Synergistic antimicrobial activity of N-methyl substituted pyrrolidinium–based ionic liquids and melittin against Gram-positive and Gram-negative bacteria

Article abstract of DOI:10.1007/s00253-020-10989-y

Abstract: In pharmaceutical industry, the prodrug approaches and drug-drug conjugates are being now vastly used to optimize the efficacy of the drugs for multipurpose. The combination or conjugation of antimicrobials agents with natural antimicrobials may

Full text of DOI:10.1007/s00253-020-10989-y

Applied Microbiology and Biotechnology
https://doi.org/10.1007/s00253-020-10989-y
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Synergistic antimicrobial activity of N-methyl substituted
pyrrolidinium–based ionic liquids and melittin
against Gram-positive and Gram-negative bacteria
Juhi Saraswat1 & Badr Aldahmash2 & Suliman Yousef AlOmar3 & Khalid Imtiyaz4 & M. Moshahid Alam Rizvi4
&
Rajan Patel1
Received: 7 July 2020 /Revised: 13 October 2020 /Accepted: 28 October 2020
#
Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract
In pharmaceutical industry, the prodrug approaches and drug-drug conjugates are being now vastly used to optimize the efficacy
of the drugs for multipurpose. The combination or conjugation of antimicrobials agents with natural antimicrobials may lead to
better synergistic antimicrobial activity. Currently, many publications show the potential of ionic liquids (ILs) as novel antimi-
crobials and even as active pharmaceutical ingredients. The current study showed the synthesis of novel pyrrolidinium-based ILs
(Cx, x = 4, 6, 8, 10, 12) and their antibacterial activity alone and in combination with antimicrobial peptide, melittin (MEL),
against clinically relevant microorganism, E. coli and S. aureus. The cytotoxicity of synthesized ILs was administered on HEK
293 cell line using MTT assay. The obtained results showed the dependency of antibacterial activity of ILs on alkyl chain length
(C4 < C6 < C8 < C10 < C12). The remarkable improvement in the antibacterial efficiency of MEL was seen with ILs; however,
antibacterial effect is more pronounced with IL having large alkyl chain length (C8, C10, and C12) at their minimal concentration
with MEL to disrupt the cell membrane. In addition, the binding study and haemocompatibility results showed favourable
biocompatibility and stability which could potentially improve its utility for the biomedical field.
Key points
• The combination of melittin and pyrrolidinium-based ILs showed improved antibacterial activity against E. coli and S. aureus
which may be used for developing new antibacterial agents.
• Moreover, the cytotoxicity and haemocompatibility results showed excellent biocompatibility of the combinations on human
cell line and human serum albumin, respectively, which could potentially improve its utility for the biomedical field.
.
.
.
.
Keywords Ionic liquid Melittin Antibacterial activity MTT assay Haemocompatibility
Introduction
Supplementary Information The online version contains
supplementary material available at https://doi.org/10.1007/s00253-020-
10989-y.
The World Health Organization (WHO) report published in
2019 calls for urgent action to avert the antimicrobial resis-
tance crisis. About 700,000 deaths due to drug-resistance dis-
eases take place each year as per the recent WHO report
(Organization WH 2019). Bacterial resistance to multiple con-
ventional antimicrobial agents has become a significant global
public health threat. Rising antibiotic resistance is seriously
threatening the vast medical advancements over the past 70
years (Chemical 2014; Payne 2008; Spellberg et al. 2013).
Bacteria acquire innate or intrinsic resistance to conventional
drugs due to various biological or climatic changes in the
environment (Khameneh et al. 2016). Besides, exponential
growth in the bacterial resistance towards antibiotics is due
* Rajan Patel
rpatel@jmi.ac.in
1
Biophysical Chemistry Laboratory, Centre for Interdisciplinary
Research in Basic Sciences, Jamia Millia Islamia, New
Delhi 110025, India
2
3
4
Department of Zoology, College of Science, King Saud University,
P.O. Box 2455, Riyadh 11451, Saudi Arabia
Doping Research Chair, Department of Zoology, College of Science,
King Saud University, Riyadh 11451, Kingdom of Saudi Arabia
Department of Bioscience, Jamia Millia Islamia, New Delhi 110025,
India

Appl Microbiol Biotechnol
to the intensive cliche of antibiotics in human medication or an
environment comprising agriculture and aquaculture (Hughes
and Karlén 2014). Bacterial resistance against conventional
antibiotics in hospitals suppresses the immunity of the patients
resulting in ineffective treatment of infections (Nascimento
et al. 2000; WHO AR 2014). Hence, there is an urgent need
for an effective alternative to the antibacterial drug.
toxicity features which limit their use in pharmaceutical indus-
tries. Therefore, we have synthesized pyrrole-based ILs as
previously reported pyrrolidinium-based ILs to possess lesser
toxicity as compared to imidazolium, ammonium-based ILs
(Qin et al. 2017). ILs possessing versatile characteristic such
as low vapour pressure, low melting point, and tuneable na-
ture have gained the interest of researchers to use them in
various fields such as drug delivery (Wani et al. 2019), organic
solvent, biomedical field, and pharmaceutical industry
(Maurya et al. 2019).
Previously, we have synthesized pyrrolidinium-based ILs
containing bis(trifluoromethane)sulfonyl imide, ((CF3SO2)2N− )
as a counter ion and N-alkyl substituted pyrrole ring as a cation
moiety. The study showed a remarkable improvement in the
antibacterial activity of melittin in the presence of ILs containing
longer chain length (Saraswat et al. 2020). Therefore, in search of
a better, cost-effective, and efficient therapeutic agent, we fo-
cused our study to further find some more combinations of anti-
bacterial activity of MEL with pyrrolidinium-based ILs which
may furnish us with antibacterial agents that might show the best
antibacterial activity. Hence, we have synthesized a range of
pyrrolidinium-based ILs having N-alkyl substituted pyrrole ring
as a cation moiety and Br- as a counter ion and accessed the effect
of ILs on the antibacterial activity of MEL against a couple of
microorganisms, E. coli and S. Aureus, thus creating a more
effective approach for the treatment of multidrug resistance. To
the best of our knowledge, the present combination has not been
used earlier as a potential candidate for the treatment of bacterial
resistance.
Antimicrobial peptides (AMPs) because of their promising
and effective antibacterial property against a broad-spectrum
microorganism represent promising alternative therapeutic
agents over the conventional antibiotics (Regmi et al. 2017).
In general, AMPs are known for targeting cell membranes,
and the tendency to disrupt the bacterial membrane contrib-
utes towards the antimicrobial activity of AMPs. Previously,
several studies showed that the combination of the short and
synthetic cationic peptide with conventional antibacterial
drugs transcends the antibacterial activity which helps to com-
bat the evolution of resistance in bacteria (Maisetta et al. 2009)
(Regmi et al. 2017). On the other hand, the combinations of
the peptide with antibiotics minimize the noxious effect in the
course of the medical procedure involved in the treatment of
severe infections caused by microorganisms. Various groups
of researchers showed the combined effect of other peptides
and drugs on the bacterium and concluded with the same
results (Khara et al. 2014). A collaborative effect of peptides
and conventional antibacterial drugs may conceivably amelio-
rate the efficacy of antibacterial agents against several bacte-
rial infections which may intercept the evolution of new resis-
tance against peptide and antibacterial drugs (Akbari et al.
2019). The unique feature of AMPs containing tryptophane
(Trp) as one of its residues helps them to bind with the cell
membrane via various intermolecular interactions such as hy-
drogen bond and electrostatic interactions, with the interfacial
region of the cell membrane showing more antimicrobial po-
tency of AMP (Khawam and Flanagan 2006). Trp residue in
AMP displays indestructible membrane-disruptive activity,
and this feature provides Trp-containing peptides with the
unique capability to link with the interfacial region of the cell
membranes for their good antibacterial activity as compared to
AMPs without Trp residue (Khawam and Flanagan 2006).
Therefore, as Trp-containing AMPs show enhanced anti-
bacterial activity, here, we have chosen a cationic peptide,
melittin (MEL), as a cationic short peptide that has one Trp
(amino acid) as an antibiotic. MEL isolated from bee venom
displays strong lytic activity against prokaryotic and eukary-
otic cells. The action mechanism of MEL involves the pore
formation into cells membrane through permeabilization
which leads to leakage of cytoplasmic content and finally cell
death (Akbari et al. 2019). The second antibiotic we selected
was pyrrolidinium-based ionic liquids (ILs) (Zhang et al.
2016a),(Qin et al. 2017). Recently, ionic liquids (ILs) emerge
as good antimicrobials agents (Garcia et al. 2013). Despite
having wonderful characteristic features, ILs possess some
Materials and methods
Materials
Melittin (purity ≥ 65%), pyrene (purity ≥ 99%), resazurin, and
Luria Bertani Broth (LBB) were obtained from Sigma Aldrich,
USA. 1-Methylpyrrolidine, bromoalkanes (1-bromobutane, 1-
bromohexane, 1-bromooctane, 1-bromodecane, 1-
bromododecane), ethyl acetate, ethanol, CDCl3, and acetone of
analytical grade were used and were purchased from MERK.
Quality control strains, Escherichia coli (MTCC 40), and
Staphylococcus aureus (MTCC 87) were purchased from the
microbial-type culture collection and gene bank (MTCC),
CSIR-Institute of Microbial Technology, Chandigarh, India.
Milli-Q water was used throughout the experiments.
Bacterial strains
LBB media was employed for a culture of microorganisms,
E. coli and S. aureus. The culture was freshly grown at 37o C
up to the log phase under an aerobic condition for 16–18 h.
The concentration of the bacterial inoculums was determined

