Available via license: CC BY
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TYPE Original Research
PUBLISHED 15 December 2023
DOI 10.3389/fmicb.2023.1304198
OPEN ACCESS
EDITED BY
Corina Ciobanasu,
Alexandru Ioan Cuza University, Romania
REVIEWED BY
Veronica Godoy,
Northeastern University, United States
Qihui Zhou,
University of Health and Rehabilitation
Sciences, China
Yong Liu,
University of Chinese Academy of
Sciences, China
*CORRESPONDENCE
Hongping Wan
hwan@sicau.edu.cn
Xinghong Zhao
xinghong.zhao@sicau.edu.cn
†These authors have contributed equally to this
work
RECEIVED 29 September 2023
ACCEPTED 27 November 2023
PUBLISHED 15 December 2023
CITATION
Zhong X, Deng K, Yang X, Song X, Zou Y,
Zhou X, Tang H, Li L, Fu Y, Yin Z, Wan H and
Zhao X (2023) Brevicidine acts as an eective
sensitizer of outer membrane-impermeable
conventional antibiotics for Acinetobacter
baumannii treatment.
Front. Microbiol. 14:1304198.
doi: 10.3389/fmicb.2023.1304198
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©2023 Zhong, Deng, Yang, Song, Zou, Zhou,
Tang, Li, Fu, Yin, Wan and Zhao. This is an
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(CC BY). The use, distribution or reproduction
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does not comply with these terms.
Brevicidine acts as an eective
sensitizer of outer
membrane-impermeable
conventional antibiotics for
Acinetobacter baumannii
treatment
Xinyi Zhong1,2†, Kai Deng2,3† , Xiuhan Yang1,2†, Xu Song1,2,
Yuanfeng Zou1,2, Xun Zhou1,2, Huaqiao Tang1,2, Lixia Li1,2,
Yuping Fu1,2, Zhongqiong Yin1,2, Hongping Wan2,3*and
Xinghong Zhao1,2,3*
1Center for Sustainable Antimicrobials, Department of Pharmacy, Sichuan Agricultural University,
Chengdu, China, 2Center for Infectious Diseases Control (CIDC), Sichuan Agricultural University,
Chengdu, China, 3Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan
Agricultural University, Chengdu, China
The antibiotic resistance of Acinetobacter baumannii poses a significant threat to
global public health, especially those strains that are resistant to carbapenems.
Therefore, novel strategies are desperately needed for the treatment of infections
caused by antibiotic-resistant A. baumannii. In this study, we report that
brevicidine, a bacterial non-ribosomally produced cyclic lipopeptide, shows
synergistic eects with multiple outer membrane-impermeable conventional
antibiotics against A. baumannii. In particular, brevicidine, at a concentration
of 1 µM, lowered the minimum inhibitory concentration of erythromycin,
azithromycin, and rifampicin against A. baumannii strains by 32–128-fold.
Furthermore, mechanistic studies were performed by employing erythromycin
as an example of an outer membrane-impermeable conventional antibiotic,
which showed the best synergistic eects with brevicidine against the tested A.
baumannii strains in the present study. The results demonstrate that brevicidine
disrupted the outer membrane of A. baumannii at a concentration range of
0.125–4 µM in a dose-dependent manner. This capacity of brevicidine could
help the tested outer membrane-impermeable antibiotics enter A. baumannii
cells and thereafter exert their antimicrobial activity. In addition, the results show
that brevicidine–erythromycin combination exerted strong A. baumannii killing
capacity by the enhanced inhibition of adenosine triphosphate biosynthesis and
accumulation of reactive oxygen species, which are the main mechanisms causing
the death of bacteria. Interestingly, brevicidine and erythromycin combination
showed good therapeutic eects on A. baumannii-induced mouse peritonitis–
sepsis models. These findings demonstrate that brevicidine is a promising
sensitizer candidate of outer membrane-impermeable conventional antibiotics for
treating A. baumannii infections in the post-antibiotic age.
KEYWORDS
antibiotic-resistant, brevicidine, synergistic eects, Acinetobacter baumannii,
bacteraemia model
Frontiers in Microbiology 01 frontiersin.org
Zhong et al. 10.3389/fmicb.2023.1304198
Introduction
Acinetobacter baumannii is a nosocomial Gram-negative
pathogen that is responsible for hospital-acquired infections,
such as respiratory tract infections, bacteremia, urinary tract
infections, surgical wound infections, and meningitis (Peleg
et al., 2008;Fishbain and Peleg, 2010;Whiteway et al., 2022).
Resistance to the last-resort antibiotic carbapenem makes this
bacterium an urgent threat to public health and a member of the
most problematic nosocomial ESKAPE pathogens. Carbapenem-
resistant A. baumannii has been classified by the World Health
Organization (WHO) as a class of bacterium for which research
and development of new therapeutic strategies are critically needed
(Tacconelli et al., 2018). Unfortunately, the fact is that the number
of newly approved first-in-class antibiotics has been steadily
decreasing in the past two decades, especially the number of
antibiotics for the treatment of infections caused by Gram-negative
pathogens (Batta et al., 2020;Brown and Wobst, 2021;Butler
et al., 2023). Therefore, novel therapeutic strategies are desperately
needed for the treatment of infections caused by antibiotic-resistant
A. baumannii. The development of antibiotic sensitizers is an
effective strategy to restore the antimicrobial activity of antibiotics
against resistant pathogens.
Brevicidine (Bre), a bacterial non-ribosomally produced cyclic
lipopeptide, was found in Brevibacillus laterosporus DSM25 by
genome mining. Previous studies have shown that brevicidine
has potent and selective antimicrobial activity against gram-
negative pathogens, including Enterobacter cloacae,Escherichia coli,
Pseudomonas aeruginosa, and Klebsiella pneumoniae (Li et al.,
2018;Zhao et al., 2020a;Zhao and Kuipers, 2021a), which are
members of the critical pathogens listed by WHO (Tacconelli
et al., 2018). However, brevicidine showed much less antimicrobial
activity against Acinetobacter baumannii, which is an important
member of the critical pathogens listed by WHO (Li et al., 2018;
Tacconelli et al., 2018;Zhao and Kuipers, 2021a). Our previous
study demonstrates that brevicidine exerts its potent bactericidal
activity against E. coli by interacting with LPS on the outer
membrane and targeting phosphatidylglycerol and cardiolipin on
the inner membrane, thereby dissipating the proton motive force
of bacteria, which results in metabolic perturbations (Zhao et al.,
2023). Considering a different mechanism of action of brevicidine
from conventional antibiotics, we hypothesized that it could serve
as an antibiotic sensitizer for conventional antibiotics in controlling
infections caused by A. baumannii.
