Journal of Ethnopharmacology 264 (2021) 113266

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Synergistic activity of bile salts and their derivatives in combination with

conventional antimicrobial agents against Acinetobacter baumannii
Verica Aleksić Sabo a, Dušan Škorić b, Suzana Jovanović-Šanta b, Isidora Nikolić a,
Csanádi János b, Petar Knežević a, *
a
Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 3, 21 000, Novi Sad, Vojvodina, Serbia
b
Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 3, 21 000, Novi Sad,
Vojvodina, Serbia

A R T I C L E I N F O A B S T R A C T

Keywords: Ethnopharmacological relevance: Bile traditionally was used in wound healing, having erodent, antioxidant and
Bile salts antimicrobial potential. Acinetobacter baumannii is a frequent etiological agent of wound infections, exhibiting
3-dehydro bile acid derivatives high level of resistance to conventional antibiotics.
Acinetobacter baumannii
Aim of the study: To determine the effect of selected bile acid sodium salts and their 3-dehydro (i.e. 3-oxo) de­
Polymyxin B
rivatives, as well as their combinations with commercial antibiotics against A. baumanniia, to confirm bile
Ciprofloxacin
Gentamicin ethnopharmacological application in wound healing from aspect of microbiology.
Synergy Materials and methods: The sensitivity of reference and multidrug resistant (MDR) A. baumannii strains to bile
DOSY NMR salts, their derivatives and conventional antibiotics were examined by a microtiter plate method. The interaction
of bile salts/derivatives and antibiotics was examined by a checkerboard method and time kill curve method. The
interaction of bile salts with ciprofloxacin in terms of micelles formation was examined by DOSY NMR technique.
Results: The bile salts sodium deoxycholate (Na-DCA) and sodium chenodeoxycholate (Na-CDCA), as well as their
derivatives sodium 3-dehydro-deoxycholate (Na-3DH-DCA) and sodium 3-dehydro-chenodeoxycholate (Na-3DH-
CDCA), potentiate antibiotic activity and resensitize A. baumannii. The bile salts and their derivatives enhance
A. baumannii sensitivity to antibiotics, particularly those that should penetrate cell to exhibit activity. The sodium
salts of bile acid derivatives, namely Na-3DH-DCA and Na-3DH-CDCA, showed synergy against both reference
and MDR strain in combination with ciprofloxacin or gentamicin, while synergy with gentamicin was obtained in
all combinations, regardless of bile salt type and bacterial strains. The synergy with Na-3DH-CDCA was further
confirmed by the time-kill curve method, as bacterial number decreased after 12 h. NMR experiment revealed
that this bile salt derivative and ciprofloxacin form co-aggregates when bile salts concentration was higher than
critical micelle concentrations (CMC), which indicate the possibility that bile salts enhance ciprofloxacin cell
penetration by membrane destabilization, contributing to the synergy.
Conclusion: The synergistic interactions between bile salts or derivatives with ciprofloxacin and particularly
gentamicin represent a promising strategy for the treatment of A. baumannii wound infections.

1. Introduction
Prior to antibiotic discovery, many naturally available agents were
used to heal wound and burn infections. Paulus Aegineta (607–690) was
an encyclopedist from the Graeco-Roman period, who recorded that
among several other agents, ox bile is an erodent (Forrest, 1982). Ero­
dents are still used for wound healing to remove dead tissue, improve
healing, and decrease probability of infections (Clasper, 2014). In an
English book Bald’s Leechbook from Anglo-Saxon period (10th century) it
is indicated that lump in eye should be treated with crushed garlic

Abbreviations: MDR, multidrug resistant; Na-CA, sodium cholate; Na-DCA, sodium deoxycholate; Na-CDCA, sodium chenodeoxycholate; Na-3DH-CA, sodium 3-
dehydro-cholate; Na-3DH-DCA, sodium 3-dehydro-deoxycholate; Na-3DH-CDCA, sodium 3-dehydro-chenodeoxycholate; TTC, triphenyl-tetrazolium chloride; MIC,
minimal inhibitory concentration; FIC, fractional inhibitory concentration; FICI, fractional inhibitory concentration index; FICIm, minimum value of FICI; DOSY,
Diffusion-ordered spectroscopy; NMR, Nuclear magnetic resonance; CMC, critical micelle concentrations.
* Corresponding author.
E-mail address: petar.knezevic@dbe.uns.ac.rs (P. Knežević).

https://doi.org/10.1016/j.jep.2020.113266
Received 30 June 2020; Received in revised form 3 August 2020; Accepted 8 August 2020
Available online 15 August 2020
0378-8741/© 2020 Elsevier B.V. All rights reserved.
V. Aleksić Sabo et al. Journal of Ethnopharmacology 264 (2021) 113266

