A comparative evaluation of antibacterial activities of imidazolium-, pyridinium-, and phosphonium-based ionic liquids containing octyl side chains.

Antibacterial activity is an essential property of ionic liquids. In this work, a comprehensive study has been performed on the antibacterial activity of ionic liquids to be utilized for further research and applications. Eighteen ionic liquids viz. Octyl Imidazolium, octyl Pyridinium, quaternary phosphonium-based cations containing bromide, sodium methane sulphonates, bis(trifluoromethane sulfonyl) imide, dichloroacetate, tetrafluoroborate, hydrogen sulfate were prepared and characterized with the help of different spectroscopic techniques. All these samples of ionic liquids were tested for their antibacterial activity against the most commonly occurring bacteria in the environment, i.e., Enterobacter aerogenes (E. aerogenes), Proteus vulgaris (P. vulgaris), Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli), and Streptococcus pyogenes (S. pyogenes). Most of the ionic liquids show good antibacterial properties, and imidazolium-based ionic liquids were even more antibacterial as compared to positive control. It was observed that a unique combination of cation and anion is essential to achieve desired antibacterial properties. The mechanism of antibacterial activity was further investigated using density functional theory calculations. A good correlation was found between experimental and theoretical studies.

Introduction

The Healthcare community faces the major challenge of an enhanced number of deaths in hospitalized patients due to microbial infections [1]. These increasing numbers of microbial infections have now gained the attention of researchers worldwide, as evident from the growing number of publications on microbial resistance and its effects on the health of the human population [2]. Bacterial infections can be controlled by sanitization [3], sterilization [4], and by the use of biocides, but the bacteria have developed resistance against them. For example, E. coli is responsible for both complicated and uncomplicated UTIs. But, some German populations have developed resistance against cotrimoxazole, which was once used as the first line of defense against UTIs [5].

Ionic liquids (ILs) with inherent antimicrobic properties and tunable nature have now emerged as active pharmaceutical ingredients (API) [6, 7, 8]. ILs mainly consist of an organic cation like imidazolium, pyridinium, quaternary ammonium, and phosphonium which can be combined with various anions to form a diverse set of molecules with distinct properties which can be further changed by the functionalization of either cation or anion [9]. The length of alkyl side chains of ILs directly affects the cytotoxicity, interaction with bovine serum albumin, and toxicity for microbial growth [10]. ILs possess unique physicochemical properties like high viscosity and density as compared to water [11], reduced biodegradability [12], low melting point (<100 °C) [9, 13], high thermal, chemical, and electrochemical stability [11, 14, 15, 16], non-volatile [17], low toxicity [6] solubility in most of the solvents and negligible vapor pressure [18].

Due to their distinctive properties ILs have drawn considerable research interest. The applications of ILs includes antibiofilm [19, 20, 21], anti-corrosion [22, 23], separation [24, 25, 26, 27, 28, 29, 30], bio-catalysis [31], drug delivery [32, 33] and electrochemistry [34]. Several type of ILs are being studied for their antibacterial properties. Some major classes include, 1-butyl-1-methylpyrrolidinium trifluoromethyl sulfonate [22], octyl and hexyl imidazolium bromide [1], -alkyl-vinyl imidazolium bromide monomer and polymer [35], butylimidazolium bromide [36], cinnamyl imidazole [37], pyridinium-based ILs [18, 38], novel phenethyl imidazolium-based ILs [39], amino thiazolyl-functionalized phosphonium ILs [40], quaternary ammonium-based ILs [41] and those derived from choline and geranate [17].

This work reported the synthesis and characterization of pyridinium, phosphonium, and imidazolium-based ionic liquids with different anions. This study compares the effect of different cations containing octyl side chains on antibacterial activity. These ionic liquids were tested for antibacterial activity against S. pyogenes, E. coli, K. pneumoniae, E. aerogenes, P. vulgaris, P. aeruginosa. The ILs based on imidazolium show better antibacterial activity than all other tested cations.

Experimental

Materials and instrument

All analytical grade reagents were used without modifications during the synthesis. The chemicals used included, 1-methylimidazole (DAEJUNG, >99%), pyridine (Sigma-Aldrich, ∼99%), tri-octyl phosphine (MACKLIN, 92.23%), octyl bromide (MACKLIN, >95%) methane sulphonic acid (Sigma-Aldrich, 99%), dichloroacetic acid (Sigma-Aldrich, >98%), potassium hydrogen sulfate (MERCK, 35–37%), lithium bis(trifluoromethane-sulfonyl) imide (MERCK, 99%), sodium tetrafluoroborate (Sigma-Aldrich, 98%) and sodium hydroxide (Sigma-Aldrich, >98%). Solvents like methanol (CH3OH), chloroform (CHCl3), acetonitrile (CH3CN), n-hexane, and ethyl acetate were dried by distillation before use. The FTIR analysis was performed on the Brucker ATR-alpha FTIR instrument in 4000–550 cm−1 to identify the functional groups present in synthesized ionic liquids. The NMR analysis was performed on Brucker NMR (400 MHz) using CDCl3 as a solvent.

Synthesis

Octyl imidazolium based ionic liquids

1-Methyl-3-octylimidazolium bromide [C8mim][Br]

The [C8mim][Br] was synthesized according to the literature [42]. Briefly, 1-Methylimidazole (5 g, 60 mmol) and octyl bromide (11.74 g, 61 mmol) were added to 60 mL acetonitrile (Scheme 1). The reaction mixture was refluxed for 48 h. After 48 h, the solvent was evaporated by a rotary evaporator. The solid product obtained was dried in a vacuum oven at 50 °C for 12 h. Product (S1-01) was obtained as yellow oily liquid with ∼81% yield.

Scheme 1

Synthesis of imidazolium-based ILs.

FTIR (cm): 3120 (CH str aromatic), 2924 and 2854 (aliphatic CH str.), 1650 (C=N str.), 1569 (C=C str.), 1463 (CH bending), (1165 C–N str.), 752 (octyl CH bending).

H-NMR (ppm) (CDCl): 0.37–0.47 (m, 3H; CH3), 0.78–0.86 (m, 2H; CH2), 1.44–1.47 (m, 10H; CH2), 3.60–3.70 (m, 3H; NCH3), 3.87–3.90 (m, 2H; NCH2), 7.21–7.22 (s, 1H; CH = CH), 7.360–7.367 (s, 1H; CH = CH), 9.529–9.531 (s, 1H; N–CH–N). C-NMR: 13.66 (CH2CH), 19.13 (CHCH3), 31.84 (CHCH2CH3), 48.84(NCH3), 36.31 (NCH2CH2), 123.86 (NCH), 122.67 (NCH), 136.94 (NCHN).

