Synergetic Antibacterial Activities of Novel aryl 1,2,3-triazole-Modified Lignocellulosic Materials

Figure 1:Triazole derivatives grafted onto Kraft pulp.

The development of new antibacterial materials continues to be an area of intense investigations due to bacterial resistance. The use of a new class of bioactive agents, with a structure and a mode of action different from conventional biocides is promising for the inhibition of resistant microorganisms. For this purpose, 1,2,3-triazoles, widely known for their antimicrobial properties [1-3], are basic substrates for the design and synthesis of a wide variety of compounds of pharmacological interest. Their introduction into polymers such as chitosan [4,5] or starch [6] led to new materials with a broad spectrum of antibacterial activities. In this way, our group has previously described the synthesis of new antibacterial triazolated materials, derivatives on cellulosic fibres of Kraft pulp. The triazolated materials were developed by grafting different aryl azide onto propargylated kraft pulp fibres [7] (Figure 1).

From two inactive substrates, propagylated Kraft pulp fibres and aryl azide compounds respectively, the original developed materials acquired their antimicrobial potential after a cycloaddition reaction and the formation of a triazole link. It was demonstrated that the nature of the substituent R’ of the aryl group clearly influenced the antibacterial effectiveness of the developed materials. As an example, after 24 hours of contact between the prepared materials and the bacterial strains, the materials bearing the methoxyl (R’=OCH₃) and acetyl (R’=COCH₃) substituents showed a bacteriostatic activity against S. aureus and the material bearing the ethoxycarbonyl substituent (R’=COOEt) shows bactericidal activity. The materials carrying hydroxymethyl (R’=CH₂OH) and hydroxyethyl (R’=CH₂CH₂OH) substituents exhibited bacteriostatic activity against E. coli (Figure 2).

Figure 2:Bacterial growth of E. coli and S. aureus in the presence different actives materials at t=24h.

The materials we’ve developed have interesting antibacterial properties, blocking the growth of bacteria and limiting their proliferation. However, after 24h of contact, none of these materials was active on both strains at the same time. We therefore continued the study by preparing mixtures of materials, one with activity against E. coli and the other with activity against S. aureus at t24, and then proceeded to evaluate their antibacterial activity. In addition, and to assess the potential of these new combinations of modified lignocellulosic materials, the activity against a mixture of the two bacteria will also be carried out (Figure 3).

Figure 3:Scheme of the strategy implemented to study the synergistic effect.

General methods

All solvents and reagents were commercially available and, unless otherwise stated, were used as received. 4-aminoacétophenone (99%), ethyl-4-aminobenzoate (98%), 4-aminobenzyl alcohol (98%), 2(4-aminophenyl) ethanol (97%), propargyl bromide (80%) and sodium azide (99%) were purchased from Alfa Aesar (Karlsruhe, Germany). Copper (II) sulfate pentahydrate (98%), sodium nitrite (99%) and sodium ascorbate (98%) were purchased from Aldrich (Lyon, France). Reactions were monitored by thinlayer chromatography (TLC) on 0.2mm silica gel precoated 60 F254 (MerckChimie, Fontenay-sous-bois, France) plates and revealed with an ultraviolet light source at 254nm. The bleached hardwood Kraft pulp was received in wet laps from a Northeastern Canada mill.

1H NMR spectra were recorded at 400.13MHz with a Bruker DPX-400 spectrometer. A Perkin Elmer 1000 FTIR spectrometer equipped with the Spectrum software was used to perform FTIR analysis. XPS experiments were carried out using a Kratos Axis Ultra spectrometer that provided elemental composition information (atomic percentage) within a depth of a few nanometers from the sample surface.

Synthesis

The triazolated materials were developed by grafting different aryl azide onto propargylated kraft pulp fibres [7].

