Antibacterial and Synergy of Clavine Alkaloid Lysergol and its Derivatives Against Nalidixic Acid-Resistant Escherichia coli

Article abstract of DOI:10.1111/cbdd.12103

Antibacterial activity of lysergol (1) and its semi-synthetic derivatives (2-14) and their synergy with the conventional antibiotic nalidixic acid (NA) against nalidixic acid-sensitive (NASEC) and nalidixic acid-resistant (NAREC) strains of Escherichia coli were evaluated. Lysergol (1) and derivatives (2-14) did not possess antibacterial activity of their own, but in combination, they significantly reduced the minimum inhibitory concentration (MIC) of NA. All the derivatives showed two- to eightfold reduction in the MIC of NA against NAREC and NASEC. Further, lysergol (1) and its derivatives 10 and 11 brought down eightfold reductions in the MIC of tetracycline (TET) against multidrug-resistant clinical isolate of E. coli (MDREC). Treatment of these strains with the combinations of antibiotics and lysergol and its derivatives 10 and 11 (at reduced concentrations) significantly decreased the viability of cells. In an another observation, lysergol and its derivatives 10 and 11 inhibited ATP-dependent efflux pumps, which was evident by ATPase inhibition and down-regulation of multidrug ABC transporter ATP-binding protein (yojI) gene. These results may be of great help in antibacterial drug development from a very common, inexpensive, and non-toxic natural product. Lysergol (1) and derivatives (2-14) did not possess antibacterial activity of their own, but in combination, they significantly reduced the minimum inhibitory concentration (MIC) of Nalidixic acid as well as tetracycline. Lysergol and its derivatives 10 and 11 inhibited ATP-dependent efflux pumps, which was evident by ATPase inhibition and down-regulation of multidrug ABC transporter ATP-binding protein (yojI) gene.

Full text of DOI:10.1111/cbdd.12103

Received Date : 08-May-2012
Revised Date : 09-Oct-2012
Accepted Date : 10-Dec-2012
Article type : Research Article
Antibacterial and synergy of clavine alkaloid lysergol and its
derivatives against nalidixic acid resistant Escherichia coli
Synergistic antibacterial potentials of clavine alkaloids
Anupam Maurya#1, Gaurav R. Dwivedi#2, Mahendra P. Darokar2, Santosh
Kumar Srivastava1*
1Medicinal Chemistry Department; 2 Molecular Bioprospection Department;
CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow-226015, India
#both the authors contributed equally
*Address of Corresponding author:
Dr. Santosh Kumar Srivastava
Medicinal Chemistry Department
Central Institute of Medicinal & Aromatic Plants
(Council of Scientific & Industrial Research)
P.O.-CIMAP, Kukrail Picnic Spot Road, Lucknow-226015 (U.P.) India
Tel.: +91-522-2718581; Fax: +91-522-2342666
E-mail: santoshkumar_1955@yahoo.com
Abstract
Antibacterial activity of lysergol (1) and its semi-synthetic derivatives (2-14) and their synergy
with the conventional antibiotic nalidixic acid (NA) against nalidixic acid sensitive (NASEC)
and nalidixic acid resistant (NAREC) strains of Escherichia coli was evaluated. Lysergol (1) and
derivatives (2-14) did not possess antibacterial activity of their own, but in combination, they
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© 2013 John Wiley & Sons A/S