Appl Microbiol Biotechnol
by measuring the optical density (OD600) using an
Analytikjena 210 (Germany) spectrophotometer. The culture
of OD600 = 0.5 for each microorganism was used to determine
the antibacterial efficacy of the synthesized ILs and their effect
on the antibacterial property of melittin (an antimicrobial pep-
tide). The final working concentration of the bacterial inocu-
lum was adjusted to 106–105 CFU/mL.
(FT-IR) spectra were recorded on Bruker tensor 37 FT-IR
spectrophotometer in the IR range of 400–4000 cm-1 and mass
spectra for each synthesized IL were recorded on LC-MS/MS
(water), Xevo TDQ, ACQUITY UPLC-H PLUS in the range
of 150–700 m/e.
Determination of surface-active parameters of ILs
Preparation of testing reagent
The surface-active behaviour of newly synthesized
pyrrolidinium-based ILs were determined using surface tensi-
ometry and pyrene probe fluorescence spectroscopy (the
detailed methodology is given in supplementary material).
Surface tension measurement was performed as described pre-
viously (Saraswat et al. 2020; Wani et al. 2019).
Figure S22(a–e) shows the plot between γ values of each IL
against the concentration where inflexion point in the graph
signifies the CMC value (Behera and Pandey 2007; Bhat et al.
Resazurin (dye) was used to evaluate the minimum inhibition
concentration (MIC) values of the individual compounds. The
solution of resazurin (15%) was prepared by dissolving
0.015 g in distilled water. The solution was vortexed and
was filtered using a 0.22-μm filter. The solution of resazurin
was kept at 4 °C and was stored for its use maximum for 4
weeks.
2019). Various other interfacial parameters such as Гmax, Amin
,
Synthesis of pyrrolidinium based-ILs [Pyr Cx]Br- (x = 4,
Πcmc, and P were determined, and their respective values are
given in Table S1. The critical micellar concentration (CMC)
values of ILs were further confirmed using pyrene probe fluo-
rescence spectroscopy as described earlier (Wani et al. 2019).
Figure S23(a–e) represents the fluorescence spectra of pyrene
(dilute solution of concentration 1 × 10-6 mol L-1) in the ab-
sence and presence of a varied concentration of synthesized
ILs (the detailed methodology is given in supplementary ma-
terial, Fig. S24(a–e)).
6, 8, 10, 12)
IL N-alkyl-N-methylpyrrolidinium bromide was synthesized
in our laboratory using a route as described earlier (Qin et al.
2017; Saraswat et al. 2020). To prepare reaction mixture,
bromoalkane (butylbromide, hexylbromide, octylbromide,
decylbromide, and dodecylbromide) (5.48 g, 0.04 mol) were
mixed with N-methylpyrrolidine (3.4 g, 0.04 mol). Ethyl ace-
tate (15 mL) was added to the reaction mixture in round bot-
tom flask as shown in Scheme 1. The reaction mixture was
refluxed and stirred continuously and refluxed for 48 h. The
course of the reaction was monitored using thin-layer chroma-
tography (TLC) in which TLC plate was visualized under UV
(254 nm) light. After refluxing, the obtained ILs were thor-
oughly washed with ethyl acetate. The solvent was evaporated
and the obtained product was kept under high vacuum at an
elevated temperature of about 60–70 °C to remove moisture
content if any.
Cytotoxicity
The synthesized ILs were screened for their toxicity effect on
normal cell line human embryonic kidney (HEK-293) cell line
using MTT assay. The cell line, HEK-293, was precured from
the National Centre for Cell Science (NCCS), Pune, India.
The procedure involved the 10,000 cells/well was grown into
the flat-bottom 96-well plates (150 μL/well) in triplicates,
allowed to attach and grow. Fresh culture of cells was pre-
pared in T-75 culture flask in Dulbecco’s Modified Eagle
Medium (DMEM Himedia) containing 10% foetal bovine se-
rum, and penicillium/streptomycin (Himedia), at 37 °C and
5% CO2 in a humidified chamber (Nuaire CO2 incubator,
USA), and the incubation of 24 h was given to the sample.
Subsequently, the prepared set of the sample in the plate was
treated with various concentrations of the synthesized ILs
Structural characterization
1H nuclear magnetic resonance (NMR) spectra were recorded
on Bruker Avance (Delta) operating at 400 MHz, and 13C
NMR spectra were recorded with a spectrometer (Delta) op-
erating at 100 MHz. The Fourier transform infrared transform
R-Br, R=Cx,
x= 4,6,8,10,12
Scheme 1 Scheme of synthesis
of pyrrolidinium-based ILs with
Br- as the counter ion
Br-
N
N
Ethyl acetate, RT, 48 hrs
Cx CH3
CH3
1-methylpyrrolidine
1-alkyl-1-methyl-pyrrolidinium bromide
(Intermediate product)

