Hemibastadin Alkaloid Analogues as Potential Anti-Biofilm Leads against Multi-Species Biofilms ()
1. Introduction
Biofouling is defined as the rapid colonization of microorganisms (bacteria, microalgae, etc.) on living or artificial surfaces, causing health risks and significant economic damage in industrial or marine environments [1]-[3]. Any surface of human interest can serve as a starting ground for biofilm development, and although the control of the development of planktonic bacterial communities is well known and mastered, bacteria within a biofilm are much more resistant to antibiotics and/or biocides (up to 1000-fold increased resistance). In this context, the development of original compounds that specifically target biofilm formation is greatly needed in view of the rational use of antibiotics and/or biocides. Such biofilm inhibitors should have the potential to be used as a preventive treatment on a wide diversity of medical and/or industrial surfaces.
Figure 1. General structure of hemibastadins, a representative example of the 1st generation, and the structure of targeted compounds.
Some of the anti-biofilm techniques that are tested today are bioinspired by the observation of sessile marine macroorganisms such as sponges, corals, or macroalgae [1]-[4]. These species are constantly exposed to undesirable bacterial colonization (e.g., biofouling) and can maintain unfouled exterior surfaces with their arsenal of secondary metabolites possessing antifouling properties [5]-[7]. However, these natural products are only obtained in small quantities, are generally toxic, and are not stable over time [8]-[19]. In this context and in a biomimetic approach, our previous preliminary investigations aimed at the discovery of new bioactive compounds showed that bromotyrosine analogues possessing a central triazole ring were good candidates as antibiofilm compounds [20] [21]. Among all classes studied, we found that hemibastadin analogues possessing a central 1,2,3-triazolic ring were the most potent (Figure 1). Conjugation of the western moiety to the eastern moiety was assumed through the triazole core. This could be achieved by a click reaction according to the copper (I)-catalyzed 1,3-dipolar cycloaddition of organic azides and alkynes, leading to the formation of 1,4-disubstituted 1,2,3-triazoles [22]. This methodology was attractive because it is a highly efficient process in bond formations among diverse building blocks that usually proceeds in 6 - 36 h at ambient temperature in water with a variety of organic co-solvents (tert-butanol, ethanol, DMSO, THF, or CH3CN) [22] [23]. Pursuing these investigations, we are now interested in the advanced SAR studies of hemibastadin derivatives, and we wish to report here the synthesis and biological results of an advanced library of hemibastadin analogues as potential antibiofilm compounds on different marine bacterial strains, and validation of their efficiency with an original model of multi-species biofilm.
2. Experimental Section
2.1. Chemistry
Preparation of intermediary carboxylic acids 3a, 3b was performed according to the methodology described in our previous reports [20] [21]. These compounds were obtained in excellent yield in two steps. Treatment of halogeno compounds 1a, 1b with sodium azide in dimethylformamide afforded the corresponding azides 2a and 2b. Synthesis of the targeted carboxylic acids 3a and 3b was then achieved by performing the copper (I)-catalyzed 1,3-dipolar cycloaddition of the organic azides with propargylic acid, resulting in the formation of 1,2,3-triazoles. Ethanol was chosen as a co-solvent to allow an easier workup and better purity of products as in our previous work [17]. In practice, propargylic acid was added at room temperature to a solution of the appropriate azide 2a, 2b, CuSO4/sodium ascorbate in a water/ethanol mixture (50/50), and the reaction time was optimized at 12 hours at room temperature. Access to the different bromotyramine analogues 4a-h was then achieved by a peptide coupling step using EDC/HOBt methodology [22]. All amides were obtained in good yields. Scheme 1 summarizes the different steps involved in the preparation of the amides.
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Scheme 1. Synthesis of hemibastadin analogues.