There are different bile salt-based complex systems (polymers or

Fig. 1. Bile acid salts as biplanar amphiphiles with convex hydrophobic (β) and
concave hydrophilic (α) face.
(Allium sativum L.) and another Allium species, combined with wine and
oxgall (bovine bile) (Cockayne, 1864–1866). In traditional Chinese
medicine, during the Tang dynasty (618–907 CE), pig, wild boar and
human bile diluted with alcohol were used for burns and wounds
dressing as artificial skin with antioxidant (and probably antimicrobial)
properties. In addition, in Chinese traditional medicine, dog and mouse
bile were used for wound and burn healing (Wang and Carey, 2014). The
traditional use of bile indicates its potential antimicrobial activity
against wound infecting bacteria, which has not been exploited yet to a
great extent.
Bile is a complex aqueous secretion that originates from hepatocytes
and consists of ~95% water in which a number of endogenous constit­
uents are dissolved, among which most dominant are bile salts (Boyer,
2013). Bile acids (conjugated as well as non-conjugated) are known to
be amphiphiles. Their anions self-associate in aqueous solution to form
micelles. This property enables them to perform their main physiolog­
ical role – the formation of mixed micelles with lipid components of food
(phospholipids, fatty acids and monoacylglycerols) during digestion
process (Monte et al., 2009). It is important to emphasize that the
structure of these molecules significantly differs from the structures of
most other surfactants, since bile acids are biplanar amphiphiles with
convex hydrophobic (β) and concave hydrophilic (α) face (Fig. 1),
compared to hydrophilic head - hydrophobic tail type of surfactants
(Hofmann et al., 1984). The special type of amphiphilicity, jointly with
other physicochemical properties, highlighted bile acids and their de­
rivatives as promising agents in the field of drug delivery. This is espe­
cially useful in case of difficult drug delivery if the drug is purely soluble
or degradable; in such manner drugs absorption and efficiency could be
increased.
complexes) that can be used as drug delivery carriers: mixed micelles,
bilosomes, polymers and conjugates (Faustino et al., 2016). One of the
most promising is the formation of mixed micelles with drug molecules
which enables better permeability of lipophilic drug molecules through
biological membranes (Poša and Kuhajda, 2010a, 2010b; Sayye­
d-Ahmad et al., 2010; Poša et al., 2012). Further, it is possible to make
conjugates of bile acids covalently bonded to biologically active com­
pound (Kramer et al., 1997). Finally, it is possible to undermine mem­
brane stability with bile acid anions in order to deliver the drug
molecule into the pathogen cell. High membrane toxicity of bile acids
and toxicity in the gastrointestinal tract of patients (Petruzzelli et al.,
2007) could be a problem with the application in medicine. Conse­
quently, there is a lot of interest in finding bile acid derivatives or for­
mulations with lower membrane toxicity. One of the promising solutions
for this problem is to use oxo bile acids, which could be obtained by
oxidation of one or more hydroxyl groups on the steroidal skeleton (Poša
and Kuhajda, 2010a, 2010b). The oxo derivatives are naturally present
in organisms as a result of biotranformation of bile salts by gut micro­
biota (Ridlon et al., 2016).
The aim of the study is to determine the effect of selected bile acid
sodium salts and their 3-dehydro (i.e. 3-oxo) derivatives, as well as their
combinations with commercial antibiotics, against A. baumannii. As a
model bacterium we chose Acinetobacter baumannii from several reasons:
(1) being a gram-negative bacterium, it is more challenging for treat­
ment than gram-positive bacteria; (2) the bacterium is frequent etio­
logical agent of wound infections, particularly war-related wound
infections, because of presence in the environment and easy wound/
burn contamination (CDC, 2004); (3) it is an important nosocomial
pathogen that prolongs the length of hospital stays and increases health
care costs (Johnson et al., 2007; Trottier et al., 2007); (4) bacteremia
caused by A. baumannii has a high mortality risk (Gkrania-Klotsasal and
Hershow, 2006; Jerassy et al., 2006; Robenshtok et al., 2006) and (5)
A. baumannii has a remarkable ability to develop or acquire resistance to
various antimicrobial agents, frequently being multidrug and pandrug
resistant (Peleg et al., 2008). Thus, the novel antimicrobial agents and
strategies for combating MDR A. baumannii infections are urgently
required and bile salts and their derivatives may offer a solution.
2. Materials and methods
2.1. Bacterial strains
In the study, six Acinetobacter baumannii strains were used: the
reference strain ATCC 19606 from the American Type Culture Collection
(Rockville, MD, USA) and five previously characterized MDR wound
isolates (Aba-4156, Aba-4803, Aba-4914, Aba-5055, Aba-6673) (Aleksic
et al., 2014). Of particular importance was MDR strain Aba-4914, as its
MICs were above the cut-off for A. baumannii strains for all applied

Table 1
Sensitivity of A. baumannii wound isolates to bile salts and antibiotics.
MIC of antibacterial agents Acinetobacter baumannii strains