Appl Microbiol Biotechnol
ranging from 9.3 to 300 μM. Later, after 48 h of treatment, the
medium was removed and cells were incubated with 20 μL of
MTT (5 mg/ml in PBS) in fresh medium for 4 h at 37 °C.
Solubilization of formazan crystals in DMSO (150 μL/well)
was done which was formed by mitochondrial reduction of
MTT, and quantification of results was done after an incuba-
tion period of 15 min by recording the absorbance at 540 nm
on the iMark Microplate Reader (Bio-Rad). The percentage of
cell viability was calculated as a fraction of control by using
Eq. 1 (Maurya et al. 2019).
μM, 100 μM, 200 μM, 250 μM) was added vertically in
columns 2–6. Subsequently, the same experimental procedure
was followed for [Pyr C6 ]Br- (10 μM, 50 μM, 100 μM, 200
μM, 250 μM), [Pyr C8]Br- (5 μM, 10 μM, 50 μM, 100 μM,
200 μM), [Pyr C10]Br- (5 μM,10 μM, 20 μM, 50 μM, 100
μM), and [Pyr C12]Br- (1 μM, 2 μM, 5 μM,10 μM, 20 μM).
Lastly, 10 μL of inoculums whose concentration was adjusted
to 106–105 CFU/mL added to each well except SC. The plate
was incubated at 37 °C for 16-18 h. After complete incuba-
tion, the quantification of results was done by recording the
optical density (OD) at 600 nm which was recorded on iMark
Microplate Reader (Bio-Rad) for each plate. Each test was
performed in the triplicate set.
OD sample mean
%cell viability ¼
 100
ð1Þ
OD control mean
Impact of ILs on MEL’s secondary structure
Antibacterial Activity
Minimum inhibition concentration determination (MIC)
The changes in the secondary structure of MEL induced by
ILs were monitored in the far-UV region (200–250 nm) using
a quartz cuvette of the path length of 0.1 cm (Patel et al. 2018).
The ellipticity value at wavelengths 222 nm and 207 nm were
used to calculate the R value using Eq. 2 (Morrisett et al. 1973;
Zhang et al. 2016b).
Microbiological toxicity of synthesized ILs against E. coli and
S. aureus was assessed through the determination of MIC
values by two-fold serial dilution method according to
Clinical and Laboratory Standards Institute guidelines
(CSLI) (Clinical, Institute LS 2012). MEL and ILs were
gradiently diluted with LBB row-wise into the 96-well plate
against E. coli and S. aureus. The final concentration range for
MEL was from 5 to 0.03 μM, whereas for ILs [Pyr C4]Br-,
480 to 0.23 mM, [Pyr C6]Br- (from 160 to 0.10 mM), [Pyr
C8]Br-(from 14.88 to 0.007 mM), [Pyr C10]Br- (from 4.32 to
0.002 mM), Pyr C12]Br-(from 0.31 to 0.003 mM). After dilu-
tion, 10 μL of bacterial inoculums was added including
growth control (GC) (broth with inoculums) except for sterile
control (SC) (broth without inoculums). The working concen-
tration of inoculum was adjusted to 106–105 CFU/mL. The
plate prepared was subjected to incubation at 37 °C for 16–18
h in an orbital shaker. After incubation, 10 μL of 15% solution
of resazurin dye was added to each well in 96-well plate.
Again, incubation was given to the plate for another 2–4 h
for the observation of colour change. After complete incuba-
tion, well with no colour change (blue colour of resazurin
remained unchanged) before a complete change of colour
(blue colour of resazurin was changed to pink) was recorded
as the MIC value of the corresponding compound.
½θŠ
222
R ¼
ð2Þ
½θŠ
222
where [θ]207 and [θ]222 are the experimentally observed abso-
lute mean residue ellipticity at 207 nm and 222 nm,
respectively.
Haemocompatibility assay
For haemocompatibility observation of IL-MEL compositions,
steady-state fluorescence, absorption, and CD spectroscopy
were employed. Samples prepared for haemocompatibility as-
say includes 5 μM HSA alone and in the presence of 10 μM
MEL. A separate set of samples was prepared which contained
MEL, HSA, and ILs where ILs, [Pyr C4 ]Br- (50 μM); [Pyr
C6]Br- (25 μM); [Pyr C8 ]Br- (20 μM); and [Pyr C10 ]Br- (10
μM); [Pyr C12 ]Br- (5 μM) were varied and kept the concen-
tration of MEL and HSA constant in each sample set. All
samples were incubated at 300 K for 30 min. The fluorescence
spectra of each sample were obtained at 298 K on a fluores-
cence spectrophotometer (Cary Eclipse spectrophotometer
(Varian, USA)) fitted with 150 W xenon lamp. The tempera-
ture of the sample holder was controlled using a Peltier control
temperature device. The sample was excited at 280 nm and
emission was recorded between 290 and 500 nm keeping the
slit width 5 nm constant for all the spectral scans. Absorption
spectra of the same set of samples were obtained between 200
and 400 nm with a slit width of 1 nm on a UV-Vis spectro-
photometer (Analytik Jena Specord-250 spectrophotometer,
Germany) and kept the slit width 1 nm constant of all spectral
Biological activity of MEL in presence of ILs
The antibacterial activity of MEL was accessed in the pres-
ence of a range of synthesized ILs as previously reported
(Omoya and Ajayi 2016). The experimental procedure in-
cludes the two-fold serial dilution of 10 μM MEL along the
column in 96-well plate ranging from 5 to 0.03 μM for mi-
croorganisms, E. coli and S aureus. The selected concentra-
tion of each ILs was added to plate; [Pyr C4]Br- (20 μM, 50