Preparation of propargylated Kraft pulp fibres

10g of Kraft pulp were suspended in 153mL of NaOH solution (7.4g) and the mixture was stirred 15 min, at room temperature leading to a dispersion of the Kraft pulp. Propargyl bromide (60mL, 61.68mmol, 10eq./AGU) of propargyl bromide was then added. The reaction medium was homogenized then maintained at room temperature, without stirring. After 96h, the mixture was filtered and washed with hot water (2x400mL) and hot ethanol (3x400mL). The product was dried at 60 °C.

Preparation of the aryl azides

To a solution of arylamine (44mmol) in ethyl acetate (80mL) and water (10mL), was added at 0 °C, 24mL of hydrochloric acid 37%. The reaction was stirred 15min and sodium nitrite (88mmol, 2eq.) in water (15mL) was added dropwise. The solution was stirred 30min before the dropwise addition of an aqueous sodium azide solution (88mmol, 2eq. in 15mL of water). Upon completion of addition, the reaction was stirred for an additional 30min. The reaction mixture was then diluted with water (80mL), extracted with ethyl acetate (2x80mL). The combined organic layers were washed with saturated solution of NaHCO₃ (80mL), then with water (80mL) and dried over MgSO₄.

Preparation of grafted material

11.5mmol of aryl azides were solubilized in THF (40mL) and then propagylated Kraft pulp fibres (1g, DS: 0.32) were added. Copper sulfate pentahydrate (0.05eq; 0.29mmol) and sodium ascorbate (0.1eq.; 0.58mmol) dissolved in a minimum of water, were introduced. After 72h at room temperature and with stirring, modified fibres were washed with THF (2x50mL), water (2x50mL) and ethanol (2x50mL), then dried at 60 °C.

Spectroscopic data

All physicochemical properties coincided with literature data [7].

Antibacterial essays

Gram-positive bacteria (S. aureus CIP76.25) and Gram-negative bacteria (E. coli CIP54.8T) were purchased by the Institute Pasteur Paris (Paris, France). The different bacterial strains were inoculated into liquid tryptic soy (pancreatic casein extract 17g/L, soy flour papaic digest 3g/L, dextrose 2.5g/L, NaCl 5g/L, and K₂HPO₄ 2.5g/L) and incubated at 37 °C overnight under aerobic conditions. The stock solution was further diluted to give a working suspension of approximately 2x106CFU/mL.

To evaluate the antibacterial effect of the different Kraft Pulp fibres surfaces, a protocol based on AATCC100 standard was implemented [8]. Sterile samples of Kraft Pulp fibres (8mm diameter) were impregnated with 50μL of bacterial suspension at a cell density of approximately 2x10⁶ CFU/mL. To determine the CFU number initially deposited onto disks (t=0), each sample was transferred into 1mL of extraction solution, composed of Triton X-100 0.5% (v/v) and physiological saline water (NaCl 0,9%). After 2h of gentle stirring at room temperature, serial dilutions of each extraction solution were performed, and each dilution was spread on tryptic soy agar plates using an automatic easy SPIRAL® plater (Interscience, St Nom la Bretêche, France). After incubation at 37 °C for 24h, plates were counted to determine total CFU per mL. Each experiment was performed in triplicate and was conducted along with necessary control, untreated Kraft Pulp (KP).

For the experiment, a sample was processed immediately after bacterial impregnation (t=0) and other samples were also processed in the same conditions after 24h at 37 °C under aerobic conditions.

Table 1:Different prepared mixtures.

The aryl azides were prepared from corresponding arylamine via diazonium salt intermediate, and then were coupled to propagylated kraft pulp by a cycloaddition reaction according to our previous work [7]. Homogeneous mixtures were prepared from obtained materials (Table 1). The mixtures were composed by 50% (wt %) of one material having activity against E. coli (R’=CH₂OH or CH₂CH₂OH) and 50% (wt %) of material active against S. aureus (R’=OCH₃, COCH₃ or COOEt) after 24 hours.

The antibacterial activity of different mixtures was evaluated against the Gram (+) S. aureus CIP 7625 and the Gram (−) E. coli CIP54.8T bacteria.