General Methods: All commercially available chemicals and reagents were used without any further purification. 1H and 13C NMR spectra were recorded at 400 and 100 MHz, respectively, on a Brüker Avance 400 MHz spectrometer. The spectra were recorded in CDCl3 and D6-acetone as solvents. Multiplicity was indicated as follows: s (singlet); d (doublet); t (triplet); m (multiplet); dd (doublet of doublets), etc. Coupling constants (J) were given in Hz. Chemical shifts are reported in δ relative to TMS as an internal standard. Mass spectra were measured on an ion trap mass spectrometer fitted with an ESI interface (Esquire 6000, Brüker Daltonics).
Typical procedure for the preparation of azides 2a, 2b
A mixture of the appropriate halide (1 equiv) and NaN3 (2.6 equiv) in DMF was stirred for 5 h at 90˚C. The reaction temperature was then allowed to warm to room temperature, and the reaction mixture was diluted with Et2O. The organic phase was washed with brine and water, dried over Na2SO4, and concentrated under vacuum. The azide products were directly used for the next reaction without further purification.
Typical procedure for preparation of triazoles 3a, 3b
Appropriate azide (1 equiv.) and propiolic acid (1.5 equiv.) were dissolved in a 1:2 mixture of water and EtOH. To this was added CuSO4. 5H2O (0.04 equiv.) and sodium ascorbate (0.08 equiv.). The resultant mixture was stirred at room temperature for 12 h, at which time TLC revealed complete conversion. The reaction solution was diluted with water and extracted three times with CHCl3. The reaction solution was diluted with brine and extracted three times with EtOAc. The organic layers were washed with water, dried over Na2SO4, and evaporated under vacuum. Crude triazoles were purified by silica gel column chromatography using a mixture of EtOAc/cyclohexane as the mobile phase.
Typical procedure for preparation of amide compounds 4a-h
To a stirred solution of the acid (1.28 mmol) in CH2Cl2/MeOH (9 ml/1ml), 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (1.2 eq) and Hydroxybenzotriazole (1.2 eq) were added. The mixture was stirred at room temperature under nitrogen for 5 min, and the appropriate amine (1 eq) and diisopropylethylamine (100 μL) were added. The reaction was stirred at room temperature for 48 hours. A saturated NaCl solution was added and the organic layer was extracted three times with CH2Cl2. The solvent was removed in vacuo and the residue was purified by flash chromatography (SiO2) using CH2Cl2/MeOH as eluent to yield compounds.
2.2. Biology
Bacterial strains
Six marine Gram-negative bacterial strains Pseudoalteromonas ulvae (TC14), Pseudoalteromonas lipolytica (TC8), Paracoccus sp. (4M6), Persicivirga (Nonlabens) mediterranea TC4, Shewanella sp. TC10, and Shewanella sp. TC11 [23].
TC14 was isolated in June 2010 in Little Bay of Toulon (1 m depth, Mediterranean Sea, France) [23]. The strain 4M6 was provided by the LBCM (Université de Bretagne Sud). It was isolated on glass slides immersed for 6 h at 1 m depth in March 2000 in the Morbihan Gulf (Bailleron Island, 47_3403700N-2_4405400W, Atlantic Ocean) [24]. TC8 was isolated in February 2008 in Little Bay of Toulon (1 m depth, Mediterranean Sea, France) [25]. TC4, TC10, and TC11 have also been isolated from various surfaces immersed in Little Bay of Toulon (1 m depth, Mediterranean Sea, France). TC10 harbored the pX5 plasmid encoding GFP (constructed by Dr. Aurore Puymège).