ATCC 19606 Aba-4156 Aba-4803 Aba-4914 Aba-5055 Aba-6673

Bile salts (mg mL− 1) Na-CA 10.1 1.4 20.1 8.0 8.0 6.4
Na-DCA 0.8 0.5 0.5 0.5 0.7 1.0
Na-CDCA 1.0 0.5 1.0 1.4 1.0 2.0
Bile salts derivatives (mg mL− 1) Na-3DH-CA 16.0 5.7 16.0 11.3 16.0 16.0
Na-3DH-DCA 2.0 0.7 1.0 2.8 2.0 2.0
Na-3DH-CDCA 2.0 1.0 1.0 2.0 1.4 2.8
Antibiotics (μg mL− 1) CIP 0.35 45.3 45.3 64.0 64.0 45.3
PMB 0.5 4.0 0.7 4.0 4.0 0.7
GEN 16.0 >256.0 64.0 >256.0 16.0 64.0

MIC – minimal inhibitory concentration, Na-CA - sodium-cholate, Na-DCA - sodium-deoxycholate, Na-CDCA-sodium-chenodeoxycholate, Na-3DH-CA - sodium-3-
dehydrocholate, Na-3DH-DCA - sodium-3-dehydrodeoxycholate, Na-3DH-CDCA - sodium-3-dehydrochenodeoxycholate, CIP-ciprofloxacin, PMB-polymyxin B, GEN-
gentamicin.

2
V. Aleksić Sabo et al.
Fig. 2. Structure of tested bile acid sodium salts (Na-CA, Na-DCA and Na-
CDCA) and their 3-dehydro derivatives (Na-3DH-CA, Na-3DH-DCA and Na-
3DH-CDCA).
antibiotics, so this strain was used further as a model, along with
ATCC19606 (Table 1) (Aleksic et al., 2014; Peleg et al., 2008). The
strains were stored in Luria-Bertani broth supplemented with glycerol
(10% v/v) at − 70 ◦ C and for the antibacterial tests the strains were
grown also in LB and incubated overnight at 37 ◦ C. For all experiments,
Mueller-Hinton agar or broth was used, unless stated otherwise.
2.2. Antimicrobial agents
In the study, as alternative antimicrobial agents bile salts: sodium
cholate (Na-CA), sodium deoxycholate (Na-DCA), and sodium cheno­
deoxycholate (Na-CDCA), as well as their 3-dehydro derivatives: sodium
3-dehydro-cholate (Na-3DH-CA), sodium 3-dehydro-deoxycholate (Na-
3DH-DCA), and sodium 3-dehydro-chenodeoxycholate (Na-3DH-CDCA)
were used (Fig. 2). 3-Dehydro derivatives were synthesized following
literature procedures (Tserng, 1978), so free acids were converted to
sodium salts by titration with an equimolar amount of
sodium-hydroxide solution, followed by freeze-drying of solutions.
Three frequently used antibiotics were included in the study, for syn­
ergism tests and method quality control: ciprofloxacin, polymyxin B and
gentamicin (Table 1).
2.3. Broth microdilution susceptibility testing
The activity of bile salts and their derivatives as alternative anti­
bacterial agents was determined by the broth microdilution suscepti­
bility testing method according to CLSI (2007). The two-fold dilutions of
bile salts in sterile distilled water were prepared in the 96-well micro­
titer plates and mixed with inoculated double-strength Mueller-Hinton
broth in a ratio of 1:1 (v/v). The final bacterial count was approx. 1 ×
106 CFU mL− 1. The final concentrations of tested bile salts were in a
range from 32 mg/mL to 0.25 mg/mL. Microtiter plates were incubated
overnight at 37 ◦ C, wells were supplemented with TTC solution to obtain
final conc. 0.1% and the microtiter plates were incubated for additional
2 h at 37 ◦ C until red color of formazan appeared in the control wells.
The minimum concentration of bile salt required to prevent the
appearance of red color, i.e. formation of formazan, was considered a
MIC. The experiment was performed in triplicate and in two indepen­
dent occasions. The results are represented as geometrical means of
replications.
2.4. Broth microdilution checkerboard method
The activity of antimicrobial agent combinations – conventional
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Journal of Ethnopharmacology 264 (2021) 113266
antibiotics and bile salts/bile salt derivatives – was determined by a
modified two-dimensional microdilution (checkerboard) method
(Wagner and Ulrich-Merzenich, 2009; Knezevic et al., 2016). As test
agents in combinations, three antibiotics and two bile salts were used
(Table 1). Antibiotics ciprofloxacin, polymyxin B and gentamicin were
selected according to their previously determined MICs. The antibiotics
were combined with Na-DCA, Na-CDCA, Na-3DH-DCA and
Na-3DH-CDCA, selected according to detected anti-A. baumannii effect
in the present study. The plates were prepared to obtain the final bile salt
concentrations in the range from 1 × MIC to 1/128 × MIC (decreasing
vertically), while the final antibiotic concentrations ranged from 1 ×
MIC to 1/32 × MIC (decreasing horizontally). Based on the tested con­
centrations of the agents in combination, it was possible to detect
different types of interactions (synergistic, additive or indifferent), with
an exception of antagonistic, which was not of interest in this study.
After the preparation of antimicrobial agent combinations in the mi­
crotiter plates, bacterial suspensions of the tested strains were prepared
as described above. The microtiter plates were incubated overnight at
37 ◦ C, and the results were read in the manner described above with
addition of TTC. The obtained MIC values of the tested combinations
were used to determine the fractional inhibitory concentration index
(FICI), according to the formula and the interpretation of the interaction
type between the antimicrobial agents. The interactions between the
antimicrobial agents in combination were estimated after determining
the MIC values by calculating the FICI for each combination of antimi­
crobial agents according to the following formulas:
FIC of antimicrobial agent A = MIC of agent A in combination / MIC of agent
A alone
FIC of antimicrobial agent B = MIC of agent B in combination / MIC of agent
B alone
FICI = FIC of agent A + FIC of agent B
The obtained FICI values were interpreted as follows: synergistic
effect if FICI ≤0.5; additive effect if 0.5 <FICI ≤1.0, and indifferent
effect if 1.0 <FICI ≤4.0 (Minato et al., 2018).
All experiments were performed in at least two independent repeti­
tions, and the calculated FICI were shown graphically with the corre­
sponding standard deviation by the isobolograms (Berenbaum, 1989)
constructed using KaleidaGraph 4.0 (Synergy Software KaleidaGraph,
USA).
2.5. Time-kill method
Determination of the kinetics of bacterial growth inhibition was done
using the time-kill assay (Verma, 2007; Knezevic et al., 2016). This test
was performed as a confirmation of detected synergistic interactions
between conventional and alternative antimicrobial agents. The change
in the bacterial count during the incubation period was monitored in
four test tubes in parallel. The content of the tubes with MH broth was as
follows: (I) only bacterium, strain ATCC 19606 (1 × 108 CFU mL− 1); (II)
bacterium and subinhibitory antibiotic concentration that gave a syn­
ergistic effect in combination with bile salt (CIP = 0.125 μg mL− 1, PMB
= 0.25 μg mL− 1 and GEN = 0.5 μg mL− 1); (III) bacterium and bile salt
subinhibitory concentration that expressed a synergistic effect in com­
bination with an antibiotic (Na-3DH-DCA = 0.5 mg/mL or
Na-3DH-CDCA = 0.5 mg/mL); and (IV) bacterium and subinhibitory
concentrations of antibiotic and bile salt.
The final volume of each tube, i.e. each treatment was 10 mL. The
tubes were incubated at 37 ◦ C for 24 h, and the bacterial count was
determined after 0, 12 and 24 h of incubation by inoculation of the MH
agar with appropriate dilutions using the spread plate method. The
plates were incubated at 37 ◦ C overnight and the bacterial colonies were
counted. The experiment was carried out in two repetitions in two in­
dependent experiments, and the results were expressed as the mean log
V. Aleksić Sabo et al. Journal of Ethnopharmacology 264 (2021) 113266