In this study, we evaluated the antimicrobial activity of
11 conventional antibiotics in combination with brevicidine
against antibiotic-resistant A. baumannii. The results show that
brevicidine exhibited synergistic effects with multiple outer
membrane-impermeable conventional antibiotics, including
erythromycin, azithromycin, rifampicin, vancomycin, and
meropenem, against the tested antibiotic-resistant A. baumannii
strains. Subsequently, the mechanism by which brevicidine
sensitizes A. baumannii to outer membrane-impermeable
conventional antibiotics was investigated by employing multiple
fluorescent probes. The results demonstrate that brevicidine
sensitizes antibiotic-resistant A. baumannii to outer membrane-
impermeable conventional antibiotics by disrupting the bacterial
outer membrane and thereafter promotes the entry of antibiotics
for treating the pathogens, which results in enhanced metabolic
perturbations, including the accumulation of reactive oxygen
species (ROS) in bacteria and inhibition of adenosine triphosphate
(ATP) synthesis. Finally, the mouse peritonitis–sepsis models
demonstrate that brevicidine has potent synergistic effects with
erythromycin, a member of outer membrane-impermeable
conventional antibiotics, in vivo. This study provides an alternative
antibiotic sensitizer for controlling infections caused by the critical
antibiotic-resistant pathogen, carbapenem-resistant A. baumannii.
Materials and methods
Ethical statement
All animal experiments conformed to the Guide for the Care
and Use of Laboratory Animals from the National Institutes of
Health, and all procedures were approved by the Animal Research
Committee of Sichuan Agricultural University, Sichuan, China.
Purification of brevicidine
Methods for the purification of brevicidine have been described
in detail in a previous study (Zhao et al., 2020a), and the purity
of purified brevicidine in trace amounts was analyzed by high-
performance liquid chromatography, which showed more than
99% purity (Zhao et al., 2023). Brevicidine was dissolved in Mili-Q
water at a concentration of 2.56 mM as the mother solution.
Antibiotics used in this study
Nisin (CAS#: 1414-45-5, ≥95%) was purchased from Handary
S.A. (Brussels, Belgium); tetracycline (CAS#: 64-75-5, ≥900
mcg/mg), amikacin (CAS#: 39831-55-5, ≥98%), erythromycin
(CAS#: 114-07-8, 850 µg/mg), azithromycin (CAS#: 83905-01-5,
≥98%), rifampicin (CAS#: 13292-46-1, ≥97%), penicillin G (CAS#:
113-98-4, ≥1500 µ/mg), and ampicillin (CAS#: 69-52-3, ≥98%)
were purchased from Hefei Bomei Biotechnology Co., Ltd (China);
vancomycin (CAS#: 1404-93-9, 900 µg/mg) was purchased from
Shanghai Aladdin Biochemical Technology Co., Ltd (China); and
meropenem (CAS#: 119478-59-7, ≥98%) was purchased from
Shanghai Macklin Biochemical Co., Ltd (China). Polymyxin B
(CAS#: 1405-20-5, ≥6,000 µ/mg) was purchased from Beijing
Solarbio Science & Technology Co., Ltd (China). Tetracycline,
erythromycin, azithromycin, rifampicin, vancomycin, penicillin G,
and meropenem were dissolved in dimethyl sulfoxide (DMSO) at
a concentration of 25.6 mM for preparing the mother solution,
while amikacin, nisin, polymyxin B, and ampicillin were dissolved
in Mili-Q water at a concentration of 25.6 mM for preparing the
mother solution. The final concentration of DMSO in the testing
culture is 1% or less (v/v), which is safe for the growth of bacteria.
In addition, the control experiments were treated with the relevant
solvent as controls.
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Zhong et al. 10.3389/fmicb.2023.1304198
Bacterial strains used and growth
conditions
A. baumannii ATCC 17978 and A. baumannii ATCC19606
were purchased from American Type Culture Collection (ATCC).
The clinical A. baumannii strain was isolated from Chengdu,
Sichuan Province of China. All bacterial strains were inoculated
in LB and incubated at 37◦C with aeration at 220 revolutions per
minute (rpm) for preparing the overnight cultures.
Synergy assay
The synergistic effect of brevicidine with conventional
antibiotics was monitored by employing a MIC assay as the
method described in previous studies (Wiegand et al., 2008;
Zhao and Kuipers, 2021b;Ding et al., 2023). In brief, antibiotics
were 2-fold serially diluted, while brevicidine was added at a
certain concentration. Indicator strains were added at a final
concentration of 5 ×105cfu/ml. After incubation at 37◦C
for 20 h, the OD600 of plates was determined. The fractional
inhibitory concentration index (FICI) was calculated using the
following formula: FICI =(MIC brevicidine in combination with
antibiotic/MIC brevicidine) +(MIC antibiotic in combination with
brevicidine/MIC antibiotic). The FICI value suggests synergistic
(≤0.5), addictive (>0.5–1), no interaction (1–4), and antagonism
(>4) effects of the two compounds (Doern, 2014).
Outer membrane permeability assay
This assay was performed according to the procedure described
in previous studies (Song et al., 2020;Xia et al., 2021). To investigate
the influence of brevicidine on the integrity of the outer membrane,
the fluorescent probe N-phenyl-1-naphthylamine (NPN, Aladdin)
was employed. A fresh culture of A. baumannii ATCC 17978
was pelleted at 4,000 g for 5 min and washed three times with
10 mM HEPES containing 10 mM glucose (GHEPES, pH 7.2). After
normalization of the cell density to an OD600 of 0.2 in GHEPES,
NPN was added at a final concentration of 30 µM and incubated
for 30 min in the dark for probe fluorescence to stabilize. After
the cell suspension (190 µ) was added to a 96-well microplate,
compounds (10 µl) were added, with the antimicrobials added after
approximately 20 s, and fluorescence was monitored for 25 min.