significantly reduced the minimum inhibitory concentration (MIC) of NA. All the derivatives
showed two to eight folds reduction in the MIC of NA against NAREC and NASEC. Further,
lysergol (1) and its derivatives 10 and 11 brought down eight fold reductions in the MIC of
tetracycline (TET) against multidrug resistant clinical isolate of E. coli (MDREC). Treatment of
these strains with the combinations of antibiotics and lysergol and its derivatives 10 and 11 (at
reduced concentrations) significantly decreased the viability of cells. In an another observation,
lysergol and its derivatives 10 and 11 inhibited ATP dependent efflux pumps, which was evident
by ATPase inhibition and down regulation of multidrug ABC transporter ATP binding protein
(yohI) gene. These results may be of great help in antibacterial drug development from a very
common, inexpensive and non toxic natural product.
Keywords: Lysergol; acyl/aryl analog; bioenhancer; multidrug resistant, efflux pumps.
Introduction
The consumption of antibiotics by man is increasing at an alarming rate. Out of the total
antibiotics we use, 20%-50% of it is unnecessary depending on the class of antibiotic [1]. This
may be due to (i) reduced absorption in the gut membrane when taken orally (ii) restrictive
uptake by the target microbe or (iii) operation of efflux pump leading to indiscriminate extrusion
of the antibiotics. So the major amounts of the antibiotics we apply are wasted and only a minor
percentage is being targeted to the infective microbes. Further, the unutilized antibiotic amount
remains as a load in the body and environment acting as a selection pressure facilitating
emergence of drug resistance in parasites and their predominance, ultimately leading to failure of
antibiotics against resistant infections [2, 3].
One of the ways, which has been feasible to reduce drug dosage, has been synergism
between two therapeutic agents. However, if both have the antibiotic property, still the problem
of continued selection pressure on microbes is likely to continue. So, we thought of searching
only those molecules, which by them are not microbicidal but when present with an antibiotic,
enhances its activity and availability (bioenhancers) [2, 4]. In this way these molecules
(bioenhancers) will not exert any selection pressure for mutants to emerge resistant against them
and on the other hand could reduce the dosage of antibiotics so that their ill effects are
minimized and the resistance development process will be substantially delayed ultimately
leading to enhanced life-span of the novel and existing antibiotics. Such antibiotics facilitators
should have novel properties like non-toxic to human, animal or plants should be effective at a
very low concentration in a combination, should be easy to formulate and most importantly
enhance uptake/absorption and activity of the antibiotics. This can lead in developing judicious
and strategic concentrations of antibiotics with specific bioenhancers to improve availability of
the drug right up to the target for effectively controlling the infectious organisms [2, 4].
Drug resistant E. coli has become the most common cause of many life-threatening
diseases such as infection of the bloodstream. Recent data from the U.S. National Healthcare
Safety Network indicate that gram-negative bacteria are responsible for more than 30% of
hospital-acquired infections of which E. coli predominate in cases of urinary tract infections
(45%) [5]. The present study primarily deals with drug resistance reversal potential of lysergol
(1) and its semi-synthetic derivatives with the conventional antibiotic, nalidixic acid against
© 2013 John Wiley & Sons A/S