Appl Microbiol Biotechnol
scans. Additionally, far-UV-CD spectra of the same set of
samples were recorded with a quartz cuvette of 0.1-cm path
length on a spectropolarimeter (Jasco-715spectropolarimeter)
equipped with a microcomputer at 298 K. The spectropolarim-
eter was calibrated using D-10-camphorsulfonic acid. The tem-
perature of the sample holder was maintained using a water
bath with an accuracy of 0.1 °C to the spectropolarimeter.
Tetramethylsilane (TMS) was used as a reference solvent.
The NMR data are reported as follows: chemical shift, multi-
plicity (s = singlet, br s = broad singlet, d = doublet, t = triplet,
q = quartet, m = multiplet, dd = doublet of doublet), coupling
constants in Hertz and integration. TLC analysis was per-
formed on commercially prepared F-254 silica gel plates and
visualized under UV irradiation. To further confirm the struc-
ture, liquid chromatography-mass spectroscopy (LCMS) mass
spectra and Fourier transform infrared FT-IR was done.
1. 1-Butyl-1-methylpyrrolidin-1-ium bromide, [Pyr C4]Br-.
The product was obtained as a brown oil. 1H NMR (400 MHz,
CDCl3) δ 3.71–3.64 (m, 4H), 3.52–3.48 (m, 2H), 3.13 (s, 3H),
2.62 (s, 1H), 2.17–2.15 (m, 4H), 1. 67–1.59 (m, 2H), 1.35–
1.25 (m, 2H), 0.84 (t, J = 7.4 Hz, 3H); 13C NMR (100 MHz,
CDCl3) δ 64.5, 64.0, 48.7, 25.9, 21.7, 19.7, 13.7; FT-IR
(4000-400 cm-1) 2998 cm-1 [ν(N − C)], 2915 cm-1 [ν(C −
Binding study
The effect of synthesized ILs on the conformation of MEL
was studied using various spectroscopic and molecular
docking (MD) methods. Initially, MD was employed to deter-
mine the binding mode and binding site on MEL. MD was
performed using AutoDock 1.5.6 software. After docking, the
conformations were ranked and placed according to their in-
creasing binding energy obtained from molecular docking.
Amongst all five ILs, the best combination of MEL-ILs was
selected based on the highest binding energy. Further, the
interaction study of MEL with [Pyr C12]Br- (IL with the
highest binding energy obtained from MD) was studied using
various spectroscopic techniques, viz. fluorescence, UV-visi-
ble, and CD spectroscopy.
The fluorescence spectra of MEL (5 μM) in Tris buffer (10
mM, 7.2 pH) alone and in the presence of a varied concentra-
tion of [Pyr C12]Br- (1.42 μM to 20.97 μM) at 298 K, 303 K,
308 K, and 313 K was recorded on the same instrument as
described earlier (Gbaj 2018; Patel et al. 2015) (the detailed
methodology is given in supplementary materials). The UV
spectra of MEL (5 μM) alone and in the presence of a varied
concentration of [Pyr C12]Br- (from 1.42 to 20.97 μM) was
recorded using the same UV spectrophotometer as earlier de-
scribed at 298 K. For recording the absorbance, quartz cuvette
of 1-cm path length was used and the spectra were recorded in
the range of 200–400 nm (Patel et al. 2015; Sharma et al.
2017).
Â
Ã
H) (stretch)], 1485 cm-1
ν
, 1192 cm-1 [ν(C − C) (Bend)],
ðCH2Þ
888 cm-1 [ν(C − H) (Bend)], 3320 cm-1 is due to absorbed mois-
ture. LCMS (ESI) [M + H]+ calculated for [C9H20BrN]
221.0779, found 222.2706. (detailed spectra is given in Fig.
S1-4)
2. 1-Hexyl-1-methylpyrrolidin-1-ium bromide, [Pyr
C6]Br-. The product was obtained as brown oil. 1H NMR
(400 MHz, CDCl3) δ 3. 72–3.61 (m, 4H), 3.50–3.46 (m,
2H), 3.11 (s, 3H), 2.70 (s, 1H), 2.20–2.17 (m, 4H), 1.69–
1.61 (m, 2H), 1.30–1.23 (m, 2H), 1.22–1.15 (m, 3H), 0.75
(t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 64.5,
64.3, 48.7, 31.3, 26.1, 24.0, 22.4, 21.7, 13.9; FT-IR (4000-400
cm-1) 2998 cm-1 [ν(N − C)], 2915 cm-1 [ν(C − H) (stretch)], 1485
Â
Ã
cm-1
ν
, 1192 cm-1 [ν(C − C) (Bend)], 88 8 cm-1 [ν(C −
ðCH2Þ
(Bend)], 3320 cm-1 is due to absorbed moisture. LCMS
H)
(ESI) [M + H] + calculated for [C11H24BrN] 249.1092, found
250.00 (detailed spectra is given in Fig. S5-8).
3. 1-Methyl-1-octylpyrrolidin-1-ium bromide, [Pyr C8]Br.-
The product was obtained as a brown oil. 1H NMR (400
MHz, CDCl3) δ 3.80–3.68 (m, 4H), 3.56–3.52 (m, 2H),
3.21 (s, 3H), 2.56 (s, 1H), 2.24 (s, 4H), 1.75–1.67 (m, 2H),
1.35–1.19 (m, 9H), 0.81 (t, J = 7.1 Hz, 3H); 13C NMR (100
MHz, CDCl3) δ 64.5, 64.3, 48.8, 31.7, 29.2, 29.1, 26.4, 24.1,
22.6, 21.7, 14.1; FT-IR (4000-400 cm-1) 2998 cm-1 [ν(N − C)],
Results
Â
Ã
Structural characterization
2915 cm-1 [ν(C − H) (stretch)], 1485 cm-1
ν
, 1192 cm-1
ðCH2Þ
[ν(C − C) (Bend)], 888 cm-1 [ν(C − H) (Bend)], 3320 cm-1 is due
to absorbed moisture. LCMS (ESI) [M + H] + calculated for
[C13H28BrN] 277.1405, found 278.3205 (detailed spectra are
given in Fig. S9-12).
The chemical structure of synthesized ILs, 1-butyl-1-
methylpyrrolidinium bromide [Pyr C4]Br-, 1-hexyl-1-
methylpyrrolidinium bromide [Pyr C6]Br-, 1-methyl-1-
octylpyrrolidiniu bromide [Pyr C8]Br-, 1-decyl-1-
methylpyrrolidinium bromide [Pyr C10]Br-, and 1-dodecyl-1-
methylpyrrolidinium bromide [Pyr C12]Br-. (shown in Fig.
S21), were synthesized in our laboratory using a route as
shown in Scheme 1. 1H NMR, 13C NMR in CDCl3 /DMSO-
d6 (solvents), FT-IR, and mass spectra confirmed the chemical
structure of the synthesized compounds. Chemical shift values
for protons and carbons are reported in ppm.
4. 1-Decyl-1-methylpyrrolidin-1-ium bromide, [Pyr
C10]Br-. The product was obtained as a brown oil. 1H NMR
(400 MHz, CDCl3) δ 3.68–3.56 (m, 4H), 3.44–3.40 (m, 2H),
3.10 (s, 3H), 2.87–2.83 (m, 1H), 2.18–2.10 (m, 4H), 1.64–
1.56 (m, 2H), 1.25–1.19 (m, 3H), 1.13–1.10 (m, 10H), 0.70 (t,
J = 6.8 Hz, 3H) 13C NMR (100 MHz, CDCl3) δ 64.5, 64.2,
48.7, 31.8, 29.4, 29.4, 29.2, 26.4, 24.1, 22.6, 21.7, 14.1; FT-IR