Fluorescence was recorded by using a Thermo Scientific Varioskan
Flash spectral scanning multimode microplate reader with an
excitation wavelength of 350 nm and an emission wavelength of
420 nm.
Membrane integrity assay
This assay was performed according to the procedure described
in a previous study (Zhao et al., 2020b). A fresh culture of A.
baumannii ATCC 17978 was pelleted at 4,000g for 5 min and
washed three times with Mueller Hinton Broth (MHB). After
normalization of the cell density to an OD600 of 0.2, propidium
iodide was loaded at a final concentration 2.5 µg/ml and incubated
for 10 min in the dark for probe fluorescence to stabilize. After
the cell suspension was added to a 96-well microplate, brevicidine
(0.125–4 µM), tetracycline (2 µM), or polymyxin B (2 µM) were
added, with the antimicrobials added after approximately 20 s,
and fluorescence was monitored for 120 min. Fluorescence was
recorded by using a Thermo Scientific Varioskan Flash multimode
microplate reader with an excitation wavelength of 533 nm and an
emission wavelength of 617 nm.
DiSC3(5) assay
A. baumannii ATCC 17978 was grown to an OD600 of 0.8. The
culture was pelleted at 4,000 ×g for 5 min and washed three times
with MHB. The cell density was normalized to an OD600 of 0.2,
loaded with 2 µM DiSC3(5) dye, and incubated for 30 min in the
dark for probe fluorescence to stabilize. After incubation, the cell
suspension was added to a 96-well microplate and incubated for
15 min. After that, the cells were treated with brevicidine (0.125–
4µM), tetracycline (2 µM), or polymyxin B (2 µM). Fluorescence
was monitored for 55 min, with the compounds added after
approximately 20 s. Fluorescence was recorded by using a Thermo
Scientific Varioskan Flash multimode microplate reader with an
excitation wavelength of 622 nm and an emission wavelength of
670 nm.
Fluorescence microscopy assay
A. baumannii ATCC 17978 was grown to an OD600 of 0.8 in
MHB. The culture was pelleted at 4,000 g for 5 min and washed
three times with MHB. After normalization of the cell density to
an OD600 of 0.2 in MHB, A. baumannii cells were then challenged
with bre vicidine (4 µM), brevicidine (2 µM), brevicidine (1 µM),
erythromycin (0.25 µM), brevicidine (4 µM) plus erythromycin
(0.25 µM), brevicidine (2 µM) plus erythromycin (0.125 µM),
brevicidine (1 µM) plus erythromycin (0.625 µM), tetracycline
(2 µM), or polymyxin B (2 µM). After incubation at 37◦C for 5 min,
cells were collected by centrifugation. Subsequently, NucGreen and
EthD-III (LIVE/DEAD Bacterial Viability Kit, Solarbio, catalog no.
EX3000) were added to the above cells. After incubation at room
temperature for 15 min, cells were washed three times with MHB.
Then, the cell suspensions were loaded on 1.5% agarose pads and
analyzed by a Nikon 80i microscope (Japan).
Time-killing assay
This assay was performed according to a previously described
procedure (Ling et al., 2015;Zhao and Kuipers, 2021c;Zhao
et al., 2021b,c;Zhan et al., 2023). An overnight culture of A.
baumannii ATCC 17978 was diluted 50-fold in MHB and incubated
at 37◦C with aeration at 220 rpm. Bacteria were grown to an
OD600 of 0.8, and then, the concentration of cells was adjusted
to ≈1×107cells per ml. A. baumannii were then challenged
with bre vicidine (4 µM), brevicidine (2 µM), brevicidine (1 µM),
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Zhong et al. 10.3389/fmicb.2023.1304198
erythromycin (0.25 µM), brevicidine (4 µM) plus erythromycin
(0.25 µM), brevicidine (2 µM) plus erythromycin (0.125 µM),
brevicidine (1 µM) plus erythromycin (0.625 µM), or polymyxin
B (4 µM) in culture tubes at 37◦C and 220 rpm. Non-treated A.
baumannii was used as untreated control. At desired time points,
200 µl aliquots were taken, centrifuged at 6,000 g for 5 min, and
resuspended in 200 µl of MHB. Overall, 10-fold serially diluted
samples were plated on MHA plates. After incubation at 37◦C
overnight, colonies were counted and c.f.u. per mL was calculated.
ATP measurement
The intracellular ATP levels were measured using a commercial
Enhanced ATP Assay Kit (Beyotime, catalog no. S0027). A fresh
culture of A. baumannii was pelleted at 4,000 g for 5 min and
washed three times with MHB. The cell density was normalized
to an OD600 of 0.2 and loaded with different components. At
1 h post treatment, cells were collected and lysed with grinding
beads. After centrifugation at 12,000 g for 10 min, 100 µl of the
supernatant was taken out, mixed with 100 µl of detecting solution,
and incubated for 5 min at room temperature. Luminescence was
measured with a Thermo Scientific Varioskan Flash multimode
microplate reader. Carbonyl cyanide m-chlorophenyl hydrazone
(CCCP, Sigma–Aldrich, CAS:555-60-2) (20 µg/ml) was used as a
positive control. The relative ATP levels were calculated using
the measured luminescence values vs. the luminescence value of
untreated cells.
Determination of reactive oxygen species
(ROS)
The levels of ROS in A. baumannii treated with different
components were measured by employing the fluorescent probe
2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Hu et al., 2023;Li
et al., 2023), following the manufacturer’s instruction (Beyotime,
catalog no. S0033S). In brief, a fresh culture of A. baumannii was
pelleted at 4,000 g for 5 min and washed t hree times with MHB.