Anti-adhesion bioassays: [23]
Bacterial strains were grown on Väätänen Nine-Salts Solution (VNSS). When the stationary phase was reached, the bacterial suspension was centrifuged. Cells were then diluted in sterile artificial sea water (ASW) and introduced into microtiter plates (sterile black PS; Nunc, Fisher Scientific, France) with tested molecules at eight concentrations (2, 5, 10, 20, 50, 100, 150, and 200 µM) in three replicates in the presence of three controls: 1) non-specific staining control, 2) adhesion control, and 3) positive control. The maximum percentage of solvent (final concentration = DMSO 2%) used for the dilution of biocides was also tested in triplicate as an additional control. After incubation during an optimized adhesion time (approximately 15 h), the non-adhered bacteria were eliminated and the adhered cells were quantified after SYTO® 61 (Molecular Probes® Invitrogen, France) (1 µM) staining. A percent of inhibition was calculated per well:
(Mean FIi − nsCi)/(Mean Fic − Mean B) × 100
with FIi as the fluorescence intensity in a treated well (tested compound + bacteria + SYTO® 61), FIc as the fluorescence intensity in a control well (bacteria + SYTO® 61), nsCi as the nonspecific control (tested compound without bacteria + SYTO® 61), and B as the blank, i.e., stain control (only SYTO® 61). A sigmoid dose–response curve was obtained when the percentage of inhibition was plotted with the log of compound concentrations, after calculation of the mean (n = 3) and standard deviation (SD) per triplicate for each concentration. EC50 values were then calculated for each compound using GraphPad Prism® (GraphPad Software, USA). This software also allowed the performance of statistical tests dedicated to the analysis of two variables simultaneously, such as the difference between strains and between biocides (two-way ANOVA). Significant differences were accepted when p < 0.05.
Toxicity tests: [25]
After growth on VNSS, bacterial strains were picked up during the exponential phase. The microtiter plates (sterile transparent PS) were filled as described in the protocol of the antiadhesion assay but using VNSS instead of ASW to allow bacterial growth. The bacterial growth was monitored by measuring the turbidity (OD600nm) every hour for 7 hours. Then, resazurin (50 μM) was added to all the wells, and fluorescence was measured after 2 h to quantify the percent of bacterial viability. The same methodology used with SYTO® 61 was applied to calculate the percent of viability after resazurin staining. Only compounds with EC50 lower than 200 μM were tested, and experiments were performed at a concentration of 100 µM of each compound using ethanol 50% as a positive antibacterial reference.
Multispecies biofilm formation
This test using TC4, TC11, and TC10 was conducted by strictly following the procedure previously described in the MAPIEM laboratory [26] [27].
Bacterial labelling: This was performed with TC4, TC11, and TC10. TC10-pX5-GFP was obtained by conjugation using E. coli WM3064 as a 113 donor strain. Briefly, post-exponential phase culture of WM3064 transformed with pX5-GFP 114 was mated with post-exponential phase Shewanella sp. TC10 in a 1:1 ratio on VNSS plates 115 containing 100 μg/ml of DAP (2,6-diaminopimelic acid) (Sigma-Aldrich, Saint Louis, 116 Missouri, USA) overnight at 20˚C. Shewanella sp. TC10 transconjugants were selected on 117 VNSS DAP-free agar plates containing 6 μg/mL of chloramphenicol.
Anti-multispecies biofilm assay: To test the capability of the molecule to inhibit multispecies biofilm, this test was conducted with three species: TC4, TC11, and TC10-pX5-GFP on a 24 h biofilm. After growth in VNSS, bacteria in the post-exponential growth phase were suspended in ASW and inoculated into 24-well plates (Corning Incorporated Costar®) containing a sterilized glass coverslip in each well to a final OD600nm of 0.3 (0.1 per strain). This test was performed with and without 100 µM of the molecule previously determined to be the most effective. Controls included single-species biofilms formed under the same conditions. After 24 h, cells were fixed using 3.7% Formalin for 15 min. For the immunostaining, samples were blocked with 3% BSA (Acros Organics, Gelle, Belgium) in PBS 1X overnight at 4˚C. The primary antibodies were added for 1h in 190 BSA 3% at 1/300 for Chicken-anti-TC4, 1/100 for Goat-anti-TC5, and 1/300 for Rabbit-anti-191 TC11. After a second blocking step of 2h at room temperature, the secondary antibodies were 192 added in BSA 3% to a concentration of 1/2000 for Goat anti-Chicken IgY (H + L) conjugated to 193 FITC (Invitrogen™, Waltham, Massachusetts, USA) for 1h, to a concentration of 1/500 for 194 Goat anti-Chicken IgY (H + L) conjugated to Alexa Fluor 405 (Abcam, Cambridge, United 195 Kingdom) for 1h, to a concentration of 1/1000 for Donkey anti-Goat IgG (H + L) coupled to 196 Alexa Fluor 633 (Invitrogen™, Waltham, Massachusetts, USA) for 1h, to a concentration of 45 μL/mL for Donkey anti-Rabbit IgG coupled to Alexa Fluor 594 for 30 min and to a concentration of 1/200 for Mouse anti-rabbit IgG coupled to CruzFluor™ 488 for 1h. Finally, the coverslips were mounted with a drop of ProLong™ Diamond Antifade before observation 200 using CLSM.