Fig. 3. Isobolograms presenting effects of bile salts (Na-DCA and Na-CDCA) and their derivatives (Na-3DH-DCA and Na-3DH-CDCA) combination with antibiotics
(ciprofloxacin, polymyxin B and gentamicin) against A. baumannii reference strain ATCC 19606. Red stars denote point of synergism. FICIm, minimum value of FICI in
the tested combinations is shown. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.).

value of the bacterial count with the corresponding standard deviation
(mean ± standard deviation).
The interaction was considered synergistic and effective against
A. baumannii isolates if the initial bacterial count after 24 h of incuba­
tion, using a combination of bile salt and antibiotic was reduced by ≥ 2
log, compared to a more effective agent when administered alone (either
bile salt or antibiotic) (Knezevic et al., 2013).
2.6. NMR measurements
All NMR measurements were performed on a Bruker AVANCE III HD
400 MHz spectrometer, equipped with Prodigy cooled probe head at
298 K. For DOSY measurements the standard Bruker ledbpgp2s pulse
program was used.
3. Results and discussion
The bile salts expressed bacteriostatic activity against A. baumannii
wound isolates in the concentrations from 0.5 mg/mL to 20.1 mg/mL,
depending on bile salt and bacterial strain (Table 1). Compounds with
the lowest potential were sodium cholate (Na-CA) and its 3-dehydro
derivative (Na-3DH-DCA), as their MICs were very high. Sodium salts
of DCA and CDCA, namely Na-DCA and Na-CDCA, and their 3-dehydro
derivatives (Na-3DH-DCA and Na-3DH-CDCA) were more potent. The

Fig. 4. Isobolograms presenting effects of bile salts (Na-DCA and Na-CDCA) and their derivatives (Na-3DH-DCA and Na-3DH-CDCA) combination with antibiotics
(ciprofloxacin, polymyxin B and gentamicin) against MDR A. baumannii wound isolate Aba-4914. Red stars denote point of synergism. FICIm, minimum value of FICI
in the tested combinations is shown. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