The cell density was normalized to an OD600 of 1.0 and loaded with
DCFH-DA at a final concentration of 10 µM, and the mixture was
incubated at 37◦C for 30 min. After washing three times with MHB,
190 µl of probe-labeled bacterial cells, followed by 10 µl of different
components, were added to a 96-well plate. Fluorescence was
recorded by using a Thermo Scientific Varioskan Flash multimode
microplate reader with the excitation wavelength of 488 nm and
the emission wavelength of 525 nm. The antioxidant N-acetyl-L-
cysteine (NAC, 6mM) was used as a control to neutralize the
production of ROS.
Mouse peritonitis–sepsis models
To assess the in vivo bioavailability of brevicidine and
erythromycin combination in A. baumannii-induced mouse
peritonitis–sepsis model, 70 BALB/c male mice (n=10 per group)
were infected intraperitoneally with A. baumannii (ATCC 17978)
at a dose of 2 ×109c.f.u. per mouse that leads to 80% of death. At
1 h post-infection, mice were treated with brevicidine (5 mg/kg),
erythromycin (5 mg/kg), brevicidine (5 mg/kg) plus erythromycin
(1.25 mg/kg), brevicidine (5 mg/kg) plus erythromycin (2.5 mg/kg),
brevicidine (5 mg/kg) plus erythromycin (5 mg/kg), or 0.9% NaCl
via intravenous injection. Mice without A. baumannii infections
were used as the non-infection control. The survival rates of
different groups were monitored for 7 days.
To gain a deeper insight into the synergistic effect of brevicidine
and erythromycin in vivo, 36 BALB/c male mice (n=6 per group)
were infected intraperitoneally with A. baumannii (ATCC 17978)
at a dose of 1 ×109c.f.u. per mouse that does not lead to death. At
1 h post-infection, mice were treated with brevicidine (5 mg/kg),
erythromycin (5 mg/kg), brevicidine (5 mg/kg) plus erythromycin
(1.25 mg/kg), brevicidine (5 mg/kg) plus erythromycin (2.5 mg/kg),
brevicidine (5 mg/kg) plus erythromycin (5 mg/kg), or 0.9% NaCl
via intravenous injection. Mice without A. baumannii infections
were used as the non-infection control. At 24 h post-infection,
organs, including the heart, liver, spleen, lung, and kidney, were
collected to measure the bacterial load.
Statistical analysis
GraphPad Prism 8.0 was used to fit the data in Figures 1,3,
4B–G. The statistical significance of the data was assessed using a
two-tailed Student’s t-test with GraphPad Prism 8.0. Correlation
analyses were evaluated by Pearson r2test, ns: p>0.05, ∗p<0.05,
∗∗p<0.01, ∗∗∗ p<0.001, and ∗∗∗∗p<0.0001.
Results and discussion
Brevicidine shows good synergistic eects
with outer membrane-impermeable
conventional antibiotics against A.
baumannii
To assess the synergistic effects of brevicidine with 11
conventional antibiotics, a synergy assay was performed
according to the method described in previous studies with
mild modifications (Wiegand et al., 2008;Cochrane and Vederas,
2014;Zhao et al., 2021a). Nisin and vancomycin are lipid II-
targeting antibiotics that show potent antimicrobial activity against
Gram-positive pathogens (Breukink et al., 2003), while they are
less active against most Gram-negative pathogens due to the outer
membrane barrier. Rifampicin is an RNA-inhibiting antibiotic and
is usually used in combination with other antibiotics (Campbell
et al., 2001). Erythromycin (Ery) and azithromycin are antibiotics
that exert their bacteriostatic antimicrobial activity by targeting
the bacterial 50S ribosomal subunit (Tanaka et al., 1973;Parnham
et al., 2014), while tetracycline (Tet) and amikacin are antibiotics
that exert their antimicrobial activity by targeting the bacterial
30S ribosomal subunit (Speer et al., 1992;Alangaden et al., 1998).
Penicillin G, ampicillin, and meropenem are bacteria cell wall
synthesis inhibitors, and these antibiotics have good antimicrobial
activity against gram-negative pathogens; however, bacterial
resistance is common for these antibiotics (Sumita et al., 1992;
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FIGURE 1
Brevicidine sensitizes A. baumannii to conventional antibiotics by disrupting the outer membrane and proton motive force. (A) NPN fluorescence in
A. baumannii ATCC17978 cells upon exposure to brevicidine at a concentration range of 0.125–4 µM. PolyB and Tet were used as outer membrane
disruption and non-outer membrane disruption antibiotic controls, respectively. Data are presented as means (n=3). (B) A. baumannii ATCC17978
cells pretreated with propidium iodide were exposed to brevicidine at a concentration range of 0.125–4 µM, and the extent of membrane leakage
was visualized as an increase in fluorescence. PolyB and Tet were used as membrane disruption and non-membrane disruption antibiotic controls,
respectively. Data are presented as means (n=3). (C) DiSC3(5) fluorescence in A. baumannii ATCC17978 cells upon exposure to brevicidine at a
concentration range of 0.125–4 µM. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and Tet were used as proton motive force disruption and
non-proton motive force disruption compound controls, respectively. Data are presented as means (n=3).
Sugimoto et al., 2002;Ono et al., 2005). Polymyxin B (PolyB) is
a bactericidal antibiotic that targets the Gram-negative bacteria
envelope (Trimble et al., 2016).
The synergy test results showed that brevicidine has synergistic
effects with tested outer membrane-impermeable conventional
antibiotics against A. baumannii (Tables 1–3). The increasing
synergistic effects were observed with erythromycin, showing
a decrease in minimum inhibitory concentration (MIC) of
128-fold against all tested A. baumannii strains, including
a clinically isolated carbapenem-resistant A. baumannii strain
(Tables 1–3). A previous study showed that a cathelicidin-derived
membrane-targeting peptide D-11 has shown synergistic effects
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FIGURE 2
Fluorescence microscopy images of A. baumannii ATCC17978 cells, which were challenged with brevicidine (4 µM), brevicidine (2 µM), brevicidine
(1 µM), erythromycin (0.25 µM), brevicidine (4 µM) plus erythromycin (0.25 µM), brevicidine (2 µM) plus erythromycin (0.125 µM), brevicidine (1 µM)
plus erythromycin (0.625 µM), tetracycline, or polymyxin B (2 µM) for 5 min. Green denotes a cell with an intact membrane, whereas red denotes a
cell with a compromised membrane.
with erythromycin against Klebsiella pneumoniae; the MIC of
erythromycin decreased 64-fold, from 128 µM to 4 µM, in the
presence of D-11 at a concentration of 4 µM (Cebrián et al., 2021).
