DECIPHERING THE ANTIBIOTIC

A D J U VA N T A C T I V I T Y O F C H O L I C

ACID-DERIVED AMPHIPHILES

A G A I N S T G R A M - N E G AT I V E B A C T E R I A

DISSERTATION SUBMITTED TO

REGIONAL CENTRE FOR BIOTECHNOLOGY

IN FULFILMENT OF REQUIREMENT
FOR AWARD OF THE DEGREE OF

MASTER OF SCIENCE
in
BIOTECHNOLOGY
by

BHARTI
UNDER THE GUIDANCE OF
Dr. AVINASH BAJAJ
SEPTEMBER 2021
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Antibiotic Adjuvant activity of Cholic Acid-derived Dimeric Amphiphiles
against Gram-negative Bacteria”, is being submitted for the award degree of
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 Contents
ABSTRACT.......................................................................................VI
INTRODUCTION................................................................................1
REVIEW OF LITERATURE...............................................................5
MATERIAL AND METHODS..........................................................11
RESULT AND DISCUSSION...........................................................18
CONCLUSION...................................................................................31
REFERENCES...................................................................................32

v
 Abstract

Antibiotic therapy is the prime mode of treatment for majority of bacterial

infections. However, overprescribed use of antibiotics has led to evolution of

Multi-drug resistant pathogens. Anti-resistance agents called antibiotic adjuvants

have gained much attention due to their ability to rejuvenate existing antibiotics

and inability of bacteria to gain resistance against them. Hydrophobic antibiotics

such as macrolides which are effective against gram positive bacteria (GPB) but

are ineffective against gram negative bacteria (GNB) despite their target being

present in both, cannot cross the GNB outer membrane (OM) because of

Lipopolysaccharide (LPS) barrier. Amphiphilic molecules that have the ability to

permeabilize the OM of GNB can be an effective approach to repurpose these

antibiotics against GNB. Bile acid having an amphiphilic backbone can be

modified with charged head groups such as amino acids arming it with cationic

functionality. These bile acid-derived cationic amphiphiles can serve as

membrane permeabilizing agents which can allow the entry of hydrophobic

antibiotics within the GNB and thus result in bacterial elimination. Herein, we

present the role of Cholic Acid-derived dimeric amphiphiles as antibiotic

adjuvants against GNB via disrupting its membrane. We also validated the

adjuvant Dimer 3 and Erythromycin (Macrolide) combination having bactericidal

property in murine wound infection model.

vi
 Chapter 1

Introduction

Alexander Fleming’s Nobel wining discovery for penicillin revolutionized

medicine. In 1945 he himself warned that overuse of the drug may result in

natural selection of resistant bacteria. 1 Formerly deadly bacterial infections have

been rendered as easily treatable ones after the use of antibiotics in medical

practice. Major Antibiotic classes that have been used in clinic include: Folate

synthesis inhibitors, Cell wall synthesis inhibitors, Protein synthesis inhibitors and

Nucleic acid synthesis inhibitors.2 Misuse, over prescription and discontinued

courses have transformed bacterial population such that many antibiotics have

drastically lost their efficacy.3 Thus we can say cure is the catalyst. Infectious

disease society of America (IDSA) has classified a fraction of superbugs as

ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,

Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp)

pathogens.4 These display new paradigms of resistance against existing

antibiotics.

However, certain mechanisms provide intrinsic resistance to certain bacteria

which render them immune to a particular class of antibiotic action. An important

example of this is the outer membrane (OM) of Gram-negative bacteria (GNB)

which is impermeable to hydrophobic antibiotics such as macrolides, conferring

them intrinsic resistance against these drugs. 5 In contrast to Gram-positive

bacteria (GPB) having only one membrane, GNB possess an outer membrane as

1
well.6 The OM of GNB is highly asymmetrical as it comprises of inner leaflet

composed of phospholipids and the outer leaflet composed of LPS. Apart from

this, their extraordinary potential to form biofilms results in persister cell

proliferation which is a major cause of nosocomial infections. GNB such as

Acinetobacter baumannii and Pseudomonas aeruginosa have been prioritized as

critical pathogens by World Health Organization (WHO).7

An approach to combat the ineffectiveness of such antibiotics to traverse the OM

of GNB is the usage of “membrane permeabilizer as antibiotic adjuvants.” 8

Antibiotic adjuvants are molecules which when administered with ineffective

antibiotic, enhance its antibacterial property thereby mitigating bacterial

infection.9 Adjuvants can act as anti-resistance as well as anti-virulent agents via