1-Methyl-3-octylimidazolium methanesulphonate [C8mim][MeSO3]

1-Methyl-octylimidazolium methanesulphonate was synthesized by anion metathesis reaction of [C8mim][Br] and sodium methanesulphonate in methanol (Scheme 1). Briefly, [C8mim][Br] (2.75 g, 10 mmol) and sodium methanesulphonate (4 g, 11.76 mmol) were added to 60 mL methanol and kept on stirring for 12 h. The solvent was evaporated with a rotary evaporator. The NaBr formed during the reaction was removed by solvent extraction using chloroform and ethyl acetate. The product (S1-02) was then dried under vacuum at 50 °C. The yield of the product is ∼71%.

FTIR (cm): 3120 (CH str. aromatic), 2924 and 2854 (aliphatic CH str.), 1569 (C=C str.), 1463 (CH bending), 1166 (C–N str.), 1040 (S=O str.), 767 (octyl CH bending).

H NMR (400 MHz, CDCl): 0.96 (m, 3H; CH3), 1.38 (m, 10H; CH2), 1.89 (m, 2H; CH2), 2.75 (s, 3H; (SO3)CH3), 4.06 (m, 3H; NCH3), 4.29 (m, 2H; NCH2), 7.60 (s, 1H; CH = CH), 7.70 (s, 1H; CH = CH), C-NMR: 13.65 (CH2CH), 19.63 (CHCH3), 32.35 (CHCH2CH3), 36.51 (CH3S), 40.05 (NCH3), 49.80 (NCH2CH2), 122.56 (NCH), 124.17 (NCH), 137.88 (NCHN).

1-Methyl-3-octyl-imidazolium Bis(trifluoromethane-sulfonyl) imide [C8mim][Tf2N]

The [C8mim][Br] (0.3 g, 1 mmol) and lithium bis(trifluoromethane-sulfonyl) imide (0.313 g, 1.1 mmol) were added in two necks round bottom flask containing 60 mL methanol and was evacuated to create an inert atmosphere. The reaction mixture was stirred at room temperature for 12 h (Scheme 1). The LiBr formed during the metathesis reaction was removed by solvent extraction followed by filtration. The solvent was evaporated to obtain the pure product (S1-03) and dried overnight under a vacuum at 40 °C. The yield of obtained product is ∼75%.

FTIR (cm): 3120 (CH str. aromatic), 2929 and 2854 (aliphatic CH str.), 1569 (C=C str.), 1463 (CH bending), 1358 (S=O asymmetric str.), 1180 (S=O symmetric str.), 1166 (C–N str.), and 739 (octyl CH bending).

H NMR (400 MHz, CDCl): 0.856–0.822 (m, 3H; CH3), 1.281–1.218 (m, 10H; CH2), 1.836 (m, 2H; CH2), 3.971–3.954 (m, 3H; NCH3), 4.173–4.136 (m, 2H; NCH2), 7.266 (s, 1H; CH = CH), 7.330 (s, 1H; CH = CH), 9.157 (s, 1H; N–CH–N). C-NMR: 13.65 (CH2CH), 19.63 (CHCH3), 32.35 (CHCH2CH3), 36.51 (CH3S), 40.05 (NCH3), 49.80 (NCH2CH2), 122.56 (NCH), 124.17 (NCH), 137.88 (NCHN).

1-Methyl-3-octylimidazolium dichloroacetate [C8mim][CHCl2CO2]

The [C8mim][Br] (0.5 g, 12 mmol) was added to 60 mL acetonitrile and mixed with sodium dichloroacetate (0.3 g, 2 mmol), followed by overnight stirring at room temperature (Scheme 1). The NaBr formed was removed by solvent extraction followed by filtration. The solvent was evaporated to obtain the pure product (S1-04) with a 96% yield.

FTIR (cm): 3120 (CH str. aromatic), 2929 and 2854 (aliphatic CH str.), 1644 (C=O str.), 1571 (C=C str.), 1463 (CH bending), 1358 (C–O str.), 1165 (C–N str.), 740 (Octyl CH bending).

H NMR (400 MHz, CDCl): 0.94 (m, 3H; CH3), 1.37 (m, 10H; CH2), 1.552 (1H, s CHCl2COO), 1.92 (m, 2H; CH2), 4.37 (m, 3H; NCH3), 4.07 (m, 2H; NCH2), 7.78 (s, 1H; CH = CH), 7.84 (s, 1H; CH = CH), 9.88 (s, 1H; N–CH–N). C-NMR: 13.66 (CH2CH), 19.13 (CHCH3), 31.84 (CHCH2CH3), 48.84 (NCH3), 36.31 (NCH2CH2), 67 (CHCl2COO) 123.86 (NCH), 122.67 (NCH), 136.94 (NCHN), 167 (CHCl2COO).

1-Methyl-3-octylimidazolium tetrafluoroborate [C8mim][BF4]

The [C8mim][Br] (0.75 g, 2.72 mmol) solution was slowly added into solution of NaBF4 (0.3 g, 2.7 mmol) in 60 mL acetonitrile. The reaction mixture was stirred overnight under an inert atmosphere at room temperature (Scheme 1). The NaBr formed was separated through filtration and solvent extraction. The solvent was evaporated with a rotary evaporator and dried under a vacuum to obtain the pure product (S1-05) with a 70% yield.

FTIR (cm): 3120 (CH str. aromatic), 2924 and 2854 (aliphatic CH str.), 1569 (C=C str.), 1463 (CH bending), 1165 (C–N str.), 1049 (BF4 anion), 752 (octyl CH bending).

H NMR (400 MHz, CDCl): 0.86 (m, 3H; CH3), 1.28 (m, 10H; CH2), 1.87 (m, 2H; CH2), 3.94 (m, 3H; NCH3), 4.17 (m, 2H; NCH2), 7.36 (s, 1H; CH = CH), 7.42 (s, 1H; CH = CH), 8.75 (s, 1H; N–CH–N). C-NMR: 13.65 (CH2CH), 19.63 (CHCH3), 32.35 (CHCH2CH3), 40.05 (NCH3), 49.80 (NCH2CH2), 122.56 (NCH), 124.17 (NCH), 137.88 (NCHN).