In this regard, brevicidine is much more capable than D-11 because
the MIC of erythromycin decreased 128-fold, from 8/16 µM to
0.0625/0.125 µM, in the presence of brevicidine at a concentration
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Zhong et al. 10.3389/fmicb.2023.1304198
FIGURE 3
Brevicidine and outer membrane-impermeable conventional antibiotic (erythromycin) combination shows augmented A. baumannii killing capacity
via enhanced inhibition of ATP synthesis and accumulation of ROS. (A) Time killing curve of brevicidine (4 µM), brevicidine (2 µM), brevicidine (1 µM),
erythromycin (0.25 µM), brevicidine (4 µM) plus erythromycin (0.25 µM), brevicidine (2 µM) plus erythromycin (0.125 µM), brevicidine (1 µM) plus
erythromycin (0.625 µM), and polymyxin B (4 µM) against A. baumannii.(B) Relative ATP concentration of A. baumannii cells treated with brevicidine
(4 µM), brevicidine (2 µM), brevicidine (1 µM), erythromycin (0.25 µM), brevicidine (4 µM) plus erythromycin (0.25 µM), brevicidine (2 µM) plus
erythromycin (0.125 µM), brevicidine (1 µM) plus erythromycin (0.625 µM), CCCP, 20 mg/ml, and tetracycline (2 µM) for 1 h. All data were presented
as means ±standard deviation (n=3). Correlation analyses were evaluated by Pearson r2test. *p<0.05; ***p<0.001. (C) Accumulation of ROS in A.
baumannii cells treated with brevicidine (4 µM), brevicidine (2 µM), brevicidine (1 µM), erythromycin (0.25µM), brevicidine (4 µM) plus erythromycin
(0.25 µM), brevicidine (2 µM) plus erythromycin (0.125 µM), brevicidine (1 µM) plus erythromycin (0.625µM), brevicidine (2 µM) plus erythromycin
(0.125 µM), N-Acetyl-L-cysteine (NAC, 6 mM), polymyxin B (2 µM), and tetracycline (2 µM). NAC, antioxidant N-Acetyl-L-cysteine.
of 1/2 µM. Strong synergistic effects were also found between
brevicidine and rifampicin/azithromycin, with a decrease in MIC of
32–128-fold against tested A. baumannii strains (Tables 1–3). This
response is expected because rifampicin and azithromycin have
shown synergistic effects with multiple membrane-active peptides
(Cochrane and Vederas, 2014;Song et al., 2020;Cebrián et al.,
2021;Xia et al., 2021). However, only weak synergistic effects
were observed between brevicidine and the two tested anti-30S
ribosomal subunit antibiotics, namely, tetracycline and amikacin.
Moderate synergistic effects were observed between brevicidine
and vancomycin against two A. baumannii reference strains
(ATCC17978 and ATCC19606), with a decrease in MIC of 8-fold
(Tables 1,2). Notably, the antimicrobial activity of vancomycin
against the clinically isolated carbapenem-resistant A. baumannii
strain increased 64-fold in the presence of brevicidine at a
concentration of 2 µM (Table 3). Interestingly, the antimicrobial
activity of nisin increased 8-fold in the presence of brevicidine,
which was unexpected due to its large molecular size (3.4 kDa).
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FIGURE 4
Brevicidine shows a good synergistic eect with conventional antibiotic (erythromycin) in mouse peritonitis–sepsis models. (A) Schemes of the
experimental protocol for the mouse peritonitis–sepsis models. (B) Survival rates of mice in the mouse peritonitis–sepsis model (n=10). Increased
survival rates of mice for 7 days by a dose that leads to 80% of death of A. baumannii (2.0 ×109c.f.u.), treated with brevicidine (5 mg/kg), brevicidine
(5 mg/kg) plus erythromycin (1.25 mg/kg), brevicidine (5 mg/kg) plus erythromycin (2.5 mg/kg), or brevicidine (5 mg/kg) plus erythromycin (5 mg/kg)
are shown. (C–G) Brevicidine and erythromycin combination significantly reduced the bacterial load of organs of the mouse peritonitis–sepsis
model. At 24 h post-infection, the mice (n=6) were euthanized by cervical dislocation. Bacterial loads (Log10 c.f.u. per gram of A. baumannii) of the
heart (C), liver (D), spleen (E), lung (F), and kidney (G) were counted. All data were presented as means ±standard deviation (n=6). Correlation
analyses were evaluated by Pearson r2test. ns, no significance; *p<0.05; **p<0.01; ***p<0.001; and ****p<0.0001.
Li et al. reported that a series of outer-membrane-acting peptides
have shown good synergistic effects with lipid II-targeting peptide
antibiotics, vancomycin and nisin, against A. baumannii strains
(Li et al., 2021). Our results show that brevicidine exhibited
comparable synergistic effects with vancomycin than these peptides
with vancomycin.
Brevicidine showed good synergistic effects with meropenem,
a member of carbapenem antibiotics, against two A. baumannii
reference strains (ATCC17978, ATCC19606), with a decrease in
MIC of 8-fold (Tables 1,2). Notably, brevicidine restored the
antimicrobial activity of meropenem against a clinically isolated
carbapenem-resistant A. baumannii strain (Table 3). However, no
synergistic effects were observed with penicillin G or ampicillin
because these two antibiotics are outer membrane permeable,
and the strains of the above two antibiotics carry beta-lactamase.
Brevicidine showed no synergistic effects with polymyxin B, which
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TABLE 1 Synergy between brevicidine and antibiotics against A. baumannii ATCC17978.