multifaceted mechanisms (Fig.1). Several reports have shown efficacy of

membrane sensitizers to rejuvenate the antimicrobial properties of hydrophobic

antibiotics against GNB.10

Despite having incremental advantage these molecules have not yet been reported

to incorporate some fatal GNB into Erythromycin spectra.

2
Figure 1 A. Pictorial representation of pathogenesis of bacterial infection on

wound. Disruption of epithelial barrier allows the bacteria to penetrate inside the

host. Bacterial multiplication and biofilm formation results in host immune

3
evasion and causes inflammation. B. Major classes of antibiotic adjuvants which

act via blocking the resistance and virulence mechanisms of pathogens.

To tackle this, molecules having facial amphiphilicity and cationic properties such

as Bile acids serving as membrane permeabilizers came into picture. 11 Bile acids

are synthesized in the liver via cholesterol metabolism. 12 The presence of steroidal

skeleton and hydroxyl groups make the scaffold amphiphilic. Several studies have

reported Bile acid derivatives as potential antibacterial agents. 13 Chemical

conjugation of head groups can provide cationic properties to bile acid derivatives

making them selective against negatively charged GNB membrane. Yadav et al

displayed the antibacterial property of Cholic acid (CA) monomers against GNB

but the molecules were having relatively high hemolytic value. 14 To address this

CA-derived dimeric molecule having charged head groups have been synthesized.

In this study we present the adjuvant activity of CA dimers in combination with

Erythromycin against GNB and their mechanistic study. We also report the

efficacy of this combination against murine wound infection.

4
 Chapter 2

Review of Literature

Antibiotic resistance among microbes threatens the achievements of modern

medicine, particularly the six species of ESKAPE pathogens. The post antibiotic

era is not far but now. Most of the lifesaving medical practice such as

chemotherapy, surgery and transplant are effective due to existing antibiotic

therapy.15 But given the evolution of drug resistance pathogens diminishes the

reliability on these treatments. However, to overcome the drug resistant pathogens

antibiotic adjuvants emerged as a boon. Antibiotic adjuvants that suppress the

intrinsic resistance mechanisms can aid in expanding the spectrum of antibiotics

which are ineffective against certain pathogens. 16 Apart from that adjuvants can

lower the dosage of antibiotics thereby mitigating the cytotoxic side effects of a

drug. Adjuvant therapy has an edge over other conventional methods as bacteria

have least evolutionary pressure to develop resistance against compounds which

are not bactericidal. The only clinically approved antibiotic adjuvant is the b-

lactamase inhibitor. b-lactamase is an enzyme that cleaves the b-lactam ring of

lactam antibiotics thereby rendering bacteria resistant against these antibiotics. 17

Aminoglycoside-modifying enzymes (AME) are responsible for aminoglycoside

resistance. Several AME inhibitors have been reported but not been clinically

approved.18

5
Bacteria express efflux pumps which maintain the cellular concentration of

antibiotic below its efficacious levels. A class of adjuvants which can inhibit

efflux pumps can enhance the bactericidal effects of a drug by increased uptake.19

Targeting LPS of GNB is an effective approach to sensitize antibiotics which are

used against GPB but are ineffective against GNB. 20-23 These compounds include

cationic and amphiphilic membrane destabilizers which interact with polyanionic

LPS. OM permeabilizers include molecules such as polymyxins, cationic CA-

derivatives or polyamines. These compounds act in a mechanism similar to those

of antimicrobial peptides.24

Antimicrobial peptides are components of host immune system which are released

in response to microbial infection. These molecules act via various mechanisms

such as barrel formation, carpet (detergent-like mechanism) and toroidal pore

formation.25 The main challenge faced by AMPs is their instability. To address

this various AMP-like compounds have been reported not only to be bactericidal

but also having the ability to enhance antibiotic efficacy against MDR bacteria. 26