4
V. Aleksić Sabo et al.
Fig. 5. Time-kill curves showing effect of sub-inhibitory concentrations combinations of antibiotics (ciprofloxacin, polymyxin B and gentamicin) and bile salts Na-
3DH-DCA (left) and Na-3DH-CDCA (right) against A. baumannii ATCC19606 (CIP = 0.125 μg mL− 1, PMB = 0.25 μg mL− 1, GEN = 0.5 μg mL− 1); (Na-3DH-DCA = 0.5
mg/mL, Na-3DH-CDCA = 0.5 mg/mL).
most active was Na-DCA, since all MDR strains had MICs in range 0.5–1
mg/mL. The slightly better activity of unmodified bile salts is probably a
result of their higher toxicity to membranes, which additionally
contribute to bile salt anti-A. baumannii activity. It is known that
replacing hydroxyl groups in bile salts with oxo groups produces
significantly less surface active and less lipophilic bile salts with higher
CMC and consequently with diminished membrane toxicity (Chen et al.,
2012). The effect of Na-3DH-DCA was considerable (0.7–2.0 mg/mL),
but also of Na-CDCA (0.5–2.0 mg/mL) and Na-3DH-CDCA (1.0–2.8
mg/mL). The determined concentrations were in accordance with pre­
viously reported A. baumannii sensitivity to Bile Salts No. 3 (mixture of
sodium cholate and sodium deoxycholate), which are usually used for
selective isolation of Acinetobacter spp. in order to inhibit other sensitive
bacteria at concentration 0.125% (Jawad et al., 1994). However, the
study evaluating selective media for isolation of Acinetobacter spp.
showed that the Leeds medium that does not contain bile salts is better
for this purpose than Herella or Holton’s agar, which contain bile salts
that inhibit some species/strains of Acinetobacter. Our results indicate
moderate to high antimicrobial effect of single bile salts or their de­
rivatives against A. baumannii, so these agents were further examined in
combination with antibiotics.
Combined effect of bile salts and antibiotics against strain ATCC
19606 and MDR 4914 are presented in Figs. 3 and 4, respectively. In
combination of Na-DCA or Na-CDCA with ciprofloxacin against
A. baumannii ATCC 19606 the synergistic effect was confirmed (FICIm =
0.42 and FICIm = 0.46, respectively). However, the same combination
was indifferent or additive against MDR strain (FICIm = 1.03 and FICIm
= 0.56). This difference indicates that the synergy with bile salts is not
universal, but dependent on strain properties. However, bile salts de­
rivatives, Na-3DH-DCA and Na-3DH-CDCA acted synergistically with
ciprofloxacin against both ATCC19606 (FICIm = 0.39 and FICIm = 0.27)
and MDR4914 strains (FICIm = 0.39 and FICIm = 0.38, respectively).
Interestingly, polymyxin B showed synergistic activity against MDR
strain (FICIm in range 0.26–0.42), regardless to applied salt, while syn­
ergy was absent with ATCC19606 strain (FICIm in range 0.59–1.00). This
difference can be ascribed to variation of lipopolysaccharide structure
among the strains, particularly O-side chains, since polymyxin B in­
teracts with lipid A in the lipopolysaccharides of outer membrane,
crossing the outer membrane through a “self-promoted uptake” mech­
anism and then interacts with the cytoplasmic membrane causing cell
death (Trimble et al., 2016). Another explanation can be based on the
difference in MICs, since ATCC19606 was already sensitive to low
concentration of polymyxine B. As MIC for MDR 4914 was 4 μg mL− 1,
the synergistic interactions decreased effective dose of this antibiotic,
making it susceptible with MIC under cutoff for resistance (<4 μg mL− 1).
However, because of the obtained variation of synergy with polymyxin B
it seems as less appropriate agent for combining with bile salts, but
re-sensitization of MDR strains should be noticed and studied further.
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Journal of Ethnopharmacology 264 (2021) 113266
The best results were obtained with gentamicin, as synergy was
detected with bile salts and their derivatives against both strains. The
salts Na-DCA and Na-CDCA gave obvious synergy with gentamicin
against both ATCC19606 (FICIm = 0.16 for both salts) and MDR wound
isolate (FICIm = 0.25 and FICIm = 0.13). As MIC cutoff for gentamicin
resistance is ≥ 16 μg mL− 1, it is obvious that in the synergistic combi­
nations MIC was significantly reduced, below cutoff, indicating that bile
salts and their derivatives can resensitize A. baumannii to this amino­
glycoside antibiotic. The synergy was achieved when Na-3DH-DCA or
Na-3DH-CDCA were combined with gentamicin against reference strain
(FICIm = 0.06 and FICIm = 0.16) (Fig. 3). The effect was similar against