Family/antibiotic MICaat brevicidine concentrationsaof MMD FICI
0 0.25 0.5 1
Tetracyclines
Tetracycline 2 2 1 1 2x 0.63
Aminoglycosides
Amikacin 8 4 4 2 4x 0.5
Macrolides
Erythromycin 8 1 0.5 0.06 128x 0.19
Azithromycin 16 2 1 0.5 32x 0.19
Rifamycins
Rifampicin 2 1 0.25 0.06 32x 0.25
Lipid II targeting peptide antibiotics
Vancomycin 32 16 8 4 8x 0.38
Nisin 4 2 2 0.5 8x 0.38
β-Lactams
Penicillin G 128 128 64 64 2x 0.63
Ampicillin 256 256 128 128 2x 0.63
Meropenem 4 2 0.5 0.5 8x 0.25
Polymyxins
Polymyxin B 1 1 1 1 0 1.25
MIC, minimum inhibitory concentration; MMD, maximum MIC decrease; FICI, fractional inhibitory concentration index [FICI =(MIC brevicidine in combination with antibiotic/MIC
brevicidine) +(MIC antibiotic in combination with brevicidine/MIC antibiotic)].
aall values reported in µM. Brevicidine showed a MIC of 4 µM against A. baumannii ATCC17978.
is a bactericidal antibiotic that exerts its antimicrobial activity
against Gram-negative bacteria by targeting the cell envelope
(Trimble et al., 2016). This can be further explained by the
fluorescent probe assays. Brevicidine shows synergistic effects with
some tested antibiotics due to its outer membrane disruption
ability; however, polymyxin B can pass outer membrane by itself.
Together, these results demonstrate that brevicidine is an effective
sensitizer of A. baumannii to outer membrane-impermeable
conventional antibiotics, such as erythromycin, azithromycin,
rifampicin, vancomycin, and meropenem.
Brevicidine sensitizes A. baumannii to
conventional antibiotics by disrupting the
outer membrane and proton motive force
To investigate the influence of brevicidine on A. baumannii
membrane, three fluorescent probe assays were performed.
N-Phenyl-1-naphthylamine (NPN) is a gram-negative bacterial
impermeable fluorescent probe due to the outer membrane
barrier (Helander and Mattila-Sandholm, 2000). If the integrity
of the outer membrane is disrupted, this fluorescent probe
can reach the phospholipid layer, resulting in a significant
increase in fluorescence. The results show that the fluorescence
signal was increased by the addition of brevicidine at a
concentration range of 0.125–4 µM in a dose-dependent manner
(Figure 1A), demonstrating that brevicidine disrupted the
outer membrane of A. baumannii at sub-MIC concentrations.
This capacity of brevicidine could promote outer membrane-
impermeable antibiotics, such as erythromycin, azithromycin,
and vancomycin, enter A. baumannii and thereafter exert their
antimicrobial activity.
Propidium iodide (PI) is a membrane-permeant fluorescent
probe. If the membrane integrity is disrupted, PI can enter
bacteria and bind to nucleic acid, resulting in a prominent
increase in fluorescence (Zhao et al., 2020b). The fluorescence
signal indicated no significant change after the addition of
brevicidine at a concentration range of 0.125–4 µM during 2 h of
monitoring (Figure 1B), demonstrating that brevicidine sensitizes
A. baumannii to outer membrane-impermeable conventional
antibiotics via disrupting the cytoplasmic membrane.
Our previous study shows that brevicidine can disrupt the
proton motive force of E. coli (Zhao and Kuipers, 2021a;Zhao et al.,
2023). To test the effect of brevicidine on the proton motive force of
A. baumannii, a DiSC3(5) (3,3′-dipropylthiadicarbocyanine iodide)
fluorescent probe assay was performed. DiSC3(5) accumulates in
the cytoplasmic membrane of bacteria in response to the 1ψ
component of the proton motive force (Wu et al., 1999;Stokes
et al., 2020). When the transmembrane 1pH potential of bacteria
is disrupted, cells compensate by increasing the 1ψ, resulting
in enhanced DiSC3(5) uptake into the cytoplasmic membrane
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TABLE 2 Synergy between brevicidine and antibiotics against A. baumannii ATCC19606.
Bacteria/antibiotic MICaat brevicidine concentrationsaof MMD FICI
0 0.25 0.5 1
Tetracyclines
Tetracycline 2 2 2 0.5 4x 0.5
Aminoglycosides
Amikacin 8 8 8 2 4x 0.5
Macrolides
Erythromycin 16 2 2 0.13 128x 0.19
Azithromycin 128 16 16 1 128x 0.19
Rifamycins
Rifampicin 1 0.25 0.06 0.02 64x 0.19
Lipid II targeting peptide antibiotics
Vancomycin 64 16 16 4 16x 0.31
Nisin 4 2 2 0.5 8x 0.38
β-Lactams
Penicillin G 256 256 256 128 2x 0.75
Ampicillin 256 256 256 256 0 1.25
Meropenem 8 2 1 1 8x 0.25
Polymyxins
Polymyxin B 1 1 1 1 0 1.25
MIC, minimum inhibitory concentration; MMD, maximum MIC decrease; FICI, fractional inhibitory concentration index [FICI =(MIC brevicidine in combination with antibiotic/MIC
brevicidine) +(MIC antibiotic in combination with brevicidine/MIC antibiotic)].
aall values reported in µM. Brevicidine showed a MIC value of 4 µM against A. baumannii ATCC19606 strains.
and therefore decreased fluorescence (Wu et al., 1999;Stokes
et al., 2020). The fluorescence signal of DiSC3(5) was decreased
(Figure 1C) after treatment with brevicidine at a concentration
range of 0.125–4 µM in a dose-dependent manner (Figure 1C),
indicating that brevicidine dissipated the proton motive force of
A. baumannii.
To investigate the influence of brevicidine and outer
membrane-impermeable conventional antibiotic combination
on the membrane integrity of A. baumannii, membrane
permeability assays were performed by using a commercial
LIVE/DEAD Bacterial Viability kit, which contains NucGreen
and EthD-III. Cells with an intact membrane will stain
green, whereas cells with a compromised membrane will
stain red. Polymyxin B (PolyB) was used as a membrane
disruption antibiotic control, while tetracycline was used as
an antibiotic control without membrane disruption. After
incubation for 5 min, green cells were obser ved for brevicidine
or brevicidine-erythromycin combination treated A. baumannii,
demonstrating that brevicidine-erythromycin combination
shows enhanced antimicrobial activity, which does not occur
via disrupting the cytoplasmic membrane integrity of A.
baumannii (Figure 2), which is consistent with the results of
PI fluorescent probe assay (Figure 1B). These results demonstrate
that erythromycin, a 50S ribosomal subunit targeting bacteriostatic
antibiotic, did not influence the effect of brevicidine on the
cytoplasmic membrane.