Small peptides having cationic functionality have also been shown to display

adjuvant properties. Zeng et al, synthesized and characterized hexadecapeptides

which altered their secondary structure in different medium. One of the

compounds, zp16 was found to act synergistically with vancomycin and

teicoplanin against Klebsiella pneumoniae.27 Their compound showed more

specificity towards bacterial cell as compared to mammalian cells (Fig. 2A).

Recently Konai et al reported a lysine based small molecule, D-LANA-14 which

6
was shown to be membrane active. D-LANA-14 along with obsolete antibiotics

tetracycline or rifampicin showed effective synergism against growing planktonic

cells of meropenem-resistant A. baumannii and P. aeruginosa clinical.28 The

combination of D-LANA with Rifampicin was observed to have antibiofilm

properties (Fig. 2B). Zhu and group screened the adjuvant properties of short

linear antibacterial peptides (SLAPs) which are derivatives of non-ribosomal

peptides. SLAP-S25 was shown to display a broad-spectrum synergistic activity

with major classes of antibiotics against MDR-GN pathogens (Fig. 2C).29

FDA approved drugs have also been shown to display potent adjuvant activity

against MDR bacteria. Chen Xu et al showed that econazole which is a known

anti-fungal compound can act as effective colistin adjuvant that resensitize

colistin resistant Enterobacteriaceae. It permeabilizes the membrane by

dissipating membrane potential (Fig. 2D).30

Apart from the small molecule and peptide-based adjuvants, several cationic

polymers have also been reported to show enhance the permeability of antibiotics

for synergistic antimicrobial therapy. Gupta et al designed Oxanorbornene

polymer series having variable linker length and R-group attached to the terminal

amine. It was observed that with increase in the octanol water partition coefficient

value, membrane sensitizing ability of the polymer enhanced. 31 Ding et al

demonstrated the adjuvant properties of a guanidinium-functionalized

polycarbonate pEt_20 containing 20 repeating units with an ethyl spacer group.

Mechanistic studies revealed that the polymer acts via membrane translocation

7
rather than membrane disruption and acts via binding to several intracellular

targets. The polymer was able to resensitize Rifampicin against GNB.32

CA has been shown to act as an appropriate scaffold for the synthesis of GNB

membrane sensitizer.13 Savage and group demonstrated that proper arrangements

of primary amines or guanidine groups on CA scaffold can be used to design

GNB sensitizers and same can allow the use of GPB specific antibiotics against

GNB.33 Singla et al engineered bile acid oligomers having facial amphiphilic

topology and activity of the oligomers was based on specific linkers. Their lead

compounds 9a and 15a having free amine group in their linker displayed additive

effects while 9a was observed to show synergism with amikacin against GPB.34

Our research group has reported the antibacterial efficacy of CA-derived

monomers against GNB.14 CA-derived dimeric compounds have been shown to

have increased specificity towards bacterial membrane although the activity

against GNB reduced. So, we evaluated the adjuvant activity of these molecules

in combination of Erythromycin and performed mechanistic studies for the same.

8
Figure 2: Different adjuvant and antibiotic combinations for enhanced anti-

bacterial properties. A. (left) Chequerboard depicting the synergy of Zp16 and

9
Vancomycin; (Right) Specificity of FITC labelled Zp16 towards Bacterial cells.

B. Anti-biofilm activity of D-LANA and Rifampicin combination against

Acinetobacter baumannii. C. Synergistic activity of SLAP25 with Colistin against

GNB. D. Chequerboard showing synergistic activity of Econazole with Colistin

against GNB.