Data Extraction From Images and Statistics
At least three replicates and five pictures per replicate were performed and used for data extraction. The pictures were acquired by CLSM. The biovolume was determined with the COMSTAT2 software [28] [29]. Results were obtained using automatic thresholding.
3. Results and Discussion
3.1. Chemistry
Structures of the obtained compounds are summarized in Table 1.
Table 1. Selected 1,4-disubstituted 1,2,3-triazoles.
|
n |
R1 |
R2 |
X |
Compound (yield)* |
0 |
H |
CH3 |
H |
4a (65%) |
0 |
CH3 |
CH3 |
H |
4b (73%) |
0 |
H |
H |
H |
4c (68%) |
1 |
H |
CH3 |
H |
4d (78%) |
1 |
CH3 |
CH3 |
H |
4e (85%) |
1 |
H |
H |
H |
4f (57%) |
1 |
H |
H |
Br |
4g (85%) |
2 |
H |
H |
Br |
4h (88%) |
*yield of peptide coupling step (EDC/HOBt).
1-(3-bromo-4-hydroxyphenethyl)-N-(4-methoxyphenyl)-1H-1,2,3-triazole-4-carboxamide (4a)
This compound was obtained from acid (2) and p-anisidine as a white solid (65%). mp 214˚C - 216˚C.
1H NMR (400 MHz, DMSO-d6) δ 10.30 (s, 1H), 10.10 (s, 1H), 8.61 (s, 1H), 7.72 (d, J = 8.9 Hz, 2H), 7.35 (s, 1H), 6.96 (s, 1H), 6.93 (d, J = 8.2, 1H), 6.84 (d, J = 8.5 Hz, 2H), 4.66 (t, J = 7.1 Hz, 2H), 3.74 (s, 3H), 3.11 (t, J = 7.1 Hz, 2H). 13C NMR (100 MHz, DMSO-D6) δ 158.3, 154.5, 149.8, 143.4, 133.5, 131.51, 130.50, 129.7, 127.6, 122.6 (2C), 115.4 (2C), 112.9, 111.2, 56.57, 51.2, 34.5. Found MS (ESI, m/z) for C18H17N4O3Br: [M + H]+, 416.84 [M + H + 2]+, 418.84.
1-(3-bromo-4-methoxyphenethyl)-N-(4-methoxyphenyl)-1H-1,2,3-triazole-4-carboxamide (4b)
This compound was obtained from acid (1) and p-anisidine as a brown solid (73%). mp > 250˚C.
1H NMR (400 MHz, DMSO-D6) δ 10.30 (s, 1H), 8.64 (s, 1H), 7.72 (d, J = 9.1 Hz, 2H), 7.49 (s, 1H), 7.14 (d, J = 8.3 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H), 6.91 (d, J = 9.1 Hz, 2H), 4.69 (t, J = 7.2 Hz, 2H), 3.8 (s, 3H), 3.74 (s, 3H), 3.17 (t, J = 7.2 Hz, 2H). 13C NMR (100 MHz, DMSO-D6) δ 158.3, 154.2, 153.3, 143.4, 133.3, 130.5, 129.9 (2C), 127.4, 122.6, 116.7 (2C), 115.4, 109.6, 55.7, 51.3, 49.06, 34.7. Found MS (ESI, m/z) for C19H19N4O3Br: 430.87 [M + H]+, 432.87 [M + H + 2]+.