Brevicidine and outer membrane-
impermeable conventional antibiotic
(erythromycin) combination shows
augmented A. baumannii killing capacity via
enhanced ATP synthesis inhibition and ROS
accumulation
To assess the killing capacity of brevicidine and outer
membrane-impermeable conventional antibiotic (erythromycin)
combination, a time-killing assay was performed as the method
described in previous studies (Ling et al., 2015;Zhao et al., 2021b,c).
The results show that brevicidine and erythromycin combination
significantly enhanced the killing capacity of each against A.
baumannii (Figure 3A).
The results shown in Figure 1C indicate that brevicidine
dissipated the proton motive force of A. baumannii. The proton
motive force is essential for the generation of ATP, which is
an essential bioactive compound for live bacteria (Bakker and
Mangerich, 1981;Ahmed and Booth, 1983;Li et al., 2020). The
dissipation of the proton motive force of A. baumannii will inhibit
or even terminate the process of ATP biosynthesis. To investigate
the effect of brevicidine and erythromycin combination on the
ATP level of A. baumannii, the intracellular ATP levels of different
treatments were measured by a commercial Enhanced ATP Assay
Kit (Beyotime, catalog no. S0027). Compared with untreated cells,
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TABLE 3 Synergy between brevicidine and antibiotics against a clinically isolated carbapenem-resistant A. baumannii stain.
Antibiotic MICaat brevicidine concentrationsaof MMD FICI
0 0.5 1 2
Tetracyclines
Tetracycline 4 4 2 1 4x 0.5
Aminoglycosides
Amikacin 8 8 4 2 4x 0.5
Macrolides
Erythromycin 16 0.5 0.25 0.13 128x 0.14
Azithromycin 128 8 4 2 64x 0.16
Rifamycins
Rifampicin 0.5 0.02 0.01 0.01 64x 0.14
Lipid II targeting peptide antibiotics
Vancomycin 32 16 4 0.5 64x 0.25
Nisin 4 1 0.5 0.5 8x 0.25
β-Lactams
Penicillin G 256 256 256 128 2x 0.75
Ampicillin 256 256 256 256 0 1.06
Meropenem 16 2 2 0.5 32x 0.19
Polymyxins
Polymyxin B 1 1 1 1 0 1.25
MIC, minimum inhibitory concentration; MMD, maximum MIC decrease; FICI, fractional inhibitory concentration index [FICI =(MIC brevicidine in combination with antibiotic/MIC
brevicidine) +(MIC antibiotic in combination with brevicidine/MIC antibiotic)].
aall values reported in µM. The MIC value of brevicidine against the tested clinically isolated carbapenem-resistant A. baumannii stain was 8 µM.
the ATP levels of 4 and 2 µM brevicidine-treated cells were
significantly decreased (Figure 3B). Erythromycin did not influence
the ATP level of A. baumannii at a concentration of 0.25 µM,
expectably, because this concentration is much lower than its MIC.
Surprisingly, the ATP synthesis inhibition capacity of brevicidine
was significantly enhanced in combination with erythromycin at
relatively low concentrations (0.0625–0.25 µM) (Figure 3B). This
effect might be contributed to the protein synthesis ability of
erythromycin. Two previously reported membrane-active peptide
antibiotic sensitizers, namely, SLAP-S25 and D-11, have also shown
ATP synthesis inhibition activity (Song et al., 2020;Xia et al., 2021).
However, these studies failed to investigate if relative antibiotics
would enhance the ATP synthesis inhibition activity of these
membrane-active peptide antibiotic sensitizers.
The proton motive force plays a vital role in the removal of
reactive oxygen species (ROS) in bacteria (Berry et al., 2018;Zhao
et al., 2023). The accumulation of ROS is an important mechanism
in which antimicrobials exert their bacterial killing capacity (Guridi
et al., 2015;Clauss-Lendzian et al., 2018;Yu et al., 2020;Liu et al.,
2023). To determine the intracellular ROS levels of A. baumannii
after treatment with different concentrations of brevicidine or
brevicidine–erythromycin combination, a 2′,7′-dichlorofluorescein
diacetate (DCFH-DA) fluorescent probe-based assay was employed
(Zhao and Kuipers, 2021a;Zhao et al., 2023). DCFH-DA is a
bacterial cell-permeable non-fluorescent probe. This molecule can
be de-esterified intracellularly, and the de-esterified product turns
to highly fluorescent 2′,7′-dichlorofluorescein upon oxidation by
ROS. The results show that brevicidine caused ROS accumulation
in a dose-dependent manner (Figure 3C). However, erythromycin
did not cause ROS accumulation at a concentration of 0.25 µM.
Interestingly, the ROS accumulation capacity of brevicidine was
significantly enhanced in combination with erythromycin at
relatively low concentrations (0.0625–0.25 µM) (Figure 3C), which
is reasonably an important synergistic mechanism of brevicidine
and erythromycin.