MDR strain (FICIm = 0.34 and FICIm = 0.31) (Fig. 4).
The checkerboard results obtained for antibiotics in combination
with Na-3DH-DCA or Na-3DH-CDCA against strain ATCC19606 were
further confirmed by time-kill curve method (Fig. 5), as these bile salt
derivatives are less toxic to human cells (Chen et al., 2012). The results
showed that combinations of bile salts derivatives acted synergistically
with ciprofloxacin and gentamicin and that are very potent, since bac­
terial number was decreased by more than 2 log after 12 h of incubation.
In contrary, polymyxin B did not decrease bacterial number, confirming
lack of synergy.
The reason of better effect of gentamicin and ciprofloxacin in com­
parison to polymyxin B is probably different mechanism of action of
these agents. As indicated above, polymyxin B interacts with cell
membrane, while ciprofloxacin and gentamicin should penetrate into
cells and interact with DNA gyrase and ribosomes, respectively (Zhao
et al., 1997; Tangy et al., 1985). Thus, it seems that bile salts enhance
drug penetration into the cells. Using A. baumannii as a model, Lopez
et al. (2017) recently confirmed that 0.5% of bile salts mixture (cholic
acid sodium salt 50% and deoxycholic acid sodium salt 50%) decreased
MICs for ciprofloxacin from 0.5 μg mL− 1 to 0.25 μg mL− 1, which is in
accordance with our findings. Previously it was confirmed that 0.5% of
bile salts from ox bladder can increase sensitivity of Lactobacillus plan­
tarum to aminoglycosides, which was ascribed to the permeabilization of
the cell membrane (Elkins and Mullis, 2004). In this context, examining
effect of oleanolic acid on aminoglycoside antibiotics against
A. baumannii, Shin and Park (2015) found that oleanolic acid increase
A. baumannii permeability to gentamicin through changes in membrane
lipid composition and in ATP synthesis. The authors indicated, based on
structural similarity of bile salts and oleanolic acid, that findings of
Elkins and Mullis (2004) are consequence of membrane
permeabilization.
To gain more information about exhibited synergism between anti­
biotics and bile acid sodium salts, we performed DOSY NMR experi­
ments. The main goal of these experiments was to determine whether
the antibiotic ciprofloxacin forms co-aggregates with 3-dehydro bile
acid anions or not, to elucidate reasons underlying synergy. Ciproflox­
acin was chosen for this experiment because it is the smallest molecule
V. Aleksić Sabo et al.
Fig. 6. 1H DOSY NMR of 2 mM (0.735 mg/mL) ciprofloxacin (A), 25 mM (10.31 mg/mL) 3DH-CDCA (B) and mixture of 2 mM (0.735 mg/mL) ciprofloxacin and 25
mM (10.31 mg/mL) 3DH-CDCA (C).
among three tested antibiotics, thus the probability of forming mixed
aggregates with bile acid anions seems to be the most certain. Further,
A. baumannii was resensitized to ciprofloxacin in combination with bile
salts and we postulated that synergistic effect is mediated via co-
aggregates formation of bile acids and this antibiotic.
Poša (2011) previously determined CMCs of 3DH-DCA, 3DH-CDCA
and 3DH-CA, which were 20, 17 and 48 mM, i.e. 8.25, 7.01, 20.67 mg
mL-1, respectively. Thus, series of solutions in D2O were made with 2
mM, i.e. 0.735 mg mL-1 of ciprofloxacin hydrochloride and 3-oxo bile
acid sodium salts, in concentrations below and above CMC. On Fig. 6
DOSY NMR spectra are shown for the mixtures of ciprofloxacin hydro­
chloride and 3DH-CDCA (2 mM and 25 mM), with the most noticeable
micelles formation. As it can be seen, log D value for ciprofloxacin hy­
drochloride at 2 mM is − 9.30, while log D for 3DH-CDCA at 25 mM
(10.31 mg mL-1; above CMC) is − 9.44. In mixture of 2 mM of cipro­
floxacin hydrochloride and 25 mM 3DH-CDCA there is one diffusion
front at the same log D value, as for bile salts above CMC (− 9.44). The
change in log D value for ciprofloxacin hydrochloride signals indicates
that this molecule is incorporated in the micelles of bile salts when they
are present in the solution. The same behavior of ciprofloxacin hydro­
chloride was noticed with other two 3-dehydro bile salts. It has been
shown in the NMR experiments that co-aggregates are formed only when
bile acid sodium salt concentration is above CMC. Accordingly, in the
synergism experiments, where concentrations of bile salts were lower
than CMCs, bile salts probably did not form co-aggregates, but enhance
ciprofloxacin penetration into the cells via membrane destabilization.
4. Conclusion
The bile salts, as endogenous non-toxic molecules, and their de­