1-Methyl-3-octylimidazolium hydrogen sulfate [C8mim][HSO4]

The [C8mim][Br] (0.5 g, 1.8 mmol) solution was added slowly into the solution of potassium hydrogen sulfate (0.25 g, 1.8 mmol) in distilled water and stirred overnight at room temperature to obtain the product (S1-06) (Scheme 1). The solvent was rotary evaporated, and the KBr formed was separated through solvent extraction followed by filtration. The product was dried under a vacuum. The yield was 73%.

FTIR (cm): 3120 (CH str. aromatic), 2958 and 2854 (aliphatic CH str.), 1567 (C=C str.), 1463 (CH bending), 1165 (C–N str.), 1047 (S=O str.), 752 (octyl CH bending).

H NMR (400 MHz, CDCl): 0.74 (m, 3H; CH3), 1.12 (m, 10H; CH2), 1.68 (m, 2H; CH2), 1.87 (s, 1H; (SO4)H), 2.48 (m, 3H; NCH3), 4.13 (m, 2H; NCH2), 7.57 (s, 1H; CH = CH), 7.71 (s, 1H; CH = CH), 9.06 (s, 1H; N–CH–N). C-NMR: 13.65 (CH2CH), 19.63 (CHCH3), 32.35 (CHCH2CH3), 40.05(NCH3), 49.80 (NCH2CH2), 122.56 (NCH), 124.17 (NCH), 137.88 (NCHN).

Octyl pyridinium based ionic liquids

Octyl pyridinium bromide [C8py][Br]

The pyridine (5 g, 63 mmol) and octyl bromide (12.28 g, 63.2mmol) were added to 60 mL acetonitrile and refluxed for 48 h. After 48 h, the solvent was evaporated through the rotary evaporator to obtain the brown-colored crystalline product (S2-01). The product was dried in a vacuum oven at 50 °C for 12 h. The product obtained in 95% yield.

FTIR (cm): 3120 (CH str. aromatic), 2924 and 2855 (aliphatic CH str.), 1633 (C=N str.), 1569 (C=C str.), 1486 (CH bending), 1172 (C–N str.), 774 (octyl CH bending).

H NMR (400 MHz, CDCl): 0.85–0.91 (m, CH3), 1.28–1.32 (m, 10H; CH2), 1.90–1.99 (m, 2H; CH2), 4.88–4.99 (m, 2H; NCH2), 8.10–8.20 (m, 1H; CH pyr-m), 8.55–8.65 (m, 1H; CH pyr-p), 9.45–9.55 (m, 1H; CH pyr-o). C-NMR: 13.66 (CH2CH), 19.13 (CHCH3), 31.84 (CHCH2CH3), 36.31 (NCH2CH2), 128.82 (CH pyr-m), 144.70 (CH pyr-p), 146.35 (CH pyr-o).

Octyl pyridinium methanesulphonate [C8py][MeSO3]

The 40 mL solution of [C8py][Br] (0.57 g, 2 mmol) in methanol was slowly added to a 40 mL solution of sodium methanesulphonate (0.25 g, 2.1 mmol) and stirred overnight at room temperature to obtain the product (S2-03) (Scheme 2). The NaBr formed was removed by solvent extraction followed by filtration. The solvent was evaporated by a rotary evaporator followed by drying under a vacuum. The yield was 73%.

Scheme 2

Synthesis of pyridinium-based ILs.

FTIR (cm): 3120 (CH str. aromatic), 2924 and 2855 (aliphatic CH str.), 1633 (C=N str.), 1569 (C=C str.), 1487 (CH bending), 1174 (C–N str.), 1042 (S=O str.), 774 (octyl CH bending).

H NMR (400 MHz, CDCl): 0.85–0.91 (m, 3H; oct-CH3), 1.28–1.32 (m, 10H; CH2), 1.90–1.99 (m, 2H; CH2), 2.41 (s, 3H; CH3SO3), 4.88–4.99 (m, 2H; NCH2), 8.10–8.20 (m, 1H; CH pyr-p), 8.55–8.65 (m, 1H; CH pyr-m), 9.45–9.55 (m, 1H; CH pyr-o). C-NMR: 13.66 (CH2CH), 19.63 (CHCH3), 32.84 (CHCH2CH3), 36.31 (NCH2CH2), 36.51 (CH3S), 128.82 (CH pyr-m), 144.70 (CH pyr-p), 146.35 (CH pyr-o).

Octyl pyridinium Bis(trifluoromethane-sulfonyl)imide [C8py][Tf2N]

The 30 mL solution of [C8py][Br] (0.3 g, 1.1 mmol) in methanol was slowly added into evacuated 30 mL Li [Tf2N] (0.311 g 1.1 mmol) solution in methanol, followed by overnight stirring under an inert atmosphere (Scheme 2). The LiBr formed was removed by solvent extraction followed by filtration. The remaining solvents were evaporated by a rotary evaporator to obtain the pure product (S2-03). The product was dried overnight in a vacuum oven at 40 °C. The yield was 75%.

FTIR (cm): 3120 (CH str. aromatic), 2924 and 2855 (aliphatic CH str.), 1633 (C=N str.), 1569 (C=C str.), 1345 and 1052 (S=O symmetric and asymmetric str.), 1172 (C–N str.), 774 (octyl CH str.).

H NMR (400 MHz, CDCl): 0.863–0.817 (m, 3H; oct-CH3), 1.448–1.227 (m, 10H; CH2), 1.964–1.86 (m, 2H; CH2), 2.940 (s, 3H; CH3–C pyr-m), 4.905–4.853 (m, 2H; N–CH2), 7.886–7.860 (m, 1H; CH pyr-m), 7.984–7.939 (m, 1H; CH pyr-p), 8.365–8.313 (m, 1H CH pyr-m), 9.652–9.632 (m, 1H; CH pyr-o). C-NMR: 13.66 (CH2CH), 19.63 (CHCH3), 32.84 (CHCH2CH3), 36.31 (NCH2CH2), 36.51 (CH3S), 128.82 (CH pyr-m), 144.70 (CH pyr-p), 146.35 (CH pyr-o).

Octyl pyridinium dichloroacetate [C8py][CHCl2CO2]

The solution of [C8py][Br] (0.54 g, 1.9 mmol) was slowly added to a solution of sodium dichloroacetate (0.3 g, 2 mmol) in 60 mL acetonitrile followed by overnight stirring at room temperature (Scheme 2). The NaBr formed was separated by solvent extraction followed by filtration. The product (S2-04) formed is dried overnight in a vacuum oven. The yield was 90%.