10
 Chapter 3

Material and Methods

3.1 Materials Required

Luria Bertani (LB) Broth Miller (M1245), Tryptone soya (TS) broth (M011),

bacteriological agar powder (GRM026), Erythromycin (CMS528) vancomycin

hydrochloride (CMS21) were purchased from HiMedia. Trypsin-EDTA

(59418C), Ciprofloxacin (17850), Propidium iodide (P1470), 2′,7’-

dichlorofluorescein diacetate (35845), N-Phenyl-1-naphthylamine (104043),

Lipopolysaccharides from Escherichia coli O111:B4 (L2630) were purchased

from Sigma and LIVE/DEAD® BacLightTM bacterial viability and counting kit

from Invitrogen. MgCl2 from Merck (13446-4-9) and NaCl from Qualigens

(7647-14-5)

3.2 Hemolytic assay

The series of ten compounds was screened for testing their hemolytic activity

against human red blood cells. 3-4 mL of blood was drawn in EDTA-coated

tubes. RBCs were pelleted down using ficoll hipaque, washed twice with

phosphate buffer saline (PBS) and diluted in 1X PBS (1:50). 3 different

concentrations (50, 100, 200 μg/mL) of 10 compounds were prepared. Equal

volumes of each compound (3 replicates for each concentration) were incubated

with equal volume of diluted RBCs at 37 ˚C at 3 different time points (1 h, 3 h, 6

h). After each time point RBCs were pelleted down by short spin and 100 μL of

11
the supernatant was added in 96 well plate. 1% Triton X 100 was taken as positive

control while 1X PBS was taken as negative control. Absorbance was recorded at

541 nm in SpectraMax® i3x Multi-Mode Detection Platform, Molecular Devices.

Formula used for percentage hemolytic

(OD 541 Sample−OD 541 Blank )

¿ X (100)
(OD 541 0.2 % TritonX 100−OD 541 Blank )

3.3 Minimum Inhibitory concentration

The compounds were screened against GNB: E. coli (MTCC443), K. pnuemoniae

(MTCC3384), and P. aerigunosa (MTCC1688). The antibacterial activity was

expressed as minimum inhibitory concentration (MIC 99). Log phase cultures of all

strains grown in LB broth having 10 5- 106 CFU/mL bacterial cells were treated

with different concentration of our compounds and different antibiotics

(Ciprofloxacin, Erythromycin and Vancomycin). The highest concentration being

128 μg/mL which was serially diluted with LB broth along the abscissa of a 96

well plate and lowest being 0.25 μg/mL. The plates were incubated for 18 h at

37˚C, and MIC99 was recorded as the compound/antibiotic concentration at which

O.D. at 600nm was similar to the Blank (LB broth alone taken as sterility

control). Values were recorded after performing at least three independent

biological experiments performed in four technical replicates.

3.2 Chequerboard assay

Log phase cultures of all strains grown in LB broth having 10 5- 106 CFU/mL

bacterial cells were treated with different combinations of Erythromycin and

Dimer (2 and 3). Micro dilutions were prepared in LB broth. Multichannel dams
12
were used to prepare serial dilutions. Erythromycin was diluted along ordinate

and amphiphile was diluted along abscissa in a 96 well plate. The MIC was

recorded as wells with the lowest concentrations of drugs with absorbance similar

to sterility control (LB broth only) at 600nm. after incubation at 37 °C for 18 h.

The FIC index (FICI) was calculated according to the following formula:

FICI = (MICErythromycin in combination / MICErythromycin alone) + (MICDimer 3 in combination / MICDimer 3 alone)

3.3 Bactericidal time-kill kinetic study

Ability of amphiphile and Erythromycin combination was tested for being

bactericidal using time- kill kinetics standard protocol. GNB culture was grown

up to log phase and then centrifuged at 5000 rpm for 10 minutes. The cells were

resuspended in LB media and OD was adjusted to 10 6 cells/ mL. Cells were

dispensed in tubes and treated with following groups at 37˚C: Untreated, Dimer 3

(8 μg/mL), Erythromycin (32 μg/mL), Combination: Dimer 3 (4 μg/mL) +

Erythromycin (16 μg/mL). Same concentrations were used for all further assays.