Appl Microbiol Biotechnol
(4000-400 cm-1) 2998 cm-1 [ν(N − C)], 2915 cm-1 [ν(C −
supporting information). The results obtained from tensiome-
try suggest that the variation in the alkyl chain at N-atom in the
pyrrole ring can modify not only the micellization behaviour
of ILs but also lead to functional differences in both efficacy
and antibacterial activity of ILs.
Â
Ã
H) (stretch)], 1485 cm-1
ν
, 1192 cm-1 [ν(C − C) (Bend)],
ðCH2Þ
888 cm-1 [ν(C − H) (Bend)], 3320 cm-1 is due to absorbed mois-
ture. LCMS (ESI) [M + H] + calculated for [C15H32BrN]
305.1718, found 305.2522 (detailed spectra is given in Fig.
S13-16)
5. 1-Dodecyl-1-methylpyrrolidin-1-ium bromide, [Pyr
C12]Br-. The product was obtained as a brown oil. 1H NMR
(400 MHz, CDCl3) δ 3.52–3.46 (m, 4H), 3.28–3.24 (m, 2H),
3.01 (s, 3H), 2.25–2.23 (m, 3H), 1.75–1.63 (m, 2H), 1.32–
1.24 (m, 19H), 0.86 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 64.8, 64.5, 48.5, 48.2, 31.8, 29.5, 29.4, 29.3, 28.9,
26.1, 23.8, 22.6, 21.4, 14.1; FT-IR (4000-400 cm-1) 2998 cm-
Cytotoxicity
The cell toxicity of synthesized ILs was administered on HEK
293 cell line, using various concentrations (9.3–300 μM).
Good response in terms of cell viability (value ranges between
80 and 95%) was shown by all ILs at lower concentrations up
to150 μM. The cell viability at a lower concentration of all the
five ILs showed almost similar value, while at higher concen-
trations there was a decrease seen in the cell viability at around
150–300 μM for [Pyr C12]Br- (value ranges from 85 to 90%)
as shown in Fig. 1(a). This suggests that all five synthesized
ILs were non-toxic to normal HEK cell line at lower concen-
tration ranging up to 150 μM. These results were compared
with the data already been reported and were found that the
synthesized pyrrolidinium-based ILs possess lesser toxicity as
compared to the reported imidazolium-, pyridinium-, and
ammonium-based ILs (Qin et al. 2017). This finding was fur-
ther used in in vitro studies to further choose the concentration
range which may induce lesser damage to the human cell and
could result in better antibacterial property.
Â
Ã
1 [ν(N − C)], 2915 cm-1 [ν(C − H) (stretch)], 1485 cm-1
ν
,
ðCH2Þ
1192 cm-1 [ν(C − C) (Bend)], 888 cm-1 [ν(C − H) (Bend)], 3320
cm-1 is due to absorbed moisture. LCMS (ESI) [M + H] +
calculated for [C17H36BrN] 333.2031, found 333.3132 (de-
tailed spectra is given in Fig. S17-20).
Determination of surface-active parameters of ILs
The optimum biological effect of ILs is attributed to various
surface-active properties of ILs, viz. adsorption, hydrophobic-
ity, micellization, migration to the cell wall, solubility,
etc.(Saraswat et al. 2020). The variability in the antimicrobial
efficacy of ILs is associated with a bunch of various surface-
active parameters such as critical micelle concentration
(CMC), the minimum area per molecule (Amin), surface excess
concentration (Гmax), and an index of surface reduction
(Πcmc), which in terms of numerical data describes the phys-
icochemical behaviour of ILs as reported by several research
groups (Garcia et al. 2013; Garcia et al. 2017; Saraswat et al.
2020). Therefore, to optimize the biological effect of synthe-
sized ILs, the surface activity of synthesized pyrrole-based ILs
in aqueous medium was studied using surface tension data.
The details are given in supplementary material.
The surface tension, γ, was recorded and was plotted
against the concentration of ILs in the water at 298 K as shown
in Fig. S22(a–e). CMC, Amin, Гmax, and P values were deter-
mined using the surface tension data. The obtained CMC
values and other parameters are listed in Table S1. The pres-
ence of varied carbon chain length at N atom in cation signif-
icantly showed variation in the antibacterial activity which
Antibacterial activity
MIC determination
The synthesized ILs were screened for their antibacterial prop-
erty by investigating minimum inhibition concentration
(MIC) against using E. coli and S. aureus. MIC is defined as
the minimum concentration required for the inhibition of 50%
of the total bacterial growth after 24 h. The obtained MIC
values are summarized in Fig. 1(b). The MIC values suggest
that ILs with longer alkyl chain are more susceptible to E. coli
and S. aureus as compared to ILs with shorter alkyl chain
length, hence showing the larger MIC value. The structural
similarity of pyrrolidinium-based ILs with imidazolium-based
ILs containing quaternary ammonium cation makes them also
a good candidate as an antibacterial agent. Imidazolium-based
ILs are known for their activity towards membrane at the
target site mainly at the cytoplasmic membrane. ILs are the
amphiphilic molecules that have a tendency of impairment
with negatively charged entity mainly the phosphate group
present in a cell membrane through electrostatic forces which
favours the adsorption onto the cell through hydrophobic-
hydrophobic interaction. Hydrophobic-hydrophobic interac-
tion allows the insertion of ILs into the cell membrane via
pore formation (poration) leading to the rupturing of the cell
was elucidated based on numerical data of CMC, Amin, Гmax
,
the free energy of adsorption (ΔGads), and standard free ener-
gy for micellization (ΔGmic) shown in Table S1. From
Table S1, a higher magnitude of ΔGads as compared to
ΔGmic indicate that the adsorption is a most favoured process
than micellization at the air-water interface. Also, the value of
ΔGads has increased with the elongation of alkyl chain length
which further advocates the increasing spontaneity of higher
analogs to get adsorb on the surface (details are given in

Appl Microbiol Biotechnol
Fig. 1 (a) Cytotoxicity assay of synthesized pyrrole-based ILs with Br- as counter ion at various concentration. (b) Showing the MIC values of [Pyr
C4]Br-, [Pyr C6]Br-, [Pyr C8]Br-, [Pyr C10]Br-, [Pyr C12]Br-, and MEL against E. coli and S. aureus
wall with leakage of electrolyte out of the cell and ultimately
to cell death (Florio et al. 2019).
microorganism as compared to ILs with smaller alkyl chain
lengths.
Mean MIC values of homologues of pyrrolidinium-based
ILs against a couple of microorganisms are listed in Fig. 1(b)
which show the decreasing trend with the increasing carbon
atoms in the chain as previously reported for various surface-
active ILs (Garcia et al. 2017). The obtained MIC values for
each IL and MEL are listed in Fig. 1(b) which clearly show
that the MIC values of ILs against E. coli (Gram-negative)
were greater as compared to the MIC values against S. aureus
(Gram-positive). The difference in susceptibility towards the
action of the pyrrolidinium ILs against E. coli and S. aureus
can be deduced based on the structural difference between
Gram-negative and Gram-positive bacteria cells.
Structurally, Gram-positive bacteria comprise a much thicker
porous membrane of about 20–70 nm made up of peptidogly-
can layer interlinked with each other by a negatively charged
teichoic acid which is responsible for its porosity. On the
contrary, the Gram-negative bacterial membrane comprises
two layers: the outer layer being negatively charged whose
thickness is about 7–8 nm and is made up of lipopolysaccha-
rides, whereas the inner membrane is relatively thin (2–7 nm)
which is made up of peptidoglycan (Qin et al. 2017).
Therefore, it is supposed that the hydrophobic part of IL is
more likely to insert into the porous cell, peptidoglycan mem-
brane resulting in the disruption of the cell membrane and cell
death. In the case of Gram-negative bacteria, the outer mem-
brane protects the cell from the entry of foreign body such as
surfactant molecule which will ultimately disrupt the inner
peptidoglycan layer in the cell. In Gram-positive bacteria,
the outer layer of peptidoglycan may absorb the surfactant
molecule by hydrophobic interaction and carry it to the inner
cell membrane leading to disruption of the cell wall.
Figure 1(b) shows the MIC values of ILs in the form of a
bar graph which clearly shows the dependence of alkyl chain
length of ILs on microbiological toxicity against clinical mi-
croorganism, E. coli and S. aureus. [Pyr C12]Br- and [Pyr
C10]Br- showed the maximum efficacy against both the
The biological effect of ILs also depends on the diverse
physicochemical properties such as critical micelle concentra-
tion, hydrophobicity, adsorption, accumulation at the cell
membrane, solubility, and transport in the medium. The max-
imum surface area (Гmax) and the minimum area (Amin) occu-
pied by a single surfactant molecule at the air-water interface
obtained from Gibb’s adsorption isotherm give an idea about
the surface arrangement of surfactant molecules. The greater
value of Гmax and a smaller value of Amin depict the denser
arrangement of molecules at the interface. From Table S1, the
value of Гmax increases with increasing the chain length, indi-
cating the increase in the compactness at the interface which
was further supported by the Amin value. The decreasing value
of Amin with increasing chain length suggests the increase in
the packing of the molecule at the interface due to the de-
creased repulsive force between the IL molecules that is the
ILs with longer chain length forms the more compact mono-
layers at the interface. For higher homologues, CMC value
decreased which indicates that the accumulating concentration
of IL at cell membrane becomes less. Further, the free energy
of adsorption (ΔGoads ) for each IL was calculated and is listed
in Table S1 which shows the increase with increasing hydro-
phobic chain length. Also, the value of ΔGoads was found to be
higher as compared to the value of ΔGomic suggesting the
favoured adsorption on micellization. Further negative in-
crease in the value of ΔGaods with longer chain length shows
an increase in the migration rate of higher analogs at the cell
membrane (Garcia et al. 2013; (Qin et al. 2017). All physico-
chemical parameters for ILs examined collectively show the
highest tendency of ILs with longer alkyl chain length to get
adsorbed on the cell membrane and hence show the relative
higher antibacterial activity for higher analogs. Apart from
this, there are several other factors such as adsorption of ILs
on the cell membrane subsequently penetrating the living bac-
terial cell and altering the permeability of the cell wall which
are responsible for their best performance (Garcia et al. 2013).