FTIR (cm): 3120 (CH str. aromatic), 2925 and 2855 (aliphatic CH str.), 1633 (C=N str.), 1569 (C=C str.), 1486 (CH bending), 1368 (C–O), 1172 (C–N str.), 772 (octyl CH bending).

H NMR (400 MHz, CDCl): 0.94 (m, 3H; CH3), 1.37 (m, 10H; CH2), 1.552 (s, 1H; CHCl2COO), 1.92 (m, 2H; CH2), 7.863 (t, 1H; NCH2), 8.903 (m, 1H; CH pyr-m), 8.991 (m, 1H; CH pyr-p), 9.074 (m, 1H; CH pyr-o). C-NMR: 13.66 (CH2CH), 19.13 (CHCH3), 31.84 (CHCH2CH3), 36.31 (NCH2CH2), 67 (CHCl2COO), 128.82 (CH pyr-m), 144.70 (CH pyr-p), 146.35 (CH pyr-o), 167(CHCl2COO).

Octyl pyridinium tetrafluoroborate [C8py][BF4]

The 30 mL solution of [C8py][Br] (0.7 g, 2.5 mmol) was added into evacuated 30 mL solution of sodium tetrafluoroborate (0.3 g, 2.7 mmol) followed by overnight stirring under nitrogen atmosphere at room temperature (Scheme 2). The NaBr formed was separated by solvent extraction followed by filtration. The solvent was evaporated to obtain the product (S2-05) and dried overnight in a vacuum oven. The yield was 70%.

FTIR (cm): 3120 (CH str. aromatic), 2926 and 2855 (aliphatic CH str.), 1633 (C=N str.), 1569 (C=C str.), 1489 (CH bending), 1172 (C–N str.), 1033 (BF4 anion), 774 (octyl CH bending).

H NMR (400 MHz, CDCl): 0.804–0.770 (m, 3H; oct-CH3), 1.260–1.166 (m, 10H; CH2), 1.927–1.891 (m, 2H; CH2), 2.596 (s, 3H; CH3 pyr-p), 4.511–4.473 (m, 2H; N–CH2), 7.794–7.778 (m, 1H; CH pyr-m), 8.658–8.641 (2H, m, CHpyr-o). C-NMR: 13.66 (CH2CH), 19.63 (CHCH3), 32.84 (CHCH2CH3), 36.31 (NCH2CH2), 128.82 (CH pyr-m), 144.70 (CH pyr-p), 146.35 (CH pyr-o).

Octyl pyridinium hydrogen sulfate [C8py][HSO4]

The solution of [C8py][Br] (0.6g, 2.3 mmol) was slowly added to the solution of potassium hydrogen sulfate (0.3g, 2.2 mmol) and stirred overnight at room temperature (Scheme 2). The KBr formed during the anion metathesis reaction was separated by solvent extraction followed by filtration. The solvent was evaporated by a rotary evaporator, and the product was vacuum dried to obtain the product (S2-06) with ∼75% yield.

FTIR (cm): 3120 (CH str. aromatic), 2923 and 2854 (aliphatic CH str.), 1632 (C=N str.), 1569 (C=C str.), 1486 (CH bending), 1169 (C–N str.), 1046 (S=O str.), 774 (octyl CH bending).

H NMR (400 MHz, CDCl): 0.74 (m, 3H; CH3), 1.12 (m, 10H; CH2), 1.68 (m, 2H; CH2), 1.87 (s, 1H; (SO4)H), 2.48 (m, 3H; NCH3), 4.13 (m, 2H; NCH2), 7.57 (s, 1H; CH = CH), 7.71 (s, 1H; CH = CH), 9.06 (s, 1H; N–CH–N). C-NMR: 13.66 (CH2CH), 19.63 (CHCH3), 32.84 (CHCH2CH3), 36.31 (NCH2CH2), 128.82 (CH pyr-m), 144.70 (CH pyr-p), 146.35 (CH pyr-o).

Phosphonium based ionic liquids

Mono-butyl tri-octyl phosphonium bromide [P4888][Br]

The tri-octyl phosphine (5 g, 13.5 mmol, and 6.02 mL) was mixed with butyl bromide (1.85 g, 13.5 mmol) in a round bottom flask containing acetonitrile and evacuated to create an inert atmosphere and refluxed at 120 °C. When TLC confirmed the completion of the reaction, the solvents were removed by evaporation to obtain the product (S3-01) as a dark yellow oily liquid. It was dried in a vacuum oven at 50 °C for 12 h. The yield was 91%.

FTIR (cm): 2960 and 2854 (CH str.), 1463 (CH bending), 746 (octyl and butyl CH bending).

H NMR (400 MHz, CDCl): 0.913–0.942 (t, 3H; CH2CH), 1.395–1.499 (m, 2H; CHCH3), 2.152–2.211 (m, 2H; CHCH2CH3), 2.508–2.515 (t, 2H; P–CH). C-NMR: 13.685 (CH2CH), 17.633 (CHCH3), 23.093 (CHCH2CH3), 23.761 (P– CH).

Mono-butyl tri-octyl phosphonium methanesulphonate [P4888][MeSO3]

The solution of [P4888][Br] (1 g, 1.9 mmol) in 40 mL methanol was slowly added to the solution of sodium methanesulphonate (0.23 g, 1.9 mmol) in 40 mL methanol, followed by overnight stirring at room temperature to obtain the product (S3-02) (Scheme 3). After the completion of the reaction, the NaBr formed was removed by solvent extraction, and unreacted methanol was removed by evaporation. The product was then dried in a vacuum oven. The yield was 73%.

Scheme 3

Synthesis of phosphonium-based ILs.

FTIR (cm): 2920 and 2854 (CH str.), 1461 (CH bending), 1236 and 1161 (symmetric and asymmetric CH str.), 749 (octyl and butyl CH bending).

H NMR (400 MHz, CDCl): 0.913–0.942 (t, 3H; CH2CH), 1.395–1.499 (m, 2H; CHCH3), 2.152–2.211 (m, 2H; CHCH2CH3), 2.508–2.515 (t, 2H; P–CH). C-NMR: 13.685 (CH2CH), 17.633 (CHCH3), 23.093 (CHCH2CH3), 23.761 (P–CH), 36.51 (CH3S).