After each time point (0, 2, 4, 8, 12 and 24 h), Bacterial suspension was serially

diluted, and plating was done on LB agar plates. Further, plates were incubated at

37C for 16-18 h, and colonies were counted and bacterial load expressed as log 10

CFU/mL as a function of time. Data were represented as means ± standard errors

of the means (SEMs) from three biological replicates.

3.4 Outer membrane permeabilizing assay

To decipher the ability of Dimer 3 to permeate the OM a dye-based assay was

performed. Log Phase bacterial culture was washed and OD was adjusted to 0.1 in

13
1X PBS. NPN dye (N- phenyl-Naphthylamine) dye was added to a 0.1 OD 10mL

bacterial suspension to a final concentration of 10 μM. Fluorescence was

measured up till saturation at an excitation value of 350 nm and emission value of

420 nm. After saturation is obtained, cells were treated with respective

concentration of compound, antibiotic and combination for 15 minutes. Then, the

suspension was transferred into the 96 well plates and kinetics was set up in

Spectramax M5 multimode microplate reader (Molecular Devices, Sunnyvale,

CA, (USA) for 30 min. The assay was conducted in three biological replicates and

data are presented as Means±SEMs from three biological replicates.

3.5 Propidium iodide uptake assay

Bacterial membrane distortion by amphiphile Dimer 3 and combination was

investigated using a propidium iodide uptake assay. Cells were grown until the

log phase and harvested at 5000rpm. Cells were resuspended into 1X PBS having

108 cells/mL and treated for 1 h. After treatment cells were stained with PI (10

µg/mL) and again incubated for 10 min. Incubated cells were washed twice with

PBS and finally resuspended into 400µl 1X PBS. Resuspended samples were

analyzed by flow cytometry using BD FACSVerse flow cytometer (BD

Biosciences, San Jose, CA) under the propidium iodide channel. Percentage of PI-

positive cells were noted and data were expressed as Mean±SEMs from the three

biological replicates of VRE.

3.6 Planktonic cells live/dead staining

14
To illustrate the live and dead cells in planktonic culture, a LIVE/DEAD

BacLight bacterial viability and counting kit was used. Cells were grown to the

log phase and harvested at 5000rpm for 10 min. These cells were further

resuspended into PBS and OD adjusted to 10 8 cells/mL and treated for 1 h. After

1 h, untreated and treated cells were stained with two-component (SYTO-9 and

PI) of LIVE/DEAD Baclight bacterial viability and counting kit. Both the

component of dye mixed into a 1:1 ratio and 1µl of this mix was added into the

treated and untreated cells and incubated for 15 min. After incubation, cells were

washed with PBS and finally resuspended into 100 µl PBS. 10 µl of suspension

was put on the glass slide and covered with a cover glass. After sealing the dried

cover glass with the transparent nail enamel, these slides were observed under an

inverted confocal microscope (Leica TCS SP8; Leica Germany) under a 63X oil

objective in fluorescein isothiocyanate (FITC-for SYTO-9, green) and tetramethyl

rhodamine isocyanate (TRITC- for PI, red) channels.

3.7 Biofilm degradation assay

Antibiofilm activity of Dimer 3 and Erythromycin combination was tested against

P. aeruginosa and E. coli Biofilms. Around 7-8 heat-sterilized coverslips

(18x18mm) were dipped into TSB media with 1% glucose (12mL) in each petri

plate containing P. aeruginosa inoculum (10µl of 108 cells/mL) and incubated at

37˚C for 24 h while E. coli cells were incubated for 48 h. Condensed biofilms

were treated for 12 h and then stained with SYTO-9 and PI of LIVE/DEAD

Baclight bacterial viability and counting kit. 1µl of 1:1 mix was added into the

treated and untreated cells and incubated for 15 min. After incubation biofilms

15
were washed with PBS and placed inverted on slide. Coverslip was sealed using

nail paint and visualized under an inverted confocal microscope (Leica TCS SP8;

Leica Germany) under a 63X oil objective in fluorescein isothiocyanate (FITC-for

SYTO-9, green) and tetramethylrhodamine isocyanate (TRITC- for PI, red)

channels.