Appl Microbiol Biotechnol
Table 1 Showing the best combination of synthesized pyrrolidinium
based ILs with MEL
Biological activity of MEL in presence of ILs
Figures 2, 3, 4, 5, and 6 (a–b) show the effect of various
concentrations of each synthesized ILs on the microbial
property of MEL in terms of the growth curves. The effect
of ILs on the antibacterial activity of MELwas studied by
examining the inhibition of E. coli and S. aureus. The results
showed a remarkable decrease in the MIC value of MEL in
the presence of ILs specially ILs with longer chain length,
viz. [Pyr C8]Br-, [Pyr C10]Br- and [Pyr C12]Br-. The MIC
value of MEL was found to be 2.50 μM and 2.30 μM against
E. coli and S. aureus, respectively. Figure 2(a) shows the
effect of [Pyr C4]Br- on the MIC value of MEL; it was seen
that with increasing concentration of IL in combination with
MEL the inhibition of bacterial burden was increased. The
combination of 1.24 μM MEL with 250 μM [Pyr C4]Br-
showed the reduction in the bacterial growth when tested
against E. coli, whereas 50 μM [Pyr C4]Br- in combination
with 1.27 μM MEL was tested against S. aureus which is
shown in Fig. 2(b). Similarly, a composition of MEL and
[Pyr C6]Br- was tested. The improved MIC values were ob-
tained as compared to the combination of [Pyr C4]Br- with
MEL and MEL alone. The reduction in the bacterial burden
was observed in case of 200 μM [Pyr C6]Br- and 1.22 μM
MEL shown in Fig. 3(a) against E. coli, whereas 10 μM [Pyr
C6 ]Br- when combined with 0.61 μM MEL is shown in Fig.
3(b) against S. aureus. Yet another, the combination of MEL
with [Pyr C8]Br- was tested which further showed improved
results in terms of a decrease in MIC value of MEL. 100 μM
solution of [Pyr C8]Br- with 0.77 μM MEL is shown in Fig.
4(a) against E. coli, and 100 μM combination of [Pyr C8]Br-
mixed with 1.25 μM MEL is shown in Fig. 4(b) against
S. aureus. The fusion of 20 μM [Pyr C10]Br- combined with
0.62 μM MEL against E. coli and combination of 20 μM [Pyr
C10]Br- with 0.64 μM MEL against E. coli and S. aureus as
shown in Fig. 5(a–b), respectively, shows the much more
improved results as compared to the combination tested with
ILs having butyl, hexyl, and octyl chain lengths. All synthe-
sized ILs in combination with MEL showed improved results
especially with increasing chain length in terms of inhibition
of bacterial cells. The best response in terms of the maximum
inhibition is shown in Fig. 6(a–b) when susceptible E. coli was
treated with a combination of 0.52 μM MEL and 10 μM [Pyr
C12]Br- and S. aureus strains when tested with a combination
of 0.63 μM MEL and 10 μM [Pyr C12]Br- for 16–18 h. All
combinations are listed in Table 1.
Microorganisms
MIC (μM)
MEL
In presence of [Pyr C4]Br-
Alone
50 μM
250 μM
E. coli (MTCC 40)
S. aureus (MTCC 87)
Microorganisms
2.50
-
1.24
-
2.30
1.27
MEL
Alone
2.50
In presence of [Pyr C6]Br-
10 μM
-
250 μM
E. coli (MTCC 40)
S. aureus (MTCC 87)
Microorganisms
1.22
2.30
0.61
-
MEL
Alone
2.50
In presence of [Pyr C8]Br-
100 μM
E. coli (MTCC 40)
S. aureus (MTCC 87)
Microorganisms
0.77
2.30
0.77
MEL
Alone
2.50
In presence of [Pyr C10]Br-
20 μM
E. coli (MTCC 40)
S. aureus (MTCC 87)
Microorganisms
0.62
2.30
0.64
MEL
Alone
2.50
In presence of [Pyr C12]Br-
10 μM
0.52
E. coli (MTCC 40)
S. aureus (MTCC 87)
2.30
0.63
(MDR) of bacteria. Therefore, we decided to design a
combination of peptide and ILs. Since MEL is a natural
known antibiotic and that was selected due to its high
solubility in water. The second antibacterial agent, we
have selected in this study was pyrrolidinium based IL
whose biological activity was examined separately.
Numerous reasons are there that are responsible for the
development of bacterial resistance. One being most gen-
erous is the frequent and higher dosage of antibiotics.
Therefore, we emphasized to increase the antibacterial
potential of MEL in the presence of synthesized ILs.
This might reduce the intake of MEL as well as will
reduce the risk of resistance development against bacterial
strain as well. The best results were obtained in terms of
bacterial growth inhibition.
The overuse of antibiotics is a reason for severe prob-
lems. The combination approach opens up the floor for
the researchers to work in the direction where they may
curtail the side effect associated with the prolonged use
of antibiotics and their high dosage. The combination of
two or more antibiotics is nowadays a recent topic of
research amongst the researchers to develop an efficient
antibiotic to render the multidrug resistance (MDR)
(Issam et al. 2015). The distinctive feature of ILs to get
adsorbed on the surface makes them an eligible
The results obtained from the synergistic study of ILs
showed an improved MIC value of MEL against both the
strain. Amongst all five ILs, the presence of [Pyr C8]Br-,
[Pyr C10]Br- and, [Pyr C12]Br- with MEL showed a re-
markable improvement in the MIC value of MEL. The
combination of antibiotics is one way to overcome the
problem associated with the rising multidrug resistance