Mono-butyl tri-octyl phosphonium Bis(trifluoromethane-sulfonyl) imide [P4888][Tf2N]

The [P4888][Br] (1.0 g, 1.9 mmol) was dissolved in 30 mL methanol to form a solution. The Bis(trifluoromethane-sulfonyl) imide (0.5 g, 1.8 mmol) was added in two necks round bottom flask containing 30 mL methanol. Air in the flask was removed to create an inert atmosphere. The solution of [P4888][Br] was added to the evacuated flask, followed by overnight stirring under a nitrogen atmosphere at room temperature (Scheme 3). The LiBr formed was separated by solvent extraction followed by filtration. The rotary evaporator removed the excess solvent to obtain the product (S3-03) and further dried overnight in a vacuum oven at 40 °C. The yield was 83%.

FTIR (cm): 2929 and 2854 (CH str.), 1463 (CH bending), 1347 and 1056 (S=O str.), 746 (octyl and butyl CH bending).

H NMR (400 MHz, CDCl): 0.913–0.942 (t, 3H; CH2CH), 1.395–1.499 (m, 2H; CHCH3), 2.152–2.211 (m, 2H; CHCH2CH3), 2.508–2.515 (t, 2H; P–CH). C-NMR: 13.685 (CH2CH), 17.633 (CHCH3), 23.093 (CHCH2CH3), 23.761 (P–CH), 36.51 (CH3S).

Mono-butyl tri-octyl phosphonium dichloroacetate [P4888][CHCl2CO2]

The sodium dichloroacetate (0.3 g, 2 mmol) solution was added into an evacuated solution of [P4888][Br] (1 g, 1.9 mmol) in 80 mL acetonitrile which was followed by overnight stirring under an inert atmosphere at room temperature (Scheme 3). The NaBr formed was filtered, and the solvent was evaporated. Pure product (S3-04) was obtained by solvent extraction and filtration followed by overnight drying in a vacuum oven. The yield was 85%.

FTIR (cm): 2923 and 2854 (CH str.), 1463 (CH bending), 1651 (C=O str.), 1376 (C–O str.), 711 (octyl and butyl CH bending).

H NMR (400 MHz, CDCl): 0.913–0.942 (t, 3H; CH2CH), 1.395–1.499 (m, 2H; CHCH3), 2.152–2.211 (m, 2H; CHCH2CH3), 2.508–2.515 (t, 2H; P–CH). C-NMR: 13.685 (CH2CH), 17.633 (CHCH3), 23.093 (CHCH2CH3), 23.761 (P–CH), 67 (CHCl2COO), 167(CHCl2COO).

Mono-butyl tri-octyl phosphonium tetrafluoroborate [P4888][BF4]

The [P4888][Br] (1 g, 1.9 mmol) was dissolved in 30 mL acetonitrile to form a solution. The sodium tetrafluoroborate (0.2 g, 1.8 mmol) was also dissolved in 30 mL acetonitrile in a round bottom flask and evacuated to remove excess air. The [P4888][Br] solution was added to evacuated sodium tetrafluoroborate solution, followed by overnight stirring under a nitrogen atmosphere at room temperature (Scheme 3). The NaBr formed was separated by filtration, and excess solvent was evaporated. The product (S3-05) was obtained by overnight drying in a vacuum oven. The yield was 70%.

FTIR (cm): 2924 and 2855 (CH str.), 1463 (CH bending), 1049 (BF4 ion), 751 (octyl and butyl CH bending).

H NMR (400 MHz, CDCl): 0.913–0.942 (t, 3H; CH2CH), 1.395–1.499 (m, 2H; CHCH3), 2.152–2.211 (m, 2H; CHCH2CH3), 2.508–2.515 (t, 2H; P–CH). C-NMR: 13.685 (CH2CH), 17.633 (CHCH3), 23.093 (CHCH2CH3), 23.761 (P–CH).

Mono-butyl tri-octyl phosphonium hydrogen sulfate [P4888][HSO4]

The [P4888][Br] (1 g, 1.9 mmol) was dissolved in 30 mL distilled water to form solution A. The potassium hydrogen sulfate (0.2 g, 1.4 mmol) was dissolved in 30 mL of distilled water to obtain solution B. Both solutions A and B, were mixed and kept on stirring for 12 h (Scheme 3). The KBr formed was removed by solvent extraction using chloroform followed by filtration. The pure product (S3-06) is obtained by overnight drying in a vacuum oven. The yield was 71%.

FTIR (cm): 2923 and 2854 (CH str.), 1460 (CH bending), 1236 and 1161 (S=O str.), 719 (octyl and butyl CH str.).

H NMR (400 MHz, CDCl): 0.913–0.942 (t, 3H; CH2CH), 1.395–1.499 (m, 2H; CHCH3), 2.152–2.211 (m, 2H; CHCH2CH3), 2.508–2.515 (t, 2H; P–CH). C-NMR: 13.685 (CH2CH), 17.633 (CHCH3), 23.093 (CHCH2CH3), 23.761 (P–CH).

Antibacterial activity

The samples of equal density and potency were prepared using distilled water. Each sample was tested in triplicate. Eighteen samples were used to check their antibacterial activity against pathogenic bacteria. New bacterial culture of 24 h was used to make the lawn in Petri dishes. About 100 μl of each equalized bacterial sample was spread on Petri dishes. Using a sterilized borer, 3 mm wells were made at equal distances. Positive and negative controls were used in equal v/v ratio on every plate. About 25 μl of sample solutions were poured into each well and then incubated for 24 h at 37 °C. The distilled water was used as negative control and levofloxacin as the positive control [43].

Computational studies

Density functional theory studies

All the ILs were modeled, and their geometry optimizations were performed at the B3LYP/6-31g level of Density Functional Theory methods [44] using the Gaussian 09 program. The geometry optimization was performed in the vacuum keeping the overall charge zero for all the ILs. For validation purposes, frequency calculations were performed on the optimized geometries using the same level of the DFT method used for optimization. As the application of these ILs was to act as inhibitors for the protein, single-point energy on all the optimized geometries was calculated in the aqueous medium with the same method and basis set enhanced by polarization functions for all atoms except hydrogen.