NASEC and NAREC strains of E. coli. The most active combinations were validated by
checkerboard and time kill assays against NASEC and NAREC as well as with another antibiotic
tetracycline against MDREC. Further efflux pump inhibition assay, ATPase inhibitory and real
time expression analysis were carried out to find the possible mode of action of lysergol and its
derivatives.
Material and methods
General experimental procedures
1
13
The NMR spectra were recorded on Avance 300 MHz spectrometer (Bruker). The H and C
NMR chemical shifts were referenced to the solvent peaks. MS were recorded on hyphenated
LC-PDA-MS (Prominence LC and mass MS-2010EV, Shimadzu). The compounds were first
vizualized on TLC plates (silica gel 60F254, Merck) under UV illumination at 254 and 365 nm
and then sprayed with Dragon Droff’s reagent.
Antibacterial agents
The conventional antibiotics nalidixic acid and tetracycline were purchased from Sigma (purity
≥98%). Lysergol was isolated and identified from the seeds of I. muricata as described in the
previous reports [6, 7]. Further, acyl and aryl derivatives of lysergol were prepared according to
the procedure given below. The purities of isolated and semi-synthetic derivatives were ≥95 %
(HPLC).
Chemical derivatization of lysergol (1)
Lysergol (50 mg) was dissolved in pyridine (1.5 mL) and to this solution respective acyl / aryl
chloride were separately added in a 1:1.5 ratio using 4-dimethylaminopyridine (DMAP) as
catalyst (Figure 1). The airtight reaction mixture was kept overnight at room temperature and the
progress of the reaction was monitored by TLC. After completion of the reaction, ice cold water
was added and the mixture was extracted with chloroform (3 x 20 mL). The pooled chloroform
extract was washed with water until neutral and dried over anhydrous Na2SO4 and evaporated
under vacuum. This chloroform extract was further purified by flash chromatography over Silica
gel to yield the respective derivatives in 75–90% yields. All the derivatives (2-14) were
characterized on the basis of their 1H, 13C NMR and mass spectroscopic data (see supplementary
file).
Bacterial strains
E. coli strains, nalidixic acid resistant DH5α (MTCC 1652) NAREC was procured from MTCC,
IMTEC Chandigarh, India, while nalidixic acid sensitive strain CA8000 (NASEC) was donated
to CIMAP repository by Dr. Sushil Kumar, Ex Director CIMAP Lucknow, India [8]. The well
characterized multi drug resistant clinical isolate-KG4 (MDREC) was gifted by Dr. Mastan
Singh and Dr M.K. Gupta, King George Medical University, Lucknow [9].
© 2013 John Wiley & Sons A/S

Media
Standard Mueller-Hinton agar and broth (MHA and MHB, Hi-Media, Mumbai, India) were used
as bacterial culture media. MHB was used for susceptibility testing. Colony counts were
determined using MHA plates.
Susceptibility and Synergy testing
The test compounds were diluted into final concentrations of 1000 to 1.9 μg/mL and tested
against DH5α, CA8000, and MDREC strains of E. coli. The MIC values were determined by 2-
fold serial dilution broth assay [10, 11] with starting inoculums of 5×105 cfu/ml, incubated at
37°C for 24 h and detected from the observatory data as per CLSI guidelines [12] using nalidixic
acid as positive control. They were determined in duplicate, with concentrations ranging from
0.39 to 200 μg/mL of nalidixic acid.
The combination studies were performed at concentrations ranging from 0.39 to 200 μg/mL of
nalidixic acid in combination with 10 μg/mL of each test compound [3, 13]. The microtitre plates
(Genaxy) were inoculated with 10 μL of diluted overnight grown culture of the test organism
with a titre equivalent to 0.5 McFarland standards. The inoculated microtitre plates were then
incubated at 37°C for 24 h. The final bacterial inoculum in each well was 5x105 CFU/mL. The
results were recorded in terms of reduction. The reported MIC values (μg/mL) and fold
reductions are the mean of 3 experiments in replicate. Results were recorded in the term of fold
reduction.
In addition, synergy studies of most active combinations were also performed by broth
checkerboard method [14]. Cation-adjusted Mueller-Hinton broth (150µL) was added to each
well of the 96-well plate. The last two columns of wells served as controls for E. coli growth and
plate sterility. The final concentrations ranged from 1.56 to 800 µg/mL for tetracycline and from
1.25 to 160 µg/mL for test compounds. Thus, each of the 80 wells had a unique combination of
antibiotics and test compounds. The final bacterial inoculum in each well was 5x105 CFU/ml
except the negative control. The plates were incubated at 37°C for 24 hours. The MIC was
recorded as the last dilution without any turbidity as per CLSI guidelines.
Time kill studies
The time kill study of nalidixic acid and tetracycline alone and in combination with lysergol (1)
and its derivatives 10 and 11 against E.coli strains was conducted at MIC, 2MIC and 4MIC
concentrations using a method described previously by Eliopoulus and Moellering [15]. Each
analysis was done in triplicate with a control without test sample. Time kill curves were derived
by plotting log10 CFU/ml against time (h). Time kill kinetics was also studied in combinations of
antibiotics and test compounds at the reduced concentrations at which maximum synergy was
observed.
© 2013 John Wiley & Sons A/S