1-(3-bromo-4-hydroxyphenethyl)-N-(4-hydroxyphenyl)-1H-1,2,3-triazole-4-carboxamide (4c)
This compound was obtained from acid (2) and 4-aminophenol as a brown solid (68%). mp > 250˚C.
1H NMR (400 MHz, DMSO-D6) δ 10.17 (s, 1H), 10.10 (s, 1H), 9.24 (s, 1H), 8.59 (s, 1H), 7.57 (d, J = 8.9 Hz, 1H), 7.37 (s, 1H), 6.96 (d, J = 8.3 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H), 6.72 (d, J = 8.9 Hz, 2H), 4.68 (t, J = 7.1 Hz, 2H), 3.16 (t, J = 7.1 Hz, 2H). 13C NMR (100 MHz, DMSO-D6) δ 158.3, 154.2, 153.3, 143.4, 133.3, 130.5, 129.9 (2C), 127.4, 122.6, 116.7 (2C), 115.4, 109.6, 51.3, 34.7. Found MS (ESI, m/z) 403.02 [M + H]+, 405.02 [M + H + 2]+.
1-(3-bromo-4-hydroxyphenethyl)-N-(4-methoxybenzyl)-1H-1,2,3-triazole-4-carboxamide (4d)
This compound was obtained from acid (2) and 4-methoxybenzylamine as a white solid (78%). mp 162˚C - 164˚C.
1H NMR (400 MHz, DMSO-d6): δ 10.10 (s, 1H, OH), 8.97 (t, J = 6.2 Hz, 1H, NHCO), 8.48 (s, 1H), 7.33 (d, J = 2.1 Hz, 1H), 7.24 (d, J = 8.7 Hz, 1H), 6.95 (dd, J = 8.3 Hz, 1H), 6.87 (d, J = 8.7 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 4.61 (t, J = 7.1 Hz, 2H), 4.36 (d, J = 6.2 Hz, 2H), 3.72 (s, 3H), 3.08 (t, J = 7.1 Hz, 2H). 13C NMR (100 MHz, DMSO-D6) δ 159.9, 156.6, 153.1, 143.1, 133.3, 132.1, 129.9, 129.4, 129.1 (2C), 126.8, 116.6, 114.1 (2C), 109.6, 55.5, 51.2, 41.8, 34.6. Found MS (ESI, m/z) for C19H19N4O3Br: 431.03 [M + H]+, 433.02 [M + H + 2]+.
1-(3-bromo-4-methoxyphenethyl)-N-(4-methoxybenzyl)-1H-1,2,3-triazole-4-carboxamide (4e)
This compound was obtained from acid (1) and 4-methoxybenzylamine (85%). mp 141˚C - 143˚C.
1H NMR (400 MHz, DMSO-d6) δ 8.98 (t, J = 6.3 Hz, 1H, NHCO), 8.5 (s, 1H), 7.45 (s, 1H), 7.23 (d, J = 6.3 Hz, 2H), 7.11 (s, 1H), 7.00 (d, J = 8.5 Hz, 1H), 6.87 (d, J = 8.7 Hz, 2H), 4.64 (t, J = 7.1 Hz, 2H), 4.34 (d, J = 6.2 Hz, 2H), 3.79 (s, 3H), 3.72 (s, 3H), 3.13 (t, J = 7.1 Hz, 2H). 13C NMR (100 MHz, DMSO-D6) δ 158.7, 154.7, 143.3, 133.4, 132.2, 131.6, 130.2, 129.8 (2C), 129.2, 126.9, 114.16 (2C), 113.07, 110.9, 56.6, 55.6, 51.2, 41.8, 34.5. Found MS (ESI, m/z) for C20H21N4O3Br: 445.12 [M + H]+, 447.12 [M + H + 2]+.
1-(3-bromo-4-hydroxyphenethyl)-N-(4-hydroxybenzyl)-1H-1,2,3-triazole-4-carboxamide (4f)
This compound was obtained from acid (2) and 4-hydroxybenzylamine (57%). mp 173˚C - 175˚C.
