volovenko YM
    : Acylation of 2-methylbenzazoles by carboxylic acid esters. Ukrainskii Khimicheskii Zhurnal 43: 163-165, 1977 (in language of original).
    OpenUrl
  17. ↵
    1. Ballantine JA,
    2. Beer RJS,
    3. Crutchley DJ,
    4. Dodd GM,
    5. Palmer DR
    : The synthesis of violacein and related compounds. Proc Chem Soc pp. 232-233, 1968.
  18. ↵
    1. McClean KH,
    2. Winson MK,
    3. Fish L,
    4. Taylor A,
    5. Chhabra SR,
    6. Camara M,
    7. Daykin M,
    8. Lamb JH,
    9. Swift S,
    10. Bycroft BW,
    11. Stewart GSAB,
    12. Williams P
    : Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acyl homoserine lactones. Microbiology 143: 3703-3711, 1997.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Lithgow JK,
    2. Danino VE,
    3. Jones J,
    4. Downie JA
    : Analysis of N-acyl homoserine-lactone quorum-sensing molecules made by different strains and biovars of Rhizobium leguminosarum containing different symbiotic plasmids. Plant and Soil 232: 3-12, 2001.
    OpenUrlCrossRef
    1. Molina L,
    2. Constantinescu F,
    3. Michel L,
    4. Reimmann C,
    5. Duffy B,
    6. Defago G
    : Degradation of pathogen quorum-sensing molecules by soil bacteria: a preventive and curative biological control mechanism. FEMS Microbiol Ecol 45: 71-81, 2003.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. McClean RJC,
    2. Pierson LS,
    3. Fuqua CA
    : Simple screening protocol for the identification of quorum signal antagonists. J Microbiol Methods 58: 351-360, 2004
    OpenUrlCrossRefPubMed
  22. ↵
    1. Szabó MA,
    2. Varga GZ,
    3. Hohmann J,
    4. Schelz Zs,
    5. Szegedi E,
    6. Amaral L,
    7. Molnar J
    : Inhibition of quorum-sensing signals by essential oils. Phytother Res 24: 782-786, 2010.
    OpenUrlPubMed
  23. ↵
    1. Clinical and Laboratory Standards Institute
    . Performance Standards for Antimicrobial Susceptibility Testing; Seventeenth Informational Supplement. CLSI document 27(1): M100-S17, 2007.
    OpenUrl
  24. ↵
    1. Viveiros M,
    2. Martins M,
    3. Couto I,
    4. Rodrigues L,
    5. Spengler G,
    6. Martins A,
    7. Kristiansen JE,
    8. Molnar J,
    9. Amaral L
    : New Methods for the Identification of Efflux-mediated MDR Bacteria; Genetic Assessment of Regulators and Efflux Pump Constituents, Characterization of Efflux Systems and Screening for Inhibitors of Efflux Pumps: Current Drug Targets 9: 760-778, 2008.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Viveiros M,
    2. Rodrigues L,
    3. Martins M,
    4. Couto I,
    5. Spengler G,
    6. Martins A,
    7. Amaral L
    : Evaluation of efflux activity of bacteria by a semi-automated fluorometric system. Methods Mol Biol 642: 159-172, 2010.
    OpenUrlPubMed
  26. ↵
    1. Takács D,
    2. Cerca P,
    3. Martins A,
    4. Riedl Zs,
    5. Hajós Gy,
    6. Molnár J,
    7. Viveiros M,
    8. Couto I,
    9. Amaral L
    : Evaluation of forty new phenothiazine derivatives for activity against intrinsic efflux pump systems of reference Escherichia coli, Salmonella Enteritidis, Enterococcus faecalis and Staphylococcus aureus strains. In Vivo 25: 719-724, 2011.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Varga GZ,
    2. Szabó MA,
    3. Hohmann J,
    4. et al
    : Quorum Sensing Inhibition by Phenothiazines and Related Compounds Lett Drug Design Discovery 8: 133-137, 2011.
    OpenUrl
  28. ↵
    1. Singh G,
    2. Wu B,
    3. Baek MS,
    4. Camargo A,
    5. Nquyen A,
    6. Slusher NA,
    7. Srinivasan R,
    8. Wiener-Kronish JP,
    9. Lynch SV
    : Secretion of Pseudomonas aeruginosa type III cytotoxins is dependent on Pseudomonas quinolone signal concentration. Microb Pathog 49: 196-203, 2010.
    OpenUrlCrossRefPubMed
    1. He YW,
    2. Wu J,
    3. Cha JS,
    4. Zhang LH
    : Rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae produces multiple DSF-family signals in regulation of virulence factor production. BMC Microbiol 10: 187, 2010.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Shen DK,
    2. Filopon D,
    3. Chaker H,
    4. Boullanger S,
    5. Derouazi M,
    6. Polack B,
    7. Toussaint B
    : High-cell-density regulation of the Pseudomonas aeruginosa type III secretion system: implications for tryptophan catabolites. Microbiology 154(Pt8): 2195-208, 2008.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Drew D,
    2. Klepsch MM,
    3. Newstead S,
    4. Flaig R,
    5. De Gier JW,
    6. Iwata S,
    7. Beis K
    : The structure of the efflux pump AcrB in complex with bile acid. Mol Membr Biol 25: 677-82, 2008.
    OpenUrlCrossRefPubMed
    1. Nikaido E,
    2. Yamaguchi A,
    3. Nishino K
    : AcrAB multidrug efflux pump regulation in Salmonella enterica serovar Typhimurium by RamA in response to environmental signals. J Biol Chem 283: 24245-53, 2008.
    OpenUrlAbstract/FREE Full Text
    1. Rosenberg EY,
    2. Bertenthal D,
    3. Nilles ML,
    4. Bertrand KP,
    5. Nikaido H
    : Bile salts and fatty acids induce the expression of Escherichia coli AcrAB multidrug efflux pump through their interaction with Rob regulatory protein. Mol Microbiol 48: 1609-19, 2003.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Thanassi DG,
    2. Cheng LW,
    3. Nikaido H
    : Active efflux of bile salts by Escherichia coli. J Bacteriol 179: 2512-2518, 1997.
    OpenUrlAbstract/FREE Full Text
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Inhibition of Quorum Sensing and Efflux Pump System by Trifluoromethyl Ketone Proton Pump Inhibitors
ZOLTÁN G. VARGA, ANA ARMADA, PEDRO CERCA, LEONARD AMARAL, MIOR A.A. MIOR AHMAD SUBKI, MICHAEL A. SAVKA, ERNŐ SZEGEDI, MASAMI KAWASE, NOBORU MOTOHASHI, JOSEPH MOLNÁR
In Vivo Mar 2012, 26 (2) 277-285;
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