3.8 Effect of external LPS and Cations

Serially diluted Dimer 3 in multichannel dams was incubated with 50 μg/mL of

LPS (0.25 μg/mL of cations) for 30 minutes at 37˚C and added in 96 well plate

along abscissa. Further Erythromycin was diluted in multichannel dams and

dispensed along ordinate in same plate. Log phase bacterial suspension was

adjusted to an OD of 0.1 and it was further diluted 10 times. Final 10 5 cells/mL

were added to the plate and incubated for 16-18 h at 37˚C. MIC of Erythromycin

in presence of different concentration of Dimer 3 incubated with LPS or cations

was plotted.

3.9 Reactive Oxygen Species determination

Log phase bacterial culture was harvested at 5000 rpm for 10 min. 10 8 CFU/mL of

cells were treated with Dimer 3 and Erythromycin combination and incubated at

37˚C for 1 h and then centrifuged for 10 minutes at 10000 rpm. Obtained

supernatant was treated with 10 μM of DCFH-DA (Dichlorofluorescein diacetate)

for 1 h. The Reactive Oxygen Species (ROS) formed was measured using

fluorescence spectroscopy with excitation / emission at 485 nm / 535 nm.

16
3.10 Animal studies: Wound infection model

We have explored the antimicrobial efficacy of our potential adjuvant and

Erythromycin combination by using the wound infection model. This model is

already standardized in our lab. Wound infection of a bacterial strain (E. coli and

P. aeruginosa) to BALB/c mice was given. After 18 h of infection, the mice were

divided into different groups each group having 5 mice. The first group was

treated with saline (thrice a day), the second group mice were treated with Dimer

3 (20mg/kg, thrice a day), the third group was treated with Erythromycin

(20mg/kg, thrice a day) and fourth group was treated with a combination of Dimer

3 (10mg/kg) and Erythromycin (10 mg/kg), thrice a day. This treatment continued

for 3 consecutive days and at 4th day, all mice were sacrificed and bacterial load

was quantified by CFUs analysis, the log 10 CFU change was calculated in

respective groups.

17
 Chapter 4

Result and Discussion

4.1 In-vitro screening and bactericidal property of combination

Series of CA-derived dimeric molecules was screened against human RBCs to

check their hemolytic activity. Dimer 2 and Dimer 3 having a linker length of 2

and 3 respectively were found to have minimal toxic profile (Table 1). Further

studies were conducted on these two molecules. These molecules were found to

be minimal bactericidal against E. coli (MTCC443), P. aeruginosa (MTCC1688),

and K. pneumoneae (MTCC3384) with an MIC value above 32 mg/mL. Given

that previous research displayed potent adjuvant-like properties of CA derivatives

31
, we subsequently performed a chequerboard assay to examine the potency of

Dimer 2 and 3 in combination with a hydrophobic macrolide, Erythromycin (Fig.

4.1 B). Fractional inhibitory Concentration (FIC) Index was used as a criterion to

define synergism. FIC<0.5 was said to display synergism. Both Dimers along

with Erythromycin displayed an FIC index as low as 0.043 - 0.078 (Fig. 4.1 D).

18
Table 1. Cytotoxic profile of cholic acid-derived dimeric amphiphiles.

19
20
21
Figure 4.1: Antimicrobial properties of CA-derived dimers in combination

with Erythromycin

A. Chemical structures of CA-derived dimeric (Dimer 1 to 8) and monomers

(CAP1 and CAP3) amphiphiles. B. Chequerboard microdilution assay in presence

of different combinations of Erythromycin and Dimer 3 against GNB (E. coli, P.

aeruginosa and K. pnuemoniae). C. Synergy curves obtained showing reduction

in MIC99 of Erythromycin with increasing Dimer 3 concentration against the 3

bacteria. D. FIC index of Dimer 2 and Dimer 3 with Erythromycin against E.

coli, P. aeruginosa and K. pnuemoniae. E. FIC index values of Dimer 3 and

Erythromycin interaction against clinical isolates of E. coli.