A suspension of bacteria was deposited onto disks prepared from different samples and then incubated at 37 °C. After 24 hours, the total CFU per mL was determined. The untreated Kraft pulp used as controls showed no antibacterial activity against the two bacterial strains used in this study.

In the case of E. coli (Figure 4), from a mixture of two materials, one bacteriostatic and the other inactive, we obtained materials that exhibited a bactericidal effect, with inhibition rates of 70%, 100%, 52% and 45% for mixtures A, B, D, and E respectively. The mixtures C and F presented a bacteriostatic activity. For S. aureus (Figure 5), a bactericidal effect was also observed for mixtures A, B and D with 60%, 10% and 45% inhibition.

Figure 4:Bacterial growth (log CFU/mL) of E. coli in the presence of the untreated Kraft Pulp (KP), the mixture A, B, C, D, E and F.

Figure 5:Bacterial growth (log CFU/mL) of S. aureus CIP 76.25 in the presence of the untreated Kraft Pulp (KP), the mixture A, B, C, D, E and F.

It was interesting to note that we have developed material A, which has the highest activity against both bacteria, S. aureus and E. coli with an inhibition rate of the order of 60%. This material was obtained from 50% of material bearing the hydroxymethyl substituent (bacteriostatic against E. coli and inactive against S. aureus), and 50% of material bearing the ethoxycarbonyl substituent (inactive against E. coli and bactericidal against S. aureus) (Figure 6).

Figure 6:Comparison of the antibacterial activity of the materials of the hydroxymethyl and ethoxycarbonyl substituents and the material A, against E. coli and S. aureus.

These results indicated that there was a synergistic effect between a bacteriostatic material and another inactive to lead to a bactericidal material. Indeed, generally, the association of two active compounds can lead to four types of interactions: indifferent, additive, synergistic or antagonistic. In addition to these interactions, where the two molecules have an antibacterial action, it exists a synergy between two molecules in their action, while one of the compounds has no activity on the bacterium [9]. This is the case for example of the effect of the combination of β-lactame and inhibitor of β-lactamase. In our case, we obtained the same synergistic effect with the mixing of two materials, one active against the bacterium and the other inactive. To our knowledge, this type of synergy between two 1,2,3-triazole derivatives grafted on solid support has never been described in the literature.

However, when a material is in the environment, it is often contaminated by several bacteria [10]. In some cases, the bacteria may become more virulent and more resistant to antimicrobials when mixed with other bacteria. Ramsey et al. [11] have shown, for example, that Acinobacter actinomycetemcomitans, a gramnegative bacterium, displayed a higher growth rate and greater virulence when co-cultivated with S. gordonii. This was due to its ability to metabolize the lactate produced by S. gordonii, which allowed it to grow faster at an early stage and persist. Therefore, we decided to investigate the antibacterial activity of the different materials elaborated on a mixture of the two bacterial strains S. aureus and E. coli. For this, we prepared a solution containing 105CFU of each bacterium in Trypticase Soy (TS) nutrient medium. 50μL of this suspension were deposited on three disks of the same sample. Since E. coli and S. aureus colonies were visually different, the colonies of each bacterium were counted at t₀ and t₂₄.

In the presence of a mixture of the two bacteria, E. coli and S. aureus (Figure 7), the antibacterial activity of materials A, B, C, D and E was comparable to that obtained against both bacteria E. coli and S. aureus, each tested separately. For material F, we observed a bactericidal effect with a 100% inhibition rate against E. coli. In fact, one goes from a bacteriostatic effect when E. coli is tested separately, to a bactericidal effect when it is tested mixed with S. aureus.

Figure 7:Antibacterial activity of different materials (A, B, C, D, E and F) on a mixture of E. coli and S. aureus.

In the case of S. aureus, a bactericidal activity was observed for materials A and B. A bacteriostatic activity was highlighted for materials C, D and E equivalent to the activity observed when the bacteria were tested separately. For material F, a bactericidal effect was shown with an inhibition rate of 55%. Again, it switched from a bacteriostatic effect when S. aureus was tested separately, to a bactericidal effect when tested mixed with E. coli.