Ethidium bromide efflux studies
The fluorometric determination of ethidium bromide efflux was performed as described
previously [16]. Bacterial (MDREC) culture was grown to reach optical density (OD) of 0.6 at
600 nm. The cells were collected by centrifugation and washed with PBS. The suspension (0.3
OD) was exposed to 5 µg/mL ethidium bromide for 60 min at 25°C in the presence of lysergol
(1) and its derivatives 10 and 11 at 10 µg/mL. The cells were harvested by centrifugation and
resuspended in fresh buffer. Loss of fluorescence was recorded for 30 min at 1 min intervals at
an excitation and emission wavelength of 530 nm and 585 nm respectively using
spectrofluorometer (FLUO star omega, BMG Labtech Germany).
ATPase inhibitory activity of phytomolecules
The bacterial membrane protein was isolated by the method described earlier [17]. ATPase
assay was carried out using quantichromTM ATPase Assay Kit (BioAssay Systems, USA) and
ATPase activity was estimated by measuring liberated inorganic phosphate (Pi)
spectrophotometrically (18).
qRT– PCR analysis of the ATP dependent efflux pump protein of E. coli
The transcriptional profile of the multidrug ABC transporter ATP binding protein (yohI) gene
was analyzed in treated and non treated cells of MDREC by the method described earlier [19].
Cells were grown to mid-log phase in the presence of sub-inhibitory concentration (1/4 MIC) of
tetracycline, lysergol (1) and its derivatives 10 and 11 alone and in combination. The real-time
quantification of the RNA templates was analyzed by SYBR GreenER qPCR super mix
(Invitrogen, USA) using 7900HT fast real time PCR system (Applied Biosystems, USA).
Observations were recorded in terms of LogRQ after normalization of indigenous gene (gapdh)
expression.
Results and discussion
Although, few antibiotics for gram-positive bacteria are in the drug discovery pipeline, but there
are none for the gram-negative bacteria. It has been found that efflux pumps are the main cause
of development of multi drug resistance (MDR); hence there is urgent need for the search of
efflux pump inhibitors and synergistic combinations of drugs, which can reverse the
phenomenon of MDR [20, 21].
The present study was aimed to isolate and identify plant molecules, which can reverse the
phenomenon of multidrug resistance specifically in human pathogenic class of gram-negative
bacteria. Clavine alkaloid lysergol was isolated and identified from the seeds of I. muricata as
described in the previous reports [6, 7].
© 2013 John Wiley & Sons A/S