Our two candidate dimers were able to restore the efficiency of Erythromycin

which alone displayed an MIC value of 128 μg/mL against the three GNB. Further

to elucidate the activity of Dimer 3 and Erythromycin combination in inhibiting

bacterial growth, time dependent studies were performed. Growth curve

monitored for 12 h did not show any increase in absorbance at 600nm in presence

of combination [16 ug/mL Erythromycin + 4 ug/mL Dimer 3] while individual

compounds allowed bacterial growth (Fig. 4.2 A and 4.2 B). In addition, time kill

assay revealed complete clearance of bacterial colonies after few hours of

combination treatment as compared to untreated or individual treatment (Fig. 4.2

C and 4.2 D). These findings suggested that Dimer 3 was a potent antibiotic

adjuvant for restoring the efficacy of Erythromycin (which is only used for GPB)

against GNB.

22
Figure 4.2 Kinetics of bacterial growth inhibition in due to Dimer 3 and

Erythromycin Synergy

23
A. P. aeruginosa and B. E. coli Growth curve monitored for 12 h displaying the

bacterial growth inhibiting properties of Dimer 3 and Erythromycin combination.

C. Time killing curves of P. aeruginosa and D. E. coli displaying that

combination is able to eliminate bacterial colonies.

4.2 Mechanism of Dimer 3 combined with Erythromycin

We hypothesized that Dimer 3 which is armed with cationic head groups acts via

disrupting the OM of GNB. Thus, we evaluated the ability of Dimer 3 alone and

in combination with Erythromycin to disrupt the OM integrity using a

hydrophobic, fluorescent probe, 1-N-phenylnaphthylamine (NPN). We observed

increased disruption by Dimer 3 and in combination (Fig 4.3 A and 4.3 B).

Further to acquire insights into potential target we tested the interaction between

Dimer 3 and LPS which is the most abundant component of GNB-OM. Upon

addition of exogenous LPS at a concentration of 0.05 mg/mL the synergism

between Erythromycin and amphiphile reduced. Dimer 3 interacted with the

external LPS which diminished its action against GNB-OM LPS thus reducing the

permeation of Erythromycin inside the cell (Fig. 4.3 C to 4.3 F).

LPS comprises of a negatively charged domain Lipid A. The molecules of Lipid

A are bridged via divalent cations resulting in reduced permeability due to closed

packing.35 Thus, the effect of divalent cations like Mg 2+ on the activity of Dimer 3

as adjuvant was examined in a similar manner. We observed that in presence of

0.25 mg/mL of MgCl2 Dimer 3 could not restore the activity of Erythromycin

24
(Fig. 4.3 G and 4.3 H). These findings suggest that Dimer 3 probably binds at the

cation binding site present in Lipid A moiety of LPS.

25
Figure 4.3 Outer membrane-targeting mechanism of Dimer 3

A. Increase in permeability of OM of P. aeruginosa and B. E. coli probed with

NPN dye upon treatment with Dimer 3. C, D. Reduction in ability of Dimer 3 to

synergize with Erythromycin upon external LPS administration against P.

aeruginosa and E, F. E. coli. G. Effect of divalent cations on the MIC of

Erythromycin in presence of Dimer 3 against P. aeruginosa and H. E. coli.

Ability of Dimer 3 to permeabilize the bacterial membrane was also evaluated

using Propidium Iodide (PI) uptake. It was found that after one hour of treatment

with Dimer 3 and Erythromycin combination the percentage of PI positive cells

increased. PI being a membrane impermeable DNA binding dye can only trespass

compromised membranes (Fig. 4.4 B and 4.4 C) This was also evaluated using

live dead imaging. Planktonic culture when treated with amphiphile along with

Erythromycin were found to have more PI-stained cells as compared to untreated

culture (Fig. 4.4A). However, Dimer 3 alone was able to increase the PI uptake

within bacterial cells.