These results indicated that the materials could inhibit bacterial growth when both bacteria are present in the community. In the case of material F (Figure 8), the activity increases, it became bactericidal when the bacteria were mixed while it was bacteriostatic, when the tests were carried out on the strains separately.

Figure 8:Comparison of the antibacterial activity of the mixture F against E. coli and S. aureus separately and in mixture.

In this study, we investigated the antibacterial activity of mixture of materials prepared by grafting various aryl triazoles onto lignocellulosic fibres of kraft pulp, using a reaction of Click Chemistry, the Copper (I) -Catalyzed Alkyne-Azide 1,3-dipolar Cycloaddition.

The study of the antibacterial activity of materials mixtures, one having an activity against E. coli and the other an activity against S. aureus after 24 hours of contact, showed a synergistic effect. Thus, we have developed a new bactericidal material “A” with an inhibition rate up to 100% against E. coli and 60% against S. aureus, from bacteriostatic material, and another inactive against the same strain.

By testing the activity of the different materials on a mixture of the two strains, E. coli and S. aureus, a marked improvement of the antibacterial activity was observed in the case of the material F, who had a bacteriostatic effect against E. coli and S. aureus when tested separately. The activity switched to 100% inhibition for E. coli and 55% against S. aureus when they were together.

1. Kant R, Kumar D, Agarwal D, Devi GR, Tilak R, et al. (2016) Synthesis of newer 1,2,3-triazole linked chalcone and flavone hybrid compounds and evaluation of their antimicrobial and cytotoxic activities. Eur J Med Chem 113, 34-49.
2. Dheer D, Singh V, Shankar R, (2017) Medicinal attributes of 1,2,3-triazoles: Current developments. Bioorg Chem 71: 30-54.
3. Ashok D, Gundu S, Aamate VK, Devulapally MG (2018) Conventional and microwave-assisted synthesis of new indole-tethered benzimidazole-based 1,2,3-triazoles and evaluation of their antimycobacterial, antioxidant and antimicrobial activities. Mol divers 22(4): 769-778.
4. Li Q, Tan W, Zhang C, Gu G, Guo Z (2015) Novel triazolyl-functionalized chitosan derivatives with different chain lengths of aliphatic alcohol substituent: Design, synthesis, and antifungal activity. Carbohyd Res 418: 44-49.
5. Li Q, Tan W, Zhang C, Gu G, Guo Z (2016) Synthesis of water-soluble chitosan derivatives with halogeno-1,2,3-triazole and their antifungal activity. Int J Biol Macromol 91: 623-629.
6. Tan W, Li Q, Wang H, Liu Y, Zhang J, et al. (2016) Synthesis, characterization, and antibacterial property of novel starch derivatives with 1,2,3-triazole. Carbohyd Polym 142: 1-7.
7. Khaldi Z, Besse C, TaKeki JKN, Ouk TS, Gloaguen V, Zerrouki R (2019) Synthesis, characterization, and antibacterial activities of a new lignocellulosic material carrying aryl triazole moiety. Polym Adv Technol 30(2): 344-350.
8. Sjollema J, Zaat SAJ, Fontaine V, Ramstedt M, Luginbuehl R, et al. (2018) In vitro methods for the evaluation of antimicrobial surface designs. Acta Biomater 70: 12-24.
9. Denes E, Hidri N (2009) Synergie et antagonisme en antibiothérapieAntibiotic synergy and antagonism. Antibiotiques 11(2): 106-115.
10. Asfahl KL, Schuster M (2017) Social interactions in bacterial cell-cell signaling. FEMS Microbiol Rev 41(1): 92-107.
11. Ramsey MM, Rumbaugh KP, Whiteley M (2011) Metabolite cross-feeding enhances virulence in a model polymicrobial infection. PLoS Pathog 7(3): e1002012.