Molecular docking studies

Molecular docking was performed to understand the binding affinity between the target protein, β-lactamase, and the designed ILs [45, 46, 47, 48]. As the β-lactamase enzymes are involved in the antibiotic resistance by hydrolyzing the peptide bond of the β-lactam ring, therefore the β-lactamase enzymes of the six gram-positive and gram-negative bacteria; E. coli (PDB ID: 5A92) [49] E. aerogenes (PDB ID: 5KID) [50], K. pneumonia (PDB ID: 6MGX) [51], P. vulgaris (PDB ID: 1HZO), P. aeruginosa (PDB ID: 4GZB) [52], and S. pneumoniae (PDB ID: 1RPS) [53] are used for molecular docking. For docking purposes, optimized geometry of all the ILs geometries was used. Molecular docking studies were performed using PyRx software where the optimized ILs and the proteins (PDB structure) were loaded, and molecular docking was performed after the selection of the predicted protein chains. Before docking, all the protein structure's energy was minimised where protonation and water molecules were removed. Default settings were maintained for all parameters during the docking process. As a result of docking, nine different conformations were generated for every pair of protein and ligand. The conformation with the highest Binding Affinity was selected for evaluation.

Result and discussion

ILs based on imidazolium, pyridinium, and phosphonium with different anions (bromide, hydrogen sulfate, dichloroacetate, bis(trifluoromethane sulfonyl)imide, tetrafluoroborate, and methanesulphonate, etc.) were prepared and characterized with the help of characterization techniques (TLC, ATR-FTIR, NMR, etc.) at different levels during and after the synthesis.

Imidazolium-based ionic liquids

The Methyl imidazole (1) was reacted with octyl bromide under reflux for 48 h in acetonitrile as a solvent to give S1-01. 1H-NMR confirmed the successful synthesis of S1-01 as the peaks for aromatic proton (NCHN) shifts from 7.385 ppm in (1) to 9.53 ppm in product S1-01. The quaternization of (1) is confirmed by shifting the –CH2 peak from 3.35 ppm in octyl bromide to 3.87–3.90 ppm in product S1-01. FTIR of S1-01 indicates quaternarization of methyl imidazole with octyl bromide. The following peaks were observed in FTIR of S1-01; 3120 cm−1 (aromatic CH stretch), 2924 cm−1, and 2854 cm−1 (aliphatic CH stretch) for CH2 and CH3, respectively. 1650 cm−1 (C=N stretch), 1569 cm−1 (C=C stretch), 1463 cm−1 (CH2 bend), 1165 cm−1 (C–N stretch), 752 cm−1 (long-chain CH2 of octyl group).

FTIR indicated the formation of S1-02 due to band appearance at 1040 cm−1 responsible for S=O stretch. This characteristic band appears due to the exchange of anions. The formation of S1-03 was confirmed by FTIR due to the appearance of peaks at 1358 cm−1, indicating asymmetric stretching vibration of S=O and at 1180 cm−1 symmetric stretching vibration of S=O. The C=O and C–O stretching bands at 1644 cm−1 and 1358 cm−1, respectively, indicate the formation of S1-04. The formation of S1-05 was confirmed by the presence strong band at 1049 cm−1 due to [BF4] anion. A peek at 1047 cm−1 due to S=O shows the formation of S1-06.

Pyridinium based ionic liquids

The pyridine (2) was reacted with octyl bromide under reflux for 48 h to give S2-01 as the product (Scheme 2). 1H-NMR confirmed the formation of S2-01 as the peaks for aromatic protons (Pyr-o/Pyr-m/Pyr-p) shifts downfield from 8.613/7.277/7.65 ppm in (2) to 9.45–9.55/8.1–8.2/8.55–8.65 ppm in product S2-01. The peak due to the –CH2 group of octyl bromide also shifts from 3.35 ppm to 4.88–4.99 ppm after quaternization of (2). FTIR also confirmed the quaternization of (2) with octyl bromide. Following bands are appeared in FTIR of S2-01; 3120 cm−1 (aromatic CH stretch), 2924 cm−1 and 2855 cm−1 (aliphatic CH stretch) for CH2 and CH3 respectively, 1633 cm−1 (C=N stretch), 1569 cm−1 (C=C stretch), 1486 cm−1 (CH2 bend), 1172 cm−1 (C–N stretch), 774 cm−1 (long-chain CH2 of octyl group).

FTIR for S2-02 shows S=O stretching vibration at 1042 cm−1, indicating the exchange of methane sulphonate anion. In FTIR for S2-03, the bands at 1345 cm−1 and 1052 cm−1 are due to S=O stretching. The C=O and C–O stretching bands appear at 1633 cm−1 and 1368cm−1, respectively, indicating the formation of S2-04. The formation of S2-05 was confirmed by the presence of a strong band at 1033 cm−1 due to [BF4] anion. The peak at 1046 cm−1 appears due to S=O stretching, indicating S2-06 formation.

Phosphonium based ionic liquids

The tri-octyl phosphine (3) was reacted with butyl bromide under an inert atmosphere at 120 °C for 48 h to give S3-01 as the product. 1H-NMR confirmed the quaternization of (3) as the –CH2 peak shifts from 3.35 ppm in butyl bromide to 2.508–2.525 ppm in product S3-01. FTIR of S3-01 also confirms the quaternarization of tri-octyl phosphine with butyl bromide. Following bands appeared in FTIR of S3-01; 2960 cm−1 and 2854 cm−1 (CH stretch), 1463 cm−1 (CH2 bend), and 746 cm−1 (long-chain CH2 in butyl and octyl groups attached to phosphorous).

The S=O stretching vibration at 1347 cm−1 and 1056 cm−1 in FTIR of S3-03 confirms its formation by replacing bromide anion. The C=O and C–O stretching vibration at 1651 cm−1 and 1376 cm−1, respectively, indicate the formation of S3-04 by replacing dichloroacetate with bromide anion. The formation of S3-05 was confirmed by the presence of a band at 1049 cm−1 due to [BF4] anion. The S=O stretching vibrations at 1236 cm−1 and 1161 cm−1 indicate the formation of S3-06.

Compared to other living organisms, the short generation time of bacteria had indirectly led the researchers to investigate the antibacterial activity of ILs [54]. The toxicity of pyridinium, phosphonium, and imidazolium-based ILs increases with alkyl chain length in their quaternary ammonium salts [55, 56]. In this study, 18 different ILs were synthesized to check their potential toxicity and effectiveness as antibacterial agents against gram-positive and gram-negative bacterial strains like E. aerogenes, P. vulgaris, K. pneumoniae, P. aeruginosa, E. coli, and S. pyogenes.

The antibacterial activity of these synthesized ILs was evaluated by the diameter of the inhibition zone on agar plates. This zone is defined as an area on the plate where the growth of the bacteria was prevented.