1H NMR (400 MHz, DMSO-D6) δ 10.08 (s, 1H, OH), 9.25 (s, 1H, OH), 8.88 (t, J = 6.3 Hz, 1H, NHCO), 8.47 (s, 1H), 7.34 (d, J = 2.1 Hz, 1H), 7.11 (d, J = 8.2 Hz, 1H), 6.94 (d, J = 8.3 Hz, 2H), 6.82 (d, J = 8.3, 1H), 6.69 (d, J = 8.3 Hz, 2H), 4.60 (t, J = 7.1 Hz, 2H), 4.31 (d, J = 5.6 Hz, 2H), 3.79 (s, 3H), 3.07 (t, J = 7.1 Hz, 4H). 13C NMR (100 MHz, DMSO-D6) δ 159.9, 156.6, 153.1, 143.1, 133.3, 130.2, 129.9, 129.4 (2C), 129.1, 126.8, 116.6 (2C), 115.4, 109.6, 51.2, 40.4, 34.6. Found MS (ESI, m/z) for C18H17N4O3Br: 417.01 [M + H]+; 419.01 [M + H + 2]+.
N-(3,5-dibromo-4-hydroxybenzyl)-1-(3-bromo-4-hydroxyphenethyl)-1H-1,2,3-triazole-4-carboxamide (4g)
This compound was obtained from acid (2) and 3,5-dibromo-4-hydroxybenzylamine (85%). mp 214˚C - 216˚C.
1H NMR (400 MHz, DMSO-D6) δ 10.09 (s, 1H, OH), 9.83 (s, 1H, OH), 9.10 (t, J = 6.3 Hz, 1H, NHCO), 8.50 (s, 1H), 7.47 (s, 1H), 7.31 (s, 1H), 6.96 (d, J = 7.8 Hz, 1H), 6.83 (d, J= 8.3, 1H), 4.61 (t, J = 7.2 Hz, 2H), 4.32 (d, J = 6.2 Hz, 2H), 3.07 (t, J = 7.3 Hz, 2H). 13C NMR (100 MHz, DMSO-D6) δ 160.2, 153.3, 150.1, 142.9, 133.5, 131.8, 130.0, 129.4, 127.0, 116.9, 112.2, 109.5, 79.6, 51.3, 34.5, 29.5. Found MS (ESI, m/z) for C18H15N4O3Br3: 572.8 [M + H]+, 574.8 [M + H + 2]+.
1-(3-bromo-4-methoxyphenethyl)-N-(4-hydroxyphenethyl)-1H-1,2,3-triazole-4-carboxamide (4h)
This compound was obtained from acid (2) and 3,5-dibromotyramine (88%). mp 171˚C - 173˚C.
1H NMR (400 MHz, DMSO-D6) δ 10.09 (s, 1H, OH), 9.83 (s, 1H, OH), 9.10 (t, J = 6.3 Hz, 1H, NHCO), 8.50 (s, 1H), 7.47 (s, 1H), 7.31 (s, 1H), 6.96 (d, J = 7.8 Hz, 1H), 6.83 (d, J= 8.3, 1H), 4.61 (t, J = 7.2 Hz, 2H), 4.32 (d, J = 6.2 Hz, 2H), 3.07 (t, J = 7.3 Hz, 2H). 13C NMR (100 MHz, DMSO-D6) δ 162.8, 160.2, 149.3, 143.1, 134.4, 133.3, 132.6, 130.1, 129.4, 126.7, 116.7, 112.2, 109.5, 51.1, 36.2, 34.6, 33.5. Found MS (ESI, m/z) for C19H17N4O3Br3: 586.89 [M + H]+; 588.89, [M + H + 2]+.