Brevicidine and outer membrane-
impermeable conventional antibiotic
(erythromycin) combination shows good
therapeutic eects in mouse
peritonitis–sepsis models
Given the attractive synergistic effects between brevicidine
and outer membrane-impermeable conventional antibiotics, we
investigated the potential of brevicidine as an antibiotic sensitizer
in mouse peritonitis–sepsis models (Figure 4A). To assess the
protection effect of brevicidine–erythromycin combination on
A. baumannii-induced mouse peritonitis–sepsis models, mice
were infected intraperitoneally with A. baumannii at a dose
of 2 ×109c.f.u. per mouse that leads to 80% of death. At 1 h
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post-infection, drugs were introduced at single intravenous
doses (Figure 4A). Erythromycin showed no protective effect
on A. baumannii-induced mouse peritonitis–sepsis models at a
dose of 5 mg/kg (Figure 4B). Brevicidine reduced the death rate
of A. baumannii-infected mice from 80% to 60%. Excitingly,
brevicidine and erythromycin showed a good synergistic effect
on A. baumannii-induced mouse peritonitis–sepsis models, and
only one mouse died in the 5 mg/kg brevicidine and 2.5/1.25
mg/kg erythromycin combination-treated group. Notably, all of
the mice survived under a single dose of 5 mg/kg brevicidine
plus 5 mg/kg erythromycin combination treatment (Figure 4B).
These results demonstrate that brevicidine–erythromycin
combination has a much stronger therapeutic efficacy than either
of them alone.
To get a deeper insight into the synergistic effect of
brevicidine–erythromycin combination in vivo, mice were infected
intraperitoneally with A. baumannii at a dose of 1×109c.f.u. per
mouse that does not lead to death at 24 h post-infection. At 1 h
post-infection, drugs were introduced at a single intravenous dose
(Figure 4A). At 24 h post-infection, all mice were sacrificed, and
the hearts, livers, spleens, lungs, and kidneys were harvested for
bacterial load measurement. Compared with the untreated group,
erythromycin (5 mg/kg) showed no significant influence on the
bacterial load of all organs investigated (Figures 4C–G), which
could explain why it had no protection effect on the infected
mice (Figure 4B). Brevicidine reduced the bacterial load of all
organs investigated, and it significantly reduced (p<0.001) the
bacterial load of the liver and spleen (Figures 4C–G), which is
consistent with the finding that brevicidine slightly reduced the
death rate of A. baumannii-infected mice (Figure 4B). Compared
with the untreated group, brevicidine–erythromycin (5 mg/kg:1.25,
2.5, or 5 mg/kg) combination significantly (p< 0.0001) reduced
the bacterial load of all organs investigated (Figures 4C–G). In
addition, compared with the brevicidine alone or the erythromycin
alone treated groups, brevicidine–erythromycin (5 mg/kg:1.25, 2.5,
or 5 mg/kg) combination significantly (p<0.01) reduced the
bacterial load of all organs investigated (Figures 4C–G). Under a
dose of 5 mg/kg brevicidine treatment, the bacterial load reduction
capacity of brevicidine–erythromycin combination shows in an
erythromycin dose-dependent manner (Figures 4C–G). Together,
these findings demonstrate the potential of brevicidine as a
novel antibiotic sensitizer for the treatment of difficult-to-treat A.
baumannii infections in the post-antibiotic age.
Conclusion
In this study, we show that brevicidine, a bacterial non-
ribosomally produced cyclic lipopeptide, has a synergistic effect
with multiple outer membrane-impermeable conventional
antibiotics, such as erythromycin, azithromycin, rifampicin,
vancomycin, and meropenem, against the tested A. baumannii
strains, including a clinically isolated carbapenem-resistant
A. baumannii stain. Furthermore, mechanistic studies were
performed by using erythromycin as an outer membrane-
impermeable antibiotic example, which showed the best synergistic
effects with brevicidine against the tested A. baumannii strains
in the present study. The results demonstrate that brevicidine
can disrupt the outer membrane of A. baumannii, which helps
the tested outer membrane-impermeable antibiotics enter A.
baumannii cells and thereafter exert their antimicrobial activity.
In addition, the results show that brevicidine–erythromycin
combination has potent ATP biosynthesis inhibition and ROS
accumulation capacities that are the main mechanisms causing
death of bacteria. Notably, brevicidine and erythromycin showed
good synergistic effects in mouse peritonitis–sepsis models. These
findings demonstrate that brevicidine is a promising sensitizer
candidate of outer membrane-impermeable conventional
antibiotics for the treatment of A. baumannii infections in the
post-antibiotic age.
Data availability statement
The original contributions presented in the study are included
in the article/supplementary material, further inquiries can be
directed to the corresponding authors.
Ethics statement
The animal study was approved by the Animal Research
Committee of Sichuan Agricultural University. The study
was conducted in accordance with the local legislation and
institutional requirements.
Author contributions
XZhon: Conceptualization, Formal analysis, Investigation,
Methodology, Visualization, Writing – original draft. KD:
Conceptualization, Formal analysis, Investigation, Methodology,
Visualization, Writing – original draft. XY: Conceptualization,
Formal analysis, Investigation, Methodology, Visualization,
Writing – original draft. XS: Investigation, Validation,
Visualization, Formal analysis, Writing – review & editing.
YZ: Investigation, Validation, Visualization, Methodology, Writing
– review & editing. XZhou: Investigation, Validation, Visualization,
Methodology, Writing – review & editing. HT: Investigation,
Validation, Visualization, Formal analysis, Writing – review &
editing. LL: Investigation, Validation, Visualization, Methodology,
Writing – review & editing. YF: Investigation, Validation,
Methodology, Writing – review & editing. ZY: Investigation,
Supervision, Visualization, Methodology, Writing – review &
editing. HW: Conceptualization, Supervision, Writing – original
draft, Writing – review & editing. XZha: Conceptualization,
Supervision, Writing – original draft, Writing – review & editing.
Funding
The author(s) declare financial support was received for the
research, authorship, and/or publication of this article. This work
was supported by the “1000-Talent Program” in Sichuan Province
[XZha (2287) and HW (1923)]. XZha was supported by the Science
and Technology Project of Sichuan Province (2022YFH0057). HW
was supported by the National Natural Science Foundation of
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Zhong et al. 10.3389/fmicb.2023.1304198
China (32102689) and the Science and Technology Project of
Sichuan Province (2022YFH0062, 2022ZYD0068).
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
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