Appl Microbiol Biotechnol
Fig. 2 Showing the effect of [Pyr C4]Br-concentration on antibacterial activity of MEL against (a) E. coli and (b) S. aureus
candidate to be used as a substitute compound for the
development of combination drug as discussed previous-
ly based on the parameters listed in Table S1.
N-position in IL did not affect the secondary structure of the
peptide, MEL (Reinhardt et al. 2014).
Haemocompatibility assay
Impact of ILs on MEL’s secondary structure
For the clinical application of any antibacterial agent,
haemocompatibility is an important aspect to be taken care
of; hence, haemocompatibility test was performed on the
key component of blood, human serum albumin, (HSA) (a
transport protein). HSA plays an important role in biological
functions in the body. It maintains the stability of the circula-
tory system (Patel et al. 2018). Therefore, the interaction of
HSA with MEL and MEL-ILs was investigated. Intrinsic
fluorophore (tryptophan, tyrosine) in HSA is responsible for
the fluorescence signal and is often used to assess the struc-
tural change in the protein of any while interacting with ligand
(drug, ILs, or other material) (Kumari et al. 2014; Maurya
et al. 2016; Patel et al. 2015). Figure 8(a) shows the fluores-
cence emission spectra of HSA, where a maximum fluores-
Before going into the application part of the non-covalent
conjugate of MEL and synthesized pyrrole-based ILs as an
antimicrobial agent, the conformational study was performed
using far-UV-CD spectroscopy. The conformational change
in the secondary structure of MEL was evaluated in the pres-
ence of ILs. The far-UV-CD spectra were recorded to study
the effect of ILs on the secondary structure of MEL. The CD
spectra of MEL alone and in the presence of ILs were recorded
as shown in Fig. 7(a–e). MEL showed a two-negative peak at
around 208 and 222 nm which are evidence of α-helical struc-
ture; the obtained spectra agrees with the reported spectra
(Zhang et al. 2016b). The spectra were used to determine the
molar ellipticity ratio, R. In Table S3, the value of R of con-
jugates was found to be approximately similar to the R-value
of pure MEL which demonstrates that alkyl chain attached at
cence intensity was observed at 342 nm wavelength (λmax
)
which is a signatory peak for the presence of Trp residue in
HSA. Further, the fluorescence emission spectra of HSA in
Fig. 3 Showing the effect of [Pyr C6]Br-concentration on antibacterial activity of MEL against (a) E. coli and (b) S. aureus

Appl Microbiol Biotechnol
Fig. 4 Showing the effect of [Pyr C8]Br- concentration on antibacterial activity of MEL against (a) E. coli and (b) S. aureus
the presence and absence of MEL and its combination with
ILs were recorded. Fluorescence emission spectra of HSA in
the presence of MEL and its combination with ILs do not
show any shift in the λmax value, which reveals that the polar-
ity of Trp remains unchanged in the presence of MEL and its
combinations with ILs. However, the fluorescence emission
intensity of HSA showed an increase in intensity suggesting
the addition of fluorescence intensity of MEL due to the pres-
ence of one Trp molecule; however, fluorescence intensity
decreased (quenching) with the addition of the conjugate to
HSA. This may be due to several mechanisms that occur such
as energy transfer, the formation of the complex, and the re-
action in excited state and collision. In contrast, the increasing
fluorescence intensity of HSA in the presence of MEL may be
due to the interaction of MEL with HSA by electrostatic at-
traction leading to change in the microenvironment of Trp.
UV-Vis spectra of the same set of samples were recorded to
administer the structural changes and complex formation. The
absorption spectra showed the characteristic band for α-helix
structure in the 200–240 nm range. Figure 8(b) shows no shift
in the UV-Vis absorption band which indicates that the sec-
ondary structure of HSA remains unchanged in the presence
of MEL and its conjugates with ILs. To administer any change
in the secondary structure, far-UV-CD spectra of the same set
samples were also obtained as shown in Fig. 8(c). From Fig.
8(c), the increasing negative ellipticity of HSA in the presence
of MEL and its conjugates reveals that MEL, as well as its
conjugates, do not disturb the secondary structure of HSA;
rather, they stabilize the secondary structure of HSA. The
results procured from far-UV-CD spectra were found to be
competent with the results obtained from the fluorescence
and UV-visible spectroscopy. Therefore, it can be concluded
from the above results that the non-covalent conjugate is
haemocompatible and can be used for developing therapeutic
drugs against which could work efficiently against bacterial
resistance.
Binding study
To study the complex formation between MEL and synthe-
sized IL, the binding study was performed. Initially, molecular
docking was performed which showed the participation of van
der Waal and π-cation interaction between MEL and synthe-
sized ILs. The amino acids involved in van der Waal
Fig. 5 Showing the effect of [Pyr C10]Br-concentration on antibacterial activity of MEL against (a) E. coli and (b) S. aureus

Appl Microbiol Biotechnol
Fig. 6 Showing the effect of [Pyr C12]Br- concentration on antibacterial activity of MEL against (a) E. coli and (b) S. aureus
interactions were Lys23, Arg22, and Ser18, whereas Trp19
was involved in π-cation interaction. Obtained Gibb’s free
energy of complexation is listed in Table S2. From
Table S2, the value of Gibb’s free energy indicates that the
complex formation between the MEL and ILs was spontane-
ous. The increasing negative magnitude of free energy with
increasing alkyl chain length in ILs implies the increasing
spontaneity of the complex formation with higher analogs.
Based on Gibb’s free energy obtained from molecular
docking, the ILs were ranked with the most possible stable
conformation. Figure S25(a–b) shows the most stable and
favourable three-dimensional and two-dimensional depiction
of the conformation of MEL-[Pyr C12]Br-. Further, based on
the mathematical value (Table S2) and stability, the most
favourable IL, [Pyr C12]Br-, was selected for binding study
with MEL. Fluorescence and UV-Vis spectroscopy was
employed to study the complex formation and feasibility of
complex formation between MEL and [Pyr C12]Br- (Maurya
et al. 2016; Patel et al. 2015). Firstly, fluorescence measure-
ment was done which showed a progressive decrease in the
fluorescence intensity (Fig. S26(a)) of MEL at λmax 353 nm
on the addition of the increasing concentration of [Pyr C12]Br-.
Figure S26(b) shows the plot between Fo/F and concentration
of [Pyr C12]Br-, where slope corresponds to Stern-Volmer
constant, Ksv. The Ksv value was found to be decreasing with
increasing temperature which suggests the process of
quenching was static. Further, binding parameters such as
binding constant (Kb) and the number of binding sites (n)
(details are given in supporting information, Fig. S27. (a))
were evaluated. The values are listed in Table 2. The value
of Kb suggests the strong binding between [Pyr C12]Br- with
MEL. Besides, the value of n indicates 1:1 binding occurring
amongst MEL and [Pyr C12]Br-. On increasing the tempera-
ture, the value of Kb decreased suggests that at the higher
temperature the complex destabilized. The thermodynamic
parameters such as free energy change (ΔG), the enthalpy
change (ΔH), and entropy change (ΔS) were also calculated
which suggests the involvement of van der Waal interaction
and hydrogen bonding in the complex formation (Ross and
Subramanian 1981) (detailed explanation is given in
supporting information, Fig. S27. (b)). Moreover, the UV-
Vis spectrum was obtained to collect insightful information
about the complex formation between the MEL and [Pyr
C12]Br-. Figure S28(a) shows the absorption spectra of MEL
alone and in the presence of [Pyr C12]Br-. The maximum
absorption of MEL was obtained around 273 nm. The Kb
was also determined which is in good agreement with fluores-
cence results (details are given in supporting information)
Discussion
The combination of two or more antibiotic drugs is one way to
combat bacterial resistance and is termed as ‘combination
therapy’. The emergence to develop new antibacterial agents
has been a current topic of concern for the researchers where
combination therapy had been considered as an effective strat-
egy to prevent antibacterial resistance. Infection due to multi-
drug resistance has become a burden worldwide, and patients
treated with traditional antibiotics has become challenging and
ineffective (Kadri et al. 2019). Traditional antibiotics are
known for their molecular targets such as protein synthesis,
cell wall synthesis, DNA replication, and various other bio-
molecular processes inside the bacterial cell (Sköld 2011;
Walsh 2000). These molecular targets are modified by various
modification processes such as gene transfer from resistive to
non-resistive cells through plasma exchange, inactivating the
antibiotics, or evacuate antibiotics out of the cell with the help
of ABC transporter (Wink 2012). The advantage of the com-
bination of antimicrobial drug over traditional antibiotics is
that it affects molecular targets such as biomembrane, pro-
teins, and DNA. This might help to conquer the antibiotic-
resistance mechanisms, to expand the antibacterial spectrum,
reduced toxicity, and reinstate the efficiency of antibiotics
(Chan et al. 2011).