3.2. Antibiofilm Activity
Compounds 4a-h were assessed for their antibiofilm activity against three strains of gram-negative bacteria (Pseudoalteromonas ulvae (TC14), Pseudoalteromonas lipolytica (TC8), Paracoccus sp. (4M6)) [25]. In an initial screening process, all compounds were tested for their ability to modulate biofilm formation at a concentration of 200 μM by using our previous method adapted from Leroy et al., using the specific fluorophore Syto®61 [25] [30]. In order to clarify structure-activity relationships, effective concentrations to inhibit 50% of bacterial adhesion (expressed as EC50) were determined for active compounds. Different elements were considered to obtain information about structure-activity relationships: length of the chain at the eastern region (n = 0, 1, 2), O-methylation on the aromatic rings, and finally, degree of bromination. Antibiofilm activities are summarized in Table 2 below.
Table 2. biological screening against bacterial biofilms of Paracoccus sp. (4M6), Pseudoalteromonas lipolytica (TC8), Pseudoalteromonas ulvae (TC14). Results are expressed as the effective concentration to inhibit 50% of biofilm formation (EC50) in micromoles/L (µM). Data represent means ± standard deviation values from three independent experiments.
Compound |
TC8 |
4M6 |
TC14 |
% of adhesiona |
EC50 |
% of adhesion |
EC50 |
% of adhesion |
EC50 |
4a |
60.8 ± 14.4 |
>200 |
67.9 ± 5.9 |
>200 |
34.3 ± 3.3 |
65.2 ± 0.9 |
4b |
ND |
NT |
ND |
NT |
ND |
NT |
4c |
68.7 ± 10.7 |
>200 |
32.2 ± |
79.0 ± 17.4 |
22.9 ± 16.1 |
90.6 ± 0.1 |
4d |
55.5 ± 6.7 |
>200 |
44.2 ± 2.3 |
174.3 ± 18.0 |
32 .3 ± 8.2 |
88.2 ± 11.7 |
4e |
ND |
NT |
ND |
NT |
ND |
NT |
4f |
65.3 ± 5.4 |
>200 |
66.2± 5.3 |
>200 |
39.6 ± 1 |
156.0 ± 15.1 |
4g |
14.1 ± 11.7 |
91.5 ± 27.0 |
44.2 ± 2.3 |
27.1 ± 4.9 |
15.1 ± 1.4 |
32.3 ± 1.2 |
4h |
36.2 ± 5.3 |
60.4 ± 4.1 |
11.8 ± 2.6 |
28.8 ± 8.2 |
19.6 ± 11.6 |
26.7 ± 7.8 |
Ampicillin |
NT |
17.9 ± 1.0 |
NT |
144.1 ± 0.6 |
NT |
9.3 ± 1.0 |
a. Percentage of adhesion at 200 µM compared to untreated samples. ND: adhesion > 95%. NT not tested.
Results indicated that the degree of methylation was of primary relevance for assessing the activity. The efficiency of compounds decreased when the two hydroxyl groups were methylated (4b, 4e when compared to 4a and 4d). On the other hand, it appeared that the degree of bromination of the eastern moiety (4g, 4h) was of primary importance in affording an interesting biological response as antibiofilm compounds, with EC50 ranging from 90 to 25 µM toward the different strains.
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Figure 2. Effect of compounds 4g and 4h at a concentration of 100 μM on the bacterial growth of Pseudoalteromonas lipolytica TC8 (up, left), Paracoccus sp. 4M6 (up, right), Pseudoalteromonas ulvae TC14 (down, left). Results are expressed by measurement of optical density at 600 nm over 6 hours. Ampicillin (5 μM) was used as a bactericidal control, and an untreated sample was used as a positive reference. (Down, right): effect of compounds 4g and 4h at a concentration of 100 μM on the viability of the three strains. Data are expressed as % of viable bacteria when compared to an untreated sample with 100% viability. All values are the mean of three replicates.