rivatives can enhance A. baumannii sensitivity to antibiotics, particularly
to antibiotics that should penetrate cell to exhibit activity. The sodium 3-
dehydro-deoxycholate (Na-3DH-DCA) and sodium 3-dehydro-cheno­
deoxycholate (Na-3DH-CDCA) showed synergy against both reference
and MDR strains in combination with ciprofloxacin or gentamicin, while
synergy with gentamicin was obtained regardless of salt type and bac­
terial strains. Taking into account the strains’ origin, it is obvious that
the obtained synergistic interactions should be further examined in vivo
and in clinical trials, particularly with gentamicin. The synergistic
concentrations can be easily obtained topically for treatment of infected
wounds, so should be further examined using corresponding in vivo
models.
6
Journal of Ethnopharmacology 264 (2021) 113266
Acknowledgements
The authors acknowledge financial support of the Ministry of Edu­
cation, Science and Technological Development of the Republic of
Serbia (Grant No. 451-03-68/2020–14/200125), as well as HEMOFARM
AD, STADA Group for kind donation of ciprofloxacin hydrochloride for
NMR experiments.
References
Aleksic, V., Mimica-Dukic, N., Simin, N., Stankovic Nedeljkovic, N., Knezevic, P., 2014.
Synergistic effect of Myrtus communis L. essential oils and conventional antibiotics
against multi-drug resistant Acinetobacter baumannii wound isolates. Phytomedicine
21, 1666–1674. https://doi.org/10.1016/j.phymed.2014.08.013.
Berenbaum, M., 1989. What is synergy? Pharmacol. Rev. 41, 93–141.
Boyer, J.L., 2013. Bile formation and secretion. Comp. Physiol. 3, 1035–1078. https://
doi.org/10.1002/cphy.c120027.
CDC, 2004. Acinetobacter baumannii infections among patients at military medical
facilities treating injured U.S. service members. J. Am. Med. Assoc. 292, 2964–2966,
2002-2004 (Reprinted from MMWR, vol 53, pg 1063-1066, 2004).
Chen, G., Yang, L., Zhang, H., Tucker, I.G., Fawcett, J.P., 2012. Effect of ketocholate
derivatives on methotrexate uptake in Caco-2 cell monolayers. Int. J. Pharm. 433,
89–93. https://doi.org/10.1016/j.ijpharm.2012.04.077.
Clasper, J., 2014. The management of military wounds in Middle ages. In: Kirkham, A.,
Warr, C. (Eds.), Wounds in Middle Ages. Taylor & Francis, London, p. 23.
Clinical and Laboratory Standards Institute, 2007. Performance standards for
antimicrobial susceptibility testing. In: Seventh Informational Supplement CLSI
Document M100-S17. Clinical and Laboratory Standards Institute, 940 West Valley
Road, Suite 1400, Wayne, Pennsylvania, USA, 19087-1898.
Cockayne, O., 1864–1866. Leechdoms, wortcunning and starcraft: being a collection of
documents, for the most part never before printed, illustrating the history of science
before the Norman conquest. In: Rolls Series 35th, 3. Longman, Green, Longman,
Roberts, and Green, London, United Kingdom. https://doi.org/10.5962/bhl.
title.58724.
Elkins, C.A., Mullis, L.B., 2004. Bile-mediated aminoglycoside sensitivity in Lactobacillus
species likely results from increased membrane permeability attributable to cholic
acid. Appl. Environ. Microbiol. 70, 7200–7209. https://doi.org/10.1128/
AEM.70.12.7200-7209.2004.
Faustino, C., Serafim, C., Rijo, P., Pinto Reis, C., 2016. Bile acids and bile acid
derivatives: use in drug delivery systems and as therapeutic agents. Expet Opin. Drug
Deliv. 13, 1133–1148. https://doi.org/10.1080/17425247.2016.1178233.
Forrest, R.D., 1982. Early history of wound treatment. J. R. Soc. Med. 75, 198–205.
Hofmann, A.F., Roda, A., 1984. Physicochemical properties of bile acids and their
relationship to biological properties: an overview of the problem. J. Lipid Res. 25,
1477–1489.
Jawad, A., Hawkey, P.M., Heritage, J., Snelling, A.M., 1994. Description of Leeds
Acinetobacter Medium, a new selective and differential medium for isolation of
clinically important Acinetobacter spp., and comparison with Herellea agar and
Holton’s agar. J. Clin. Microbiol. 32, 2353–2358. https://doi.org/10.1128/
JCM.32.10.2353-2358.1994.
Knezevic, P., Aleksic, V., Simin, N., Svircev, E., Petrovic, A., Mimica-Dukic, N., 2016.
Antimicrobial activity of Eucalyptus camaldulensis essential oils and their interactions
with conventional antimicrobial agents against multi-drug resistant Acinetobacter
baumannii. J. Ethnopharmacol. 178, 125–136. https://doi.org/10.1016/j.
jep.2015.12.008.
V. Aleksić Sabo et al.
Knezevic, P., Curcin, S., Aleksic, V., Petrusic, M., Vlaski, L., 2013. Phage-antibiotic
synergism: a possible approach to combating Pseudomonas aeruginosa. Res.