Antibacterial activity evaluation of the clavine alkaloid lysergol against one nalidixic acid
sensitive (NASEC) and one nalidixic acid resistant (NAREC) strains of E. coli, showed that
lysergol do not posses antibacterial activity (MIC 1000 µg/mL). But when 10 µg/mL of alkaloid
was tested separately in combination with nalidixic acid, it showed significant synergistic
activity. It remarkably reduced the MIC of nalidixic acid by eight folds against the NAREC and
four folds against the NASEC. This potential synergistic effect of lysergol prompted us to
prepare some new derivatives for their enhanced activity. A total of thirteen semi synthetic acyl
and aryl derivatives (2-14) of lysergol (1) were prepared as described in the material and method
section. All the derivatives (2-14) except 2 were found to be new and characterized on the basis
1
of their H, 13C NMR and mass spectroscopic data. The purities of isolated clavine alkaloid
lysergol (1) and semi-synthetic derivatives (2-14) were ≥95 % (HPLC). Further, these derivatives
when evaluated for their synergistic potential against the NASEC and NAREC strains of E. coli,
it was observed that two aryl derivatives 10 and 11 reduced the MIC of nalidixic acid by eight
folds against the NASEC, which is actually two times to that of lysergol. On comparing these
observations with the known drug resistance reversal agent, reserpine [22, 23], it was observed
that clavine alkaloids lysergol and most of the derivatives exhibited two to four times better
activity than the reserpine (Table 1).
The effective concentrations of lysergol (1) and its derivatives 10 and 11 were determined
through checkerboard assay and found to be 10 µg/mL (Table 2; see suppl. file). Further, these
observations were validated by using another antibiotic tetracycline against MDREC and similar
types of data were collected wherein reduction in the MIC of tetracycline up to 8 folds was
recorded (Table 2). Similar type of results were also reported earlier where it was found that
some alkaloids did not possessed antibacterial activity but in combinations they were able to
reduce the dose of partner drugs many folds [13, 22].
The treatment of NASEC, NAREC with NA at MIC, 2MIC and 4MIC concentrations reduced
the viability of E. coli significantly (Figure 2A, 3A). However, the bactericidal activity of NA
was achieved at very low concentration when tested in combination with lysergol (1) and its
derivatives 10 and 11 (Figure 2B, 3B). Similar type of data was also recorded when another
antibiotic tetracycline (TET) was tested against multidrug resistant strain MDREC of E.coli
(Figure 4A, 4B). Our observations are in accordance with earlier reports wherein bactericidal
activity was achieved at sub-inhibitory concentrations when tested in combinations [23, 24].
In order to understand possible mechanism of action of lysergol (1) and its two derivatives 10
and 11, they were subjected to fluorescence based ethidium bromide efflux assay using
multidrug resistant strain MDREC of E.coli. As shown in Figure 5, significant decrease in
fluorescence was observed in non treated control cells. While in presence of lysergol (1) and its
two derivatives 10 and 11, the loss of fluorescence was significantly reduced, reflecting a strong
interference with ethidium bromide efflux by these compounds. Accumulation and efflux of
© 2013 John Wiley & Sons A/S

ethidium bromide are good indicators of the involvement of efflux pumps in the resistance
mechanism, particularly in Gram negative bacteria such as E. coli [25, 26].
Further, to understand whether these compounds interfere with ATP dependent efflux pump,
they were evaluated for ATPase inhibitory activity in MDREC. It was found that lysergol (1) and
its derivatives 10 and 11 significantly inhibited ATPase activity (Figure 6) in terms of liberated
inorganic phosphate (Pi) indicating involvement of these compounds in the inhibition of ATP
dependent efflux pumps. Similar studies have been reported earlier for understanding the
mechanism of action of known drugs such as reserpine, ouabain and phenothiazine [27].
The involvement of these compounds in the inhibition of ATP dependent efflux pump was
further validated by studying the expression of multidrug ABC transporter ATP binding protein
(yohI) gene upon the treatment of MDREC by lysergol (1) and its derivatives 10 and 11. As
evident from the Figure 7, up regulation of this gene was observed when the cell were treated
with tetracycline (1/4 MIC). While, this gene was found to be significantly down regulated upon
the treatment of MDREC with lysergol (1) and its derivatives 10 and 11. Similarly, expression of
yojI gene was found to be down regulated in presence of the combinations of tetracycline and
test compounds at the concentrations where maximum synergy was achieved (Figure 7).
Tetracycline is known to induce higher expression of different efflux pump genes [19, 28] and
inhibition/modulation of this activity by the compounds reported in this study indicates towards
their potential as drug resistance reversal agents.
Conclusions
From the above results, it may be concluded that lysergol (1) and its derivatives 10 and 11 are
the novel efflux pump inhibitors being reported for the first time. Inhibition of the efflux pump
yojI by these efflux pump inhibitors can be useful in: (i) lowering the dose of antibiotics; (ii)
reducing the drug resistance development frequency; and (iii) increasing the efficacy of
antibiotics against multidrug resistant E.coli strains. These results may be of great help in the
development of inexpensive and dose economic antibacterial drug formulations from a very
common and widely distributed herb, I. muricata.
ACKNOWLEDGEMENTS
Financial support for from CSIR-CIMAP and fellowship to AM and GRD from CSIR, New
Delhi is gratefully acknowledged. We also thank all of our colleagues for their assistance in
completion of this study. It is stated that there is no conflict of interest among the authors.
© 2013 John Wiley & Sons A/S