The ILs based on octyl-imidazolium showed the highest antibacterial activity against all strains of the bacteria; some ILs were even more effective than positive control levofloxacin. Table 1 and Figure 1 show that the ILs based on octyl imidazolium were effective against all bacterial strains. The IL S1-02 showed the highest inhibition values of 27 mm for E. aerogenes and 24 mm against P. aeruginosa, while IL S1-06 showed no inhibition against S. pneumoniae. The ILs based on quaternary salts of pyridinium showed antibacterial activity against E. aerogenes and K. pneumoniae. It is clear from Figure 2 and Table 2 that ILs S2-04 and S2-05 show the highest inhibition against K. pneumoniae with an IZ value of 20 mm Table 3 and Figure 3 show the bacterial inhibition for ILs based on quaternary salts of phosphonium with the highest IZ value of 14 mm against E. aerogenes. All phosphonium-based ILs also showed effectiveness against E. coli. Overall, all 18 synthesized ILs showed inhibition effectiveness, but ILs based on imidazolium are more effective than others. The order of effectiveness is as follows: ILs (Imidazolium) > ILs (phosphonium) ∼ ILs (Pyridinium).

Table 1

Antibacterial activity of octyl imidazolium-based ILs.

ILs	Bacterial Strains
	E. coli	E. aerogenes	K. pneumoniae	P. vulgaris	P. aeruginosa	S. pneumoniae	S. pyogenes
- control	0	0	0	0	0	0	0
+ control	17	27	12	25	27	25	30
S1-01	21	25	19	20	25	22	14
S1-02	21	27	20	18	24	17	13
S1-03	15	17	17	10	17	20	20
S1-04	19	17	16	17	20	23	15
S1-05	18	22	15	19	15	17	20
S1-06	12	17	17	17	11	0	20

Figure 1

Antibacterial activity of imidazolium-based ILs.

Figure 2

Antibacterial activity of pyridinium-based ILs.

Table 2

Anti-bacterial activity of octyl pyridinium-based ILs.

ILs	Bacterial Strains
	E. aerogenes	K. pneumoniae
S2-01	0	0
S2-02	16	14
S2-03	13	16
S2-04	20	15
S2-05	20	18
S2-06	18	14

Table 3

Anti-bacterial activities of Phosphonium-based ILs.

ILs	Bacterial Strains
	E. coli	E. aerogenes
S3-01	0	9
S3-02	12	14
S3-03	9	9
S3-04	11	13
S3-05	10	14
S3-06	10	13

Figure 3

Antibacterial activity of phosphonium-based ILs.

In-silico antibacterial activity

The antibacterial activity can also be analyzed using in-silico methods like Molecular Docking studies. The strong binding energy of ligand (drug or inhibitors) with the protein shows the maximum inhibition or higher antibacterial activity. Therefore, in this research, molecular docking studies were performed on all the 18 ionic liquids with the β-Lactamase protein of six bacteria; E. coli (PDB ID: 5A92), E. aerogenes (PDB ID: 5KID), K. pneumonia (PDB ID: 6MGX), P. vulgaris (PDB ID: 1HZO), P. aeruginosa (PDB ID: 4GZB), and S. pneumoniae (PDB ID: 1RPS). Details of the docking protocol are mentioned in the Computational details; however, docking results are presented in Table 4.

Table 4

Molecular docking scores of Binding affinity in Kcal/mol (RMSD values).

IONIC LIQUIDS			BACTERIAL STRAINS
Cations	Anions	Label	E. coli	E. aerogenes	K. pneumoniae	P. vulgaris	P. aeruginosa	S. pneumoniae
Imidazolium	Br	S1-01	-4 (5.14)	-4.6 (2.08)	-4.5 (34.34)	-4.5 (3.84)	-4.7 (2.54)	-4.3 (45.4)
	[CH3SO3]	S1-02	-4.9 (4.2)	-5 (4.38)	-5.1 (2.42)	-4.8 (4.47)	-4.8 (7.11)	-5.2 (25.63)
	[Tf2N]	S1-03	-6.6 (2.06)	-7.7 (2.70)	-6.3 (3.78)	-6.8 (5.73)	-7.7 (2.88)	-7.7 (5.46)
	[CHCl2CO2]	S1-04	-4.9 (2.64)	-5.3 (2.91)	-4.9 (2.58)	-4.7 (6.94)	-5.0 (5.23)	-4.7 (2.99)
	[BF4]	S1-05	-4.8 (3.14)	-3.5 (2.09)	-3.2 (2.09)	-3.2 (2.10)	-3.2 (2.11)	-4.0 (2.09)
	[HSO4]	S1-06	-5.5 (6.99)	-5.4 (2.36)	-5.3 (3.81)	-6.4 (2.01)	-6 (6.67)	-6 (4.56)
Pyridinium	Br	S2-01	-4.6 (3.28)	-5.0 (2.73)	-4.6 (3.25)	-4.5 (3.48)	-4.9 (6.06)	-5.3 (6.64)
	[CH3SO3]	S2-02	-4.9 (5.43)	-5.8 (6.49)	-5.2 (3.50)	-4.7 (3.40)	-5.8 (4.76)	-5.7 (5.75)
	[Tf2N]	S2-03	-7.1 (4.73)	-7.3 (3.99)	-6.4 (5.25)	-6.7 (2.12)	-7.5 (2.07)	-8.4 (2.23)
	[CHCl2CO2]	S2-04	-5.6 (2.73)	-5.7 (2.14)	-5.0 (2.49)	-5.0 (5.56)	-5.3 (6.17)	-6.1 (2.29)
	[BF4]	S2-05	-3.5 (1.84)	-3.5 (2.11)	-3.3 (2.10)	-3.2 (2.26)	-3.2 (1.82)	-3.7 (1.82)
	[HSO4]	S2-06	-5.2 (2.31)	-5.6 (4.37)	-5.2 (18.75)	-5.1 (6.39)	-6.1 (6.37)	-6.2 (2.71)
Phosphonium	Br	S3-01	-3.9 (7.59)	-4.9 (2.03)	-4.1 (3.76)	-3.9 (5.07)	-4.4 (4.15)	-4.9 (5.41)
	[CH3SO3]	S3-02	-4.5 (6.40)	-4.8 (2.01)	-4.5 (5.94)	-4.1 (5.41)	-4.5 (3.41)	-5.9 (2.64)
	[Tf2N]	S3-03	-5.2 (3.75)	-6 (3.30)	-4.9 (3.75)	-5.1 (8.95)	-5.7 (3.45)	-5.1 (4.81)
	[CHCl2CO2]	S3-04	-4.5 (31.75)	-4.9 (2.82)	-4.2 (6.173)	-4.6 (7.37)	-4.7 (5.54)	-4.5 (6.26)
	[BF4]	S3-05	-3.5 (2.10)	-3.5 (2.10)	-3.3 (19.09)	-3.2 (2.44)	-3.2 (1.83)	-3.7 (2.60)
	[HSO4]	S3-06	-5 (5.40)	-6.1 (7.29)	-4.9 (2.032)	-5.1 (3.01)	-5.7 (5.61)	-4.6 (6.87)

Molecular docking results for the imidazolium-based ionic liquids show that the IL S1-01 shows potent inhibition with all the gram-positive and gram-negative bacteria's ranging binding affinity values from -6.3 to -7.7 kcal/mol. All the binding pockets and their interactions are presented in Table 5 and Figure 4.