Microbiol. 164, 55–60. https://doi.org/10.1016/j.resmic.2012.08.008.
Kramer, W., Wess, G., Enhsen, A., Falk, E., Hoffmann, A., Neckermann, G., Schubert, G.,
Urmann, M., 1997. Modified bile acids as carriers for peptides and drugs. J. Contr.
Release 46 (1–2), 17–30. https://doi.org/10.1016/S0168-3659(96)01599-4.
López, M., Blasco, L., Gato, E., Perez, A., Fernández-Garcia, L., Martínez-Martinez, L.,
Fernández-Cuenca, F., Rodríguez-Baño, J., Pascual, A., Bou, G., Tomás, M., 2017.
Response to bile salts in clinical strains of Acinetobacter baumannii lacking the
AdeABC efflux pump: virulence associated with quorum sensing. Front. Cell. Infect.
Microbiol. 7, 143–155. https://doi.org/10.3389/fcimb.2017.00143.
Minato, Y., Dawadi, S., Kordus, S.L., Sivanandam, A., Aldrich, C.C., Baughn, A.D., 2018.
Mutual potentiation drives synergy between trimethoprim and sulfamethoxazole.
Nat. Commun. 9, 1003–1010. https://doi.org/10.1038/s41467-018-03447-x.
Monte, M.J., Garcia Marin, J.J., Antelo, A., Vazquez Tato, J., 2009. Bile acids: chemistry,
physiology, and pathophysiology. World J. Gastroenterol. 15, 804–816. https://doi.
org/10.3748/wjg.15.804.
Peleg, A.J., Seifert, H., Paterson, D.L., 2008. Acinetobacter baumannii: emergence of a
successful pathogen. Clin. Microbiol. Rev. 21, 538–582. https://doi.org/10.1128/
CMR.00058-07.
Petruzzelli, M., Vacca, M., Moschetta, A., Sasso, R.C., Palasciano, G., Van Erpecum, K.J.,
Portincasa, P., 2007. Intestinal mucosal damage caused by non-steroidal anti-
inflammatory drugs: Role of bile salts. Clin. Biochem. 40, 503–510. https://doi.org/
10.1016/j.clinbiochem.2007.01.015.
Poša, M., 2011. QSPR study of the effect of steroidal hydroxy and oxo substituents on the
critical, micellar concentration of bile acids. Steroids 76, 85–93. https://doi.org/
10.1016/j.steroids.2010.09.003.
Poša, M., Csanádi, J., Kövér, K., Guzsvány, V., Batta, G., 2012. Molecular interactions
between selected sodium salts of bile acids and morphine hydrochloride. Colloids
Surf., B 94, 317–323. https://doi.org/10.1016/j.colsurfb.2012.02.014.
Poša, M., Kuhajda, K., 2010a. Influence of bile acids on the adsorption of lidocaine and
verapamil in an in vitro experiment. J. Serb. Chem. Soc. 75, 433–440. https://doi.
org/10.2298/JSC080810029P.
Journal of Ethnopharmacology 264 (2021) 113266
Poša, M., Kuhajda, K., 2010b. Hydrophobicity and hemolytic potential of oxo derivatives
of cholic, deoxycholic and chenodeoxycholic acids. Steroids 75, 424–431. https://
doi.org/10.1016/j.steroids.2010.02.008.
Ridlon, J.M., Harris, S.C., Bhowmik, S., Kang, D.J., Hylemon, P.B., 2016. Consequences
of bile salt biotransformations by intestinal bacteria. Gut Microb. 7, 22–39. https://
doi.org/10.1080/19490976.2015.1127483.
Sayyed-Ahmad, A., Lichtenberger, L.M., Gorfe, A.A., 2010. Structure and dynamics of
cholic acid and dodecylphosphocholine− cholic acid aggregates. Langmuir 26 (16),
13407–13414. https://doi.org/10.1021/la102106t.
Shin, B., Park, W., 2015. Synergistic effect of oleanolic acid on aminoglycoside
antibiotics against Acinetobacter baumannii. PLoS One 10 (9), e0137751. https://doi.
org/10.1371/journal.pone.0137751.
Synergy Software KaleidaGraph, Version 4.0, 2457 Perkiomen Avenue, PA 19606 USA.
Tangy, F., Moukkadem, M., Vindimian, E., Capmau, M.-L., Le Goffic, F., 1985.
Mechanism of action of gentamicin components. Eur. J. Biochem. 147, 381–386.
Trimble, M.J., Mlynárčik, P., Kolář, M., Hancock, R.E., 2016. Polymyxin: alternative
mechanisms of action and resistance. CSH Perspect. Med. 6 (10), a025288. https://
doi.org/10.1101/cshperspect.a025288.
Tserng, K.Y., 1978. A convenient synthesis of 3-keto bile acids by selective oxidation of
bile acids with silver carbonate-Celite. J. Lipid Res. 19, 501–504.
Verma, P., 2007. Methods for determining bactericidal activity and antimicrobial
interactions: synergy testing, time-kill curves, and population analysis. In:
Schwalbe, R., Steele-Moore, L., Goodwin, A.C. (Eds.), Antimicrobial Susceptibility
Testing Protocols. CRC Press, pp. 275–299.
Wang, D.Q., Carey, M.C., 2014. Therapeutic uses of animal biles in traditional Chinese
medicine: An ethnopharmacological, biophysical, chemical and medicinal review.
World J. Gastroenterol. 20, 9952–9975. https://doi.org/10.3748/wjg.v20.i29.9952.
Zhao, X., Xu, C., Domagala, J., Drlica, K., 1997. DNA topoisomerase targets of the
fluoroquinolones: a strategy for avoiding bacterial resistance. Proc. Natl. Acad. Sci.
U.S.A. 94, 13991e13996. https://doi:10.1073/pnas.94.25.13991.