Appl Microbiol Biotechnol
Fig. 7 Far-UV-CD spectra of MEL (30 μM) in the presence and absence of different concentrations of (a) [Pyr C4]Br-, (b) [Pyr C6]Br-], (c) [Pyr C8]Br-],
(d) [Pyr C10]Br-, and (e) [Pyr C12]Br-] in 10 mM Tris buffer at 298 K and at pH 7.2
Earlier studies showed the synergistic effect of MEL with
some antibiotics (Akbari et al. 2019). Recent studies related to
synergism between MEL and doripenem the MIC value of
MEL and doripenem decreased up to 61.6- and 51.3-fold,
respectively, against A. baumannii, where they reported that
the consumption of MEL has reduced to 61.6 folds (Issam
et al. 2015). This subsequently causes a decrease in its cyto-
toxicity. Another study revealed the combination of MEL
with colistin where it did not show any additive effect against
bacterial burden (Akbari et al. 2019). This study showed the
application of MEL-doripenem as an antibacterial agent in the
treatment of infections caused by A. baumannii. Therefore,
there was an urgent need to develop the combination with
MEL to achieve an agent that could work more than 61.6-fold
against Gram-negative and Gram-positive bacteria.
Our previous study relates to developing a combination of
two antibiotics, MEL and IL (best result was obtained with
−
[PyrC12NTf3 ]), where the reduction in bacterial burden was
found to be up to 95%. This was the remarkable achievement
in the study and reached to a combination which might reduce

Appl Microbiol Biotechnol
Fig. 8 (a) Absorption spectra, (b) fluorescence emission spectra, (c) far-UV-CD spectra of HSA (5 μM) and MEL (10 μM) in absence and presence
of different concentrations of [Pyr C12] Br- at 298 K and at pH 7.2
the dose intake of MEL and will retrench the toxic effect. ILs
are known for its adsorption characteristic whereas MEL for
its pore formation mechanism. Our present study involved the
synthesis of pyrrole-based ILs having Br-as a counter ion. The
best antibacterial combination was obtained when MEL was
combined with IL having octyl, decyl, and dodecyl carbon
chain. The reduction in bacterial burden was up to 90–92%
which showed comparatively lesser efficiency to our previous
results (Saraswat et al. 2020). The increased biological effi-
ciency of the conjugate in the previous study may be due to
the more hydrophobic counter (CF3SO2)2N− attached as com-
pared with the Br- with the cation moiety in the present study.
A previous study showed that the increased toxic effect of IL
with (CF3SO2)2 N– counterion is may be due to the increased
hydrophobicity and fluorine atom present in the counter ion.
In our study, we have used ILs contained Br– as a counter ion
and due to this, the cytotoxicity assay results showed less
toxicity behavior of used ILs. Therefore, we tried the series
of pyrrole-based ILs having Br– as a counter ion as an effec-
tive antibacterial agent with MEL.
The inhibition of bacterial burden up to 90–92% was ob-
served when treated with MEL in combination with [Pyr
C12]Br-. The presence of IL with MEL showed exceptional
improvement in the MIC value of MEL. This will not only
Table 2 Stern-Volmer constant (Ksv), binding constant (Ka), number of binding sites n, and relative thermodynamic parameters for the [Pyr C12]Br- and
MEL system at different temperatures and 7.2 pH
Temp (K)
Ksv
n
Kb
ΔS
ΔH
ΔG
Ka, UV-Vis
(Lmol-1 s-1)
(Lmol-1)
(kJK-1 mol-1)
(kJmol-1)
(kJmol-1)
(Lmol-1)
298
303
308
313
7.78 × 104
4.03 × 104
3.45 × 104
2.57 × 104
0.92
0.76
0.66
0.57
6.20 × 104
0.25 × 104
0.09 × 104
0.01 × 104
− 795.68
− 262.04
− 24.93
− 20.95
− 16.97
− 12.99
30.46 × 104

Appl Microbiol Biotechnol
decrease the dosage of either MEL or IL but will also exclude
the chances of getting side effects. This will result in the for-
mation of MEL-IL, as a potential therapeutic agent for the
treatment of infections occurring due to E. coli and
S. aureus. Improved antimicrobial efficiency of MEL-IL con-
jugate is more likely due to their target site on the cell wall.
The action mechanism of IL is still unknown, but MEL is
known for its pore-forming tendency on the cell wall. The
improved efficacy of MEL-IL may be due to the reason that
IL may be adding more absorption efficiency to MEL so that it
may get more adsorbed on the cell membrane. The surface
tension data and MIC data showed the dependence of MIC
value on the surface activity of synthesized ILs. ILs with lon-
ger alkyl chain length has the highest tendency to get adsorbed
on the cell membrane. Subsequently, MEL which is non-
covalently attached to ILs could create a pore into the cell
membrane and helps in entering of IL into the cell leading to
leakage of cytoplasmic material and cell death. ILs with MEL
showed a significant effect on the microbial activity of MEL.
In the present study, all ILs with shorter chain length showed
improved results in terms of MIC value of MEL but the best
synergism was found when MEL and [Pyr C12]Br- within the
tested range of concentrations. MEL-ILs conjugate adsorbs
more on the cell wall, creating the pore inside the outer mem-
brane of the bacterial cell due to increased absorption with
increasing hydrophobicity, increased migration rate, and dis-
persed charge. The aforesaid mode of action is more like to
administrate the penetration of MEL to reach the cell wall
leading to disruption of the cell wall and cell death. The de-
velopment of bacterial resistance is due to the genetic and
climatic modifications. The gene exchange from resistive to
non-resistive bacterial cells favours bacterial resistance
(Munita and Arias 2016) which is more likely to stop the
plasma exchange between the cells and might help in decreas-
ing bacterial resistance.
toxicity, in turn, will decrease the chances of developing re-
sistance towards bacterial strains. The combination of MEL
and ILs display a possible cost-effective and eco-friendly an-
tibacterial composition within the tested concentration range.
Besides, the haemocompatibility assay suggested the compat-
ibility of MEL-IL conjugate with human albumin. Also, the
binding studies suggested the stability of the composition
formed. Overall, their pharmacological and safety properties
suggest that the conjugates of MEL with ILs will hopefully
lead to the development of a therapeutic agent in the area of
healthcare for the treatment of infections and will exclude the
risk of bacterial resistance.
Acknowledgements This research was supported by the Researchers
Supporting Project number (RSP-2020/35), King Saud University,
Riyadh, Saudi Arabia. Dr. Rajan Patel greatly acknowledges the financial
support from the Science and Engineering Research Board (EEQ/2016/
000339) New Delhi, India. Juhi Saraswat is also thankful to the ICMR,
New Delhi, India, for providing a research grant (F. No.45/19/2019-BIO/
BMS).
Author contribution JS performed the whole experimental part and wrote
the manuscript.
KI performed the cytotoxicity experiment in the study.
MA wrote the cytotoxicity part in the manuscript.
SYA and BA help to revise the manuscript.
RP is the corresponding author.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical statement This article does not contain any studies with human
participants or animals performed by any of the authors.
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