Table 5

Molecular docking results: important interactions for the high binding affinity models.

IONIC LIQUIDS			BACTERIAL STRAINS
Cations	Anions	Label	E. coli	E. aerogenes	K. pneumoniae	P. vulgaris	P. aeruginosa	S. pneumoniae
Imidazolium	[Tf2N]	S1-03	Lys-73	Met-217, Phe-122	SerB-217, Gly-B219, Lys-B211	Thr-216, Asn-132	Ser-64, Ser-319	Arg-652, Val-662
Pyridinium	[Tf2N]	S2-03	Ser-237	Thr-316, Asn-340	His-A189, His-B189, Asn-A220, Asn-B220,His-A250, His-B250	Ser-130, Ser-237	Tyr-151, Asn-347	Lys-420, Arg-654
Phosphonium	[CH3SO3]	S3-02	-	-	-	-	-	Gly-664, Pro-697
	[Tf2N]	S3-03	Ser-130, Ser-237	-	Lys-211, His-260	Lys-147	Ser-319, Asn-344, Asn-347	-
	[HSO4]	S3-06	-	Ser-65, Tyr-151, Ser-315	-	-	-	-

Figure 4

Molecular docking results of imidazolium-based IL S1-03 with bacteria.

The proposed reaction mechanism for inhibiting β-lactamase with IL S1-03 is presented in Figure 5, showing the maximum inhibition capacity. It has been observed in Figure 4 (S1-03-ILP3) that Ser and Lys are present at the binding sight showing maximum interaction. According to the proposed reaction mechanism, the simultaneous transfer of protons occurs (Figure 5), resulting in the formation of positively charged Lys and direct bonding of Ser with the anion part of ionic liquid.

Figure 5

Proposed reaction mechanism for β-lactamase inhibition with S1-03.

On comparing the docking results for the pyridinium-based ILs, the same behavior was observed as for imidazolium-based ILs. The IL S2-03 shows the maximum inhibition with all the six bacteria with the binding affinity values ranging from -6.4 to -8.4 (Figure 6).

Figure 6

Molecular docking results of pyridinium-based ILs S2-03 with bacteria.

On the other hand, when the molecular docking results for phosphonium-based ionic liquids were analyzed, slightly different behavior was observed; the IL S3-03 shows maximum inhibition for E. coli, K. pneumonia, P. vulgaris, and P. aeruginosa, however, S3-06 shows maximum inhibition for E. aerogenes and S3-02 shows maximum inhibition for S. pneumonia (Figure 7).

Figure 7

Molecular docking results of phosphonium-based ILs with bacteria.

Analyzing the molecular docking results, it was also observed that the anionic part of imidazolium and pyridinium-based ILs interacts with the amino acids present at the active site; however, in the case of phosphonium based ILs, the cationic part of ILs shows interaction at the binding site. On comparing all the molecular docking results of the three schemes of imidazolium, pyridinium, and phosphonium-based ILs, it was observed that the pyridinium-based ILs would show strong binding affinity values and has maximum inhibition capacity relative to the other two schemes of ILs.

Conclusions

In brief, 18 ILs based on octyl pyridinium, octyl imidazolium, and mono-butyl-tri-octyl phosphonium cations and anions like bromide, sodium methane sulphonate, lithium bis(trifluoro-methane-sulfonyl)imide, sodium dichloroacetate, and sodium tetrafluoroborate, and potassium hydrogen sulfate were prepared as effective antibacterial agents. All the synthesized ILs were characterized by FTIR and NMR. The antibacterial activity of these ILs was tested by the agar well diffusion method against both gram-positive and gram-negative bacteria like E. aerogenes, P. vulgaris, K. pneumoniae, P. aeruginosa, E. coli, and S. pyogenes.

The ILs based on imidazolium showed the highest antibacterial activity against all tested bacteria and are more antibacterial than positive control levofloxacin. The IL S1-02 showed the highest inhibition with an IZ value of 27 mm for E. aerogenes and 24 mm against P. aeruginosa, while IL S1-06 showed no inhibition against S. pneumoniae. ILs based on pyridinium showed activity against E. aerogenes and K. pneumoniae, with ILs S2-04 and S2-05 showing the highest inhibition against K. pneumoniae with an IZ value of 20 mm. In contrast, the ILs based on phosphonium showed activity against E. coli and E. aerogenes, with the highest IZ value of 14 mm against E. aerogenes. The order of effectiveness is as follows: ILs (Imidazolium) > ILs (phosphonium) ∼ ILs (Pyridinium). The molecular docking results show that IL S1-03 shows potent inhibition with all bacteria with binding affinity values ranging from -6.3 to -7.7 kcal mol−1. From pyridinium-based ILs, the IL S2-03 shows the maximum inhibition with all the six bacteria with the binding affinity values ranging from -6.4 to -8.4 kcal mol−1. While in the case of phosphonium-based ILs, the IL S3-03 shows maximum inhibition for E. coli, K. pneumonia, P. vulgaris, and P. aeruginosa, however, S3-06 shows maximum inhibition for E. aerogenes and S3-02 shows maximum inhibition for S. pneumonia.

Declarations

Author contribution statement

Rabia Hassan: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Muhammad Asad Asghar, Arshemah Qaisar: Contributed reagents, materials, analysis tools or data; Wrote the paper.

Mudassir Iqbal: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Uzma Habib: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Bashir Ahmad: Performed the experiments; Analyzed and interpreted the data.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors and proceed further with the article.

Data availability statement

Data included in article/supplementary material/referenced in article.

Declaration of interests statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.