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Figures Captions
Figure 1. Chemical derivatization of lysergol (1)
Figure 2: Time–kill curves of NASEC showing the dose dependent bactericidal effect of (A)
nalidixic acid; (B) nalidixic acid in combination with lysergol and its derivatives Der-10 and
Der-11.
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Figure 3. Time–kill curves of NAREC showing the dose dependent bactericidal effect of (A)
nalidixic acid; (B) nalidixic acid in combination with lysergol and its derivatives Der-10 and
Der-11.
Figure 4. Time–kill curves of MDREC showing the dose dependent bactericidal effect of (A)
Tetracycline: (B) Tetracycline in combination with lysergol and its derivatives Der-10 and Der-
11.
Figure 5. Inhibition of ethidium bromide efflux by lysergol and its derivatives Der-10 and Der-
11 in MDREC .
Figure 6. Inhibition of ATPase by lysergol and its derivatives Der-10 and Der-11 in MDREC .
Figure 7. Expression level of yojI mRNA in MDREC upon the induction by tetracycline alone
and lysergol and its derivatives Der-10 and Der-11 alone and in combinations.
Table 1. MIC of lysergol/derivatives (2-14) alone and in combination with Nalidixic acid
Compounds
MIC of compounds alone MIC of Nalidixic acid in combination
against E. coli (µg/mL) with compounds (10 µg/mL)
Fold
Fold
CA8000
CA8000
DH5α
DH5α
reduction
reduction
Nalidixic acid
Lys-1
6.25
1000
1000
100
-
-
-
1000
1000
1.56
1.56
4
4
12.5
25
8
4
Der-2
2
4
4
4
4
4
4
8
8
2
4
8
4
4
2
4
8
8
Der-3
Der-4
Der-5
Der-6
Der-7
Der-8
Der-9
Der-10
Der-11
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
3.125
1.56
1.56
1.56
1.56
1.56
1.56
0.78
0.78
50
25
12.5
25
25
50
25
12.8
12.5
© 2013 John Wiley & Sons A/S

2
2
4
2
2
2
4
2
Der-12
1000
1000
1000
500
1000
1000
1000
500
3.125
3.125
1.56
50
50
25
50
Der-13
Der-14
Reserpine* (RES)
3.125
*Known drug resistance reversal agent
Table 2. Reduction in the minimum inhibitory concentrations of antibiotics in combination with
plant compounds against drug sensitive and resistant strains of E.coli through checker board
assay.
Plant compounds
(µg/mL)
MIC of tetracycline
with/without plant
compounds against
MDREC
MIC of NA with/without plant
compounds against
NASEC
NAREC
Lys-1 (160)
Lys-1 (10)
1.56/6.25
12.5/100
12.5/100
50/100
100/800
100/800
1.56/6.25
200/800
100/800
100/800
400/800
100/800
100/800
200/800
400/800
400/800
800/800
Lys-1 (1.25)
Der-10 (160)
Der-10 (10)
Der-10 (1.25)
Der.-11(160)
Der-11(10)
3.125/6.25
0.78/6.25
12.5/100
12.5/100
50/100
0.78/6.25
3.125/6.25
0.78/6.25
0.78/6.25
3.125/6.25
3.125/6.25
12.5/100
12.5/100
25/100
Der-11(1.25)
50/100
Reserpine (RES, 160)
Reserpine (10)
3.125/6.25
6.25/6.25
50/100
Reserpine (1.25)
100/100
© 2013 John Wiley & Sons A/S

© 2013 John Wiley & Sons A/S

© 2013 John Wiley & Sons A/S

© 2013 John Wiley & Sons A/S

© 2013 John Wiley & Sons A/S