26
Figure 4.4 Membrane permeabilization and ROS production due to Dimer 3.

A. Fluorescent micrographs obtained after SYTO-PI staining witnessing the

membrane permeabilizing nature of Dimer 3. B. Percentage of PI- positive E. coli

and C. P. aeruginosa cells analyzed via flow cytometry. D. Increase in production

27
of ROS in P. aeruginosa and E. E. coli upon treatment with combination of

Dimer 3 and Erythromycin measured using fluorescent intensity of DCFH-DA.

ROS are involved in a variety of cellular dysfunction mechanisms. 36 To evaluate

the property of Dimer 3 in inducing intracellular ROS formation, we performed a

2’,7’- Dichlorofluorescein diacetate (DCFH-DA) dye-based assay. The

fluorescence intensity was found to be increased in treated sample as compared to

the untreated one. (Fig. 4.4 D and 4.4 E) These results are consistent with the fact

that AMPs also induce ROS generation within the pathogen for mitigating

infection. Thus, Dimer 3 acts in a mechanism similar to AMPs and trigger ROS

generation which enhances bacterial death when used in combination with

antibiotic.37

4.3 Anti-Biofilm activity of Dimer 3 and Erythromycin combination:

Bacterial Biofilms are associated with persistent microbial infections. These are

multilayered aggregates of microbes embedded within extracellular polymeric

substances (EPS), making it a challenge for drug permeation. 38 These structures

when formed on medical devices significantly enhance the risk of nosocomial

infections and enhance the possibility of infection spread. To address this, we

tested anti-biofilm property of Dimer 3 in combination with Erythromycin.

Condensed biofilms for E. coli and P. aeruginosa were treated with combination

for 12 h and visualized under confocal microscope after SYTO9/PI staining.

Obtained results demonstrate that Dimer 3 and Erythromycin when used in

combination displayed potent anti-biofilm activity against GNB biofilms.

28
Figure 4.5 Fluorescence micrographs of untreated Biofilms and after 12 h

treatment with Dimer 3 and Erythromycin combination witnessing potent anti-

biofilm properties of this combination against E. coli and P. aeruginosa biofilms.

29
4.4 Dimer 3 restores Erythromycin against GNB in-vivo

Therapeutic potential of Dimer 3 was investigated in wound infection model. E.

coli or P. aeruginosa cells were applied on a circular wound created in male

BALB/C. One group was administered with normal saline, two of the groups

received individual treatment of Dimer 3 and Erythromycin and fourth group was

treated with a combination of both. After 3 days of treatment. At fourth day the

wound area of the skin was surgically cut, homogenized and spread plated on LB

agar plates. The bacterial counts were assessed by counting the CFU. While

treatment with Dimer 3 and Erythromycin alone could not reduce the bacterial

load, their combination was observed to display around 2 log fold reduction in

CFU as compared to untreated and individually treated groups. These

observations reveal the potency of Dimer 3 in restoring sensitivity towards

Erythromycin (Fig. 4.6).

30
Figure 4.6. In-vivo efficacy of Dimer 3 and Erythromycin combination

against GNB wound infection.

A. Schematic presentation showing the design of animal studies where dimer 3

and Erythromycin combination is tested for its antibacterial activity. B. Bacterial

load reduced on the wound treated with the combination in E. coli and C. P.

aeruginosa.

31
 Conclusion

Over the years the challenge to overcome antimicrobial resistance is increasing.

In addition to bacterial evolution against conventional drugs intrinsic resistance

amongst GNB has threatened the clinical utility of the available antibiotics. In this

study we present the role of CA-derived dimers as membrane permeabilizing

antimicrobial adjuvant which can resensitize Erythromycin against GNB.

Mechanistic investigation revealed that “Dimer 3” interacts with the LPS of the

OM of GNB and induced the production of intracellular ROS. The synergistic

activity of Dimer 3 and Erythromycin combination against GNB was not only

bactericidal but also eliminated bacterial Biofilms. This combination also showed

strong anti-bacterial effect in vivo by reducing bacterial load in mice wound

infection. In conclusion our study confirms the effective potential of Dimer 3 to

act as antibiotic adjuvant by permeabilizing OM of GNB.

32
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