In order to verify whether the compounds 4g and 4h exhibited specific antibiofilm activity or if this observation was simply related to a general toxic effect on the bacteria, a growth inhibition and viability assay was performed. Active compounds 4g and 4h were tested for their capacity to inhibit the growth of the three strains TC14, TC8, and 4M6 over 6 hours. Experiments were performed at the high concentration of 100 μM and using ampicillin at a concentration of 5 µM as a reference. We have already shown that the antibiofilm activity of ampicillin was directly connected to an antibacterial and general toxic effect on these different strains (especially against TC8 and TC14), but in contrast, the results presented in Figure 2 showed that when compared to untreated samples, the compounds 4g and 4h exhibit no effect on the bacterial growth of the TC14 strain. For viability, the same methodology used for the antiadhesion assay with Syto®61 was applied using the resazurin test at the concentration of 100 μM.
This suggested that their anti-biofilm activities were not directly connected to antibacterial effect, in contrast to ampicillin, which is toxic especially towards TC14 and TC8. In regard to these results, and as most biofilm communities are composed of multiple different bacteria living in close proximity, compounds 4g and 4h, which were identified as the more potent and non-toxic compounds, were selected for a test against a multispecific biofilm. Such multispecies biofilms could be used as a routine model to test newer biocidal agents. For this purpose, we have developed a model of three bacterial species Persicivirga (Nonlabens) mediterranea TC4, Shewanella sp. TC10 and Shewanella sp. TC11 [23] [26]. In practice, a preliminary screening of toxicity of compounds 4g, 4h at a concentration of 100 µM showed no effect on growth and viability of TC10 and TC11 strains, while the TC4 strain was more sensitive to exposure to compounds 4g and 4h. Additionally, we can note that the known antibiotic ampicillin presented the same profile against TC4, while being less active on the two other strains (Figure 3).
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Figure 3. Left, effect of compounds 4g and 4h at a concentration of 100 μM on the viability of the 3 strains using ampicillin (5 μM) and ethanol as references. Data are expressed as % of viable bacteria compared to an untreated sample with 100% viability. All values are the mean of three replicates. Right, biovolumes measured at 24 h for each strain in three-species biofilms. Results report the untreated samples (black) and samples treated with compounds 4g and 4h at 100 μM (grey). All values are the mean of three replicates.
For the multispecies experiment, TC4, TC10, and TC11 were inoculated in the same proportion onto glass coverslips, and the biofilms were studied at 24 h using confocal laser scanning microscopy (CLSM). Experiments were conducted with compounds 4g and 4h at 100 µM and without compounds as a reference. Figure 4 shows the CLSM pictures.
Measurement of the biovolumes of each strain within the multispecies biofilm with and without compounds 4g and 4h revealed that the two compounds reduced by around 100% the formation of the biofilm of the three strains. No significant differences can be noted between the results on TC4, for which 4g and 4h are toxic, with those observed for the two other strains. The results of this measurement are confirmed by examination of confocal laser scanning microscopy images, which showed a total inhibition of the biofilm growth. Taken together, our results strongly indicate that compound 4g and 4h act as potent and specific antibiofilm agents against Gram-negative bacteria. Since the growth and viability of the different strains were poorly affected by exposure to the compounds (except TC4), we can consider them as non-toxic on the different isolated strains as well as on multispecies biofilms.
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Figure 4. Confocal laser scanning microscopy images of the multispecies biofilm composed of Persicivirga (Nonlabens) mediterranea TC4, Shewanella sp. TC10, and Shewanella sp. TC11 on coverslips at 24 hours. Images show the biofilm without treatment as a reference (up) and treated with 100 μM of compound 4g (middle) and 4h (down). Images of each strain have been extracted, and the overlay of the three strains is shown on the right.
4. Conclusion
In summary, we have used click chemistry to investigate the chemical diversity of a library of hemibastadin analogues based on a triazole-amide framework. In the present paper, we have designed new analogues active against biofilm growth of gram-negative bacteria. Finally, the low toxicity of the more potent anti-biofilm leads allows us to focus on future interest in the development of these molecules as non-toxic anti-biofilm compounds for their potential use as non-toxic co-biocide or co-antibiotic in view of rational eradication of persistent biofilms.
Acknowledgements
We are especially grateful to the LBCM laboratory (Université de Bretagne Sud).